Therefore, PDW may serve as a potential marker for predicting SAB. However, homogeneous and multiethnic studies with larger sample sizes are warranted to validate our findings due to several limitations in this meta-analysis.Numerous studies have indicated that mean platelet volume (MPV) and platelet distribution width (PDW) were correlated with spontaneous abortion (SAB), but the results were contradictory. Hence, we performed a meta-analysis to assess the association of MPV and PDW with SAB. We systematically searched on China National Knowledge Internet, PubMed, and Embase up to May 2022 to retrieve eligible studies. The synthesized standard mean difference (SMD) with a 95% confidence interval (CI) was used to evaluate the correlation of MPV and PDW with SAB. A total of 20 studies were finally included in this meta-analysis. The pooled analysis results showed that PDW was markedly increased in patients with SAB versus women with a healthy pregnancy (SMD = 1.03; 95% CI: 0.51–1.54;   HThe compatible contact between the placenta and maternal circulation is a vital determinant for a successful pregnancy . ConversMean platelet volume (MPV) and platelet distribution width (PDW), which are routinely detected in clinical laboratories, have been determined as parameters of platelet activation ,15. Intr22.1The conducted research is not related to either human or animal use, so ethical approval and informed consent are unnecessary.2.2Two researchers independently conducted the literature collection by searching PubMed, Embase, and China National Knowledge Internet up to May 2022. Search terms included “MPV, PDW or ” and “abortion or miscarriage or pregnancy loss.” In addition, the references of retrieved studies were screened to identify additional eligible studies omitted by electronic search. The detailed search strategies for each database were presented in Supplement 1.2.3The studies synchronously satisfying the inclusion criteria mentioned below were eligible: (1) had a full-text organization structure; (2) investigated the association of MPV or PDW with SAB and reported the mean MPV or PDW and their corresponding SD in patients and women with healthy pregnancy; and (3) published in English or Chinese. For studies with overlapping data, the study with the larger sample size was selected. In contrast, studies were excluded if they met any of the following criteria: (1) they did not assess the association of MPV or PDW with SAB; (2) they did not provide data about the mean MPV or PDW and their corresponding SD; or (3) they were academic dissertations, review articles, editorial articles, meta-analyses, or meeting abstracts.2.4The Newcastle-Ottawa scale (NOS) was applied to appraise study quality . The NOS2.5Two researchers extracted the data independently based on the inclusion criteria. Extracted data were fed into a collection table and examined by a third author. Disagreements were removed via discussion. In this meta-analysis, the extracted data included the study author, publication year, country, sample size, age of pregnant women, gestational age, type of abortion, mean MPV value, mean PDW value, and SD of MPV and PDW.2.6Q test (χ2) and I2 test were used to assess the statistical heterogeneity among the studies. The random-effect model was selected to calculate the SMD when there was significant heterogeneity. p < 0.05 was considered to indicate a statistically significant difference. Subgroup analyses and meta-regression analyses based on sample size, country, age of pregnant women, gestational age, and recurrent miscarriage were carried out to explore the sources of heterogeneity [The statistical analysis was conducted using STATA version 12.0  and RevMan version 5.3 software. The standard mean difference (SMD) and its 95% confidence interval (CI) were utilized to evaluate the pooled MPV and PDW values. When the SMD was above 0 and the 95% CI did not cross 0, the MPV or PDW value was considered to be increased in the SAB group versus the control group. Otherwise, the combination of SMD <0 and the 95% CI not crossing 0 indicated a decrease in the MPV or PDW value in the SAB group versus the control group. Additionally, when the 95% CI crossed 0, the MPV or PDW value was considered to be equal between the two groups. The Cochrane ogeneity . Sensitiogeneity .33.1n = 14) came from Turkey, and the others were conducted in China. The sample size of the included studies ranged from 80 to 2192. A total of 13 studies focused on recurrent miscarriage. Among the included studies, 18 studies reported MPV measurements, while 14 studies reported PDW. The methodological quality of the studies was evaluated by NOS, and the scores ranged from 6 to 7, implying that the included studies had a relatively acceptable quality. The detailed information about study characteristics is summarized in Through electronic and manual searches, we first obtained 77 articles. Then, academic dissertations, duplicate articles, studies with unmatched themes, studies lacking data about MPV or PDW, and articles without full text were excluded; thus, 57 studies were excluded, and 20 studies were finally included in this meta-analysis ,32,33,343.2I2 = 98.0; p < 0.00001), we used random-effect models to perform the meta-analysis for PDW. The result of the overall pooled analysis showed that the PDW level was markedly increased in the SAB group versus the healthy pregnancy group , China, age of pregnant women (≤30 years), gestational age (<28 weeks), and recurrent abortion, but not in the other subgroups. Meanwhile, significant heterogeneity was still observed in all the subgroup analyses. However, the results of meta-regression analyses suggested that the study country may be one of the major sources of heterogeneity for the pooled estimation of the correlation between PDW and SAB.Fourteen studies involving 2,076 patients and 2,303 subjects with healthy pregnancy explored the relationship of PDW with spontaneous miscarriage ,30,31,323.3I2 = 97.0%; p < 0.00001) were applied in the meta-analysis for MPV. The pooled result showed that there were no significant differences in MPV levels between patients with SAB and control subjects , whereas there were no significant differences in MPV value .Pregnancy loss represents a serious reproductive health problem, which brings huge emotional and physical damage to thousands of families . The etiN > 200 and N ≤ 200), China, biological age (≤30 year), gestational age (<28 week), but not in the other subgroups. The limitation of case numbers in individual subgroups may be partly responsible for these contradictory results. Of note, our sensitivity analysis showed that the overall estimation of PDW did not substantially alter with a single study sequentially excluded, and no evident publication bias for studies referring to PDW was identified, which suggested the validity of our meta-analysis to a degree. In addition to the hypercoagulation state, an excessive maternal inflammatory response is another crucial cause of spontaneous pregnancy loss [The concentrations of coagulation factors are often altered during pregnancy, leading to a state of hypercoagulation . Moreovency loss . The plancy loss . Remarkancy loss ,39. HencMPV is a parameter of evaluating platelet size and has also been identified as a marker of platelet activation  and systThere are several limitations in our meta-analysis, so the results should be interpreted cautiously. First of all, the clinical heterogeneity among the included studies exists in this meta-analysis, such as definitions of spontaneous miscarriage, age of patients and control subjects, gestational age, thromboembolism-precipitating risk, methods of measuring PDW and MPV, and testing time. Thus, it is hard to exclude the possibility that the pooled results could be biased by heterogenic factors. Second, all of the included studies collected data with the retrospective method, which may be accompanied by a high risk of selection bias. Third, this study only compared the levels of PDW between women with pregnancy loss and healthy subjects, while the cut-off values of high PDW levels were not determined due to the unavailability of relevant data in the included studies. This drawback makes it difficult to use PDW as a clinical tool to predict spontaneous pregnancy loss. Fourth, publication bias might exist since only studies published in English and Chinese were considered in this meta-analysis, notwithstanding the funnel plot and Egger’s test indicating no evident publication bias. Fifth, all the included studies only enrolled Chinese or Turks, so it is arbitrary to generalize our conclusion to other ethnicities. Last but not least, trial sequential analysis (TSA) can help to evaluate whether the least sample size required for obtaining a stable and robust conclusion is enough . Of note5In summary, PDW was markedly increased in patients with SAB versus women with healthy pregnancies, while there were no significant differences in MPV between patients with SAB and women with healthy pregnancies. Therefore, PDW may be used as a potential marker for predicting SAB. However, homogeneous and multiethnic studies with larger sample sizes are warranted to validate our findings due to several limitations in this meta-analysis.


TP53, PTPRB, PLCG1, KDR as well as FLT4 amplification have been observed in AS. There is a potential therapeutic value of inhibition of the VEGF pathway against angiosarcoma. Our case first described a patient with two sites of cutaneous angiosarcomas (cASs) that responded differently to anlotinib. And genetic analysis revealed that those two sites had different FLT4 variants, suggesting that FLT4 amplification could be the cause of anlotinib non-response.Angiosarcoma (AS) is a rare, clinically aggressive tumor with limited treatment options and a poor prognosis. Mutations involving the angiogenesis-related genes Angiosarcomas (ASs) are rare, aggressive tumors of vascular or lymphatic endothelial cell origin that account for about 2%–3% of all adult soft tissue sarcomas. They can arise from anywhere on the body, with cutaneous angiosarcomas  involving the head and neck, particularly the scalp, followed by the breast, extremity, trunk, liver, and other sites , 2. ASs When compared to Western countries, approximately 50%–60% of cutaneous angiosarcomas (cASs) originate in the head and neck, with Asians involving up to 90% of the scalp and face . cASs arSurgical resection with wide excisional margins is the main therapy for R0 resection cASs, but due to insufficient surgical excision and the infiltrative growth pattern, local recurrences or metastases are common, particularly in head and neck locations, with 5-year overall survival of 10%–15% , 12. SevMYC, KLT4, FLT4, and recurrent mutations in TP53, PLCG1 (R707), and PTPRB, as well as genetic alterations in the mitogen-activated protein kinase (MAPK) pathway, have been reported in ASs . The sequencing results showed that the primary lesions only had the EK2/IRS1 Table 1.FLT4 F131S mutation to regorafenib was not ruled out because of its extracellular location and complicated genomic profiles  1, VEGFR3, VEGFR2/KDR, platelet-derived growth factor receptor a (PDGFR-a), c-Kit, and fibroblast growth factor receptor (FGFR) 1–3 and inhibits tumor angiogenesis and tumor cell proliferation. It has been approved as a third-line treatment for patients with advanced non-small-cell lung cancer (NSCLC)  that targets vascular endothelial growth factor receptor ( (NSCLC) . Based o (NSCLC) . A phase (NSCLC) . More tr (NSCLC) . So far, (NSCLC)  and card (NSCLC)  or combi (NSCLC) . In the FLT4 gene encodes VEGFR3 and is involved in lymphatic differentiation. It has been reported that inappropriate genetic abnormalities of VEGFR signaling are critical in the genesis of angiosarcomas . The FLT1 gene encodes VEGFR1 and is a member of the VEGFR family. The point mutation c.542G>A was found in FLT1’s Ig-like domain 2, which is a high-affinity ligand-binding region  and endometrial cancer (37%) being the most common  for the publication of any potentially identifiable images or data included in this article.YiG treated and observed the patient. YuG, JM, and YiG collected the clinical information, diagnostic information, therapeutic information, and images of the patients. JL performed the histopathological and immunohistochemical examinations. YiG, YuG, JM, and JL prepared the manuscript and the literature search. YJ, XY, and TS performed data analysis and interpretation. YiG and JM reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.Authors YJ, XY, and TS are employed by Jiangsu Simcere Diagnostics Co., Ltd.The remaining 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.


