金合欢素的降血脂及抗动脉粥样硬化作用及机制初探

The hypolipidemic and anti-atherosclerotic effects of acacetin and its mechanism of action in mice

本文旨在研究金合欢素对小鼠血脂代谢及动脉粥样硬化的影响并初步探索其作用机制。利用不同浓度的金合欢素处理人肝癌细胞HepG2后,采用RT-qPCR和Western blot方法分别检测LDLR mRNA水平和蛋白表达情况以及SREBP-2的蛋白表达情况。C57BL/6J小鼠给予金合欢素灌胃处理5周,全自动生化分析仪检测血清中总胆固醇(total cholesterol, TC)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol, LDL-C)、高密度脂蛋白胆固醇(high-density lipoprotein cholesterol, HDL-C)和甘油三酯(triglyceride, TG)等指标, Western blot检测低密度脂蛋白受体(LDL receptor, LDLR)和固醇调节元件结合蛋白2 (sterol-regulatory element binding protein-2, SREBP-2)蛋白表达水平。载脂蛋白ApoE基因敲除(apolipoprotein E knockout, ApoE KO)小鼠灌胃给药12周后,全自动生化分析仪检测小鼠血清中的血脂水平等指标;油红O染色观察主动脉全长及主动脉根部斑块中脂质沉积情况; RT-qPCR和Western blot分别检测LDLR mRNA水平和蛋白表达情况以及SREBP-2蛋白水平; 16S rDNA测序分析小鼠肠道内容物的菌群组成变化情况(动物实验经医药生物技术研究所实验动物伦理审查委员会批准)。体外实验结果表明,金合欢素显著上调LDLR mRNA和蛋白水平,同时在蛋白水平增加转录因子SREBP-2的表达。体内实验结果表明,金合欢素与对照组相比可以显著降低C57BL/6J小鼠血清中TC和LDL-C水平。机制研究显示,金合欢素显著提高肝组织中的LDLR水平,同时升高SREBP-2的蛋白水平。ApoE KO小鼠的血脂结果及斑块形态学分析发现,金合欢素组与高脂饮食组间的差异没有统计学意义。肠道菌群分析结果显示,金合欢素处理组肠道菌群的组成与高脂饮食组相比存在结构差异。总之,金合欢素可能通过调控转录因子SREBP-2来增加LDLR的表达,进而降低C57BL/6J小鼠血清中LDL-C水平,调节小鼠的血脂代谢,同时对ApoE KO小鼠的肠道菌群也有影响,提示其可能具有潜在抑制动脉粥样硬化的作用。

The purpose of this research is to investigate the effects of acacetin on serum lipid metabolism and atherosclerosis in mice and explore its molecular mechanism.HepG2 cells were treated with different concentrations of acacetin.The expression of LDL receptor(LDLR)and sterol-regulatory element binding protein-2(SREBP-2)were detected by RT-qPCR and/or Western blot.C57BL/6 J mice were given acacetin(50 mg·kg-1)for 5 weeks by gavage.Serum total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C)and triglyceride(TG)were analyzed by an automatic biochemical analyzer.The expression of LDLR or SREBP-2 was detected by Western blot.After 12 weeks of intragastric administration of acacetin(30 mg·kg-1)in apolipoprotein E knockout(ApoE KO)mice,the serum lipid levels were determined by an automatic biochemical analyzer.The lipid deposition in aortic plaque(en face)and aortic root plaque were stained with oil red O.The expression of LDLR and SREBP-2 were detected by RT-qPCR and/or Western blot.The intestinal content microflora was analyzed by 16S rDNA sequencing(All animal studies were approved by the Animal Experimentation Ethics Committee of Institute of Medicinal Biotechnology,CAMS&PUMC).In vitro results indicated that acacetin significantly up-regulated LDLR mRNA and protein levels,and stimulated LDLR transcription factor SREBP-2 protein expression.As indicated from in vivo studies,compared with control group,acacetin significantly decreased the serum levels of TC and LDL-C in C57BL/6 J mice by 34%and 57%(P<0.01),respectively.Furthermore,mechanic study showed that acacetin significantly increased the protein expression of hepatic LDLR and SREBP-2.Although the results of serum lipid profiles,hepatic LDLR/SREBP-2 expression and area of atherosclerotic lesions in aorta and aortic root in ApoE KO mice showed differences between acacetin and high-fat diet group,the differences did not reach statistical significance.Nevertheless,acacetin exhibited a profound influence on the composition of the intestinal microbiota as indicated by 16s rDNA sequencing analysis.In conclusion,these results demonstrated that acacetin can decrease the serum lipid levels in C57BL/6 J mice through up-regulation of hepatic LDLR and SREBP-2,and alter gut microflora in high-fat diet fed Apo KO mice.This study suggests the possibility that acacetin has a potential role in inhibiting the progression of atherosclerosis.