Responsible for the acute infectious disease porcine epidemic diarrhea (PED), PED virus (PEDV) induces severe diarrhea and high mortality in infected piglets and thus severely harms the productivity and economic efficiency of pig farms. In our study, we aimed to investigate and analyze the recent status and incidence pattern of PEDV infection in some areas of Shandong Province, China. We collected 176 clinical samples of PED from pig farms in different regions of Shandong Province during 2019–2021. PEDV, TGEV, and PORV were detected using RT-PCR. The full-length sequences of positive PEDV S genes were amplified, the sequences were analyzed with MEGA X and DNAStar, and a histopathological examination of typical PEDV-positive cases was performed. RT-PCR revealed positivity rates of 37.5% (66/176) for PEDV, 6.82% (12/176) for transmissible gastroenteritis virus, and 3.98% (7/176) for pig rotavirus. The test results for the years 2019, 2020, and 2021 were counted separately, PEDV positivity rates for the years were 34.88% (15/43), 39.33% (35/89), and 36.36% (16/44), respectively. Histopathological examination revealed atrophied, broken, and detached duodenal and jejunal intestinal villi, as typical of PED, and severe congestion of the intestinal submucosa. Moreover, the results of our study clearly indicate that the G2 subtype is prevalent as the dominant strain of PEDV in Shandong Province, where its rates of morbidity and mortality continue to be high. Based on a systematic investigation and analysis of PEDV's molecular epidemiology across Shandong Province, our results enrich current epidemiological data regarding PEDV and provide some scientific basis for preventing and controlling the disease. Porcine epidemic diarrhea (PED) is a common viral disease in raising pigs, one with high morbidity, high mortality, and complex causes that are difficult to control –4. AmongPED virus (PEDV), a member of the genus A coronavirus in the Coronaviridae family, is a linear single-stranded, positive-strand RNA virus ~28 kb in length . In the In our study, we collected 176 clinical samples of PED from pig farms in different regions of In Shandong Province, China during 2019–2021 in order to investigate the prevalence of PEDV there. We performed phylogenetic analysis, comparative nucleotide analysis, and comparative analysis of deduced amino acid sequences with the aim of clarifying the epidemiology of PEDV in Shandong Province.During 2019–2021, we collected 176 intestinal and fecal samples from pigs with diarrheal symptoms on pig farms in localities throughout Shandong Province . The colThe RNA isolater Total RNA Extraction Reagent R401-01 (Vazyme) was added to the tissue homogenate supernatant, and total RNA was extracted from the intestinal and fecal samples according to the instructions. The primers were synthesized by Sangon; PEDV-positive RT-PCR amplification products were sent to Sangon for sequencing. The sequences were compared using MegAlign in DNAStar and analyzed for nucleotide and deduced amino acid homology with a selection of reference strains entered in the NCBI's database . MEGA X The digestive tracts of PEDV-positive pigs were fixed in 10% formalin and removed after 48 h to produce conventional paraffin sections for pathological histological examination using hematoxylin-eosin (HE) staining as a means to observe lesions.In practice, severe diarrhea with vomiting , severe RT-PCR revealed positivity rates of 37.5% (66/176) for PEDV, 6.82% (12/176) for TGEV, and 3.98% (7/176) for PoRV. Among them, one case of mixed infection of PEDV and TGEV was found, but no case of mixed infection of PEDV and PoRV was found. When the test results for the years 2019, 2020, and 2021 were counted separately, PEDV positivity rates for the years were 34.88% (15/43), 39.33% (35/89), and 36.36% (16/44), respectively .A total of 12 PEDV S gene sequences were obtained after sequencing , all of A comparative nucleotide analysis of the 12 isolates with the classical vaccine strain CV777 using MegAlign in DNAStar revealed that all isolates had varying degrees of base mutations, deletions, and insertions. In particular, all had three consecutive nt (TTG) insertions at position 164, all had nine consecutive nt (GGGTGTTAA) insertions at position 177, and all had one G insertion at position 206. The SDHY-YT strain had three consecutive nt (ACC) insertions at position 419, whereas the other 11 strains all had AAC insertions. All isolates had an A deletion at position 218 and three consecutive nt (GAA) deletions at positions 480–482 .A comparison of the deduced amino acid sequences of the 12 isolates with those of CV777 using MegAlign revealed multiple deduced amino acid mutations, deletions, and insertions as well. By contrast, all isolates had four consecutive deduced amino acid (QGVN) insertions at positions 59–62, the SDHY-YT strain had a T insertion at position 140, and all other isolates had an N insertion. All 12 isolates additionally had a G deletion at position 160 .Analysis using MegAlign in DNAStar revealed that the nucleotide homology among the 12 isolates ranged from 97.2 to 99.9%, while the deduced amino acid homology ranged from 95.9 to 99.7%. Compared with the reference strains, the nucleotide homology was in the range of 93.0–99.1%, while the deduced amino acid homology was in the range of 90.9–99.4%. Compared with the classical vaccine strain CV777, the nucleotide sequence homology was in the range of 93.1–93.9%, while the deduced amino acid sequence homology was in the range of 91.2–93.0% .To understand the current genotypes of the dominant strains of PEDV in Shandong Province, we constructed a genetic evolutionary tree  using thNext, our antigenic index prediction analysis of the vaccine strain CV777 S protein and the 12 isolated S proteins using the Jameson–Wolf method with Protean in DNAStar revealed that the S protein's overall antigenic index was high, with a significant difference in antigenicity within ~20–280 aa and a less significant difference elsewhere .Histopathological analysis revealed that the duodenal villi had atrophied and detached, and that the submucosa was congested . The colCoping with PED continues to be a daunting challenge for pig farming in China . Studies748SNIGVCK) within PEDV; however, two AA substitutions  occurred at the SS6 antigenic epitope S764QSGQVKI. Antigenic index predictions showed a more pronounced difference in antigenicity at 20 aa to 280 aa, with little difference at the remaining positions, which suggests that PEDV S protein changes primarily concentrated in the S1 gene region. In general, the current epidemic strains of PEDV in Shandong Province have high affinity and more obvious mutations in the S1 gene.In past research, the S gene of PEDV, the primary structural protein gene, has shown significant variation. The S gene sequences of the prevalent strains were systematically analyzed to accurately capture current trends in the strains and identify markers of PEDV variation . The resDespite the continued high-intensity vaccination of pigs against PED in Shandong Province, the disease can nevertheless occur after multiple vaccinations. In recent years, the incidence of PED in China has shown that the prevalent strains are the main cause of PED, while the incidence and mortality rates have increased year after year . In our The results of this study clearly indicate that PEDV is the main pathogen causing diarrheal symptoms in pigs, the G2 subtype is the dominant strain of PEDV in Shandong Province, where its rates of morbidity and mortality continue to be high. At present, the most prevalent strain of PEDV in some areas of the province, however, is only distantly related to the vaccine strain CV777. Using the most prevalent strain for vaccines against PED is therefore recommended. In addition, all 12 PEDV strains S1-IDMP isolated in this study had S58_S58insQGVN-N135dup-D158_I159del-like mutations, which require ongoing attention.ON890168 to ON988085-ON988096).The sequences of the isolated strains in this study have been uploaded to GenBanK .Author FM was employed by company Huayun (Shandong) Inspection and Quarantine Service Co. 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.


