KLF5沉默通过抑制NF-κB信号通路减少氧化应激条件下心肌细胞炎症因子分泌
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摘要
目的研究沉默Krüpple样因子5(KLF5)对氧化应激条件下心肌细胞炎症因子分泌的影响及机制。方法心肌H9c2细胞用过氧化氢处理,qRT-PCR和Western blot检测细胞中KLF5 mRNA和蛋白表达水平。用KLF5 shRNA慢病毒感染H9c2细胞,给予过氧化氢处理后,qRT-PCR和Western blot检测细胞中KLF5表达水平。MTT检测沉默KLF5对过氧化氢处理的心肌细胞活性的影响,同时用qRT-PCR法检测肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)mRNA水平,Western blot检测细胞中核因子-κBp65(NF-κBp65)蛋白水平。用NF-κB激活剂处理沉默KLF5的心肌细胞,MTT检测其在过氧化氢处理后细胞活性,qRT-PCR法检测过氧化氢处理后细胞中TNF-α、IL-1β水平。结果过氧化氢诱导H9c2细胞中KLF5 mRNA和蛋白表达。KLF5 shRNA慢病毒感染可下调氧化应激条件下H9c2细胞中KLF5的表达。过氧化氢能够下调H9c2细胞活性,提高细胞分泌的TNF-α、IL-1β水平,促进细胞中NF-κBp65蛋白表达。沉默KLF5能够提高过氧化氢条件下H9c2细胞经活性,减少细胞分泌TNF-α、IL-1β,抑制细胞中NF-κBp65蛋白表达。NF-κB激活剂可以逆转沉默KLF5对过氧化氢处理的心肌细胞增殖活性、炎症因子分泌的影响。结论氧化应激诱导心肌细胞表达KLF5,沉默其表达可以通过抑制NF-κB通路减少心肌细胞中炎症因子表达。
This study was designed to study the effect and mechanism of KLF5 silence on the secretion of inflammatory factors in myocardial cells under oxidative stress. The H9c2 cells of myocardium, infected with or without KLF5 shRNA lentivirus, were treated with hydrogen peroxide, then qRT-PCR and Western blot were used to detect the level of KLF5 mRNA and protein expression in the cells. Furthermore, MTT was used to detect the effect of KLF5 silence on the activity of cardiac myocytes treated by hydrogen peroxide, at the same time, the levels of TNF-α and IL-1β was detected by qRT-PCR. Western blot was used to detect the level of NF-κB p65 protein in the cells. On the other hand, myocardial cells of silent KLF5 were treated with NF-κB activator, followed by hydrogen peroxide treatment. Then the activity of the cells were detected by MTT, the levels of TNF-α and IL-1β in the cells were detected by qRT-PCR. Data showed thathydrogen peroxide induced the expression of KLF5 mRNA and protein in H9 c2 cells. KLF5 shRNAlentivirus infection could down-regulate the expression of KLF5 in H9 c2 cells under oxidative stress; hydrogen peroxide could down-regulate the activity of H9 c2 cells,increase the level of TNF-α and IL-1β secreted by cells, promote the expression of NF-κB p65 protein in cells.Silencing KLF5 could increase the activity of H9 c2 cells under hydrogen peroxide, decrease the secretion of TNF-αand IL-1β, and inhibit the expression of NF-κB p65 protein. NF-κB activator can reverse the effect of KLF5 silence on the proliferation and the secretion of inflammatory factors of cardiomyocytes treated by hydrogen peroxide.In conclusion, oxidative stress induces the expression of KLF5 in cardiac myocytes, and silencing KLF5 expression can reduce the levels of inflammatory factors by inhibiting the NF-κB pathway.
