过表达CHIP对缺氧/复氧诱导下RIPK3介导神经细胞程序性死亡的影响

目的 探讨热休克蛋白70羧基末端相互作用蛋白(Carboxylterminus of Hsp70 interacting protein,CHIP)过表达对缺氧(Hypoxia,H)/复氧(Reoxygenation,R)诱导下受体相互作用蛋白激酶3(Receptor-interacting protein kinase 3,RIPK3)介导的神经细胞程序性死亡的影响。方法 将体外培养的小鼠脑神经瘤(Mouse neuroblastoma N2a cells,Neuro-2a)细胞分为正常组(正常培养)、H/R组(构建H/R模型)、H/R+空载体组(转染pcDNA3.1空载体质粒后构建H/R模型)和H/R+CHIP过表达组(转染pcDNA3.1-CHIP过表达质粒后构建H/R模型); 采用实时荧光量PCR(Real-time fluorescence quantitative PCR,qPCR)检测细胞中CHIP mRNA相对表达水平; 免疫印迹法(Western blotting)检测细胞中CHIP,RIPK3和混合系列蛋白激酶样结构域(Mixed lineage kinase domain-like protein,MLKL)蛋白相对表达水平; 细胞计数试剂盒-8(Cell counting kit-8,CCK-8)法检测细胞存活率; 试剂盒检测细胞乳酸脱氢酶(Lactate dehydrogenase,LDH)漏出量和白细胞介素-6(Interleukin-6,IL-6)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、超氧化物歧化酶(Superoxide dismutase,SOD)、丙二醛(Malondialdehyde,MDA)水平; 2,7-二氯二氢荧光素二乙酸酯(2,7-Dichlorodi hydrofluorescein diacetate,DCFH-DA)法检测活性氧(Reactive oxygen species,ROS)水平。结果 与正常组比较,H/R组细胞中CHIP mRNA,CHIP蛋白表达水平和细胞存活率以及SOD水平均明显降低,而LDH漏出量和RIPK3,MLKL蛋白表达以及IL-6,TNF-α,ROS,MDA水平均明显升高(P<0.05); 与H/R组比较,H/R+空载体组中上述各指标水平均无明显变化(P>0.05); 与H/R+空载体组比较,H/R+CHIP过表达组细胞中CHIP mRNA,CHIP蛋白表达水平和细胞存活率以及SOD水平均明显升高,而LDH漏出量和RIPK3,MLKL蛋白表达以及IL-6,TNF-α,ROS,MDA水平均明显降低(P<0.05)。结论 CHIP过表达可通过抑制RIPK3介导的神经细胞程序性坏死来减轻H/R诱导的神经细胞损伤。

Effects of CHIP overexpression on RIPK3-mediated programmed cell death induced by hypoxia/reoxygenation

ObjectiveTo investigate the effect of carboxylterminus of the Hsp70 interacting protein(CHIP)overexpression on receptor interacting protein kinase 3(RIPK3)-mediated programmed cell death induced by hypoxia(H)/reoxygenation(R).Methods Neuro-2a cells were divided into normal group(normal culture), H/R group(H/R model), H/R + empty vector group(H/R model was constructedafter transfection of pcDNA3.1 empty vector plasmid)and H/R + CHIP overexpression group(H/R model was constructed after transfection of pcDNA 3.1-CHIP overexpression plasmid), the expression of CHIP mRNA was detected by real-time quantitative PCR(qPCR), Western blotting was used to detect the expression levels of CHIP, RIPK3 and mixed lineage kinase domain-like protein(MLKL), the cell survival rate was detected by cell counting kit-8(CCK-8), the leakage of lactate dehydrogenase(LDH)and the levels of interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), superoxide dismutase(SOD)and malondialdehyde(MDA)were detected by the kit, and the level of reactive oxygen species(ROS)was measured by 2,7-dichlorodihydrofluorescein diacetate(DCFH-DA).Results Compared with those in normal group, the CHIP mRNA and CHIP protein expressions, cell survival rate and SOD level in H/R group were significantly lower, while LDH leakage, RIPK3 andMLKL protein expressions, IL-6, TNF-α, ROS and MDA levels in H/R group were significantly higher(P<0.05); compared with those in H/R group, the above indexes in H/R + empty body group had no significant difference(P>0.05); compared with those in H/R + empty vector group, the CHIP mRNA and CHIP protein expressions, cell survival rate and SOD level in H/R + CHIP overexpression group were significantly higher, while LDH leakage, RIPK3 and MLKL protein expressions, IL-6, TNF-α, ROS and MDA levels in H/R group were significantly lower(P<0.05).Conclusion CHIP overexpression can reduce H/R-induced neuronal injury by inhibiting RIPK3-mediated programmed necrosis.