Wnt5a对缺氧复氧导致的心肌细胞氧化损伤的影响
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摘要
目的探讨Wnt5a对缺氧复氧导致的心肌细胞氧化损伤的影响。方法以心肌细胞HCM为研究对象,分为:正常组、模型组、siRNA control组、Wnt5a siRNA组。其中正常组细胞正常培养,模型组、si RNA control组、Wnt5a siRNA组细胞按照缺氧复氧处理,siRNA control组、Wnt5a siRNA组细胞分别用转染Wnt5a siRNA和siRNA control后的HCM细胞。Western blot和RT-PCR检测Wnt5a mRNA和蛋白水平,噻唑蓝(MTT)检测细胞增殖活力,流式细胞术检测细胞凋亡,硫代巴比妥酸比色法检测丙二醛(MDA)水平,黄嘌呤氧化法检测超氧化物歧化酶(SOD)活性,二硝基苯肼显色法检测上清中乳酸脱氢酶(LDH)活性。结果模型组心肌细胞中Wnt5a mRNA和蛋白水平、细胞凋亡率、MDA水平、LDH水平均明显高于正常组(P<0.05),而细胞存活率、SOD水平均明显低于正常组(P<0.05)。si RNA control组细胞中Wnt5a mRNA和蛋白水平、细胞凋亡率、MDA水平、LDH水平、SOD水平及细胞存活率与模型组相比没有明显差异。Wnt5a siRNA组Wnt5a mRNA和蛋白水平、细胞凋亡率、MDA水平、LDH水平均明显低于模型组(P<0.05),而细胞存活率、SOD水平均明显高于模型组(P<0.05)。结论 Wnt5a在缺氧复氧心肌细胞中表达升高,干扰Wnt5a表达能够降低缺氧复氧诱导的心肌细胞氧化损伤,减少细胞凋亡。
Objective To discuss the influence of Wnt5a on cardiomyocyte oxidative damage induced by hypoxia/reoxygenation. Methods The human cardiomyocytes(HCM) were taken as study objects and divided into normal group, model group, siRNA control group and Wnt5a siRNA group. HCM were normally cultured in normal group, and given hypoxia/reoxygenation treatment in model group, siRNA control group and Wnt5a siRNA group.HCM were transfected with Wnt5a siRNA or siRNA control respectively in siRNA control group and Wnt5a siRNA group. The mRNA and protein levels of Wnt5a in HCM were detected by using Western blotting assay and RTPCR, HCM proliferation activity was detected by using methyl thiazolyl tetrazolium test(MTT), and HCM apoptosis was detected by using flow cytometry. The level of MDA was detected by using thiobarbituric acid(TBA) method,activity of SOD was detected by using xanthineoxidation(XTO) method, and activity of LDH was detected by using dinitrophenylhydrazine(DNPH) method. Results The mRNA and protein levels of Wnt5a, HCM apoptosis rate and levels of MDA and LDH were significantly higher(P<0.05), and HCM survival rate and SOD level were significantly lower in model group than those in normal group(P<0.05). The mRNA and protein levels of Wnt5a, HCM apoptosis rate, levels of MDA, LDH and SOD and HCM survival rate had no significant difference between siRNA control group and model group. The mRNA and protein levels of Wnt5a, HCM apoptosis rate and levels of MDA and LDH were significantly lower(P<0.05), and HCM survival rate and SOD level were significantly higher in Wnt5a siRNA group than those in model group(P<0.05). Conclusion The expression of Wnt5a increases in hypoxia/reoxygenation HCM, and intervention of Wnt5a can reduce the oxidative damage and apoptosis of HCM induced by hypoxia/reoxygenation.
Objective To discuss the influence of Wnt5a on cardiomyocyte oxidative damage induced by hypoxia/reoxygenation. Methods The human cardiomyocytes(HCM) were taken as study objects and divided into normal group, model group, siRNA control group and Wnt5a siRNA group. HCM were normally cultured in normal group, and given hypoxia/reoxygenation treatment in model group, siRNA control group and Wnt5a siRNA group.HCM were transfected with Wnt5a siRNA or siRNA control respectively in siRNA control group and Wnt5a siRNA group. The mRNA and protein levels of Wnt5a in HCM were detected by using Western blotting assay and RTPCR, HCM proliferation activity was detected by using methyl thiazolyl tetrazolium test(MTT), and HCM apoptosis was detected by using flow cytometry. The level of MDA was detected by using thiobarbituric acid(TBA) method,activity of SOD was detected by using xanthineoxidation(XTO) method, and activity of LDH was detected by using dinitrophenylhydrazine(DNPH) method. Results The mRNA and protein levels of Wnt5a, HCM apoptosis rate and levels of MDA and LDH were significantly higher(P<0.05), and HCM survival rate and SOD level were significantly lower in model group than those in normal group(P<0.05). The mRNA and protein levels of Wnt5a, HCM apoptosis rate, levels of MDA, LDH and SOD and HCM survival rate had no significant difference between siRNA control group and model group. The mRNA and protein levels of Wnt5a, HCM apoptosis rate and levels of MDA and LDH were significantly lower(P<0.05), and HCM survival rate and SOD level were significantly higher in Wnt5a siRNA group than those in model group(P<0.05). Conclusion The expression of Wnt5a increases in hypoxia/reoxygenation HCM, and intervention of Wnt5a can reduce the oxidative damage and apoptosis of HCM induced by hypoxia/reoxygenation.
