冠心康对microRNA-126和VEC的调控及抗动脉粥样硬化的作用机制研究

Research of Mechanism in Guanxikang Anti-Atherosclerosis Based on Regulation of microRNA-126 and VEC

目的:观察冠心康对小鼠主动脉及血管内皮细胞miRNA-126及其下游相关信号GRS16、CXCL12、CXCR4、VCAM-1表达的调控,探索中医药抗动脉粥样硬化的作用机制。方法:体外细胞实验中,将小鼠血管内皮细胞随机分为5组,即对照组、模型组、中药高剂量含药血清组、中药低剂量含药血清组。不同组别VEC分别经不同含药血清干预后,观察细胞增殖和凋亡情况,采用qRT-PCR和Western-blot检测VEC中pri-miR-126、RGS16、CXCL12、CXCR4、VCAM-1 mRNA和蛋白表达;体内实验采用HE染色检测各组小鼠主动脉横截面病理损伤程度,采用qRT-PCR和Western-blot检测主动脉pri-miR-126、RGS16、CXCL12、CXCR4、VCAM-1 mRNA和蛋白表达。结果:体外实验结果显示,与对照组相比,中药高低剂量组的VEC凋亡率和增殖率明显优于模型组(P均<0.05),中药高剂量组和中药低剂量组VEC增殖率比较差异不明显(P>0.05);与对照组相比,中药高低剂量组VEC中miR-126、RGS16、CXCL12、CXCR4和VCAM-1 mRNA表达明显优于模型组(P<0.05),中药高剂量组和中药低剂量组间比较差异不明显(P>0.05);与对照组相比,中药高低剂量组VEC中RGS16、CXCL12、CXCR4和VCAM-1蛋白表达明显优于模型组(P<0.05),中药高剂量组和中药低剂量组间比较差异不明显(P>0.05)。体内实验结果显示,小鼠主动脉HE染色结果,对照组小鼠主动脉血管管径厚薄均匀,内膜光滑平整,无动脉粥样硬化病灶,模型组小鼠血管管径厚薄不均匀,斑块形成明显,血管管壁厚度、AS斑块截面积显著大于其他各组。中药高低剂量组小鼠主动脉各层结构正常,炎性细胞浸润较轻,病变轻,AS斑块较小,病变程度明显轻于模型组。与对照组相比,中药高低剂量组主动脉miR-126、RGS16、CXCL12、CXCR4和VCAM-1 mRNA表达明显优于模型组(P<0.05),中药高剂量组和中药低剂量组间比较差异不明显(P>0.05);与对照组相比,中药高低剂量组主动脉RGS16、CXCL12、CXCR4和VCAM-1蛋白表达明显优于模型组(P<0.05),中药高剂量组和中药低剂量组间比较差异不明显(P>0.05)。结论:冠心康抑制动脉粥样硬化斑块的形成的作用机制可能与调控miRNA-126及其下游相关信号GRS16、CXCL12、CXCR4、VCAM-1表达有关。

Objective:To observe the regulation of miRNA-126 and its downstream related signals GRS16, CXCL12, CXCR4 and VCAM-1 in mice aorta and vascular endothelial cells and explore the mechanism of anti-atherosclerosis of traditional Chinese medicine. Methods:In vitro cell experiments, mice vascular endothelial cells were randomly divided into 5 groups, namely, control group, model group, Chinese medicine high-dose drug-containing serum group and traditional Chinese medicine low-dose drug-containing serum group. Different groups’ VEC were treated with different drug-containing serum to observe cell proliferation and apoptosis. qRT-PCR and Western-blot were used to detect pri-miR-126, RGS16, CXCL12, CXCR4, VCAM-1 mRNA and protein in VEC. In vivo experiments,HE staining was used to detect the degree of pathological damage in the aorta of each group. The expressions of pri-miR-126, RGS16, CXCL12, CXCR4, VCAM-1 mRNA and protein in aorta were detected by qRT-PCR and Western-blot. Results:The results of in vitro experiments showed that compared with the control group, the apoptotic rate and proliferation rate of VEC in the high-and low-dose groups of Chinese medicine were significantly better than those in the model group(P<0.05). The proliferation rate of VEC in the high-dose group and the low-dose group of traditional Chinese medicine was not different(P>0.05). Compared with the control group, the expressions of miR-126, RGS16, CXCL12, CXCR4 and VCAM-1 mRNA in the VEC of the high-and low-dose Chinese medicine group was significantly better than that of the model group(P<0.05). There was no significant difference between the low-dose group of traditional Chinese medicine(P>0.05). Compared with the control group, the expressions of RGS16, CXCL12, CXCR4 and VCAM-1 in VEC were significantly better than those in the model group(P<0.05). There was no significant difference between the high-dose group and the low-dose group(P>0.05). The results of in vivo experiments showed that the aortic HE staining of the mice in the control group was uniform in the aortic vessel diameter. The endometrium was smooth and flat, and there was no atherosclerotic lesion. The vascular diameter of the model group was uneven. The formation was obvious, and the thickness of the vessel wall and the cross-sectional area of the AS plaque were significantly larger than those of the other groups. In the high-low dose group of Chinese medicine, the aortic layers of the mice were normal, the inflammatory cells infiltrated lightly, the lesions were light, the AS plaques were small, and the lesions were significantly lighter than those of the model group. Compared with the control group, the mRNA expressions of aorta miR-126, RGS16, CXCL12, CXCR4 and VCAM-1 in the high-and low-dose groups of Chinese medicine were significantly better than those in the model group(P<0.05) and the difference between the high-dose Chinese medicine group and the low-dose Chinese medicine group. It was not obvious(P>0.05). Compared with the control group, the expressions of RGS16, CXCL12, CXCR4 and VCAM-1 in the aorta of the Chinese medicine group were significantly better than those in the model group(P<0.05). The high and low dose groups of traditional Chinese medicine had no significant difference between the groups(P>0.05). Conclusion:The mechanism of Guanxinkang to inhibit the formation of atherosclerotic plaque may be related to the regulation of miRNA-126 and its downstream related signals GRS16, CXCL12, CXCR4 and VCAM-1 expressions.