The possibility of permanent cardiovascular damage causing cardiovascular long COVID has been suggested; however, data are insufficient. This study investigated the prevalence of cardiovascular disorders, particularly in patients with cardiovascular long COVID using multi-modality imaging.A total of 584 patients admitted to the hospital due to COVID-19 between January 2020 and September 2021 were initially considered. Upon outpatient follow-up, 52 (9%) were suspected to have cardiovascular long COVID, had complaints of chest pain, dyspnea, or palpitations, and were finally enrolled in this study. This study is registered with the Japanese University Hospital Medical Information Network (UMIN 000047978).p = 0.014) and in-hospital cardiac events  was significantly higher in patients with cardiovascular disorders than in those without. A multivariate logistic regression analysis revealed that a severe condition  and in-hospital cardiac events  were independent risk factors of cardiovascular disorders in cardiovascular long COVID patients.Of 52 patients with long COVID who were followed up in the outpatient clinic for cardiovascular symptoms, cardiovascular disorders were present in 27% (14/52). Among them, 15% (8/52) had myocardial injury, 8% (4/52) pulmonary embolisms, and 4% (2/52) both. The incidence of a severe condition (36% [5/14] vs. 8% [3/38], Suspicion of cardiovascular involvement in patients with cardiovascular long COVID in this study was approximately 30%. A severe condition during hospitalization and in-hospital cardiac events were risk factors of a cardiovascular sequalae in CV long COVID patients. The impact of coronavirus disease 2019 (COVID-19) on healthcare systems and economies is undoubtedly one of the worst disasters experienced by humans in recent decades. Worldwide, millions have survived COVID-19 . Other nWe analyzed all patients admitted to Nihon University Itabashi Hospital diagnosed with COVID-19; this comprised a total of 584 patients hospitalized at our hospital from January 2020 until September 2021. During their hospital stay, we informed all 584 COVID-19 patients to visit our cardiology outpatient clinic if they had any CV long COVID symptoms  after discharge. As a result, 9% (52/584) visited the cardiology outpatient clinic because of CV long COVID symptoms after discharge and were enrolled in the study . Patient2, pacemaker implantation, or allergy to gadolinium contrast media). CMR and SPECT/CT were chosen for the detailed examinations of this protocol because myocardial injury , chest X-ray, and echocardiography, were performed. The ECGs were evaluated by an experienced cardiologist according to the Sokolow-Lyon voltage criterion , 11. If l injury , 15 and l injury  are comm2 × 360 mm2; acquisition matrix size, 266 × 180; reconstruction matrix size, 384 × 384; SENSE factor 2; and inversion time, 200–300 ms) 15 min after contrast agent administration  in three long-axis slices  and a stack of short-axis slices completely encompassing the left ventricle. The CMR images were interpreted by experienced observers using commercial post processing software . Myocardial LGE was independently identified by 3 observers . Discrepancies were resolved through a consensus. The diagnosis of myocardial injury was based on confirmation of the presence of a CMR-late gadolinium enhancement (LGE) by multiple physicians in accordance with previous studies  was performed with the patient in the supine position. Planar imaging was performed in multiple projections . The images were reconstructed in the transaxial, coronal, and sagittal views and reviewed for perfusion defects. The mismatched perfusion defects in this study were based on mismatches between the CT and scintigraphy images. The PE diagnosis was made by multiple physicians using the imaging findings of segmental or subsegmental perfusion defects in areas where no abnormalities were observed on CT images, in accordance with a previous study , Clinical Frailty Scale (CFS), history of the illness, smoking, and drinking. After the initial outpatient visit, follow-up visits were ordered every 1–3 months for up to one year. Follow-up visits to assess the duration of the symptoms were conducted in the outpatient cardiology clinic of Nihon University Itabashi Hospital. All participants were interviewed face-to-face by a trained cardiologist, who assessed the presence or absence of chest pain, palpitations, and dyspnea, which have a high cardiovascular specificity among the long COVID symptoms reported in previous studies , using st-test or Mann–Whitney U-test. Between-group differences in the categorical variables were evaluated using the chi-square test. Comparisons of continuous and categorical variables among the three groups  were performed using the Kruskal–Wallis test and chi-square test, respectively. A logistic regression analysis was performed to evaluate the odds ratio (OR) of the risk factors and associated long COVID symptoms. Due to the small number of cases, a multivariate analysis was performed using a propensity score based on the age, BMI, and prevalence of atrial fibrillation. A Spearman’s rank correlation was used to assess the association between the follow-up period and duration of the long COVID symptoms. All statistical analyses were performed using SPSS Statistics 24 software , and a p-value <0.05 was considered significant.Continuous variables were reported as the mean ± standard deviation (SD) or median (interquartile range [IQR]). Categorical variables were reported as the percentage and number of patients. Differences in the continuous variables between the two groups (presence/absence of cardiovascular disorders) were evaluated using the unpaired Student’s Of the 52 patients who visited the cardiology outpatient clinic, a screening examination with an ECG, Chest X-ray, and echocardiography identified RAVI in 9, LAVI in 7, and RAVI/LAVI in 23 patients. SPECT/CT was performed in all 9 patients with RAVI and 4 patients who had RAVI/LAVI but contraindications to CMR. CMR and SPECT/CT were performed in the remaining 19 RAVI/LAVI patients; CMR was performed in 6 of the 7 patients with LAVI (1 was excluded due to a contraindication to CMR). Screening examinations identified the presence of cardiovascular disorders in 27% (14/52), i.e., 15% (8/52) had myocardial injury, 8% (4/52) PEs, and 4% (2/52) both . There wp = 0.07) and had a higher BMI  and higher prevalence of atrial fibrillation (AF) . There were no differences in the sex, CFS, smoking and drinking history, prevalence of diabetes mellitus, dyslipidemia, hypertension, hyperuricemia, chronic kidney disease, history of coronary artery disease, heart failure, strokes, chronic obstructive lung disease/asthma, and malignant tumors between the two groups. The status and therapy profiles are shown in p = 0.014), and in-hospital cardiac events  was significantly higher in patients with cardiovascular disorders. There was no significant difference between the two groups regarding drug treatment, such as with steroids, antiviral drugs, antibody cocktails, antirheumatic drugs, and anticoagulants. In terms of the peak biomarker values during the hospitalization, there were no differences in the other biomarkers, such as the troponin I, N-terminal pro-brain natriuretic peptide (NTproBNP), and D-dimer. In patients with cardiovascular disorders, when qualitatively comparing the subgroups with myocardial injury, PEs, and both, the patients with PEs were more frequently female. The incidence of using oxygen therapy, in-hospital cardiac events, and the use of anti-rheumatic drugs were higher in the patients with myocardial injury or both myocardial injury and PEs than in those with a PE alone.The baseline characteristics are summarized in p = 0.017) and in-hospital cardiac events  were independent risk factors of cardiovascular disorders in long COVID patients.Univariate and multivariate logistic regression analyses evaluated the factors associated with cardiovascular disorders . A severp = 0.07), and the duration of the long COVID symptoms from the onset of the illness tended to be longer  in the group with cardiovascular disorders . No deaths or hospitalizations due to cardiovascular events occurred during the observation period.The time from the onset to hospitalization in this cohort of patients was a median of 5 days , the length of the hospitalization was a median of 13 days , and the time from discharge to the initial outpatient visit was a median of 21 days , respectively. The aforementioned total time from the onset to the initial outpatient visit for long COVID symptoms was a median of 40 days . The long COVID symptoms are shown in isorders . A closeIn this study, we investigated the cardiovascular manifestations of CV long COVID patients by multi-modality imaging methods. The major findings of our study were as follows: (1) 9% of COVID-19 patients presented to the cardiology outpatient clinic after discharge for CV long COVID symptoms; (2) 27% of CV long COVID patients had some cardiovascular disorders. Of those, 15% had myocardial injury, 8% had PE, and 4% had both complications; (3) an in-hospital severe condition and cardiac events were independent risk factors of a cardiovascular disorder in CV long COVID patients; and (4) during the observation period, CV long COVID patients who did or did not have cardiovascular disorders had no major adverse events.In our registry, approximately 10% of the patients had CV long COVID symptoms  and visited the cardiology outpatient clinic after discharge. In a meta-analysis regarding long COVID, the median (IQR) proportion of COVID-19 survivors experiencing at least one long COVID episode at 1 month was 54.0% , at 2–5 months, 55.0% , and at 6 or more months, 54.0%  . DyspneaIn CV long COVID patients, 15% had a myocardial injury. Several studies on echocardiography abnormalities have been conducted since the beginning of the COVID-19 epidemic , 23. WhiDuring a median follow-up of 163 days , no deaths or hospitalizations due to cardiovascular events were observed among the patients. The duration of symptoms was 11 weeks  in patients with cardiovascular disorders and 6 weeks  in those without cardiovascular disorders. In this study, even those who visited the cardiology outpatient clinic with CV long COVID symptoms improved within approximately 12 weeks on average, and no major adverse events occurred during the observation period. Weber et al. reported that among the COVID-19 patients who survived their index hospitalization, the incremental mortality through 1 year was low, even among troponin-positive patients . HoweverThis study had several limitations. First, as it was conducted in a prospective observational fashion, causal relationships could not be established. Because of the selection bias that existed in this cohort due to the presence of those who did not accept the physician’s suggestion of an outpatient visit, the small proportion of women and patients in this study with a history of heart failure and coronary artery disease with little or no left ventricular systolic dysfunction, unmeasured confounders should be considered when interpreting the results. In particular, our results were based on the data obtained through specific screening examinations, which have a different sensitivity and specificity in identifying a cardiac pathology at the cardiology outpatient clinic; therefore, because symptomatic potential pulmonary disease and asymptomatic potential cardiac disease might be missed, our results regarding the use of imaging modalities and clinical outcomes between facilities should be considered descriptive. Second, due to the low number of clinical events, this study included a small number of patients, and we could not perform a statistical analysis of the factors associated with myocardial injury and PEs. In addition, the number of clinical events may not have been large enough to obtain statistical significance. Third, in the CMR analysis of this study, mainly a qualitative evaluation of the LGE was performed, but T1mapping, T2mapping, and a strain analysis were not performed. The purpose of this study was to clarify the incidence of overt myocardial injury in patients with long COVID. Therefore, subtle left ventricular and right ventricular dysfunction might have been missed. Finally, the clinical data before the COVID-19 hospitalization was lacking; hence, whether cardiovascular disorders existed before the hospitalization could not be determined.The suspicion of cardiovascular involvement in CV long COVID patients was approximately 30%. A severe condition during hospitalization and in-hospital cardiac events were risk factors of a cardiovascular sequelae in CV long COVID patients.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Institutional Review Board (IRB) of Nihon University Itabashi Hospital (RK-210209-1). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.NM, KH, YG, and YO contributed to the conception and design of the study. NH, TN, YT, KF, RA, YW, YE, MM, and KK organized the database. NM performed the statistical analysis and wrote the first draft of the manuscript. HF and AY wrote sections of the manuscript. All authors contributed to the manuscript revision, read, and approved the submitted version.


