靶向TLR4的siRNA慢病毒感染减少过氧化氢诱导的心肌H9C2细胞凋亡及氧化损伤
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摘要
目的:研究沉默TLR4对过氧化氢(H_2O_2)诱导的心肌H9C2细胞凋亡及氧化损伤的影响。方法:用H_2O_2处理心肌H9C2细胞,qRT-PCR和Western blot检测细胞中Toll样受体4(TLR4) mRNA和蛋白水平。用siRNA TLR4慢病毒感染心肌H9C2细胞,H_2O_2诱导以后,流式细胞术检测细胞凋亡,Western blot检测细胞中凋亡蛋白Bcl-2相关X蛋白(Bax)和剪切型Caspase-3(Cleaved Caspase-3)水平,硫代巴比妥酸法检测细胞中丙二醛(MDA)水平,黄嘌呤氧化法检测细胞中超氧化物歧化酶(SOD)活性,二硝基苯肼显色法检测上清中乳酸脱氢酶(LDH)含量,二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)法检测活性氧(ROS)水平,ELISA法检测培养液上清中TNF-α、IL-6水平。结果:H_2O_2诱导处理以后的心肌H9C2细胞中TLR4mRNA和蛋白水平均明显高于未经诱导的心肌H9C2细胞(P<0. 05)。siRNA TLR4慢病毒感染后可以降低心肌H9C2细胞中TLR4 mRNA和蛋白水平。H_2O_2诱导后的心肌H9C2细胞凋亡率升高,Bax和Cleaved Caspase-3蛋白水平升高,细胞中SOD活性降低,MDA水平升高,ROS水平也升高,同时细胞培养液中LDH水平升高,细胞分泌的TNF-α、IL-6增多,与未经诱导的心肌H9C2细胞比较,差异有统计学意义(P<0. 05)。敲低TLR4后的心肌H9C2细胞经H_2O_2诱导处理以后,细胞凋亡率降低,细胞中Bax和Cleaved Caspase-3蛋白水平降低,细胞中SOD活性升高,MDA水平降低,ROS水平也降低,同时细胞培养液中LDH水平降低,细胞分泌的TNF-α、IL-6减少,与单纯经H_2O_2诱导处理的心肌H9C2细胞比较,差异有统计学意义(P<0. 05)。结论:沉默TLR4减弱H_2O_2诱导的心肌H9C2细胞凋亡及氧化损伤,减少细胞中ROS的聚集,减少心肌H9C2细胞分泌炎症因子。
Objective: Study the effect of silent TLR4 on cardiomyocyte apoptosis and oxidative damage induced by H_2O_2.Methods: Cardiac myocytes treated with H_2O_2,qRT-PCR and Western blot were used to detected the level of TLR4 mRNA and protein in the cells,myocardium cells infected with siRNA TLR4 lentivirus,after H_2O_2 induced,cell apoptosis was detected by flow cytometry,Western blot detected the level of apoptotic protein Bax and Cleaved Caspase-3 in the cells,thiobarbituric acid assay was used to detect the level of MDA in cells,xanthine oxidation assay was used to detect the activity of SOD in cells,the content of LDH was detected by dinitrophenylhydrazine colorimetry assay in the supernatant,the level of ROS was detected by DCFH-DA,the level of TNF-α and IL-6 in the supernatant of the culture liquid were detected by ELISA method. Results: The level of TLR4 mRNA and protein in cardiac myocytes after H_2O_2 treatment were significantly higher than that of uninduced cardiomyocytes( P<0. 05). The level of TLR4 mRNA and protein in cardiac myocytes reduced after siRNA TLR4 lentivirus infection. The apoptosis rate of cardiac myocytes increased after H_2O_2 induction,the levels of Bax and Cleaved Caspase-3 protein elevated,the activity of SOD in the cells reduced,the level of MDA elevated,the level of ROS risied,the level of LDH increased in the cell culture medium,the levels of TNF-α and IL-6 secreted by cells increased,compared with uninduced cardiac myocytes,the difference were statistically significant( P < 0. 05). Cardiac myocytes with knocked down of TLR4 after H_2O_2 induced,the rate of apoptosis reduced,the level of Bax and Cleaved Caspase-3 protein in the cells decreased,the activity of SOD increased,the level of MDA decreased,the level of ROS decreased,the level of LDH in cell culture solution reduced,TNF-α and IL-6 decreased,compared with the myocardial cells treated with H_2O_2 alone,the difference were statistically significant( P<0. 05). Conclusion: Silence TLR4 reduce the aggregation of ROS and reduce the secretion of inflammatory factors,attenuated the apoptosis and oxidative damage induced by H_2O_2 in cardiac myocytes.
