PTEN对缺氧复氧心肌H9c2细胞凋亡、氧化损伤及免疫炎症因子水平的影响

Effects of PTEN on apoptosis,oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation

目的:研究第10号染色体缺失的磷酸酶及张力蛋白同源物(PTEN)对缺氧复氧条件下心肌H9c2细胞凋亡、氧化损伤及免疫炎症因子水平的影响。方法:用H9c2细胞构建缺氧复氧模型。细胞转染PTEN小干扰RNA(siRNA)和阴性对照siRNA,缺氧复氧处理后,RT-PCR和Western blot分别测定细胞中PTEN的mRNA和蛋白水平。MTT法测定细胞活力,流式细胞术测定细胞凋亡,用黄嘌呤氧化法测定细胞中超氧化物歧化酶(SOD)活性,用硫代巴比妥酸法测定丙二醛(MDA)含量,用二硝基苯肼法测定培养液上清中乳酸脱氢酶(LDH)活性,ELISA测定培养液上清中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和白细胞介素6(IL-6)水平,Western blot法测定细胞中cleaved caspase-3、Bax和Fas L的蛋白水平。结果:缺氧复氧处理后H9c2细胞中PTEN的mRNA和蛋白水平均明显升高(P <0. 05)。转染PTEN siRNA后的细胞中PTEN的mRNA和蛋白水平明显下降(P <0. 05)。缺氧复氧处理后H9c2细胞活力下降,凋亡率升高,细胞中cleaved caspase-3、Bax和Fas L的蛋白水平升高,MDA水平也升高,SOD活性下降,培养液上清中LDH、TNF-α、IL-1β和IL-6的水平升高(P <0. 05),而下调PTEN可以部分拮抗缺氧复氧对H9c2细胞活力、凋亡率、MDA水平、SOD水平及培养液上清中LDH、TNF-α、IL-1β和IL-6水平的影响。结论:下调PTEN可以减轻缺氧复氧诱导的心肌H9c2细胞氧化损伤,减少H9c2细胞凋亡,降低TNF-α、IL-1β和IL-6的水平。

AIM:To investigate the effects of phosphatase and tensin homolog deleted on chromosome 10(PTEN)on the apoptosis,oxidative damage and immune inflammatory factors in myocardial H9c2 cells with anoxia/reoxygenation(A/R).METHODS:The H9c2 cells were used to establish a model of A/R.The H9c2 cells were transfected with PTEN small interfering RNA(siRNA)and negative control.After A/R,the expression of PTEN at mRNA and protein levels was determined by RT-PCR and Western blot,respectively.The cell viability was measured by MTT assay.The apoptosis was analyzed by flow cytometry.Xanthine oxidase method was used to determine superoxide dismutase(SOD)activity.The content of malondialdehyde(MDA)was detected by thiobarbituric acid method.The lactate dehydrogenase(LDH)activity in the supernatant was evaluated by 4-dinitrophenylhydrazine method.The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and IL-6 in culture supernatant were examined by ELISA.The protein levels of cleaved caspase-3,Bax and FasL in the cells were determined by Western blot.RESULTS:After A/R,the expression of PTEN at mRNA and protein levels was significantly increased in the H9c2 cells(P<0.05).The mRNA and protein levels of PTEN were decreased significantly after transfection with PTEN siRNA(P<0.05).The viability of H9c2 cells was decreased after A/R,while the apoptotic rate was increased.The protein levels of cleaved caspase-3,Bax and FasL were increased in the cells.The MDA level was elevated,the activity of SOD was decreased,and the levels of LDH,TNF-α,IL-1βand IL-6 in the culture supernatant were increased(P<0.05).Down-regulation of PTEN partly antagonized the effects of A/R on the viability,apoptotic rate,MDA content,SOD activity,and the levels of LDH,TNF-α,IL-1βand IL-6 in culture supernatant.CONCLUSION:Down-regulation of PTEN attenuates oxidative damage induced by A/R,reduces apoptosis and secretion levels of TNF-α,IL-1βand IL-6 in the H9c2 cells.