This study was designed to study the effect and mechanism of KLF5 silence on the secretion of inflammatory factors in myocardial cells under oxidative stress. The H9c2 cells of myocardium, infected with or without KLF5 shRNA lentivirus, were treated with hydrogen peroxide, then qRT-PCR and Western blot were used to detect the level of KLF5 mRNA and protein expression in the cells. Furthermore, MTT was used to detect the effect of KLF5 silence on the activity of cardiac myocytes treated by hydrogen peroxide, at the same time, the levels of TNF-α and IL-1β was detected by qRT-PCR. Western blot was used to detect the level of NF-κB p65 protein in the cells. On the other hand, myocardial cells of silent KLF5 were treated with NF-κB activator, followed by hydrogen peroxide treatment. Then the activity of the cells were detected by MTT, the levels of TNF-α and IL-1β in the cells were detected by qRT-PCR. Data showed thathydrogen peroxide induced the expression of KLF5 mRNA and protein in H9 c2 cells. KLF5 shRNAlentivirus infection could down-regulate the expression of KLF5 in H9 c2 cells under oxidative stress; hydrogen peroxide could down-regulate the activity of H9 c2 cells,increase the level of TNF-α and IL-1β secreted by cells, promote the expression of NF-κB p65 protein in cells.Silencing KLF5 could increase the activity of H9 c2 cells under hydrogen peroxide, decrease the secretion of TNF-αand IL-1β, and inhibit the expression of NF-κB p65 protein. NF-κB activator can reverse the effect of KLF5 silence on the proliferation and the secretion of inflammatory factors of cardiomyocytes treated by hydrogen peroxide.In conclusion, oxidative stress induces the expression of KLF5 in cardiac myocytes, and silencing KLF5 expression can reduce the levels of inflammatory factors by inhibiting the NF-κB pathway.
引文
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[24]陈轶霞,龙玲,骆学农.痘病毒锚蛋白重复序列蛋白对NF-кB信号通路的影响[J].中国免疫学杂志,2017,33(5):765-768.
[25]Xu X,Qi X,Shao Y,et al.High glucose inducedmacrophage activation through TGF-β-activated kinase 1signaling pathway[J].Inflamm Res,2016,65(8):1-10.
[26]Ma D,Zhang RN,Wen Y,et al.1,25(OH)2D3-induced interaction of vitamin D receptor with p50 subunit of NF-κB suppresses the interaction between KLF5 and p50,contributing to inhibition of LPS-induced macrophage proliferation[J].Biochem Biophys Res Commun,2016,482(2):366.1033
[2]Essick EE,Ouchi N,Wilson RM,et al.Adiponectin mediates cardioprotection in oxidative stress-induced cardiac myocyte remodeling[J].Am J Physiol Heart Circ Physiol,2011,301(3):984-93.
[3]Liao X,Haldar SM,Lu Y,et al.Kruppel-like factor 4regulates pressure-induced cardiac hypertrophy[J].J Mol Cell Cardiol,2010,49(2):334-338.
[4]Clerk A,Kemp TJ,Zoumpoulidou G,et al.Cardiac myocyte gene expression profiling during H2O2-induced apoptosis[J].Physiol Genomics,2007,29(2):118-127.
[5]欧思琳,李茹冰.NF-κB在动脉粥样硬化疾病中的研究进展[J].中国免疫学杂志,2018,34(7):1095-1101.
[6]Sur I,Undén AB,Toftg?rd R.Human Kruppel-like factor5/KLF5:synergy with NF-kappaB/Rel factors and expression in human skin and hair follicles.[J].Eur J Cell Biol,2002,81(6):323-334.
[7]Rong L,Shi P,Zhi N,et al.Mifepristone suppresses basal triple-negative breast cancer stem cells by downregulating KLF5 expression[J].Theranostics,2016,6(4):533-544.
[8]Shindo T,Manabe I,Fukushima Y,et al.Krüppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin II signaling and an essential regulator of cardiovascular remodeling[J].Nat Med,2002,8(8):856-863.
[9]Drosatos K,Pollak NM,Pol C,et al.Cardiac myocyte KLF5regulates ppara expression and cardiac function[J].Circ Res,2016,118(2):241-253.
