基于PPARγ通路的冠心病痰瘀互结证小鼠发病机制研究

Study on pathogenesis of mice model of coronary heart disease with phlegm-blood stasis syndrome based on PPARγ pathway

目的基于过氧化物酶增殖物激活受体γ(PPARγ)通路探讨冠心病痰瘀互结证小鼠模型的发病机制。方法健康C57BL/6J雄性小鼠随机分为对照组和假手术组,载脂蛋白E基因敲除(ApoE-/-)雄性小鼠随机分为血瘀组、痰浊组、痰瘀互结组。痰浊组和痰瘀互结组小鼠给予高脂饲料喂养12周,其余各组小鼠给予普通饲料喂养12周。在8周末时,对血瘀组和痰瘀互结组小鼠进行冠状动脉左前降支结扎,假手术组小鼠行穿线不结扎。酶联免疫法测定血清白细胞介素-6(IL-6)、内皮素(ET)、血管紧张素II(Ang II)、PPARγ水平,免疫印迹法检测肝脏组织PPARγ、ATP结合盒转运蛋白A1(ABCA1)、CD36蛋白表达水平,免疫组化方法检测主动脉基质金属蛋白酶-9(MMP-9)、CD40、核转录因子-κB(NF-κB)蛋白表达水平。结果与假手术组比较,各组小鼠血清中IL-6水平未出现显著变化,痰瘀互结组小鼠血清ET水平显著升高(P<0.01),血瘀组小鼠血清中Ang II水平显著升高(P<0.05),痰浊组和痰瘀互结组小鼠血清中Ang II水平显著升高(P<0.01),PPARγ水平降低;在肝脏组织中,血瘀组、痰浊组和痰瘀互结组小鼠PPARγ、ABCA1蛋白表达水平显著降低(P<0.01),CD36蛋白表达水平升高;主动脉组织中CD40、MMP-9、NF-κB蛋白表达水平显著升高(P<0.01)。结论冠心病痰瘀互结证发病过程中会造成较为严重的动脉粥样硬化斑块,可能是通过激活PPARγ通路实现的。

Objective To explore the pathogenesis of phlegm-blood stasis syndrome in mice model of coronary heart disease based on PPAR gamma pathway.Methods Healthy SPF C57 BL/6 J mice were used in the control group and the sham operation group,and ApoE-/-mice were used in the blood stasis group,phlegm turbid group and phlegm-blood stasis group.The phlegm turbid group and the phlegm-blood stasis group were fed with high-fat diet for 12 weeks,and the other groups were fed with normal feed for 12 weeks.At the end of the 8 th week,the left anterior descending coronary artery was ligated in the blood stasis group and the phlegm-blood stasis group.The sham operation group was not ligated.The levels of IL-6,ET,Ang II and PPARγin serum were measured by enzyme linked immunosorbent assay,the levels of PPARγ,ABCA1 and CD36 protein in liver tissue were detected by Western blotting,and the levels of CD40,MMP-9 and NF-κB protein in aorta were detected by immunohistochemistry.Results Compared with sham operation group,there was no significant change in serum IL-6,the content of serum ET in the group of phlegm and blood stasis was increased significantly(P<0.01),the content of Ang II in blood stasis group was increased significantly(P<0.05),the content of serum Ang II in phlegm turbid group and phlegm-blood stasis group was increased significantly(P<0.01),and the content of PPARγwas decreased.In liver tissue,the expression levels of PPARγand ABCA1 protein in blood stasis group,phlegm turbid group and phlegm-blood stasis group were decreased significantly(P<0.01),the expression of CD36 protein was increased.CD40,MMP-9 and NF-κB levels in aorta tissue were increased significantly(P<0.01).Conclusion The phlegm-blood stasis syndrome of coronary heart disease can cause more serious atherosclerotic plaque in the course of its onset.Its mechanism may be through activating PPARγpathway.