Still today, information about the canine perinatal period is lacking, even if this period has many challenges. One of the essential key systems for the correct final fetal development and for the neonatal adaptation is the hypothalamic–pituitary–adrenal axis. The final products of this axis are cortisol and dehydroepiandrosterone (DHEA). The aim of the present study was to investigate DHEA concentrations in claws and hair of dead newborn puppies, classified as premature puppies (PRE-P), stillborn puppies (STILL-P) and puppies that died from the 1st to the 30th day of life (NEON-P). Results showed higher DHEA concentrations in claws of PRE-P puppies when compared with other categories. DHEA concentrations in hair did not reveal any statistical difference. The findings of the present study confirm that higher DHEA levels are produced during intrauterine life when compared with subsequent phases, also in puppies that died. The use of claws confirms their potential for long-term hormonal studies in puppies. These results provide useful information about the canine perinatal period.p < 0.05), whilst no significant differences were found in hair for all the studied factors. The results confirm the hypothesis that higher amounts of DHEA are produced during the intrauterine life in dogs, also in puppies that will die soon after birth.The latest intrauterine fetal developmental stage and the neonatal period represent the most challenging phases for mammalian offspring. Toward the term of pregnancy, during parturition, and after birth, the hypothalamic–pituitary–adrenal axis (HPA) is a key system regulating several physiologic processes, through the production of cortisol and dehydroepiandrosterone (DHEA). This study was aimed to assess DHEA concentrations in hair and claws of 126 spontaneously dead newborn puppies, classified as premature puppies (PRE-P), stillborn puppies (STILL-P) and puppies that died from the 1st to the 30th day of life (NEON-P). The possible influence of newborn sex, breed body size, and timing of death on DHEA concentrations in both matrices was evaluated. Claw DHEA concentrations were higher in the PRE-P group when compared to STILL-P and NEON-P puppies ( In veterinary medicine, the perinatal period spans from the last intrauterine fetal developmental stage until the end of the neonatal period. This phase is full of challenges for the mammalian offspring, and remains largely unstudied in canine species. At term of pregnancy, several physiologic processes are regulated from the fetal hypothalamic–pituitary–adrenal (HPA) axis, such as the fetal lung final maturation , the resIn response to the adrenocorticotrophic hormone, adrenals produce cortisol and dehydroepiandrosterone (DHEA), two hormones characterized by opposite effects . AlthougAlthough cortisol, as the endpoint of stress, has broadly been investigated in literature, up to now, less data have been available on the biologic functions of DHEA, both in human and companion animals. However, recent papers have also reported that secretion of DHEA ) is sometimes elicited after an acute stressor or after the challenge with the adrenocorticotrophic hormone, in humans ,11. AddiIn human fetuses and newborns, HPA activity was previously investigated through neonatal blood  or cord To overcome these issues, cortisol concentrations were recently studied in hair; overall, this matrix has proven to be suitable for the detection of cortisol, both in humans ,16 and aThe property of accumulating hormones during a long time-window allows to reduce the number of samplings, respecting animal welfare. Another advantage is the possibility to store samples without specific precautions, wrapped in paper envelopes and kept at room temperature until the analysis, as previously reported ,24,25.Both cortisol and DHEA were detected in fingernails of adult humans , and DHELastly, hair ,26 and nFor these reasons, with the purpose of improving knowledge about the perinatal period in dog and about the related perinatal HPA axis activity, the aims of the present study were: (a) to assess the concentrations of DHEA in hair and claws in newborn puppies spontaneously dead along the perinatal period; (b) to assess the possible influence of newborn sex, breed size, and timing of death on DHEA concentrations in hair and claws in dead puppies; (c) to assess the possible correlation between hair and claw DHEA concentrations in dead puppies.The study was approved by the Università degli Studi di Milano Ethical Committee (OPBA) with protocol OPBA_147_2019. All the owners and breeders signed a written informed consent form for the collection of hair and claws from dead puppies for research purposes.The study was performed on purebred puppies spontaneously dead and without gross physical malformations. After sex recording, puppies were classified as belonging to small-sized breeds (bodyweight ≤ 10 kg), or to a merged class of medium-large-sized breeds (bodyweight >10 kg), according to maternal body weight.Based on the timing of death, puppies were grouped as follows: (1) premature puppies (PRE-P) when puppies were delivered between 2 and 1 weeks before the expected date of whelping, calculated on the base of ultrasonographic prediction of parturition date ; (2) stiSoon after death, a proper amount of hair at least 20 mg) and all claw tips were carefully sampled from each puppy. Hair was shaved closest to the skin always from the same area [ mg and aHair and claw strands were washed in 3-mL isopropanol to ensure the removal of any steroids on their surface. Hair and claws DHEA were extracted with 3-mL (min 0.12 mL/mg) of methanol and measured using a solid-phase microtiter RIA assay, as described before ,25,29.In brief, a 96-well microtiter plate  was coated with goat anti-rabbit γ-globulin serum diluted 1:1000 in 0.15 mM sodium acetate buffer, pH 9, and the plate was incubated overnight at 4 °C. The plate was then washed twice with phosphate-buffered saline (PBS), 0.05 M, pH 7.5 (RIA buffer), pH 7.4, and incubated overnight at 4 °C with 200 μL of the anti-DHEA serum diluted 1:80,000. The rabbit anti-DHEA antibody used was obtained from Analytical Antibodies . Cross-reactivities of this antibody with other steroids are as follows: DHEA 100%, 5-Androsten-3β,17β-diol 9.2%, epiandrosterone 2.8%, 5α-Androstane-3β,17β-diol 0.6%, testosterone 0.1%, androstenedione 0.1%, DHEA sulfate 0.04%, cortisol < 0.001%. After washing the plate with RIA buffer, the standards (5–200 pg/well), the quality control extract, the test extracts, and the tracer  were added, and the plate was incubated overnight at 4 °C. Bound hormone was separated from free hormone by decanting and washing the wells in RIA buffer. After addition of 200 μL of scintillation cocktail , the plate was counted on a β-counter . Intra- and interassay CV were 3.7 and 11.6%, respectively. The sensitivity of the assay, calculated as the interpolated dose of the response to a concentration of zero minus the statistical error, was 12.4 pg/mL.p < 0.05 .Data regarding DHEA concentrations in hair and claws were statistically analyzed by ANOVA, to assess (a) possible differences in hair and claw concentrations of DHEA, according to newborn sex and to breed size; (b) possible differences in hair or claw concentrations of DHEA according to newborn timing of death. Separate analyses were performed in order to assess the influence of single effects alone, avoiding the possible influence of factors interaction. The possible correlation between hair and claw DHEA concentrations was assessed by Spearman’s correlation test. The significance was set at The number of puppies enrolled in the study was 126, belonging to many canine breeds; there were 60 (47.6%) females and 66 (52.4%) males.According to maternal body size, 53 puppies (42.1%) were classified as belonging to small-size breeds and 73 (57.9%) to medium-large-sized breeds.Based on the timing of death, 22 puppies (17.5%) were PRE-P, 78 (61.9%) were STILL-P, and 26 (20.6%) were NEON-P.Data regarding hair and claw DHEA concentrations in the 126 puppies, grouped according to sex and breed body size, are summarized in Data about hair and claws DHEA concentrations in the 126 puppies grouped according to the timing of death are reported in The Spearman correlation test did not show significant correlations between DHEA concentrations in hair and claws.The present study showed data about DHEA concentrations in hair and claws of premature born dead puppies, stillborn puppies, and newborn puppies spontaneously dead in the first 1–30 days of age, providing further evidence of the usefulness of alternative and non-invasive matrices for perinatal hormonal studies in canine species, as previously reported ,24,25.Because body hair in dogs appears at 44–46 days post-coitum, with the completion being reported at 52–54 days , it is rp < 0.05) in PRE-P when compared to STILL-P or NEON-P, in accordance with a previous study on cortisol concentrations in claws of dead puppies [The most relevant result of the present study was the significant effect of the timing of death on DHEA concentrations in claws, significantly higher ( puppies . Recogni puppies . These s puppies .However, when measurement of DHEA concentrations is performed after the exposure to a stressor, results can have variable patterns: in a recent report, indeed, 60% of human newborns experienced a drop in salivary concentrations of DHEA after a stressor, whereas the remaining 40% showed an increased salivary DHEA concentration . MoreoveConcerning the concentrations of DHEA in newborn dogs, a previous study , performAnother hypothesis relies on the report that in humans, the fetal steroid synthesis seems to be deviated towards DHEA rather than cortisol as early as the 8th–10th week until the 32nd–36th week of pregnancy . Given tIn the present study, other parameters, such as newborn sex and breed body size, were evaluated for their possible influence on DHEA concentrations in the claws and hair of dead newborn puppies. Concerning neonatal sex, no statistical differences were found, in agreement with a recent report on live newborn puppies , which rA final comment regards the absence of significant differences in hair DHEA concentrations according to age at death. This result is different from data previously reported for cortisol , in whicThis hypothesis could also be supported by the lack of correlation between the DHEA concentrations in coat and claws, suggesting a possible different pattern of DHEA accumulation in dogs during the perinatal period.This study, beside confirming the usefulness of the claws as matrix for evaluating the retrospective long-term accumulation of hormones in newborn dogs, showed that DHEA concentrations in claws of spontaneously dead puppies were significantly higher in puppies delivered prematurely compared to stillborn and to puppies that died within 30 days of age, confirming the hypothesis that higher amounts of this steroid hormone are produced during intrauterine life, and also in puppies that die soon after birth.