[10]Takeda N,Manabe I,Uchino Y,et al.Cardiac fibroblasts are essential for the adaptive response of the murine heart to pressure overload[J].J Clin Invest,2010,120(1):254-265.
[11]Bell KN,Shroyer NF.Krüpple-like factor 5 is required for proper maintenance of adult intestinal crypt cellular proliferation[J].Dig Dis Sci,2015,60(1):86-100.
[12]Kumar M,Kasala ER,Bodduluru LN,et al.Baicalein protects isoproterenol induced myocardial ischemic injury in male Wistar rats by mitigating oxidative stress and inflammation[J].Inflamm Res,2016,65(8):1-10.
[13]Hu X,Ma R,Lu J,et al.IL-23 Promotes Myocardial I/Rinjury by increasing the inflammatory responses and oxidative stress reactions[J].Cell Physiol Biochem,2016,38(6):2163-2172.
[14]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.
[15]Chuo CH,Devine SM,Scammells PJ,et al.VCP746,a novel A1 adenosine receptor biased agonist,reduces hypertrophy in a rat neonatal cardiac myocyte model[J].Clin Exp Pharmacol Physiol,2016,43(10):976-982.
[16]Nakano M,Knowlton AA,Dibbs Z,et al.Tumor necrosis factor-alpha confers resistance to hypoxic injury in the adult mammalian cardiac myocyte[J].Circulation,2002,97(14):1392-1400.
[17]Kumar A,Kumar A,Paladugu B,et al.Transforming growth factor-beta1 blocks in vitro cardiac myocyte depression induced by tumor necrosis factor-alpha,interleukin-1beta,and human septic shock serum[J].Crit Care Med,2007,35(2):358-364.
[18]Zwijnenburg PJ,Van der Poll T,Florquin S,et al.IL-1receptor type 1 gene-deficient mice demonstrate an impaired host defense against pneumococcal meningitis[J].J Immunol,2003,170(9):4724-30.
[19]Pascual V,Allantaz F,Arce E,et al.Role of interleukin-1(IL-1)in the pathogenesis of systemic onset juvenile idiopathic arthritis and clinical response to IL-1blockade[J].J Exp Med,2005,201(9):1479-1486.
[20]Bell JR,Curl CL,Harding TW,et al.Male and female hypertrophic rat cardiac myocyte functional responses to ischemic stress and?2-adrenergic challenge are different[J].Biol Sex Differ,2016,7(1):1-15.
[21]Lappas M.KLF5 regulates infection-and inflammationinduced pro-labour mediators in human myometrium[J].Reproduction,2015,149(5):413-424.
[22]Lee HL,Park MH,Hong JE,et al.Inhibitory effect of snake venom toxin on NF-κB activity prevents human cervical cancer cell growth via increase of death receptor 3 and 5expression[J].Arch Toxicol,2016,90(2):463-477.
[23]梁伟杰,陈美姬,何洁仪,等.ATP敏感性钾通道的开放通过抑制TLR4/NF-κB通路对抗高糖引起的H9c2心肌细胞损伤和炎症[J].中国病理生理杂志,2016,32(7):1153-1160.
[24]陈轶霞,龙玲,骆学农.痘病毒锚蛋白重复序列蛋白对NF-кB信号通路的影响[J].中国免疫学杂志,2017,33(5):765-768.
[25]Xu X,Qi X,Shao Y,et al.High glucose inducedmacrophage activation through TGF-β-activated kinase 1signaling pathway[J].Inflamm Res,2016,65(8):1-10.
[26]Ma D,Zhang RN,Wen Y,et al.1,25(OH)2D3-induced interaction of vitamin D receptor with p50 subunit of NF-κB suppresses the interaction between KLF5 and p50,contributing to inhibition of LPS-induced macrophage proliferation[J].Biochem Biophys Res Commun,2016,482(2):366.1033