基于PI3K/AKT信号通路的谷红注射液抗H9c2心肌细胞缺氧/复氧损伤的机制研究

目的基于PI3K/AKT信号通路,探讨谷红注射液(guhong injection,GHI)对H9c2心肌细胞缺氧/复氧(H/R)损伤的保护作用。方法培养H9c2心肌细胞,CCK-8比色法筛选GHI实验剂量。将细胞随机分为8组:正常组、模型组、GHI低、中、高剂量组(30、60和90μL·mL^-1)、阳性药组(维拉帕米注射液7.5μL·mL^-1)、GHI+LY294002(PI3K抑制剂)组和LY294002组。除正常组外,其他组均建立缺氧4 h复氧16 h的H/R损伤模型。ELISA检测各组细胞内肌酸激酶同工酶(CKMB)、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量,Western blot法检测各组细胞内p-PI3K、PI3K、p-AKT、AKT及GSK-3β蛋白表达水平。结果与模型组相比,各给药组LDH、CK-MB均降低(P<0.05),MDA降低(P<0.01)、SOD升高(P<0.01)。p-PI3K/PI3K、p-AKT/AKT及GSK-3β蛋白表达水平均升高(P<0.01)。给予LY294002后,与模型组相比,LDH、CK-MB、MDA及SOD无显著性差异,p-PI3K/PI3K、p-AKT/AKT及GSK-3β蛋白表达水平均降低(P<0.01)。结论GHI可以明显减轻H/R对H9c2心肌细胞造成的损伤,表现出抗氧化应激与抗凋亡的作用,其作用机制可能与PI3K/AKT信号通路有关。

Mechanism of Guhong Injection against Hypoxia/Reoxygenation Injury of H9c2 Cardiomyocytes Based on PI3K/AKT Signaling Pathway

OBJECTIVE To explore the protectiveness of Guhong injection(GHI)on H9c2 cardiomyocytes injured by hypoxia/reoxygenation(H/R)based on PI3K/AKT signaling pathway.METHODS H9c2 cardiomyocytes were cultured and the optimal GHI doses were screened by CCK-8.The cells were randomly subjected into 8 groups:control group,model group,low,medium and high GHI dose groups(30,60 and 90μL·mL^-1 respectively),positive drug group(verapamil injection 7.5μL·mL^-1),GHI+LY294002(PI3K inhibitor)group and LY294002 group.Cells were established H/R model with hypoxia for 4 h and reoxygenation for 16 h excepted the control group.The CK-MB,LDH,MDA content and SOD activities in each group were detected by Elisa,and the expression levels of p-PI3K,PI3K,p-AKT,AKT and GSK-3βin each group were detected by Western blot.RESULTS Compared each drug-treated group with the model group,LDH and CK-MB contents were decreased(P<0.05),MDA content was decreased(P<0.01),and SOD activity was increased(P<0.01).Meanwhile,the expression levels of p-PI3K/PI3K,p-AKT/AKT,and GSK-3βprotein were elevated(P<0.01).Compared with the model group,LDH,CK-MB,MDA and SOD activity in LY294002 group were no significant difference,while the expression levels of p-PI3K/PI3K,p-AKT/AKT and GSK-3βprotein decreased(P<0.01).CONCLUSION GHI could represent anti-oxidative and anti-apoptotic effect which reduced the damage of injured H9c2 myocardial cells caused by H/R.These effects might be related to the PI3K/AKT signaling pathway.