Dentin conditioning can affect the adhesion of stem cells in endodontic regenerative treatments. This study aimed to assess the effects of the most commonly used endodontic irrigants, namely, ethylenediaminetetraacetic acid (EDTA), MTAD, and QMix in comparison with Er:YAG laser  on the adhesion of stem cells from the apical papilla (SCAPs) to dentin. Forty dentin specimens were prepared and subjected to different treatments in 5 groups (n=8) of control, irrigation with EDTA for 1 minute, irrigation with MTAD for 5 minutes, irrigation with QMix for 5 minutes, and Er:YAG laser irradiation. SCAPs were isolated from third molar tooth buds that two-thirds of their roots had formed. The cells were cultured on dentin specimens for 3 days and were counted using scanning electron microscopy (SEM). P<0.05). All other modalities induced cell adhesion with no significant difference with each other (P>0.05). MTAD resulted in significantly lower adhesion of cells to dentin compared with other groups (Despite many favorable properties, MTAD cannot serve as an optimal irrigant in endodontic regenerative procedures since it inhibits the adhesion of SCAPs to dentin and impairs an important step in tissue engineering. Key words:Endodontic Regeneration, Er-YAG laser, MTAD, QMix, EDTA, SCAP, stem cell adhesion. Endodontic regenerative procedures are biologically-based processes designed for predicTable replacement of the lost or injured tooth structures such as dentin, root, or dentin-pulp complex. Ideally, the lost tooth structures should be replaced with tissues of the same origin in order to reinstate their normal physiological functions ,2.In an optimal, successful regeneration treatment, it is imperative to induce the adhesion and attachment of dental pulp stem cells or other types of stem cells to dentin. Presence of smear layer on the root canal walls may inhibit or impair the adhesion of stem cells  to dentin. Smear layer is a 1-5-µm increment of denatured debris created by instrumentation, which is composed of dentin chips, odontoblastic processes, non-specific mineral and organic compounds, and microorganisms ,3. The aQMix is a novel endodontic irrigant composed of polyamino carboxylic acid as chelator [17% ethylenediaminetetraacetic acid (EDTA)], biguanide as an antimicrobial agent (2% chlorhexidine), deionized water, and surfactant. It can effectively eliminate the smear layer and expose the dentinal tubules. In addition to smear layer removal, it has optimal antimicrobial properties for elimination of resistant bacteria such as Enterococcus faecalis . EvidencEDTA serves as a chelator; it binds to calcium ions and releases them from the crystalline structure of hydroxyapatite. Thus, it degrades the mineral phase of dentin. A high number of growth factors are present in demineralized extracellular matrix such as transforming growth factor-B1, fibroblast growth factor-2, bone morphogenetic protein-2, platelet-derived growth factor, placental growth factor, epidermal growth factor, and angiogenic factors such as vascular endothelial growth factor. The key role of these growth factors in promotion of tissue regeneration has been well documented .MTAD was first introduced by Torabinejad and Johnson in Loma Linda University in 2003. It is composed of an aqueous phase containing 3% doxycycline as a broad-spectrum antibiotic, 4.25% citric acid as a chelator and demineralizing agent, detergent, and 0.5% polysorbate 80 (tween 80) as surfactant. Doxycycline hydrochloride has been used instead of free-base doxycycline monohydrate in the composition of MTAD due to its higher water solubility . MTAD isLaser irradiation has also been used for smear layer removal and dentin conditioning. Nd:YAG and CO2 lasers can eliminate the smear layer, uncover the dentinal tubules, and expose the collagen fibers with minimal change in the diameter of the tubules . A recenTreatment of specimens.-The study protocol was approved by the ethics committee of Hamadan University of Medical Sciences (IR.UMSHA.REC.1396.685). Twenty single-rooted extracted teeth of patients presenting to the School of Dentistry of Hamadan University of Medical Sciences were used for this study. The teeth had been extracted for purposes not related to this study.Immediately after extraction, the teeth were gently scrubbed with a sterile brush to eliminate blood, saliva and the residual soft tissue. They were then rinsed with sterile saline. Each tooth was sectioned at two points with a diamond disc and low-speed hand-piece under sterile water coolant. The first section was made at 5 mm distance from the cementoenamel junction, and the second section was made at 2 mm distance from the first section. For the purpose of standardization of specimens in terms of tubular density and quality of root dentin at different distances from the cementoenamel junction, only the segment in the middle was used for this study. Next, the specimens were sectioned along their longitudinal axis in buccolingual direction to expose the pulpal wall. Two specimens were prepared from each tooth. To prevent possible contamination from the pulp, a cylindrical bur attached to a low-speed hand-piece was held parallel to the longitudinal axis of the root to remove 0.5 mm from the pulpal wall (in contact with the pulp tissue). All 40 specimens were immersed in 5.25% sodium hypochlorite and rinsed with sterile saline for 1 minute. The specimens were then randomized into five groups as follows:(I) Control group: The specimens in this group were not irrigated with any irrigant and were not subjected to Er:YAG laser irradiation either.(II) EDTA group: The specimens were immersed in 17% EDTA for 1 minute.(III) MTAD group: The specimens were immersed in MTAD for 5 minutes.(IV) QMix group: The specimens were immersed in QMix for 5 minutes.(V) Laser group: The specimens were subjected to Er:YAG laser irradiation with 2.94 µm wavelength, 25 mJ energy per pulse, 15 Hz frequency, and 20 seconds of pulse duration. Laser was irradiated perpendicular to the specimen surface ,14.Culture of SCAPs.-The apical papilla tissue was collected from the third molars of young adults between 16 to 18 years. A minimum of two-thirds of the third molar roots had formed. Written informed consent was obtained from the donors. The collected tissue was diced into equal-size pieces that were digested by using 3 mg/cc collagenase type I and 4 mg/cc dispase at 37°C for 30 minutes. The tissue suspension was filtered through a filter with 70 µm diameter and homogenized. The cells were then cultured in culture plates with 25 cm diameter in modified Eagle’s medium containing 15% to 20% fetal bovine serum, ascorbic acid 2-phosphate, penicillin, streptomycin, and Fungizone, and incubated in a humid incubator at 37°C and 5% CO2. After reaching 80% confluence, the cells were passaged and the cell content was assigned to three flasks .-et al.  were flat; while, weakly attached cells with limited pseudopod-like processes were round in shape. The specimens in the MTAD group showed the minimum number of flat cells with a mean value of 8 cells attached to each specimen. Although the control, laser, EDTA and QMix groups had the highest number of flat cells in an orderly manner, the difference among them was not significant in this respect (Root canal disinfection is the first step in any regenerative process. The second step involves the transfer of the SCAPs from the apical papilla into the root canal after induction of bleeding . Type ofThis study compared the effects of dentin conditioning with EDTA, MTAD, QMix and Er:YAG laser on the attachment of SCAPs. The results indicated that application of different irrigants and Er:YAG laser irradiation changed the behavior of SCAPs. Irrigation with MTAD significantly inhibited cell attachment while the other three modalities induced cell attachment.et al. (et al. (According to the literature, MTAD has lower cytotoxicity than 17% EDTA, 5.25% NaOCl, and QMix; in other words, it reportedly has a higher biocompatibility than the conventional irrigants ,18,19. A (et al.  indicateThe chelating agents such as EDTA eliminate the mineral part of the smear layer and create a clean dentin surface with open dentinal tubules, which is an ideal substrate for the attachment of SCAPs.EDTA is extensively used after root canal disinfection with NaOCl, and significantly supports the viability, attachment, and differentiation of stem cells. In fact, EDTA is capable of reversing the adverse effects of NaOCl . It has et al. found, dentin microhardness significantly reduced with increasing time of application of EDTA more than 1 min(In this study, the specimens of MTAD and QMix groups irrigated for 5 min, but irrigation time for EDTA group was 1 min. As De-Deus han 1 min. Beside han 1 min,26.Another study indicated that negative effect of EDTA on cell viability increased with increasing application time. Whereaset al., (Er:YAG laser, which has recently gained increasing popularity among the clinicians, has a wavelength of 2.94 µm, which is in the near-infrared range, and is well absorbed by water. Thus, it does not increase the temperature of the target tissue following irradiation. Lasers are extensively used for root canal disinfection in endodontic treatments. Naghsh et al.,  reportedet al., ,30. Er:Yet al., -30.et al. (et al. (Divito et al.  demonstr (et al.  assessedet al. (In addition to antimicrobial properties, QMix can effectively eliminate the smear layer. Evidence shows that QMix is more biocompatible than EDTA, NaOCl, and chlorhexidine -8. Accoret al.  showed tet al. . A recenet al. .To the best of the authors’ knowledge, no study has been conducted on cell adhesion to dentin following irrigation with QMix. However, the results of the available studies regarding the cytotoxicity of QMix, EDTA, and MTAD are controversial. In the present study, QMix was compared with other commonly used irrigants, and the results indicated that QMix is a promising irrigant and can contribute to a successful outcome in regenerative treatments.The present study indicated that although smear layer removal capability, biocompatibility, and antimicrobial activity are ideal properties required for dentin conditioning in a successful regenerative process, these properties are not enough since MTAD is an antimicrobial chelator with optimal biocompatibility; however, it resulted in minimum cell adhesion in the present study and also the limited studies available on this topic ,21. Irri


