鳖甲煎丸缓解氧化三甲胺加重的BDL大鼠肝纤维化的分子机制OA

Objective To establish a model to evaluate the therapeutic effect of Biejiajian pills(BJJP)on bile duct ligation(BDL)-induced liver fibrosis in rats,and to investigate the molecular mechanisms by which BJJP alleviates trimethlylamine oxide(TMAO)-aggravated liver fibrosis in BDL rats.Methods A rat model of therapeutic BJJP for BDL-induced hepatic fibrosis was established.Thirty rats were divided randomly into Sham,BDL,and BJJP low-dose(-L),medium-dose(-M),and high-dose(-H)groups.Collagen-fiber deposition was analyzed by Masson and Sirius Red staining.Expression levels of α-smooth muscle actin(α-SMA)and type Ⅰ collagen α1 chain(COL1A1)proteins in liver tissue in each group were detected by immunohistochemical staining and Western blot.Feces were collected from rats in the Sham,BDL,the BJJP-H groups for 16S rRNA high-throughput microbial sequencing and non-targeted metabolomics analysis.Serum and feces levels of trimethylamine(TMA)and TMAO were determined in the Sham,BDL,and BJJP-H groups,respectively,by non-targeted and targeted metabolomics technologies.To investigate the therapeutic effects of BJJP on TMAO-aggravated hepatic fibrosis and its potential molecular mechanisms,a further 20 Sprague Dawley rats were divided randomly into Sham,BDL,BDL+TMAO,and BDL+TMAO+BJJP-H groups.Changes in histomorphology and collagen fibers in liver tissue were detected by hematoxylin-eosin,Masson,and Sirius Red staining.Expression levels of α-SMA,flavin-containing monooxygenase 3(FMO3),phosphorylated(p-)protein kinase B(AKT),and p-phosphatidylinositol 3-kinase(PI3K)in each group were analyzed quantitatively by Western blot.Results Compared with the Sham group,liver levels of collagen fiber,α-SMA and COL1A1 protein were increased in the BDL group(P＜0.000 1,P＜0.000 1,P＜0.001).Compared with the BDL group,levels of collagen fiber in the BJJP-L,BJJP-M,and BJJP-H groups were significantly decreased(P＜0.05,P＜0.05,P＜0.001),as well as the levels of α-SMA protein and COL1A1 protein(all P＜0.05).BJJP improved the intestinal flora and metabolic disorders in BDL rats,and reduced feces levels of TMA and serum levels of TMAO(all P＜0.05).Compared with the Sham group,rats in the BDL group exhibited disorganized hepatic cord arrangement,evident hepatocyte swelling,and extensive collagen-fiber deposition,along with up-regulation of α-SMA,FMO3,p-AKT,and p-PI3K protein expression(all P＜0.05).The above indicators in liver tissues of rats were further increased in the BDL+TMAO group compared with the BDL group(P＜0.001,P＜0.05,P＜0.000 1,P＜0.05).Compared with the BDL+TMAO group,rats in the BDL+TMAO+BJJP-H group exhibited more orderly hepatocellular arrangement,reduced collagen-fiber deposition(P＜0.000 1),and decreased protein expression of α-SMA,FMO3,p-AKT,and p-PI3K(P＜0.001,P＜0.01,P＜0.001,P＜0.05).Conclusions BJJP can improve hepatic fibrosis while TMAO can aggravate hepatic fibrosis in BDL rats.BJJP may alleviate the hepatic fibrosis process in BDL rats by regulating intestinal flora TMAO.