Multi-target Molecular Mechanism And ER-mediated Signaling Pathway Of Phytoestrogen-like Effect Of Siwu Decoction

Estrogen has a wide range of functions in the human body,which plays important physiological functions in many systems of the human body.Similar to estrogen,phytoestrogen is mainly derived from plants and can act as active ingredients.Up to now,many traditional Chinese medicine prescriptions have been used in gynecological clinics and played important roles.Research on their pharmacological mechanisms has found that multiple systems of the human body have significant physiological functions.Si-WuTang is known as the "First Recipe in Gynecology",which is composed of Danggui(Angelica sinensis),Shudihuang(Rehmannia glutinosa),Baishao(Paeonia lactiflora Pall)and Chuanxiong(Conioselinum anthriscoides)and has the effects of nourishing blood and regulating menstruation.Exiting studies have shown that the effects of Si-Wu-Tang are similar to pseudoestrogen and anti-estrogens.This paper starts with the phytoestrogen effects of Si-Wu-Tang,and discuss its two-way regulation from two aspects.On the one hand,this study uses breast cancer cells to explore the molecular mechanism of the antiestrogen-like effect of Si-Wu-Tang,and uses network pharmacology and molecular docking to screen out the key targets of Si-Wu-Tang for non-triple-negative breast cancer.And then,breast cancer(MCF-7)cells are used for in vitro verification.On the other hand,this study uses female SD rats to explore the molecular mechanism of estrogen-like effect of Si-Wu-Tang,and applied network pharmacology combined with literature research to find the key pathway targets of Si-Wu-Tang-estrogen,and carried out Validated by in vivo experiments.The research is organized as follows:(1)The establishment of target database of Si-Wu-Tang ingredientsThe effective blood components of Si-Wu-Tang are collected from the TCMSP database,including 2 active ingredients of Shudihuang,13 active ingredients of Baishao,7 active ingredients of Chuanxiong and 2 active ingredients of Danggui.A total of 121 main components of Si-Wu-Tang that meet the standards are collected from the ETCM database,including 16 active components of Baishao,53 active components of Chuanxiong,51 active components of Danggui,and 1 active component of Shudihuang.Then,collecting the target information of each component from the TCMSP database.At the same time,the SwissTarget database is used to find targets that match the chemical structure of the components.Integrating the targets of the two databases for use.(2)The collection of targets that are related to non-triple negative breast cancerCollecting the targets that are related to breast cancer from OMIM and GeneCards,and screening related chips from the GEO database for differential analysis and integration.A total of 3131 non-TNBC related genes are obtained.(3)Screening of key targets of "Si-Wu-Tang-Non-Triple Negative Breast Cancer"Integrating the data targets of "Si-Wu-Tang-Non-Triple Negative Breast Cancer",obtaining 79 related genes,and constructing a protein interaction network.The top ten targets with "degree" value are:IL6,AKT1,JUN,PTGS2,MAPK8,CASP3,ESR1,CAT,MMP9 and PPARG.Through further screening through oncomine tumor database and Kaplan-Meier survival analysis website,PTGS2,ESR1,CAT,and PPARG are confirmed as key targets for follow-up verification.(4)Molecular docking verification of the key targets of "Si-Wu-Tang-Non-Triple Negative Breast Cancer"The chemical components of Si-Wu-Tang have good bindings to the targets.The docking score of Myricanone and PPARG is-8.8,the docking score of(+)-catechin and CAT is-7.8,and the docking score of Beta-sitosterol and PTGS2 is-9.2,The docking score of(+)-catechin and ESR1 is-8.0.(5)Verification of Anti-estrogen-like targets of Si-Wu-Tang on MCF-7 cellsAfter the intervention,the proliferation of MCF-7 cells is significantly reduced,and the inhibitory effect of Si-Wu-Tang is the most significant when the concentration of SiWu-Tang is 5 mg/mL.The results of RT-PCR test show that the mRNA expression levels of PTGS2,ESR1 and PPARG increased(P<0.01)and the mRNA expression level of CAT decreased in Si-Wu-Tang groups compared with control groups.Therefore,Si-Wu-Tang can exert an anti-estrogen-like effect on MCF-7 cells by regulating targeting sites such as PTGS2,ESR1,PPARG,and CAT.