In addition, it increased the activity of SOD enzymes  post exercise, 48 and 96 h after irradiation. However, PBMT did not increase CAT activity  post exercise. We did not find any difference in TAC or GPx biomarkers. Conclusion: Low to moderate certainty evidence shows that PBMT is a resource that can reduce oxidative damage and increase enzymatic antioxidant activity post exercise. We found evidence to support that one session of PBMT can modulate the redox metabolism.Oxidative stress induced by exercise has been a research field in constant growth, due to its relationship with the processes of fatigue, decreased production of muscle strength, and its ability to cause damage to the cell. In this context, photobiomodulation therapy (PBMT) has emerged as a resource capable of improving performance, while reducing muscle fatigue and muscle damage. To analyze the effects of PBMT about exercise-induced oxidative stress and compare with placebo therapy. Data Sources: Databases such as PubMed, EMBASE, CINAHL, CENTRAL, PeDro, and Virtual Health Library, which include Lilacs, Medline, and SciELO, were searched to find published studies. Study Selection: There was no year or language restriction; randomized clinical trials with healthy subjects that compared the application (before or after exercise) of PBMT to placebo therapy were included. Study Design: Systematic review with meta-analysis. Level of Evidence: 1. Data Extraction: Data on the characteristics of the volunteers, study design, intervention parameters, exercise protocol and oxidative stress biomarkers were extracted. The risk of bias and the certainty of the evidence were assessed using the PEDro scale and the GRADE system, respectively. Results: Eight studies (n = 140 participants) were eligible for this review, with moderate to excellent methodological quality. In particular, PBMT was able to reduce damage to lipids post exercise (SMD = −0.72, CI 95% −1.42 to −0.02, I In the current literature, it is already well evidenced that the production of reactive oxygen species (ROS) is associated with the development of pathogens such as cancer, cardiovascular diseases, diabetes mellitus, hypertension, and several neurodegenerative diseases ,2,3,4. TIn recent decades, new studies have shown that physical exercise can induce oxidative stress ,8,9,10. Recently, among the various resources researched to reduce the harmful effects of exercise, low-level laser therapy, also called photobiomodulation therapy (PBMT), has gained prominence . The PBMTwo years later, in 2008, the first randomized controlled trial (RCT) investigating the use of PBMT to improve athletic performance was published . In the Recently, a systematic review  sought tThus, there is a need for a new systematic review of randomized trials with human subjects verifying the PBMT effects on exercise, specifically on its impact on oxidative damage and antioxidant defense biomarkers. Seeking to fill this gap in the literature, this systematic review aims to summarize the effects of PBMT on exercise-induced oxidative stress in healthy individuals.The process of searching for and selecting articles met all the steps suggested by the Preferred Reporting Items for Systematic Review and Metanalysis Protocols–PRISMA-P . The proA literature review was developed without restrictions on the language of publication, from the earliest record up to September 2021 in electronic databases: The Cochrane Central Register of Controlled Trials (CENTRAL), Physiotherapy Evidence Database–PEDro, PubMed and Virtual Health Library (VHL) which include Lilacs, Medline and SciELO, Embase and Cumulative Index to Nursing and Allied Health Literature (CINAHL).The main keywords DeCS/MeSH search terms used in the English language were: “oxidative stress”, “oxidative”, “stress”, “low-level light therapy”, “phototherapy”, “photobiomodulation”, “laser therapy” and “exercise”; for each concept, keywords were combined with the ‘AND/OR’ operator  irradiation applied at any time (before or post exercise) of the physical exercise proposedComparisons  ●Placebo therapyOutcome measures  ●Oxidative stress biomarkers (damage oxidative and antioxidant activi-ty)The data extracted from the eligible studies were about the characteristics of the participants , interventions (PBMT) compared to control and/or placebo groups, exercise protocol enrolled on , the moment of irradiation (before or after an exercise session), PBMT application parameters and assessment of oxidative stress using indirect biomarkers.Data extraction was performed by a reviewer (JVF) and summarized through tabulation. All tabulated data were later reviewed by another reviewer (MVF). In the case of data that were not reported in the article, the corresponding author was contacted by email.The meta-analysis performed by one author (JVF) was reviewed by a statistician (TDM), using RevMan review management software  to summarize the treatment effect of PBMT on modulation of oxidative stress. Meta-analysis was only performed for those studies that compared PBMT to a placebo/sham group due to many comparisons. When a study had two or more groups submitted to PBMT but with different parameters or different application times (before or after exercise) compared to placebo, each group was considered as an independent study.Pooled-effect estimates on continuous outcomes were conducted using post exercise values mean and standard deviations (SDs) in each of the time-points  for each group and were expressed as mean difference (MD) or standard mean difference (SMD). When the data values were not expressed in SD, SD values were calculated using methods recommended in the Cochrane Handbook for Systematic Reviews of Interventions . As an e2 statistic, with thresholds set as I2 = 25% (low), I2 = 50% (moderate), and I2 = 75% (high). The significance level was set a priori at p < 0.05.Calculations were done using a random-effects model. A forest plot was created to present the MD or SMD with 95% confidence intervals (CIs). The heterogeneity between studies using the IThe quality of the RCTs was classified by two researchers (JVF and MVF) using the PEDro scale, which is based on concealed allocation, intention-to-treat analysis, and adequacy of follow-up . The Phy2 value was greater than 50%); indirection ; imprecision (downgraded if less than 400 participants were included in the comparison for continuous data) and other factors . If a study only evaluated the activity of a biomarker at a given time point, we considered the data inconsistent and imprecise (less than 400 participants for continuous outcomes and less than 300 participants for dichotomous outcomes), providing “low-certainty evidence,” which could be downgraded to “very low-certainty evidence” if we identified further limitations in relation to the risk of bias , in 75% and in all studies, respectively. The catalase (CAT) was analyzed for four studies (50%) ,40,41,420–5 min) ,40,41,42exercise ,40,42; 5 h after ,40,41,42 h after ,37,41,42The studies evaluated the risk of bias through the PEDro scale. All included studies were considered excellent quality methodologically, with low risk of bias with scores varying from eight to ten points (mean was 9.13 points). The complete score of each of the studies is presented in 2 = 42%, p = 0.02, see 2 = 59%, p = 0.01, see 2 = 57%, p = 0.002, see 2 = 0%, p = 0.05, see The antioxidant activity was measured through SOD in five trials ,38,40,422 = 79%, p = 0.64, see 2 = 0%, p = 0.007, see 2 > 90%), but we did report them descriptively  to which the individual is submitted and the magnitude of the damage caused. Moreover, PBMT increased the enzymatic antioxidant activity in SOD and CAT, but not for GPx. This effect size seems to be increased in subjects exposed to high intensity training more than in healthy individuals . Furthermore, the analysis of one of the trials demonstrated that PBMT was not able to change TAC levels in the short-term.We observed that the vast majority of included studies (86%) used the application of PBMT before exercising. Other systematic reviews ,24 prese2•−) and hydrogen peroxide (H2O2), occurs predominantly by the contraction of skeletal muscles performed during physical exercise [2O2 results in strength decline, and that this occurs due to the cytotoxicity of H2O2 which is capable of forming the hydroxyl radical (·OH). The hydroxyl radical is highly reactive, has a strong oxidizing potential and is capable of damaging the muscle cell structure, resulting in lipid peroxidation and protein carbonylation [Currently, it is known that the production of ROS, as superoxide radical , whose main role is to maintain redox homeostasis. Based on pooled studies with a low risk of bias ,38,40,42 of H2O2 . We obse of H2O2 ,42. Ther of H2O2  supportsp > 0.05) in the activities of antioxidant enzymes at certain time-points , most laboratory studies of PBMT which examined various types of animal models, diseases, or injuries, as well as exercises, observed the modulation of PBMT on antioxidant enzymes [Based on our results and current literature ,26,52, P enzymes ,57,58,59 enzymes .p < 0.05), but between interventions (PBMT versus placebo) there was no statistically significant difference for lipid damage at any time-point [PBMT has a dose-response effect, so it is important to identify what doses are used for the proposed objective . In geneme-point . Therefome-point ,63.2 > 50%). Among the studies included in this review, studies carried out with athletes showed a greater effect size [It is known that the level of oxidative damage can be influenced by the type, time, duration of exercise, age, level of physical activity, nutritional status, and antioxidant capacity of each individual ,13,14,15ect size ,40,42 anect size ,64. In aect size . TherefoTo the authors’ knowledge, this is the first systematic review with meta-analysis to investigate the effects of PBMT on exercise-induced oxidative stress in humans. Its strengths are related to a comprehensive search in several databases. The search, without language and year restriction, included an evaluation of methodological quality by the PEDro Scale, the use of GRADE system to assess the certainty of evidence and a prospectively registered protocol. Performing a statistical analysis should also be considered as a strong point since currently, the relationship between oxidative stress and PBMT is a field that few researchers have investigated.However, this review is not free of limitations. We observed inconsistency or imprecision in some comparisons due to the low number of trials and small sample size (>10 volunteers per group) in some studies . Second of all, the various healthy populations have been mixed, and future studies should be conducted to investigate whether there is a difference in the effect of PBMT on redox metabolism between specific populations . In addition, our results have important implications for the practitioners of sports and musculoskeletal rehabilitation, since PBMT can modulate oxidative stress and maintain redox homeostasis which can contribute to shorter muscle recovery time, minimize the deficits in the ability to produce strength after exercise and reduce induced muscle damage by exercise.In conclusion, our results are based on the low and moderate certainty of evidence with studies of good to excellent methodological quality according to the PEDro scale, suggesting that PBMT is an effective approach to enhance antioxidant activity and minimize exercise-induced oxidative damage. Therefore, we encourage future studies to be carried out with a high methodological quality to identify the effect of PBMT in modulation of oxidative stress in the long-term in aerobic and anaerobic training programs.