Carassius auratus) were fed with C-CTX1 enriched flesh, and their livers were analyzed by nuclear magnetic resonance (NMR). Altered concentrations of glucose, glycogen, taurine, alanine, and lactate were observed. Furthermore, an increase in liver size was noted in animals fed a toxic diet. In addition, increased ammonia concentrations in the aquarium with toxic feed were detected. These results suggest that C-CTX1 ingestion leads to changes in liver metabolomic, which are related to the glucose-alanine cycle. This is the first evidence of CTXs-mediated alterations in liver metabolomic, and a step to further elucidate the metabolism of these toxins within fish.Ciguatoxins (CTXs) are potent neurological marine toxins able to cause ciguatera poisoning (CP) in humans via the food web. Although considered one of the most important food-borne diseases in the world, little is known about how CTXs are metabolized in fish, and the possible alteration of hepatic metabolomic linked to these toxins. To clarify this aspect, goldfish (Carassius auratus) fed C-CTX1 using nuclear magnetic resonance (NMR)-based metabolomics. Thirteen goldfish were treated with C-CTX1-enriched flesh and sampled on days 1, 8, 15, 29, 36, and 43. On day 43, two individuals, referred to as ‘Detox’, were isolated until days 102 and 121 to evaluate the possible recovery after returning to a commercial feed. At each sampling, hepatic tissue was weighed to calculate the hepatosomatic index (HSI) and analyzed for the metabolomics study; animals fed toxic flesh showed a higher HSI, even greater in the ‘Detox’ individuals. Furthermore, altered concentrations of alanine, lactate, taurine, glucose, and glycogen were observed in animals with the toxic diet. These disturbances could be related to an increase in ammonium ion (NH4+) production. An increase in ammonia (NH3) concentration in water was observed in the aquarium where the fish ingested toxic meat compared to the non-toxic aquarium. All these changes may be rationalized by the relationship between CTXs and the glucose–alanine cycle.Ciguatera poisoning (CP) is a foodborne disease known for centuries; however, little research has been conducted on the effects of ciguatoxins (CTXs) on fish metabolism. The main objective of this study was to assess different hepatic compounds observed in goldfish ( Ciguatoxins (CTXs) are polyether compounds responsible for ciguatera poisoning (CP), which is one of the most important fish and shellfish-borne diseases worldwide . Three fCurrently, the metabolization of the CTX inside the organisms is not clear. The liver is considered the primary organ for the detoxification and storage of lipophilic compounds  and the Metabolomics allows the detection of variations in metabolites among tissue types throughout an organism. This strategy has made it possible to study the disturbances caused by environmental changes and toxic exposures. In the last few years, many studies have been conducted to observe the alterations caused by xenobiotics in organisms, showing its great value for toxicological studies, as reviewed by Bonvallot et al. , and refCarassius auratus), a member of the Cyprinidae family which includes other carps such as the common carp (Cyprinus carpio) and Crucian carps (genus Carassius), has been used as model fish species. It is an omnivorous fish with a high resistance to temperature fluctuations, low dissolved oxygen levels, and a high tolerance range for pH. These characteristics, among others, make it a widely used species as a model for the scientific community, reviewed by Blanco et al. [C. auratus to assess the metabolomics alterations caused by pollutants [In this study, the goldfish  and ammonium ion (NH4+). Both forms exist in equilibrium (NH3 + H3O+ ↔ NH4+ + H2O), and normally the general term ammonia will refer to both forms. In freshwater fish species, NH3 diffuses easily through biological membranes, while these membranes are usually less permeable to ionic compounds; therefore, toxic effects are related to the accumulation of NH3 in the environment. However, within the organism, it is the high levels of NH4+ that present the strongest hazard [In the experimental survey, the control of the ammonia parameter is critical for the survival of fish. In aqueous solutions, ammonia is present as unionized ammonia . The first, described in this section, was to evaluate the liver metabolomics changes of fish fed with C-CTX1-contaminated flesh. The second, described in Two experimental trials were carried out with goldfish (n = 26) were obtained from a local commercial fish distributor from Las Palmas de Gran Canaria, Spain. Detailed procedure and flesh CTX-like toxicity results of this research have been previously published [For the first experiment, adult goldfish individuals (ublished .n = 13) and control (n = 13) groups. On the first day of the experiment, three individuals from the control group were sacrificed to determine the normal liver metabolomics profile of goldfish fed with commercial feed. After that, the experimental group was fed flesh from an amberjack naturally contaminated with C-CTX1, as confirmed by liquid-chromatography–mass-spectrometry (LC-MS/MS) (0.270 ng·g−1) by the University of Vigo; whereas, the control group was fed with non-toxic amberjack flesh showing a CTX level below the limit of detection (LOD) observed by a neuroblastoma (Neuro-2a)-cell-based assay (CBA) [−1 in their food every day; each feeding was supervised to verify the correct ingestion of the corresponding daily dose.Briefly, after a period of acclimation, fish were arbitrarily separated into two groups: the experimental (ay (CBA) . To ensuFish were sampled after 1, 8, 15, 29, 36, and 43 days of daily toxic feeding. Control and experimental fish were analyzed in each sampling and livers from both groups were collected for metabolomics study. Each sampling was performed 24 h post-feeding and before any new exposure. After sacrifice, fish were immediately dissected, and the liver was frozen at −80 °C for the metabolomics determination . All the measures of body weight (nearest g) and length (nearest cm) were collected before freezing.On day 43 of the experiment, three fish showed severe symptoms while feeding and one of them was sampled and analyzed. The other two fish were isolated to assess a possible recovery after changing their diet back to commercial feed, one up to day 102 and the other one until day 121 of the experiment. These animals were identified as ‘Detox’.The experimental protocol was approved by the Committee for Animal Welfare of the University of Las Palmas de Gran Canaria and by the Department of Agriculture, Livestock, Fisheries and Water of the Canary Islands Government (code no. OEBA-ULPGC 28/2018).Immediately after sampling, the liver was weighed, to the nearest 0.001 g, to determine the HSI to obtain information on liver condition and energy content. HSI was calculated as:C. auratus  from the experimental (n = 52) and control (n = 52) were extracted by addition of 1.5 mL acetonitrile/water (1:1). The tissue was homogenized using a SONICS ultrasonic processor equipped with a 3 mm ultrasound probe in 30 sec pulse periods up to 8 min followed by cooling on dry ice for 15 min. After centrifugation for 15 min at 15,000× g at 4 °C, 1.0 mL of the supernatants was transferred to empty vials and concentrated in a Thermo Scientific Savant SpeedVac Vacuum Concentrator  at room temperature. The dry samples were reconstituted in phosphate buffer (600 μL of a 0.2 M solution) containing D2O, NaN3 (3 mM), and TSP (1 mM). The 1H NMR and HSQC spectral data were acquired at 298 K on spectrometers Bruker Avance-500  and Avance-600 equipped with a TCI cryoprobe, respectively. For each experiment, 60 transients were collected into 64 k data points using a spectral width of 7507.5 Hz (acquisition time of 2.18 s) and a relaxation delay of 1 s. All spectra were processed with the software MestReNova . Afterward, all spectra were phase- and baseline-corrected. Finally, the chemical shifts were referenced to the TSP signal at 0.0 ppm. Each spectrum was normalized using the TSP signal as a standard for comparative analyses.Liver tissue samples of 1H and 13C NMR chemical shifts obtained from the HSQC experiments of the selected representative samples (toxic and non-toxic) were analyzed using the online COLMAR 13C-1H HSQC metabolomics database (http://spin.ccic.ohio-state.edu/index.php/hsqc/index (last accessed on 28 September 2022); Campus Chemical Instrument Center of The Ohio State University) [Metabolic content signal analysis of 2D HSQC was performed to determine the toxic effects of the CTXs-enriched diet on the liver metabolic pathways of the treatment and control groups. The versity)  to ident3) concentration alterations. Fish (n = 4) from one aquarium received toxic raw amberjack flesh, whereas the other one (n = 4) received non-toxic raw amberjack flesh. The day before the beginning of the experiment, biofilter materials were collected, mixed, and divided into the two filters, to guarantee the same conditions in both aquaria.Two aquaria with similar fish biomass , amount of water, filters, types, and amount of biofilter materials  were used to determine the possible ammonia . Two testers were used: freshwater low-range ammonia colorimeter—checker® HC , and medium-range ammonia colorimeter—checker® HC , depending on the ammonia levels. The method followed by these checkers is an adaptation of the ASTM Manual of Water and Environmental Technology, D1426, Nessler Method.Ammonia levels were recorded 14 days out of the 16 days of the experiment. Measurements were conducted after 24 h post-feeding. On days 5 and 12, the individuals were not fed, receiving food 6 days a week to study the possible variation in the ammonia concentration. Ammonia levels were determined with a freshwater ammonia colorimeter—checker3–N) present in the water. According to the manufacturer, to convert the results in ammonia levels (ppm), the following formula was used:The ammonia checker used displays the amount  of ammonia nitrogen . Besides NHTo evaluate the HSI values, data analysis was conducted using PASW Statistics software 18.0 for Windows .p-value ≤ 0.05 was considered statistically significant.The non-parametric test Kruskal–Wallis was used to compare the HSI values according to the food received, due to the small number of individuals analyzed. As usual, a n = 11) showed a higher HSI than the group fed non-toxic flesh (n = 10)   . The twoC. auratus were extracted and reconstituted in phosphate buffer, containing D2O, NaN3, and TSA as internal standards for NMR analysis, as described in the experimental section. Firstly, 2D HSQC experiments were performed in both the control and treatment groups  than the toxic one (0.000 ± 0.05 ppm). The non-toxic aquarium kept a higher value for the first two days of the experimental diet, but after the third ingestion of toxic food, the levels of the toxic aquarium were greater than the non-toxic one . The differences between the two aquaria increased gradually, with a maximum of 3.399 on day 9. That day, due to the high ammonia levels, a water change was done in both aquaria. In the first days after the renewal of water, the ammonia levels decreased gradually. Three days later, the ammonia levels in the toxic aquarium increased again, whereas in the non-toxic aquarium, the concentration reached 0.000 ± 0.05 ppm. The ammonia levels continued increasing until the end of the experiment.The ammonia levels are shown in + Cl−-dependent transporters [+ ions are required to elicit taurine transport [+ in neuronal cells [The experimental results demonstrate that the presence of CTXs in fish diet causes a sudden increment of taurine in the fish liver that remains at stable levels along the feeding experiment. In general terms, the concentration of taurine is determined by parameters such as biosynthesis , taurinesporters . It regusporters . In partransport . The impransport . Fish-baal cells ; hence, On the other hand, nutritional studies with marine fish species have revealed that diets supplemented with taurine restored the activity of amino acid catabolic and gluconeogenic enzymes and hexokinase to control levels ,40. DespDiplodus sargus) [Scophthalmus maxima) [The efficiency of carbohydrate metabolism in fish mainly depends on the enzyme activity, insulin receptors, rate of glucose transport, and regulation efficiency of hepatic glucose utilization . Dietary sargus) . Moreove maxima) . Under t4+, as NH3 in water, is detected in treated fish.Most of the observed metabolic alterations seem to be related to the glucose–alanine cycle , which cC. auratus showing CTXs toxic syndrome on day 43 were fed with a commercial diet for at least 60 additional days. A comparison of 1H NMR liver metabolic profiles revealed that the toxic effects are sustained with time and that recovery of initial metabolic levels is not produced in the period analyzed (Specimens of analyzed . However4+ concentration is observed. Therefore, an increment of NH3 levels was produced in the aquarium with fish fed with toxic flesh, shown in 3 concentrations decreased in the non-toxic aquarium. This could be explained by the absence of feeding on the previous day, and therefore the reduction in ammonia excretion. In the toxic aquarium, no decline was observed on that day, probably because the animals continued to excrete NH3 because of CTXs detoxification. The differences between the two aquaria increased gradually, with a maximum of 3.4 ppm after 9 days in which a water renewal was performed (3 levels was appreciated. This reduction is produced by a more efficient transformation into other nitrogen compounds by the bacteria of the aquaria, considering that these bacteria were under higher ammonia concentrations the previous days. Nonetheless, it can be observed that the tendency of NH3 increment continues in the water renewal cycles.The accumulation of CTXs in the diet of fish involves an important metabolic alteration in the liver. Indeed, previous results published by Sanchez-Henao et al.  showed terformed . In the Increased levels of glucose and glycogen in the hepatic tissue could be the reason why animals fed toxic flesh showed a higher HSI than the non-toxic group , since tCarassius auratus has been described as an excellent model species for studies on CTXs accumulation in tissues and the analysis of their toxic effects. Due to this fact, in this work we have been able to analyze the effects of these toxins in the liver, which is considered the target organ for detoxification. Thus, from the metabolic point of view, CTXs cause an important disruption in concentrations of taurine, alanine, and lactate, as well as glucose and glycogen in the liver. These alterations affected the hepatic size, with an increment of the HSI in animals that ingested toxic flesh. In addition, a relevant increment of NH3 levels was detected in aquaria with fish fed with CTXs. Although further research should be done, all these parameters can be rationalized through the relationship of CTXs to the glucose–alanine cycle.


