补阳还五汤通过调节血管平滑肌细胞Cx43作用于动脉粥样硬化的研究

The research of Buyang Huanwu Tang anti-atherosclerosis through regulating Cx43 of HUVSMC

目的:研究补阳还五汤抗同型半胱氨酸(Hcy)诱导血管平滑肌细胞(HUVSMC)增殖的作用及其机制。方法:以14.9g/kg补阳还五汤剂量连续灌胃SD大鼠1周,提取补阳还五汤含药血清,并与胎牛血清(FBS)按一定比例,配置成20%(20%补阳)、10%(10%补阳+10%FBS)、5%(5%补阳+15%FBS)补阳还五汤含药血清组组。血管平滑肌细胞长满培养瓶或培养板后,用同型半胱氨酸分组造模,将细胞随机分为10组:空白组、模型组、乙酰半胱氨酸(NAC)组、20%、10%、5%补阳还五汤含药血清组、NF-kB阻断剂PDTC组、MAPK阻断剂SB20组、ERK阻断剂PD98组、PKC阻断剂Stau组。蛋白质印迹法(Western blot)测定血管平滑肌细胞Cx43蛋白的表达量;RT-PCR技术检测各组血管平滑肌细胞Cx43 mRNA水平;建立血管内皮-平滑肌细胞共培养体系,Western blot分别检测内皮细胞和平滑肌细胞内Cx43的表达;电镜检测血管内皮-平滑肌细胞共培养体系细胞缝隙连接的变化。结果:Western blot实验中,平滑肌细胞与共培养体系中的内皮细胞、平滑肌细胞的Cx43蛋白表达水平,模型组与空白组比较,模型组中Cx43蛋白表达量均显著升高;乙酰半胱氨酸组,20%、10%、5%补阳还五汤含药血清组与模型组比较,Cx43蛋白表达显著下调。RT-PCR实验中,模型组与空白组比较,模型组中Cx43mRNA表达量显著升高;乙酰半胱氨酸组、20%、10%、5%补阳还五汤含药血清组与模型组比较,Cx43mRNA表达量显著降低。电镜检测细胞缝隙连接实验中:模型组与空白组比较,内皮-平滑肌细胞之间形成的缝隙连接遭到破坏,而20%补阳还五汤含药血清组却能形成与正常组相似的连接。结论:补阳还五汤可能通过下调Cx43蛋白以及其mRNA的表达,改善细胞之间的缝隙连接,减少血管平滑肌细胞增殖而发挥抗动脉粥样硬化的作用。

Objective：To explore the effect and mechanism of anti- cell proliferation of Buyang Huanwu Tang（BHT） by Hcy in human aortic vascular smooth muscle ceils. Methods： BHT 14.9g/kg were administered （ig） to SD rats for a week, and after administration, the serums of rats were collected. The drug groups in FBS were prepared, 20% BHT group （20% BHT in rat＇s serum）, 10% BHT group （10% BHT in rat- serum was added to 10% FBS）, 5% BHT group （5% BHT in rat＇s serum was added to 15% FBS）. HUVSMC were induced by Hcy and randomiy divided into 10 groups ： control group, model group, NAC group ,20%, 10% and 5 % BHT group, inhibitor PDTC, SB20, PD98, Stau group. The expression of Cx43 and Cx43 mRNA of HUVSMC were determined. Co-culture system of HUVSMC and HUVEC was built, The expression of Cx43 in endothelial ceils and smooth muscle ceils in co-culture system were detected respectively. Electron microscopy showed the changes of gap junction between vascular endothelial cells and smooth muscle cells. Results： HUVSMC and HUVEC in the co-culture system and HUVSMC alone, compared with control group, Cx43 proteins in model group expression have significantly increased. 20%, 10% ,5% BHT and NAC groups could decrease Cx43 protein level. In the experiment of RT-PCR, compared with control group , Cx43 mRNA expres- sions in model group have significantly increased. 20% ,10% ,5% BHT and NAC groups could decrease it. Compared with control group, cells gap junction were destroyed. 20% BHT group was similar to the control group. Conclusion： BHT could reduce the expression of Cx43 protein and its mRNA level, improve the gap junction between ceils , decrease the ceil proliferation to play the role of resistance to atheroscle-