基于AMPK调控ACC/CPT1信号通路介导脂肪酸代谢探究化瘀祛痰方含药血清对HepG2细胞脂质沉积的影响

Effect of Serum Containing Huayu Qutan Prescription(化瘀祛痰方)on Lipid Deposition in HepG2 Cells Based on AMPK Regulation of Fatty Acid Metabolism Mediated by ACC/CPT1 Signaling Pathway

目的 观察化瘀祛痰方含药血清对HepG2细胞脂质沉积的影响,并探究其作用机制。方法 采用1 mmol/L油酸棕榈酸混合液诱导HepG2细胞24 h构建脂质沉积模型,分别给予化瘀祛痰方含药血清、AMP依赖的蛋白激酶[Adenosine 5’-monophosphate(AMP)-activated protein kinase, AMPK]抑制剂及抑制剂+中药联合干预,油红O染色观察脂质沉积情况,试剂盒检测细胞中甘油三酯(triglyceride, TG)含量,蛋白印迹法检测AMPK、p-AMPK、乙酰辅酶A羧化酶(Acetyl CoA Carboxylase, ACC)、p-ACC、肉碱棕榈酰基转移酶-1(Carnitine Palmitoyltransferase-1,CPT1)蛋白相对表达量,以及实时荧光定量法检测细胞中AMPK、ACC、CPT1 mRNA相对表达水平。结果 与模型组相比,化瘀祛痰方含药血清组细胞中脂滴数目减少、TG水平降低,细胞中p-AMPK、CPT1蛋白及CPT1 mRNA相对表达量升高,ACC、p-ACC蛋白及ACC mRNA相对表达量显著降低(P<0.01),AMPK蛋白及基因相对表达水平无明显差别(P>0.05);AMPK抑制剂组细胞中脂滴及TG含量升高,AMPK、CPT1蛋白及mRNA相对表达量降低,ACC、p-ACC蛋白及ACC mRNA相对表达量升高(P<0.05或P<0.01);与AMPK抑制剂相比,抑制剂+中药含药血清组细胞中脂滴数目减少、TG水平降低,AMPK、p-AMPK蛋白及AMPK mRNA相对表达水平无显著差异(P>0.05),ACC、p-ACC蛋白及ACC mRNA相对表达量降低、CPT1则相对表达升高(P<0.05或P<0.01)。结论 化瘀祛痰方含药血清可能通过激活AMPK使其磷酸化,并调控ACC/CPT1信号通路,抑制脂肪酸合成,促进脂肪酸氧化,从而降低HepG2细胞内的甘油三酯水平及脂质沉积。

Objective To observe the effect of serum containing Huayu Qutan Prescription(化瘀祛痰方)on lipid deposition in HepG2 cells and explore its mechanism.Methods HepG2 cells were induced to construct lipid deposition model by 1 mmol/L oleic palmitic acid mixed solution.The HepG2 cells were treated with serum containing Huayu Qutan Prescription,Adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)inhibitor and inhibitor+serum containing Huayu Qutan Prescription,respectively.Lipid deposition was observed by oil-red O staining,while triglyceride(TG)content in cells was detected by kit.The protein expressions of AMPK,p-AMPK,acetyl CoA carboxylase(ACC),p-ACC and carnitine palmitoyltransferase-1(CPT1)were detected by western blot,and the genes were detected by Realtime PCR.Results Compared with those of the model group,the number of lipid droplets and TG level in serum containing Huayu Qutan Prescription group were decreased and the relative expressions of p-AMPK,CPT1 protein and CPT1 mRNA were increased,and the contents of ACC,p-ACC protein and ACC mRNA were significantly decreased(P<0.01).While there were no significant differences in AMPK protein or gene levels(P>0.05)compared with those of the model group.In AMPK inhibitor group,the lipid droplets and TG were increased,the relative expressions of AMPK and CPT1 protein and mRNA were decreased,and the relative expressions of ACC,p-ACC protein and ACC mRNA increased(P<0.05 or P<0.01).Compared with those of the AMPK inhibitor group,the lipid droplets and TG in the inhibitor+Huayu Qutan Prescription group decreased,and there was no significant difference in AMPK,p-AMPK protein or AMPK gene(P>0.05).The relative expressions of ACC,p-ACC protein and ACC mRNA were decreased,and the protein and gene levels of CPT1 increased(P<0.05 or P<0.01).Conclusions Serum containing Huayu Qutan Prescription can activate AMPK to phosphorylation,regulate ACC/CPT1 signaling pathway,inhibit fatty acid synthesis and promote fatty acid oxidation,thereby reducing triglyceride level and lipid deposition in liver cells.