The low carbon martensitic stainless AWS 410NiMo steel has in its chemical composition 13% chromium, 4% nickel, and 0.4% molybdenum (wt.%) and is used in turbine recovery, rotors, and high-pressure steam pump housings due to its resistance to impact at low temperatures, as well as to corrosion and cavitation. Those applications of the AWS 410NiMo steel frequently demand repair, which is performed by welding or cladding. Arc welding is a well-established technique for joining materials and presents several parameters that influence the mechanical performance of the weld bead. Although numerous welding processes exist, optimizing welding parameters for specific applications and materials is always challenging. The present work deals with a systematic study to verify the correlation between the pulsed fluxed core arc welding (FCAW) parameters, namely pulse current and frequency, welding speed, and contact tip work distance (CTWD), and the bead morphology, microstructure formation, residual stress, and hardness of the martensitic clad. The substrate used was the AISI 1020 steel, and the AWS 410NiMo steel was the filler metal for clad deposition. From the initial nine (9) samples, three (3) were selected for in-depth characterization. Lower heat input resulted in lower dilution, more elevated hardness, and lower compressive residual stresses. Therefore, the results highlight the need for selecting the proper heat input, even when using a pulsed FCAW procedure, to achieve the desired performance of the clad. In the present case, a higher heat input appears to be more advantageous owing to the lower convexity index, smooth hardness transition between fusion and heat-affected zones in addition to more elevated compressive stresses. The main component of a hydroelectric plant, the hydraulic turbine, converts the waterfall’s kinetic energy into mechanical energy, which is converted by a generator into electrical energy. These components are subjected to various mechanical and chemical stresses and are mainly worn by cavitation . The hydThe turbine runners were initially manufactured from C-Mn steel castings for structural applications , classifThese turbine runners are subjected to critical cyclic loads generated from the transient operation during service, slurry erosion resultant from the repeated impact of solid particles carried by liquids, and complex hydraulic phenomena, such as high-frequency pressure fluctuations ,8,9. ForPowder bed fusion (PBF) processes ,15 usingWith the development of new electronic power supplies for arc welding, it is currently possible to use pulsed arc welding, which provides better control of the electric arc with less energy input, thus resulting in few distortions of the welded parts. Although the pulsed technique presents a high number of parameters, if adjusted correctly, it provides better weld quality than conventional arc welding .The fluxed core arc welding process (FCAW) is a fusion welding process that employs tubular wire. The heat necessary for joining is provided by an electric arc established between the part and a continuously fed wire. The tubular wire has a flux composed of inorganic and metallic materials that exhibit several functions, among which the improvement of the characteristics of the electric arc, the transfer of the weld metal, the protection of the fusion bath, and in some cases, the addition of alloy elements, in addition to acting as slag former. The chemical composition of the flux also influences the mechanical properties of the deposited metal . The appAlthough the welding process may be employed for cladding and repairing components back to their integrity, the weld bead geometry and properties play a vital role in determining the final mechanical properties of the entire workpiece. Therefore, the bead geometry can be used as a quality measure, i.e., bead width and height, depth of penetration, and convexity index (ratio between width and height). These features are dependent on welding parameters, such as wire feed rate, welding current, welding speed, plate thickness, base current (Ib), peak current (Ip), base time (tb), and peak time (tp). Therefore, it is essential to set up proper welding parameters to produce adequate process stability and suitable weld bead geometry ,22.Recent works ,24,25 haThe present work deals for the first time with the interplay of the pulsed FCAW process variables  with fundamental microstructural features, such as phase formation and distribution, weld bead morphology, residual stresses, and the resulting mechanical properties of the clad. AISI 1020 steel was applied as substrate, and the low carbon martensitic stainless steel 410NiMo was used for cladding. The evolution of the base metal microstructure, heat-affected zones (HAZ), diluted zones, and bead on plate clads with different deposition conditions was systematically discussed using optical and electron microscopy, electron backscatter diffraction (EBSD), X-ray stress analyses, and micro-hardness profiles. The relevance of adjusting the welding heat input by understanding the microstructure formation and the resulting mechanical properties is demonstrated.Plates of AISI 1020 steel  with dimensions of 185 × 63.5 × 12.7 mm were used as the substrate for welding. This steel is widely used in gears, camshafts, and bolts due to its ease of machining and ductility. A corrosion-resistant 410NiMo   was selected as cladding material. The filler metal was the AWS EC410NiMo tubular wire with 1.2 mm diameter . −1, a direct current positive electrode, Ar + 2 vol.% O2 shielding gas with a flow rate of 18 L·min−1. The stable arc length was 2.0 mm. The welding position consisted of 1G/PA, and the torch was fixed at 90 degrees. An electronic multiprocess inversal 450A IMC  welding power supply was used for depositing beads using pulsed current. All specimens used were free of grease, oil, and contaminants. They were heated in a furnace at 200 °C, taken to the welding bench, and welded when the temperature was 150 °C. That procedure was applied to all the samples. The pulsed FCAW process was used, and the following welding conditions were kept constant: a weld bead with a mean peak current of 305 ± 60 A, peak time of 10 ms, wire feed rate of 8.5 m·minAfter conducting preliminary tests using as variables the average pulse current, the pulse frequency, the welding speed, and the nozzle contact distance piece, three levels were chosen for all process parameters. Due to the numerous variables, the Taguchi method was a practical approach to obtain the correlation among parameters, reducing the number of experiments . Therefo−1 was estimated with Equation (1) from the ISO/TR 17671-1 [−1. The K factor was equal to 0.8 because the same standard is recommended to FCAW. The mean current value (Im) and root mean square (Irms) value of current can be measured during welding, and both are related to the weld bead formation [m, welding penetration, Irms, and the weld width [The heat input Q in J·mm 17671-1 . U correormation . Furtherld width .(1)Q=k 3 solution  for 10 s. The weld beads were analyzed in terms of geometry, surface visual appearance, and microstructural observation. The regions analyzed in the deposited clad were the base metal (BM), heat-affected zone (HAZ), fusion zone (FZ).The samples were cut near half of the weld bead’s length. They were metallographically prepared using a standard procedure of mechanical grinding and polishing steps, according to the ASTM E3 standard recommendations . The etcThe measurements of the reinforcement height (r), bead width (b), and penetration depth (p) of the weld beads, as shown in ® Quanta 650 FEG  and a Tescan® Mira3 . Backscattered electron (BSE) micrographs and energy dispersive spectroscopy (EDXS) analysis were carried out using an acceleration voltage of 20 kV, with a working distance of 15 mm, and a spot size of 30 nm. In addition, the EBSD measurements were carried out using an acceleration voltage of 20 keV and a step size between 0.2 and 2.0 µm. The EBSD data were analyzed using the HKL Channel 5 system from Oxford . Large maps showing the transition between BM, HAZ, and FZ were measured with a step size of 0.75 and 2 µm. In addition, localized maps with a step size of 0.2 µm were used to indicate the type of microstructure, grain morphologies, and boundaries.The electron microscopy work was conducted with the SEM/EBSD FEIThe test specimens were removed from the central region of the weld bead. Samples were also conventionally prepared metallographically and etched with a 4% Nital etching for 1 min to reveal the fused zone, the heat-affected zone, and the base metal.Vickers hardness linear profiles were made using the Leco LM100-AT  hardness system with a 5 N load and 200 µm indentation distances. The hardness profiles were measured through the base metal (BM), heat-affected zone (HAZ), and fusion zone (FZ), as shown in α radiation (1.78897 Å wavelength) and using a beam size of 2 mm × 2 mm. Only one point of interest was measured on each sample at the midpoint along the weld bead top surface. 