Based On "Intestinal Flora-inflammatory Microenvironment-autophagy" To Explore The Mechanism Of Juanduyin Therapy For Endometriosis

Objective: The substance composition of Juandauyin was preliminarily analyzed,and the pharmacodynamic mechanism of Juandauyin was explored based on intestinal flora and autophagy.Methods: In the first chapter,HPLC-MS was used to analyze the main components of Juantayin decoction.Chapter 2 In vivo experiment to explore the relationship between the therapeutic mechanism of Juan-tong-yin and "intestinal flora-inflammatory microenvironment-autophagy".Stool samples from endometriosis and non-endometriosis patients were examined by 16 S r DNA to understand the characteristics of intestinal flora in EMs patients.The EMs rat model was replicated by autologous endometrial transplantation and divided into sham operation group,model group and Juan-tong-yin group.Juan-tong-yin was given gavage intervention to observe the overall efficacy of Juan-tong-yin.16 S r DNA was used to detect intestinal flora among the three groups,and immunohistochemistry was used to detect the expression of colon tissue compact link protein(ZO-1)among the three groups.The contents of endotoxin(LPS)in serum and peritoneal fluid of the three groups were detected by Elisa,and endometrial autophagy in the three groups was observed by transmission electron microscopy.Chapter 3 predicts the mechanism of Juantayin treatment of EMS through network pharmacology.It was verified that the therapeutic mechanism target of Juantaiyin was related to autophagy of endoplasmic reticulum stressed cells.In Chapter 4,cell experiments verified that the action mechanism of Juantaijiu therapy on EMs is related to endoplasmic reticulum stress-autophagy inflammation microenvironment.Endoplasmic reticulum stress cell autophagy marker proteins PERK,LC3 B and Beclin-1 were detected by immunohistochemistry in endometrial samples of EMs patients and non-patients to understand the autophagy levels of EMs patients and verify the pathogenesis of EMs predicted by mesh drugs.The endometrial tissues of EMs patients were obtained for isolation and culture of primary cells in vitro.CCK-8,scratch and Transwell chamber tests were used to observe the therapeutic phenotype of Juan-tong-yin drug-containing serum,and endoplasmic reticulum stress autophagy inhibitor GSK was used for response verification.The autophagy flux of cells in each group was observed by transfection with autophagy double-labeled fluorescent adenovirus.The expressions of PERK,LC3BⅡ/Ⅰ,Beclin-1,ATF4,NLRP3,and IL-18,which are key proteins in the autophagy pathway of endoplasmic reticulum stressed cells and inflammatory microenvironment,were detected by WB and immunofluorescence assay..Results: A total of 241 compounds were identified in Chapter 1,and the top 20 compounds were: Protocatechualdehyde,tryptophan,isopsoralen,4-methoxysalicylic acid,choline,L-phenylalanine,neopsoralen isoflavones,rosinic acid,L-proline,cryptotanshinone,malic acid,Paeoniflorin,dragon blood B,2-isopropyl malic acid,caffeic acid,7,4’-dihydroxyflavone,L-leucine,glycyrrhizin,tanshinone ⅡA,4-methylcatechol.Chapter 2 The proportion and diversity of Firmicutes/Bacteroides in intestinal flora of EMs patients decreased,and most of the different bacteria were inflammatory pathogens.The above phenotypes exist in EMs rats,and Juantian intervention can increase the ratio of firmicutes/Bacteroides and increase the diversity of bacteria,while the differential bacteria decrease in inflammatory pathogens.LPS in serum and peritoneal fluid of EMs rats increased,ZO-1 expression in colon decreased,autophagosome in ectopic endometrial tissue decreased,and the above indexes could be reversed after Juantal intervention,with statistical significance.In Chapter 3,the network pharmacology suggested that the Juan-tong-yin extract had 288 gene targets and 961 modes of action.GO and KEGG analysis predicted that Juan-tong-yin could treat EMs through cell migration,cell adhesion,cell death,immune inflammation and other pathways,in which autophagy and endoplasmic reticulum unfolded protein reaction played an important role.Chapter 4 The protein expressions of PERK and LC3 B in endometrial tissues of EMs patients were significantly decreased.Primary EMs endometrial mesenchymal cells(ESCs)were successfully isolated,and the proliferation,invasion and metastasis of ESCs could be reduced after the intervention of Juantal decoction with drug-containing serum,and the above phenotypes could be restored after pretreatment with GSK.Further detection showed that PERK,LC3BⅡ/Ⅰ,Beclin-1,ATF4 proteins were up-regulated,NLRP3,IL-18 proteins were down-regulated,and the expression of these proteins could be restored after pretreatment with GSK.Conclusion(s): The effective components of Juan-tong-yin decoction are complex and diverse,and it has a multi-pathway and multi-target therapeutic mechanism.Juantayin component exposed in intestine can improve the inflammatory microenvironment by adjusting intestinal flora to treat EMs;The juantayin component absorbed into the blood and reached the target tissue can improve the local inflammatory microenvironment of the tissue by activating autophagy to treat EMs.