牛磺熊去氧胆酸预防高脂饮食小鼠胆囊结石的研究

Tauroursodeoxycholic acid prevents gallbladder stone formation in high-fat fed mice

目的:探讨牛磺熊去氧胆酸(tauroursodeoxycholic acid,TUDCA)对高脂饮食小鼠胆囊胆固醇结石形成的抑制作用。方法:将30只SPF级雄性c57bl/6小鼠随机分为成石饲料喂养(lithogenic diet, LD)组和TUDCA(5 g/kg LD)喂养组。喂养8周后取血清、胆囊、肝脏、小肠,并观察胆囊内结石或结晶形成情况。检测肠道菌群的分布和结构变化。检测小鼠胆囊胆汁成分、血清脂质及肝脏脂质含量,采用定量实时聚合酶联反应检测肝脏及小肠胆汁酸和脂质代谢相关基因mRNA相对表达量。结果:在LD组小鼠胆囊内均可发现成型的胆固醇结石并伴有大量结晶形成,成石率100%。在TUDCA组小鼠胆囊内未发现结石,仅在3只小鼠胆囊内发现有结晶形成。TUDCA组肠道菌群厚壁菌门丰度(0.55%±0.06%)高于LD组(0.28%±0.02%)(P<0.01)。TUDCA组拟杆菌门丰度(0.05%±0.01%)低于LD组(0.30%±0.01%)(P<0.001)。厚壁菌门/拟杆菌门比值上升3.13倍。TUDCA组血清总胆固醇[(2.36±0.11) mmol/L]低于LD组[(5.79±0.43) mmol/L](P<0.01),肝脏总胆固醇[(4.12±0.21) mg/g]也低于LD组[(83.17±3.06) mg/g](P<0.001)。TUDCA组肝脏的胆固醇转运基因ATP结合盒G5和G8(6.32±0.26和8.12±0.86)与LD组相比(9.48±0.64和16.76±1.61)均下降(P<0.01),TUDCA组胆盐输出泵蛋白编码基因11 (15.74±1.06)、乙酰辅酶A乙酰基转移酶2 (2.04±0.02)、Cyp27(36.41±0.77)的表达量显著高于LD组(3.87±0.71、1.47±0.09和28.70±1.09)(P<0.001)。TUDCA组胆囊胆固醇饱和指数(1.06±0.15)也低于LD组(2.40±0.36)(P<0.01)。结论:TUDCA通过改善高脂饮食小鼠肠道菌群丰度和分布,抑制小肠吸收及合成脂质,同时降低肝脏胆固醇合成和分泌,最终降低高脂饮食小鼠胆囊结石成石率。

Objective To investigate the effect of tauroursodeoxycholic acid(TUDCA) on prevention of cholesterol gallstones formation in high-fat fed mice. Methods Thirty SPF grade male c57 bl/6 mice were randomly divided into lithogenic diet(LD) group and TUDCA group. The mice in TUDCA group were fed 5 g TUDCA/1 000 g LD. After 8 weeks of feeding, serum, gallbladder, liver and small intestine were collected and the formation of gallstones or crystals in gall-bladder was examined. Composition of intestinal microbiota was examined. Gallbladder bile lipid composition, serum lipid and liver lipid in mice were detected. Expression of genes involved in bile acid and lipid metabolism of liver and small intestine was detected by quantitative real-time PCR. Results Cholesterol gallstones with cholesterol crystal formed in all mice of LD group(100%). Only cholesterol crystals were found in three mice without any gallstone in TUDCA group. The abundance of intestinal Firmicutes in TUDCA group(0.55%±0.06%) was higher than that in LD group(0.28%±0.02%)(P<0.01), while the abundance of Bacteroides in TUDCA group(0.05% ±0.01%) was lower than that in LD group(0.30%±0.01%)(P<0.001). Compared with LD group, the ratio of Firmicutes/Bacteroides in TUDCA group increased by 3.13 times.Serum total cholesterol levels in TUDCA group [(2.36±0.11)mmol/L] decreased compared with that in LD group [(5.79 ±0.43)mmol/L](P<0.01). Hepatic total cholesterol levels in TUDCA group [(4.12 ±0.21) mg/g] decreased significantly compared with that in LD group significantly [(83.17±3.06) mg/g](P<0.001). mRNA expression level of Abcg5 and Abcg8 in liver of TUDCA group(6.32±0.26, 8.12±0.86) decreased significantly compared with that in LD group(9.48±0.64, 16.76±1.61)(P<0.01). The expression level of Abcb11(15.74±1.06), Acat2(2.04±0.02), Cyp27(36.41±0.77) in the liver of TUDCA group increased compared with those in LD group(3.87±0.71, 1.47±0.09, 28.70±1.09)(P<0.001). Gallbladder cholesterol saturation index in TUDCA group(1.06±0.15) also decreased compared with that in LD group(2.40±0.36)(P<0.01).Conclusions TUDCA might inhibit the absorption and synthesis of lipids in small intestine by improving intestinal microbiota of high-fat fed mice, so as to reduce gallstone formation in the mice.