PI3K／Akt信号通路在乳化异氟醚预先给药减轻乳鼠心肌细胞缺氧复氧损伤中的作用

Role of PI3K/Akt signal pathway in anoxia-reoxygenation injury attenuated by emulsified isoflurane pretreatment in neonatal rat cardiomyocytes

目的评价磷脂酰肌醇-3激酶／蛋白质丝氨酸苏氨酸激酶（PI3K／Akt）信号通路在乳化异氟醚预先给药减轻乳鼠心肌细胞缺氧复氧损伤中的作用。方法Wistar乳鼠60只，乳鼠心肌细胞于24孔培养板原代培养后，采用缺氧2h复氧2h建立缺氧复氧模型。乳鼠心肌细胞随机分为6组，每组8孔，正常对照组（C组）：按正常条件继续培养4h；A／R组：制备缺氧复氧模型；EI组：于细胞缺氧前即刻在培养液中加入乳化异氟醚，终浓度为1．68mmol／L；EI＋wortmannin（PI3K特异性抑制剂）组（EIW组）：于细胞缺氧前即刻在培养液中加入乳化异氟醚和wortmannin，终浓度分别为1．68mmol／L和100nmol／L；wortmannin组（W组）：于细胞缺氧前即刻在培养液中加入wortmannin，终浓度为100nmol／L；脂肪乳组（F组）：与细胞缺氧前即刻在培养液中加入30％脂肪乳，终浓度为0．05％。复氧2h时，取细胞培养上清液测定乳酸脱氢酶（LDH）活性、肌钙蛋白I（cTnI）浓度，心肌细胞匀浆后测定超氧化物歧化酶（SOD）活性与丙二醛（MDA）含量，并测定心肌细胞磷酸化Akt（p-Akt）的表达水平。结果与C组比较，其余5组上清液LDH活性、cTnI浓度和心肌细胞MDA含量升高，心肌细胞SOD活性降低（P〈0．05）；与A／R组比较，EI组和EIW组上清液LDH活性、cTnI浓度和心肌细胞MDA含量降低，心肌细胞SOD活性升高，F组除心肌细胞SOD活性升高外（P〈0．05），其余指标差异均无统计学意义（P〉0．05），W组上述指标差异无统计学意义（P〉0．05）；与EI组比较，EIW组、W组和F组上清液LDH活性、cTnI浓度和心肌细胞MDA含量升高，心肌细胞SOD活性降低（P〈0．05）。C组、EIW组、H／R组、F组和EI组心肌细胞p-Akt表达水平依次升高（P〈0．05）。结论乳化异氟醚预先给药减轻乳鼠心肌细胞缺氧复氧损伤可能与进一步激活PI3K／Akt信号通路有关。

Objective To evaluate the role of PI3K/Akt signal pathway in anoxia-reoxygenation （A/R） injury attenuated by emulsified isoflurane pretreatment in neonatal rat cardiomyocytes. Methods The cardiomyocytes of 60 neonatal Wistar rats （1-3 days old） were incubated in 24 well culture plates and randomly assigned into 6 groups （ n = 8 each） ： control group （group C） ; A/R group ; EI group; EI ＋ wortmannin （specific inhibitor of PI3K） group （group EIW） ; wortmannin group （group W） ; fat emulsion group （group F）. In group C, the cardiomyocytes were cultured for 4 h. In group A/R, the cardiomyocytes were exposed to 95% N2-5% CO2 saturated ischemic solution for 2 h followed by 2 h reoxygenation with 95 % O2-5 % CO2 saturated reperfusion solution. In group EI, emulsified isofiurane was added to the culture medium immediately before cardiomyocyte hypoxia with the final concentration of 1.68 mmol/L. In group EIW, emulsified isoflurane and wortmannin were added to the culture medium in combination immediately before cardiomyocyte hypoxia with the final concentrations of 1.68 mmol/L and 100 nmol/L respectively. In group W and F, wortmannin and 30% fat emulsion were added to the culture medium immediately before cardiomyocyte hypoxia with the final concentration of 100 nmol/L and 0.05% respectively. The supematant was taken from culture medium to measure LDH activity and cTnI concentration, and myocardial cell homogenate was prepared to measure SOD activity and MDA content and to detect phosphorylation-Akt （p-Akt） expression by Western blot at the end of 2 h reoxygenation. Results The supernatant LDH activity, cTnI concentration and cardiomyocyte MDA content were significantly higher, while SOD activity lower in the other 5 groups than in group C （ P 〈 0.05）. The supernatant LDH activity, cTnI concentration and cardiomyocyte MDA content were significantly lower, while cardiomyocyte SOD activity higher in group EI and EIW, and cardiomyocyte SOD activity higher in group F than in group A/R （P 〈 0.05）, there was no significant difference in the above parameters between group W and A/R （P 〉 0.05）. The supernatant LDH activity, cTnI concentration and cardiomyocyte MDA content were significantly higher, while cardiomyocyte SOD lower in group EIW, W, and F than in group EI （ P 〈 0.05）. The cardiomyocyte p-Akt expression was significantly increased in ascending order from group C, EIW, A/R, F to group EI （P 〈 0.05 ）. Conclusion Emulsified isoflurane pretreatment can attenuate A/R injury in neonatal rat cardiomyocytes via activation of PI3K/Akt signal pathway.