Rhabdomyosarcoma (RMS) is the most common soft tissue tumor in children, and its most common pathological types include embryonal RMS and alveolar RMS. In contrast, spindle cell RMS (SRMS) is a rare type. Moreover, the tongue is a rare primary site of RMS, and infancy is a rare age at onset.CDK4 and BRCA1 genes, both with unknown significance and a possible relation to RMS, were detected. In Case 2, three gene variants of unknown significance that were possibly associated with RMS—TRIP13, APC, and RAD54L—were identified.Two infants were diagnosed with lingual RMS at 3 and 5 months after birth, respectively, and were admitted to Beijing Children’s Hospital. The pathological type in both cases was SRMS. Both were classified as low-risk and were treated with surgery and chemotherapy. Case 1 was in complete remission at the latest follow-up, and Case 2 had a relapse 10 months after stopping chemotherapy, achieving complete remission after the multimodal treatment of chemotherapy, surgery, and radiotherapy. The venous blood gene test of the two infants did not indicate a pathogenic mutation or a possible pathogenic mutation related to RMS. In Case 1, variants of the Lingual RMS in infants is rare. Its clinical manifestations lack specificity, and early recognition is complex. The success and timing of local treatment are important prognostic factors. Genetic testing may be helpful for the early detection of tumor susceptibility and the estimation of prognosis. Rhabdomyosarcoma (RMS) is the most common soft tissue tumor in children, accounting for approximately 6.5% of childhood tumors. Its most common primary sites are the head, neck, and genitourinary system, followed by the limbs and trunk, among others . RMS of A 4-month-old girl was admitted to our hospital with a mass of the tongue that had been previously resected. The mass was 3–4 mm in diameter and was discovered on the left lingual margin of the tongue more than 1 month after birth. Consultation with a doctor was not sought, and the tumor gradually increased to approximately 6–8 mm in diameter 2 months later. The tumor did not considerably affect drinking. Subsequently, she was admitted to a local hospital and was preliminarily diagnosed with “mucoceles on the left margin of the tongue”, which was surgically resected. The pathologic diagnosis was highly differentiated SRMS. The neoplastic cells were short and fusiform, with atypia and mitosis, and they grew infiltratingly into the surrounding muscle tissue. The immunohistochemical results were Desmin (+), Myogenin (+), MyoD1 (+), Ki-67 (10%), Myoglobin (+), CD34 (+), S-100 (−), SMA (−), and TFE3 (−).No abnormalities in the birth, maternal pregnancy, or family histories of the patient were noted.Physical examination revealed some tissue missing at the left lingual margin related to surgery, and no other abnormality was observed. Her body weight was 6.9 kg at admission (age 4 months old), which was normal. Laboratory examinations, including the blood routine, C-reactive protein, electrolyte, and lactic dehydrogenase levels, liver and kidney function, and coagulation function, revealed no abnormalities. Enhanced magnetic resonance imaging (MRI) indicated that the soft tissue in the right palatal and parapharyngeal spaces was slightly thicker than that in the contralateral space, and the strengthening was slightly more evident. Chest computed tomography (CT), abdominal ultrasound, cranial MRI, a bone marrow smear, and a bone scan revealed no metastatic lesions. The infant was finally diagnosed with SRMS of the lingual surface. The clinical TNM staging was T1aN0M0, and the Intergroup Rhabdomyosarcoma Studies (IRS) staging was II; thus, the tumor was classified as low risk.The infant started receiving chemotherapy after she was diagnosed at our hospital, more than 1 month postoperatively. The treatment included four courses of vincristine, actinomycin D, and cyclophosphamide (the VAC regimen), followed by four courses of vincristine and actinomycin D (the VA regimen). Subsequently, chemotherapy was stopped, and she did not receive radiotherapy. At her last review, she had been followed up for 26 months, and the tumor was in complete remission without metastasis. She had normal height, weight, and language development, and she could eat normally without any abnormal taste.in situ hybridization (FISH) of NCOA2 gene-related translocations was negative.A 3-month-old girl was admitted to our hospital after experiencing choking for 2 months. A mass at the root of the tongue was detected upon physical examination Figure 1No abnormalities in the birth, maternal pregnancy, or family histories of the patient were noted.Additionally, no evident abnormalities were detected upon physical examination. The body weight was 5.5 kg at admission (age 3 months old). Laboratory examinations, including the blood routine, C-reactive protein, electrolyte, and lactic dehydrogenase levels, liver and kidney function, and coagulation function, also revealed no abnormalities. The maxillofacial-enhanced MRI performed in our hospital indicated that the base of the tongue was thick, and uneven lamellar enhancement of a fat-saturated T2 signal was observed; this was considered a postoperative change. Chest CT, abdominal ultrasound, cranial MRI, bone marrow smear, and bone scan indicated no metastatic lesions. Finally, the infant was diagnosed with SRMS of the tongue, with a clinical TNM staging of T1aN0M0 and an IRS staging of I; the tumor was thereby classified as low risk.The patient started receiving chemotherapy once she was diagnosed, which began 2 months postoperatively. The chemotherapy included four courses of the VAC regimen, followed by four courses of the VA regimen. She did not receive radiotherapy. The maxillofacial-enhanced MRI performed 10 months after the completion of chemotherapy indicated that the range of nodular and patchy fat-saturated T2 signal in the tongue’s body and base increased, demonstrating uneven and obvious enhancement with an unclear boundary involving the geniohyoid, left geniohyoid, and mylohyoid muscles 3. BilatWith the informed consent of the patients’ parents, exome–custom capture sequencing was performed to identify single-gene diseases. Target sequences were captured, sequenced, and analyzed in the exon coding region of more than 6,100 genes known to be associated with human diseases, as well as in the 5-bp region of adjacent introns (excluding the untranslated region). Peripheral venous blood samples of patients and their parents were collected, and genomic deoxyribonucleic acid was extracted to classify variant pathogenicity. The classification of variations was as follows: pathogenic, possible pathogenic, unknown significance, possible benign, and benign variations.CDK4 (NM000075.3), named C. 806C > T (p.Ala269VAL), from the mother that is not included in the Clinvar/HGMD database and whose software forecasts are inconsistent; and 2) heterozygous mutation of BRCA1 (NM007294.3), named C. 3350T > C , from the father that is not included in the Clinvar/HGDD database and is predicted as benign by the software. In Case 2, the following three variants of unknown significance were detected and may possibly be associated with the disease: 1) heterozygous variation of TRIP13 (NM004237.3), named C. 572C > T , from the mother that is not included in the Clinvar/HGMD database and is predicted as deleterious by the software; 2) heterozygous variation of APC (NM000038.5), named C. 8437A > G (p.THR2813ALA), from the mother that is not included in the Clinvar/HGMD database and is predicted as inconsistent by the software; and 3) heterozygous variation of RAD54L (NM003579.3), named C. 689T > C , from the father that is not included in the Clinvar/HGMD database and is predicted as deleterious by the software. In Case 2, the following three variants that are possibly associated with the disease were also detected: two RAB3GAP2 variants and one MMACHC variant.The results revealed no pathogenetic or possible pathogenetic variation in the two cases, which was consistent with the genealogical inheritance pattern and related to the phenotype of the infants. In Case 1, the following two variants of unclear significance that are possibly related to the disease were detected: 1) heterozygous variation of The clinical manifestations of tongue RMS are its lack of specificity and complex recognition in its early stage, which may lead to misdiagnosis. In the early stage, the tumor expands and infiltrates the muscle from which it arises, presenting first as a well-demarcated nodule or polypoid lesion with a soft or gummy consistency. When these lesions grow rapidly, they may cause dyspnea, dysphagia, and cough, including acute respiratory obstruction . In CasePrevious studies have demonstrated age as a strong, independent risk factor affecting the prognosis of RMS, even after considering other factors such as group, stage, and tumor histology. Infants aged <1 year and adolescents aged >10 years who were diagnosed with RMS had worse survival than children aged 1–9 years . Among tPreviously, young children were assumed to have no tolerance for radiation therapy; however, when radiation is omitted, even in those with stage I disease, the risk of recurrence is high, with local recurrence being the most common, thus confirming the need for radiation therapy. Therefore, brachytherapy, proton therapy, intensity modulated radiation therapy, and other emerging precision radiotherapy methods may improve both the prognosis of the infant and radiotherapy tolerance . Case 2 VGLL2, SBF, TEAD1, and NCOA2 gene fusions had a better prognosis, whereas patients with a MYOD1 gene mutation demonstrated stronger tumor aggression and higher mortality, regardless of age. Considering that this distinct molecular subtype is characterized by more aggressive biologic behavior than other genetic subtypes of SRMS and ScRMS, the MYOD1 genotype has been recommended to be used as a molecular marker in both subclassification and prognostication of SRMS/ScRMS , SRMS and ScRMS were combined and classified as an independent subtype of RMS , accountMS/ScRMS . The twoTP53  . Local aTP53 . Additiosyndrome . Some geCDK4 gene (Case 1) was located at 12q14, encoding the cell cycle regulator CDK4, and its amplification was observed in ARMS, breast cancer, non-small cell lung cancer, and glioblastoma multiforme (BRCA1 gene (Case 1), located at 17q21, is a tumor suppressor gene, and its mutation can be observed in breast cancer and ovarian cancer (TRIP13 gene (Case 2) is located on 5p15 and can be highly expressed in various malignant tumors, although no RMS has been reported (APC gene (Case 2), located at 5q21-22, is also a tumor-suppressor gene, and its inactivation or mutation is associated with colorectal cancer, myeloma, RMS, and adenocarcinoma (RAD54L gene (Case 2) was located at 1p32, and its mutation may be associated with breast cancer, colon cancer, lymphoma, and meningioma . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the minor(s)’ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.XM conceived the idea for this study and managed the cases. NX collected clinical data. PY and YS analyzed and interpreted the patients’ data. LF performed the histological examination. TY analyzed the MRI images. RG performed the single gene disease exome-custom capture sequencing. PY, NX, and XM were major contributors in the writing of the manuscript. All authors contributed to the article and approved the submitted version.