2ψ method covering a sin2ψ-range from 0 to 0.8 and using a step size of 0.16 in sin2ψ. The diffraction elastic constants (DEC) were calculated based on the Eshelby–Kroener approach [Samples of the 410NiMo stainless steel clad with 58 mm in length and 4.4 mm in width were prepared for residual stress analysis using X-ray diffraction. The longitudinal and transversal stress components were determined using a PANALYTICAL MRD-XL  diffractometer equipped with Co-Kapproach .Wide weld beads in clads are recommended to reduce the number of passes, thus increasing the process’s productivity. However, the recommended values for CI should be around 30% ,22. The For each level of average welding current , the test closer to 30% in the convexity index was chosen to perform the microstructural analyses and residual stress. For the average current of 170 A, sample 2 was selected with a convexity index of 36.80%. Sample 6 was selected for the current of 200 A with a convexity index of 33.95%. Finally, sample 9 was selected for the current of 230 A with a convexity index of 30.55%. The heat input increased from samples 2 to 9. Quasi-polygonal ferrite with grain size around 30 µm forms the microstructure of the AISI 1020 steel substrate. The microstructure is shown in the inverse pole figure (IPF) map in In Different metallurgical zones were formed in the weld bead due to the effect of the heat input on the recrystallization of the microstructure and phase transformation. A well-defined FZ, a CGHAZ, an FGHAZ, an ICHAZ, and a SCHAZ were observed for all conditions. No considerable morphological differences were observed for the ferrite and martensite phases for the investigated samples 2, 6, and 9. The FZ consisted of a needle-like mixed with a lath-type morphology, The formed microstructure observed was mainly lath-type, such as martensite or bainite, in agreement with the literature ,19,32. FThe deposited clad (stainless steel) and the substrate (mild steel) had different chemical compositions . Figure The results of weld bead morphology relating the welding parameters with the resulting morphology and weld bead characteristics are presented in p-value, lower than the level of significance (0.05), rejecting the null hypothesis. Therefore, when the lines have a slight variation, there is also a p-value higher than the significance level, thus concluding that this particular input variable does not significantly influence the response. Statistical analyses were carried out for the parameters obtained in all clads produced according to the L9 orthogonal array shown in For the statistical generation of each response variable, the following equations for mathematical relationships were formulated.Width (mm):Reinforcement (mm):Penetration (mm):Penetration area (mm²):Reinforcement area (mm²):Dilution (%):Convexity Index (%):The obtained hardness line profiles for the pulsed FCAW strategies agreed well with the literature for fusion welding techniques. Cladding with 410NiMo deposited by thermal spray in CA6NM martensitic stainless steel achieved microhardness values between 311 and 431 Vickers . The harResidual stresses may be determined across weldments by several methods, such as diffraction techniques, hole drilling, and global contour methods ,41. HoleContour methods enable the determination of high-resolution maps of stress normal to the surface in a wide range of materials. The main requirement is that the planar section must be cut carefully through the material under residual stresses. Mechanical methods tend to create cutting stresses and reduce out-of-plane deflections while being measured. Hence, electric discharge wire machining (EDWM) is commonly used to diminish artifacts. The residual stresses can be determined by imposing the measured displacements with the negative sign within a Finite Element model. However, EDWM cannot be utilized to cut non-conductive materials. The limitations of the contour method involve its destructive nature and the interpretation of data. It considers that the residual stress state is independent of the length coordinate . EDWM sectioning also ignores displacements of material points parallel to the cutting plane. It would require subsequent sectioning through different planes to reconstruct multiple stress components.Lab X-ray stress analyses are versatile, phase selective, suit a wide range of crystalline materials, assess the multiaxiality of the stress state, and enable the separation of macro-and micro-residual stresses. However, X-ray diffraction yields information from only the near-surface region. In the present case of claddings, this is more relevant because clads of martensitic stainless steels are used for wear and corrosion protection. Hence, compressive stresses in the surface region with a few micrometers control wear, corrosion, and even fatigue resistances in these materials. The nature of the mean residual stresses in the clads was compressive. Results showed higher compressive stress  in the transverse direction than in the longitudinal direction in all cases. The residual compressive stress values in the transverse direction increased from −302 MPa for sample 2 to −397 MPa for sample 6, and finally −529 MPa for sample 9, thus exhibiting an inverse proportionality to the heat input. Therefore, the higher the heat input, the more compressive the residual stresses.One of the challenges of welding for cladding is the proper adjustment of parameters used in the process and their effects on the properties of the weld. For the investigated clads, the deposition rate decreased to a lesser extent with the increase of the average current and more significantly with the increased welding speed. Contrastingly, the weld bead images suggested that the deposition rate increased by increasing the pulse frequency and CTWD.The ANOVA results demonstrate that the average current was the most critical factor in controlling the bead geometry. Higher current means a more pronounced Joule effect causing heating at the tip of the electrode. The higher the current, the larger is the weld width. The welding speed and CTWD also influenced the bead geometry. A lower deposition rate promotes a lower CI, while lower CI better suits the reinforcements and coating properties. The parameter study shows that fast welding speeds and low currents reduce the deposition rate. However, low currents result in high CI due to the lack of fusion and rapid solidification, tending to a circular cross-section morphology, as obtained in sample 2. However, cladding and welding applications should achieve the highest possible deposition rate, i.e., productivity.The final welding parameters influenced the thermal history and, thus, the phase transformation kinetics in the produced clads. The welding pool transforms to austenite and δ-ferrite upon solidification. The δ-ferrite decomposes into austenite at temperatures not far from the solidification one. Austenite, then, undergoes a solid-state change to a structure that depends on both the cooling rate and the hardenability of the alloy, which is controlled by its chemical composition. No δ-ferrite was observed along the grain boundaries, in agreement with the literature ,43, sinc, the HAZ can be subdivided into four regions according to the extent of grain growth and austenitization: coarse-grained zone (CGHAZ), fine-grained zone (FGHAZ), intercritical zone (ICHAZ), and over-tempered base metal [The heat-affected zone (HAZ) formed adjacent to the fusion zone. As indicated in Figure 17se metal . They alThe present phases formed after welding governed the hardness values, which steeply increased due to the FCAW process. The higher the applied current, the lower is the hardness in FZ, CGHAZ, and FGHAZ, Consequently, sample 2 showed more elevated hardness values in FZ than samples 6 and 9. The welding speed also influenced the heat input, final bead morphology, and, consequently, hardness. A slow weld speed can provide a wider bead, higher heat input, and higher dilution. The dilution also affects the morphology of the deposition, promoting a broader area of contact. The ideal convexity index should be around 30%  to a staSteels with martensitic steel clads produced by the GTAW and FCAW welding processes generally present a segregated heterogeneous molten zone with well-defined austenitic and martensitic regions . The welThe formed microstructure was mainly lath-type, such as martensite and bainite, in agreement with the literature ,46,48. AExtensive martensite and bainite formation with preferential crystallographic orientation along the direction of heat extraction in the FZ, which is characteristic of columnar grains after fusion welding, were verified in all tested conditions.−1 resulted in, respectively, 440, 387, and 368 Vickers hardness in the fusion zone near the interface. This occurred because lower heat inputs led to lower dilution, lower cooling rate, and, consequently, lower hardness values in the region near the interface in the FZ.The different hardness distributions presented an inversely proportional relationship to the applied heat input during cladding. Heat inputs of 0.43, 0.47, and 0.79 kJ·mmThe higher the heat input, the larger the grains and the more pronounced is the CGHAZ.A higher heat input led to higher compressive residual stress due to higher peak temperatures and broader temperature gradients. Those effects lead to higher thermal stresses, which cause lattice deformation, thus visible as compression stresses at the clad surface.The formation of bainitic microstructure in the CGHAZ zone is suggested by a decrement in the band contrast and was more visible in sample 9. Furthermore, it decreased the overall hardness values in the CGHAZ for samples 6 and 9.Lower heat inputs lead to lower peak temperatures, rapid cooling rates, and a more refined microstructure in the FGHAZ. In addition, the fine-grained ferrite with low quality of Kikuchi patterns leads to higher hardness values in the FGHAZ, the lower the heat input.Higher heat input is more advantageous to clad AWS 410NiMo stainless steel onto low carbon AISI 1020 steel by FCAW due to the low convexity index and smooth hardness transition between FZ and HAZs in addition to the more expressive compressive stresses.The present work investigated the influence of the pulsed FCAW welding parameters on the final clad morphology, residual stresses, microhardness distribution, and microstructural gradients through the base metal, heat-affected zones, and fusion zone consisting of the martensitic AWS 410NiMo stainless filler metal. The results allow for drawing the following conclusions:


