Vitamin D (VD) is an essential micronutrient with multiple functions for human growth, and adequate intake should be guaranteed throughout life. However, VD insufficiency is observed in infants all over the world. Low VD concentration in the breast milk of non-supplemented mothers and low compliance to VD daily supplementation are the main causes of VD insufficiency, especially in the long term. Furthermore, VD supplementation dosages are still debated and differ by country. We conducted a systematic review to compare the most recent evidence on different postnatal VD supplementation strategies, determining whether supplementation given to the mother is as effective as that administered directly to the child, and whether different dosages and administration schedules differ significantly in terms of efficacy and safety. We identified 18 randomized controlled trials (RCTs) addressing the role of infant (n = 961), maternal (n = 652) or combined infant and maternal VD supplementation (n = 260 pairs). In all studies, similar outcomes emerged in terms of efficacy and safety. According to our findings, alternative approaches of VD supplementation may be adopted, especially in cases where the adherence to daily supplementation strategies is poor. This review shows that different dosages and supplementation strategies result in similar VD sufficiency rates. Therefore, international guidelines may be revised in the future to offer multiple and different options of supplementation for specific settings and ages. Human breast milk, despite being the best choice for infant feeding, is generally lacking in proper amounts of Vitamin D (VD), with concentrations ranging from 10 to 80 IU/L in healthy lactating women ,3,4. TheFurthermore, it is known that VD serum levels are maintained by cutaneous synthesis and oral intake, while VD metabolism is both renal and hepatic . NonetheA wide variety of methods and dosages of supplementation are described in the literature. Oral supplementation of 400 IU/day to all infants from few days after birth to at least 6 months of age is generally recommended . HoweverFurthermore, a low adherence to constant daily supplementation in infants and toddlers, even in culturally and economically diverse settings, has been frequently described ,23,24,25This systematic review of the literature was conducted according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines . Three dStudies were included if they met the inclusion criteria of being conducted on healthy children aged from 0 to 36 months, born full-term to mothers on a VD sufficiency status at the time of delivery and coming from a developed country. Only randomized controlled clinical trials (RCTs) published in English between 1 January 2000 and 30 September 2021 were considered.Exclusion criteria were: (i) observational studies; (ii) RCTs with fewer than 15 mother–infant pairs; (iii) children older than 36 months; (iv) cohorts coming from less developed countries; (v) prenatal VD supplementation; (vi) baseline maternal 25(OH)D concentrations less than 10 ng/mL (severe deficiency); and (vii) absence of evaluation of VD status in children as a main outcome of the study.The titles and abstracts of articles that met the inclusion criteria were then independently screened for relevance by two authors, and the full text of potentially relevant articles was evaluated in depth by the same two investigators. Investigators were chosen for their expertise in the field, particularly in pediatric nutrition and supplementation policies.A quality assessment of the studies was conducted in order to assess their most important biases and weaknesses. The Newcastle-Ottawa Scale was used to assess the quality of the observational studies . This scFrom each selected study, the following data were extracted using a predefined database: name of the first author, year of publication, country, number of participants, route of administration, dosage and timing of VD supplementation, length of follow-up and risk ratios.The studies were then independently considered according to the following supplementation strategies: VD supplementation trials for children, for lactating mothers and for both lactating mothers and their children. Data on the accuracy and reliability of the assay for VD assessment were also extracted ,29.The primary study endpoint was to compare different methods and timing of VD supplementation on 25(OH)D serum concentrations after the intervention. The secondary endpoint was to assess any potential side effect related to the specific intervention among the different cohorts, when reported.For the final analysis, 18 articles were selected. The literature search process is summarized in Eight trials investigated the supplementation of VD in children only ,35,36,37Various supplementation regimens were assessed in these studies. Two studies evaluated the efficacy of a single high dose of 50,000 IU of VD given to infants at birth ,35. In oThe studies conducted on toddlers included two cohorts comparing the administration of VD-fortified milk formula with cow’s milk or meat-derived food ,37. BothSeven studies focused on the exclusive maternal supplementation of VD and included a total of 652 mothers  with two cohorts of children receiving a dose of 400 IU/d . Both stThree studies evaluated the efficacy of combined supplementation for mother and child ,46,47. TThis systematic review discusses the most recent evidence in the literature about the efficacy of different methods of VD supplementation on circulating VD levels to limit deficiency in infants and toddlers. No significant differences emerged in terms of safety and efficacy among proper VD supplementation regimens given directly to the mother, the child or both.The prevalence of VD deficiency observed among breastfed infants throughout the world is higher in those who lack an adequate sunlight exposure due to limited time spent outdoors or who do not follow regular VD supplementation regimens ,49,50. FThe choice of including subjects aging 12 to 36 months, rather than only infants, was made since they have different nutritional intakes, dietary habits and lifestyles. Indeed, just a few studies evaluated VD needs during the first years of life, and little is known on how the VD status changes after breastfeeding is discontinued. Additionally, the European Food Safety Authority set an adequate intake of 15 μg/d for children aged 1–17 years, not excluding the 12–24 months range of age and based on data mostly collected on adults . FurtherSeveral studies have been conducted on a possible association between the supplementation of VD and the reduced risk of developing autoimmune diseases, such as type 1 diabetes ,60,61 anThe present review suggests that exclusive high-dose supplementation provided to mothers and a proper administration given to the child can both be deemed suitable approaches for the prevention of VD deficiency in exclusively breastfed infants. Accordingly, the significant maternal VD supplementation of 6400 IU/day or >100,000 IU on a monthly schedule has been found to be able to safely supply adequate amounts of VD to breast milk , completAccording to most of the international recommendations and global consensus about the prevention of rickets, all infants should be orally supplemented with 400 IU/day of VD from birth to 12 months of age, regardless of feeding and nutrition types . The effConsidering that the compliance with daily VD supplementation in the first years of life is variable in different countries, and that rates of withdrawal and adherence change during the first months of life, an increased awareness about the correct posology and doses of VD administration is needed ,23,24. TAccording to the European Society for Pediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) guidelines, all infants during the first year of life should receive 400 IU/day oral supplementation of VD, resulting in a minimal serum concentration of 20 ng/mL . Beyond To promote compliance to VD supplements, the fortification of commonly used food products has also been suggested ,37,87. IOne of the strength of this review is the specific focus on the efficacy outcomes of the studies included. We evaluated the VD status of children not in terms of higher serum levels achieved by different cohorts, but in terms of real VD deficiency prevalence in the first years of life. Moreover, this review adds the evaluation of the safety outcomes. Additionally, the exclusion of trials that included high prenatal supplementation and/or VD deficient mothers at baseline reduced the possible risk of bias. Another strength of this study may be considered to be the inclusion of both breastfed infants and toddlers, as well as the rarely described weekly/monthly administration schedules.An important limitation of this systematic review is the impossibility to perform a formal meta-analysis using a quantitative comparison, due to the wide heterogeneity of study designs, with different dosing frequencies and amounts, different baseline VD status at randomization, inclusion criteria and follow-up length.In conclusion, different supplementation schedules given to mothers in RCTs showed similar efficacy and safety, and no adverse effects have been observed in children receiving high VD dosages, despite the fact that many of these strategies have not yet been implemented in clinical practice. VD supplementation should be revised according to the different settings, offering multiple options to families, or evaluating the best choice case-by-case, especially for subjects who are more prone to poor adherence to daily supplementation. Moreover, considering the wide variability in the causes that could lead to this poor adherence in culturally and economically diverse settings and countries, specific strategies that could help to increase adherence rates are not simple to be identified, and case-by-case reasoning should be performed.Additional dose–response studies on other types of supplementation strategies are then needed, with a specific focus on the 12–36 months range, given that dosages exceeding 400 IU/d did not provide additional benefits on the health and bone status in the first year of life. International guidelines should reconsider supplementation and milk fortification procedures in view of real-world compliance, which seems to be lower than expected, particularly beyond the first months of life.


