论文部分内容阅读
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 protective effect and mechanism of melatonin (Mel) on H9C2 cells during ischemia-reperfusion injury and to clarify the role of protein kinase C epsilon (PKCε) signaling in this process. Methods H9C2 cells were pretreated with Mel (100μmol / L) for 12 hours and then subjected to simulated ischemia-reperfusion (SIR) injury (45 min after ischemia and 4 h reperfusion). Cell viability was measured by CCK-8 and TUNEL The activity of superoxide dismutase (SOD) and the release of malondialdehyde (MDA) in cells were measured by enzymatic activity assay. The expression of PKCε, cytoplasmic PKCε, Bcl-2, Bax, Caspase-3 and gp91phox protein expression, PKCε signaling pathway in PKCε specific blocking agent εV1-2 role in this process. Results After SIR treatment, the viability of H9C2 cells was significantly decreased, the apoptosis rate was significantly increased, the translocation of PKCε membrane was decreased, the ratio of Bcl-2 / Bax was decreased, the expression of Caspase-3 and gp91phox protein was increased, the content of MDA was increased, decline. Mel pretreatment significantly increased the viability of H9C2 cells after SIR treatment, decreased the apoptosis rate, promoted the translocation of PKCε membrane, upregulated the activity of SOD and decreased the content of MDA, and decreased the expression of Caspase-3 and gp91phox. However, the use of εV1-2 to block the PKCε signaling pathway reversed the protective effects of Mel (all P <0.05). Conclusion Mel can reduce oxidative stress injury and apoptosis induced by SIR in H9C2 cells by activating PKCε protective signaling pathway.