Inflammation is a chronic, sterile, low-grade inflammation that develops with advanced age in the absence of overt infection and may contribute to the pathophysiology of sarcopenia, a progressive and generalized skeletal muscle disorder. Furthermore, a series of biomarkers linked to sarcopenia occurrence have emerged. To aid diagnostic and treatment strategies for low muscle mass in sarcopenia and other related conditions, the objective of this work was to investigate potential biomarkers associated with appendicular lean mass in community-dwelling older women. This is a cross-sectional study with 71 older women (75 ± 7 years). Dual-energy X-ray absorptiometry was used to assess body composition. Plasmatic blood levels of adipokines , tumor necrosis factor (TNF) and soluble receptors (sTNFr1 and sTNFr2), interferon (INF), brain-derived neurotrophic factor (BDNF), and interleukins  were determined by enzyme-linked immunosorbent assay. Older women with low muscle mass showed higher plasma levels of adiponectin, sTNFr1, and IL-8 compared to the regular muscle mass group. In addition, higher adiponectin plasma levels explained 14% of the lower appendicular lean mass. High adiponectin plasmatic blood levels can contribute to lower appendicular lean mass in older, community-dwelling women. According to the International Classification of Diseases ICD-10-MC), sarcopenia is a gradual and generalized muscle disease that consists of the progressive loss of muscle mass, muscle strength, and/or physical function 0-MC, sar. Low musA continuous, sterile, low-grade inflammation known as “inflammaging” may contribute to the clinical symptoms of chronic diseases ,5,6. ThuMoreover, previous research has demonstrated a negative correlation between inflammatory biomarkers and chronic conditions . In oldeIt is challenging to identify a single biomarker that can characterize sarcopenia due to the complex pathophysiology and the multiple pathways that cause muscle wasting and sarcopenia ,8. ThereThis cross-sectional study was approved by the Ethics Committee of the Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM) with number 1.461.306. All participants signed an informed consent form. The assessments were performed in the Laboratório de Fisiologia do Exercício (LAFIEX) and Laboratório de Inflamação e Metabolismo (LIM) of the UFVJM between June 2016 and June 2017.To identify older women compatible with the assessment procedures, community-dwelling older women were recruited based on their registration at the Basic Health Units (BHU) in Diamantina, Minas Gerais, Brazil. All participants were visited at home and answered a questionnaire with information about their medical history, life habits, and comorbidities. The exclusion criteria were age (<65), cognitive decline evaluated by the Mini Mental State Examination , subjectThree planned sessions included assessments. At first, they completed the clinical health interviews after signing the permission form, and the eligibility criteria were checked. On the newly scheduled day, the participants had examinations of their body composition in the morning while abstaining from food, liquids, and medicine. After a 15-min pause for rest and a standard meal, all participants started the handgrip strength test. To describe the inflammatory profile, blood samples were taken from the participants twenty-four hours after body composition .Blood was drawn from the patient’s upper limb through antecubital venipuncture and preserved in 10-mL heparin tubes. After collecting blood, the samples were centrifuged at 3000 rpm in a centrifuge for 10 min. Plasma samples were stored in the freezer −80 °C until the analysis. After six months of storage, the inflammatory blood profile was evaluated by measuring the plasma levels of biomarkers IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IFN, TNF, adiponectin, leptin, resistin, BDNF, and soluble receptors sTNFR1 and sTNFr2 by an immunoenzymatic technique (ELISA sandwich)  according to the manufacturer’s instructions .®. The participants were instructed to stay seated, with a neutral fist, an elbow flexed 90 degrees, and a neutral shoulder. The HGS measure, i.e., an isometric contraction of the dominant hand applied on the handles of the dynamometer, was expressed in kilogram force (kgf). The mean of three measurements was used for analysis [Handgrip strength (HGS) was evaluated using a Jamar dynamometeranalysis ,12.Body composition was measured using DXA with Encore Software 2005 . The same assessor performed all body composition evaluations in the morning. After screening by DXA at the ALM, total fat mass and total trunk mass were obtained.From the sum of the lean muscle masses of the arms and legs obtained by DXA, it was possible to determine the ALM ,13. AccoThe following sixteen independent variables  were included as possible independent factors associated with ALM.GPower software version 3.1.9.2, was used to calculate the sample size. We estimated the sample size from previous work, which found a correlation between adiponectin and sarcopenia of 0.24 and an effect size of 0.31 . In addit-test for parametric variables or Mann–Whitney-test for nonparametric variables were used to compare the mean differences of continuous variables among subjects with and without low ALM. Pearson or Spearman correlations analysis was used to investigate the relationship between ALM and anthropometric variables and biomarkers. The Statistical Package for the Social Sciences, version 22.0 , and MedCalc Statistical Software, version 13.1 , were used to conduct statistical analyses. Data normality was verified by the Kolmogorov–Smirnov test. Continuous variables were expressed as the mean and standard deviation (± SD). The p < 0.1) were included. Four assumptions were used in the linear regression analysis: linearity, residual distribution, homoscedasticity, and the absence of multicollinearity. Scatter plots were used to assess the linearity of the independent variables and residuals, and a histogram was used to look at the distribution of residuals. The scatter plot confirmed the homoscedasticity, which was defined by the evenly distributed residuals in the regression line. The variance inflation factor (VIF) values below 10.0 were used to define the absence of multicollinearity. Additionally, the autocorrelation of the variables was verified by the Durbin–Watson test, and the values between 1.5 and 2.5 showed that there was no autocorrelation in the data. Statistical significance was set at 5%. Univariate and stepwise multivariate linear regression were used to confirm the determinants of ALM. In each multivariate model adjusted for age, variables related to ALM in the univariate analysis  participated in the study. The characteristics of body composition, muscle strength, and biomarker plasma levels are presented in p < 0.05)  .p < 0.01) and a significant negative correlation between ALM and adiponectin , IL-8 , and sTNFr-1   of the ALM, and adiponectin explained 14% of the ALM variability  of community-dwelling older women  . In univer women .To our knowledge, this is the first study to assess the association between appendicular muscle mass using DXA and a broad panel of blood biomarkers related to sarcopenia. In our screening, low appendicular muscle mass or the risk of sarcopenia was present in 69% of the community-dwelling older women. Handgrip strength (HGS), total fat mass, and trunk fat mass were significantly lower in the low ALM group. Regarding biomarkers, plasma levels of adiponectin, sTNFr1, and IL-8 were higher in the low ALM group and demonstrated an association with adiponectin, sTNFr1, and IL-8 with ALM. In clinical terms, community-dwelling older women with less muscle mass had higher blood levels of these biomarkers . Of noteNoteworthy, older women with low ALM also presented low HGS, as in cases of confirmed sarcopenia ,2,14,15.There is growing evidence suggesting that chronic low-grade inflammation, or inflammation, could play a key role in the development of sarcopenia ,16,17,18Among all evaluated biomarkers, adiponectin, IL-8, and sTNFr1 correlated negatively with ALM . It is uThus, the findings of the study are consistent with studies that found an increase in sTNFr1 blood levels to be negatively linked to muscle mass parameters . FurtherSarcopenia is linked to the adipokines, i.e., leptin and adiponectin , which aA biomarker with antidiabetic, anti-inflammatory, and antiatherogenic properties is adiponectin ,16,32. In = 557) were more likely to have higher levels of adiponectin [p = 0.02) explaining 14% of the ALM variability , occurring independently of the total fat mass or trunk fat mass.According to a meta-analysis, including seven studies, those with sarcopenia , suggesting that trunk fat mass and adiponectin are possible predictors of appendicular muscle mass in older community-dwelling women. Thus, our findings together allow us to speculate on the involvement of adiponectin in the modulation of inflammatory control and ALM, influencing positively the balance between the anti-inflammatory  and proinflammatory biomarkers  in community-dwelling older women. However, the biomarkers were dosed systemically in our group, but possibly future research that measures the biomarkers directly in the muscle would provide more illuminating results. More research advances are needed to explore whether there is a cause-effect relationship in the interaction between muscle and biomarkers.When the model of the analysis was adjusted for age, the results remained the same  Older women with lower ALM assessed by DXA also have lower HGS. Low levels of these clinical features are directly associated with sarcopenia in older women; (2) higher adiponectin levels were associated with lower appendicular muscle mass among community-dwelling older women; (3) contribution to clinical or public health strategies aimed at early assessment and diagnosis of sarcopenia and other diseases related to low muscle mass in old women. The limitation of this study was the short observation time ; only older community-dwelling women were investigated; our findings should not be extrapolated to the population of clinical settings, long-term care facilities, or hospital environments; and we did not assess a cutoff point for adiponectin to predict ALM. Future studies that determine a cutoff point for adiponectin based on ALM in both sexes may further enhance our understanding of the association of these variables with sarcopenia.High adiponectin levels in the blood contribute to lower ALM in community-dwelling older women. Therefore, initiatives for the diagnosis and treatment of sarcopenia in community-dwelling older women may focus on this adipokine as a possible biomarker of sarcopenia.


