PI3K/Akt信号途径抑制烧伤后大鼠缺血缺氧心肌细胞凋亡

PI3K/Akt signal pathway inhibits ischemia and hypoxia-induced myocardial apoptosis in rats

目的探讨PI3K/Akt信号途径在烧伤后大鼠缺血缺氧心肌细胞凋亡中的作用及机制。方法首先建立模拟烧伤的动物模型,通过Western blot观察PI3K/Akt信号途径的活化规律,TUNEL实验观察烧伤大鼠心肌凋亡。然后建立离体培养的缺血缺氧心肌细胞模型,并分为对照组、单纯缺血缺氧组、LY294002处理组或IGF-1处理组,应用ELISA观察缺血缺氧心肌细胞凋亡。并通过DEVD荧光检测caspase-3酶活性,RT-PCR检测p53、Bax的转录活性。结果烧伤后大鼠心肌组织内PI3K和pAkt表达逐渐增加,伤后3h达高峰;烧伤3h后,大鼠心肌细胞凋亡率显著增加;应用LY294002特异性抑制PI3K/Akt通路后,大鼠心肌细胞凋亡率较对照组增加更为显著(P<0.05)。缺血缺氧导致体外培养心肌细胞凋亡增加,caspase-3活性逐渐增强,p53、Bax mRNA表达量逐渐升高;与单纯缺血缺氧组相比,应用LY294002阻断PI3K/Akt途径活化后,心肌细胞凋亡数量增加更显著(P<0.05),caspase-3活性增高更为明显(P<0.05),p53、Bax mRNA表达量均显著升高(P<0.05);用IGF-1预先激活PI3K/Akt途径后,与单纯缺血缺氧组比较,心肌细胞caspase-3活性明显降低(P<0.05)。结论PI3K/Akt信号途径在缺血缺氧心肌细胞中具有抗凋亡作用,该作用与PI3K/Akt调控促凋亡基因p53、Bax的表达,抑制caspase-3凋亡反应有关,并为心肌缺血缺氧损害的临床防治提供新思路及实验依据。

Objective To investigate the role and mechanisms of the PI3K/Akt signal pathway in the apoptosis of rat cardiomyocytes after ischemia and hypoxia due to severe burn injury. Methods Burn models of full-thick 30% TBSA burn injury were established by scalding rat back in hot water. The activation of the PI3K/ Akt signal pathway was observed by Western blotting, and myocardial apoptosis was assessed by TUNEL. Cultured rat cardiomyocytes under ischemic/hypoxic conditions were divided into the control group, the ischemia and hypoxia group, and the LY294002 or IGF-1 treatment group. The apoptosis of cardiomyocytes were assessed by ELISA. The caspase-3 activity was measured by DEVD fluorescence. The transcriptional activity of p53, Bax was assessed by RT-PCR. Results After severe burn, PI3K and pAkt expressions increased gradually in the rat myocardial tissue, and reached a peak at 3 h postburn. At 3 h postbum, the apoptosis increased signifi- cantly in rats. When the PI3K/Akt pathway was specifically inhibited by LY294002, the myocardial apoptosis increased significantly in the burn group as compared with the control group （P 〈 0.05 ）. Cultured cardiomyocytes after ischemia and hypoxia showed the following changes： increased apoptosis, gradually enhanced caspase-3 activity, gradually increased p53 and Bax mRNA expressions. Compared with the ischemia and hypoxia group, blockade of the PI3K/Akt pathway with LY294002 increased the number of apoptotic cardiomyo-cytes, caspase-3 activity, and p53 and Box mRNA expression significantly （P 〈 0.05）. After activation of the PI3K/Akt pathway with IGF-1, easpase-3 activity decreased significantly （P 〈 0.05）, as compared with the isehemia and hypoxia group. Conclusion The PI3K/Akt signal pathway plays anti-apoptotie role in ischemie/hypoxie eardiomyoeytes. PI3K/Akt pathway regulating p53 and Box expression is probably the mechanism of antiapoptosis, which can suppress the activity of easpase-3 and decrease the number of apoptotie cardiomyocytes.