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
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摘要
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 H9 c2 cells with anoxia/reoxygenation( A/R). METHODS: The H9 c2 cells were used to establish a model of A/R. The H9 c2 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 Fas L 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 H9 c2 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 H9 c2 cells was decreased after A/R,while the apoptotic rate was increased. The protein levels of cleaved caspase-3,Bax and Fas L were increased in the cells. The MDA level was elevated,the activity of SOD was decreased,and the levels of LDH,TNF-α,IL-1β and 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-1β and 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-1β and IL-6 in the H9 c2 cells.
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 H9 c2 cells with anoxia/reoxygenation( A/R). METHODS: The H9 c2 cells were used to establish a model of A/R. The H9 c2 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 Fas L 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 H9 c2 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 H9 c2 cells was decreased after A/R,while the apoptotic rate was increased. The protein levels of cleaved caspase-3,Bax and Fas L were increased in the cells. The MDA level was elevated,the activity of SOD was decreased,and the levels of LDH,TNF-α,IL-1β and 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-1β and 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-1β and IL-6 in the H9 c2 cells.
引文
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[11]Chen JH,Zhang P,Chen WD,et al.ATM-mediated PTEN phosphorylation promotes PTEN nuclear translocation and autophagy in response to DNA-damaging agents in cancer cells[J].Autophagy,2015,11(2):239-252.
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[18]Liu L,Hu X,Cai G Y,et al.High glucose-induced hypertrophy of mesangial cells is reversed by connexin43overexpression via PTEN/Akt/m TOR signaling[J].Nephrol Dialysis Transpl,2012,27(1):90-100.
[19]周裔忠.磷酸酶-张力蛋白同源物-PTEN与心肌细胞钙/心肌肥厚关系的研究[D].重庆:第三军医大学,2005.
[20]符丽珍,黄茂芹,劳之勇,等.miR-221在H2O2诱导的大鼠心肌细胞损伤中的作用及机制研究[J].中国比较医学杂志,2017,27(5):83-88.
[21]Ruan H,Li J,Ren S,et al.Inducible and cardiac specific PTEN inactivation protects ischemia/reperfusion injury[J].J Mol Cell Cardiol,2009,46(2):193-200.
[22]张军,代文静,周敬群,等.抗衰老Klotho蛋白减轻大鼠乳鼠心肌细胞缺氧/复氧损伤[J].中国病理生理杂志,2015,31(6):980-987.
[23]Wen XR,Fu YY,Liu HZ,et al.Neuroprotection of sevoflurane against ischemia/reperfusion-induced brain injury through inhibiting JNK3/caspase-3 by enhancing Akt signaling pathway[J].Mol Neurobiol,2016,53(3):1661-1671.
[24]Zheng M,Song D,Luo Z,et al.Effect of puerarin on expression of Fas/Fas L mRNA in pulmonary injury induced by ischemia-reperfusion in rabbits.[J].Nat Product Commun,2015,10(2):253-256.
[25]厉小雪,徐水凌,张婷婷,等.槲皮素诱导MCF-7细胞凋亡及其与Fas/Fas L通路的相关性研究[J].中国病理生理杂志,2015,31(8):1437-1443.
[26]Melo MT,Oliveira IMD,Grivicich I,et al.Diphenyl diselenide protects cultured MCF-7 cells against tamoxifen-induced oxidative DNA damage[J].Biomed Pharmacother,2013,67(4):329-335.
[27]左长鹏,王芳,纵静,等.人参皂苷Rb1对H9c2细胞缺氧复氧的作用[J].中国老年学杂志,2017,37(10):2341-2344.
[28]Zhao M,Sun L,Yu XJ,et al.Acetylcholine mediates AMPK-dependent autophagic cytoprotection in H9c2 cells during hypoxia/reoxygenation injury[J].Cell Physiol Biochem,2013,32(3):601-613.
[29]Liang JC.San-Huang-Xie-Xin-Tang protects cardiomyocytes against hypoxia/reoxygenation injury via inhibition of oxidative stress-induced apoptosis[J].J Nat Med,2012,66(2):311-320.
[30]Lin L,Wu XD,Davey AK,et al.The anti-inflammatory effect of baicalin on hypoxia/reoxygenation and TNF-αinduced injury in cultural rat cardiomyocytes[J].Phytother Res,2010,24(3):429-437.
[2]任安立,李靖凯,周冬冬.程序化细胞死亡因子5对缺氧/复氧诱导的心肌细胞凋亡和自噬的影响及机制[J].中国病理生理杂志,2017,33(2):251-256.
[3]Yu L,Li F,Zhao G,et al.Protective effect of berberine against myocardial ischemia reperfusion injury:role of Notch1/Hes1-PTEN/Akt signaling[J].Apoptosis,2015,20(6):796-810.
[4]付兴根,邢佑敏,韩刚,等.螺内酯对糖尿病大鼠心肌PTEN表达的影响[J].中国现代医学杂志,2014,24(11):28-33.
[5]梁超杰,刘付宝.PTEN研究的相关进展[J].肝胆外科杂志,2016,24(1):73-76.
[6]Li CM,Shen SW,Wang T,et al.Myocardial ischemic post-conditioning attenuates ischemia reperfusion injury via PTEN/Akt signal pathway[J].Int J Clin Exp Med,2015,8(9):15801-15807.
