RIP1介导的坏死性凋亡通过NF-κB通路调节肾小管上皮细胞炎症反应

RIP1-mediated necroptosis regulates inflammatory response of renal tu⁃bular epithelial cells via NF-κB signaling pathway

目的:探讨受体相互作用蛋白1(RIP1)介导的坏死性凋亡对肾小管上皮细胞炎症的影响及其机制。方法:体外培养人肾小管上皮HK-2细胞,应用肿瘤坏死因子α(TNF-α)联合Z-VAD-FMK刺激24 h。乳酸脱氢酶(LDH)细胞毒性实验检测细胞坏死百分比,Western blot检测观察RIP1、IKK-α和NF-κB p65的表达,Western blot和ELISA测定白细胞介素1β(IL-1β)和单核细胞趋化蛋白1(MCP-1)的水平,real-time PCR检测NF-κB p65的mRNA表达水平。进一步应用RIP1抑制剂necrostatin-1(Nec-1)和NF-κB特异性抑制剂吡咯烷二硫代氨基甲酸铵(PDTC)进行干预,检测上述指标。结果:与对照组比较,TNF-α联合Z-VAD-FMK刺激组(T/Z组)的HK-2细胞中cleaved RIP1蛋白水平明显升高,IKK-α和NF-κB p65的蛋白水平明显增高,LDH释放明显增多(P<0. 01)。Western blot和ELISA检测炎症相关指标IL-1β和MCP-1的水平均有明显升高,real-time PCR检测NF-κB p65的mRNA表达水平也有明显增高。给予Nec-1或PDTC刺激后(T/Z+N组或T/Z+P组),LDH释放及炎症相关指标IL-1β和MCP-1的蛋白水平明显降低,同时给予以上2种刺激后(T/Z+P/N组)IL-1β和MCP-1的蛋白水平进一步降低(P<0. 05)。结论:T/Z条件下,RIP1介导的坏死性凋亡在肾小管上皮细胞炎症反应中发挥重要作用。这一作用可能是部分通过调节NF-κB信号通路活化实现的。

AIM:To investigate the effect of receptor-interacting protein 1(RIP1)-mediated necroptosis on human kidney proximal tubular cell inflammation and its related mechanisms.METHODS:Human kidney proximal tubu⁃lar HK-2 cells were cultured in vitro,and stimulated with tumot tumor necrosis factor-α(TNF-α)and Z-VAD-FMK for 24 h.Lactate dehydrogenase(LDH)cytotoxicity assay was used to detect the percentage of necrosis.Western blot was used to detect the protein expression of RIP1,IKK-αand NF-κB p65.The protein levels of interleukin-1β(IL-1β)and mono⁃cyte chemoattractant protein-1(MCP-1)were determined by Western blot and ELISA.Real-time PCR was used to detect the mRNA expression level of NF-κB p65.Furthermore,the RIP1 inhibitor necrostatin-1(Nec-1)and the NF-κB specific inhibitor ammonium pyrrolidinedithiocarbamate(PDTC)were used,and the above indicators were also detected.RE⁃SULTS:Compared with control group,the protein level of RIP1 was increased in TNF-αcombined with Z-VAD-FMK stimulation group(T/Z group).The protein levels of IKK-αand NF-κB p65 were obviously increased,and the release of LDH was increased(P<0.01).Western blot and ELISA showed that the expression levels of IL-1βand MCP-1 were signif⁃icantly increased(P<0.01).Real-time PCR showed that the mRNA expression level of NF-κB p65 was also obviously in⁃creased.After Nec-1 or PDTC stimulation(T/Z+N group or T/Z+P group),the release of LDH,and the expression levels of inflammation-related indicators IL-1βand MCP-1 were significantly decreased.The protein expression levels of IL-1βand MCP-1 were further reduced after treatment with the above 2 stimulati(T/Z+P/N group).CONCLUSION:Under T/Z condition,RIP1-mediated necroptosis plays an important role in renal tubular inflammatory response,which may be partly achieved by regulating the activation of NF-κB signaling pathway.