Study On The Mechanism Of Alisthenol B Acetate Prevents Postmenopausal Atherosclerosis By Regulating Gut Microbiota And Intestinal Estrogen Receptor

Objective:To explore the correlation between the down-regulation of estrogen receptors,the changes of gut microbiota and vascular damage in postmenopausal women,and to explore whether AlisolB-23-acetate(AB23 A)can prevent postmenopausal atherosclerosis by regulating the gut microbiota and intestinal estrogen receptors and to explore the possible mechanism.Methods:Chapter 1:Aortic vessels were collected from premenopausal(<45 years old)and postmenopausal(>55 years old)women with aortic replacement surgery,and feces were collected from premenopausal(<45 years old)normal women and postmenopausal(>55 years old)women with coronary heart disease.16SrRNA gene high-throughput sequencing was used to analyze the gut microbiota in female feces,HE staining was used to observe the pathological changes of female aortic vessels,transmission electron microscopy was used to observe the ultrastructural changes in female blood vessels,and immunohistochemical staining was used to detect NLRP3,Caspase-1,IL-1β,ERα,ERβ,GPER SRC,PI3K and p-AKT expression in female blood vessels.The correlation between aortic inflammation,estrogen receptors expression and intestinal flora composition in premenopausal and postmenopausal women were analyzed.Chapter 2:Using LDLR-/-mice combined with a high-fat diet to replicate the atherosclerosis model.Bilateral ovariectomy was performed on atherosclerosis model mice to replicate the postmenopausal atherosclerosis model(model group).Meanwhile,C57BL/6 mice with normal diet or high fat diet groups and LDLR-/-mice with normal diet group were set.The AB23A treatment experiment,the fecal bacteria transplantation experiment,and the AB23A and antibiotics combined application experiment were conducted in postmenopausal atherosclerosis model mice.At the end of the experimental period,biochemical kits were used to detect the levels of TC,TG,LDL-c and HDL-c in the serum of mice to evaluate blood lipids,ELISA kits were used to detect the levels of IL-1β,1L-6 and TNF-α in the serum of mice to evaluate blood inflammation,oil red O staining was used to detect the positive area in aorta and in aortic root slices of mice to evaluate atherosclerotic injury,masson staining was used to detect collagen in aortic root slices of mice to evaluate fiber cap area,immunohistochemical staining was used to detect the expression levels of NLRP3 and IL-1β in aortic root slices and colon of mice to evaluate tissue inflammation,HE staining was used to detect colon tissue morphology of mice to evaluate histopathological damage,immunohistofluorescence staining was used to detect the expression levels of Claudin-1,occluduin and ZO-1 in colon of mice to evaluate the state of tight junctions,and western blotting was used to detect the protein expression levels of NLRP3,IL-1β,Claudin-1,Occludin and ZO-1 in colon of mice to further confirm the inflammation and the state of tight junctions.16SrRNA gene high-throughput sequencing was used to analyze the gut microbiota in mice feces.The correlations between the atherosclerosis-related symptoms,gut microbiota and tight junctions were analyzed in the mice in model mouse AB23A treated experiment.Chapter 3:The AB23A treatment experiment was conducted in postmenopausal atherosclerosis model mice.At the end of the experimental period,ELISA kit was used to detect the estrogen level in the serum of mice,immunohistochemical staining was used to detect the expression levels of ERα,ERβ,GPER and SRC in the colon of mice,immunohistochemical staining was used to detect the expression levels of PI3K and p-AKT in the colon of mice,and western blotting was used to detect the protein expression levels of ER,ERβ,GPER,SRC,PI3K,AKT and p-AKT in colon tissue of mice.Using cholesterol-loaded Caco-2 cells to replicate the intestinal epithelial cell injury model.Using AB23A and combining with ERa inhibitor MPP,ERβ inhibitor PHTPP,GPER Inhibitor G-15,PI3K inhibitor LY294002 or AKT inhibitor 10-DEBC to inhibit the corresponding protein or,combining with ERa siRNA,ERβsiRNA,GPER siRNA or SRC siRNA to silence the expression of corresponding gene,to treated Caco-2 cells.Different controls and groups were set in different experiments.MTT method was used to detect cell viability,oil red O staining was used to evaluate lipid accumulation,immunofluorescence method was used to detect SRC,ERα,ERβ,GPER,PI3K,p-AKT,Claudin-1,Occludin and ZO-1 expressed alone or together in cells,western blotting was used to detect the protein expression of SRC,ERα,ERβ,GPER,NLRP3,IL-β,PI3K,p-AKT,Claudin-1,Occludin,and ZO-1 in cells,and immunoprecipitation method was used to detect the interaction between GPER and SRC.Results:Chapter 1:Compared with premenopausal women,postmenopausal women showed more severe damage to endothelium and smooth muscle of the blood vessels,and showed increased expression of NLRP3,Caspase-1,and IL-1β,while showed increased expression of ERα,ERβ,GPER,SRC,PI3K and AKT.Compared with normal premenopausal women,the abundance and diversity of the gut microbiota in the feces of postmenopausal women with coronary heart disease were significantly decreased,and the relative abundance of species at a specific level changed significantly.For instance,at the phylum level,the relative levels of Firmicutes,Tenericutes,and Fusobacteria were significantly reduced in the feces of postmenopausal women,while the relative levels of Verrucomicrobia and Bacteroidetes were significantly increased;at the genus level,the relative levels of Clostridium and Faecalibacterium were significantly decreased,while the relative levels of Shigella,Lactobacillus and Bacteroides were significantly increased.Correlation analysis showed that there was a significant correlation between the aortic inflammation,estrogen receptor expression and gut microbiota composition in women before and after menopause.Chapter 2:Compared with atherosclerosis model mice,the levels of