基于AMPK/ACC信号通路探讨夏枯草提取物调节ZDF大鼠脂代谢的机制

Mechanism of Prunellae Spica Extracts on Lipid Metabolism in ZDF Rats Based on AMPK/ACC Signalling Pathway

目的:观察夏枯草提取物(Prunellae Spica extracts,PS)对Zucker糖尿病肥胖(ZDF)模型大鼠肝腺苷酸活化蛋白激酶(AMPK)/乙酰辅酶A羧化酶(ACC)信号通路的影响以探讨其改善大鼠脂代谢的机制。方法:32只雄性ZDF(fa/fa)2型糖尿病模型大鼠随机分为模型组、二甲双胍组(180 mg·kg^(-1)·d^(-1))和PS低、高剂量组(12.25,24.5 mg·kg^(-1)·d^(-1)),每组8只。8只Zuker Lean(ZL)大鼠设为正常组。于给药0,4,8周测体质量及空腹血糖。给药8周后,腹主动脉取血,离心抽取血清-20℃冻存,取肝组织-80℃冻存,及4%多聚甲醛固定,石蜡包埋。放射免疫法测血清甘油三酯(TG),胆固醇(CHO),低密度脂蛋白胆固醇(LDL-C)及游离脂肪酸(FFA)。油红O染色观察肝细胞脂滴含量。实时荧光定量聚合酶链式反应(Real-time PCR)检测肝脏AMPKα_2及ACC mRNA表达。免疫组化法测肝细胞磷酸化(p)-AMPKα蛋白表达。结果:与正常组比较,模型组大鼠血清TG,CHO,LDL-C及FFA升高,肝细胞脂滴增加,肝AMPKα_2 mRNA表达减少而ACC1和ACC2 mRNA表达增加,p-AMPKα蛋白表达减少(P<0.05,P<0.01)。与模型组比较,PS低、高剂量组可明显降低ZDF大鼠血清TG,CHO,LDL-C及FFA,减少肝细胞脂滴,上调肝AMPKα_2 mRNA且下调ACC1和ACC2 mRNA表达,上调p-AMPKα蛋白表达(P<0.05,P<0.01)。结论:PS能有效改善2型糖尿病ZDF大鼠肝脏脂代谢紊乱,其机制可能与调节肝AMPK/ACC信号通路有关。

Objective: To observe the effect of Prunellae Spica extracts( PS) on the lipid metabolism in Zuker Diabetes Fatty( ZDF) rats based on AMP-activated protein kinase/acetyl Co A carboxylase( AMPK/ACC)signaling pathway. Method: The 32 male ZDF( fa/fa) type 2 diabetic rats were randomly divided into model group,metformin group( 180 mg·kg^-1·d^-1),and low and high-dose PS groups( 12. 25,24. 5 mg·kg^-1·d^-1),with 8 in each group. 8 male Zuker Lean( ZL) rats were selected as normal group. Body weight and fasting blood glucose were monitored at the 0th,4th and 8th weeks after administration. After 8 weeks,abdominal aorta blood was collected,serum was frozen at -20 ℃ by centrifugation,liver tissue was frozen at -80 ℃,fixed with 4%paraformaldehyde and embedded in paraffin. Serum triglyceride( TG),cholesterol( CHO),low density lipoprotein cholesterol(LDL-C) and free fatty acid( FFA) levels were measured by radioimmunoassay. Fat droplets in hepatocytes were measured by oil red O staining. Gene expressions of AMP-activated protein kinase-alpha 2( AMPKα2),Acetyl Co A carboxylase( ACC) in liver were detected by real-time polymerase chain reaction( Realtime PCR). Protein expressions of p-AMPKα were observed by immuno-histochemical( IHC) method. Result:Compared with the normal group,the T2DM model group showed significant increases in serum levels of TG,CHO,LDL-C,FFA and lipid droplets in hepatocytes. AMPKα2 mRNA expression was decreased,while ACC1 and ACC2 mRNA expressions were increased significantly. p-AMPKα protein expression in liver was decreased significantly( P <0. 05,P< 0. 01). Compared with the model group,low and high-dose Ps groups showed significant decreases in serum levels of TG,CHO,LDL-C,FFA and lipid droplets in hepatocytes in ZDF rats,up-regulation in mRNA expression of AMPKα2,down-regulation in mRNA expressions of ACC1 and ACC2,and up-regulation in protein expression of p-AMPKα( P< 0. 05,P <0. 01). Conclusion: PS can effectively improve liver lipid metabolism in ZDF rats. Its mechanism may be related to the regulation of AMPK/ACC signaling pathway in liver.