Maytenus senegalensis is one of the medicinal plants widely used in traditional medicine to treat infectious and inflammatory diseases in Africa, there is a lack of safety data regarding its use. Therefore, the study aimed to asselss the safety and tolerability of the antimalarial herbal remedy M. senegalensis. Material and Methods: The study design was an open-label, single-arm, dose-escalation. Twelve eligible male healthy Tanzanians aged 18 to 45 years were enrolled in four study dose groups. Volunteers’ safety and tolerability post-investigational-product administration were monitored on days 0 to 7,14, and 56. Results: There were no deaths or serious adverse events in any of the study groups, nor any adverse events that resulted in premature discontinuation. The significant mean changes observed in WBC (p = 0.003), Neutrophils (p = 0.02), Lymphocytes (p = 0.001), Eosinophils (p = 0.009), Alanine aminotransferase (p = 0.002), Creatinine (p = 0.03) and Total bilirubin (p = 0.004) laboratory parameters were not associated with any signs of toxicity or clinical symptoms. Conclusions: M. senegalensis was demonstrated to be safe and tolerable when administered at a dose of 800 mg every eight hours a day for four days. This study design may be adapted to evaluate other herbal remedies.Background: Though  Maytenus senegalensis (Lam.) Exell is one of the medicinal plants belonging to the Celastraceae family. It is a shrub or tree that grows up to 15 m high and is widely distributed in most African countries as well as Arabia, Afghanistan and India [M. senegalensis contain several active substances, such as triterpenes, sterols, saponosids, alkaloids and tannins [ tannins ,3. M. senegalensis is reported to be anti-inflammatory [M. senegalensis is one of the most important medicinal plants for the treatment of malaria, fever, pain and chronic diseases [M. senegalensis have also been reported from other African countries, such as Benin, Ivory Coast, Kenya, Sudan, Zambia and Zimbabwe [Pharmacological assays have confirmed that most of these substances have biological activities. ammatory ,3, antimammatory ,4,5, antammatory , antibacammatory ,9,10,11.diseases . TraditiZimbabwe ,13,14. M. senegalensis ethanol extract at different doses in mice. The M. senegalensis extract was found to be nontoxic, and the oral median lethal dose in mice was determined to be greater than 1600 mg/kg body weight [M. senegalensis at a dose of 5000 mg/kg body weight was well tolerated, without causing visible signs of toxicity or mortality in mice [M. senegalensis ethanol extract can be categorized as a nontoxic crude drug that acts harmlessly under normal traditional usage without causing acute toxic symptoms and complications.Studies have been conducted to determine the acute toxicity of y weight . In anot in mice . Thus, MM. senegalensis obtained from clinical trials necessitated the need to conduct this study. There is a claim that products with a long history of popular use are generally safe when used properly at common therapeutic doses . The lacM. senegalensis in healthy Tanzanian adult volunteers. This study may demonstrate a new development strategy for identifying novel antimalarial drugs that can counteract the emergence of antimalarial resistance through exploiting the rich natural biodiversity of Africa to accelerate malaria elimination [This study was conducted to evaluate the safety and tolerability of antimalarial herbal remedy mination ,18,19. MM. senegalensis in healthy adult Tanzanian volunteers . The study design was an open-label, single-arm, dose-escalation study to evaluate the safety and tolerability of antimalarial herbal remedy M. senegalensis and followed by a safety evaluation for seven days after the administration. The volunteers of group 2 were enrolled next in the study and given 600 mg of M. senegalensis and followed by a safety assessment for seven days after administration. Then, the volunteers from group 3 were enrolled in the study and given 800 mg of M. senegalensis every 8 h for 4 days, followed by a safety evaluation for 7 days after the first administration. Lastly, volunteers of group 4 were enrolled in the study and given 800 mg of M. senegalensis every 8 h for 4 days , with three volunteers in each group. The volunteers from group 1 were enrolled first in the study, given 400 mg of r 4 days . Study vAll medications other than the tested product were recorded as concomitant medications. Neither routine activities nor dieting were restricted during the trial period.M. senegalensis was administered under direct observed treatment (DOT). As a precaution, women were not included in the study to reduce the risk of reprotoxicity, because preclinical data on reproduction toxicity were not available.All study volunteers were hospitalized for 7 days. The dose of The trial was carried out from June to September 2021 at the Bagamoyo Clinical Trial Facility (BCTF), located on the Kingani Estate, 74 km north of Dar es Salaam within the coastal region.2 and who were long-term residents of the study area and agreed to release medical information and to be attended by a study clinician prior to and during the study. Other inclusion criteria were the volunteer’s agreement to provide contact information for a third-party household member or close friend to the study team, available by mobile phone 24 h during the study period, agreement not to participate in another clinical trial and donate blood during the study period, health that complied with the study protocol procedures and agreement to undergo HIV, hepatitis B (HBV) and hepatitis C (HCV) tests, demonstrated understanding of the study, signed written informed consent and free of malaria parasitemia by blood smear at enrolment. The study population included healthy men aged 18 to 45 years with a Body Mass Index (BMI) ranging from 18 to 30 kg/mVolunteers were excluded if they had any of the following criteria: received an investigational malaria drug in the last 5 years, participation in any other clinical study involving investigational medicinal products within 30 days prior to the onset of the study, history of arrhythmias, prolonged QT-interval or another cardiac disease, clinically significant abnormalities in electrocardiogram (ECG) at screening, a positive family history of cardiac disease in a first- or second-degree relative at the age of 50, a history of psychiatric disease, suffering from any chronic illness , any confirmed or suspected immunosuppressive or immune-deficient condition, history of drug or alcohol abuse, use of chronic immunosuppressive drugs or other immune-modifying drugs within three months of study onset except inhaled and topical corticosteroids. Other exclusion criteria were any clinically significant deviation from the normal range in biochemistry or hematology, blood tests or urine analysis; positive HIV, hepatitis B virus or hepatitis C virus tests; suspicion of having clinically active TB history or physical examination with a positive QuantiFERON-TB Gold Test in-tube assay; symptoms, physical signs, and laboratory values suggestive of systemic disorders that could muddle the interpretation of the study results or jeopardize the health of volunteers. The last exclusion criterion was any medical, social or occupational reason that, in the judgment of the study clinician, was a contraindication to participation or impaired the ability to give informed consent, increased the risk to the volunteer due to participation in the study, affected the ability of the volunteer to participate in the study or impaired interpretation of the study data. The study aimed to assess the safety and tolerability of an antimalarial herbal remedy. Therefore, the calculation of statistical power and sample size was not performed. The sample size for the study was set at 12 volunteers. The study was carried out using adapted dose escalation rules for the traditional 3 + 3 design, which is commonly used in oncology phase 1 clinical trials . The regM. senegalensis were harvested on 9 June 2020 from Bugabo buzi village, Misenye District in the Kagera Region. The region has two rain seasons: heavy rain from March to May and short rains from September to December. The plant was identified by Haji O. Selemani, a botanist from the University of Dar es Salaam, with Voucher Specimen Number 5672, which was deposited at the University Herbarium. The root barks were then debarked to obtain the root barks, which were air-dried under shade at room temperature for two weeks and then pulverized by a grinding mill. The pulverized root backs (20 kg) were extracted three times by cold percolation at room temperature using 80% ethanol and filtered by Macherey–Nagel filter paper. The filtrates were distilled under reduced pressure at 40 °C to give 2.24 kg of brown gummy extract.The root barks of The presence of an active ingredient (Pristimerin) for the treatment of malaria was qualitatively determined using the Thin Layer Chromatography (TLC) method. The extract (10 mg) and Pristimerin (1 mg) were separately dissolved in methanol (1 mL). The solutions were then spotted on a TLC and eluted with dichloromethane/methanol at 99:1. The Pristimerin in the sample was observed at Rf 0.3, matching the standard Pristimerin . The extract was granulated by mixing it with starch at a ratio of 2:1 (Extract 2: Starch 1) and packed in hard shell capsules. Each capsule contained 300 mg of powder (200 mg of extract and 100 mg of starch). These capsules were packed in blister bags containing 100 capsules .A trial batch of investigational products was analyzed at the Institute of Traditional Medicine-MUHAS, Tanzania Bureau Standards & Government Chemist Laboratory Authority (GCLA) to minimize the risks of misidentification . The tesThe balance between efficacy and safety was considered during the selection of a dose for the estimation of the human equivalent dose (HED). The toxicity of drugs increases with increasing doses. According to a previous study, a dose of 100 mg/kg body weight was safe and was able to clear parasites by almost 100%; it was also demonstrated that it was safe at doses as high as 1600 mg/kg body weight . TherefoThe safety of the tested product was evaluated through taking medical history, physical examination, vital signs, 12-lead ECG, clinical laboratory tests and incidence of AEs. Physical examinations were performed by study clinicians and included the examination of the following: general appearance, eyes, ears, nose, throat, chest/respiratory, heart/cardiovascular, gastrointestinal/liver, musculoskeletal/extremities, dermatological/skin, thyroid/neck, lymph nodes, and neurological/psychiatric systems.The vital signs observed during the study included systolic blood pressure, diastolic blood pressure, pulse rate, and body temperature at baseline and 0, 1, 2, 3, 4, 5, 6, 7, 14, 28 and 56 days. A 12-lead ECG was performed at baseline and at 3, 7, 14, 28 and 56 days.Safety laboratory tests (including hematology and chemistry) were performed at baseline and at 3, 7, 14 and 28 days. A total of 1 mL of venous blood sample was collected