Colon ascendens stent peritonitis (CASP) surgery induces a leakage of intestinal contents which may cause polymicrobial sepsis related to post-operative failure of remote multi-organs  and possible death from systemic syndromes. Mechanisms underlying such phenomena remain unclear. This article aims to elucidate the mechanisms underlying the CASP-model sepsis by analyzing real-world GEO data  generated from mice spleen 12 hours after a CASP-surgery in septic MyD88-deficient and wildtype mice, compared with untreated wildtype mice. Firstly, we identify and characterize 21 KO MyD88-associated signaling pathways, on which true key regulators  are marked, which were coordinately, significantly, and differentially expressed at the systems-level, thus providing massive potential biomarkers that warrant experimental validations in the future. Secondly, we observe the full range of polymicrobial  sepsis triggered by the CASP-surgery by comparing the coordinated up- or down-regulations of true regulators among the experimental treatments born by the three data under study. Finally, we discuss the observed phenomena of “systemic syndrome”, “cytokine storm” and “KO MyD88 attenuation”, as well as the proposed hypothesis of “spleen-mediated immune-cell infiltration”. Together, our results provide novel insights into a better understanding of innate immune responses triggered by the CASP-model sepsis in both wildtype and MyD88-deficient mice at the systems-level in a broader vision. This may serve as a model for humans and ultimately guide formulating the research paradigms and composite strategies for the early diagnosis and prevention of sepsis. The CASThe real-world GSE24327 data were generated from samples recovered from spleens 12 hours after the CASP-model sepsis in MyD88-deficient and wildtype mice , we designed three subtypes of data according to the original bench-experiments (vs. septic WT) for comparing septic null (Myd88–/–) with septic wildtype mice; GSE24327_B (b. septic KO MyD88 vs. untreated WT) for comparing septic null (Myd88–/–) with untreated wildtype mice; and GSE24327_C (c. septic WT vs. untreated WT) for comparing septic wildtype with untreated wildtype mice.We downloaded GSE24327 data from https://github.com/allenaigit/pathwayko/. Briefly, the PathwayKO package is systems software, rather than a standalone application algorithm, which comprises multiple modules with diverse dependencies, allowing users to conduct integrated processes in a pipeline fashion, such as (I) preprocessing, (II) ROC-AUC calculating, (III) statistics analyzing, and (IV) visualizing. Users who might lack knowledge and/or experience would seek help from professionals and consult the offered users’ manual to understand our interpretations. It currently incorporates the state-of-the-art methods of pathway analysis, statistics analysis, and visualizing analysis. The non-topology-based methods of pathway analysis include SAFE  for multiple testing , and type command lines:$ cd/home/PathwayKO_demo_runPathwayKO_demo_run$ R> library(devtools)> devtools::install_githubTo get started with the package, the user needs to run a demo:$ cd/home/PathwayKO_demo_runPathwayKO_demo_run$ R> library(pathwayko)> pathwayko_demohttps://github.com/allenaigit/pathwayko/tree/main/inst/docs/Users_manual.pdf.Batch-execution is conducted with parallel computations, which produces resulting output directories, each containing a group of desired output-files. The user completed a desired job by following instructions in the User’s manual, publicly available at https://cistrome.shinyapps.io/timer) (We estimated the correlation between the expression level of key regulators and the infiltration level of core immune cells (CICs)  in human cancer cohorts by utilizing cohort database TIMER2.0 (o/timer) .A), (B) and (C) under study. Note that via the subsequent analyses throughout the current study.Top-panel Figure 1via the PathwayKO package benchmarked on each data under study  from the (C) treatment (septic wildtype vs. untreated wildtype). Only 1 pathway is identified as insignificant (pGFdr >0.05) from the (A) treatment (septic KO MyD88 mice vs. septic wildtype mice). Only 2 pathways are identified as insignificant (pGFdr >0.05) from the (B) treatment (septic KO MyD88 mice vs. untreated wildtype mice). These findings suggest that such insignificant pathways are likely dependent on MyD88-deficiency, i.e., the KO MyD88 deficiency has attenuated signaling. For instance, the number of true DEGs on the Toll-like signaling pathway is remarkably decreased from 52 in (C) treatment to 50 in (B) treatment, and to 34 in (A) treatment  the (C) treatment provokes the strongest innate immune responses at the systems-level at 12 hours after the CASP-surgery, 2) the (A) or (B) treatment causes relatively weaker innate immune responses at the systems-level at 12 hours after the CASP-surgery, and 3) some regulators on the target signaling pathways are dependent on the KO MyD88-deficiency, but others are independent of it.There are common features of comparisons on the target signaling pathways: Case 1: The Toll-like receptor signaling pathway (mmu04620) is the basal target pathway that is solely responsible for the KO MyD88 phenotype (pGFdr <0.0001) identified from all three data (see A), 50 in (B) and 52 in (C) treatment (see C) treatment, MyD88 is up-regulated, where the majority of ligands, receptors, adaptors and transducers  are up-regulated. Only a few  are down-regulated. Therefore, the downstream effectors  are collectively up-regulated at the systems-level.Firstly, in the (A) treatment, the majority of regulators are down-regulated, except for the few  that remain up-regulated. In particular, members of the major axis  are drastically down-regulated and the downstream effectors  are drastically attenuated at the systems-level. These data suggest that the KO MyD88-deficiency had attenuated the expression level of major inflammatory effectors  at the systems-level in deficient mice.Secondly, for instance, in the  is a composite target pathway, which embeds the Toll-like signaling pathway, B-cell receptor signaling pathway, T-cell receptor signaling pathway, and RIG-I-like receptor signaling pathway (pGFdr <0.0001) target pathway identified from all three data (A), 18 in (B) and 34 in (C) treatment (see C) treatment, MyD88 is up-regulated, of which the majority of ligands, receptors, adaptors and transducers , MIP-2 (CXCL2), SDF-1α (CXCL12) and ICAM (CD54)) are up-regulated. Only a few  are down-regulated. These key regulators are related to inflammation, survival, myeloiesis, B-cell lymphopoiesis, lymphocyte adhesion and T-cell co-stimulation at the systems-level.Firstly, in the (A) treatment, the majority of regulators  are down-regulated except for a few  that are up-regulated. These regulators are related to inflammation, survival, myeloiesis and B-cell lymphopoiesis at the systems-level.Secondly, in the  is a composite target pathway, which embeds the Toll-like signaling pathway, TNF signaling pathway, and p53 signaling pathway (pGFdr <0.0001) target pathway from all three data (A), 39 in (B) and 80 in (C) treatment (see C) treatment: 1) on the classic MAPK pathway, regulators  are up-regulated, while a few  are down-regulated, and 2) on the JNK/p38 MAPK pathway, the majority of regulators  are up-regulated, while only a few  are down-regulated. These key regulators positively contribute to cell cycle regulation, such as proliferation, differentiation, inflammation, and anti-apoptosis at the systems-level.Firstly, in the (A) treatment: 1) on the classic MAPK pathway, regulators  are up-regulated, while a few  are down-regulated, and 2) on the JNK/p38 MAPK pathway, regulators  are up-regulated, while a few  are down-regulated at the systems-level.Secondly, for instance, in the  is a composite target pathway, which embeds the Toll-like signaling pathway, NFκB signaling pathway and MAPK signaling pathway (pGFdr <0.0001) target pathway from all three data (A), 56 in (B), and 63 in (C) treatment (see (C) treatment, MyD88 is up-regulated, around which the majority of regulators  are up-regulated. Only a few regulators  are down-regulated. The downstream effectors  are accordingly up-regulated. Note that ASC means antibody secreting cells  treatment, only a few regulators  are down-regulated, while a few  are up-regulated. The downstream effectors  are accordingly down-regulated at the systems-level.Secondly, in the Case 5: The PD-L1 expression and PD-1 checkpoint pathway in cancer (mmu05235) is a composite target signaling pathway, which embeds the Toll-like signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, HIF-1 signaling pathway, and T-cell receptor signaling pathway (pGFdr <0.0001) target pathway identified from all three data (A), 27 in (B), and 37 in (C) treatment (see C) treatment: 1) on the Toll-like receptor signaling pathway, a few regulators  are up-regulated, while only NFAT is down-regulated; 2) on the HIF-1 signaling pathway, HIF-1α is up-regulated; 3) on the MAPK signaling pathway, only EGFR and AP-1 are up-regulated; 4) on the PI3K-AKT signaling pathway, a few regulators  are up-regulated. All of these key regulators positively contribute to the increased production of PD-L1; and 5) on the T-cell receptor signaling pathway, the major regulators  are up-regulated, while a few  are down-regulated. All of these key regulators positively contribute to the increased apoptosis but decreased cell cycle progression, IL-2 production, T-cell activation, and effector T-cell development at the systems-level.Firstly, in the (A) treatment: 1) on the Toll-like receptor signaling pathway, a few regulators  are up-regulated, while a few (MyD88 and TRAF6) are down-regulated; 2) on the HIF-1 signaling pathway, only HIF-1α is down-regulated; 3) on the MAPK signaling pathway, only EGFR and AP-1 are down-regulated, while only ERK is up-regulated; and 4) on the PI3K-AKT signaling pathway, a few regulators  are down-regulated. All of these key regulators contribute to the decreased production of PD-L1 and 5) on the T-cell receptor signaling pathway, a few regulators  are down-regulated, while a few  are up-regulated. All of these key regulators inversely contribute to the increased apoptosis and decreased cell cycle progression, IL-2 production, T-cell activation, and effector T-cell development at the systems-level.Secondly, in the (Tuberculosis (pGFdr <0.0001 or 0.05) from the (A) and (B) treatment, respectively; only one pathway (Herpes simplex virus 1) and two pathways  are identified as insignificant (pGFdr >0.05) from the (A) and (B) treatment, respectively. All of these pathways are identified as significant (pGFdr <0.0001) from the (C) treatment that compared septic wildtype with untreated wildtype mice. These data suggest possible death from systemic syndromes, which increase the difficulty of early diagnosis and prevention of sepsis because it is unlikely that such systemic syndromes can be precisely attributed to a specific type  of infections.Each infection-related signaling pathway embeds the basal Toll-like receptor signaling pathway coupled with more than one of the other composite signaling pathways  treatment (septic KO MyD88 vs. untreated wildtype)  and the interferons  are categorized in the  correlated with the infiltration level of CICs. Some examples are highlighted (via the axis of intestine (colon)–heart–spleen–liver–kidney–lung, etc.).To examine possible infiltration of the core immune cells (CICs)  that would be recruited by the top 50-ranked regulators intersected across all of the three data see Table 2,vs. septic WT (GSE24327_A); (b). septic KO MyD88 vs. untreated WT (GSE24327_B); and (c). septic WT vs. untreated WT (GSE24327_C). These data were generated