JNK信号通路在异氟烷预处理减轻脑缺血再灌注损伤中的作用

Role of c-Jun N-terminal kinase signaling pathway in isoflurane preconditioning against cerebral ische-mia/reperfusion injm-y

目的评价c—Jun氨基末端激酶（Jun N—terminal kinase，JNK）信号通路在异氟烷（isoflurane，ISO）预处理减轻脑缺血再灌注（ischemia／reperfusion，I／R）损伤中的作用，探讨JNK信号通路与细胞凋亡的关系。方法采用四血管阻断法制作全脑缺血再灌注模型。选择清洁级雄性SD大鼠40只，按随机数字表法将其分为假手术组（s组）、缺血再灌注伤组（I／R组）、JNK信号通路抑制剂SP600125＋I／R组（SP＋I／R组）、ISO预处理组（ISO组）和ISO预处理＋JNK信号通路抑制剂SP600125组（ISO＋sP组），每组8只。预处理方法为连续5d吸入15∥LISO，1h／d。末次预处理24h后行I／R，72h后取脑组织作常规HE染色观察其组织形态学变化，并用TUNEL法检测海马中神经细胞的凋亡率，用免疫组化染色检测caspase-3在海马神经细胞中的表达，采用Westernblot法检测海马组织P—JNK蛋白表达。结果与S组比较，其余各组的脑损伤评分、神经细胞凋亡率、caspase-3表达均升高（P〈0．05），I／R组的P—JNK蛋白表达升高（P〈0．05），而SP＋I／R组、ISO组、ISO＋SP组的p—JNK蛋白表达与s组比较差异无统计学意义（P〉0．05）；与I／R组比较，SP＋I／R组、ISO组、ISO＋SP组的脑损伤评分、神经细胞凋亡率、caspase-3表达、P—JNK蛋白表达均降低（P〈0．05）；与SP＋I／R组和ISO组比较，ISO＋SP组脑损伤评分、神经细胞凋亡率、caspase-3表达进一步降低（P〈0．05），P—JNK蛋白表达差异无统计学意义；SP＋I／R组和ISO组比较各项指标差异无统计学意义。结论ISO预处理可通过抑制JNK信号通路、下调caspase-3的表达来减轻脑缺血再灌注损伤。

Objective To evaluate the role of c-Jun N-terminal kinase （JNK） signaling pathway in isoflurane （ISO） preconditioning against cerebral ischemia/reperfusion （I/R） injury and investigate the relationship between JNK signaling pathway and apoptosis. Methods Global cerebral I/R models were made by 4-artery occlusion technique. Forty male SD rats of clean grade were divided into sham operation group （S group）, I/R injury group （I/R group）, SP600125 （an inhibitor of JNK signaling pathway） ＋ I/R group （ SP ＋ I/R group）, ISO preconditioning group （ ISO group）, and ISO preconditioning ＋ SP600125 group （ISO ＋ SP group） according to the random number table. Preconditioning protocol was successive inhalation of 15 g/L ISO for 5 days, 1 h/d. I/R was induced at 24 hours after the end of preconditioning. Brain tissues were harvested at 72 hours later to take histomorphological examination by HE staining as well as detect apoptosis of hippocampal nerve cells by TUNEL method, expression of caspase-3 in hippocampal nerve cells by immuno-histochemistry, and expression of protein p-JNK in hippocampal tissues by Western blot. Results Compared with S group, brain injury score, apoptosis ratio of nerve cells, and expression of caspase-3 were significantly increased in the other groups （ P 〈 0.05 ）.Moreover, p-JNK protein had a higher expression in IR group than in S group （ P 〈 0.05 ） , but no significant difference was observed in SP ＋ I/R group, ISO group, and ISO ＋ SP group as compared with S group （ P 〉 0.05 ）. Compared with IZR group, brain injury score, apoptosis ratio of nerve cells, expres- sion of caspase-3, and expression of p-JNK protein were all declined in SP ＋ IVR group, 1SO group, and ISO ＋ SP group （P 〈 0.05 ）. Moreover, brain injury score, apoptosis ratio of nerve cells, and expression of caspase-3 had further decline in ISO ＋ SP group as compared with SP ＋ I/R group and ISO group （ P 〈 0.05 ）, but the difference in expression of p-JNK protein was insignificant among the three groups. Compared with SP ＋ I/R group, no significant changes of each index were found in ISO group. Conclusion ISO preconditioning alleviates cerebral 1/R injury through down-regulating expression of caspase-3 and inhibiting JNK signaling pathway.