新型免疫抑制剂浅蓝霉素A诱导肾损伤炎症的作用

Role of inflammation induced by the novel immunosuppressant caerulomycin A in kidney injury

目的探究高浓度浅蓝霉素A(Cae A)对人肾小管上皮细胞HK2的影响,并探讨NOD样受体热蛋白结构域相关蛋白3(NLRP3)在此过程中发挥的作用。方法采用MTT法测定不同浓度的Cae A对HK2细胞活力的影响;采用实时定量PCR,Western blotting以及免疫荧光法检测高浓度Cae A对肾损伤分子1(KIM-1)以及NLRP3表达情况的影响;同时用实时定量PCR方法,检测高浓度Cae A对白细胞介素-1β(IL-1β)、白细胞介素18(IL-18)、白细胞介素33(IL-33)、单核细胞趋化蛋白-1(MCP-1)、肿瘤坏死因子α(TNF-α)的mRNA表达情况;随后将HK2细胞分为对照组、高浓度Cae A组和高浓度Cae A加NLRP3抑制剂CY-09组,Western blotting检测KIM-1和NLRP3蛋白的表达变化。结果MTT的结果显示,高浓度Cae A可以抑制HK2细胞活力;实时定量PCR,Western blotting和免疫荧光实验表明,高浓度Cae A可以上调KIM-1和NLRP3的水平,以及IL-1β、IL-18、IL-33、MCP-1、TNF-α的mRNA表达水平,而CY-09能显著下调NLRP3和KIM-1的蛋白水平。结论高浓度的Cae A能明显抑制HK2细胞的活力,诱导HK2细胞发生损伤及炎症反应,有显著的肾毒性,而且该肾毒性是通过激活NLRP3通路来实现的。

Objective To investigate the effect of high concentration of Caerulomycin A(Cae A)on HK2 in renal tubular epithelial cells and to explore the role of cytoplasmic nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)in this process.Methods The effect of different concentrations of Cae A on the viability of HK2 cells was determined by MTT;the expression of kidney injury molecule(KIM-1)and NLRP3 was detected by real-time quantitative PCR,Western blot and immunofluorescence,while the effect of Cae A on the mRNA expression of IL-1β,IL-18,IL-33,MCP-1,TNF-αwas also measured by real-time quantitative PCR.HK2 cells were divided into control group,high concentration of Cae A group and high concentration of Cae A plus NLRP3 inhibitor CY-09 group,and the expression of KIM-1 and NLRP3 protein was detected by Western blot.Results The results of MTT showed that high concentration of Cae A could inhibit HK2 cell viability.Real-time quantitative PCR,Western blot and immunofluorescence assays showed that high concentration of Cae A upregulated the expression of KIM-1 and NLRP3,as well as the mRNA levels of IL-1β,IL-18,IL-33,MCP-1,TNF-α,while CY-09 could down-regulate the expression of NLRP3 and KIM-1.Conclusion High concentration of Cae A significantly inhibited the viability of HK2 cells and induced damage and inflammatory response to HK2 with some nephrotoxicity that might be achieved via NLRP3 pathway.