from samples recovered from spleen at 12 hours after the CASP-surgery in mice, which were used for mRNAs profiling on microarray  through blood circulation in the body. The recruitment of infiltrating CICs fighting pathogens leaked from gut contents would result in severe sepsis progression across remote multi-organs after the CASP-surgery, contributing to the newly observed phenomena: “systemic syndrome” in (C) treatment, “cytokine storm” in (B) treatment, and “KO MyD88-attenuation” in (A) treatment in the current study, which are discussed as follows.Based on the 21 KO MyD88-associated target signaling pathways, we conceptualize the systemic mechanisms underlying the innate immune responses triggered by the CASP-model sepsis Table 2 C) treatment (compared septic wildtype mice with untreated wildtype mice) displays SIRS because of the strongest innate immune responses (pGFdr <0.0001) signaling pathways (“Systemic inflammatory response syndrome (SIRS)” has been perceived to be an inflammatory state affecting the whole body, responding to an infectious or noninfectious insult. It should have both pro- and anti-inflammatory components although its definition is simply referred to an “inflammatory” response . The (C)esponses . Moreovepathways Table 1,pathways , provideA), (B) and (C) treatments (1) ligands, receptors and adaptors (MyD88 and Lepr); 2) signal transducers ; 3) transcriptional factors ; and 4) downstream effectors, such as interleukins , chemokines , and colony-stimulating factors (Csf3r and Csf2rb). Our data highlight these core regulators intersected across all data (see AandBandC in B) treatment (see Bonly in A), (B) and (C) treatments. We term such newly observed facts as “cytokine-storm” (B) treatment that compared septic KO MyD88 mice with untreated wildtype mice  treatment compared septic wildtype mice with untreated wildtype mice, which produced 648 DEGs (A) treatment that compared septic KO MyD88 mice with septic wildtype mice produced 408 DEGs (B) treatment that compared septic KO MyD88 mice with untreated wildtype mice produced 266 DEGs (C) treatment produced much more DEGs than the KO MyD88 deficient mice in the (A) and (B) treatment, respectively. We term these newly observed facts as “MyD88-deficiency attenuation” (The (nuation” . We concp<0.001) correlation . Such a positive feedback loop is also initiated in MyD88-deficient mice (B) at the early stage of sepsis progression (B), leading to a low mortality rate  of the immune system . The strthe body 9. Such ity rate . We recoThis study aimed to elucidate systemic mechanisms underlying the standard CASP-model sepsis in the body through top-down data driven analysis on high-throughput functional genomics data. The novelty of this article roots in six aspects. 1) The in-house PathwayKO package is effective in conducting cutting-edge knockout pathway enrichment analysis in order to identify and assess the entirety of knockout signaling pathways at the systems-level. 2) The real-world GEO data  generated from the spleen 12 hours after the CASP-surgery safeguard unbiased choice of samples for the CASP-model sepsis in mice (both MyD88-deficient and wildtype). Therefore, we can discuss the full range of polymicrobial  sepsis in provoking severe innate immune responses, resulting in failure of distant multi-organs  at the systems-level. 3) The 21 target KO MyD88-associated signaling pathways create a broader vision, providing novel insights into mechanisms underlying the CASP-model sepsis. 4) The true key regulators  that were coordinately, significantly, and differentially expressed at the systems-level, as marked on those true target signaling pathways, provide massive potential biomarkers that warrant experimental validations in the future. 5) Based on the newly observed facts in the CASP-model sepsis, we discuss “systemic syndrome”, “cytokine storm”, and “KO MyD88 attenuation”, as well as the proposed hypothesis of “spleen-mediated immune-cell infiltration” at the systems-level. 6) Our results provide novel angles in a broader vision towards a better understanding of the CASP-model sepsis in mice, which may serve as a model for humans, to ultimately guide formulating the research paradigms and composite strategies for the early diagnosis and prevention of severe sepsis.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE24327.Publicly available datasets were analyzed in this study. This data can be found here: HA and YA designed the project. HA conducted computations and analyzed data. HA, BL, FM, and YA interpreted results, wrote manuscript and approved the final manuscript.This work was supported by a grant-in-aid from National Science and Technology Major Programs of China (2014ZX0801105B-002) and Supercomputing Program of National Natural Science Foundation of China (No. U1501501-534) to YA.Author HA was employed by SINOMACH-IT.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.


Fructus Ligustri Lucidi  and Ecliptae Herba , is commonly used in clinical practice to prevent and treat liver diseases. Modern pharmacological studies have shown that both EH and FLL can inhibit HCC proliferation. However, the pharmacological mechanism, potential targets, and clinical value of EZW in inhibiting HCC have not been fully elucidated. We used multilevel databases (Gene Expression Omnibus (GEO), Traditional Chinese Medicine Systems Pharmacology (TCMSP), High-throughput Experiment- and Reference-guided database (HERB), and SwissTargetPrediction) to show that EZW suppresses HCC through 19 active components acting on 66 potential targets. Enrichment analysis revealed that EZW mainly regulates HCC progression through various metabolic pathways, the cell cycle, and cellular senescence. Furthermore, we used The Cancer Genome Atlas (TCGA)-LIHC database to analyze the expression patterns and clinical characteristics of cellular senescence-related genes and identified CDK1, CDK4, CHEK1, and G6PD as key therapeutic molecular targets in EZW-suppressed HCC. Molecular docking revealed that EZW could exert its anti-HCC effect by binding various active components to the above cellular senescence-related genes and regulating their activities. In conclusion, we systematically revealed the potential pharmacological mechanisms and molecular targets of EZW against HCC based on multilevel data integration and a molecular docking strategy.As a multicomponent, multitarget empirical therapy, traditional Chinese medicine (TCM) has been used clinically in Asia for thousands of years. Due to this unique feature, TCM therapy is considered a promising therapeutic strategy for the treatment of hepatocellular carcinoma (HCC). Er-Zhi-Wan (EZW), a well-known TCM formula containing two herbs,  Worldwide, liver cancer is one of the most common cancers, and its morbidity and mortality are on the rise , 2. It hin vitro and in vivo [The pathological mechanism of HCC is very complex, and multiple targets and signaling pathways are involved in its development . This co in vivo –18. Due Fructus Ligustri Lucidi  and Ecliptae Herba . According to Chinese medicinal theory, EZW is commonly used clinically in China for the treatment of liver-kidney yin deficiency syndrome (LKYDS), which is a pathological and diagnostic pattern caused by an imbalance of yin and yang and is more common in primary liver cancer, diabetes, and hypertension [Er-Zhi-Wan (EZW), a well-known TCM formula, was first recorded in the ‘Fu Shou Jing Fang’ in the Ming Dynasty . It consrtension . Therefortension  uncoverertension  revealedrtension . HoweverSince TCM prescriptions comprise many kinds of herbs and contain many kinds of ingredients, it is difficult to systematically and comprehensively study the pharmacological mechanism of TCM with the existing experimental methods. With the successful establishment of multiple biological databases and the rapid development of systems biology, the emergence of network pharmacology has brought great opportunities for breakthroughs in TCM research , 24. To To identify which genes are involved in the progression of HCC, we analyzed the Gene Expression Omnibus (GEO) dataset GSE84402 to identify genes that were differentially expressed in 14 pairs of HCC tissues and corresponding noncancerous tissues. As shown in the volcano plot, a total of 1199 differentially expressed genes (DEGs) were identified in these 14 pairs of liver tissues, of which 632 genes were upregulated and 567 genes were downregulated in cancerous tissues compared with noncancerous liver tissues . It is sTo further investigate the underlying molecular mechanisms involved in HCC progression, we performed enrichment analysis on 1199 DEGs, including GO and KEGG analyses. We used bubble plots to display the top 20 GO  and KEGGIn addition, the results of the top 20 KEGG pathways indicated that the progression of HCC is closely related to 6 categories, including metabolism, cellular processes, human diseases, genetic information processing, organismal systems, and environmental information processing. In particular, 12 of the 20 pathways were involved in the metabolism category, including “Metabolic pathways (ko01100)”, “Fatty acid degradation (ko00071)”, “Retinol metabolism (ko00830)”, “Tryptophan metabolism (ko00380)”, “Tyrosine metabolism (ko00350)”, “Alanine, aspartate and glutamate metabolism (ko00250)", "Valine, leucine and isoleucine degradation (ko00280)”, “Drug metabolism – cytochrome P450 (ko00982)”, “Caffeine metabolism (ko00232)”, “Metabolism of xenobiotics by cytochrome P450 (ko00980)”, “Carbon metabolism (ko01200)”, and “Biosynthesis of amino acids (ko01230)”. Notably, metabolic pathways (ko01100) were enriched with multiple downregulated DEGs, including multiple metabolic enzymes . The heatmap results showed that abnormal changes in various metabolic enzymes occurred during the progression of HCC, which suggested that abnormal expression of metabolic enzymes affected the development of HCC . FurtherFructus Ligustri Lucidi and Ecliptae Herba in equal proportions. According to the two criteria of drug-likeness (DL) ≥ 0.18 and oral bioavailability (OB) ≥ 30% [EZW is produced by mixing B) ≥ 30% , 28, a tB) ≥ 30% . TherefoWe identified potential targets for these 19 active compounds through two target prediction databases, High throughput Experiment- and Reference-guided (HERB) and SwissTargetPrediction. In the HERB database, flavonoids  possessed more targets, but targets failed to be identified for 4 active compounds  . SimilarTo explore which pathological targets EZW acts on to treat HCC, we performed Venn diagram analysis of 1199 DEGs in HCC with potential therapeutic targets of the active components in EZW. As shown in To further understand the interconnections between the herbs, active compounds, and potential therapeutic targets for HCC, we generated an herb-compound-target (H-C-T) network . The resTo explore hub genes in the PPI network, we performed a topological analysis of this network. The PPI network was entered into Cytoscape software to calculate the topological parameters of the nodes in the network. We calculated the top 10 hub genes in the PPI network using a different approach via the CytoHubba plugin and visualized the network via Cytoscape. These calculation methods included the degree method, maximum neighborhood component (MNC), maximal clique centrality (MCC), and density of maximum neighborhood component (DMNC). The results showed that regardless of the calculation method, the genes in the MCODE1 and MCODE2 components of the top ten hub genes were highly enriched, and mainly included CDK1, CDK4, CCNB1, CCNB2, CCNA2, PCNA, AURKA, and AURKB .To fully reveal the mechanism underlying the treatment of HCC by EZW, we performed GO and KEGG pathway analyses. The top 25 GO terms revealed the functions of