清热化痰活血方药稳定ApoE基因敲除小鼠主动脉易损斑块及与蛋白酶体通路的关系

Relationship between stabilization effect of Qingre Huatan Huoxue Formula on vulnerable plaques of aorta artery and UPP in ApoE knockout mice

目的:观察清热化痰活血方对Apo E基因敲除小鼠主动脉粥样硬化易损斑块的稳定作用及对泛素通路的影响。方法:Apo E基因敲除小鼠80只,自8周龄时予以高脂饮食,随机分为空白对照组、蛋白酶体抑制剂组、蛋白酶体抑制剂加清热化痰活血方组及清热化痰活血方组,每组20只。自13周始给药,分别给予0.9%氯化钠溶液10mg·kg-1·d-1,硼替佐米3.0mg·kg-1·d-1,硼替佐米3.0mg·kg-1·d-1加清热化痰活血方180mg·kg-1·d-1及清热化痰活血方180mg·kg-1·d-1,17周后评估主动脉病理形态学改变,免疫荧光法测定主动脉斑块中单核细胞/巨噬细胞-2(MOMA-2)的表达、免疫组化法测定NF-кB的P65亚基的表达及CD40L表达,荧光探针的方法测定超氧化物阴离子自由基的表达。结果:蛋白酶体抑制剂加清热化痰活血方组与蛋白酶体抑制剂组比较主动脉的MOMA-2的荧光表达、CD40L的表达、NF-кB的P65亚基的表达、超氧化物阴离子自由基的表达均无显著变化,但斑块的脂质核含量有显著下降(P<0.01);清热化痰活血方组与其他各组比较,主动脉的MOMA-2的荧光表达、NF-кB的P65亚基及CD40L的表达、超氧化物阴离子自由基及CD40L的表达、斑块的脂质核含量均有显著下降(P<0.01)。结论:清热化痰活血方可以显著抑制主动脉动脉粥样硬化的炎性因子的表达,其机制与部分抑制泛素蛋白酶体通路有关,蛋白酶体可能是中药作用的一个靶点。

Objective：To observe the stabilization effect of Qingre Huatan Huoxue Formula（QHHF）on vulnerable plaques of aorta artery and ubiquitin-proteasome pathway（UPP） in ApoE knockout mice.Methods：80 male ApoE knockout mice were fed with high-fat diet since 8 weeks of age.They were randomly divided into control group,proteasome inhibitor group,proteasome inhibitor combined with QHHF group and QHHF group,and with 20 mice in each group.Mice in all the groups received gavage administration with normal saline（10mg·kg^（-1）·d^（-1））,bortezomib（3.0mg·kg^（-1）·d^（-1））,bortezomib（3.0mg·kg^（-1）·d^（-1））combined with QHHF（180mg·kg^（-1）·d^（-1）） and QHHF（180mg·kg^（-1）·d^（-1））respectively since the 13 th week.After 17 weeks,the pathologic morphological changes of aorta was evaluated,the expression of Monocyte/macrophages 2（MOMA-2） in aorta plaque was measured by using immunofluorescence method,the expression of nuclear factor-kappa B（NF-κB/p65） and CD40 L was determined by using immunohistochemical method,and expression of super oxide anion radical were detected by using fluorescent probe technique.Results：The difference in expression of MOMA-2,CD40 L,NF-κB/p65 and super oxide anion radical between proteasome inhibitor combined with QHHF group and proteasome inhibitor group was not significant,but the content of lipid in plaques of mice in proteasome inhibitor combined with QHHF group was decreased significantly（P〈0.01）.Compared with the other groups,the expression of MOMA-2,CD40 L,NF-κB/p65,super oxide anion radical and the content of lipid in plaques of mice in QHHF group were all decreased significantly（P〈0.01）.Conclusion：QHHF could inhibit the expression of inflammatory factors of aortic atherosclerosis obviously,and the mechanism might relate with the partial inhibition effect on UPP which might be one of traditional Chinese medicine targets.