补中益气汤调控Fibulin 5促进成纤维细胞活化改善痔病OA

Objective To explore the molecular mechanism by which Buzhong Yiqi Decoction(BYD)ameliorates hemorrhoidal disease.Methods(1)Animal experiment:A hemorrhoidal disease model was established in rats using the topical application of glacial acetic acid.Twenty SD rats were randomly divided into a control group,a model group,and low-,medium-,and high-dose BYD groups,with 4 rats per group.Interventions lasted for 7 consecutive days.Pathological changes in perianal tissues were observed by hematoxylin and eosin(HE)staining.The expression of Fibulin 5 and α-SMA in perianal tissues was detected by immunohistochemistry.(2)Cell experiments:① Primary fibroblasts were extracted from hemorrhoidal tissue samples of patients with Grade Ⅲ prolapsed internal hemorrhoids.Cells were cultured in drug-containing serum media with different concentrations of BYD(10%,20%,30%).The effects of BYD-containing serum on the proliferation and migration of primary fibroblasts were evaluated using the Cell Counting Kit-8(CCK-8)assay and cell scratch wound healing assay,respectively.② Fibroblasts were divided into a control group,a model group,and a BYD group.The protein and gene expression levels of Fibulin 5,LTBP-4,α-SMA,and CollagenⅠ were detected by Western Blot and real-time quantitative polymerase chain reaction(RT-qPCR),respectively.③ Fibulin 5 was knocked down in fibroblasts using siRNA technology.Cells were divided into a control group,a model group,a BYD group,and a BYD+siRNA group.The protein and gene expression levels of Fibulin 5,LTBP-4,α-SMA,and Collagen Ⅰ were detected by Western Blot and RT-qPCR,respectively.Results(1)Results of animal experiments:BYD improved the degree of pathological lesions in the perianal tissues of model rats.The expression levels of Fibulin 5 and α-SMA in the perianal tissues of the model group were lower than those in the control group(P＜0.05),while the expression levels in the BYD group were higher than those in the model group(P＜0.05).(2)Results of cell experiments:① The proliferation and migration abilities of primary fibroblasts from hemorrhoid patients were lower than those of the control group(P＜0.01).The proliferation and migration abilities in the BYD group were higher than those in the model group(P＜0.01).② The protein and gene expression levels of Fibulin 5,LTBP-4,α-SMA,and CollagenⅠ in fibroblasts of the model group were lower than those in the control group(P＜0.05).The expression levels of these proteins and genes in the BYD group were higher than those in the model group(P＜0.05).③ After knocking down Fibulin 5,the promoting effect of BYD on the expression of Fibulin 5,LTBP-4,α-SMA,and CollagenⅠ proteins and genes was reversed(P＜0.05 or P＜0.01).Conclusion BYD may ameliorate hemorrhoidal lesions by upregulating the expression of Fibulin 5 and promoting fibroblast activation.