the potential therapeutic targets of EZW for the treatment of HCC. Annotation analysis of CCs revealed that these therapeutic targets were mainly involved in “cyclin-dependent protein (GO:0000307)”, “serine/threonine protein kinase complex (GO:1902554)”, and “condensed chromosome (GO:0000793)” . For MF In the KEGG pathway enrichment analysis, the results indicated that EZW exerted its effect on HCC mainly through the following pathways: “Cell cycle (ko04110)”, “Progesterone-mediated oocyte maturation (ko04914)”, “Steroid hormone biosynthesis (ko00140)”, “p53 signaling pathway (ko04115)”, “Cellular senescence (ko04218)”, “TNF signaling pathway (ko04668)”, “Metabolism of xenobiotics by cytochrome P450 (ko00980)”, “IL-17 signaling pathway (ko04657)”, “Hepatitis B (ko05161)”, and “FoxO signaling pathway (ko04068)” . These tTo further identify the molecular mechanisms involved in the suppression of HCC by EZW, we captured these therapeutic targets through the Metascape database for pathway and process enrichment analysis. We present the correlations between different enriched terms in a network graph, and the results revealed that “PID FOXM1 pathway”, “Cell cycle”, “steroid metabolic process”, “SUMOylation”, and “cell cycle G2/M phase transition” were significantly enriched , 7E.Based on the above enrichment analysis, we speculate that cell cycle pathways play a key role in the effects of EZW treatment on HCC. However, recent studies have shown that cellular senescence permanently inhibits the proliferative capacity of cells and induces irreversible cell cycle arrest, which is considered a promising strategy for the treatment of cancer . Our KEGTo fully characterize which senescence-related genes are involved as therapeutic targets in the treatment of HCC by EZW, we first identified 153 cellular senescence-induced genes and 121 cellular senescence-inhibited genes from the cellular senescence gene database CellAge . SubsequUnivariate Cox proportional hazards regression analysis revealed that seven cellular senescence-related genes were associated with the prognosis of HCC, and CDK1, CDK4, G6PD, AURKA, MMP9, IGFBP3, and CHEK1 were considered risk factors  . FurtherThe above seven senescence-related genes were analyzed by least absolute shrinkage and selection operator (LASSO) Cox regression analysis to establish a cellular senescence-related signature for predicting survival. A 4-gene signature was constructed according to the optimum λ value , 8F. We We analyzed the clinical characteristics of the 4 cellular senescence-related genes  involved in the treatment of HCC by EZW to further evaluate the clinical application value of EZW in HCC treatment. To investigate the role of these genes in LIHC, we assessed RNA-seq data obtained from 374 HCC patient tissues and 50 normal tissues using transcriptional data from the TCGA-LIHC database. The results showed that the levels of all four genes were significantly higher in tumor tissues than in normal liver tissues (P < 0.05), which was consistent with the previous GEO dataset results . The traThe previous results suggest that CDK1, CDK4, CHEK1, and G6PD are key therapeutic targets through which EZW suppresses HCC. Based on the H-C-T network analysis, EZW inhibits HCC progression through multiple active components acting on these targets. To further reveal how these active ingredients act on these targets, we used a molecular docking strategy to simulate their binding modes and calculated the binding energies to infer the affinity of these active ingredients to the targets. According to the prediction results obtained using molecular docking, a variety of active ingredients  could bind to these key targets  with low binding energies . As showHCC is a common malignancy that usually arises in the context of chronic liver disease. Despite modern strategies for patient management, patients with advanced HCC have few treatment options and a poor prognosis. Moreover, the progression of HCC involves multiple mechanisms and aberrant changes in multiple pathological genes . This coin vivo. Data from Nikolaou et al. [Exploring the pathogenesis of HCC will make an important contribution to providing suitable treatment strategies in the future. In our study, HCC progression was found to be regulated by a complex gene network involving the aberrant expression of thousands of DEGs. Notably, functional enrichment analysis of these DEGs revealed that the pathogenesis of HCC is closely related to various metabolic pathways and processes of cell cycle regulation, which have been shown to be critical for the development of liver disease , 33. Amou et al.  suggest u et al.  showed tFurthermore, cyclins and cyclin-dependent kinases (CDKs) are typically involved in most metabolic processes, such as glucose metabolism, lipogenesis, amino acid metabolism and mitochondrial activity , 40. KEGThe extract of EZW contains a variety of compounds, which undoubtedly increases the difficulty of systematically mapping the pharmacological mechanism of EZW in inhibiting tumors. Therefore, identifying the active components of EZW through databases can be used to more accurately and efficiently elucidate its mechanism. After screening, the key active components of EZW were mainly flavonoids, coumarins, sterols, and natural triterpenoid carboxylic acids. Among them, the flavonoids quercetin and luteolin were identified as the common active components of the two herbs in EZW. We speculate that these two active components may be an important basis for the synergistic anti-HCC effect of the two herbs in EZW. Natural flavonoids have been identified as one of the major classes of natural anticancer agents, exerting antitumor activity through cell cycle arrest as a major mechanism in various cancer cells . FurtherAccording to the H-C-T network diagram, EZW may exert its anti-HCC effect through the action of multiple active components on multiple targets. Subsequent PPI network, MCODE, and topological analyses indicated that EZW suppresses HCC through the regulation of multiple cyclins  and cyclin-dependent kinases (CDK1 and CDK4), which were defined as hub genes. Further KEGG enrichment analysis depicting the top 25 pathways revealed that the cell cycle pathway ranked first, and our aforementioned results indicated that this pathway plays a key role in HCC progression, suggesting that this pathway is an important mechanism by which EZW inhibits HCC. However, the pharmacological mechanism of EZW also involves the p53 signaling pathway, which has been shown to play a central role in regulating the cell division cycle . These fInterestingly, the pharmacological mechanism by which EZW inhibits HCC also involves the process of cellular senescence. Cellular senescence constitutes a permanent state of cell cycle arrest in proliferating cells induced by different stresses and has been recognized in recent years as an important mechanism for preventing tumor cell proliferation . TCM witin vivo and significantly improves survival [These data strongly suggest that CDK1, CDK4, CHEK1, and G6PD are potential prognostic biomarkers in HCC and key therapeutic targets for EZW to suppress HCC based on the cellular senescence process. Wu et al.  reportedsurvival . Previousurvival . Alteredsurvival . Althougin silico simulations were used in this study to elucidate the interaction mode between the active components of EZW and these molecular targets. Since the extract of EZW contains various compounds, systematically depicting a clear pharmacological mechanism of how EZW inhibits tumors remains a challenge. The results of molecular docking revealed that multiple active compounds of EZW could be well combined in the pockets of CDK1, CDK4, CHEK1, and G6PD. We speculate that these active compounds may inhibit their activities by targeting these molecular targets, thereby disturbing the cell cycle and metabolism to suppress HCC.Our study suggests that EZW can be used to treat HCC by acting on these molecular targets, but its intrinsic mechanism needs to be further elucidated. Therefore, Overall, we elucidated the potential pharmacological mechanisms and molecular targets of EZWs in HCC therapy by integrating multiple databases and performing molecular docking analysis. EZW can regulate tumor progression through multiple metabolic pathways, the cell cycle, and cellular senescence. In particular, TCM-induced cellular senescence may become a promising cancer treatment strategy. TCGA data analysis showed that CXP may improve the prognosis and clinical outcomes of HCC patients by regulating cellular senescence-related genes, revealing its clinical application value. In addition, the CDK1, CDK4, CHEK1, and G6PD genes were identified as key therapeutic targets for EZW in the treatment of HCC. Our results suggest that EZW can suppress tumors by disrupting the cell cycle, senescence, and metabolism through these therapeutic targets. To our knowledge, this is the first systematic pharmacological study of EZW in HCC therapy. Therefore, although there are still limitations in this study, this study provides innovative research methods and breakthroughs for TCM research.https://old.tcmsp-e.com/tcmsp.php). First, the Chinese terms “Mo-Han-Lian” and “ Nü-Zhen-Zi” of Ecliptae Herba and Fructus Ligustri Lucidi were separately entered into the database to determine their components and retrieve their pharmacokinetic data. Here, we selected two pharmacokinetic parameters as screening criteria to identify the active components in these herbs based on previous studies [https://pubchem.ncbi.nlm.nih.gov/).We determined the chemical components of EZW through the Traditional Chinese Medicine Systems Pharmacology Database  is a high-throughput experiment- and reference-guided database of traditional Chinese medicine. SwissTargetPrediction (http://www.swisstargetprediction.ch/) is an online database that predicts targets of biologically active small molecules. To identify the pathological genes associated with HCC progression, we downloaded GSE84402 from the GEO (http://www.ncbi.nlm.nih.gov/geo/) database. The expression data obtained from 14 pairs of human HCC tissues and corresponding noncancerous tissues were analyzed using the “limma” R package of Bioconductor (https://bioconductor.org/packages/release/bioc/html/limma.html), and the differentially expressed genes of HCC were screened with false discovery rate (FDR) < 0.05 and |log2FC| > 1. Subsequently, the “ggplot2” and “ComplexHeatmap” packages in R language were used for volcano plot and heatmap visualization, respectively. Finally, the potential targets of EZW and the DEGs of HCC were subjected to Venn diagram analysis to obtain overlapping targets. These overlapping targets are the therapeutic targets of EZW in the treatment of HCC.First, we screened the potential therapeutic targets of EZW’s active ingredients based on the HERB and SwissTargetPrediction databases. HERB . Statistical analysis and visualization of gene expression between adjacent nontumor tissues (n = 50) and tumor tissues (n = 374) were performed using the “ggplot2” R package. The list of cellular senescence-related genes was obtained from the cellular senescence gene database CellAge (https://genomics.senescence.info/cells/), including 153 senescence-induced genes and 121 senescence-inhibited genes.The RNA sequencing (RNA-Seq) expression profile dataset of 374 HCC patients, which included data on clinicopathological characteristics and survival, was downloaded from The Cancer Genome Atlas , an online data analysis and visualization platform for KEGG pathway and GO enrichment analyses, was used. OmicShare can be also used for Venn diagrams, heatmaps, network building, volcano map analysis, and more. In addition, we used the Metascape (https://metascape.org/gp/index.html#/main/step1) database for pathway and process enrichment analysis and generated network maps.OmicShare (A list of 66 therapeutic targets for EZW in the treatment of HCC was entered into the Metascape database (species limited to “Homo sapiens”) to generate a protein-protein interaction (PPI) network. Moreover, the Molecular Complex Detection (MCODE) algorithm was used to identify densely connected network components if the network contained 3 to 500 proteins, as these MCODEs are likely to represent densely connected regions in large PPI networks of molecular complexes. Subsequently, we performed visualization, hub gene analysis, and topological analysis of the raw data (cys format file) of the PPI network obtained by Metascape analysis using Cytoscape software. Through the CytoHubba plugin of Cytoscape software, hub gene analysis was performed on the network, including analysis methods such as degree, MNC, MCC, and DMNC. In addition, the parameters of topological features can be calculated by the Cytoscape plugin Network Analyzer, including “degree”, “intercentrality”, “closeness centrality”, “clustering coefficient”, and “topological coefficient”. The H-C-T network was analyzed and displayed through the OmicShare tool.Univariate Cox proportional hazards regression analysis was performed to identify cellular senescence-related prognostic genes (P < 0.01), and gene interactions were visualized by the R package “circlize”. Next, least absolute shrinkage and selection operator (LASSO) Cox regression was conducted with a random seed using the R package “glmnet” to construct the risk score model for predicting survival in the training cohort. Normalized expression matrices of candidate prognostic cellular senescence-related genes were set as independent variables in the regression, and the response variables were OS and patient status in the TCGA cohort. A risk score was determined for each patient based on the normalized expression level of each gene and its corresponding regression coefficient. The formula is as follows:The patients were then divided into low-risk and high-risk groups based on the median risk score.https://www.rcsb.org/), and water molecules and heteroatoms were removed by PyMOL 1.8 software. Moreover, the 3D chemical structures of the active ingredients of EZW were downloaded in SDF format from PubChem and converted to ‘pdb’ format by PyMOL 1.8. Next, the proteins and active ingredients were converted to ‘pdbqt’ format files by AutoDockTools (version 1.5.6), and the grid box feature of AutoDockTools was used to define specific docking pockets in the selected proteins to which the active ingredients could bind. Once all data were prepared, the command prompt was used to perform molecular docking analysis and visualize the docking results with PyMOL.The X-ray crystal structures of the selected proteins were obtained from the Protein Data Bank  analysis were drawn using the R package “survminer”. Two-tailed P values < 0.05 were considered statistically significant.Publicly available datasets were analyzed in this study. This data can be found here: TCMSP, TCGA, GEO, etc.Supplementary File 1Supplementary File 2Supplementary File 3Supplementary File 4Supplementary File 5


He's Yangchao formula (HSYC) has been clinically proven to be effective in treating diminished ovarian reserve (DOR). However, the underlying molecular mechanisms of HSYC in DOR are unclear. in vivo.  This study aims to predict the underlying mechanisms of He's Yangchao formula (HSYC) against DOR through network pharmacology strategies and verify  Systematic network pharmacology was used to speculate the bioactive components, potential targets, and the underlying mechanism of HSYC in the treatment of DOR. Then, the CTX-induced DOR mouse model was established to verify the effect of HSYC against DOR and the possible molecular mechanisms as predicted in the network pharmacology approach. In vivo experiments showed that HSYC could promote the recovery of the estrous cycle and increase the number of primordial, growing follicles and corpora lutea. Besides, The results of qRT-PCR showed HSYC could regulate the expression of AKT1, ESR1, P53, and IL6 in DOR mice.  A total of 44 active components and 423 potential targets were obtained in HSYC. In addition, 91 targets of DOR were also screened. The identified hub genes were AKT1, ESR1, IL6, and P53. Further molecular docking showed that the four hub targets were well-bound with their corresponding compounds.  It was demonstrated that HSYC could increase ovarian reserves, and AKT1, ESR1, IL6, and P53 may play an essential role in this effect, which provided a new reference for the current lack of active interventions of DOR. Diminished ovarian reserve (DOR) defines as a decrease in the quantity and quantity of oocytes, which is a major cause of female infertility and assisted reproductive technology (ART) failure –3. Its oThe decline of ovarian reserve is a continuous and gradual process. The premature depletion of ovarian reserve eventually results in premature ovarian insufficiency, which is a more severe condition that makes it much harder to treat. Therefore, rapid treatment is crucial for DOR women. Various treatment strategies are being used to fight against DOR, including estrogen supplementation, dehydroepiandrosterone (DHEA), ovulation induction therapy, ART, and ovum donation. DHEA is an endogenous steroid that originates from the zona reticularis of the adrenal cortex and the ovarian theca cells in women. It is produced by the conversion of cholesterol, acting an important role in the formation of estradiol in peripheral tissues . DHEA isHe's TCM Gynecology,” which is the famous school doctrine of Gynecology of TCM in China and has lasted for more than 170 years [Traditional Medicine has been included in the influential global medical compendium by the World Health Organization. Traditional Chinese Medicine (TCM) is a branch of Traditional Medicine and is an essential element of China's national health policies. It exerts beneficial therapeutic efficacy and reduces side effects through various herbal combinations, thereby preventing and treating various diseases . As show70 years . HSYC ef70 years . Howeverin vivo experiments were performed to validate the molecular mechanism. Compared with western medicine, TCM is characterized by multicomponent and multitarget and exerts its effect on multiple biological processes to treat diseases via its diverse bioactive components, which act on multiple targets . NetworkAsparagi Radix, Radix Puerariae, Angelicae Sinensis Radix, Platycladi Semen, Cuscutae Semen, Cistanches Herba, Rubi Fructus, and Paeoniae Radix Alba) were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform ). TCMSP is a complete library of Chinese herbal ingredients containing more than 36,000 chemical molecules, ingredients-related targets, and their relationships with diseases [in vivo, which is called the pharmacokinetics of TCM. Oral bioavailability (OB) and drug-like (DL) are the important parameters of ADME [https://www.tcmip.cn/ETCM/index.php/Home/Index), SymMap (https://www.symmap.org/ (accessed on 15 March 2020)), and PubChem (https://pubchem.ncbi.nlm.nih.gov (accessed on 15 March 2020)) were used to retrieve and predict the potential targets of bioactive components in HSYC. The obtained potential targets, including the name, gene ID, and organism, were normalized based on the UniProt protein database (https://www.uniprot.org (accessed on 18 March 2020)).All bioactive compounds of 8 herbs in HSYC  is a database for collecting a comprehensive compendium of human genes. The keyword “diminished ovarian reserve” was used to search GeneCards database (accessed on 18 March 2020). Next, “diminished ovarian reserve” was used as a keyword to search DisGeNET (https://www.disgenet.org (accessed on 18 March 2020)). In addition, we searched the National Centre for Biotechnology Information Gene ).The DOR-related targets were collected from three databases. GeneCards (https://string-db.org (accessed on 18 March 2020)) with the cut-off criterion of combined score > 0.9 and the species “Homo sapiens.” In addition, the targets of the active compounds in HSYC were matched with the DOR targets to obtain the intersected genes. Then, the intersected genes of compounds-DOR were submitted to the STRING database to structure another PPI network.Based on the above analyses, a visual interaction network based on the herbs-compounds-targets interactions was established by Cytoscape software. Meanwhile, to show the mechanism of action of HSYC, the protein-protein interaction (PPI) network of potential targets of the bioactive compounds in HSYC was constructed by the STRING database  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis and annotation were performed using the R package “clusterProfiler” to characterize the pathways according to the intersected genes of bioactive components and DOR. GO terms with Bonferroni value <0.05 and KEGG pathways with Then, a visual herb-component-target-pathway interaction network based on interactions between the herbs, components, DOR-related targets, and pathways was established by Cytoscape software to holistically explain the mechanism of HSYC in the treatment of DOR.The intersected genes of the three subnetworks based on three topology parameters, including degree, betweenness, and closeness, were intersected with the 22 potential therapeutic targets to get hub genes. The crystal structures of protein receptors of hub targets were obtained from RCSB Protein Data Bank (https://www.rcsb.org/(accessed on 29 March 2020)). PyMol 2.4.0 was used to remove the original ligand, solvent molecules, and redundant protein chains and add polar hydrogen for the crystal structures of protein receptors. Then AutoDock Tools 1.5.6 was used to compute Gasteiger charges and determine the center and size of the docking box for further docking. The 3D structure of the bioactive compounds was obtained from the PubChem database. The structure was assigned MMFF94 force field and charges, made energy minimization and added polar hydrogen. AutoDock Vina, a freely available open-source program for molecular docking, was used to evaluate the binding of the bioactive compounds and their corresponding targets. Before molecular docking, all the protein structures and the bioactive compounds were converted to PDBQT format for further docking using AutoDock Tools 1.5.6. Then the bioactive compounds were docked into their corresponding proteins using AutoDock Vina. Finally, the binding affinity was calculated by AutoDock Vina and visualized the docking result by the open-source version PyMol 2.4.0 software.n = 6/group): control, DOR, low (Low-HSYC), moderate (Mod-HSYC), high (High-HSYC), and DHEA. A DOR mice model induced by cyclophosphamide (CTX) was established based on the previous study [Experimental Methodology of Pharmacology compiled by Wei et al. [Thirty-six female C57BL/6 mice (8 weeks old) were purchased from Shanghai SIPPR-Bk Lab Animal Company, with the license number SCXK (Shanghai) 2018–0006. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Zhejiang Chinese Medical University . The mice were maintained under standard conditions  with ad libitum access to rodent chow and water. After 1-week adaptation, mice were randomly divided into six groups  and analyzed under a light microscope . The estrous cycle was determined according to the cytological appearance as previously reported .