Ardisia is widely used as a hepatoprotective and anti-inflammatory agent in China. However, the active ingredients in Japanese Ardisia and their potential mechanisms of action in the treatment of autoimmune hepatitis (AIH) are unknown. The pharmacodynamic substance and mechanism of action of Japanese Ardisia in the treatment of AIH were investigated using network pharmacology and molecular docking technology in this study. Following that, the effects of Japanese Ardisia were evaluated using the concanavalin A (Con A)-induced acute liver injury rat model. The active ingredients and targets of Japanese Ardisia were searched using the Traditional Chinese Medicine Systems Pharmacology database, and hepatitis-related therapeutic targets were identified through GeneCards and Online Mendelian Inheritance in Man databases. A compound–target network was then constructed using Cytoscape software, and enrichment analysis was performed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Molecular docking technology was used to simulate the docking of key targets, and the AIH rat model was used to validate the expression of key targets. Nineteen active chemical components and 143 key target genes were identified. GO enrichment analysis revealed that the treatment of AIH with Japanese Ardisia mainly involved DNA–binding transcription factor binding, RNA polymerase II-specific DNA transcription factor binding, cytokine receptor binding, receptor-ligand activity, ubiquitin-like protein ligase binding, and cytokine activity. In the KEGG enrichment analysis, 165 pathways were identified, including the lipid and atherosclerotic pathway, IL-17 signaling pathway, TNF signaling pathway, hepatitis B pathway, and the AGE–RAGE signaling pathway in diabetic complications. These pathways may be the key to effective AIH treatment with Japanese Ardisia. Molecular docking showed that quercetin and kaempferol have good binding to AKT1, IL6, VEGFA, and CASP3. Animal experiments demonstrated that Japanese Ardisia could increase the expression of AKT1 and decrease the expression of CASP3 protein, as well as IL-6, in rat liver tissues. This study identified multiple molecular targets and pathways for Japanese Ardisia in the treatment of AIH. At the same time, the effectiveness of Japanese Ardisia in treating AIH was verified by animal experiments.Japanese  Autoimmune hepatitis (AIH) is a common liver disease worldwide, seen in both men and women, but predominantly in women. According to epidemiological surveys, the incidence of AIH ranges from 0.67 to 2.0/100,000 people per year, and the prevalence ranges from 4.0 to 42.9/100,000 .The pathogenesis of AIH is not completely understood. AIH is thought to be caused by genetic factors, molecular mimetic mechanisms, immune damage, and a variety of physical and chemical factors ,9,10,11.Ardisia, known as Ardisia japonica or marlberry, is used as a medicinal plant in traditional Chinese medicine. It grows very slowly, and its leaves have a similar appearance to tea leaves. Bright red berries appear under the leaves in autumn, and therefore, it is also called ‘aidicha’ or ‘yedizhu’ in Chinese. Japanese Ardisia is mainly grown in the southern provinces of China, such as Hunan and Guangxi, where it is a popular medicinal herb used in Chinese folk medicine. The Chinese ancient medicine book ‘Compendium of Materia Medica’ records that A. japonica has the effect of ‘detoxification and promoting blood circulation’. The pharmacodynamic components of A. japonica are saponins, coumarins, benzoquinones, and flavonoids [A japonica is commonly used to treat chronic bronchitis, pulmonary tuberculosis, tuberculous pleurisy, and acute icteric hepatitis. Meanwhile, A. japonica has shown remarkable curative effects in the treatment of chronic hepatitis [Japanese avonoids ,14,15. Iavonoids ,17. In cArdisia in the treatment of AIH through network pharmacology and molecular docking analysis of the interaction between drug molecules and AIH-related targets, and to provide a theoretical basis for clinical research. The specific flow chart is shown in Cyberpharmacology can effectively reveal the material basis and mechanism of action of Chinese medicine by systematically and integrally exploring the relationship between drugs and diseases . Thus, tArdisia were obtained by searching the TCMSP database. Nineteen molecules with OB ≥ 30% and DL ≥ 0.18 were identified as bioactive compounds [Oral bioavailability (OB) is the fraction of an orally administered drug that reaches systemic circulation. This is an important consideration for bioactive molecules used as therapeutic agents. Drug likeness (DL) qualitatively assesses the capacity of a molecule to become an orally administered drug based on its bioavailability . The maiompounds , as showArdisia were obtained from the TCMSP database . After screening to remove invalid gene IDs, the targets downloaded from the GeneCards , OMIM databases , and TTD  were crossed with those from the TCMSP database to obtain potential targets for the treatment of AIH in Japanese Ardisia. The obtained data are presented as a Venn diagram  to construct the PPI network (https://www.r-project.org/ accessed on 15 November 2021) are shown in Ardisia active ingredient targets and autoimmune hepatitis disease targets were imported into Cytoscape 3.7.0  for topological analysis. The key targets were sorted according to the degree value, with higher degree values indicating nodes that were more central in the network and more important. Through the PPI protein interaction network, the disease-related targets are found, and the degree value is greater than the median by screening twice to find key targets. Thus, the key target genes for autoimmune hepatitis treatment mainly included AKT1, IL6, VEGFA, CASP3, JUN, MYC, etc. , and the common targets between Japanese Ardisia, its active components, and autoimmune hepatitis were visualized. The data were merged into a component-target network diagram via the merge function in Cytoscape 3.7.0 to obtain the network diagram of ‘Japanese Ardisia–component–gene–autoimmune hepatitis’  and AutoDock software (https://autodock.scripps.edu/ accessed on 16 November 2021) were used to visualize the docking results in the form of two-dimensional and three-dimensional diagrams , and AKT1 expression levels were significantly lower (p < 0.01) in the model group compared to the normal control group. CASP3 protein expression was significantly lower (p < 0.01), AKT1 protein expression was significantly higher (p < 0.05), and IL-6 protein expression was significantly lower (p < 0.05) in the Japanese Ardisia administration group after 10 days compared to the model group.Western blot analyses are shown in AIH is a chronic inflammatory disease characterized by abnormally high levels of serum autoantibodies, hypergammaglobulinemia, and serum transaminases . With thChinese medicine’s multi-component and multi-target properties, and thus its holistic and systemic action characteristics, make it unique in its advantages and potential for complex diseases, but this complexity has also limited its application and development.Ardisia for the treatment of AIH.Cyberpharmacology analyzes drug action at the systemic level and reveals the synergistic mechanism of drug action on the human body, which fits well with the dialectical and holistic view of Chinese medicine theory, and it is expected to bring a breakthrough to Chinese medicine research characterized by a holistic approach, providing new methodological support for Chinese medicine to move from empirical to theoretical science ,25,26. WArdisia. The TCMSP database analysis resulted in the screening of 19 active ingredients. 