into a K3 EDTA vacutainer tube (Greiner Bio-one) and a Z-serum clot activator tube (Greiner Bio-one) for hematology and biochemistry analysis, respectively. For hematological analysis, a fully automated 5-part differential hematology analyzer  was used to analyze samples within 8 h of blood draw. The results printed out were double-checked by two independent, qualified technicians before being handed out to clinicians for clinical management. For biochemical analysis, samples were allowed to stand at room temperature for 30 min before separating the serum by centrifugation at 3000 rpm for 10 min. The serum was analyzed using Cobas Integra 400 Plus  within 24 h after the blood draw. If the testing was to be delayed, serum for biochemical analyses was stored at −20 °C and subjected to a single freeze–thaw cycle at the time of analysis, and whole EDTA blood for hematological analyses was stored at 4–8 °C and analyzed within 24 h after the blood draw.For the quality assurance of the analyzers, internal quality control (IQC) and external quality assurance (EQA) quality assessments were performed on the main and back-up analyzers according to standard operating procedures. IQC was carried out daily using quality control materials supplied by the respective analyzer’s manufacturer. EQA was performed monthly using materials supplied by an independent EQA firm (the College of American Pathologists (CAP) for the biochemistry analyzer and the United Kingdom National External Quality Assessment Scheme (UK NEQAS) for the hematology analyzer). Only the analyzers that passed the IQC and EQA assessments were used for clinical sample analysis.3/L), red blood cell count (4.39 to 6.5 × 106/L), hemoglobin (12.6 to 17.3 g/dL), platelets (107 to 396.2 × 103/L), neutrophil counts (1.18 to 5.46 × 103/L), lymphocyte counts (1.19 to 3.4 × 103/L), and eosinophil counts (0 to 0.78 × 103/L) were all examined. The analysis of liver function tests included AST (12.3 to 74.7 U/L), ALT (3.5 to 46.8 U/L) and total bilirubin (3.1–31.1μmol/L). The serum creatinine level (49 to 95.3 μmol/L) was measured as a function of the kidney.White blood cell count  guidelines . The corSolicited adverse events were monitored and recorded up to day 7, while unsolicited adverse events were monitored and recorded up to day 28 after the first administration of the tested product.All study volunteers were monitored throughout the study for serious adverse events (SAEs). The study clinicians were responsible for evaluating all AEs in terms of intensity , duration, severity, outcome, and relationship to the study drug. The Standard Treatment Guidelines & National Essential Medicines List, Tanzania Mainland (STG/NEMLIT-2017); Division of AIDS (DAIDS) Table for Grading the Severity of Adult and Pediatric Adverse Events ; US FDA, Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials: Guidance for Industry (September 2007); and Common Terminology Criteria for Adverse Events (CTCAE)  were used to develop the grading criteria for adverse events.p-value ≤ 0.05 was considered significant.Case report forms were created to collect clinical and biological data for each volunteer. Data were first recorded on a paper CRF and then transcribed into the Castor Electronic Data Capture (EDC) system and saved in an electronic case report form (eCRF). Furthermore, data from the castor EDC were extracted and transferred to Stata Statistical Software version 15 for further analysis. Means and standard deviations were reported for continuous numbers and percentages for categorical variables. The comparison of the different laboratory parameter means over time was done using Friedman’s test. A Only 12 of the 30 study volunteers who had been screened were eligible for randomization. These 12 eligible volunteers were randomized to four study groups , with three (3) volunteers in each study group. The 12 enrolled volunteers completed the study and were included in the analysis. 2. The 12 study volunteers enrolled in the study were African males with a mean age of 27.70 ± 6.41 years. The average body mass index (BMI) of these volunteers was 22.77 ± 2.87 kg/mThere were no deaths or serious adverse events in any of the study groups or adverse events that led to premature discontinuation of the study volunteers. Furthermore, no solicited or unsolicited clinical adverse events (AEs) were reported in the first 28 days of groups 1, 2, 3 and 4. All reported events were laboratory adverse events. A total of 21 AEs were reported throughout the study. According to In addition, laboratory data, vital signs, physical findings, and 12-lead electrocardiogram parameters did not show any relevant changes over time that might be related to the study drug. Clinical review of the laboratory, 12-lead electrocardiogram, vital signs, and physical examination data did not indicate any safety concerns.p = 0.05 for group 2 and p = 0.003 for groups 3 and 4). However, the highest mean value observed was within the normal range.p = 0.05), neutrophil count (NEUT-N) for group 3 (p = 0.02), lymphocyte count (LYMP_N) for group 3 (p = 0.001) and eosinophils count (EOS_N) for group 3 (p = 0.009). However, the observed changes were within the normal range.Furthermore, there were statistically significant changes in the mean value of platelets (PLT) for group 1 . The highest mean value of alanine aminotransferase observed on day seven was slightly out of the normal range. However, the mean value of alanine aminotransferase on day 14 was within the normal range.p = 0.03) for groups 3 and 4, which were slightly outside of the normal range for creatinine. Furthermore, there was a decreasing trend in the mean value of total bilirubin from the baseline for groups 3 and 4. The mean value of total bilirubin decreased up to day three and then started to return to baseline. These decreasing changes are statistically significant (p = 0.004). However, the observed changes in the total bilirubin mean values were within the normal range. The statistically significant changes observed in mean values from baseline for white blood cells, platelets, neutrophil counts, lymphocyte counts, eosinophil counts, alanine aminotransferase, total bilirubin, and creatinine were not associated with any signs of toxicity or clinical symptoms.There were statistically significant changes in the mean value for creatinine  in healthy adult male volunteers. There were no deaths or serious adverse events or adverse events considered clinically relevant or resulting in tested product discontinuation or study withdrawal. Furthermore, no solicited or unsolicited clinical adverse events (AEs) were reported in the first 28 days post administration of the tested product. All AEs reported were laboratory adverse events and were graded either 1 or 2 in severity.M. senegalensis. Preclinical animal studies have shown that orally administered M. senegalensis root extracts are well tolerated, without evidence of toxicity or death, in experimental mice [All reported adverse events were transient and resolved without any clinical sequelae. The clinical review of physical examination, vital signs, and 12-lead ECG data did not reveal safety signals. The findings of this study are supported by the good pre-clinical safety data of tal mice ,15. M. senegalensis to be anti-inflammatory [In this study, there was a significant increase in the mean value of white blood cells and their differentials  for the group that received the highest dose of the tested product. This finding suggests the potential immunomodulatory effects of the products tested. WBC and its differences are known for a protective role against foreign bodies and infectious pathogens through the production, transport and distribution of antibodies in the immune response . Plants ammatory ,3, antimammatory ,4,5, antammatory ,9,10,11.Hemoglobin inside RBC is essential in transferring respiratory gases. It transports oxygen to cells and carbon dioxide to the lungs . The finThe results of the study showed that statistically significant changes in the mean value of platelets in the group that received the highest dose of the tested product. Platelets are fragments of large bone marrow cells that function to aid blood clotting and initiate the repair of the walls of blood vessels . This fiMaytenus senegalese induced a transient change in ALT levels in healthy adult male volunteers. This study finding is consistent with the findings of a preclinical animal study, which revealed a transient dose-dependent increase in ALT activity following administration of an 800 mg/kg dose of M. senegalensis to male rats [AST and ALT levels are the predominant blood markers of liver injury. In this study, there was an increasing trend in mean values of the ALT parameter for the study group that received the highest dose of the tested product up to day seven after administration, and then it started to return to baseline. This finding implies that the administration of ale rats . The level of creatinine is one of the indicators that play an important role in determining the synthetic and excretory functions of the kidney. A significant increase in serum creatinine after administration of the tested product may be an indication of renal failure, especially with regard to glomerular filtration, which directly affects the rate of clearance of waste substances by the kidney . FurtherBilirubin is the breakdown of hemoglobin in red blood cells and is a by-product of hemolysis (RBC destruction). The measurement of bilirubin allows the evaluation of liver function and hemolytic anemia . In thisM. senegalensis as an antiparasitic agent. In addition, there is a lack of pharmacokinetic data for the M. senegalensis extract. The availability of pharmacokinetics data for herbal remedy extracts is crucial for the prediction of potential herb–drug interactions and herbal pharmacological efficacy [M. senegalensis extract are needed in the future. However, it would be a challenge due to the chemical complexity [The study was limited by the small number of male volunteers enrolled. More research should be done to investigate the effects of gender differences. Nevertheless, the information presented here lays a foundation for future follow-up studies to evaluate the efficacy of efficacy . Pharmacmplexity .M. senegalensis, was demonstrated to be safe, tolerable, and free of any untoward toxic effects in healthy male volunteers when administered at a dose of 800 mg every 8 h for four days. The statistically significant changes in the mean values from baseline for white blood cells, neutrophils, lymphocytes, eosinophils, alanine aminotransferase, total bilirubin, and creatinine were not associated with any toxicity or clinical symptoms. In summary, an antimalarial herbal remedy, The design of this study may be adapted to evaluate other herbal products with medicinal potentials and fast-track finding solutions for malaria and other health problems in Africa. Therefore, the findings of this study contribute to the effort to develop medical evidence and fill the scientific gap created by the lack of safety data on most herbal remedies.