μm thick sections. The number of follicles was observed by hematoxylin and eosin (HE) staining according to the methods previously described [The ovarian tissues were fixed in paraformaldehyde, embedded in paraffin, and then cut into 5 escribed . All HE µg of DNA sample was used for the PCR amplification of related genes according to the reaction conditions as follows: 95°C for 4 min, 38 cycles of 94°C 30 s, 60°C 30 s, 72°C 30 s. Primers were synthesized by Sangon  and listed in −ΔΔCt method. β-Actin was used as the internal reference gene.Total RNA was extracted from ovarian tissues using TRIzol reagent . The first-strand cDNA was reversely transcribed using a Bestar qPCR RT Kit . 1 t-test, and differences across six groups were analyzed using one-way ANOVA. A significant level was set at P < 0.05.Animal data were expressed as the mean ± standard deviation (SD) from 6 mice. Statistical analysis was conducted in GraphPad Prism 6.0. Differences between groups were analyzed using an unpaired A total of 44 kinds of bioactive components in HSYC with good ADME properties were obtained. The detailed information on bioactive compounds is described in The PPI network of targets in HSYC with the highest confidence was constructed using the STRING database, including 288 nodes and 1395 edges see . The colA total of 91 DOR-related targets were obtained from the human genomic database. The number of DOR-related targets in GeneCards, DisGeNET, and NCBI Gene was 44, 43, and 28, respectively . Twenty-R package. KEGG pathway analysis revealed that the targets of the PPI network were assigned to 89 KEGG pathways based on P < 0.01. GO annotation showed that the targets of the PPI network were classified into 1139 biological process (BP) terms, 20 cellular component (CC) terms, and 43 molecular function (MF) terms according to P < 0.01 . If the affinity is less than zero, it means that the ligand can spontaneously bind to the receptor. The smaller the value is, the easier the bioactive component is to bind to the receptor. As shown in Then, a herb-compound-target-pathway network see  was consP < 0.05). In addition, The mice's estrous cycle was continuously measured to evaluate the protective effects of HSYC on ovarian function. The diestrus stage of the DOR group was significantly prolonged during the 28 days of experiments, as presented in As shown in P < 0.05). This result shows that high-dose HSYC can prevent the loss of different types of follicles caused by CTX. Both HSYC and DHEA can significantly increase the number of growing follicles and the number of corpus luteum (P < 0.05), and the effect of HSYC is better than that of DHEA .The histological analysis of the ovaries is presented in Figures P < 0.05); the expressions then elevated after HSYC and DHEA treatment . In addition, the expressions of IL6 and P53 have the opposite tendency. The mRNA expressions of IL6 and P53 were upregulated in DOR mice, while HSYC and DHEA could significantly inhibit their expression levels . Moreover, in inhibiting P53 mRNA expression, high-dose HSYC is more significant than DHEA (P < 0.05).In contrast to the control group, the mRNA expressions of AKT1 and ESR1 declined in the DOR group , encoded by the gene ESR1 [The four hub targets, AKT1, ESR1, P53, and IL6, were created as 3D graphs of docking and analysis. Further molecular docking showed that the four hub targets were well-bound with their corresponding compounds. AKT1 is the important gene in the PI3K-AKT signaling pathway. Also, AKT1 is a multifunctional protein regulating cell growth, survival, and proliferation . It playcultures . AKT1−/−cultures . As the ene ESR1 , 44. ESRene ESR1 . Severalene ESR1 , 47. Proene ESR1 . Many stene ESR1 . P53 canene ESR1 . Similarene ESR1 . Besidesene ESR1 , 52. CliNext, we studied the efficiency and mechanism of HSYC on DOR mice through animal experiments. A regular estrous cycle that lasts for 4 to 6 days in mice can be a key index of the integrity of the HPO reproductive axis . In the The results of qRT-PCR suggested that the expression of AKT1 and ESR1 was downregulated, and the expression of P53 and IL6 was upregulated in DOR mice. HSYC could promote the expression of AKT1 and ESR1 and inhibit the expression of P53 and IL6. These are consistent with previous reports. These results were consistent with the results predicted by network pharmacology and molecular docking.in vitro experiments, other possible pathways should be performed through in vivo and in vitro experiments in the future.As with any study, this study did have some limitations. First, due to public databases being updated in real-time, this study can only partially elucidate the molecular mechanism by which HSYC improves ovarian reserve. Second, only four hub genes were verified by molecular docking, and Taken together, HSYC may ameliorate ovarian reserve against CTX-induced DOR via the regulation of multiple targets. Our study further suggested that combined network pharmacology prediction and experimental validation study may offer a valuable tool to characterize the action mechanism of TCM.


Background: Hepatocellular carcinoma is one of the most common cancers with the characteristics of invasion and high mortality. Current forms of prevention remain severe. Scutellaria barbata is widely used in traditional Chinese medicine treatment of various tumors. This study explored the mechanism of Scutellaria barbata in the treatment of hepatocellular carcinoma by network pharmacology and bioinformatics.Methods: The active ingredients of Scutellaria barbata and potential targets for the treatment of hepatocellular carcinoma were collected by network pharmacology. The protein interaction network was constructed to screen the core targets, and the association between the core targets and diseases was further verified by bioinformatics methods. Finally, the active ingredients corresponding to the targets closely related to the disease were screened for AMDE characteristics analysis. Molecular docking of drug-like ingredients with corresponding targets was performed. We used CCK-8 kit to determine the effect of active ingredients on cell proliferation.Results: 29 candidate active ingredients and 461 related targets of Scutellaria barbata were screened. A total of 8238 potential therapeutic targets for hepatocellular carcinoma were indentified. Finally, 373 potential targets for the treatment of HCC were obtained. The active ingredients: wogonin, Rhamnazin, eriodictyol, quercetin, baicalein, and luteolin, etc. The core targets were CDK1, CDK4, SRC, and E2F1. A total of 3056 GO enrichment entries were obtained, and 180 enrichment results were obtained by KEGG pathway analysis. Genes were mainly enriched in PI3K-Akt signaling pathway, IL-17 signaling pathway, TNF signaling pathway, apoptosis pathway, and hepatocellular carcinoma pathway. Molecular docking results showed that the screened compounds had strong binding ability with the corresponding target proteins. CCK8 assays showed that Rhamnazin and Luteolin suppressed the proliferation of HCC cells significantly compared with controls.Conclusion: This study revealed that the mechanism of Scutellaria barbata in the treatment of hepatocellular carcinoma may be that the active ingredients inhibit the expression of core genes and block the PI3K-AKT signaling pathway to inhibit the proliferation, and migration and induce apoptosis of cancer cells. Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death in many parts of the world . Hepatecin vivo animal experiments and in vitro drug screening, but these methods are usually costly and labor-intensive. Over the past decade, the explosion of omics data has created opportunities for computational anticancer drug prediction and improved the efficiency of drug discovery . According to the pharmacokinetic parameters: oral bioavailability (OB) ≥ 30%, and drug-like index (DL) ≥ 0.18, the eligible candidate active ingredients were screened, and the related targets of the candidate active ingredients screened in the TCMSP database were collected. The selected candidate active ingredients were retrieved in Pubchem database (https://pubchem. ncbi. nlm. nih. gov/), and the SDF file with 2D structure was downloaded. Then, the Swiss Target Prediction database (http://www.swisstargetprediction.ch/) was imported to predict the target of active ingredients. Finally, the targets collected from the Swiss Target Prediction and TCMSP databases were merged, and the repetitive values were removed as the targets of candidate active ingredients of Scutellaria barbata.All chemical constituents of https://db.idrblab.org/ttd), GeneCards database (https://www.genecards.org/), Disgenet database (http://www.disgenet.org/web/DisGeNET/menu/home), and OMIM database (https://www.omim.org/), and all selected potential therapeutic targets for hepatocellular carcinoma were combined to remove duplicate values as targets for hepatocellular carcinoma.The keywords “Hepatocellular carcinoma” and “HCC” were searched in the TTD database , the protein type was set as “Homo sapiens”, the confidence was set to >0.9, and the other parameters remained the default, to obtain the protein interaction relationship. The results were imported into Cytoscape 3.9.0 software, and the key genes of TOP10 were screened by the “MCC” algorithm with high prediction accuracy for key proteins in the PPI network in the “CytoHubba” plug-in. The GEPIA database (http://gepia.cancer-pku.cn/) was used to verify the mRNA expression levels of the selected key genes in HCC and normal liver tissues, and the total survival time of 10 key genes overexpression in HCC patients was analyzed, with 50% up and downregulated of gene expression as the analysis standard. In this study, HCC patients were divided into two groups according to the median expression of core genes. Log-rank p < 0.05 was considered statistically significant. The protein expression levels of key genes in normal liver tissues and HCC tissues were detected by The Human Protein Atlas (HPA) database (https://www.proteinatlas.org/). Finally, the mRNA and protein expression levels in HCC and normal liver tissues were different, and the key genes with statistically significant overall survival in HCC patients were used as the core genes for further analysis.To clarify the interaction between active ingredients and targets. The target of p < 0.05 was used as the screening index .The gene information of potential targets for drug treatment of hepatocellular carcinoma was analyzed using the “Clusterprofiler” package of R software for GO functional enrichment and KEGG pathway analysis. Scutellaria barbata is orally taken after decoction in traditional clinical use. Therefore, combined with clinical application, the ingredients absorbed by oral administration are the effective ingredients of Scutellaria barbata in the treatment of hepatocellular carcinoma. Ideal oral drug molecules should conform to Lipinski’s five rules of physical and chemical properties; which predicts the drug similarity of a compound with certain biological activity. According to Lipinski’s five rules, drug-like compounds used for oral administration should exhibit the following characteristics: MW ≤ 500; mlogP ≤4.5; number of rotatable bonds ≤5; hydrogen bond acceptors <10; and hydrogen bond donor <5; The Swiss ADME network service platform (http://www.swissadme.ch/)) was used to analyze the ADME characteristics of TCM ingredients interacting with the core target genes according to the ingredient-target interaction information in the “Targets Information” in the “Related Targets” section of TCMSP database. Because the expected action site of the screened compounds is the liver, it is hoped that the active ingredients should not penetrate the blood-brain barrier (BBB permeant) to reduce the adverse reactions caused by the later development of active ingredients. Finally, compounds conforming to the Lipinski's five rules and not penetrating the BBB were selected for further molecular docking with the interacting target proteins.There are many