基于肝脏代谢组学研究柴胡皂苷A对小鼠酒精性脂肪肝保护作用

Hepatoprotective Effect of Saikosaponin A on Alcoholic Fatty Liver Mice Based on Metabonomics

目的:基于代谢组学探讨柴胡皂苷A(SSa)改善酒精饮食诱导下小鼠酒精性脂肪肝(AFL)的作用机制。方法:将C57BL/6J雄性小鼠分为饮食对照(NC)组、模型(EtOH)组、不同剂量SSa干预(Et+SA-L、Et+SA-M、Et+SA-H,5、10、20 mg·kg^(-1))组,每组6只。除对照组外,各组小鼠使用含5%乙醇的流质饮食诱导10 d构建AFL模型,自乙醇饲料喂养第4天起给药至造模结束。应用酶联免疫吸附法检测小鼠血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、三酰甘油(TG)、总胆固醇(TC)和小鼠肝脏丙二醛(MDA)含量水平。采用苏木精-伊红(HE)染色观察肝脏组织形态学变化。采用超高效液相色谱串联四极杆静电场轨道阱质谱法(UPLC-Q-Exactive MS)建立检测NC组、EtOH组、Et-SA-H组小鼠肝脏代谢谱,以偏最小二乘法-判别分析(PLS-DA)和正交偏最小二乘法-判别分析(OPLS-DA)分析模式识别,以T检验、差异倍数筛选潜在生物标志物,并借助MetaboAnalyst数据库富集代谢通路。结果:SSa减轻AFL小鼠肝体比降低AFL小鼠血清中ALT、AST、TC、TG和MDA的含量,缓解AFL小鼠肝脂肪变性。NC组、EtOH组及Et+SA-H组的肝脏代谢组学分析显示,有5个与SSa调控AFL作用相关的潜在生物标志物,主要涉及甘油磷脂代谢、糖基磷脂酰肌醇(GPI)锚定生物合成、苯丙氨酸代谢、烟酸盐与烟酰胺代谢、组氨酸代谢、脂肪酸生物合成和细胞色素P450对外源物的代谢等途径。结论:SSa调控AFL小鼠肝脏脂质代谢、降低肝脏脂肪变性、改善肝脏功能,其机制与肝脏TG代谢和肝细胞微粒体乙醇氧化代谢密切相关。

Objective:To analyze the protective effect of saikosaponin A(SSa)on alcoholic fatty liver(AFL)mice by metabonomics.Methods:Male mice of C57BL/6J were divided into normal control group(NC),model group(EtOH),and SSa groups at different doses(Et+SA-L、Et+SA-M、Et+SA-H,5,10,20 mg·kg^(-1)),six in each group.Except the NC group,rats in all other groups were given liquid diet containing 5%alcohol for 10 days to establish AFL models,and drug administration was performed on the 4th day of alcohol feeding till the end of modeling.Serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglyceride(TG),total cholesterol(TC)and liver malondialdehyde(MDA)were determined by enzyme-linked immunosorbent assay(ELISA),and HE staining was used to observe the morphological changes of liver tissue.The metabolic profiles of mouse samples from NC group,EtOH group and Et-SA-H group were established by ultra-high performance liquid chromatography tandem quadrupole electrostatic field orbittrap mass spectrometry(UPLC-Q-Exactive MS).Partial least squares-discriminant analysis(PLS-DA)and orthogonal partial least squares-discriminant analysis(OPLS-DA)were used for pattern recognition.Potential biomarkers were screened by T-test and Fold Change(FC),and metabolic pathways were enriched by MetaboAnalyst.Results:SSa reduced the liver/body weight ratio,down-regulated the serum ALT,AST,TC,TG and MDA,and alleviated the liver steatosis of AFL mice.Liver metabolomics in NC group,EtOH group and Et+SA-H group showed that there were five potential biomarkers associated with SSa regulation of AFL,mainly involving glycerol phospholipid metabolism,glycosyl phosphatidylinositol(GPI)anchored biosynthesis,phenylalanine metabolism,niacin and niacinamide metabolism,histidine metabolism,fatty acid biosynthesis,cytochrome P450 in metabolism of foreign substances and other pathways.Conclusion:SSa can regulate liver lipid metabolism,reduce liver steatosis and improve liver function in AFL mice,and the specific mechanism is closely related to TG metabolism and ethanol oxidation metabolism of liver microsomes.