143 effective targets for the treatment of autoimmune diseases were identified using databases, such as GeneCards and OMIM. According to the results of the component–target network analysis, 150 target genes were associated with the drug-disease target intersection. The core genes were AKT1, IL6, VEGFA, and CASP3. AKT1, also known as protein kinase B, regulates a wide variety of cellular functions, including cell proliferation, survival, metabolism, and angiogenesis, in both normal and malignant cells [IL6 is a pro-inflammatory cytokine with a wide variety of biological functions. It is involved in physiological activities such as the inflammatory response, cellular immunity, and hematopoietic regulation. IL6 plays major roles in the differentiation of B cells into immunoglobulin-secreting cells and antibody production, the activation of T cell proliferation and differentiation, the immune response, and the promotion of inflammatory reactions [IL6 is rapidly synthesized in response to tissue injury or an inflammatory infection. This promotes the body’s defense function by stimulating the acute immune response and the hematopoietic system. When the tissue recovers its homeostasis, IL6 synthesis is discontinued [IL6 pathway is associated with liver injury and hepatocellular carcinoma [VEGFA induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis, and induces the permeabilization of blood vessels. It is essential for both physiological and pathological angiogenesis. CASP3 is a cysteine aspartate protease that participates in the activation cascade of cysteine proteases. CASP3 plays an important role in inflammation and tumor progression [This study provides key information about the anti-hepatitis effect of Japanese nt cells . IL6 is eactions . IL6 is ontinued . The conarcinoma . VEGFA igression . It is hgression , and it gression  and tumogression .Ardisia has a therapeutic effect on hepatitis via a mechanism that may be related to key molecules involved in the regulation of autoimmune hepatitis. It inhibits associated inflammatory factors by regulating various signaling pathways, downregulating the concentrations of serum hyaluronic acid and tumor necrosis factor, protecting hepatocytes from injury, reducing liver inflammation, and protecting against lipid peroxidation [Ardisia is related to the inflammatory response, cell cycle regulation, and hormone metabolism. In the KEGG enrichment analysis, 163 pathways were identified, including the lipid and atherosclerosis pathway, IL-17 signaling pathway, TNF signaling pathway, human cytomegalovirus infection pathway, fluid shear stress and atherosclerosis pathway, hepatitis B pathway, and AGE–RAGE signaling pathway in diabetic complications. It can be seen that the pathway is mainly related to oxidative stress, immune regulation, and inflammatory responses, with the AGE-RAGE signaling pathway being closely related to inflammation, which activates the MAPK and NF-KB pathways and interferes with immune and oxidative stress responses [Ardisia, which affects the secretion of certain substances by acting on specific receptors or enzymes, to achieve its therapeutic effect.Japanese xidation . Inflammxidation . GO enriesponses . TNF is esponses . The IL-Ardisia, with the key targets AKT1, IL6, VEGFA, and CASP3, the docking binding energy and the number of intermolecular hydrogen bonds were obtained. The results showed that quercetin and kaempferol had a good binding ability with the key targets and can spontaneously bind to form a stable binding conformation.The binding interaction between each compound and its receptor was scored by the molecular docking program. A lower score indicated more stable binding between the ligand and its receptor ,40. ThroArdisia is effective in the treatment of AIH. Western blot analysis revealed that Japanese Ardisia could reduce the expression of CASP3 and IL-6 while increasing the expression of AKT1. These proteins are involved in the AGE–RAGE signaling pathway in diabetic complications. According to this, the Japanese Ardisia may be able to modulate these key targets to achieve therapeutic effects on immune liver injury.Animal studies have shown that Japanese Additionally, there are also limitations to the research at this stage, as network pharmacology techniques can only predict drug composition and targets qualitatively. High-performance liquid chromatography (HPLC) or ultraviolet spectrophotometry (UV) should be used to determine the plausibility of the screened active ingredients, and this should be combined with pharmacology, pharmacodynamics, and pharmacokinetics to make the screened active ingredients and mechanism of action more convincing.http://tcmspw.com/tcmsp.php (accessed on 14 November 2021)), GeneCards (http://www.genecards.org (accessed on 14 November 2021)), Online Mendelian Inheritance in Man ), the Therapeutic Target Database , the Search Tool for Retrieval of Interacting Genes (STRING) protein interaction database (https://string-db.org (accessed on 15 November 2021)), Cytoscape 3.7.0 (http://www.cytoscape.org (accessed on 15 November 2021)), and the Protein Data Bank (PDB) protein structure database (https://www.rcsb.org/pdb (accessed on 16 November 2021)) were used in this study.The Traditional Chinese Medicine Systems Pharmacology (TCMSP) database, an analysis platform  and screened for active compounds by oral bioavailability (OB) and drug similarity (DL), as well as potential targets of action for activity, the active compounds obtained from the screening are presented in the form of a list.The components of Japanese https://www.r-project.org/ accessed on 15 November 2021) to remove duplicate regions of gene targets, the intersection of the active ingredient and disease target was obtained and plotted as a Venn diagram.GeneCards, OMIM, and TTD were used to identify hepatitis-related gene targets. Specific search parameters, such as the keyword ‘autoimmune hepatitis’, were used for data collection. Using R 4.0.4 software  network was constructed according to proximity centrality, intermediate centrality, and degree value, and the key targets were screened using Cytoscape 3.7.0. The cross-targets of Japanese The previously acquired common genes and the associated active ingredients were visualized and analyzed using Cytoscape 3.7.0 software.Ardisia for autoimmune hepatitis treatment were imported into the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. GO was selected for enrichment analysis, KEGG was selected for pathway analysis, and the p-value threshold was set at <0.05 to determine the enrichment pathways of key targets. An advanced bubble diagram was constructed from the enrichment results: the smaller the p-value, the higher the enrichment; the larger the bubble, the richer the genes were.The potential targets of Japanese AKT1), interleukin 6 (IL6), vascular endothelial growth factor A (VEGFA), and caspase 3 (CASP3) were selected as the target proteins for molecular docking experiments. The three-dimensional structures of AKT1, IL6, VEGFA, and CASP3 were downloaded from the PDB protein structure database and saved in PDB format [https://autodock.scripps.edu/ accessed on 16 November 2021) for molecular docking, and the docking results were analyzed using the PyMOL (https://pymol.org/2/ accessed on 16 November 2021) visualization tool.The two-dimensional structures of quercetin, kaempferol, and laricitrin were obtained from the TCMSP database and saved as a Mol2 file. AKT serine/threonine kinase 1 , the concentrate was centrifuged at 5000 rpm for 15 min, the supernatant was collected and concentrated to 1.5 g/mL , and the obtained concentrate was refrigerated at 4 °C [The whole herb of Japanese  at 4 °C ,44.n = 6), con A group (n = 6), and extract of Japanese Ardisia (EJA) (n = 6) groups after being fed adaptively for a week. The control and model groups were given saline, while the administration group was given 36 g/kg of Japanese Ardisia by gavage once daily for 10 days. One hour after the last dose, all groups were given 25 mg/kg con A in the tail vein, except for the control group which was given an equal amount of saline in the tail vein, rats were euthanized 12 h after con A treatment, and their livers were dissected from the rats.SPF-grade male SD (Sprague-Dawley) rats, weighing (200 ± 10) g, with six animals per cage were housed in specific pathogen-free facility with a 12 h light and 12 h dark cycle at 22 °C. All mice were randomly divided into three groups, control group  (Cat.No.C009-2-1) and aspartate transaminase (AST) (Cat.No.C010-1-1) measurements were performed according to the instructions provided by the kit supplier.All the animals were provided by Hunan Sleek Jingda Laboratory Animal Co. License No. SCXK (Xiang) 2019-0005. Animal welfare and experimental procedures followed the regulations of the Animal Ethics Committee of Guilin Medical College.Tissue removed from 2.8.2 was fixed in 4% paraformaldehyde, paraffin sections were embedded, and, finally, the pathological sections were observed by HE staining, and the pathological changes of liver tissues were observed under an electron microscope.AKT1 , CASP3 , IL-6 , β-actin antibody , and incubated them overnight at 4 °C. Then, we incubated them with goat anti-rabbit IgG (H + L) and goat anti-mouse Ig G (H + L) for 1 h at room temperature. The protein bands were visualized using an ECL chemiluminescence kit  and quantified under Image J system, version 6.0.To verify the protein expression level, we extracted protein from rat liver tissue, weighed a certain amount of liver tissue, fully lysed it with RIPA lysis solution , used SDS-PAGE electrophoresis, and then transferred it to PVDF  membrane. We then blotted and closed them at room temperature for one hour, and then we incubated them with p < 0.05 was used to indicate statistical significance.The experimental results are presented as mean ± SEM for each group, with at least three independent experiments. The differences between the treatment and normal groups were analyzed by a one-way analysis of variance using GraphPad Prism 8.0 . Ardisia, and finally determined the effect of Japanese Ardisia anti-AIH on the expression of key target proteins by Western blotting analysis. Japanese Ardisia tends to work through the IL-17 signaling pathway, the TNF signaling pathway, the AGE–RAGE signaling pathway in diabetic complications, and other targets such as AKT1, IL-6, and CASP3 to exert drug effects. The potential signaling pathways uncovered in this study lay the theoretical groundwork and point the way for future experimental validation.In summary, we first performed a network pharmacology and molecular docking approach to elucidate the anti-AIH effects and potential mechanisms of Japanese