Results: we observed a rapid decline of symptoms and a low rate of intensive care in patients treated with Taurisolo®, with a faster decline of symptoms. Conclusions: This is the first trial assessing the safety and feasibility of Taurisolo® aerosol formulation. We could argue that this treatment could act as an add-on therapy in the treatment of COVID-19 patients, owing to both its anti-inflammatory and antioxidant effects. Further controlled trials are needed, which may be of interest to evaluate the compound’s efficacy.Background: Polyphenols are the largest class of bioactive compounds in plants, which are synthesized as secondary metabolites. In the last few years, interesting studies have demonstrated the efficacy of polyphenols against coronavirus infections. Methods: we conducted a phase II multicentric clinical trial (TAEROVID-19) during the first wave of the COVID-19 pandemic in order to assess the safety and feasibility of Taurisolo The SARS-CoV-2-related epidemic, firstly reported in December 2019 in Wuhan, China, rapidly spread worldwide, becoming a pandemic, as defined by the World Health Organization (WHO) in March 2020 .SARS-CoV2 infection has a varying clinical course, including asymptomatic or pauci-symptomatic patterns or, in many cases, causing severe systemic complications. According to data from the first and second wave, about 80% of patients are pauci-symptomatic or asymptomatic, while 15% show severe patterns with pneumonia and other complications, and 5% suffer from critical illness . The firDuring the first wave, before the anti-SARS-CoV-2 vaccination, a variety of therapeutic approaches were tested, such as chloroquine, idroxychloroquine, azithromycin, lopinavir–ritonavir, favipiravir, ribavirin, interferon, remdesivir, steroids, and anti-IL6 inhibitors ,13,14,15®) has relevant antioxidant and anti-inflammatory activities in humans, in addition to a potential antiviral efficacy [Polyphenols are the largest class of bioactive compounds in plants, which are synthesized as secondary metabolites. In the last few years, interesting studies demonstrated the efficacy of polyphenols against coronavirus infections. These are in vitro studies conducted on different cell models infected with micro-organisms belonging to the coronavirus family. In general, these studies reported that in vitro polyphenols exert a marked and well-demonstrated activity against coronavirus . All theefficacy ,20,21,22efficacy . Inhalatefficacy .® aerosol formulation in hospitalized patients suffering from SARS-CoV-2 pneumonia.For all these reasons, we conducted a phase II multicentric clinical trial (TAEROVID-19) during the first wave of the COVID-19 pandemic in order to assess the safety and efficacy of Taurisolo®-based aerosol formulation on admission to intensive care units (ICU) in hospitalized patients with COVID-19-related pneumonia as an add-on therapy. All participants provided written informed consent before study participation based on the principles of the Declaration of Helsinki. The study protocol and amendments were approved by the institutional review board and independent ethics committees at each center ; the study was conducted according to Good Clinical Practice guidelines defined by the International Council for Harmonisation and local laws. The primary endpoint was the rate of ICU admission  or death.TAEROVID-19 is an Investigator-Initiated Study (IIS), a phase II clinical trial, multicentric , single arm, aimed mainly at evaluating the safety and efficacy of TaurisoloEligible patients were aged at least 18 years old with SARS-CoV-2 infection , lung involvement at admission proved by HRCT (high-resolution computed tomography) findings ranging between 10/20 and 16/20 of Chung score, and lung function mildly impaired (P/F ratio > 150), consistent with the definition of mild to moderate COVID-19 pneumonia.Patients were ineligible to be included in the study if requiring invasive mechanical ventilation at admission , requiring vasopressors or inotropes, having symptom onset more than 7 days from admission, having a secondary bacterial and/or mycotic infection, having already started a treatment with IL6 inhibitors , or with a prognosis of less than 24 h (imminent death).®  3 times per day (every 8 h) and Polygonum cuspidatum E.S  administered in 10 cc of physiological saline solution by Portable YM-252 Mesh Nebulizer whit CPAP oronasal mask AirFit F20 (ResMed). Patients were treated with study medication for a maximum of 14 days, until informed consent was withdrawn, or until hospital discharge or ICU admission or death.Patients received a nebulization of TaurisoloEligible and consenting patients were studied by collecting data about clinical, radiological, and epidemiologic data such as radiological score   as blood gasses analysis , blood count, routine lab test, , and inflammatory markers .A nasopharyngeal swab was collected at baseline (T0), at day 1 (T1), 3 (T2), 5 (T3), 7, 14, 21, and 28 with viral load measurement through Cycling threshold (Ct). A real-time polymerase chain reaction (RT-PCR) Ct, analyzed from respiratory samples taken by swab, is a semiquantitative measure of viral load widely used to monitor SARS-CoV-2 infection. Commonly, the viral genes detected include nucleocapsid (N) and envelope (E) proteins that play a key role in viral self-assembly, RNA-dependent RNA polymerase (RdRP), and spike (S) glycoprotein that interacts with the host cell’s ACE2 receptors.The Ct value is inversely proportional to the amount of viral nucleic acid in specimens  and can be used to estimate viral load.The compliance of the therapy was evaluated by considering the number and duration of dispensed therapies according to the following classification: optimal (70% of the planned treatment dispensed), good (between 30–70% of the planned treatment), poor (less than 30% of the therapy is delivered).® is a polyphenolic extract from Aglianico cultivar grapes, collected during the autumn 2018 harvest, produced at a large scale by the MB-Med Company . Briefly, grapes were extracted with hot water (50 °C); the obtained solution was filtrated, concentrated, and spray-dried, obtaining a fine powder. For the formulation used in the trial, Polygonum cuspidatum extract containing 98% resveratrol was added to Taurisolo®. The polyphenol profile was evaluated by a high-performance liquid chromatography-diode array detector  analysis using the method described by Giusti et al. [® + Polygonum cuspidatum formulation before and after aerosol. More specifically, during aerosol, the nebulized formulation was collected through a plastic pipe connected to the device and condensed into a falcon tube. The solution was then lyophilized and solubilized in methanol for the HPLC-DAD analysis. The polyphenol profile of the Taurisolo® + Polygonum cuspidatum formulation before and after aerosol is reported in ® + 4.75 mg Polygonum cuspidatum) of the formulation used in the trial.Taurisoloi et al.  and previ et al. ,28. The Safety was assessed through the description of adverse events (AE) observed on the EP. These were reported individually, and if a patient presented the same AE repeatedly during the follow-up course, only the most severe was reported. Descriptive tables with the incidence of the main AEs were therefore constructed.Aerosol therapy was considered  a high-risk procedure for aerosolization and virus dispersion. However, there are several measurements to make this risk negligible: (i) aerial isolation of the patient being treated and use of personal protective equipment as required by current recommendations on hospitalized patients; (ii) use of CPAP masks that reduce the dispersion of exhaled material. The latter point was demonstrated through a test performed at the Pharmacy Department of Federico II University. The protocol applied for this evaluation is shown below.The safety of the YM-252 (Mesh Nebulizer) and oronasal mask for CPAP AirFit F20  misting system has been tested in humans to evaluate the risk of aerosolization of the virus. For this purpose, the mask was connected to the aerosol device by a collector. Some strips of absorbent paper  have been applied to the outside of the mask, along the entire external perimeter in contact with the face, in order to create a sort of breathable seal.Folin-Ciocalteu reagent is a commercially available reagent, based on a mixture of aqueous solution and phosphomolybdate and ammonium phosphotungstate, used in analytical chemistry for the determination of phenols and polyphenols. The reduction of the reagent in an alkaline environment leads to the formation of anions, which are intensely colored blue.The reagent is sprayed on the paper strips by a special nebulizer at the end of the aerosol application. The possible presence of polyphenols on the paper is detected by the appearance of blue spots.Sample size was based on a Fleming–A’Hern design. Based on the current evidence, it was assumed that a rate for the primary outcome ≥10% would be too larger to warrant future trials but that a rate ≤1% would warrant future investigations. Using an alpha level of 0.05 and a beta level equal to 0.10, the target recruitment was equal to 46 patients with zero or only one event to be observed for further research to be considered feasible. This was the smallest number that satisfied the design criteria. However, the target sample size was not reached, and only 43 patients were evaluable for analysis. Therefore, the actual alpha level was equal to 0.062 with a power of 0.931.The study population evaluated to test the efficacy for primary and secondary endpoints consisted of all patients enrolled in the study who received at least one dose of the experimental treatment . The safety analysis was also being conducted on the EP.Standard descriptive analyses were used to describe the characteristics of the enrolled sample. Average, standard deviation (SD), median, minimum, and maximum were calculated for the numeric variables, while the categorical variables were described by absolute frequencies and percentages. The admission rate in ICU was obtained by dividing the number of subjects who underwent admission to the ICU by the total of the EP. The corresponding upper end of the 90% confidence interval was calculated for the formal verification of the hypothesis that the real admission rate to the ICU was lower than 1%.The change in the cumulative incidence of events at the end of the follow-up was also calculated. The longitudinal trajectories of the laboratory parameters and clinical data were assessed using mixed linear models (with intercept and random angular coefficient). The time was entered as a numeric variable. No interim analyses were planned. Any subgroup analyses were considered exploratory as the study was not sized for a comparison of effectiveness between different groups.n = 6; 14%) or former smokers . Most frequent comorbidities at baseline were hypertension  and diabetes   were recruited for the trial with a mean age of 61.8 ± 12.4 years (range: 20.9 to 84.5). The target sample size was not reached, and only 43 patients were evaluable for analysis. As things stand, the actual alpha level was equal to 0.062 with a power of 0.931. Mean BMI was equal to 27.9 ± 4.4 (range: 17.7 to 40.1). Thirteen patients (30.3%) were either actual smokers   while st; 17.1%) .n = 30; 76.9%), dyspnea , and cough   .All patients received at least one dose of the experimental treatment with a median treatment duration of 7 days (range: 1 to 14 days) as an add-on therapy, in addition to the usual therapeutic approaches summarized in During a median follow up of 10.5 days (range: 1 to 31 days), only one patient required ICU admission, thus rejecting the null hypothesis that the ICU admission rate was equal or higher than 10% in favor of the alternative hypothesis that the rate was equal or lower than 1%. The estimated rate was equal to 2.33% .p < 0.001) and median IL6 decreased from 22  to 4.3  (p < 0.001). Furthermore, fibrinogen levels lessened from 585  to 377  (p < 0.001). No significant reduction was observed for D-Dimer or white blood count (WBC)  to 0.5 (IQR: 0.4 to 2) (nt (WBC) .Virological data, measured by evaluation of Ct from baseline to day 5, showed a faster decreasing trend, although not enough to reach statistical significance . At T0 (p = 0.033) as well as oxygen saturation . On the contrary, the observed increase in PaO2 did not reach statistical significance   .Because radiological improvement was slower than clinical, we did not perform a second HRCT during hospitalization.Safety analysis was performed on all patients. Only one adverse event was reported in a patient with bronchial hyperreactivity (cough not controlled by sedatives), related to the mode of administration of the drug. Only one patient discontinued treatment early due to worsening respiratory failure (later died in the ICU). There was only one death in a patient with severe respiratory insufficiency, who died after being transferred to the intensive care unit because of a concomitant massive pulmonary embolism, with no radiological signs of deep vein thrombosis.This nutraceutical proved to be very safe, in terms of the occurrence of adverse effects, since only one patient out of 43 was obliged to discontinue the treatment owing to unrestrained cough related to bronchial hyperreactivity. Moreover, aerosol therapy is considered, like intubation, bronchoalveolar lavage, and oxygen therapy with high-flow nasal cannula, a high-risk procedure for aerosolization and potential viral dispersion. In our experience, conversely, we accurately demonstrated, by a colorimetric control test, that there was no dispersion.In fact, this trial demonstrated that, even if in a small group of patients, topic administration of this drug via the aerosolic route could be considered safe in COVID-19 patients. A recent paper by Ramakrishnan et al. shows that inhaled glucocorticoid budesonide, given for a short duration, might be both an effective treatment of early COVID-19 in adults, and a safe method of administration, in terms of lack of viral spreading . We found in our patients, independently from remdesivir therapy, a rapid negativization of the patient and, therefore, less contagiousness expressed in an increase in C (t) within a few days. It is interesting to analyze these data and evaluate those present in other experiences. As reported by Singanayagam et al., the duration of infectiousness following mild-to-moderate COVID-19, closely related to the level of SARS-CoV-2 RNA, declines over time progressively, with a probability of culturing virus that declined to 6% at 10 days after symptom onset .Xiaowen et al. underline that negative conversion of SARS-CoV-2 RNA takes 14 days on average (IQR: 10–18) from the first positive RT-PCR test. In this study, they found that the rate of RNA negative conversion within 7 days, 14 days, and 21 days among all patients were 10.2% , 62.7% (48.1–73.2%), and 91.2% (80.4–96.4%), respectively .Carini et al. found that the time length of negativization, calculated from the beginning of symptom onset to two consecutively negative RT-PCR test results, collected at least 24 h apart after the disappearance of symptoms, required three weeks or more in the majority of subjects. More than 70% of symptomatic patients became test-negative within four weeks; this proportion rose to 80% in the asymptomatic ones. Moreover, 29.2% of symptomatic versus 20% of asymptomatic patients became test-negative at five weeks . Those fFurthermore, the British National Institute for Health and Care Excellence (NICE) states that the aerosols produced by the patients affected by SARS-CoV-2 infections are generated from the fluid within the nebulizer chamber that does not carry the patient–viral derived particles . In fact®. Similar results were also shown in a paper by Mahat et al., who described a slower decline in serum levels of inflammatory markers in mild to moderate COVID-19 pneumonia [®, showing an increase in P/F ratio from 292 ± 60.4 at baseline to 310 ± 73 at day 7 (p = 0.033) and oxygen saturation from 95.8 ± 2 at baseline vs. 97.1 ± 1.6 at day 7 (p < 0.001). Comparing our results with what is reported in the literature, a faster decline of symptoms and a lower rate of ICU access in patients treated with Taurisolo® [In our study, we observed a low rate of ICU admission  in contrast to other authors’ experience since only one patient out of 43 (2.3%) required ICU admission. Colaneri et al. report an access rate to ICU of 6.8%, and Zhang and coworkers report a rate ranging from 6.5 to 13.7%, according to age ,36. Furtneumonia . Our res®, which contains grape pomace extract [Besides, a recent paper by Xi-Jian Dai et al. underlines that consumption of red wine played protective effects against the COVID-19, while consumption of beer and cider increased the COVID-19 risk: such a result could strengthen the potential anti-inflammatory and antioxidant utility of Taurisolo extract .®, in addition to vaccination.Our trial began at a time when COVID vaccines were not yet available. During the study, the first RNA vaccines were distributed to healthcare professionals, but such protection was not yet available to the general population. None of our enrolled patients had access to vaccination before contracting COVID-19 pneumonia. We believe in a possible “booster” role of TaurisoloFrom the literature, we know that grape pomace extract (GPE) has antiviral activity against various micro-organisms. Several mechanisms of action have been demonstrated, including down-regulation of virus entry co-receptor expression, suppression of virus replication, and reduction of induced inflammation. In addition to its direct antiviral activity, GPE was found to be effective in alleviating the pathological complications of respiratory viral infection by reducing the expression of pro-inflammatory mucins and interleukins, including IL-1β, −6, and −8 .Most observational studies assessed that the mRNA vaccines against COVID appear to be safe and highly effective tools to prevent severe disease, hospitalization, and death against all variants of concern. The protection appears to be against symptomatic and asymptomatic infection. mRNA vaccines were associated with a faster decline in viral load against several variants, including Delta, and a lower probability of viral culture positivity ,40.® in reducing the inflammatory cascade and, therefore, the severity of the disease, in addition to the vaccines, given the negligible risk profile related to its nebulization compared to the possible benefits.Based on these considerations, we hypothesize a future use of Taurisolo® nebulization could represent a solid adjuvant to reduce the time of infection and contagiousness along with vaccination, in order to reduce the spreading of the disease.Our trial suffers from some limitations: first of all, the small number of patients; then, the absence of a control group, even if it must be considered that this is a phase 2, single-arm uncontrolled study, and therefore represents a proof of concept. The lack of a control group is mainly linked to ethical reasons, in relation to the need to guarantee, especially during the first wave of COVID-19, the best possible treatment for the entire population under study, without giving up drugs that could alter the validity of our data . Finally, the dynamic changes of the pandemic through time and the development of different variants of SARS-CoV-2 limits the extrapolation of those results on the current infected population; however, we think that Taurisolo® could act as an add-on therapy in the treatment of COVID-19 patients, owing to both its anti-inflammatory and antioxidant effects [® could be used in paucisymptomatic non-hospitalized patients to accelerate negativization. Therefore, in the future, such a nutraceutical could be useful in the therapy of chronic pulmonary diseases such as COPD, bronchial asthma, and bronchiectasis.In conclusion, we could argue that aerosol therapy with Taurisolo effects . Besides


FOS, NR4A2, EGR2, CXCR4, ATF3, and SERPINE1) were found. Combined with the results of the PPI network analysis and literature search, FOS and NR4A2 were selected as the target genes for further investigation. We validated the bioinformatic results via reverse transcription PCR (RT-PCR) and Western blot analyses on human and rat samples. The expression levels of the mRNA and protein of FOS and NR4A2 were upregulated in both human and rat samples. In summary, we found that FOS may play an important role in AVF stenosis, which could be a potential therapeutic target of AVF stenosis.Arteriovenous fistula (AVF) is the most widely used hemodialysis vascular access in China. However, stenosis of the AVF limits its use. The mechanism of AVF stenosis is currently unknown. Therefore, the purpose of our study was to explore the mechanisms of AVF stenosis. In this study, we identified the differentially expressed genes (DEGs) based on the Gene Expression Omnibus (GEO) dataset (GSE39488) between venous segments of AVF and normal veins. A protein–protein interaction (PPI) network was constructed to identify hub genes of AVF stenosis. Finally, six hub genes ( Chronic kidney disease (CKD) is a condition causing irreversible destruction of renal parenchyma, with a progressive loss of kidney function over several years. Maintenance hemodialysis (MHD) is the best alternative to renal transplantation due to the shortage of human donor organs. Patients undergoing MHD need patent vascular access for optimal treatment, and autologous arteriovenous fistula (AVF) is the optimal vascular access. However, AVF stenosis, which could be attributed to a variety of factors, may limit its use. Our previous study demonstrated that hypercholesterolemia and hyperphosphatemia are independent risk factors for AVF stenosis in patients with MHD . Meanwhihttps://www.ncbi.nlm.nih.gov/geo/, accessed on 1 July 2022). In addition, online tools, such as GEO2R , Network Analyst , and the Search Tool for the Retrieval of Interacting Genes database (STRING) , have been widely used. All of these bioinformatic tools are useful for identifying hub genes related to AVF stenosis. Although some hub genes have been reported previously [Bioinformatic analysis has been used to identify novel biomarkers of many different diseases ,6. Moreoeviously , these sIn our study, GSE39488 was downloaded to screen DEGs between the control and AVF group. Thereafter, a protein–protein interaction (PPI) network of DEGs was constructed; hub genes with a higher degree were identified. All processes in this study were performed using RStudio and Cytoscape software and related online tools.The GSE39488 dataset, which contains the gene expression profile data of AVF patients, was obtained from the GEO database [p-value of <0.05 were considered to be differentially expressed.The raw data of gene expression profiles from GEO were preprocessed for background correction  and data normalization (log2 transformation) using RStudio software version 1.1.383. The DEGs between AVF tissues and normal veins were identified via Network Analyst online tool. The genes with a log2 fold-change (FC) of >1 and an adjusted STRING online database was used to construct the PPI network based on the DEGs which were previously identified. Cytoscape software version 3.7.2 was used to visualize the PPI network . ThereafA total of five patients who required AVF repair and three patients who required AVF creation were recruited from the Department of General Surgery, Xuanwu Hospital. The clinical characteristics of these patients, including age, gender, smoking, hypertension, and diabetes mellitus, were collected and compared between groups. The results presented in Our present study involved 20 male Sprague–Dawley rats  aged 6 months. The AVFs were created in an end-to-side manner between the right external jugular vein and the right common carotid artery. The animals were euthanized 7, 14, or 28 days after the surgical procedure. The normal right external jugular vein served as the control group. Heparin , dosed at 0.1 IU/g, was used to prevent acute AVF thrombosis after the operation.Isoflurane inhalation anesthesia with oxygen-enriched (40%) air was used, with isoflurane concentrations of 4% for induction and 1.5% for maintenance. After anesthesia, the animals were fixed supine on a heating blanket, and the eyes were covered with a piece of gauze. Thereafter, the surgical area was shaved and disinfected. Finally, a median neck incision was made under a YZ20T4 dissecting microscope .The surgical procedures were as follows: Firstly, the right common carotid artery was dissected carefully. After, two vascular clamps were placed proximally and distally. A 5 mm incision was made longitudinally on the wall of the common carotid artery. Meanwhile, we dissected the right sternocleidomastoid muscle and transected it from the middle. Next, the right external jugular vein was detected and clamped proximally and ligatured distally, followed by a transection from the distal. Thereafter, the artery and vein were rinsed with saline containing 100 IU/mL heparin, whereupon the end of the vein was anastomosed to the side of the artery with 11-0 running sutures. At the end of the process, heparin was injected intravenously into the external jugular vein at a dose of 0.1 IU/g body weight. After completion of the anastomosis, venous and arterial clamps were removed carefully, starting with the venous clamp. If bleeding occurred at the site of the anastomosis, gentle compression to stop bleeding was performed; otherwise, an additional suture was inserted. In the final step, after confirmation of the patency of the