compounds in traditional Chinese medicine, and https://www.rcsb.org/) database and saved it in PDB format. Water molecules and small molecular ligands were removed in PyMOL2.5.2 software. The MGLTools 1.5.7 support tool of Audock vina software is used to hydrogenate the corresponding, complete the selection of receptors, and set the docking active center (including the entire protein molecule) through the grid box function in the software. Finally, the docking was completed by vina molecular docking function to verify the screening results of network pharmacology. The smaller the molecular binding energy (affinity) is, the better the binding activity between the protein and core active ingredients.The 3D structure of the compound was downloaded from the Pubchem database, and Chem3D2019 software was used for energy optimization to obtain the 3D structure of the compound, which was stored in mol2 format. The 3D crystal structure of the target protein with resolution below 2.5 Å was download from the PDB  containing 10% fetal bovine serum (FBS), streptomycin (100 ng/ml), and penicillin (100 U/ml), cells and culture medium from Zhong Qiao Xin Zhou Biotechnology. The parameters of the incubator were 5% CO2 and 37°C.Rhamnazin (CMEC) and Luteolin (CMEC) dissolution with DMSO. When used, diluted with complete medium to a specific concentration (DMSO <0.05%) drug-containing solution.Cells were inoculated onto 96-well plates at 1 × 105/ml (100ul/well) and cultured overnight. CCK-8 kit (proteinbio) was used to cell viability detection. Cells treated with drug-containing medium for 24H. Remove the original medium and wash twice with 100ul PBS per well. Add 100ul complete medium (containing 10ul CCK-8 kit) to each well), Incubation of 2H, then use of enzyme calibration (Biotek synergy HI) detection OD value at 450 nm wavelength.p < 0.05 was considered statistically significant.The data are presented as the means ± standard deviations (SD). GraphPad Prism software was used to draw graphs. The comparison among groups were tested using one-way analysis of variance (ANOVA). The Student’s t-test was used to analyze the differences between two groups. Scutellaria barbata were screened from the TCMSP database  ≥ 30% and drug-like (DL) ≥ 0.18, 29 active ingredients of database . A totalBy using “hepatocellular carcinoma” and “HCC” as keywords, 53 targets were collected in the TTD database, 7917 targets in the GeneCards database, 559 targets in the Disgenet database and 236 targets in the OMIM database. After the repetition was removed, 8238 potential therapeutic targets for HCC were obtained.A total of 372 potential therapeutic targets for hepatocellular carcinoma were obtained by crossing the targets of active ingredients and potential therapeutic targets of hepatocellular carcinoma. Cytoscape software was used to construct the “Chinese medicine-ingredient—target-disease” regulatory network . The conp < 0.05) , and the results were imported into Cytoscape3.9.0 software. The key genes of TOP10 were obtained by the MCC′ algorithm of′ Cytohubba′ plug-in . GEPIA d < 0.05) . Combinep values of biological processes, cell components and molecular functions in GO enrichment analysis and the p values of KEGG enrichment analysis were the highest based on analysis using R language software , cell components , and molecular functions . The main enriched biological processes included response to xenobiotic stimuli, wound healing, response to oxidative stress; the main enriched cell components were plasma membrane raft, vesicle lumen, organelle outer membrane. The main enriched molecular functions included nuclear receptor activity, ligand-activated transcription factor activity, transmembrane receptor protein tyrosine kinase activity, etc. The KEGG pathway analysis was enriched to 180 enrichment results. The results of KEGG pathway enrichment analysis showed that genes were mainly enriched in the PI3K-Akt signaling pathway, IL-17 signaling pathway, TNF signaling pathway, apoptosis and other pathways in addition to various cancer pathways. The 10 pathways with the smallest software .Scutellaria barbata was found. There were five ingredients that only interacted with SRC and one ingredient that only interacted with CDK1. There are 17 ingredients interacted with core gene targets in Scutellaria barbata. The Swiss ADME network service platform was used to screen 12 compounds that conformed to the five rules of Lipinski and did not penetrate the BBB (According to the “ingredient-target” interaction information of “Targets Information” in the “Related Targets” section of the TCMSP database, one ingredient interacting with CDK1, SRC, and E2F1, nine ingredients interacting with CDK1 and SRC, and one ingredient interacting with CDK4 and CDK1 in  the BBB .AutoDock Vina software was used for molecular docking of the core gene target protein and the interaction of traditional Chinese medicine ingredients. It is generally believed that the lower the binding energy of the ligand and receptor, the better the binding force. When the binding energy is less than 0 kcal/mol, the two can spontaneously combine. When the binding energy is less than −5 kcal/mol, the binding force is better. When the binding energy is less than −7 kcal/mol, the binding configuration has strong activity. According to the molecular docking results, the binding energy of all the core active ingredients with the core target protein was ≤ −5 kcal/mol . The docAfter adding different concentrations of drugs to treat HepG2 cells and Hep3B cells, CCK-8 kit was used to detect cell activity. The experimental results showed that specific concentrations of Rhamnazin and Luteolin could inhibit the proliferation of HepG2 cells and Hep3B cells .Scutellaria barbata in the treatment of hepatocellular carcinoma were preliminarily screened. Through the construction of a protein interaction network and screening and verification of core targets, it was found that CDK1, SRC, CDK4 and E2F1 mRNA and protein expression levels were overexpressed in liver cancer tissues compared with normal liver tissues. Survival analysis showed that these genes were highly expressed in liver cancer patients and correlated with poor prognosis. According to previous studies, miR-203 inhibits the proliferation and migration of lung cancer cells and promotes apoptosis of lung cancer cells by targeting SRC expression (In vitro and in vivo studies, cell cycle progression was blocked by downregulate CDK1 in liver cancer cells in the G2-M phase (29 potential active ingredients and 372 potential targets of pression . CDK1 ispression ; it is e-M phase . Metform-M phase . miR-127-M phase . CDK4 sp-M phase . By knoc-M phase . E2F1 is-M phase . The abn-M phase . MicroRN-M phase . Celastr-M phase .The screened standard active compounds were confirmed in previous studies, and the combination of wogonin and sorafenib effectively killed human liver cancer cells by enhancing cell apoptosis and inhibiting autophagy . wogoninScutellaria barbata may play a role in the treatment of HCC through the following aspects.The above studies demonstrate that the core genes are related to the occurrence and progression of cancer, active ingredients have anticancer effects, and some active ingredients have been shown to inhibit the proliferation and migration of liver cancer cells and induce apoptosis, which demonstrates the scientific nature of this study. Therefore, combined with the analysis results, it is considered that Scutellaria barbata in the treatment of hepatocellular carcinoma may be through Rhamnazin and Hispidulin etc. Active ingredients such as baicalein inhibit the expression of core genes, regulate the cell cycle, inhibit the proliferation and migration of cancer cells, and induce apoptosis.Previous studies have shown that inhibiting the expression of core target genes can inhibit the proliferation of cancer cells and induce apoptosis of cancer cells. According to the results of survival analysis, the high expression of these genes in hepatocellular carcinoma is related to poor prognosis. The molecular docking results of the above core targets and their corresponding active ingredients show good active configurations. Combined with previous studies on the role of core genes in the occurrence and progression of cancer and the pharmacological effects of active ingredients, it is speculated that the mechanism of Scutellaria barbata may block the PI3K-Akt signaling pathway by active components such as wogonin, hispidulin and eriodictyol etc to inhibit the proliferation and migration of cancer cells, induce apoptosis of cancer cells, and play a role in the treatment of hepatocellular carcinoma.The PI3K-Akt signaling pathway plays an important role in the results of KEGG enrichment analysis. The PI3K-Akt signaling pathway is closely related to cell proliferation, cell cycle progression and apoptosis . InhibitScutellaria barbata may block the PI3K-Akt signaling pathway by active ingredients such as wogonin, hispidulin and hriodictyol etc to inhibit the proliferation and migration of cancer cells, induce apoptosis of cancer cells, and play a role in the treatment of hepatocellular carcinoma.Combined with the results of network pharmacology analysis and previous studies, it is speculated that Scutellaria barbata, used mainly for oral administration for the treatment of hepatocellular carcinoma in this study, the AMDE characteristics of the active ingredients of Scutellaria barbata were analyzed, and were in accordance with the principle of oral administration and not through the BBB and were screened to reduce the adverse reactions after later development. Finally, the further screened genes and the required active ingredients were combined with network pharmacology analysis and previous research results to explore the mechanism of Scutellaria barbata in the treatment of hepatocellular carcinoma and improve the accuracy and scientificity of the analysis results. The results of this study are mainly based on the network pharmacology method. Currently, some active ingredients screened have no pharmacological effects and are limited to the database, so there may be some differences in the actual action process of Scutellaria barbata.In this study, through the network pharmacology method to determine the core target, further screening in liver cancer tissue and normal liver tissue showed that the mRNA and protein expression levels were different, and abnormal gene expression and poor prognosis-related genes were identified in hepatocellular carcinoma patients for follow-up analysis. In addition to the clinical characteristics of Scutellaria barbata in clinical practice, the network pharmacology method and molecular docking technology were used to analyze the mechanism of active ingredients in accordance with oral drug-like rules in the treatment of hepatocellular carcinoma. A preliminary theoretical study was carried out to explore the development of active ingredients of Scutellaria barbata for HCC treatment. We expeect to provide more options for the clinical treatment of hepatocellular carcinoma.In this study, combined with the practical application of


