丹蒌片调控ERS通路诱导血管平滑肌细胞凋亡的实验研究

Experimental study on apoptosis of vascular smooth muscle cells of Danlou Tablet in regulating endoplasmic reticulum stress pathway

目的:观察丹蒌片对同型半胱氨酸(HCY)诱导增殖的血管平滑肌细胞(VSMC)凋亡及内质网应激蛋白(BIP)表达的影响。方法:取第2-5代原代培养的大鼠腹主动脉VSMC,除空白组外,均加入10-4mol/L的HCY培养24h,经过免疫组化检验特异性标志蛋白α-SMA确认,建立大鼠增殖的VSMC模型。以加入药物不同分为空白组,HCY组、瑞舒伐他汀(可定)组、丹蒌片组、丹蒌片+可定组。其中可定组、丹蒌片组、丹蒌片+可定组分别加入相应的含药血清,继续培养48h,Annexin V-PI法检测细胞凋亡率,Western blotting检测BIP蛋白表达情况。结果:免疫组化中α-SMA表达呈阳性率超过98%,提示培养细胞为VSMC;HCY组凋亡率显著低于空白组(P<0.01),与HCY组比较,可定组、丹蒌片组、丹蒌片+可定组可显著增强大鼠VSMC凋亡率(P<0.01);Western blotting检测,与空白组比较,HCY组BIP表达升高(P<0.01);与HCY组比较,可定组、丹蒌片组、丹蒌片+可定组细胞BIP蛋白表达水平明显上调(P<0.05,P<0.01),可定组、丹蒌片组、丹蒌片+可定组组间比较,BIP蛋白表达水平无明显差异。结论:丹蒌片通过上调BIP表达诱导VSMC凋亡。

Objective： To study the effect of Danlou Tablets on the apoptosis and expression of BIP of homocysteine- induced production VSMCs. Methods： Making proliferation of rat VSMC models induced by adding HCY in density of 10^-4 mol/L to the rat aortic vascular smooth muscle cells between second to fifth generations except the blank group, which were confirmed by specific marker protein α -SMA in immunohistochemistry way. Then adding serum containing drugs additionally to Rosuvastatin group, Danlou Tablet group and Danlou Tablet＋Rosuvastatin group and cultured the cells 48hs. At last, apoptosis rate and expression of BIP protein were detected by Annexin V-PI and Western blotting. Results expression of α -SMA positive rate was higher than 98% which suggested the success of culturing VSMCs. Apoptosis rate in HCY group was significantly lower than control group （P〈0.01）; Compared with HCY group, Rosuvastatin group, Danlou Tablet group, Danlou Tablet＋Rosuvastatin group could significantly enhance the rate of VSMC apoptosis （P〈0.01）. During the Western blotting progress, expression of BIP protein in HCY group was higher than control group （P〈0.01）, and so it was in Rosuvastatin group, Danlou Tablet group, Danlou Tablet＋Rosuvastatin group compared with HCY group （P〈0.05, P〈0.01）. And there was no significant difference in the expression of protein among the last three groups. Conclusion： Danlou Tablet could induce the apoptosis of vascular smooth muscle cell by up-regulating the expression of BIP protein.