基于肠道菌群-TβMCA-FXR轴探讨田黄方对老年脂代谢紊乱小鼠作用机制

Efficacy and Mechanism of Tianhuang Formula(田黄方)in Regulating Lipid Metabolism Disorders in Senile Mice Based on Gut Microbiota-TβMCA-FXR Axis

目的:从肠道菌群角度探讨中药复方田黄方改善老年高脂血症小鼠脂代谢紊乱的作用机制。方法:3 w~4 w龄雄性C57BL/6J小鼠正常饮食喂养至12月成自然衰老小鼠,喂饲高脂饮食14 w构建老年高脂血症模型,给予田黄方提取物100 mg/kg干预10 w,正常对照组予普通饲料喂养至试验结束。试剂盒检测小鼠血清和肝脏总胆固醇(TC)、三酰甘油(TG)含量、胆盐水解酶(BSH)活力,靶向代谢组学联合16S rDNA测序分析胆汁酸代谢谱以及肠道菌群结构,Western Blot、PCR法检测法尼醇X受体(FXR)、蛋白酪氨酸磷酸酶1(SHP1)、胆固醇7α-羟化酶(CYP7A1)、成纤维生长因子15(FGF15)的mRNA和蛋白表达情况。结果:与模型对照组相比,田黄方提取物100 mg/kg组小鼠肝脏TC、TG含量显著下降(P<0.01),肠道菌群发生重塑,大肠埃希菌属(Escherichia)、阿克曼菌属(Akkermansia)、拟杆菌属(Bacteroides)丰度明显减少(P<0.05或P<0.01)、BSH酶活力下降(P<0.01),肠道中游离胆汁酸脱氧胆酸(DCA)、鹅脱氧胆酸(CDCA)和石胆酸(LCA)含量降低(P<0.01);与模型对照组相比,牛磺-β-鼠胆酸(TβMCA)20 mg/kg组小鼠血清及肝脏TC、TG含量显著下降(P<0.01),回肠和肝脏Fxr、Fgf15 mRNA和蛋白表达下调(P<0.05),Cyp7a1 mRNA和蛋白表达上调(P<0.05)。结论:田黄方通过调节肠道菌群,抑制BSH活性,使TβMCA水平增加进而抑制肠道FXR、FGF15表达,同时下调肝脏FXR和SHP表达,上调CYP7A1表达,导致胆汁酸合成增加,TC降低。田黄方通过肠道菌群-TβMCA-FXR轴发挥降脂作用。

Objective:To explore the mechanism of Tianhuang Formula(THF)in alleviating lipid metabolism disorders in elderly mice with hyperlipidemia.Methods:Male C57BL/6J mice aged 3–4 weeks were fed with a normal diet until 12 months to induce naturally aging,and a high-fat diet was given for 14 weeks to construct a model of lipid metabolism disorders.The mice were administrated with THF extract(100 mg/kg)for 10 weeks,and those in the control group were fed with normal diet until the end of the experiment.The total cholesterol(TC),triglyceride(TG),and bile salt hydrolase(BSH)activity in serum and liver were measured by the corresponding assay kits.Targeted metabolomics analysis combined with 16S rDNA sequencing was employed to analyze the bile acid metabolism profile and the gut microbiota structure.Western blotting and RT-PCR were employed to respectively measure the protein and mRNA levels of farnesoid X receptor(FXR),SH2 domain-containing protein-tyrosine phosphatase-1(SHP1),cholesterol 7α-hydroxylase(CYP7A1),and fibroblast growth factor 15(FGF15)in mice.Results:Compared with the model group,THF extract decreased the content of TC and TG in mouse liver(P<0.05)and remodeled the gut microbiota.Specifically,it decreased the relative abundance of Escherichia(P<0.01)and Bacteroides(P<0.05).Furthermore,the THF extract weakened the BSH activity(P<0.01)and lowered the levels of free deoxycholic acid(P<0.01),chenodeoxycholic acid(P<0.05),and lithocholic acid(P<0.01)in the intestinal tract.Compared with the model group,taurine-β-murocholic acid(20 mg/kg)decreased the content of TC and TG in the serum and liver(P<0.01).Western blotting and PCR results showed that compared with the model group,taurine-β-murocholic acid(20 mg/kg)down-regulated the mRNA of FXR and FGF15 in the ileum and liver(P<0.05)and up-regulated those of CYP7A1(P<0.05),the protein results was similar to gene expression.Conclusion:THF may regulate the gut microbiota and inhibit the activity of BSH to increase the level of tauro-beta-muricholic acid(T-βMCA),thereby inhibiting intestinal FXR and FGF15.Meanwhile,it down-regulates the expression of FXR and SHP and up-regulates the expression of CYP7A1 in the liver,leading to increased bile acid synthesis and decreased TC.THF exerts lipid-lowering effect through the gut microbiota-T βMCA-FXR metabolism axis.