AVF, the skin was closed with a 4-0 running suture. The surgical procedure is shown in Thereafter, the abdominal cavity was accessed through a midline incision from the xiphoid process to the symphysis pubis. Retractors were placed to maintain exposure. The abdominal content was reflected to the right to expose the inferior vena cava, aorta, and right kidney. The intestines were wrapped in saline-soaked gauze to keep them moist and warm. The right ureter was then carefully isolated and ligated with 8-0 prolene suture. Finally, the skin was closed with a 4-0 running suture.Four rats were sacrificed at each time point of 7, 14, and 28 days after surgery, and the proximal external jugular vein of the AVF was resected for subsequent analysis. Each sample was equally divided into three parts, which were used for RT-PCR, Western blot, and histological analysis.S (%) = S1 represents the area of the whole vein; S2 represents the area of hyperplasia intima plus residual lumen; S3 represents the area of the residual lumen. S1 and S2 were distinguished by staining. Image J software was used to calculate S1, S2, and S3 . The RNA was then reverse-transcribed into cDNA using a Reverse Transcription Kit . The cDNA was used as a template for SYBR Green qPCR Mix , and RT-PCR reaction was performed using StepOnePlus Real-Time PCR System. GAPDH was used as the reference for mRNA analysis. The 2Proteins were extracted from the samples via radioimmunoprecipitation assay buffer . The protein lysates of 30 μg were loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk powder for 0.5 h. PVDF membranes were then probed with mouse anti-FOS antibody , anti-NR4A2 antibody , and anti-β-actin monoclonal antibody  overnight at 4 ℃. The PVDF membranes were washed with Tris-buffered saline containing Tween 20 (TBST) for 10 min × 3 and labeled with horseradish peroxidase (HRP)-conjugated secondary antibodies  for 1 h at RT. Immunoreactivities were detected by enhanced chemiluminescence . The protein content was calculated by densitometry using Image J software.p < 0.05 was considered statistically significant. GraphPad Prism software v8.0.2 was used for the visualization of the results.Statistical analyses of three independent experiments are presented as the mean ± SEM. The significance of the differences was analyzed by a one-way ANOVA test, and Log2 transformation was performed to normalize the raw data in the GSE39488 dataset. The results of data normalization are presented in p-value of <0.05 and a log FC of >1, with 47 upregulated and 15 downregulated genes. Both a heatmap  and the occurrence of AVF stenosis.To verify the results of bioinformatics analysis, RT-PCR and Western blot analysis were performed. We collected the veins from five patients with AVF stenosis (test). However, because of the nature of the patients, we only collected three normal veins as the control (NC). As shown in FOS and NR4A2. The surgical process is described in detail in FOS and NR4A2 increased with time after AVF creation  were identified using the STRING online tool and proceeded by Cytoscape software.To identify more specific and effective biomarkers in AVF stenosis, a combination of analyses, including bioinformatic, RT-PCR, and Western blot, based on the number of human and animal samples, was conducted. In our study, a GEO dataset (GSE39488), which included six outflow veins of AVF and four normal veins, was analyzed, and DEGs between the AVF and normal veins were identified using RStudio software. Next, a PPI network of DEGs was constructed. Finally, six hub genes (FOS, which is involved in the MAPK pathway, was one of the hub genes identified by our study. FOS can encode leucine zipper proteins, which can dimerize with proteins of the JUN family, thereby forming the transcription factor complex AP-1. As such, FOS have been implicated as regulators of cell proliferation, differentiation, and transformation. In some cases, the expression of FOS has also been associated with apoptotic cell death [FOS in vascular endothelium, which promotes the injury of endothelial cells and leads to vascular stenosis [At the same time, we found that ll death . Jun Xu stenosis . Zheng Jstenosis . FOS. In summary, we found that the expression level of FOS was upregulated in stenotic AVF, which is consistent with the bioinformatics results. Moreover, the expression level will upregulate with the increase in the degree of stenosis. This showed that FOS could be a therapeutic target for AVF stenosis.Meanwhile, some studies suggested that foam cell formation may also depend on the activation of JNK2 and the p38α MAPK signaling pathways . HoweverFOS leads to AVF stenosis is complex. The results from several studies indicate that FOS could activate the MAPK signaling pathway [FOS may cause AVF stenosis by the activation of the MAPK signaling pathway.The mechanism by which  pathway ,21. The  pathway . The pat pathway . Some st pathway . TherefoFOS, NR4A2, CXCR4, EGR2, SERPINE1, and ATF3. FOS, which was associated with the activation of the MAPK pathway, may be responsible for AVF stenosis.The present study identified a total of 62 DEGs between stenotic and normal veins. We identified six hub genes: There are still several limitations of our study. Firstly, the sample size of our study may be insufficient, and studies with a larger sample size may be necessary to confirm our results. Secondly, GSE39488 is performed by microarray profiling which may need to be updated. Finally, due to our limited resources, no in-depth study was performed. Future studies may be needed to clarify their relationship.


Melophagus ovinus disease is a common ectoparasitosis, which can lead to a decrease in animal production performance, product quality, and even death. Aconitum flavum Hand.-Mazz. has many pharmacological activities including insecticidal, heat-clearing, analgesic, and dehumidifying. However, there are few researches focused on the effects and related mechanism of Aconitum flavum Hand.-Mazz. in killing Melophagus ovinus. In this study, 11 alkaloids of Aconitum flavum Hand.-Mazz. were detected, and its total alkaloid activity was determined. The results showed when the total alkaloid concentration was 64 mg/ml and the treatment time was 16 h, the killing rate of Melophagus ovinus reached 100%. Through the observation of the differences in the surface of Melophagus ovinus in each experimental group, it was found that the morphology of the posterior end of the female Melophagus ovinus in the alkaloid treatment group was significantly different from that of the blank and positive control groups, and most of the epidermal tissue was obsessive and missing. Moreover, the enzyme activity determination results of 64 mg/ml group were significantly different when compared with the normal control group, while there was no significant difference in other groups. Then, the Melophagus ovinus gene library was established by the unreferenced genome transcriptome sequencing, the proteomic comparison was performed using tandem mass tag labeled protein detection technology, and finally, the samples were quantitatively analyzed by liquid chromatography-mass spectrometry tandem and bioinformatics methods. Based on the above experimental results, it was speculated that Aconitum flavum Hand.-Mazz. total alkaloids may cause the imbalance of protein disulfide isomerase expressions by affecting the regulation of Hsp40 cellular protein homeostasis and the oxidation of protein disulfide isomerase and related proteins. This would affect the selective recognition of signal sequence, the targeted transport of Sec 61, and the correct folding of the three-dimensional structure of amino acid chain, weakening the clearance of amino acid chains that cannot be correctly folded and eventually resulting in the killing of Melophagus ovinus. This study preliminarily revealed the mechanism of Aconitum flavum Hand.-Mazz. total alkaloids against Melophagus ovinus and provided a theoretical basis for the screening of Melophagus ovinus action targets and the development of new veterinary drugs. Melophagus ovinus disease is a common external parasitic disease caused by blood-sucking Melophagus ovinus parasitic on animal surfaces, which is characterized by itchy skin, chronic inflammation, and direct contact transmission. It mainly occurs in sheep, dogs, horses, etc., resulting in animal emaciation, anemia, poor growth, decreased quality of livestock products, and even death of individual animal pups. Besides, the most serious harm of Melophagus ovinus is that it can be used as a vector to spread bluetongue virus  belongs to the genus Aconitum in the family of Ranunculaceae. It mainly grows in the mountainous grassy slopes or sparse forests with an altitude of 2,000–3,700 m in Tibet, Gansu, Qinghai, Shanxi, Ningxia, Sichuan, and other provinces of China and is used as medicinal materials in these areas. According to modern pharmaceutical research, the main active components of AFHM are diester diterpenoid alkaloids. Zhang et al. used electrospray ionization tandem mass spectrometry and high-resolution electrospray ionization mass spectrometry to analyze the total alkaloids in AFHM roots and detected five main alkaloids including 12-epi-napelline, 3-deoxyaconitine, aconitine, 3-deoxyaconitine-8-linoleate, and aconitine-8-linoleate . In addition, the molecular levels of GSTs, ABC transporters, sodium ion voltage-gated channels, and pH-gated chloride channels have also been reported to be related to the mechanisms of action or resistance of ivermectin and other drugs  and 3-deoxyaconitrile (production batch number: DST200810-033) were purchased from Lemeitian Pharmaceutical/Desit Biological. Phosphate buffered saline (PBS), BCA protein concentration determination kit, catalase (CAT), peroxidase (POD), carboxylesterase (CarE), glutathione peroxidase (GSH-Px), monoamine oxidase (MAO), acetylcholinesterase (AChE), and Ca2+-Mg2+-ATPase detection kits were purchased from Solarbio Science & Technology Co., Ltd. .The roots of AFHM were purchased from Gansu Fuxinghou Biomedical Technology Co., Ltd. The specimen of AFHM (20200311) was kept in the Key Laboratory of Veterinary Pharmaceutical Development of the Ministry of Agriculture, CAAS Lanzhou Institute of Husbandry and Pharmaceutical. The The dry powder of AFHM was soaked in 95% ethanol for 24 h (mass/volume ratio was 1:10), and the extraction was repeated three times. The combined extract was concentrated under reduced pressure on a rotary evaporator at 60°C, 70 rpm/min, and 0.076 MPa to obtain the ethanol extract. Furthermore, the ethanol extract was dissolved in 2% HCl and centrifuged at 4,000 rpm for 10 min. The supernatant was filtrated twice and extracted with petroleum ether for three times. The lower aqueous solution was adjusted to pH 9.5 with alkali solution, and the obtained yellow precipitate was alkaloid A. The supernatant was extracted with equal volume chloroform for five times, and the lower layer of chloroform extract was collected and concentrated under reduced pressure to obtain the colloidal substance which was alkaloid B. The total alkaloid of AFHM was the mixture of A and B.The mixture was dissolved with 3 ml of chromatographic methanol, and the sample composition was detected by HPLC/QTOF-MS with the specific detection conditions as follows: High Performance Liquid Agilent 1290 HPLC, Waters UPLC BEH C18 column (1.7 × 2.1 μm × 100 mm), 0.1% formic acid solution as mobile phase A, acetonitrile-0.1% formic acid solution as mobile phase B, 5 μl as the injection volume, and 400 μl/min as the flow rate. The total biological base of AFHM was dissolved and diluted to 3 mg/ml with chromatographic methanol and filtered with 0.22 μM filter before use. Standard aconitine and 3-deoxyaconitine were dissolved in chromatographic methanol to prepare 0.800 and 0.379 mg/ml solutions, respectively, and then filtered by a 0.22 μM filter. The samples were used for chromatographic detection with conditions of high-performance liquid chromatography Agilent 1290 HPLC, Agilent C18 column , 0.2% phosphoric acid-0.4% triethylamine solution as mobile phase A, acetonitrile as mobile phase B, 10 μl as the injection volume, 0.8 ml/min as the flow rate and 240 nm as the detection wavelength.Melophagus ovinus with body lengths of 4–6 mm were randomly divided into seven groups: blank control group, positive control group (1% phoxim), and drug treatment groups . Each group was set with three replicates, and there were 10 Melophagus ovinus in each replicate. The observation time points of mortality of Melophagus ovinus were set to 1, 2, 4, 8, 16, 24, 48, 72, 96, 120, and 136 h.The Melophagus ovinus before and after treatment with AFHM total alkaloids. Melophagus ovinus in each group were treated with cleaning, fixing, and gold spraying, and the differences of body appearance were observed under 