(6)"Si-Wu-Tang-Estrogen" Related Pathway Mechanism AnalysisAccording to the "Siwu Decoction-estrogen" related targets,KEGG pathway analysis accords with a total of 132 pathways with p-value less than 0.05.Hormonerelated pathways include AGE-RAGE signaling pathway in diabetic complications,Prolactin signaling pathway,Endocrine resistance,Estrogen signaling pathway,and MAPK signaling pathway.Inflammation-related pathways include TNF signaling pathway,NF-kappa B signaling pathway,IL-17 signaling pathway,C-type lectin receptor signaling pathway,Th17 cell differentiation,and Hepatitis C.Immune-related pathways include Toll-like receptor signaling pathway,C-type lectin receptor signaling pathway.The pathways related to apoptosis include Platinum drug resistance,Apoptosis,Apoptosis-multiple species.Nervous system related pathway is Neurotrophin signaling pathway.Metabolism-related pathways include Tryptophan metabolism and Drug metabolism-cytochrome P450.According to the relevant literature,this paper will focuses on exploring the estrogen signal transduction pathway and the MAPK signal transduction pathway.(7)Effects of Si-Wu-Tang on estrogen signal pathway in ovary and uterus of female SD ratsThe WB results of the ovarian tissue showed that the ER? and ER? of the Si-WuTang groups are significantly increased compared with the control groups,which is consistent with the results of the estradiol group and dose-dependent.However,the GPER of Si-Wu-Tang groups is lower than that of control groups,which iss dose-dependent.The results of ovarian immunohistochemistry show that ER?,ER?,and GPER are expressed more obviously in all levels of follicles.Among them,GPER is mainly distributed on the cell membrane,and ER? and ER? are mainly distributed on the nucleus.The RT-PCR results show that the mRNA levels of ER? and ER? in Si-Wu-Tang groups are lower than those in control groups,but the mRNA level of GPER is higher than that in control groups.The expression levels of WB and mRNA measurement are different,suggesting that the regulation of estrogen receptor by Si-Wu-Tang is likely to be post-transcriptional.The results show that when Si-Wu-Tang exerted an effect on ovarian tissue,the regulation of different estrogen receptors was different,and the regulation effect on GPER was more obvious.The WB results of the uterine tissue show that the ER? and ER? of the Si-Wu-Tang groups are significantly increased compared with control groups,which is consistent with the result of the estradiol group and is dose-dependent.The GPER of the Si-Wu-Tang high-dose groups is lower than that of control groups,and the GPER of the Si-Wu-Tang low-dose groups is higher than that of control groups,which is not dose-dependent.It is indicated that the different doses of Si-Wu-Tang have different regulating effects on the expression of GPER.The results of RT-PCR show a low level of ER? mRNA and a high level of ER? mRNA.Compared with control groups,the ER? and GPER mRNA levels of Si-Wu-Tang groups are lower than those of control groups,and the ERa mRNA level of Si-Wu-Tang groups is higher than GPER.It can be seen that the effect of GPER is more obvious by regulating ERa when Si-Wu-Tang exerts an estrogen-like effect on the uterus.(8)Effects of Si-Wu-Tang on MAPK signal pathway in ovary and uterus tissues of SD young ratsThe results of WB and RT-PCR of ovarian and uterine tissues show that the expression level of EGFR is closely related to the dosage of Si-Wu-Tang;Different doses of Si-Wu-Tang have different effects on EGFR;the effects of Si-Wu-Tang is very likely to be post-transcriptional regulation;Si-Wu-Tang can exert an estrogen-like effect on ovarian tissue through the EGFR pathway mediated by estrogen receptor.The downstream targets of the MAPK pathway,MAPKp38,ERK1+2,and MEK1+2,all declined,indicating that Si-Wu-Tang needs to down-regulate the downstream targets of the MAPK pathway to play a role when exerting phytoestrogen effects.ERK1+2 is closely related to cell proliferation and anti-apoptosis.Si-Wu-Tang regulates the expression of this pathway,indicating that Si-Wu-Tang may be related to cell proliferation and apoptosis when it exerts estrogen-like effects in ovarian and uterine tissues.In summary,in order to study the two-way regulatory mechanism of the estrogenlike effect of Si-Wu-Tang,this study evaluates and explores the efficacy and molecular mechanism of Si-Wu-Tang from multiple aspects such as in vivo,in vitro,physiology,pathology,virtual screening,and experimental demonstration.On the one hand,this study uses MCF-7 cells to explore the multi-target molecular mechanism of Si-Wu-Tang after exerting anti-estrogen-like effects,which shows that Si-Wu-Tang can exert an antiestrogen-like effect on MCF-7 cells,and its mechanism is related to the regulation of targets such as PTGS2,ESR1,CAT,and PPARG.On the other hand,this study uses SD young rats to explore the molecular mechanism of Si-Wu-Tang after exerting an estrogenlike effect,which shows that Si-Wu-Tang has an estrogen-like effect on the uterus and ovarian tissues of SD young rats,and plays an effect by regulating the estrogen signaling pathway and MAPK signaling pathway.