GRP78在七氟醚预处理抑制大鼠心肌细胞凋亡中的作用

Role of GRP78 in sevoflurane preconditioning-induced inhibition of apoptosis in cardiomyocytes of rats

目的探讨葡萄糖调节蛋白78（GRP78）在七氟醚预处理抑制大鼠心肌细胞凋亡中的作用。方法采用随机数字表法将培养的大鼠心肌细胞分5组（n=30），正常对照组（c组）：细胞不经任何处理；缺氧复氧组（H／R组）：细胞在95％N2-5％CO2混合气体中缺氧2h后放回95％空气-5％CO，37℃培养箱内复氧1h；七氟醚预处理组（s组）：细胞经2．5％七氟醚孵育20min，洗脱10min后进行缺氧复氧；siRNA—GRP78组：采用siRNA—GRP78100nmol／L转染心肌细胞，24h后进行七氟醚预处理及缺氧复氧处理；siRNA对照组（siRNA组）：采用随机序列核酸siRNA转染心肌细胞，其它处理同siRNA-GRP78组。各组处理结束后，采用Westernblot法测定心肌细胞GRP78、胞浆和线粒体细胞色素C的表达；采用ELISA法测定培养液LDH和CK的活性；采用流式细胞仪测定细胞凋亡率；采用Fura-2／AM钙离子荧光探针法测定细胞内游离Ca^2＋浓度（[Ca^2＋]．）；采用荧光分光光度法测定线粒体膜通透性转运孔（mPTP）开放程度。结果与C组比较，H／R组心肌细胞GRP78和胞浆细胞色素C表达上调，培养液LDH和CK活性、细胞凋亡率、[Ca^2＋]，升高，线粒体细胞色素e表达下调（P〈0．01）；与H／R组比较，S组心肌细胞GRP78和线粒体细胞色素e表达上调，培养液LDH和CK活性、细胞凋亡率、[Ca^2＋’]，和mPTP开放程度降低，胞浆细胞色素C表达下调（P〈0．01），siRNA组上述指标差异无统计学意义（P〉0．05）；与s组比较，siRNA—GRP78组心肌细胞GRP78和线粒体细胞色素C表达下调，培养液LDH和CK活性、细胞凋亡率、[Ca^2＋]．和mPTP开放程度升高，胞浆细胞色素C表达上调（P〈0．01）。结论GRP78参与了七氟醚预处理抑制大鼠心肌细胞凋亡的作用，机制与维持细胞内Ca^2＋稳态，抑制mPTP开放有关。

Objective To investigate the role of glucose-regulated protein 78 （GRP78） in sevoflu- rane preconditioning-induced inhibition of apoptosis in cardiomyoeytes of rats. Methods The cultured car- diomyoeytes of Sprague-Dawley rats were randomly divided into 5 groups （ n = 30 each） using a random number table： control group （group C） , hypoxia-reoxgenation （H/R） group, sevoflurane preconditioning group （group S）, siRNA-GRP78 group and siRNA control group. H/R was produced by 2 h exposure of cells to 95% N2-5% CO2 in an air-tight chamber at 37℃ , followed by reoxygenation with 95% 02-5% CO2 in an air-tight chamber at 37 ℃ for 1 h. In group S, the cells were incubated with 2.5% sevoflurane for 20min, followed by 10-rain washout before H/R. In siRNA-GRP78 group, the cells were transfected with siRNA-GRP78 100 nmol/L, and 24 h later preconditioning with sevoflurane was performed and H/R was produced. In siRNA group, cells were transfeeted with siRNA, and the other treatments were similar to those previously described in siRNA-GRP78 group. After treatment in each group, the expression of GRP78 in myocardial cells and cytochrome c in cytoplasm and mitochondria was detected by Western blot. Lactic dehydrogenase （LDH） and creatine kinase （CK） activities in the culture medium of each group were deter- mined by ELISA. The apoptosis in myocardial cells was assessed by flow cytometry. Apoptotic rate was cal- culated. Intracellular free Ca2. concentration （ [ Ca^2＋ ] i ） was measured with the fluorescent probe Fura-2/ AM. The opening of mPTP was measured by fluorescence spectrophotometry. Results Compared to group C, the expression of GRP78 in myocardial cells and cytochrome c in cytoplasm was significantly up-regula- ted, LDH and CK activities in the culture medium, apoptotic rate and [ Ca^2＋ ] i were increased, and the expression of cytochrome c in mitochondria was down-regulated in H/R group. Compared to group H/R, the expression of GRP78 in myocardial cells and cytochrome c in mitochondria was significantly up-regulated, LDH and CK activities in the culture medium, apoptotic rate, [ Ca^2＋ ]i and opening of mPTP were de- creased, and the expression of cytochrome c in cytoplasm was down-regulated in group S, and no signifi- cant change was found in the parameters mentioned above in siRNA group. Compared to group S, the ex- pression of GRP78 in myocardial cells and cytochrome c in mitochondria was significantly down-regulated, LDH and CK activities in the culture medium, apoptotic rate, [ Ca^2＋ ] i and opening of mPTP were in- creased, and the expression of cytochrome c in cytoplasm was up-regulated in group siRNA-GRP78. Con- clusion GRP78 is involved in sevoflurane preconditioning-induced inhibition of apoptosis in cardiomyocytes of rats, and the mechanism is related to maintenance of intracellular Ca^2＋ stability and inhibition of opening of mPTP.