Objective: Study the effect of silent TLR4 on cardiomyocyte apoptosis and oxidative damage induced by H_2O_2.Methods: Cardiac myocytes treated with H_2O_2,qRT-PCR and Western blot were used to detected the level of TLR4 mRNA and protein in the cells,myocardium cells infected with siRNA TLR4 lentivirus,after H_2O_2 induced,cell apoptosis was detected by flow cytometry,Western blot detected the level of apoptotic protein Bax and Cleaved Caspase-3 in the cells,thiobarbituric acid assay was used to detect the level of MDA in cells,xanthine oxidation assay was used to detect the activity of SOD in cells,the content of LDH was detected by dinitrophenylhydrazine colorimetry assay in the supernatant,the level of ROS was detected by DCFH-DA,the level of TNF-α and IL-6 in the supernatant of the culture liquid were detected by ELISA method. Results: The level of TLR4 mRNA and protein in cardiac myocytes after H_2O_2 treatment were significantly higher than that of uninduced cardiomyocytes( P<0. 05). The level of TLR4 mRNA and protein in cardiac myocytes reduced after siRNA TLR4 lentivirus infection. The apoptosis rate of cardiac myocytes increased after H_2O_2 induction,the levels of Bax and Cleaved Caspase-3 protein elevated,the activity of SOD in the cells reduced,the level of MDA elevated,the level of ROS risied,the level of LDH increased in the cell culture medium,the levels of TNF-α and IL-6 secreted by cells increased,compared with uninduced cardiac myocytes,the difference were statistically significant( P < 0. 05). Cardiac myocytes with knocked down of TLR4 after H_2O_2 induced,the rate of apoptosis reduced,the level of Bax and Cleaved Caspase-3 protein in the cells decreased,the activity of SOD increased,the level of MDA decreased,the level of ROS decreased,the level of LDH in cell culture solution reduced,TNF-α and IL-6 decreased,compared with the myocardial cells treated with H_2O_2 alone,the difference were statistically significant( P<0. 05). Conclusion: Silence TLR4 reduce the aggregation of ROS and reduce the secretion of inflammatory factors,attenuated the apoptosis and oxidative damage induced by H_2O_2 in cardiac myocytes.
引文
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[22]杨雪梅,吴刚.苦参碱抑制LPS诱导巨噬细胞IL-1β、TNF-α分泌及机制研究[J].中国免疫学杂志,2016,32(6):820-824.Yang XM,Wu G.Inhibitory effect of Matrine on IL-1β,TNF-αof macrophages induced by LPS[J].Chin J Immunol,2016,32(6):820-824.
[23]刘会芳,夏雨,徐倩,等.高尿酸通过TLR4/NF-κB信号通路诱导肾小管上皮细胞上皮-间充质转化的作用研究[J].第三军医大学学报,2017,39(19):1919-1925.Liu HF,Xia Y,Xu Q,et al.High uric acid induces epithelialmesenchymal transition of renal tubular epithelial cells via TLR4/NF-κB signaling pathway[J].Acta Acad Med Mil Tert,2017,39(19):1919-1925.
[24]Putra AB,Nishi K,Shiraishi R,et al.Jellyfish collagen stimulates production of TNF-αand IL-6 by J774.1 cells through activation of NF-κB and JNK via TLR4 signaling pathway[J].Mol Immuol,2014,58(1):32-37.
[25]Jiao H,Zhang Y,Yan Z,et al.Caveolin-1 Tyr14 phosphorylation induces interaction with TLR4 in endothelial cells and mediates My D88-dependent signaling and sepsis-induced lung inflammation[J].J Immunol,2013,191(12):6191-6199.
[2]Liu XB,Yan S,Pleasure DE,et al.The vulnerability of thalamocortical circuitry to hypoxic-ischemic injury in a mouse model of periventricular leukomalacia[J].Bmc Neurosci,2016,17(1):1-9.
[3]Cui X,Song H,Jie S.Curcumin attenuates hypoxic-ischemic brain injury in neonatal rats through induction of nuclear factor erythroid-2-related factor 2 and heme oxygenase-1[J].Exp Thera Med,2017,14(2):1512-1518.
[4]Li LI,Zhang Y,Nutrition DO,et al.The progress of research of TLR4 and insulin resistance[J].Par Ent Nut,2013,33(1):70-80.
[5]Imahashi K,Morishita K,Kusuoka H,et al.Kinetics of a putative hypoxic tracer,99mTc-HL91,in normoxic,hypoxic,ischemic,and stunned myocardium.[J].J Nuclear Med Off Publ Soc Nuc Med,2000,41(6):1102-1107.
[6]Lee SM,Hutchinson M,Saint DA.The role of Toll-like receptor 4(TLR4)in cardiac ischaemic-reperfusion injury,cardioprotection and preconditioning[J].Clin Exp Phar Phys,2016,43(9):864-871.
[7]Ahmed S,Moawad M,Elhefny R,et al.Is toll like receptor 4 a common pathway hypothesis for development of lung cancer and idiopathic pulmonary fibrosis?[J].Egyptian J Chest Dis Tub,2016,65(1):289-294.
