PI3K-Akt信号通路与大鼠肝脏缺血预处理保护作用关系的研究

The Relationship between PI3K-Akt Signal Pathway and the Protective Effects of Ischemic Preconditioning in Rat Livers

目的研究PI3K-Akt信号转导系统在大鼠肝缺血再灌注(IR)及缺血预处理(IPC)后的激活规律,探讨其与IPC保护作用的关系。方法将68只SD雄性大鼠随机分成空白对照组(Sham)、缺血再灌注组(IR)、缺血预处理组(IPC)、阻断剂组(LY)、溶剂对照组(DMSO)5组,于复灌0、1/2、1、2、4h各不同时间点取材,应用免疫组化和免疫印迹法测定肝组织中Akt1、p-Akt1等信号蛋白的表达;HE染色观察肝组织显微结构;同时测定血清肝功酶水平。结果IR组大鼠在各复灌时间点上都显示明显肝细胞损伤,并随复灌时间延长加重;肝细胞质内有p-Akt1(Thr-308)表达。IPC组大鼠血清肝功酶指标在复灌0、2、4h较IR组明显降低(P<0.01),p-Akt1(Thr-308)表达更强、时程延长;同时,IPC使Akt1下游底物Bad(Ser-136)磷酸化水平增强、caspase-3的活化减少。应用PI3K特异性阻断剂LY294002阻断PI3K-Akt信号系统活化的同时,也抑制了IPC的保护作用。结论IPC通过对PI3K-Akt信号转导通路的调控发挥了对肝脏的保护作用;该信号通路的激活水平上调,可能是肝IPC保护作用的机制之一。

Objective To investigate the changes in phosphatidylinositol 3 kinase - Akt （ PI3K - Akt） signal pathway after ischemia reperfusion （IR） injury and ischemic preconditioning （IPC） , and the relationship between PI3 K - Akt and IPC. Methods Sixty - eight male SD rats were randomly divided into five groups： Sham, IR, IPC, LY and D MSO group. At 0 hour, 1/2 hour, 1 hour, 2 hours and 4 hours of reperfusion time point, blood were obtained to determine the levels of serum ferment. The liver tissue samples were prepaired for liver histology and determination of the experssion of signal protein of PI3K -Akt system, such as Akt1 and p -Akt1, by means of immunohisto - chemistry and western - blot. Results Compared with rats in sham group, the changes in serum ferment and histology indicated obvious damage after 1 hour ischemia following reperfusion and such hepatic damge become worse with time. The expressions of p - Akt1 （Thr - 308） in liver tissues were higher than that in sham group （P 〈 0.01 ）. Rats in IPC group shown a decreased damage in hepatic cell compared to rats in IR group, after 0, 2 and 4 hours of reperfusion （P 〈 0.01 ）. Meanwhile, the phosphorylation of Akt1 in IPC group shown a higher level and a longer activation period than that in IR group. Furthermore, IPC up - regulated the phosphorylation of Bad （Ser - 136） , a downstream substrate for Akt1, and down - regulated the activation of Caspase -3. LY294002, a specific blocker of PI3K, significantly blocked PI3K - Akt signal pathway and inhibited the protective effects of IPC in the liver. Conclusion The protective effects of IPC in the rat liver is through regulating PI3K - Akt signal pathway. Perhaps, the up - regulation of the activation of PI3K - Akt system is one of the mechanism through which the protective effects of IPC can realise.