lipids,serum inflammation and atherosclerotic damage in postmenopausal atherosclerosis model mice were further increased,as well as colon inflammation,tight junction damage,and pathological changes.The treatment of AB23A significantly reduced the blood lipids,serum inflammation and atherosclerotic damage in model mice,and significantly reduced colon inflammation,tight junction damage and pathological changes.Compared with atherosclerosis model mice,the abundance and diversity of the gut microbiota were significantly decreased,and the relative abundance of species at a specific level significantly changed in postmenopausal atherosclerosis model mice.The treatment of AB23A significantly increased the abundance and diversity of the gut microbiota and reversed the changing of the relative abundance of species at a specific level of model mice.For instance,the treatment of AB23 A significantly increased the relative phylum level of Firmicutes,which were significantly decreased in the feces of model mice,and significantly decreased the relative phylum level of Verrucomicrobia,which were significantly increased in the feces of model mice;at the genus level,the treatment of AB23A significantly decreased the relative level of Bacteroides and Akkermansia,which were significantly increased in the feces of model mice.Correlation analysis showed that there was a significant correlation between atherosclerosis-related symptoms,gut microbiota composition and tight junctions in mice.Fecal microbiota transplantation resulted in the abundance and diversity and the relative abundance of species at a specific level of the gut microbiota of model mice approaching the donor mice.The abundance and diversity of the gut microbiota of the recipient mice receiving the feces from the model group were significantly lower than that of the recipient mice receiving the feces from the AB23A treated group,and the same changes could be found in the relative abundance of species at a specific level.The changing of the composition of gut microbiota is sufficient to cause changing in blood lipids,serum inflammation,atherosclerosis-related symptoms,colon inflammation,tight junction damage and pathological changes.Compared with the recipient mice receiving the feces from the model group,blood lipids,serum inflammation,atherosclerosis-related symptoms,colon inflammation,tight junction damage and pathological changes were significantly reduced in mice receiving the feces from the AB23 A treated group.Antibiotics significantly inhibited the abundance and diversity of gut microbiota of mice in each group.Antibiotics treatment was significantly reduced the blood lipids,serum inflammation,atherosclerosis-related symptoms,colon inflammation,tight junction damage and pathological changes in model group mice.AB23A treatment of antibiotic-administered model mice still promoted the abundance and diversity of gut microbiota;the application of antibiotics enhanced the effects of AB23A on blood lipids,serum inflammation and atherosclerosis in model mice and enhanced the reducing effect in colon inflammation,tight junction damage and pathological changes.Chapter 3:Bilateral ovariectomy resulted in significant reduction in estrogen receptors,estrogen level and SRC expression in LDLR-/-mice with a high-fat diet.At the same time,PI3K/AKT signaling was also significantly reduced.AB23A did not affect the estrogen level in model mice,but significantly up-regulated the expression of colon estrogen receptors and SRC expression,and up-regulated PI3K/AKT signaling in model mice.Cholesterol load leads to cholesterol accumulation,increased inflammation,and tight junction damage in Caco-2 cells;AB23A reduced cholesterol accumulation,inflammation and tight junction damage in cholesterol treated Caco-2 cells,and up-regulated the expression of SRC and enhanced PI3K/AKT signaling.When the inhibitor of ERa or ERβ was used or the gene of ERa or ERβ was silenced,it does not affect the inhibition effect of AB23A in cholesterol accumulation and inflammation,does not affect the the protective effect of AB23A in tight junctions,and does not affect the the up-regulation effect of AB23A in GPER and SRC expression and the activation of PI3K/AKT signal in cholesterol treated Caco-2 cells.When GPER inhibitor or GPER siRNA were used,the inhibitory effect of AB23A on cholesterol accumulation and inflammation and the protective effect of tight junctions in cholesterol treated Caco-2 cells were cancelled,at the same time,the upregulation of SRC and the activation of PI3K/AKT signaling by AB23 A were also cancelled.When the SRC gene is silenced,the inhibitory effect of AB23A on cholesterol accumulation and inflammation and the protective effect of tight junctions in cholesterol treated Caco-2 cells were cancelled,and the activation of PI3K/AKT signals by AB23A was also cancelled,but it does not affect the up-regulation of estrogen receptor expression by AB23 A in cholesterol treated Caco-2 cells.When PI3K/AKT signal is blocked,the inhibitory effect of AB23A on cholesterol accumulation and inflammation and the protective effect of tight junctions in cholesterol treated Caco-2 cells were cancelled,but the inhibition of PI3K/AKT signal does not affect the up-regulation effect on estrogen receptor and SRC by AB23A.Conclusion:1.Increased vascular inflammation and damage in postmenopausal women have a significant correlation with the decrease of estrogen receptor signal and the disorder of gut microbiota.2.AB23A reduces intestinal inflammation and protects the intestinal barrier in postmenopausal LDLR-/-mice by regulating the gut microbiota,thereby reducing blood lipids,serum inflammation,and vascular inflammation,and inhibiting atherosclerosis development in postmenopausal mice.3.AB23A up-regulates the expression of SRC by activating GPER and downstream PI3K/AKT signals to inhibit colonic epithelial inflammation and tight junction damage,which are one of the mechanisms of AB23A on inhibiting the development of atherosclerosis in postmenopausal mice.