| AimsIn the past,we have confirmed that postmenopausal lipid metabolism abnormalities are significantly related to intestinal flora disorders.Liuwei Dihuang Fang(LWDHF)is an effective traditional Chinese medicine for the prevention and treatment of postmenopausal AS.But whether the treatment of LWDHF on postmenopausal AS is related to the regulation of intestinal flora still need to be confirmed.The estrogen level and estrogen receptor function after menopause are decreased,which lead to the decrease of antimicrobial peptide secretion in the intestine.Whether LWDHF can improve the secretion of the antimicrobial peptides by regulating the intestinal estrogen receptor and then regulate the intestinal flora to prevent postmenopausal AS is also worth exploring.Therefore,this study aimed to explore the correlation between intestinal estrogen receptors and intestinal endogenous antimicrobial peptides,intestinal flora and intestinal barrier,postmenopausal AS symptoms and circulating harmful substances to explore the mechanisms of LWDHF in preventing and treating postmenopausal AS.The study may provide experimental support and theoretical basis for the prevention and treatment of postmenopausal AS by LWDHF.MethodsPostmenopausal AS mice treatment:normal diet C57BL/6 mice were used as the control group.LDLR-/-mice with the sham operation were fed a high-fat diet as the sham group.LDLR-/-mice with bilateral ovarian resection were fed a high-fat diet as the postmenopausal AS mice(model group).Also,the postmenopausal AS mice were given 4.5g/kg or 9.0g/kg LWDHF.Estradiol(0.13mg/kg)was used as the positive control drug.The model replication and drug treatment last for 90 days.Fecal microbiota transplantation in postmenopausal AS mice:After the administration,the feces of donor mice(normal control group,model group,estradiol 0.13mg/kg group,LWDHF 9.0g/kg group)were collected every 3 days.After treatment,fecal bacteria were transplanted to recipient mice(postmenopausal AS model).The groups were as follows:normal control group→model group(Control→Model);model group→model group(Model→Model);estradiol 0.13mg/kg group→model group(E2→Model);LWDHF 9.0g/kg group→Model group(HD-LW→Model).Once every three days for 90 days.Intestinal epithelial cell injury model in vitro:Caco-2 cells were stimulated with LPS to replicate the intestinal epithelial cell injury model.LWDHF,estrogen,intestinal antimicrobial peptide IAP inhibitor phenylalanine,ERa inhibitor MPP,ERβ inhibitor PHTPP and ERa siRNA were used to treat Caco-2 cells injured by LPS.Chapter 1:Among the drug treatment or the fecal bacteria transplantation of postmenopausal AS mouse,biochemical kits were used to detect serum levels of TC,TG,LDL-C and HDL-C.ELISA kits were used to detect serum levels of IL-1β,IL-6,TNF-α and MCP-1 levels.Immunohistofluorescence staining was used to detect vascular ICAM-1 expression.Oil red O staining was used to detect the positive area in aorta and in the arterial root section.Masson staining was used to detect the collagen expression in the aortic root section.16S rRNA high-throughput sequencing was used to detect the intestinal flora in the stool.Pearman correlation analysis was used to analyze the correlation among intestinal flora alpha diversity index,AS plaque area,serum lipids and inflammatory factors.It was proposed to confirm whether LWDHF could resist the development of AS in postmenopausal mice by regulating the intestinal flora.Chapter 2:Among the drug treatment or the fecal bacteria transplantation of postmenopausal AS mouse,serum LPS and LBP levels were detected by ELISA to indirectly evaluate the intestinal barrier permeability.The expressions of colonic ZO-1,occludin and Mucin-2 protein were detected by immunohistochemistry or western blotting.The number of colonic goblet cells was detected by alcian blue staining to directly evaluate the intestinal barrier damage.In the intestinal epithelial cell injury model,the expressions of ZO-1 and occludin in the cells were detected by western blotting to evaluate the effect of the drug in vitro.It was proposed to confirm whether LWDHF could improve the intestinal barrier function of postmenopausal AS mice by regulating the intestinal flora.Chapter 3:Among the drug treatment of postmenopausal AS mice,immunofluorescence and western blotting were used to detect the expression of IAP in the duodenum.In vitro intestinal epithelial cell injury model,combined with the application of IAP inhibitor phenylalanine,western blotting was used to detect the expression of IAP,ZO-1,and occludin.It was proposed to confirm whether the protective effect of LWDHF on regulating the intestinal flora and intestinal barrier of postmenopausal AS mice was related to the up-regulation of intestinal antimicrobial peptide expression.Chapter 4:Among the drug of treatment of postmenopausal AS mice,ELISA was used to detect serum estrogen levels.Western blotting,immunohistochemistry and immunofluorescence were used to detect duodenal ERa and ERβ expression.Western blotting was used to detect duodenum intestine KLF4.In intestinal epithelial cell injury model,the treatment was combined with ERa inhibitor MPP,ERβ inhibitor PHTPP,ERa siRNA.Western blotting was used to detect the expression of IAP and KLF4.It was intended to confirm whether LWDHF up-regulated the antimicrobial peptide IAP and its transcription regulator KLF4 through intestinal ERa.ResultsChapter 1:Compared with the model group,E2 and LWDHF could significantly reduce the aorta and aortic root lesions of postmenopausal AS mice,and significantly reduce the level of collagen fibers in the aortic root;E2 and LWDHF significantly reduced the expression of ICAM-1 in the aorta of the model group,and significantly reduced the serum lipid TC,TG,LDL-C and inflammatory factors IL-6,TNF-α,IL-1β and MCP-1 levels in the model group,while increased the serum HDL-C level.The