降脂益生菌对非酒精性脂肪性肝病小鼠胆汁酸代谢及转运的影响和机制初探

Effects of cholesterol-lowering probiotics on bile acid metabolism and transport in mice with nonalcoholic fatty liver disease and the possible mechanisms

目的探讨降脂益生菌(鼠李糖乳杆菌DM9054和植物乳杆菌86066联合制剂)对非酒精性脂肪性肝病(nonalcoholic fatty liver disease, NAFLD)小鼠胆汁酸代谢及转运的影响和可能机制。方法 18只雄性FXR-/-小鼠随机分为3组(n=6):正常饮食组、高脂饮食组和高脂饮食+降脂益生菌组。其中正常饮食组给予普通饮食和生理盐水灌胃,高脂饮食组给予高脂饮食和生理盐水灌胃,高脂饮食+降脂益生菌组给予高脂饮食和降脂益生菌灌胃。所有小鼠干预12周,处死小鼠1周前行胰岛素耐量试验和腹腔注射葡萄糖耐量试验。小鼠处死后自动生化分析仪检测血脂、胆汁酸及肝功能指标;RT-PCR检测肝脏和回肠组织炎症因子相对表达量;HE染色评估肝脏和回肠组织病理情况;Western blot检测法尼醇受体(Farnesoid X receptor, FXR)通路中的成纤维细胞生长因子15(fibroblast growth factor 15,FGF15)、成纤维细胞生长因子受体4(fibroblast growth factor receptor 4,FGFR4)和小分子异源二聚体(short heterodimer partner, SHP)、胆汁酸合成限速酶胆固醇7α-羟化酶(cholesterol 7α-hydroxylase, CYP7A1)及胆汁酸转运相关的胆盐输出泵(bile salt export pump, BSEP)的蛋白表达。结果和高脂饮食组相比,高脂饮食+降脂益生菌组小鼠血清中胆汁酸含量明显下降(P=0.000 1),FGF15、FGFR4和BSEP蛋白表达水平升高(P=0.009 7、0.024 2、0.000 1),CYP7A1的蛋白表达水平降低(P=0.006 9)。此外,通过降脂益生菌干预还明显改善了高脂饮食FXR-/-小鼠的糖脂代谢紊乱(P=0.002 4)、肝脏脂肪变性、肝脏和回肠组织炎症(P=0.013 8、0.000 1、0.000 1)以及肠黏膜屏障功能(P=0.014 2)。结论降脂益生菌具有类似选择性肠道FXR激动剂的作用,能够通过调控肠道FXR-FGF15通路改善胆汁酸的代谢及转运,进而缓解高脂饮食FXR-/-小鼠的NAFLD。

Objective To investigate the effects and possible mechanisms of cholesterol-lowering probiotics(combination of Lactobacillus rhamnosus GG DM9054 and Lactobacillus plantarum WCFS1 86066) on bile acid metabolism and transport in mice with nonalcoholic fatty liver disease(NFLD). Methods Eighteen male FXR-/- mice were randomly divided into three groups(n=6): normal diet group, high fat diet group or high fat diet with probiotics group, and were respectively given normal diet and normal saline gavage, high fat diet and normal saline gavage, or high fat diet and cholesterol-lowering probiotics gavage, for 12 weeks. A week before the mice were sacrificed, insulin tolerance test and intraperitoneal glucose tolerance test were performed. After the mice were sacrificed, the automatic biochemical analyzer was used to detect blood lipids, bile acids and liver function indicators;RT-PCR was used to detect the relative expression of inflammatory factors in the liver and ileum tissue;The pathology of liver and ileum tissue was evaluated using HE staining;Western blot was used to detect fibroblast growth factor 15, fibroblast growth factor receptor 4, short molecular heterodimer in the Farnesoid X receptor pathway, bile acid synthesis rate-limiting enzyme cholesterol 7α-hydroxylase, and bile acid export related bile salt export pump protein expression. Results Compared with the high fat diet group, the intervention of cholesterol-lowering probiotics reduced bile acid level in serum(P<0.01), increased the expression levels of FGF15, FGFR4 and BSEP(all P<0.01), and decreased that of CYP7 A1(P<0.01). In addition, cholesterol-lowering probiotics also significantly improved glucose and lipid metabolism disorders(P<0.05) and liver steatosis, reduced liver and ileum inflammation(all P<0.05), and repaired intestinal barrier function in high fat diet FXR-/- mice. Conclusion Cholesterol-lowering probiotics have similar effects to selective intestinal FXR agonists. It can improve the metabolism and transport of bile acids by regulating intestinal FXR-FGF15 pathway, thereby alleviating NAFLD in high fat diet FXR-/-mice.