基于LC-MS技术非酒精性脂肪肝病痰湿证大鼠肝脏的脂质组学分析

Lipidomic analysis of the liver in rats with nonalcoholic fatty liver disease with syndrome of phlegm-dampness based on LC-MS

目的从脂质组学层面探寻非酒精性脂肪肝病(nonalcoholic fatty liver disease,NAFLD)痰湿证的脂质生物标志物以揭示NAFLD痰湿证的机理。方法以高脂饲料喂养制备NAFLD痰湿证大鼠模型,造模后痰湿证模型组大鼠的体质量、肝重明显升高(P<0.01或P<0.05),血清总胆固醇、血清甘油三酯及丙氨酸氨基转移酶、天冬氨酸氨基转移酶较正常组明显偏高(P<0.01或P<0.05);提示NAFLD造模成功。采用液相色谱—质谱联用技术对样本进行脂质组学分析,选用主成分分析和正交偏最小二乘法—判别分析(orthogonal-partial least squares-discriminant analysis,OPLS-DA)并进行置换检验,避免检验模型过拟合。后根据OPLS-DA的结果,设置变量投影重要度VIP>3.5、P<0.001且倍数变化>2或<0.5来寻找正常组与NAFLD痰湿证组大鼠肝脏脂质的代谢物信息,寻找与NAFLD痰湿证密切相关的差异性脂质生物标志物。结果肝脏提取物的总离子流色谱图分离良好;质量控制样本聚集,重复性良好;主成分分析、偏最小二乘判别分析及OPLS-DA模型验证显示组间明显聚集,模型拟合指数R 2=0.59,预测指数Q 2=-0.45。共筛选出23个潜在生物标志物,其中12个甘油三酯、一个甘油二酯呈上调趋势;磷脂酰胆碱(15∶0_20∶3)呈上调趋势,其余均呈下调趋势。结论非酒精性脂肪肝痰湿证大鼠与正常对照组大鼠脂质代谢存在显著差异,其发病机制与甘油磷脂和甘油三脂等代谢异常相关,本研究筛选出的23个脂质生物标志物或可作为NAFLD痰湿证的新的诊治依据。

Objective To explore the lipid biomarkers of phlegm dampness syndrome in nonalcoholic fatty liver disease(NAFLD)from the perspective of lipid histology to reveal the mechanism of phlegm dampness syndrome in NAFLD.Methods A rat model of NAFLD with phlegm dampness syndrome was established by feeding with high fat diet.The body weight and liver weight of rats in the model group of phlegm dampness syndrome were significantly increased,and the blood lipid indexes total cholesterol,triglyceride(P<0.01 or P<0.05),and liver lipids ALT,AST were significantly higher than those in the normal group(P<0.01 or P<0.05).The samples were analyzed by liquid chromatography-mass spectrometry for lipomics.Principal component analysis(PCA)and orthogonal partial least-squares discriminant analysis(OPLS-DA)were used for displacement testing to avoid overfitting the test model.After that,based on the results of OPLS-DA,set VIP>3.5,P<0.001,and fold change>2 or<0.5 to search for liver lipid metabolite information of rats in the normal group and the NAFLD phlegm dampness syndrome group,and to search for differential lipid biomarkers closely related to NAFLD phlegm dampness syndrome.Results The total ion chromatogram of the liver extract was well separated;QC samples were clustered with good repeatability;PCA analysis,PLS-DA analysis,and OPLS-DA model validation showed significant aggregation between groups,with a model fitting index of R 2=0.59,and a prediction index of Q 2=-0.45.A total of 23 potential biomarkers were screened out,among which 12 triglycerides and one diglyceride showed an upward trend;in phosphatidylcholine(15∶0_20∶3)showed an upward trend,while the rest showed a downward trend.Conclusion There is a significant difference in lipid metabolism between rats with non alcoholic fatty liver syndrome of phlegm dampness and the normal control group,and its pathogenesis is related to abnormal metabolism of glycerol phospholipids and triglycerides.The 23 lipid biomarkers selected in this study may serve as a new basis for diagnosis and treatment of NAFLD syndrome of phlegm dampness.