化痰祛瘀中药抗动脉粥样硬化的作用机制研究

Anti-atherosclerotic mechanism of phlegm-transforming and stasis-resolving Chinese medicinals

目的观察化痰祛瘀中药对小鼠主动脉和血管内皮细胞(VEC)微小核糖核酸126(miR-126)及其下游信号表达的调控,研究中医药抑制动脉粥样硬化(AS)的作用机制。方法在细胞实验中,将血管内皮细胞随机分为4组,即对照组、模型组、中药高剂量(86.4 g/kg)含药血清组、中药低剂量(21.6 g/kg)含药血清组。不同组别的血管内皮细胞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RT-PCR和Western blot检测血管内皮细胞中miRNA-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RT-PCR和Western blot检测主动脉miRNA-126、RGS16、CXCL12、CXCR4、VCAM-1 mRNA和蛋白表达。结果体外实验结果显示,干预后,与对照组相比,模型组、高剂量组和低剂量组的VEC凋亡率均明显升高(P<0.05),增殖率均明显降低(P<0.05),miR-126 mRNA表达均明显降低(P<0.05),RGS16、CXCL12、CXCR4、VCAM-1 mRNA和蛋白表达均明显升高(P<0.05);而与模型组相比,高剂量组和低剂量组的VEC凋亡率均明显降低(P<0.05),增殖率均明显升高,miR-126 mRNA表达均明显升高(P<0.05),RGS16、CXCL12、CXCR4、VCAM-1 mRNA和蛋白表达均明显降低(P<0.05);与高剂量组相比,低剂量组VEC的凋亡率和增殖率无统计学差异(P>0.05),miR-126 mRNA表达无统计学差异(P>0.05),RGS16、CXCL12、CXCR4、VCAM-1 mRNA和蛋白表达无统计学差异(P>0.05)。体内实验结果显示,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;中药高剂量组和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。干预后,与对照组相比,模型组、高剂量组和低剂量组主动脉中miR-126 mRNA表达均明显降低(P<0.05),RGS16、CXCL12、CXCR4、VCAM-1 mRNA和蛋白表达均明显升高(P<0.05);与模型组相比,高剂量组和低剂量组主动脉中miR-126 mRNA表达均明显升高(P<0.05),RGS16、CXCL12、CXCR4、VCAM-1mRNA和蛋白表达均明显降低(P<0.05);与高剂量组相比,低剂量组主动脉中miR-126 mRNA表达无统计学差异(P>0.05),RGS16、CXCL12、CXCR4、VCAM-1 mRNA和蛋白表达无统计学差异(P>0.05)。结论化痰祛瘀中药抑制动脉粥样硬化斑块形成的作用机制可能与调控miRNA-126,影响其下游信号GRS16、CXCL12、CXCR4、VCAM-1表达有关。

Objective To observe the regulation of miRNA-126 and its downstream signal expression in mouse aorta and vascular endothelial cells by observation of phlegm and blood stasis method to study the mechanism of Chinese medicine in inhibiting atherosclerosis. Methods In the cell experiment, the vascular endothelial cells were randomly divided into 4 groups: control group, model group, high-dose Chinese medicine-containing serum group, and low-dose Chinese medicine-containing serum group. Different groups of vascular endothelial cells were treated with different drug-containing serum to observe cell proliferation and apoptosis. RT-PCR and Western-blot assay were used to detect miRNA-126, RGS16, CXCL12, CXCR4, VCAM-1, mRNA and protein expression in vascular endothelial cells. The aortic cross-sectional pathological damage was evaluated with HE staining in vivo. The aortic miRNA-126, RGS16, CXCL12, CXCR4, VCAM-1 mRNA and protein expression were measured with RT-PCR and Western-blot methods. Results The results of in vitro experiments showed that the apoptosis rates of VEC in the model group, high-dose group and low-dose group were significantly higher than that in the control group(P<0.05), and the proliferation rate was significantly decreased(P<0.05). Compared with the model group, the apoptotic rate of VEC was significantly decreased in the high-dose group and the low-dose Chinese medicine-containing serum group(P<0.05), and the proliferation rate was significantly increased. Compared with the high-dose group, there was no significant difference in apoptotic rate and proliferation rate(P>0.05). After intervention, the expressions of miR-126 mRNA in the VEC of the model group, the high-dose group and the low-dose group were significantly lower than that of the control group(P<0.05). Compared with the model group, the expression of the miR-126 mRNA in the VEC of the high-dose group and the low-dose group was significantly increased(P<0.05). Compared with the high-dose group, there was no significant difference in the expression of miR-126 mRNA in the low-dose group(P>0.05). After intervention, the expressions of RGS16, CXCL12, CXCR4, VCAM-1 mRNA and protein in the VEC of the model group, high-dose group and low-dose group were significantly higher than those of the control group(P<0.05). Compared with the model group, the expressions of RGS16, CXCL12, CXCR4, VCAM-1 mRNA and protein were significantly decreased in high-dose group and low-dose group(P<0.05). Compared with high-dose group, there was no significant difference in RGS16, CXCL12, CXCR4, VCAM-1 mRNA and protein expression in low dose group VEC(P>0.05). The results of in vivo experiments showed that through HE staining, in the control group, the aortic vessels were even, the endometrium smooth and flat, and there was no atherosclerotic lesion;In the model group, the aortic vessels were uneven, plaque formed, and the thickness of the blood vessels and the cross-sectional area of the AS plaque were significantly larger than those of the other groups;In the high-dose group and the low-dose group, the aortic layers were normal, the inflammatory cells infiltrated lightly with mild lesions;the AS plaques were small and the lesion was significantly lighter than the model group. After intervention, the expressions of miR-126 mRNA in the arteries of the model group, high-dose group and low-dose group were significantly lower than that of the control group(P<0.05). Compared with the model group, the expression of miR-126 mRNA in the arteries was significantly increased(P<0.05). Compared with the high-dose group, there was no significant difference in the expression of miR-126 mRNA in the arteries of the low-dose group(P>0.05). After intervention, the expressions of RGS16, CXCL12, CXCR4, VCAM-1 mRNA and protein in the arteries of the model group, high-dose group and low-dose group were significantly increased compared with the control group(P<0.05). Compared with the model group, the expressions of RGS16, CXCL12, CXCR4 and VCAM-1 mRNA and protein in the arteries of the high-dose group and the low-dose group were significantly decreased(P<0.05). Compared with the high-dose group, there was no significant difference in the RGS16 and CXCL12 CXCR4 and VCAM-1 mRNA and protein expression in the arteries of the low-dose group.(P>0.05). Conclusion The mechanism of action in inhibiting the formation of atherosclerotic plaque may be related to the regulation of miRNA-126 affecting the expression of downstream signals GRS16, CXCL12, CXCR4 and VCAM-1.