Modified Sanmiaosan is an effective cure in the treatment of ulcerative colitis, but its mechanisms of action remain unclear. This study revealed the pharmacological mechanisms of Modified Sanmiaosan acting on ulcerative colitis through a pharmacology approach.  The active compounds and the targets of Modified Sanmiaosan were selected from the Traditional Chinese Medicine Systems Pharmacology database according to the absorption and metabolism. The UC-related therapeutic targets were collected from the PharmGKB database, the GeneCards database, the GADA database, and the OMIM database. The networks of “drug-component-target-disease” and “herbal-component-target” were constructed by the Cytoscape software. Protein–protein interaction network was generated by the STRING database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed by the R software. Molecular docking technology was used to identify the affinity and activity between active compounds and targets.  The 80 effective ingredients of MSM were collected. A total of 5180 UC-related genes and the 153 key targets of MSM and UC-related were obtained. JUN, Akt1, and MAPK1 were identified as the “hub targets” involved in the effects of Modified Sanmiaosan on ulcerative colitis. Hub targets were mainly involved in inflammatory response and oxidative stress. As the results of GO analysis, biological processes such as DNA-binding transcription and RNA polymerization may participate in the treatment process; KEGG pathway analysis showed that hub targets were mainly involved in IL-17 signal pathway and TNF signal pathway of ulcerative colitis. The high affinity and activity of the active compounds and targets were verified through molecular docking.  These findings demonstrate the active ingredients in Modified Sanmiaosan reduce inflammatory response by TNF and IL-17 signaling pathways to treat ulcerative colitis. Anti-inflammation and immune regulation may be the main mechanism of Modified Sanmiaosan in the treatment of ulcerative colitis. This study not only provide new insights into the development of a natural therapy for the prevention and treatment of ulcerative colitis but also proves a feasible method for discovering potential activated compounds from Chinese herbs. Ulcerative colitis (UC) is an idiopathic, chronic inflammatory disorder of the colonic mucosa, which starts in the rectum and progresses proximally in a continuous manner through the entire colon. It is characterized by chronic mucosal inflammation that recurs for life. The most prominent symptoms include hematochezia, diarrhea, and abdominal pain. Moreover, several patients with UC are affected by extraintestinal complications. The incidence and burden of UC are on the rise globally. The annual incidence of UC is between 8 and 14 cases per 100,000 persons in the Western population, and the prevalence is increasing in Asia . First-lAtractylodes lancea, Phellodendri chinensis, and Radix Achyranthis bidentatae [In TCM, clearing heat and removing dampness is a key method for UC treatment . Sanmiaodentatae .Bletilla striata and Agrimonia eupatoria were added to form Modified Sanmiaosan (MSM). MSM has been reported to have achieved a good curative effect in the treatment of UC [Atractylodes lancea has proven regulation of the nuclear factor κB (NF-κB) signaling pathway to reduce lipopolysaccharide-induced inflammatory injury of human colonic epithelial cells [Agrimonia eupatoria ameliorates dextran sodium sulfate-induced colitis through NF-κB signaling pathway [Bletilla striata effectively inhibited the release of intestinal inflammatory cytokines such as IL-6, TNF-α, and IL-1β and attenuated dysfunction of the intestinal barrier in UC mouse model induced by 3% dextran sodium sulfate [Based on the formulation of Sanmiaosan, nt of UC . Atractyal cells . Agrimon pathway . Bletill sulfate . MSM is Network pharmacology is an innovative way to explore new drug targets, which can identify active drug ingredients by investigating the relationship between drug components and disease targets. Based on the intersection of drug targets with key genes, the drug-target network can be constructed, and the potential mechanism can be explored. This method can be helpful to discern the pharmacological action and mechanism of TCM . In thisAtractylodes lancea, Phellodendri chinensis, Radix Achyranthis bidentatae, Bletilla striata, Agrimonia eupatoria) were searched in the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database (https://old.tcmsp-e.com/tcmsp.php/) by the ADME model. The ingredients with OB ≥ 30% and DL ≥ 0.18 were selected. OB represents the ability of a compound to circulate in the body after oral administration. DL is an indicator for determining the similarity of physicochemical properties of a compound with conventional drugs. The target names of all compounds were transformed into UniProt IDs using the UniProt database (http://www.uniprot.org/).The components of MSM (https://www.genecards.org), the OMIM database (https://omim.org), the PharmGKB database (https://www.pharmgkb.org), and the Genetic Association Database  [By searching the keywords “Ulcerative colitis”, the targets related to UC were collected from the GeneCards database (nih.gov) .The Venn diagram generation software Venny 2.1 was used to identify potential key targets of MSM in treating UC.http://www.cytoscape.org/). The nodes in the network represent herbs, chemicals, and potential targets; the edges indicate interactions between them. In the network, the quantity of each edge was defined as “degree.”The “drug-component-target-disease” and “herbal-component-target” networks were constructed for visualization using Cytoscape version 3.6.1 (https://string-db.org/) database to build the PPI network model. The minimum required interaction score was set as “medium confidence = 0.9,” while the other parameters remain as default. In the network, the size of the nodes represents the degree size. The higher the degree, the better the correlation between the proteins. Targets with scores in the top 30 were selected as core targets [Key targets were imported into the STRING  enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of target genes were performed using the R software (version 3.6.0); the r graphs .Download the SDF format of 3D structure of kaempferol, luteolin, and quercetin in PubChem, and then import it into ChemDraw 3D. Use the MM 2 module to minimize the energy, obtain the optimal conformation of the lowest energy, and save it as a mol 2 file. MAPK1, AKT1, and JUN were retrieved from the PDB database to obtain the protein structure; their PDB IDs are 6slg, 7nh5, and 1a02. Then, PyMOL is used for visualization. MGTools 1.5.6 is used for water removal, hydrogenation, charge calculation, and nonpolar hydrogen combination. Then, use AutoDock Vina 1.1 2 for docking the ligand with the receptor .Atractylodes lancea ingredients, 37 Phellodendri chinensis ingredients, 20 Radix Achyranthis bidentatae ingredients, 9 Bletilla striata ingredients, and 5 Agrimonia eupatoria ingredients  to construct the PPI network. In p value: < 1.0e-16), which reflects the complexity of the disease mechanism and the characteristics of TCM for disease treatment. Gene targets with a high degree in the PPI network include JUN, Akt1, and mitogen-activated protein kinase-1 (MAPK1); these genes were considered “hub targets” involved in the effects of MSM on UC  in the PPI network and the top three active ingredients  in the network were selected for molecular docking. Molecular docking showed that quercetin, kaempferol, and luteolin can closely bind three targets . The PyMOL software was used for visual analysis of molecular docking. The 3D structures of JUN, Akt1, and MAPK1 are shown in Figures In this study, network pharmacology methods were used to explore the potential targets and mechanisms of MSM in treating UC. A total of 80 active compounds were identified using the TCMSP database (OB ≥ 30% and DL ≥ 0.18) and 5180 UC-related targets were obtained from the PharmGKB database, the GeneCards database, the GADA database, and the OMIM database. The MSM targets and UC targets were intersected by using a Venn tool to obtain 153 targets, identified as “key targets” for PPI network construction and GO\KEGG analysis. The PPI network was constructed by uploading these 153 key targets to the STRING database. Targets with high degree value in the PPI network included JUN, Akt1, and MAPK1; these genes were considered “hub targets” involved in the effects of MSM on UC. GO and KEGG enrichment analyses of the 153 key targets revealed that IL-17 and TNF signaling pathways were the most enriched pathways. Cytoscape was used to generate the network showing the relationship between drug components and targets containing 80 active ingredients and 153 key targets. The treatment of diseases by TCM was mainly achieved by the synergistic action of many active compounds on multiple targets. Network pharmacology was used to reveal the underlying mechanisms with the above characteristics.β-sitosterol, and baicalein. This finding suggests that these compounds may be important for the therapeutic role of MSM in UC and warrant further investigation. As reported, quercetin is a polyphenolic flavonoid compound whose antioxidant activity has been investigated in numerous studies. It is effective in treating and preventing various diseases due to its effect on glutathione, enzyme activity, signal transduction pathways, and reactive oxygen species (ROS) production [β, IL-6, IL-17, IL-22, and TNF-α) and regulates various inflammation-related signaling pathways [β-Sitosterol is a bioactive compound found in the plant cell membrane, with a chemical structure similar to cholesterol. β-Sitosterol is known to possess various benefit actions such as immunomodulatory effect, anticancer, and anti-inflammatory activity [κB and STAT3 signaling pathways and mitigating radiation-induced enteritis by improving endothelial dysfunction, thereby preventing the development of colitis-associated cancer [As previously reported, 153 active compounds of MSM in the treatment of UC were obtained. Through network analysis, the most promising compounds were identified as quercetin, kaempferol, luteolin, oduction . Kaempfeoduction . In a nooduction . Luteolioduction . Moreovepathways . β-Sitosactivity . Variousd cancer , 22.PPI network identified JUN, Akt1, and MAPK1 as core targets. These targets involved in inflammation, apoptosis, and other processes may play an important role in the therapeutic effect of MSM in UC. JUN is a major component of the heterodimeric transcription factor AP-1 and a critical driver of cell proliferation and apoptosis . AdditioWe performed GO enrichment analysis and KEGG enrichment analysis of the 153 potential targets. GO analysis results showed that biological processes such as DNA-binding transcription activator activity, RNA polymerase II-specific cytokine receptor binding, transcription factor activity, RNA polymerase II proximal, promotor sequence-specific DNA binding, protein serine/threonine kinase activity, receptor ligand activity, and cytokine activity may play a role in the treatment of UC. KEGG pathway analysis showed that these targets were mainly involved in IL-17 and TNF signaling pathways of UC.The pathogenesis of UC consists of immuno-inflammatory pathways, including the epithelial barrier, commensal microflora, dysregulation of immunological responses, and leukocyte recruitment.α (TNF-α), IL-1, IL-6, IL-9, IL-13, and IL-33) play a significant role in upregulation, while anti-inflammatory cytokines  play a significant role in downregulation of disease progression. The imbalance of proinflammatory and anti-inflammatory cytokines is a key factor in the immune dysfunction of intestinal mucosal [Proinflammatory cytokines  in patients with UC is 2.4-fold higher compared to the general population .However, this study has limitations. The mechanism of MSM in the treatment of UC through the IL-17 and TNF signaling pathways needs to be further explored. In addition, the mechanism of MSM determined in this study needs to be further analyzed and verified through experimental research. We will further use molecular biology methods to investigate the effects and molecular mechanism of MSM in treating UC and conduct random clinical trials to identify the effectiveness of MSM in patients with UC.