[7]Tang JY,Jin P,He Q,et al.Naringenin ameliorates hypoxia/reoxygenation-induced endoplasmic reticulum stressmediated apoptosis in H9c2 myocardial cells:involvement in ATF6,IRE1αand PERK signaling activation[J].Mol Cell Biochem,2016,424(1-2):111-122.
[8]Rosser CJ,Tanaka M,Pisters LL,et al.Adenoviral-mediated PTEN transgene expression sensitizes Bcl-2-expressing prostate cancer cells to radiation[J].Cancer Gene Ther,2015,11(4):273-279.
[9]Shen HG,Li LP,Yang SJ,et al.MicroRNA-29a contributes to drug-resistance of breast cancer cells to adriamycin through PTEN/AKT/GSK3βsignaling pathway[J].Gene,2016,593(1):84-90.
[10]Ke TW,Wei PL,Yeh KT,et al.MiR-92a promotes cell metastasis of colorectal cancer through PTEN-mediated PI3K/AKT pathway[J].Ann Surg Oncol,2015,22(8):2649-2655.
[11]Chen JH,Zhang P,Chen WD,et al.ATM-mediated PTEN phosphorylation promotes PTEN nuclear translocation and autophagy in response to DNA-damaging agents in cancer cells[J].Autophagy,2015,11(2):239-252.
[12]Sun Y,Tian H,Wang L.Effects of PTEN on the proliferation and apoptosis of colorectal cancer cells via the phosphoinositol-3-kinase/Akt pathway.[J].Oncol Rep,2015,33(4):1828-1836.
[13]Punnoose EA,Ferraldeschi R,Szaferglusman E,et al.PTEN loss in circulating tumour cells correlates with PTENloss in fresh tumour tissue from castration-resistant prostate cancer patients[J].Br J Cancer,2015,113(8):1225-1233.
[14]张冀东,崔炜.抑癌基因PTEN与心血管疾病研究进展[J].中国心血管杂志,2009,14(6):486-488.
[15]Zhou YZ,Zhu SJ,Yu LJ,et al.PTEN negatively regulates isoproterenol-induced cardiac hypertrophy and effects of captopril on PTEN expression[J].Chin J Cardiol,2005,33(8):738-742.
[16]Lai Y,He S,Ma L,et al.HOTAIR functions as a competing endogenous RNA to regulate PTEN expression by inhibiting miR-19 in cardiac hypertrophy[J].Mol Cell Biochem,2017,432(1-2):1-9.
[17]江梦,夏中元,肖业达.参附注射液对糖尿病大鼠心肌缺血/再灌注损期间PTEN表达的影响[J].中国急救医学,2008,28(10):914-916.
[18]Liu L,Hu X,Cai G Y,et al.High glucose-induced hypertrophy of mesangial cells is reversed by connexin43overexpression via PTEN/Akt/m TOR signaling[J].Nephrol Dialysis Transpl,2012,27(1):90-100.
[19]周裔忠.磷酸酶-张力蛋白同源物-PTEN与心肌细胞钙/心肌肥厚关系的研究[D].重庆:第三军医大学,2005.
[20]符丽珍,黄茂芹,劳之勇,等.miR-221在H2O2诱导的大鼠心肌细胞损伤中的作用及机制研究[J].中国比较医学杂志,2017,27(5):83-88.
[21]Ruan H,Li J,Ren S,et al.Inducible and cardiac specific PTEN inactivation protects ischemia/reperfusion injury[J].J Mol Cell Cardiol,2009,46(2):193-200.
[22]张军,代文静,周敬群,等.抗衰老Klotho蛋白减轻大鼠乳鼠心肌细胞缺氧/复氧损伤[J].中国病理生理杂志,2015,31(6):980-987.
[23]Wen XR,Fu YY,Liu HZ,et al.Neuroprotection of sevoflurane against ischemia/reperfusion-induced brain injury through inhibiting JNK3/caspase-3 by enhancing Akt signaling pathway[J].Mol Neurobiol,2016,53(3):1661-1671.
[24]Zheng M,Song D,Luo Z,et al.Effect of puerarin on expression of Fas/Fas L mRNA in pulmonary injury induced by ischemia-reperfusion in rabbits.[J].Nat Product Commun,2015,10(2):253-256.
[25]厉小雪,徐水凌,张婷婷,等.槲皮素诱导MCF-7细胞凋亡及其与Fas/Fas L通路的相关性研究[J].中国病理生理杂志,2015,31(8):1437-1443.
[26]Melo MT,Oliveira IMD,Grivicich I,et al.Diphenyl diselenide protects cultured MCF-7 cells against tamoxifen-induced oxidative DNA damage[J].Biomed Pharmacother,2013,67(4):329-335.
[27]左长鹏,王芳,纵静,等.人参皂苷Rb1对H9c2细胞缺氧复氧的作用[J].中国老年学杂志,2017,37(10):2341-2344.
[28]Zhao M,Sun L,Yu XJ,et al.Acetylcholine mediates AMPK-dependent autophagic cytoprotection in H9c2 cells during hypoxia/reoxygenation injury[J].Cell Physiol Biochem,2013,32(3):601-613.
[29]Liang JC.San-Huang-Xie-Xin-Tang protects cardiomyocytes against hypoxia/reoxygenation injury via inhibition of oxidative stress-induced apoptosis[J].J Nat Med,2012,66(2):311-320.
[30]Lin L,Wu XD,Davey AK,et al.The anti-inflammatory effect of baicalin on hypoxia/reoxygenation and TNF-αinduced injury in cultural rat cardiomyocytes[J].Phytother Res,2010,24(3):429-437.