引文
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[19]Rodrigo R,Libuy M,FeliúF,et al.Oxidative stress-related biomarkers in essential hypertension and ischemia-reperfusion myocardial damage[J].Disease markers,2013,35(6):773-90.
[20]Hu H,Zhai C,Qian G,et al.Protective effects of tanshinone IIA on myocardial ischemia reperfusion injury by reducing oxidative stress,HMGB1 expression,and inflammatory reaction[J].Pharmaceutical biology,2015,53(12):1752-8.
[2]Yang Y,Sun Y,Yi W,et al.A review of melatonin as a suitable antioxidant against myocardial ischemia-reperfusion injury and clinical heart diseases[J].Journal of pineal research,2014,57(4):357-66.
[3]Ma S,Wang Y,Chen Y,et al.The role of the autophagy in myocardial ischemia/reperfusion injury[J].Biochimica et Biophysica Acta(BBA)-Molecular Basis of Disease,2015,1852(2):271-6.
[4]Wei B,Li WW,Ji J,et al.The cardioprotective effect of sodium tanshinone IIA sulfonate and the optimizing of therapeutic time window in myocardial ischemia/reperfusion injury in rats[J].Atherosc lerosis,2014,235(2):318-27.
[5]Duffy DJ,Krstic A,Schwarzl T,et al.GSK3 inhibitors regulate MYCNmRNA levels and reduce neuroblastoma cell viability through multiple mechanisms,including p53 and Wnt signaling[J].Molecular cancer therapeutics,2014,13(2):454-67.
[6]Aisagbonhi O,Rai M,Ryzhov S,et al.Experimental myocardial infarction triggers canonical Wnt signaling and endothelial-tomesenchymal transition[J].Disease models&mechanisms,2011,4(4):469-83.
[7]Wu X,Zhou S,Zhu N,et al.Resveratrol attenuates hypoxia/reoxygenation induced Ca2+overload by inhibiting the Wnt5a/Frizzled2 pathway in rat H9c2 cells[J].Molecular medicine reports,2014,10(5):2542-8.
[8]Yu H,Zhao XX,Shan XH,et al.Effect of thioredoxin-interacting protein on Wnt/β-catenin signaling pathway and diabetic myocardial infarction[J].Asian Pacific journal of tropical medicine,2015,8(11):976-82.
[9]Daskalopoulos EP,Hermans KCM,Janssen BJA,et al.Targeting the Wnt/frizzled signaling pathway after myocardial infarction:a new tool in the therapeutic toolbox?[J].Trends in cardiovascular medicine,2013,23(4):121-7.
[10]Deb A.Cell-cell interaction in the heart via Wnt/β-catenin pathway after cardiac injury[J].Cardiovascular research,2014,102(2):214-23.
[11]Assmus B,Iwasaki M,Sch?chinger V,et al.Acute myocardial infarction activates progenitor cells and increases Wnt signalling in the bone marrow[J].European heart journal,2011,33(15):1911-9.
[12]Nakamura K,Sano S,Fuster JJ,et al.Secreted frizzled-related protein5 diminishes cardiac inflammation and protects the heart from ischemia/reperfusion injury[J].Journal of Biological Chemistry,2016,291(6):2566-75.
[13]Kilian B,Mansukoski H,Barbosa FC,et al.The role of Ppt/Wnt5 in regulating cell shape and movement during zebrafish gastrulation[J].Mechanisms of development,2003,120(4):467-76.
[14]Takahashi K,Takahashi T,Suzuki T,et al.Protective effects of SEA0400,a novel and selective inhibitor of the Na+/Ca2+exchanger,on myocardial ischemia-reperfusion injuries[J].European journal of pharmacology,2003,458(1):155-62.
[15]周珊珊.Wnt-5a/Frizzled-2途径对心肌缺血再灌注损伤后心肌Ca~(2+)变化的影响的研究[D].南方医科大学,2011.
[16]Qian L,Shi J,Zhang C,et al.Downregulation of RACK1 is associated with cardiomyocyte apoptosis after myocardial ischemia/reperfusion injury in adult rats[J].In Vitro Cellular&Developmental BiologyAnimal,2016,52(3):305-13.
[17]Ramond A,Godin-Ribuot D,Ribuot C,et al.Oxidative stress mediates cardiac infarction aggravation induced by intermittent hypoxia[J].Fundamental&clinical pharmacology,2013,27(3):252-61.
[18]Ansley DM,Wang B.Oxidative stress and myocardial injury in the diabetic heart[J].The Journal of pathology,2013,229(2):232-41.
[19]Rodrigo R,Libuy M,FeliúF,et al.Oxidative stress-related biomarkers in essential hypertension and ischemia-reperfusion myocardial damage[J].Disease markers,2013,35(6):773-90.
[20]Hu H,Zhai C,Qian G,et al.Protective effects of tanshinone IIA on myocardial ischemia reperfusion injury by reducing oxidative stress,HMGB1 expression,and inflammatory reaction[J].Pharmaceutical biology,2015,53(12):1752-8.