35 × scanning electron microscope.Stereomicroscope and scanning electron microscope were used to observe the body surface differences of Melophagus ovinus with body lengths of 4–6 mm were randomly divided into seven groups as described in Section Killing effect of AFHM total alkaloids in vitro. The activities of catalase (CAT), peroxidase (POD), carboxylesterase (CarE), glutathione peroxidase (GSH-Px), monoamine oxidase (MAO), acetylcholinesterase (AChE), and Ca2+-Mg2+-ATPase in each group were determined by biochemical analysis kits after 16 h treatment .The Melophagus ovinus used for unreferenced genome transcriptome sequencing were collected and randomly divided into three groups. Guanidine isothiocyanate-phenol-chloroform-one-step method was used to extract the total RNA of Melophagus ovinus. The mRNA with poly A structure in total RNA was enriched by oligo (DT) magnetic beads, and the RNA was interrupted to a fragment with a length of about 300 bp by ion interruption. Using RNA as a template, the first strand cDNA was synthesized with six-base random primers and reverse transcriptase, and the second strand cDNA was synthesized with the first strand cDNA as a template. After the library was constructed, the library fragments were enriched by PCR amplification. And, the library was selected according to the fragment size of 450 bp. The total and effective concentrations of the library were detected using an Agilent 2100 Bioanalyzer. Then, libraries containing different index sequences  were mixed proportionally according to the effective concentration and the amount of data required. The mixed library was uniformly diluted to 2 nM, and alkali denatured to form a single chain library. After RNA extraction, purification, and library construction, these libraries were sequenced by next-generation sequencing (NGS) based on the Illumina HiSeq sequencing platform .Thirty Melophagus ovinus were randomly divided into a blank control group and an AFHM group (64 mg/ml), with three replicates in each group and 10 Melophagus ovinus in each replicate. Samples were collected when the treatment time was 16 h. The sample was ground to cell powder in liquid nitrogen and transferred to a 5-ml centrifuge tube. Four volumes of lysis buffer  were then added to the cell powder, and the mixture was sonicated three times on ice using a high-intensity ultrasonic processor (Scientz) . The remaining debris was removed by centrifugation at 12,000 g at 4°C for 10 min. Finally, the supernatant was collected, and the protein concentration was determined with the BCA kit according to the manufacturer's instructions.The The protein solution was reduced with 5 mM dithiothreitol for 30 min at 56°C and then alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. Next, the protein sample was diluted by adding 100 mM TEAB to a urea concentration <2 M. The first digestion was performed overnight at a 1:50 trypsin-to-protein mass ratio, and the second digestion was performed for 4 h at a 1:100 trypsin-to-protein mass ratio. Finally, the obtained peptides were desalted by the C18 SPE column.The TMT-based LC-MS/MS identification and data normalization analysis were performed by PTM-biolab™  and Shanghai Personal Biotechnology Co., Ltd. Tryptic peptides were first dissolved in 0.5 M TEAB. Each channel of the peptide was labeled with its respective TMT reagent  and incubated for 2 h at room temperature. Five microliters of each sample were pooled, desalted, and analyzed by MS to check labeling efficiency. Then, 5% hydroxylamine was added to the samples for quenching. The pooled samples were then desalted with Strata X C18 SPE column (Phenomenex) and dried by vacuum centrifugation.The sample was fractionated into fractions by high pH reverse-phase HPLC using an Agilent 300 Extend C18 column . Briefly, peptides were separated into 80 fractions with a gradient of 2–60% acetonitrile in 10 mM ammonium bicarbonate (pH 10) over 80 min. Then, the peptides were combined into nine fractions and dried by vacuum centrifugation.The tryptic peptides were dissolved in solvent A  and directly loaded onto a homemade reversed-phase analytical column . Peptides were separated with different concentration gradients of solvent B (0.1% formic acid in 90% acetonitrile), i.e., a gradient of 5–25% for 60 min, a gradient of 25–35% for 22 min, climbing to 80% in 4 min, and holding for 4 min. All operations were performed at a constant flow rate of 450 nl/min on an EASY-nLC 1200 UPLC system (ThermoFisher Scientific).The separated peptides were analyzed in Q ExactiveTM HF-X (ThermoFisher Scientific) with a nano-electrospray ion source. The electrospray voltage applied was 2.0 kV. The full MS scan resolution was set to 60,000 for a scan range of 3501,600 m/z. Up to 20 most abundant precursors were then selected for further MS/MS analyses with 30 s dynamic exclusion. The HCD fragmentation was performed at a normalized collision energy (NCE) of 28%. The fragments were detected in the Orbitrap at a resolution of 30,000. The fixed first mass was set as 100 m/z. The automatic gain control (AGC) target was set at 1E5, with an intensity threshold of 3.3E4 and a maximum injection time of 60 ms.PRJNA836644) and UniProt Knowledgebase concatenated with reverse decoy database. Trypsin/P was specified as a cleavage enzyme allowing up to two missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in first search and 5 ppm in main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as a fixed modification, and acetylation on protein N-terminal and oxidation on Met were specified as variable modifications. FDR was adjusted to <1%.The resulting MS/MS data were processed using the MaxQuant search engine (v.1.6.15.0). Tandem mass spectra were searched against the SRA data (Accession ID: http://www.ebi.ac.uk/GOA/). First, the identified protein IDs were converted to UniProt IDs and then mapped to GO IDs by protein IDs. If some identified proteins were not annotated by the UniProt-GOA database, the InterProScan software would be used to the annotated protein's GO functional based on the protein sequence alignment method. Then, proteins were classified by Gene Ontology annotation based on three categories: biological process, cellular component, and molecular function.The Gene Ontology, or GO, is a major bioinformatics initiative to unify the representation of genes and gene product attributes across all species. More specifically, the project aims to maintain and develop its controlled vocabulary of genes and gene product attributes, annotate genes and gene products, assimilate and disseminate annotation data, and provide tools to facilitate access to all aspects of the project data. The gene ontology covers three domains including cellular component, molecular function, and biological process. The cellular component is a component of a cell, but with the proviso that it is part of some larger object. This may be an anatomical structure  or a gene product group . Molecular function describes activities, such as catalytic or binding activities, that occur at the molecular level. GO molecular function terms represent activities rather than the entities (molecules or complexes) that perform the actions and do not specify where, when, or in which context the action takes place. The biological process is a series of events accomplished by an ordered assembly of one or more molecular functions. Biological processes and molecular functions are difficult to distinguish, but the general rule is that a process must have multiple distinct steps. Gene ontology (GO) annotation proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/interpro/) is a database that integrates diverse information about protein families, domains, and functional sites and is freely available to the public via Web-based interfaces and services. Central to the database are diagnostic models, known as signatures, by which protein sequences can be searched to determine their potential functions. InterPro can be used for large-scale analysis of whole genomes and meta-genomes, as well as in characterizing individual protein sequences.Protein domains are conserved parts of a specific protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and can normally be stabilized and folded independently. Many proteins consist of several structural domains. One domain may appear in a variety of differentially expressed proteins. Molecular evolution uses domains as building blocks that can be recombined in different arrangements to create proteins with different functions. Domains vary in length from ≈25 to 500 amino acids. The shortest domains, such as zinc fingers, are stabilized by metal ions or disulfide bridges. Domains often form functional units, such as the calcium-binding EF-hand domain of calmodulin. Because they are independently stable, domains can be “swapped” by genetic engineering between one protein and another to make chimeric proteins. Identified protein domain functional descriptions were annotated by InterProScan  based on the protein sequence alignment method, and the InterPro domain database was used. InterPro (KEGG connects known molecular interaction network information such as pathways and complexes (the “Pathway” database), genes and proteins information generated by genome projects (including the gene database) and biochemical compound and reaction information (including compound and reaction databases). These databases are different networks known as “protein network” and “chemical universe.” There are efforts in progress to add knowledge to KEGG, including adding information regarding ortholog clusters to the KEGG Orthology database. KEGG pathways mainly include metabolism, genetic information processing, environmental information processing, cellular processes, rat diseases, and drug development. Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to annotate protein pathways. First, KEGG database descriptions of proteins were annotated using KEGG online service tools KAAS. Then, KEGG online service tool KEGG mapper was used to map the annotation results to the KEGG pathway database.The cells of eukaryotic organisms are elaborately subdivided into functionally distinct membrane-bound compartments. Eukaryotic cells are mainly composed of extracellular space, cytoplasm, nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum (ER), peroxisome, vacuoles, cytoskeleton, nucleoplasm, nucleolus, nuclear matrix, and ribosomes. Bacteria also have subcellular localizations that can be separated when the cell is fractionated. The most common localizations referred to include the cytoplasm, the cytoplasmic membrane , the cell wall , and the extracellular environment. Most Gram-negative bacteria also contain an outer membrane and periplasmic space. Unlike eukaryotes, most bacteria do not contain membrane-bound organelles; however, there are some exceptions. Therefore, we used wolfpsort, a subcellular localization predication software, to predict subcellular localization. Wolfpsort is an updated version of PSORT/PSORT II for the prediction of eukaryotic sequences. Especially for protokaryon species, subcellular localization prediction software CELLO was used.p-value < 0.05 was considered to be significant. Encyclopedia of Genes and Genomes (KEGG) database was used to identify enriched pathways by a two-tailed Fisher's exact test and to test the enrichment of the differentially expressed protein against all identified proteins. The pathway with a corrected p-value <0.05 was considered to be significant. These pathways were classified into hierarchical categories according to the KEGG website. InterPro  database was used to perform protein domain enrichment analysis for each category protein, and a two-tailed Fisher's exact test was employed to test the enrichment of the differentially expressed protein against all identified proteins. Protein domains with a corrected p-value < 0.05 were considered to be significant.Proteins were classified into biological process, cellular compartment, and molecular function according to GO annotations. For each category, a two-tailed Fisher's exact test was employed to test the enrichment of the differentially expressed protein against all identified proteins. The GO with a corrected P values, and then filtered those categories enriched in at least one of the clusters with P < 0.05. These filtered P value matrixes were transformed by the function x = –log 10 . Finally, these x values were z-transformed for each functional category. These z scores were then clustered by one-way hierarchical clustering  in Genesis. Cluster memberships were visualized by a heat map using the “heatmap. 