基于AMPK信号通路探讨黄芪甲苷对db/db小鼠2型糖尿病合并非酒精性脂肪肝病的作用机制

Mechanism of AstragalosideⅣon db/db Mice with Type 2 Diabetes Mellitus and Non-alcoholic Fatty Liver Disease Based on AMPK Signaling Pathway

目的:基于网络药理学和实验验证研究黄芪甲苷(ASⅣ)对db/db小鼠2型糖尿病(T2DM)合并非酒精性脂肪肝病(NAFLD)的作用机制。方法:通过随机将24只db/db小鼠分为4组:模型组、二甲双胍组、黄芪甲苷低、高剂量组。采用6只C57小鼠作为空白组。黄芪甲苷低、高剂量组分别予黄芪甲苷0.015、0.030 g·kg^(-1)灌胃,二甲双胍组予0.067 g·kg^(-1)灌胃,空白组和模型组予等体积蒸馏水灌胃。灌胃结束后,检测小鼠空腹血糖(FBG),进行口服葡萄糖耐量试验,检测血清血脂水平及肝组织病理情况。通过网络药理学预测黄芪甲苷治疗T2DM合并NAFLD的作用靶点及富集通路,并采用分子生物学技术验证其主要相关富集通路;蛋白免疫印迹法(Western blot)检测肝组织AMPK、p-AMPK、甾醇调节元件结合蛋白-1(SREBP-1)、脂肪酸合成酶(FAS)蛋白表达。结果:与空白组比较,经ASⅣ治疗的小鼠体质量、肝质量系数、空腹血糖、血清总胆固醇、甘油三酯和低密度脂蛋白胆固醇水平降低(P<0.05,P<0.01)。肝组织的病理学显示脂质蓄积情况明显改善,影像学结果显示经黄芪甲苷治疗后,脂肪肝的程度减轻。网络药理学预测结果表明血管内皮生长因子α(VEGFA),半乳糖凝集素3(LGALS3),蛋白激酶B2(Akt2),含Rho相关卷曲螺旋蛋白激酶1(ROCK1),蛋白激酶B1(Akt1),信号传导及转录激活蛋白(STAT3),间质表皮转化因子(MET)是“药物-疾病”中的关键靶点,京都基因与基因组百科全书(KEGG)富集的结果显示AMP依赖的蛋白激酶(AMPK)信号通路与T2DM合并NAFLD密切相关。Western blot结果显示,与空白组比较,模型组中p-AMPK/AMPK表达水平明显下调,SREBP-1、FAS蛋白表达水平明显上调(P<0.01);与模型组比较,二甲双胍组、黄芪甲苷高剂量组p-AMPK/AMPK表达水平明显上调,SREBP-1、FAS蛋白表达水平明显下调(P<0.05,P<0.01)。结论:黄芪甲苷通过激活AMPK信号通路来调控脂质类蛋白的表达,从而改善脂质代谢。

Objective:To study the mechanism of astragalosideⅣ(ASⅣ)on db/db mice with type 2 diabetes mellitus(T2DM)and non-alcoholic fatty liver disease(NAFLD)based on network pharmacology and experimental validation.Method:A total of 24 db/db mice were randomly divided into four groups:model group,metformin group,and low-dose and high-dose ASⅣgroups.Six C57 mice were used as the blank group.The low-dose and high-dose ASⅣgroups were given ASⅣof 0.015 and 0.030 g·kg^(-1) by gavage,and the metformin group was given 0.067 g·kg^(-1) by gavage.The blank and model groups were given equal volumes of distilled water by gavage.After intragastric administration,fasting blood glucose(FBG)was detected,and an oral glucose tolerance test was performed.Serum lipid level and liver histopathology were detected.The target and enrichment pathway of ASⅣfor treating T2DM and NAFLD were predicted by network pharmacology,and the main enrichment pathway was verified by molecular biology techniques.The protein expressions of AMPK,p-AMPK,sterol regulatory element-binding protein-1(SREBP-1),and fatty acid synthetase(FAS)in liver tissue were detected by Western blot.Result:Compared with the blank group,the levels of body mass,liver weight coefficient,fasting blood glucose,serum total cholesterol,triglyceride,and low-density lipoprotein cholesterol in mice treated with ASⅣwere decreased(P<0.05,P<0.01).The pathology of liver tissue showed significant improvement in lipid accumulation,and imaging results showed that the degree of fatty liver was reduced after ASⅣtherapy.Network pharmacological prediction results showed that vascular endothelial growth factorα(VEGFA),galactoagglutinin 3(LGALS3),serine/threonine kinase B2(Akt2),RHO-associated coiled-coil protein kinase 1(ROCK1),serine/threonine kinase B1(Akt1),signaling and transcriptional activator protein(STAT3),and messtimal epidermal transformation factor(MET)were key targets in"drugdisease"network.The results from the Kyoto encyclopedia of genes and genomes(KEGG)enrichment showed that the AMP-dependent protein kinase(AMPK)signaling pathway was strongly associated with T2DM and NAFLD.Western blot results showed that compared with the blank group,the expression levels of p-AMPK/AMPK in the model group were significantly down-regulated,while those of SREBP-1 and FAS proteins were significantly up-regulated(P<0.01).Compared with the model group,the expression levels of p-AMPK/AMPK in the metformin group and high-dose ASⅣgroup were significantly up-regulated,while those of SREBP-1 and FAS proteins were significantly down-regulated(P<0.05,P<0.01).Conclusion:ASⅣregulates the expression of lipid proteins by activating the AMPK signaling pathway,thereby improving lipid metabolism.