蛋白激酶C epsilon信号通路介导褪黑素抗H9C2细胞缺血再灌注损伤的机制研究

Melatonin protects H9C2 cells against ischemia-reperfusion injury via protein kinase C epsilon signaling pathway

目的探讨褪黑素(Mel)在H9C2细胞缺血再灌注损伤过程中的保护作用及其机制,明确蛋白激酶C epsilon(PKCε)信号通路在此过程中的作用。方法 H9C2细胞经Mel(100μmol/L)预处理12 h后,接受模拟缺血再灌注(SIR)损伤(缺血45 min,再灌注4 h),采用CCK-8法和TUNEL法分别检测细胞活力和凋亡率,使用酶活性测定法检测细胞中超氧化物歧化酶(SOD)的活性和丙二醛(MDA)的释放量,使用Western blot法检测细胞膜PKCε、细胞质PKCε、Bcl-2、Bax、Caspase-3和gp91phox蛋白的表达情况,并使用PKCε特异性阻断剂εV1-2观察PKCε信号通路在这一过程中发挥的作用。结果经SIR处理后,H9C2细胞活力显著降低,凋亡率明显增加,PKCε膜转位减少,Bcl-2/Bax比例下调,Caspase-3与gp91phox蛋白表达量增加,细胞中MDA含量上升,SOD活力下降。Mel预处理可显著提高SIR处理后H9C2细胞活力,降低细胞凋亡率,促进PKCε膜转位,上调细胞中SOD活性及降低MDA含量,并可降低Caspase-3与gp91phox蛋白表达量。而使用εV1-2阻断PKCε信号通路可逆转Mel的上述保护作用(均P<0.05)。结论 Mel可通过激活PKCε保护性信号通路减轻SIR引起的H9C2细胞氧化应激损伤与凋亡。

Objective To investigate the underlying mechanisms of the protective effect of melatonin （Mel） on simulated ischemia/reperfusion （SIR） injury in H9C2 cells and the role of PKC epsilon （PKCε） signaling pathway in this process. Methods The cultured H9C2 cells were pretreated with Mel （ 100 μmol/L） for 12 h and then subjected to SIR （45 min ischemia, 4 h reperfusion）. Cell viability and apoptotic index were detected by CCK-8 and TUNEL methods, and the intracellular malondialdebyde （MDA） content and superoxide dismutase （SOD） activity were assessed by specialized kits. The translocation of protein kinase C epsilon （PKCe） and the expression levels of Bcl-2, Bax, Caspase-3 and oxidative stress marker gp91^phox were detected by Western blotting. PKCε specific inhibitor εV1-2 was used to study the role of PKCε in this process. Results We found that SIR treatment reduced cell viability and increased apoptotic index, inhibited PKCε translocation and reduced the Bcl-2/Bax ratio. Mel pretreatment significantly increased cell viability and reduced apoptotic index, down-regulated the MDA content and up-regulated the SOD activity. In addition, Mel pretreatment significantly increased PKCe translocation and Bcl- 2/Bax ratio and decreased the expression levels of Caspase- 3 and gp91^phox. However, these protective effects of Mel mentioned above were largely abolished by εV1-2 administration. Conclusion Mel pretreatment may protects H9C2 cells against SIR injury via attenuating oxidative stress and apoptosis in a PKCε-dependent manner.