2” function from the “gplots” R-package.To further hierarchical clustering based on differentially expressed protein functional classification , we first collated all the categories obtained after enrichment along with their P < 0.05 were considered significant.Data analysis was done by SPSS (19.0) computer-based software. Parametric one-way ANOVA with the Tukey test was used to investigate differences between groups . Values of The AFHM total alkaloids were detected by HPLC/QTOF-MS , and theMelophagus ovinus was 100%, which was the minimum AFHM concentration comparable to the killing rate of the positive control drug  in the same action time  catalyzes the degradation of the neurotransmitter acetylcholine, terminates its stimulating effect on the excitation of the postsynaptic membrane, maintains the normal transmission of nerve impulses in organisms, and plays an important role in the regulation of nerve conduction . In thisMelophagus ovinus. Among them, the CarE inhibitory activity of AFHM total alkaloids at 64 mg/ml was significantly better than other concentrations. Studies have demonstrated that organophosphorus pesticides can bind and inhibit the activity of CarE (Carboxylesterase (CarE) can catalyze the hydrolysis of endogenous and exogenous substances containing ester bond, amide bond, and thioester bond, participate in lipid transport and metabolism, and is related to the detoxification and metabolism of a variety of drugs, environmental poisons and carcinogens . It is o of CarE . TherefoMelophagus ovinus. The antioxidant effect of SOD needs the participation and coordination of CAT. Because SOD mainly catalyzes the disproportionation reaction of O2− to produce H2O2 and O2 and eliminates the damage of O2− to cells, and the generated H2O2 is further decomposed into H2O by CAT  might slightly interfere with the homeostasis of Melophagus ovinus and initiate a series mechanism of repair and maintenance. For example, low concentrations of AFHM total alkaloids could increase the expression of cytoprotective and repairing proteins by activating transcription factors and kinases, resulting in increased activity of antioxidant enzyme CAT to a certain extent.Under normal growth and metabolism, peroxidase (CAT) greatly improves the maintenance ability of intracellular redox balance of O by CAT . As shown effect ” might bin vivo. It can change electrophilic compounds into hydrophilic substances by a covalent combination of catalytic substances and their metabolites with the sulfhydryl group of GSH, degrade toxic compounds, and excrete them out of the body (Melophagus ovinus tissues in the administration group was significantly reduced (P < 0.05), as shown in GST plays a major role in the detoxification enzyme system the body . GST molMelophagus ovinus was aggravated, monoamine neurotransmitters in the body were accumulated, and the demand for oxidative decomposition of MAO was increased, resulting in the inhibition of MAO enzyme activity in experiment groups. On the other hand, nerve cells of Melophagus ovinus were damaged by the action of AFHM total alkaloids. The dysfunctional damaged cells put the oxidative decomposition of MAO in an accelerated and violent decomposition dynamic, causing the different levels of MAO enzyme activity under the action of different concentrations of AFHM total alkaloids. Besides, the MAO enzyme activity level of the AFHM total alkaloid group at a concentration of 64 mg/ml was higher than that of other AFHM groups, which further indicated that this concentration was the optimal killing concentration of AFHM total alkaloids for Melophagus ovinus. The result was corroborated with the result of the in vitro killing experiment of Melophagus ovinus.MAO is a flavin protein located on the outer membrane of mitochondria, including two subtypes of monoamine oxidase A (MAO-A) and MAO-B. It can catalyze the oxidative deamination of varieties of monoamine neurotransmitters and release hydrogen peroxide at the same time, resulting in cellular oxidative stress . Therefo2O2 as an electron acceptor and has the dual effect of eliminating the toxicity of hydrogen peroxide and phenolic amines. At the same time, it is one of the key factors for pests to resist the oxidative stress of plant secondary substances and form the adaptability of the feeding host. Therefore, to analyze and verify the function of the protective enzyme POD in insect resistance (mite), the changes in POD gene expression and enzyme activity in the host pest should be considered simultaneously , which widely exists in animals, plants, and microorganisms and can effectively reduce the oxidative damage of cells caused by ROS. It directly oxidizes phenolic or amine compounds with Haneously . As illu2+Mg2+-ATPase is widely distributed in organisms and can catalyze the hydrolysis of ATP molecules to produce ADP molecules and inorganic phosphorus. The activity of the ATP molecular enzyme can be determined by measuring the content of inorganic phosphorus. Ca2+-Mg2+-ATPase is an important enzyme protein on the cell membrane, which can actively transport Ca2+ outside the cell and ingest Mg2+. The function of Ca2+-Mg2+-ATPase is to move Mg2+ into the cell and move Ca2+out of the cell to maintain the relative stability of the internal environment .The identified protein needs to contain at least one unique peptide. We used the TMT-based quantitative proteomic approach to obtain a comprehensive view of the protein changes influenced by irradiation . We idenp value < 0.05, the threshold of change for significant up-regulation was >1.2 and <1/1.2. Finally, we detected 151 upregulated and 81 downregulated proteins  in GO classification were enriched and analyzed, respectively. The subcellular structure of proteins was annotated by the PSORTb (V3.0) software, and COG/KOG function classification statistics were carried out.The differentially expressed proteins in the comparison group were enriched at three levels of GO classification, KEGG pathway, and protein domain  during protein processing in the endoplasmic reticulum, and one protein (Hsp90) was down-regulated . It was The differentially expressed proteins in the comparison group were classified into GO, KEGG pathway, and protein domain enrichment, and they were further divided into four parts according to the differential expression multiple, called Q1–Q4, as shown in In order to clearly show the interaction relationship between different proteins, the top 50 proteins with the closest interaction relationship were selected to draw the protein-protein interaction network . Among tIn the process of separation, since AFHM contains a large amount of diterpenoid alkaloids, the separation effect of ordinary column chromatography was not ideal, we mainly used normal-phase silica gel chromatography and reverse-phase C18 chromatography for separation . Q-TOF aMelophagus ovinus, also termed the sheep ked, is a kind of wingless fly that belongs to the order Diptera, family Hippoboscidae. Melophagus ovinus is one of the most common hematophagous ectoparasites of sheep, mainly distributed in the neck, shoulder, perineum, and hind legs of animals   , 45–47, nosomes) . Nelson nosomes)  stated ting goat , Bison bd Vulpes  are not Traditional Chinese medicine, especially botanical and animal medicine, are living entities whose characteristics, such as external morphology, color, size, texture, characteristic odor of cross-section, and other microscopic characteristics of chemical composition, are largely determined by the genetic code-gene of the medicinal material. Of course, we cannot ignore the influence of environmental factors. Although genes are the source of genetic information, functional proteins are the executors of gene functions , 51. TheMelophagus ovinus was carried out. The protein extraction and bioinformatics analysis of the drug group and the control group showed that there were significant differences in the expression of some proteins, 80 proteins were up-regulated, 57 proteins were down-regulated, and 44.53% of the differential proteins existed in the cytoplasm. Some proteins that mainly exist in cells, organelles, organelle membranes, and other cellular compositions have significant differences in expression. Most of these proteins are involved in organic matter metabolism, cell metabolism, biological process regulation, primary metabolic process, nitrogen compound metabolism, anatomical structure development, and other biological processes, their main molecular functions include binding proteins, binding organic cyclic compounds, binding heterocyclic compounds, binding ions, hydrolase activity, etc. In this study, the TMT differential proteomics research method was adopted, and all differential proteins were taken as the research objects. by searching and evaluating through the retrieval and evaluation of the differential proteins, the molecular evolution relationship network of the interaction between the drugs and Melophagus ovinus was constructed. Furthermore, the mechanism of the effective parts of AFHM against Melophagus ovinus was discussed.Proteomics technology is currently used to study diseases with complex pathogenesis, such as diabetes, hypertension, and metabolic syndrome. The term proteome was originally coined by Australian scientists Wilkins and Williams in 1994 and was later defined as all proteins expressed by a genome, cell, or cell culture under specific conditions. Proteomics is the science of analyzing and describing biological proteins, protein-protein interactions, and protein modifications to systematically determine the expression of each protein in a cell or tissue, the composition of protein complexes, their interrelationships, and their abundance and modification status. In this experiment, a proteomic study of the killing effect of the AFHM total alkaloids on Melophagus ovinus in vitro during the same action time. Therefore, 64 mg/ml of AFHM total alkaloids was considered as the optimal drug concentration and was used for subsequent experiments. When the action time was 16 h and the total alkaloids concentration of AFHM was 64 mg/ml, the activity of Ca2+-Mg2+-ATPase increased significantly when compared with the blank control group (P < 0.01), while AChe, CarE, CAT, GSH-Px, MAO, and POD enzyme activities were significantly decreased (P < 0.01). Protein extraction and bioinformatics analysis of the total alkaloid treated group and control group showed that 80 proteins were up-regulated and 57 proteins were down-regulated. The COG/KOG classification mainly played the roles of post-translational modification, protein turnover and chaperone, signal transduction, intracellular transport, secretion and vesicular transport, cytoskeleton constitution, and other functions. The GO enrichment and distribution bubble diagram showed that the differential proteins related to the regulation of RNA biosynthesis, nucleic acid regulation template transcription, transcription regulation DNA template, and other biological processes were significantly different (P < 0.01). Some differential proteins were involved in the metabolic pathways of thiazoline-6-mercaptopurine, some affected the basic transcription factors of eukaryotic cells, and some might influence the cell phagocytosis process and promote the production or action pathways of ATPase. This study obtained the comprehensive effect of AFHM total alkaloids on Melophagus ovinus body proteins by using TMT technology, enzyme activity detection, and bioinformatics analysis, screening out potential drug-protein targets, providing a theoretical basis for elucidating the mechanism of AFHM killing Melophagus ovinus.In this paper, HPLC/QTOF-MS was used to detect the constituents of alkaloids extracted. Eleven compounds in the comparison database were consistent with those reported in the literatures. Aconitine was determined by HPLC, and the content was 1.2195 mg/g. About 64 mg/ml of AFHM total alkaloids could achieve a 100% insecticidal rate within 16 h, which was in line with the industry's evaluation standard of good anti-parasite effect. This concentration was the minimum AFHM total alkaloids concentration equivalent to the killing rate of positive control drug phoxim to https://www.ncbi.nlm.nih.gov/, bioproject/836644.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: XiW, BH, and SW conceived and designed the study. BH and SW conducted the experiments, XiW, ZY, and BH drafted the manuscript. XiW, YZ, FC, XX, FW, YH, CC, BW, PB, XuW, YL, HZ, and BH supervised the study. XiW, ZY, YZ, BH, and SW reviewed the methods and the results. All authors read and approved the manuscript.This work was financially supported by the Central Public-interest Scientific Institution Basel Research Fund (No. 1610322020008) and the Innovation Program of Chinese Academy of Agricultural Sciences – Veterinary Natural Medicine and Antibiotic Replacement (No. 25-LZIHPS-03). Thanks for the technical support provided by PTM Biolabs Inc (Hangzhou) and Shanghai Personal Biotechnology Co., Ltd.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.