Glycated hemoglobin (HbA1c) associates with the risk of arterial stiffness, and such association can be found between fasting blood glucose (FBG), postprandial blood glucose (PBG), triglyceride-glucose index (TyG index), and arterial stiffness. However, the results were inconsistent, longitudinal studies were sparse, and comparison of these glycemic parameters was less conducted. We aimed to explore the longitudinal relationship between HbA1c and arterial stiffness and compare the effect of the parameters.Data were collected from 2011 to 2019 in Beijing Health Management Cohort (BHMC) study. Cox proportional hazard models were fitted to investigate the association between the parameters and arterial stiffness. A generalized estimation equation (GEE) analysis was conducted to investigate the effect of repeated measurements of glycemic parameters. A receiver operating characteristic (ROC) analysis was performed to compare the predictive value of glycemic parameters for arterial stiffness.Among 3,048 subjects, 591 were diagnosed as arterial stiffness during the follow-up. The adjusted hazard ratio (HR) [95% confidence interval (CI)] for arterial stiffness of the highest quartile group of HbA1c was 1.63 (1.22–2.18), which was higher than those of FBG, PBG, and TyG index. The nonlinear association of arterial stiffness with HbA1c and PBG was proved. The robust results of the sensitivity analysis were obtained.HbA1c is an important risk factor of arterial stiffness compared with PBG, FBG, and TyG index, and has a strong predictive ability for arterial stiffness among non-diabetics and the general population. Arterial stiffness, an important pre-stage status of disease, has a dramatic effect on the progression of severe vascular diseases . Many stCurrently, many studies have explored the relationship between HbA1c and risk of arterial stiffness, but studies comparing the effect of HbA1c, FBG, PBG, and TyG index on arterial stiffness were sparse. Additionally, most of the studies were cross-sectional , 16, andIn this cohort study, we aimed to explore the longitudinal relationship between HbA1c and the risk of arterial stiffness; compare the effect of HbA1c, PBG, FBG, and TyG index on arterial stiffness; and provide effective information for population with different levels of the parameters.The Beijing Health Management Cohort (BHMC) study aims to explore the important chronic diseases among participants in Beijing. Participants were enrolled in 2011 and 2012, and followed until December 31, 2019, in this study. Of 8,917 participants, 2,555 participants with cardiovascular diseases, cancer, or atherosclerosis or without information about HbA1c were excluded. A total of 3,314 participants failed to take the final diagnostic test at the final survey. A total of 3,048 participants received at least one follow-up and were enrolled in the study Figure 1Physical and laboratory examination was conducted by trained medical professionals in the whole process based on the 1964 Declaration of Helsinki and the updated version.2). After at least 5 min rest and 30 min interval with caffeine forbidden, mean arterial pressure (MAP) was calculated as diastolic blood pressure (DBP) plus one-third of pulse pressure, and the latter was measured as systolic blood pressure (SBP) minus DBP. Participants were supposed to keep calm in the sitting position, and the equipment should be placed at the height of the heart, as well as the tested right arm. Such test should be conducted at least three times with 1–2 min interval, and the mean of the records was applied. After at least 12 h overnight fasting, the blood samples were collected and the following main parameters were tested by high-performance liquid chromatography (HPLC) : HbA1c, FBG, high-density lipoprotein cholesterol (HDL-C), hemoglobin, low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG).Participants were required to take off their shoes and heavy clothes when taking their anthropometric measurements. Body mass index (BMI) is the measure of dividing the weight (kg) by the square of the height , education level , excessive salt intake (>6 g/day), drinking status , smoking status (past/current smoker), sleep duration , and medication history of hypertension, diabetes, and hyperlipidemia was collected.We defined arterial stiffness as baPWV >1,800 m/s or ABI <0.9 –24 in thWe stratified the study population into quartiles based on HbA1c level . TyG index was calculated as ln (fasting TG (mg/dl) × FBG (mg/dl)/2). We also classified the participants into quartile groups based on the level of PBG, FBG, and TyG index. Continuous variables were summarized as mean ± standard deviation or median with interquartile range (IQR). Categorical variables were presented as numbers and proportions. We used ANOVA test for non-paired samples of normally distributed parameters and the Kruskal–Wallis test for non-parametric variables. The chi-squared test was applied for the comparison of categorical variables among 'four quartile groups of HbA1c.Three-step stepwise multivariable-adjusted Cox proportional hazard regression models were conducted to explore the association between HbA1c, PBG, FBG, and TyG index and the risk of the arterial stiffness. Model 1 was adjusted for age and gender. Model 2 was adjusted for variables in model 1, as well as education level, smoking status, drinking status, physical activity intensity, sleep duration, excessive salt intake, anemia, and medication history. Model 3 was adjusted for variables in model 2 plus BMI, MAP, LDL-C, HDL-C, and TG. As for TyG index, we excluded TG from model 3. A multivariable adjusted restricted cubic spline model with 3 knots was used to assess the dose–response relationship between HbA1c, PBG, FBG, and TyG index and the risk of arterial stiffness. Receiver operating characteristic (ROC) analysis was performed to explore the predictive ability of HbA1c, PBG, FBG, and TyG index on arterial stiffness. Considering the effect of glycemic status, ROC curves were also obtained in both diabetic and non-diabetic populations.We performed several sensitivity analyses. A generalized estimation equation (GEE) model was built to explore the association of repeated measurement of glycemic parameters and arterial stiffness. Cox regression analysis was further performed among diabetics and non-diabetics. Considering the effect of anemia on measurement of HbA1c, Cox regression analysis was conducted and the GEE model was used among individuals without anemia at baseline.p-value <0.05 was considered to be statistically significant. All statistical analyses were performed using R version 3.5.1 , SAS version 9.4  and Stata version 15 .For all analyses, a two-tailed In this cohort study, 3,048 qualified participants finished the follow-up and were enrolled in the final analysis. Of the 3,048 participants, 1,939 participants took the baseline and final examination, and their repeated measurements of glycemic parameters and outcomes in 2013–2017 were also collected. Finally, 1,939 individuals were included to explore the impact of repeated measurement of the glycemic parameters on arterial stiffness. The baseline characteristics of the whole study population in different groups stratified by HbA1c quartiles were summarized in p-value) were 1.13  for the Q2 group, 1.53  for the Q3 group and 1.63  for the Q4 group with the covariates in Model 3 controlled  in comparison to other parameters . The patients/participants provided written informed consent to participate in this study.ZH, LT, and SC came up with the idea and made the design of the study. XK contributed to the data. ZH and JW conducted the statistical analysis. ZH drafted the primary manuscript. ZH, JL, JZ, JW, XZ, YL, and ZW revised the manuscript and provided important advice for modification. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (grant numbers 82073668 and 81872708).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.