a diversity analysis of intestinal flora showed that E2 and LWDHF significantly increased the richness and diversity of the intestinal flora of the model mice;the β diversity analysis of the intestinal flora showed that the composition of the administration group’s intestinal flora was significantly different from the model group,while was similar with the normal control group.In the phylum and genus,LWDHF changed the species composition of the intestinal flora in the model group,making it tend to be similar as the control group.The correlation analysis between the a diversity index and AS plaque area,blood lipids and inflammatory factor levels showed that the richness and diversity of the intestinal flora were significantly negatively correlated with AS plaque area,serum lipid TC,TG,LDL-C and inflammatory factors IL-6,TNF-α,IL-1β and MCP-1 levels;AS plaque area was significantly positively correlated with serum lipid TC,TG,LDL-C and inflammatory factors IL-6,TNF-α,IL-1β levels.Fecal bacteria transplantation caused the composition of the intestinal flora in postmenopausal AS mice to approach the donor mice.The abundance and diversity of intestinal flora in the Model→Model group were significantly lower than that in the HD-LW→Model group.The same changes also included the relative abundance at the levels of phylum and genus.The transplantation of fecal bacteria in the LWDHF treatment group changed the structure of the intestinal flora in the model mice,which could significantly reduce blood lipids,serum inflammation,and AS plaque area.The above results confirmed that LWDHF could resist the development of AS in postmenopausal mice by regulating the intestinal flora.Chapter 2:E2 and LWDHF could significantly reduce serum LPS and LBP levels in postmenopausal AS mice.Pearman correlation analysis showed that serum LPS and LBP levels were significantly positively correlated with AS plaque area,and were significantly negatively correlated with the diversity and relative abundance of intestinal flora.E2 and LWDHF could significantly increase the expression of ZO-1,occludin,Mucin-2,and the number of goblet cells in the colon of postmenopausal AS mice.LWDHF significantly up-regulated the expression of ZO-1 and occludin in the LPS-induced Caco-2 cell injury model.The results of fecal bacteria transplantation showed that the LPS and LBP levels in the serum of mice in the Model→Model group were significantly higher than those in the Control→Model group.Compared with the Model→Model group,the serum levels of LPS and LBP in the E2→Model group and the HD-LW→Model group were significantly reduced,and the intestinal permeability markers ZO-1,occludin,Mucin-2,and the number of goblet cells increased significantly.The richness and diversity of the intestinal flora of mice in each group of fecal bacteria transplantation were significantly negatively correlated with serum LPS and LBP levels.These results indicated that LWDHF could improve the intestinal barrier function of postmenopausal AS mice by regulating the intestinal flora.Chapter 3:The results of immunofluorescence and western blotting showed that E2 and LWDHF could significantly up-regulate the expression of IAP in the intestines of postmenopausal AS mice.Different doses of LWDHF significantly up-regulated the expression of IAP in Caco-2 cells stimulated by LPS.IAP inhibitor phenylalanine significantly inhibited the up-regulation of IAP and the expression of ZO-1 and occludin in LPS-induced Caco-2 cell injury model.The correlation analysis showed that the IAP expression was significantly positively correlated with the richness and diversity of intestinal flora,and significantly negatively correlated with aortic AS plaque area,blood lipids,inflammatory factors,LPS and LBP levels.These results indicated that the effect of LWDHF on regulating the intestinal flora and intestinal barrier of postmenopausal AS mice was related to the up-regulation of intestinal antimicrobial peptide expression.Chapter 4:LWDHF had no significant effect on serum estrogen levels in postmenopausal AS mice.However,the results of western blot and immunofluorescence showed that LWDHF could significantly up-regulate ERa and ER(3 in the duodenal intestines of postmenopausal AS mice.Western blot results also showed that LWDHF significantly up-regulated the expression of KLF4 in the duodenum of postmenopausal AS mice.The same results were observed in the LPS-induced Caco-2 cell injury model.LWDHF significantly up-regulated the expressions of ERα,ERβ and KLF4 in the cells.The combination of LWDHF with ERa inhibitor MPP or the ERβ inhibitor PHTPP was applied to treat the LPS-induced Caco-2 cell injury model.The results showed that the blockade of ERa inhibited the up-regulation of KLF4 and IAP induced by LWDHF,while the blockade of ERβ did not affect the effect of LWDHF.After silencing with ERa siRNA,the effect of LWDHF on up-regulating the expression of IAP and KLF4 in the Caco-2 cell injury model induced by LPS disappeared.These results indicated that LWDHF up-regulated the antimicrobial peptide transcription regulator KLF4 through the intestinal ERa to promote the secretion of the antimicrobial peptide IAP.Conclusion1.LWDHF could reshape intestinal flora and improve the intestinal barrier function to resist the development of AS in postmenopausal mice.2.LWDHF could up-regulate IAP,reshape the structure of intestinal flora and improve the intestinal barrier function,reduce blood lipids,inflammatory factors,LPS and LBP levels,and play an anti-AS effect in postmenopausal mice.3.LWDHF could up-regulates transcription regulator KLF4 through intestinal ERa to promote the secretion of IAP,reshape the structure of the intestinal flora and improve the intestinal barrier function,and inhibit the occurrence and development of AS in postmenopausal mice. |
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