To investigate the value of intestinal flora in predicting major adverse cardiovascular and cerebrovascular events (MACCE) in patients with refractory hypertension (RH).  359 patients with RH hospitalized in our hospital from April 2020 to March 2021 were followed up for 1 year and selected for the study. These patients were divided into a MACCE group and no-MACCE group. Results were analyzed by comparing general information, the abundance of intestinal flora at the phylum level, and the abundance of intestinal flora at the species level between the two groups. The influence factors related to MACCE were evaluated using multifactor logistic regression analysis, and the value of intestinal flora in predicting MACCE was determined using receiver operating characteristic (ROC) and the area under ROC (AUC). P < 0.05). The abundances of Actinomycetes and Verrucomicrobia were higher in the MACCE group than in the no-MACCE group, while unnamed viruses were the opposite (P < 0.05). The abundances of Eubacterium eligens, Akkermansia muciniphila, Prevotella stercorea, and Eubacterium rectale were lower in the MACCE group than in the no-MACCE group, while Escherichia coli, Clostridium hathewayi, and Ruminococcus gnavus were opposite (P < 0.05). Systolic blood pressure, Actinomycetes, unnamed viruses, Verrucomicrobia, Eubacterium eligens, Akkermansia muciniphila, Prevotella stercorea, Eubacterium rectale, Escherichia coli, Clostridium hathewayi, and Ruminococcus gnavus were closely associated with MACCE in RH patients (P < 0.05). In addition, Akkermansia muciniphila had the highest AUC among the single indicator but was still lower than the AUC of the combined detection.  Systolic blood pressure was higher in the MACCE group than in the no-MACCE group ( The increases of Actinomycetes, Verrucomicrobia, Escherichia coli, Clostridium hathewayi, and Ruminococcus gnavus and the decreases of unnamed viruses, Eubacterium eligens, Akkermansia muciniphila, Prevotella stercorea, and Eubacterium rectale were associated with MACCE in RH patients, and the combined detection may provide a method and idea for predicting and preventing MACCE. In China, the morbidity of hypertension is 18.8% in people aged ≥18, while the blood pressure control rate is only 6.1%, which is not only related to medication compliance and treatment timeliness but also related to the number of patients with refractory hypertension (RH) . IntestiThe study recruited 359 patients with RH hospitalized in our hospital from April 2020 to March 2021. During the 1-year follow-up, 4 patients were lost to follow-up and 355 patients were visited, including 179 females and 176 males, aged from 38 to 86 years, with an average of 56.64 ± 13.90 years. All patients signed the written informed consent. The study was permitted by the Ethics Committee of our hospital. All patients were divided into 2 groups, including a MACCE group and no-MACCE group.Inclusion criteria are listed as follows: (i) patients meeting the diagnostic criteria of RH , (ii) paGeneral information questionnaire was performed to collect RH patient information, including age, gender, body mass index, diastolic blood pressure, systolic blood pressure, duration of disease, diabetes, hyperlipidemia, smoking, drinking, chronic renal insufficiency, and medication history.Reagents used in this study included Stool DNA Kit  and 2 × Taq PCR MasterMix . Primer synthesis was performed by Sangon Biotech . Experiment instruments included an ultraviolet spectrophotometer , polymerase chain reaction (PCR) thermocycler , automatic PCR analysis system , and ultraviolet transmission and reflection analyzer .2 g of midstream stools was collected from RH patients using a special stool collector, and DNA was extracted using a stool DNA Kit. DNA purification and concentration were analyzed by an ultraviolet spectrophotometer, and DNA integrity was determined by agarose gel electrophoresis. The OD260/OD280 of purified DNA with high purity is between 1.6 and 1.8. Then, V3-V4 variable regions of microbial 16 SrDNA were amplified by PCR with primers  with the reaction at 94°C for 3 min, 27 cycles at 94°C for 30 s, 72°C for 30 s, and 72°C for 10 min and then identified by high-throughput sequencing using an Illumina HiSeq sequencer, followed by bioinformatics analysis. The DNA was quantified by quantitative real-time PCR with a PCR thermocycler. Double barcode was introduced into the primer area for PCR amplification to prevent amplification bias and the occurrence of chimeric sequences to ensure the satisfactory concentration of amplification products in the minimum cycle number of samples.When investigating microbial diversity, operational taxa are introduced to facilitate analysis and improve efficiency. Representative sequences of the operational taxonomic unit were selected and compared with ribosomal RNA data of Greengenes Database 13-8 version based on 99% sequence similarity clustering to obtain the annotation information of species. Single sequences and chimeric sequences without duplication were taken out, and the confidence threshold was set to 70% to obtain the composition of the tested samples at phylum and species classification levels. The species abundance spectrum was evaluated according to the proportion of phylum and species in the total number of sequences.The observation indexer were as follows: (1) general information was compared between the two groups. (2) The abundance of intestinal flora at the phylum level was compared between the two groups. (3) The abundance of intestinal flora at the species level was compared between the two groups. (4) The influence factors of RH complicated with MACCE were analyzed between the two groups. (5) The predictive value of intestinal microbiota-related indexes for MACCE was assessed between the two groups.χ2 test and expressed as n (%), while variables data were compared with Student's t-tests and expressed as means ± standard deviations (SD). The factors related to MACCE were evaluated using multifactor logistic regression analysis, and the value of intestinal flora in predicting MACCE was determined using receiver operating characteristic (ROC) and the area under ROC (AUC). P < 0.05 indicated statistical significance.All data were analyzed using SPSS24.0. Attribute data were compared using the P < 0.05) , including 55 cases with cerebral hemorrhage, 62 cases with cerebral infarction, 44 cases with acute myocardial infarction, and 8 cases with cardiovascular and cerebrovascular event-related death. 186 cases (52.39%) did not occur with MACCE. The results showed that there was no significant difference in age, gender, body mass index, diastolic blood pressure, duration of disease, diabetes, hyperlipidemia, smoking, drinking, chronic renal insufficiency, and medication history when comparing the two groups . But, sy < 0.05) .P < 0.05)  .P < 0.05)  .P < 0.05) was taken as the independent variable. As shown in P < 0.05).MACCE occurrence was taken as the dependent variable, and the comparison index between the two groups (According to the ROC curve of intestinal microbiota-related indexes to predict MACCE, we found that the combined detection had the highest AUC .Diastolic blood pressure and/or systolic blood pressure still cannot be effectively controlled after RH patients sufficiently take more than 3 types of antihypertensive drugs including diuretics. It is difficult to control blood pressure in such patients, and the risk of MACCE increases accordingly . Thus, iAt the level of phylum classification, Bacteroidetes and phylum Firmicutes are the two most dominant phyla in the intestinal flora of healthy people and hypertensive people . The preAt the level of species classification, the number of Escherichia coli, Clostridium hathewayi, and Ruminococcus gnavus is higher in hypertensive patients than in healthy people . Our datIn the present study, according to the results of ROC analysis, we found that the AUC of each intestinal flora in predicting MACCE was >0.7, showing their predictive value in MACCE. In particular, the AUC of Akkermansia muciniphila was the highest compared with other intestinal flora, suggesting that Akkermansia muciniphila had the highest predictive value in MACCE among these intestinal florae. However, the AUC of combined detection of the intestinal flora was 0.926, which was significantly higher than that of other single intestinal flora, suggesting that the combined detection of the intestinal flora could improve the reliability of predicting MACCE. The reason may be that these intestinal florae are associated with the occurrence of MACCE, and the combined detection covers more mechanisms of MACCE occurrence. However, intestinal flora detection has certain requirements on hospital hardware and software configuration, which may limit its application in primary medical centers. Thus, further studies are needed to study more convenient intestinal flora detection methods.Taken together, the increased number of Actinomycetes, Verrucomicrobia, Escherichia coli, Clostridium hathewayi, and Ruminococcus gnavus and the decreased number of unnamed virus, Eubacterium eligens, Akkermansia muciniphila, Prevotella stercorea, and Eubacterium rectale were associated with MACCE in RH patients. The combined detection can provide a method and idea for predicting and preventing MACCE.


