姜黄素对H2O2致H9c2心肌细胞损伤的抗凋亡作用及对NF-κB信号通路的调节作用研究

Study on the Anti-apoptotic Effect of Curcumin on H2O2-induced H9c2 Cardiomyocyte Injury and the Regulation of NF-κB Signaling Pathway

目的:探讨姜黄素对H2O2致H9c2心肌细胞损伤的抗凋亡作用及对核因子κB(NF-κB)信号通路的调节作用。方法:将大鼠H9c2心肌细胞随机分为正常对照组、损伤模型组和姜黄素低、中、高剂量组(25、50、100μmol/L)。正常对照组不作任何干预;损伤模型组细胞以50μmol/L H2O2诱导12 h以建立损伤模型;姜黄素各剂量组经相应浓度药物预处理24 h后,再以50μmol/L H2O2诱导12 h。继续培养24 h后,采用MTT法和TUNEL法分别检测细胞的存活率和凋亡率;采用WTS-8法和显色法分别检测细胞中超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;采用免疫荧光法检测细胞中微管相关蛋白1轻链3(LC3)阳性表达的相对荧光强度;采用实时定量聚合酶链式反应法检测细胞中NF-κB p65 mRNA的表达水平;采用Western blotting法检测细胞中NF-κB p65、磷酸化NF-κB p65(p-NF-κB p65)蛋白的表达水平。结果:与正常对照组比较,损伤模型组细胞的存活率和SOD活性均显著降低,细胞凋亡率、MDA含量、LC3阳性表达相对荧光强度以及NF-κB p65 mRNA和NF-κB p65、p-NF-κB p65蛋白的表达水平以及p-NF-κB/NF-κB比值均显著升高(P<0.05)。与损伤模型组比较,姜黄素各剂量组细胞存活率和SOD活性均显著升高,细胞凋亡率、MDA含量、LC3阳性表达相对荧光强度以及NF-κB p65 mRNA和NF-κB/p65、p-NF-κB p65蛋白的表达水平以及p-NF-κB p65/NF-κB p65比值均显著降低(P<0.05)。结论:姜黄素能够提高H2O2致损伤H9c2心肌细胞的存活率、降低其凋亡率,提高心肌细胞中SOD活性并降低MDA含量;上述作用可能与调节心肌细胞NF-κB信号通路有关。

OBJECTIVE:To investigate the anti-apoptotic effect of curcumin on H2O2-induced H9c2 cardiomyocyte injury and the regulation of NF-κB signaling pathway.METHODS:H9c2 cardiomyocyte were randomly divided into normal control group,injury model group,curcumin low-dose,medium-dose and high-dose groups(25,50,100μmol/L).Normal control group didn’t received any intervention.The cells in injury model group were induced with 50μmol/L H2O2 for 12 h to establish the injury model.The cells in curcumin groups were treated with relevant concentration of drugs for 24 h,and then induced with 50μmol/L H2O2 for 12 h.After cultured for 24 h,survival rate and apoptotic rate of cells were measured by MTT method and TUNEL method;SOD activity and MDA content were determined by WTS-8 assay and color test;relative fluorescence intensity of LC3 positive expression was detected by immunofluorescence method;mRNA expression of NF-κB p65 in cells was detected by real-time PCR;Western blotting assay was used to detect the protein expression of NF-κB p65 and p-NF-κB p65 in cells.RESULTS:Compared with normal control group,survival rate and SOD activity were decreased significantly in injury model group,while apoptotic rate,MDA content,relative fluorescence intensity of LC3 positive expression,mRNA expression of NF-κB,protein expression of NF-κB p65 and p-NF-κB p65 as well as p-NF-κB/NF-κB were increased significantly(P<0.05).Compared with injury model group,survival rates and SOD activities were increased significantly in curcumin groups,while apoptotic rates,MDA contents,relative fluorescence intensities of LC3 positive expression,mRNA expression of LC3 positive cells,protein expression of NF-κB p65 and p-NF-κB p65 as well as p-NF-κB p65/NF-κB p65 were decreased significantly(P<0.05).CONCLUSIONS:Curcumin can increase the survival rate of H2O2-induced H9c2 cardiomyocyte injury,decrease its apoptotic rate,increase SOD activity and decrease MDA content in cardiomyocytes.Above effects may be related to the regulation of NF-κB signaling pathway.