温阳活血解毒方稳定ApoE^(-/-)小鼠动脉粥样硬化斑块机制研究

Mechanism Study on Yang-Warming,Blood-Activating,Toxins-Removing Recipe for Stabilizing Atherosclerotic Plaque in ApoE^(-/-)mice

【目的】探讨温阳活血解毒方防治动脉粥样硬化(AS)不稳定斑块发生发展的可能作用机制。【方法】以高脂饲料喂养50只8周龄雄性载脂蛋白E基因敲除(ApoE^(-/-))小鼠13周,复制动脉粥样硬化模型。将模型小鼠随机分为模型组、辛伐他汀组(2.6 mg·kg^(-1)·d^(-1))和中药低、中、高剂量组(14.08、28.16、56.32 g·kg^(-1)·d^(-1)),每组9只。分组后次日起各给药组开始灌胃给药,每日1次,连续灌胃给药13周;模型组灌胃等体积生理盐水。采用苏木素-伊红(HE)染色观察主动脉组织病理学变化;用干化学法在全自动生化仪上检测血清中总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)水平;分别采用蛋白免疫印迹(Western Blot)法和实时荧光定量聚合酶链式反应(RT-qPCR)法检测主动脉组织中小凹蛋白1(Caveolin^(-1))和磷酸化细胞外信号调节蛋白激酶1/2(p-ERK1/2)的蛋白及mRNA表达水平。【结果】与模型组比较,中药高剂量组及辛伐他汀组小鼠主动脉胆固醇结晶明显减少,斑块明显趋于稳定,血清中TC、TG、LDL-C水平明显降低,HDL-C水平明显升高(均P<0.01),主动脉组织中Caveolin^(-1)蛋白的表达量明显增加,p-ERK1/2蛋白的表达量明显降低(均P<0.05)。与模型组比较,各给药组主动脉组织Caveolin^(-1) m RNA、p-ERK1/2 mRNA表达水平均无明显变化,差异无统计学意义(P>0.05)。且与辛伐他汀组比较,中药高剂量组在稳定斑块、调节血脂水平、Caveolin^(-1)蛋白表达水平方面无明显差异(P>0.05)。【结论】温阳活血解毒方可调节血脂水平,增加动脉粥样硬化斑块稳定性,其机制可能与上调Caveolin^(-1)蛋白表达抑制ERK1/2信号通路的激活有关。

Objective To explore the possible therapeutic mechanism of Yang-Warming, Blood-Activating,Toxins-Removing Recipe(YBTR)for the formation and progress of unstable plaque of atherosclerosis(AS).Methods Fifty 8-week-old male ApoE^(-/-)mice were induced into atherosclerosis model by feeding with high-fat diet for 13 weeks. And then the model mice were randomly divided into model group,simvastatin group(2.6 mg·kg^(-1)·d^(-1)),low-,middle-and high-dose YBTR groups(14.08,28.16,56.32 g·kg^(-1)·d^(-1)),9 mice in each group. From the following day the medication groups were given intragastric administration of corresponding medicine for 13 continuous weeks, once a day, and the model group was given intragastric administration of the same volume of normal saline. After medication, the histopathological features of mouse aorta tissue were observed by hematoxylin eosin(HE)staining. The levels of serum total cholesterol(TC), triglyceride(TG), low density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C)were detected by automatic biochemical analyzer by dry chemistry method. The protein and m RNA expression levels of Caveolin^(-1) and phosphorylation of extracellular singal-regulated protein kinase(p-ERK1/2)in aortic tissue were detected by Western blotting assay and real-time fluorescence quantitative polymerase chain reaction(RT-qPCR),respectively. Results Compared with the model group, the cholesterol crystals were significantly reduced in the high-dose YBTR group and simvastatin group, and plaque tended to be stable obviously, the serum TC, TG and LDL-C levels were decreased,HDL-C level was increased(all P < 0.01),the protein expression level of Caveolin^(-1) in aortic tissue was increased,the expression protein level of p-ERK1/2 was decreased(all P < 0.05). Compared with the model group,the mRNA expression levels of Caveolin^(-1) in aortic tissue had no obvious changes(P>0.05). There were no statistically significant differences in lipid metabolism,plaque stability,and Caveolin^(-1) protein expression between the high-dose YBTR group and simvastatin group(P>0.05). Conclusion YBTR is effective for regulating blood lipid level and increasing atherosclerotic plaque stability,the mechanism possible is associated with the upregulation of Caveolin^(-1) expression and suppression of activation of ERK1/2 signaling pathway.