Multivariable linear regression analysis confirmed the above finding , independently of age, overweight, and central obesity. No correlation was noted between the duration of breastfeeding and other spirometric parameters. In addition to its favorable impact on the metabolic profile, prolonged exclusive breastfeeding seems to exert a propitious effect on the function of smaller airways throughout childhood.Although the propitious effects of breastfeeding on children’s health are indisputable, the impact of exclusive breastfeeding on the lung function later in life remains controversial. Our objective was to explore the possible associations between breastfeeding and the lung function of children who were exclusively breastfed for an extensive period of time. This was a cross-sectional study of children who were exclusively breastfed for more than 12 months. Demographics and anthropometric data were collected; the body mass index (BMI), % body fat, and % central obesity were calculated; and all the participants underwent standard spirometry with reversibility testing. The relationship between breastfeeding duration and spirometric parameters was assessed by Spearman’s correlation and multivariable regression, after adjustment for other confounders. Forty-six children (21 boys), aged 9.2 ± 2.4 years, with a reported breastfeeding duration of 27.5 ± 12.5 months (range 12–60 months) were included; 13% were overweight (none were obese) and 21.7% had central obesity. The average FEV1 was 104.7 ± 10.4% and the average FEF25-75 was 107.9 ± 13.3%. The duration of exclusive breastfeeding was positively correlated with FEF25-75% (r = 0.422,  Ample evidence from epidemiological and biological studies has emphasized the propitious health effects of breastfeeding . HoweverInitially, the vast majority of women commence breastfeeding soon after delivery; thus, the great challenge is, as a society and as healthcare professionals, to establish and protect the continuation of this very positive initial step. A striking paradigm, addressing this goal in the best possible way, is the implementation of the ten steps towards successful breastfeeding of the WHO and United Nations Children’s Fund (UNICEF) . Hence, In Greece, the latest national data showed an increase in breastfeeding rates ,9, althoThe protective action of human milk against respiratory and gastrointestinal infections in infancy, together with its favorable effects against the development of obesity, diabetes, and atopy in childhood and adolescence, have been well established ,10,11,12The objective of this study was to explore the possible associations between breastfeeding and the lung function of children who were exclusively breastfed for an extensive period of time, in particular for more than 12 months. We hypothesized that PEB may have a propitious impact on the usual spirometric indices, independent of other confounding factors.This cross-sectional, observational study was nested within the initiative ‘Children’s Lungs: from infancy to adolescence’ of the Pediatric Respiratory Unit of Democritus University of Thrace, Greece, which included children up to 14 years of age. The research model applied to conduct the present study was quantitative research methodology following a positivist approach, in order to test our hypothesis that PEB may have a beneficial effect on the spirometric parameters, independent of other confounding determinants. The study population was recruited from a private breastfeeding clinic in Thessaloniki, Greece, and children from 4 to 14-years-old who had been exclusively breastfed for at least 12 months were included. The lower age limit was chosen based on the possibility to perform a technically acceptable and repeatable spirometry. Children with congenital heart disease, neuromuscular disorders, chromosomal aberrations, or a history of significant prematurity (born at <34 weeks of gestation) were excluded. The parents of eligible participants were initially contacted via telephone and, if they agreed to participate, a visit to the clinic was scheduled. On the day of the visit, following a detailed explanation of the study aims and protocol by the investigators , a consent document was initially filled and signed by the parents. This document included the authorization to access and use all the relevant clinical data of the participants. The study protocol was reviewed and approved by the Ethics Committee of the Democritus University of Thrace (IRB no. 23927/2382/02.01.2017).2 (kg/m2) and assessed according to the International Obesity Task Force (IOFT) normative data [2 cutoffs, and obesity as a BMI higher than the 30 kg/m2 threshold. Waist-to-height (WtH) ratio was calculated as the ratio between waist circumference (cm) and body height (cm), and used to calculate central obesity according to the International Diabetes Federation (IDF) criteria [Participants were invited to attend the clinic at predetermined dates, once per week, between January and December 2019. After the demographics and medical history, including the duration of exclusive breastfeeding from the medical records of the clinic, were collected; weight, height, waist and hip circumference, and the triceps, biceps, subscapular, and suprailiac skinfolds were measured. The body mass index (BMI) was calculated as weight/heightive data ; overweicriteria . Body facriteria .Participants performed spirometry using a Micro 5000 spirometer  according to the ERS/ATS guidelines . Forced 2) and WtH ratio. The statistical significance for the present study was set to 0.05. Statistical analyses were performed using IBM SPSS version 27 .A priori sample size calculation was not performed due to the paucity of the relevant clinical data . Continuous variables were expressed as mean± standard deviation (SD) and range. Spearman’s correlation was used to assess the relationship between spirometric indices  and duration of exclusive breastfeeding. Multivariable linear regression was used to assess the above relationship after adjustment for confounding factors, such as sex, age, BMI , 11 were not possible to contact (change of telephone number), and 8 did not consent to participate. Thus, the study population consisted of 46 children (21 boys) aged 9.2 ± 2.4 years, of whom the demographic and somatometric characteristics are presented in Spirometric data are presented in p = 0.003). Multivariable linear regression analysis confirmed the above relationship, independent of the effect of sex, age, BMI, and WtH ratio  associated with FEF25-75% at 12 and 18 years of age; however, not with the FEV1 or FEV1/FVC ratio [In contrast, longitudinal studies have failed to show a favorable effect of breastfeeding on lung function. In the Promotion of Breastfeeding Intervention Trial (PROBIT), a breastfeeding promotion intervention had no detectable effect on the spirometric indices of the 13,557 participants after 16 years of follow-up . SimilarVC ratio .However, the previous published literature that has not found any positive associations regarding breastfeeding duration and lung function is sparse. It should be noted that in the aforementioned studies ,23,36 thThe pathways by which breastfeeding may exert its beneficial effect on lung function remain largely unknown . The effOur study has several limitations. First, it is a single-center observational study, based on a relatively small sample size. Although as discussed above, we chose to solely focus on PEB at the cost of a restricted population sample, our results cannot be generalizable. On the other hand, the true effect of exclusive breastfeeding could only be assessed by a randomized clinical trial; yet, it would be unfeasible to randomize healthy infants to receive breastmilk or formula. Second, we lack control groups; e.g., of exclusively formula-fed infants or participants who were breastfed for less than 1 year. Again, our intention was to focus on PEB; the inclusion of control groups was deemed logistically unfeasible and beyond the aims of this nested study. Finally, we did not collect and, thus, we did not adjust for other possible confounders, such as parental socioeconomical status and tobacco smoke exposure.In conclusion, the results of the present study show that PEB has a favorable effect on FEF25-75% throughout childhood, independent of known confounding factors, such as body composition, overweight, and obesity. FEF25-75% represents a sensitive parameter for assessing peripheral airway function among pediatric populations. Thus, our findings extend the existing knowledge on the propitious effects of exclusive breastfeeding, suggesting that it may also influence the function of smaller airways in the long-term. Taking into consideration our current findings, breastfeeding for more than 6 months should be regarded as a first step towards a healthy life and must be promoted, protected, and supported. Additional, well-designed studies are required to explore the mechanisms by which PEB may affect lung function in childhood and beyond.


Progressive bulbar palsy (PBP) is a type of motor neuron disease (MND). The main symptoms include dysarthria, dysphagia, tongue muscle atrophy and fasciculations. This disease is generally severe and develops rapidly. Due to the lack of effective treatment, many patients with MND in China turn to traditional Chinese medicine treatment for help. We successfully relieved dysphagia and sialorrhea in a patient with PBP for 3 years with herbal medicine and acupuncture.The patient was a 68-years-old woman with PBP and suffered from severe dysphagia and sialorrhea.Progressive bulbar palsy.Chinese herbal medicine and acupuncture.After 4 months of herbal medicine and acupuncture treatment, dysphagia and sialorrhea were relieved considerably. The patient’s condition has been stable for more than 3 years and continues to be treated with Chinese herbal medicine and acupuncture.Our case suggests that alternative therapies such as herbal medicine and acupuncture may be effective in alleviating the symptoms of MND/PBP. However, standardized clinical studies are still required to verify the effectiveness and safety. The progressive neurological deterioration involves the corticospinal tract, brainstem and anterior horn cells of the spinal cord. Death generally occurs within 2 to 4 years due to respiratory failure. The most common motor neuron disease is amyotrophic lateral sclerosis (ALS). Other forms include primary lateral sclerosis, progressive muscular atrophy, and progressive bulbar palsy (PBP). The initial symptom of MND is often extremity weakness; about 70% of patients present with this “limb-onset” disease. The remaining 25% present with dysarthria and dysphagia, and about 5% of patients present with trunk or respiratory symptoms at the onset. According to a systematic analysis of the global burden of MND in 2016, the worldwide all-age prevalence was 4.5 (4.1–5.0) per 1,00,000 people, and the all-age incidence was 0.78 per 1,00,000 person-years.Motor neuron disease (MND) is characterized by the degeneration of both upper and lower motor neurons, leading to muscle weakness and eventual paralysis. The only 2 approved drugs for the treatment of MND are riluzole and edaravone.,9 Riluzole can only prolong the median survival time by approximately 2 to 3 months in patients with ALS. It is still unclear whether edaravone therapy prolongs survival in the long term. Due to the lack of effective treatment, many patients with MND in China turn to traditional Chinese medicine (TCM) treatment. Studies have verified the effectiveness of acupuncture in improving swallowing ability after stroke.,13 It has also been reported that Chinese herbal medicine and acupuncture may be an effective treatment for MND, relieving symptoms and improving quality of life.,15 However, there are few studies that investigate the effectiveness and safety of traditional Chinese medicine in treating dysphagia and sialorrhea in patients with MND. Here, we report a case of successfully alleviating the symptoms of dysphagia and sialorrhea for a patient with PBP with Chinese herbal medicine and acupuncture for more than 3 years. The ethics committee of Guang’anmen Hospital, China Academy of Chinese Medical Sciences approved the study. The patient has provided informed consent for publication of this case and the writing of this case followed CARE guidelines.The pathophysiology of MND remains unknown, which limits the development of disease-modifying therapies.The timeline with clinical and procedural data is shown in Figure The patient was a 68-year-old lady, who in October 2016, presented with dysarthria, dysphagia, and sialorrhea of unknown origin. There was no tongue numbness, abnormal taste, hoarseness, dyspnea, diplopia or blurred vision, limb weakness or numbness, dizziness, or loss of consciousness. The local hospital did not give a precise diagnosis, and provided treatments for cerebral infarction. After treatment with antiplatelets, circulation