[8]Gao Y,Fang X,Tong Y,et al.TLR4-mediated My D88-dependent signaling pathway is activated by cerebral ischemia-reperfusion in cortex in mice[J].Biomed Phar,2009,63(6):442-450.
[9]蓝景生,张震,罗薇,等.大蒜素对心肌纤维化的影响及对TLR4/NF-κB信号通路的作用[J].中国免疫学杂志,2016,32(4):500-503.Lan JS,Zhang C,Luo W,et al.Effects of Allicin on myocardial fibrosis and TLR4/NF-κB pathway[J].Chin J Immunol,2016,32(4):500-503.
[10]Ferwerda G,Meyer-Wentrup F,Kullberg BJ,et al.Dectin-1synergizes with TLR2 and TLR4 for cytokine production in human primary monocytes and macrophages[J].Cell Microbiol,2008,10(10):2058-2066.
[11]Wu H,Liu L,Tan Q,et al.Somatostatin limits intestinal ischemiareperfusion injury in macaques via suppression of TLR4-NF-κBcytokine pathway[J].J Gastroint Surg,2009,13(5):983-993.
[12]Wang C,Sun H,Song Y,et al.Pterostilbene attenuates inflammation in rat heart subjected to ischemia-reperfusion:role of TLR4/NF-κB signaling pathway[J].Int J Clin Exp Med,2015,8(2):1737-1746.
[13]Oyama J,Blais C,Liu X,et al.Reduced myocardial ischemiareperfusion injury in Toll-like receptor 4-deficient mice[J].Circulation,2004,109(6):784-789.
[14]Andrassy M,Volz HC,Riedle N,et al.HMGB1 as a predictor of infarct transmurality and functional recovery in patients with myocardial infarction[J].J Inter Med,2011,270(3):245-253.
[15]Liu XH,Zhang ZY,Sun S,et al.Ischemic postconditioning protects myocardium from ischemia/reperfusion injury through attenuating endoplasmic reticulum stress[J].Shock,2008,30(4):422-427.
[16]Yue T,Chen J,Bao W,et al.In vivo myocardial protection from ischemia/reperfusion injury by the peroxisome proliferatoractivated receptor-γagonist rosiglitazone[J].Circulation,2001,104(21):2588-2594.
[17]Krishnamurthy P,Thal M,Verma S,et al.Interleukin-10deficiency impairs bone marrow-derived endothelial progenitor cell survival and function in ischemic myocardium[J].Circulation Res,2011,109(11):1280-1289.
[18]Liu HR,Tao L,Gao E,et al.Anti-apoptotic effects of rosiglitazone in hypercholesterolemic rabbits subjected to myocardial ischemia and reperfusion[J].Cardiovascular Res,2004,62(1):135-144.
[19]Bacaksiz A,Teker ME,Buyukpinarbasili N,et al.Does pantoprazole protect against reperfusion injury following myocardial ischemia in rats?[J].Eur Rev Med Pha Sci,2013,17(2):269-275.
[20]Zou J.Expression of TNF-αand IL-6 in tissues and serums in the early stage of myocardial ischemia-reperfusion in rat[J].J China Med Univ,2013,42(9):830-835.
[21]Dawn B,Guo Y,Rezazadeh A,et al.Tumor necrosis factor-alpha does not modulate ischemia/reperfusion injury in naive myocardium but is essential for the development of late preconditioning.[J].J Mol Cell Card,2004,37(1):51-61.
[22]杨雪梅,吴刚.苦参碱抑制LPS诱导巨噬细胞IL-1β、TNF-α分泌及机制研究[J].中国免疫学杂志,2016,32(6):820-824.Yang XM,Wu G.Inhibitory effect of Matrine on IL-1β,TNF-αof macrophages induced by LPS[J].Chin J Immunol,2016,32(6):820-824.
[23]刘会芳,夏雨,徐倩,等.高尿酸通过TLR4/NF-κB信号通路诱导肾小管上皮细胞上皮-间充质转化的作用研究[J].第三军医大学学报,2017,39(19):1919-1925.Liu HF,Xia Y,Xu Q,et al.High uric acid induces epithelialmesenchymal transition of renal tubular epithelial cells via TLR4/NF-κB signaling pathway[J].Acta Acad Med Mil Tert,2017,39(19):1919-1925.
[24]Putra AB,Nishi K,Shiraishi R,et al.Jellyfish collagen stimulates production of TNF-αand IL-6 by J774.1 cells through activation of NF-κB and JNK via TLR4 signaling pathway[J].Mol Immuol,2014,58(1):32-37.
[25]Jiao H,Zhang Y,Yan Z,et al.Caveolin-1 Tyr14 phosphorylation induces interaction with TLR4 in endothelial cells and mediates My D88-dependent signaling and sepsis-induced lung inflammation[J].J Immunol,2013,191(12):6191-6199.