异氟醚预处理对谷氨酸诱导大鼠神经元样PC12细胞凋亡的影响

Effect of isoflurane preconditioning on glutamate-induced apoptosis in rat neuronal PC12 cells

目的探讨异氟醚预处理对谷氨酸诱导大鼠神经元样PCI2细胞凋亡的影响。方法神经生长因子孵育5d的神经元样PCI2细胞，以5×10^4个／ml密度接种于6cm培养皿（3ml／皿）或6孔板（2ml／孔），采用随机数字表法，将其随机分为4组（n=18）：正常对照组（C组）、谷氨酸组（G组）、谷氨酸＋异氟醚组（GI组）和谷氨酸＋异氟醚＋三磷酸肌醇受体拮抗剂光溜海绵素组（GIX组）。C组不做任何处理；G组、GI组和GIX组均加入500／xmol／L谷氨酸，GI组和GIX组通人1．2％异氟醚2h，停止通入后10min加入谷氨酸，GIX组通人异氟醚前即刻加入光溜海绵素100nmol／L。于谷氨酸孵育20min时，每组取6皿和6孔，收集细胞，采用流式细胞术检测细胞凋亡率和线粒体膜电位（MMP），采用显微荧光测量技术检测细胞内钙离子浓度（[Ca^2＋]i）。结果与C组比较，G组和GIX组细胞凋亡率和[Ca^2＋]i升高，MMP降低（P〈0．01），GI组上述指标差异无统计学意义（P〉0．05）；与G组比较，GI组和GIX组细胞凋亡率和[Ca^2＋]i降低，MMP升高（P〈0．05或0．01）；与GI组比较，GIX组细胞凋亡率和[Ca^2＋]i升高，MMP降低（P〈0．01）。结论异氟醚预处理可抑制大鼠神经元样PC12细胞凋亡，其机制与激活内质网三磷酸肌醇受体，抑制内质网释放Ca^2＋提高MMP有关。

Objective To investigate the effect of isoflurane preconditioning on glutamate-induced apoptosis in rat neuronal PC12 cells.Methods The PC12 ceils were cultured for 5 d with nerve growth factor in vitro. The cells were seeded into 6-cm-diameter culture dishes （3 ml/dish） or 6-well plates （2 ml/well） with the density of 5 × 10^4/ml and randomly divided into 4 groups （n = 18 each）： normal control group （group C）; glutamate group （group G）;glutamate ＋ isoflurane group （group GI） and glutamate ＋ isoflurane ＋xestospongin C （an antagon of inositul trisphosphate receptors） group （group （;IX）. The neuronal PC12 ceils were exposed to glutamate 500 μmol/L in groups G, GI and GIX. The neuronal PC12 cells were exposed to 1.2% isoflurane for 2 h in groups GI and GIX. Xestospongin C was added to the culture medium immediately before isoflurane preconditioning. Glutamate was added to the culture medium at 10 min after isoflurane preconditioning in groups GI and GIX. The cells were collected from six dishes or wells in each group after being incubated with glutamate for 20 min. The apoptosis and mitoehondiral membrane potential （MMP） were assessed by flow cytometry. IntracellularCa^2＋ eoncentration （[ Ca^2＋] i）was detected by confocal fluorescence microscopy. Results Compared with group C, the apoptotic rate and [Ca^2＋]i were significantly increased and MMP was decreased in groups G and GIX （P 〈 0.01 ）, but there was no significant difference in the variables mentioned above in group GI （P 〉 0.05）. Compared with group G, the apoptotic rate and [Ca^2＋ ]i were significantly decreased and MMP was increased in groups GI and GIX （ P 〈 0.05 or 0.01）. Compared with group GI, the apoptotic rate and [Ca^2＋]i were significantly increased and MMP was decreased in group GIX （ P 〈 0.01 ）. Conclusion Isoflurane preconditioning can inhibit apoptosis in rat neuronal PCl2 cells by activating inositol trisphosphate receptors, inhibiting Ca^2＋ release from the endoplasmic reticulum and increasing MMP.