improvement and reducing her lipid, the symptoms did not improve and aggressively progressed. On January 9, 2018, the patient sought medical advice at Beijing Xuanwu Hospital, National Center for Neurological Disorders of China. Physical examination showed normal function of the advanced cortex, bilateral tongue muscle atrophy, fibrillation, normal pharyngeal reflex, uvula in the middle, and negative mandibular reflex. Muscle strength and muscular tension were normal, and the reflexes of the bilateral biceps brachii, triceps brachii, and radial membrane were hyperactive. The left palmomaxillary reflex was positive, the pathological signs of both lower limbs were negative, and the water-swallowing test result was grade 2. The mini-mental state examination score was 30. Supplementary examination showed no obvious abnormality in the blood, urine, stool, or cerebrospinal fluid. She underwent head and cervical MRI. The head MRI showed a right frontal subcortical punctate ischemic focus while the cervical MRI showed C4 to C5 and C5 to C6 intervertebral disc herniation. On electromyography images, the sensory conduction velocity of the double median nerve (finger I and finger III) had slowed and the amplitude of sensory conduction of the left median nerve (finger I) had decreased. Neuropsychological examination, carotid ultrasound, and intracranial artery ultrasound showed no obvious abnormality.1 10 mg 3 times per day were prescribed to improve nerve function. After taking riluzole for 10 months, the patient’s symptoms gradually worsened, and she stopped taking the drug.The patient was an elderly female with unclear onset and progressive aggravation of symptoms, mainly manifested as bulbar paralysis. The first consideration was neurodegenerative disease involving the bulbar for qualitative diagnosis, emphasizing PBP. The onset type of ALS was not excluded from the differential diagnosis. The patient had no evidence of involvement of the anterior horn of the spinal cord, such as limb muscle weakness, atrophy, or muscle fasciculation. The electromyography results were not that illuminating and the patient was diagnosed with MND/PBP. She began treatment with riluzole 50 mg twice per day to inhibit glutamate release. Mecobalamin 0.5 mg and vitamin BOn December 7, 2018, the patient came to our traditional Chinese medicine hospital for treatment. Symptoms at the time of her first visit were as follow: dysarthria, dysphagia, and excessive saliva, which required using a handkerchief. She had atrophy and fibrillation of tongue muscle, weakness of limbs, feeling of limb muscle fasciculation, and no apparent atrophy of limb muscles. She reported poor sleep quality, a weight loss of 5 kg due to poor nutrition in the past 6 months, constipation, normal urination, pink tongue and greasy coating, and deep and slow pulse.According to TCM theory, we determined that she had flaccid disease and the syndrome of deficiency of spleen and kidney yang. The herbal medicine prescription was formulated to strengthen the spleen and kidney, supplement qi, and warm yang. Huangqi Shengji decoction was the main prescription of Chinese herbal medicine. The specific medication and dosage were as follows: milkvetch root 80 g, cassia twig 15 g, Chinese angelica 30 g, prepared rehmannia root 10 g, debark peony root 30 g, Sichuan lovage rhizome 10 g, suberect spatholobus stem 30 g, prepared common monkshood branched root 30 g, ephedra 15 g, alum processed pinellia 15 g, thunberbg fritillaria bulb 10 g, loquat leaf 30 g, inula flower 20 g, 2-toothed achyranthes root 15 g, manchurian wild ginger 10 g, spine date seed 30 g, tuber fleeceflower stem 30 g, liquorice root 10 g, golden thread 15 g, snakegourd fruit 30 g, largehead atractylodes rhizome 30 g, deer-horn glue 6 g, human placenta 3 g, nux vomica 0.3 g. The herbal medicines were decocted with water, about 200 ml each time, twice a day. , bilateral Fengchi (GB20), Hegu (LI4), Toulinqi (GB15), Shenting (GV24), Baihui (GV20), Quchi (LI11), Gongxue , Tunyan , Fayin  and Wai Jinjin Yuye  were chosen. The positions of the acupoints are shown in Figure   at these acupoints. After twirling for 15 seconds, the needles were pulled out. 0.30 × 40 mm needles were inserted to a depth of 20 to 30 mm at Gongxue, GB20, LI4, GB15, GV24, GV20, and LI11 and were kept for 30 minutes. The GB20 was connected with electroacupuncture. A continuous wave was given; the frequency was 2 Hz, and the current intensity was 2 mA. The treatment frequency was once every other day, 3 times a week.For acupuncture treatment, unilateral Lianquan CV23) and Zhiqiang3 and ZhiOn January 18, 2019, 1 month after the combined acupuncture and herbal medicine treatment, the patient’s saliva decreased slightly, fatigue improved, and appetite increased. The other symptoms were the same as before. We slightly adjusted the herbal medicine prescription and continued the acupuncture treatment. On March 12, 2019, after 3 months of treatment, saliva decreased significantly, and the frequency of limb muscle fasciculation decreased. On April 2, 2019, after 4 months of treatment, she had less saliva, dysphagia was relieved considerably, and she had no problem eating. The strength of limbs was enhanced, and sleep condition was also improved. The change in amyotrophic lateral sclerosis functional rating scale revised score in the first 6 months of treatment is depicted in Figure  Among patients with PBP, 87% progress to definite ALS. Epidemiological statistics show that PBP accounts for 4.1% of MND in China. The onset age of PBP is generally late, mainly after 40 or 50 years of age. The main symptoms include dysarthria, dysphagia, tongue muscle atrophy, and fasciculations. This type of disease is generally severe and develops rapidly, and most patients die of respiratory muscle paralysis or lung infection within 1 to 2 years. The mechanisms underlying neurodegeneration in MND remain incompletely understood. Many cellular and molecular processes have been implicated, including toxic protein aggregation, mitochondrial dysfunction, excitotoxicity, hypermetabolism, oxidative stress, and inflammation. The only 2 approved drugs for the treatment of MND are riluzole and edaravone.,9 A 50 mg dose, twice a day for 18 months, of riluzole, a glutamatergic neurotransmission inhibitor, can delay the course of the disease and prolong the median survival time by about 2 to 3 months in patients with ALS. Edaravone, a free-radical scavenger of peroxyl radicals, showed efficacy in a small subset of people with MND in maintaining function and quality of life in the early stage.,23 It is still unclear whether edaravone therapy prolongs survival in the long term. Although promising outcomes were obtained in preclinical studies, numerous compounds investigated failed in human clinical trials,,25 and there is no available treatment to stop or reverse the progressive course of MND. Symptomatic treatments include the treatment of cramps, pain, spasticity, noninvasive ventilation for supporting respiratory function, and enteral tube feeding to support nutrition deficiencies.PBP is a form of MND, which is less common than ALS. Anticholinergic medications (amitriptyline and glycopyrronium bromide) are often used for treating sialorrhea, but there is not enough evidence proving the efficacy of these drugs in MND.,33 The patient turned to TCM for symptom relief. After 4 months of herbal medicine combined with acupuncture treatment, the dysphagia and sialorrhea were significantly reduced, and her quality of life improved markedly. She avoided a PEG and feeding tube insertion. For this patient, almost 6 years have passed since the onset of symptoms, and the treatment has been maintained for more than 3 years. The disease has not deteriorated or progressed to ALS, and a relatively good treatment effect has been achieved.The main complaint of our patient was progressive dysphagia and sialorrhea. For dysphagia, feeding tube placement and percutaneous endoscopic gastrostomy (PEG) may be necessary if the patient has poor nutrition and loses weight. For sialorrhea, botulinum toxin type-B injections to parotid and submandibular glands are mostly effective in the short term (up to 4 weeks). However, there is probably no benefit beyond this time after a single injection. Therefore, various herbal medicines are used to tonify the spleen and kidney, warm yang, and replenish qi. TCM is a complementary and alternative treatment for MND, especially in China, and includes herbal medicine, massage, acupuncture, moxibustion, and other methods, among which herbal medicine and acupuncture are most commonly used.,35 To alleviate the symptoms, 90% of patients take Chinese herbal production in Shanghai, China. In animal models, TCM improved motor function and extended survival duration by inhibiting inflammation. Herbal medicines have been testified to prolong survival duration and relieve symptoms for patients with MND in some case reports and clinical studies.,38 However, the credibility of these findings is limited by the non-RCT design, unverified outcome measures, a small sample size, or short follow-up. Thus, these reports cannot provide evidence-based support for the clinical use of TCM in the treatment of MND. A prospective registry study has been conducted in China to clarify whether TCM is an appropriate therapy for patients with MND (CARE-TCM). This study will help identify common diagnostic approaches and treatment modalities among Chinese patients with ALS receiving TCM treatment, enabling the establishment of strategies for treatment based on evidence-based medicine. To promote the application of herbal medicine as an alternative therapy in the treatment of MND, animal experiments that explain the pharmacology and toxicology and large-scale and rigorously designed high-quality clinical studies should be performed.Under TCM theory, we believe that the patient belongs to the syndrome of deficiency of spleen and kidney yang. Experiments on animals suggest that electroacupuncture treatment can help increase anti-inflammation activity in the central nervous system and respiratory system of animals with MND. Unfortunately, the number of clinical studies on acupuncture for the treatment of MND is minimal. In our case, we used neck acupuncture, which is often used for aphasia or dysphagia caused by bulbar paralysis after stroke, to solve the problem of dysphagia and sialorrhea. Its effectiveness and safety have been confirmed by many clinical studies. Commonly used acupoints include Lianquan, Wai Jinjin Yuye, Renying, Tiantu, and Fengchi.Acupuncture is a type of complementary and alternative medicine that has been widely used in China, Korea, and Japan for centuries.We do acknowledge that it is hard to determine whether the combined treatment is superior to herbal medicine or acupuncture, when used alone, in alleviating symptoms and improving quality of life of patients with MND/PBP. When herbal medicine and acupuncture are used simultaneously, the efficacy of the 2 therapies cannot be distinguished, either. In TCM hospitals, due to the rapid progression of the disease and difficulty in treatment, patients with MND are managed with multiple TCM therapies. Each therapy has its own indications and limitations. Acupuncture is good at improving dysphagia while herbal medicine is good at improving some of the associated symptoms. For acupuncture treatment, patients have to come to the hospital 3 times a week, whereas herbal medicine can be taken at home to ensure the continuity of treatment when patients are unable to visit the hospital.Our case report suggests that acupuncture combined with Huangqi Shengji decoction may alleviate dysphagia and salivation in patients with PBP. When faced with patients with MND/PBP with dysphagia and salivation symptoms in clinical practice, TCM doctors or acupuncturists can use this combined treatment and observe the therapeutic effect. Due to the low incidence rate and the complexity of TCM interventions, it is difficult to conduct standardized randomized controlled trials to investigate the efficacy and safety of this combined treatment. However, case-control, prospective cohort, or observational studies can be conducted to observe the therapeutic effects. After the preliminary evaluation of the efficacy of the combined treatment, we can proceed to interventional studies.This paper reports a case of Chinese herbal medicine combined with acupuncture in the treatment of MND/PBP that successfully alleviated dysphagia and sialorrhea. Our report suggests that alternative therapies, such as herbal medicine and acupuncture, may effectively reduce the symptoms of MND/PBP. However, standardized clinical studies are still required to verify the effectiveness and safety of this treatment.Conceptualization: Yajing Yang, Jinxia Ni.Resources: Weiqian Chang, Yukun Tian.Visualization: Shaohong Li.Writing – original draft: Wenzeng Zhu.Writing – review & editing: Siyang Peng.