【摘要】 目的探讨溶酶体在黄芩素改善高脂诱导的小鼠非酒精性脂肪性肝病中的作用。方法将体质量18～20 g雄性C57BL/6J小鼠随机分为4组:正常对照组(C,10%脂肪供能)、高脂组(H,60%脂肪供能)、高脂+黄芩素组[H+B,黄芩素:400 mg/(kg·bw)]、黄芩素对照组[B,黄芩素:400 mg/(kg·bw)]。12 w后处死小鼠并收集血清及组织样本,油红O染色观察肝脏脂质沉积,采用试剂盒测定血清中AST、ALT水平及肝脏中CathepinB活性,采用Western-blotting法测定LC3、p62、Beclin1、Cathepin B、Cathepin D、V-ATPaseB2及LAMP2蛋白表达水平,免疫荧光法观察肝细胞胞浆V-ATPaseB2与溶酶体共定位的水平。结果与正常对照组相比,高脂喂养导致小鼠肝脏严重的脂质沉积及血清AST、ALT水平明显增加;肝脏自噬标志蛋白LC3II及自噬底物蛋白p62异常升高,Beclin1降低;胞浆组织蛋白酶B的活性增加了20.8%,成熟形式组织蛋白酶D表达降低、溶酶体膜V-ATPase复合物V1亚基表达降低,胞质蛋白V-ATPase B2与溶酶体膜相关蛋白2A(LAMP2)共定位减少;黄芩素干预减轻肝脏脂质沉积、抑制溶酶体组织蛋白酶B的释放、上调成熟形式组织蛋白酶D及溶酶体膜V-ATPase复合物V1亚基表达,胞质蛋白V-ATPase B2与LAMP2共定位面积增加,肝脏损伤明显缓解。结论黄芩素可能通过减轻溶酶体损伤、促进溶酶体膜V-ATPase复合物的组装,缓解高脂喂养导致肝脏溶酶体功能紊乱及自噬抑制,从而减轻非酒精脂肪肝损伤。[营养学报,2019,41(2):183-188]更多还原

【Abstract】 Objective To investigate the role of lysosomal function in non-alcoholic fatty liver disease(NAFLD) and the protection of baicalein. Methods Male C57 BL/6 J mice(18-20 g) were randomly divided into four groups as follows:control group(C, 10% fat), high fat diet group(H, 60% fat), high fat diet plus baicalein group(H+B, baicalein,(400 mg/(kg·bw)), baicalein control group(B, 400 mg/(kg· bw)). Mice were fed for 12 weeks. Fasting serum and liver tissue samples were collected for further biochemical assays. Hepatic lipid content was observed by Oil-Red O staining. The levels of AST, ALT and Cathepin B activity were detected by corresponding assay kits. Hepatic LC3,p62,beclin1,Cathepin B, Cathepin D,V-ATPaseB2 and LAMP2 expressions were determined by Western blotting. Cytosol V-ATPase B2 and LAMP2 colocalizations were shown by immunofluorescence. Results High fat diet(HFD) feeding resulted in higher hepatic lipid deposition,leakage of aminotransferases, LC3 II and p62 accumulation and beclin1 expression reduction. Meanwhile, HFD feeding increased cathepsin B activity by 20.8% and decreased mature cathepsin D level, lysosomal V-ATPase complex V1 subunit expression and the cytosol V-ATPase B2 and LAMP2 colocalization. Such harmful effects were partially counteracted by baicalein. Conclusion Baicalein promots lysosomal V-ATPase complex assembly and consequently restores lysosomal function in HFD-fed mice. [ACTA NUTRIMENTA SINICA, 2019, 41(2):183-188]更多还原