摘要
目的:探究miR-223通过抑制NOD样受体热蛋白结构域3(NLRP3)炎症小体减轻免疫球蛋白A肾病(IgAN)大鼠肾脏损伤的作用。方法:IgA-HMC培养液培养HK2细胞建立IgAN细胞模型,并根据组别转染miR-223 mimics或pc-NLRP3重组载体,RT-PCR检测临床样本和细胞中miR-223和NLRP3基因表达,双荧光素酶报告实验检测miR-223和NLRP3的相互作用关系,Western blot检测细胞中NLRP3、接头蛋白凋亡相关点状蛋白(ASC)、胱天蛋白酶1(Caspase-1)蛋白水平。牛血清白蛋白+脂多糖+四氯化碳法建立IgAN大鼠模型,并根据组别尾静脉注射miR-223 agomir或si-NLRP3,收集尿液检测蛋白尿,取心尖血检测血清肌酸酐(SCr)和尿素氮(BUN),HE染色观察大鼠肾组织病理改变,TUNEL染色检测肾组织细胞凋亡,ELISA检测血清IL-1β、IL-18浓度,Western blot检测肾脏NLRP3、ASC、Caspase-1蛋白水平。结果:相较于健康对照组,IgAN患者中miR-223表达水平降低(P<0.05)。在HK2细胞中,与Control组相比,pIgA组miR-223表达水平降低,NLRP3基因和蛋白表达水平升高(P<0.01),同时ASC和Caspase-1蛋白水平升高(P<0.01),转染miR-223 mimics可降低NLRP3、ASC、Caspase-1蛋白水平(P<0.01),pc-NLRP3可逆转miR-223 mimics引起的NLRP3、ASC、Caspase-1蛋白水平降低(P<0.01),荧光素酶报告实验显示miR-223可靶向作用于NLRP3。在IgAN模型大鼠体内,与Control组相比,IgAN组血、尿病理指标升高(P<0.01),肾组织内出现明显的病理性改变及细胞凋亡(P<0.01),IL-1β和IL-18浓度升高(P<0.01),NLRP3炎症小体激活(P<0.01),通过尾静脉注射miR-223 agomir或si-NLRP3均可显著减轻上述各指标改变。结论:miR-223可靶向作用于NLRP3,从而减轻IgAN引起的大鼠肾脏损伤。
Objective:To investigate the role of miR-223 in alleviating renal damage in rats with immunoglobulin A nephropathy(IgAN)by inhibiting NOD-like receptor pyrin domain containing 3(NLRP3)inflammasome.Methods:IgA-HMC culture medium was used to culture HK2 cells to establish IgAN cell model,miR-223 mimics or pc-NLRP3 were transfected to corresponding group respectively,miR-223 and NLRP3 gene expressions in clinical samples and cells were detected by RT-PCR,dual luciferase reporter assay was used to test the interaction between miR-223 and NLRP3,protein levels of NLRP3,apoptosis-associated speck-like protein(ASC),Caspase-1 were measured by Western blot.IgAN rat model was established by bovine serum albumin+lipopolysaccharide+carbon tetrachloride method,miR-223 agomir or si-NLRP3 were injected to corresponding group through vena caudalis,collect urine to measure proteinuria,and take apical blood to measure serum creatine(SCr)and urea nitrogen(BUN),pathological changes of renal tissue were observed by HE staining,cell apoptosis were determined by TUNEL staining,IL-1βand IL-18 were detected by ELISA,protein levels of NLRP3,ASC,Caspase-1 in renal tissue were detected by Western blot.Results:In samples of IgAN patient,expression of miR-223 was decreased compared to samples of healthy control(P<0.05).In HK2 cells,compared with Control group,expression of miR-223 in pIgA group was decreased,gene and protein expressions of NLRP3 were increased(P<0.01),and protein levels of ASC and Caspase-1 were increased(P<0.01),transfection of miR-223 mimics can reduce protein levels of NLRP3,ASC,Caspase-1(P<0.01),transfection of pc-NLRP3 can reverse the reduction of NLRP3,ASC,Caspase-1 caused by miR-223 mimics(P<0.01).Dual luciferase reporter assay show that miR-223 can target with NLRP3.In IgAN rat model,compared with control group,blood and urine pathological parameters in IgAN group were increased(P<0.01),renal tissues emerged significant pathological changes and cell apoptosis(P<0.01),concentrations of IL-1βand IL-18 were increased(P<0.01),NLRP3 inflammasome was activated(P<0.01),tail vein injection of miR-223 agomir or si-NLRP3 significantly alleviated the above changes.Conclusion:miR-233 can target with NLRP3,to attenuate kidney damage of rats caused by IgAN.
作者
王瑾
王加强
邱洪
杨丽萍
李佳
谭翔
WANG Jin;WANG Jia-Qiang;QIU Hong;YANG Li-Ping;LI Jia;TAN Xiang(Department of Clinical Laboratory,Sichuan Academy of Medical Sciences,Sichuan Provincial People′s Hospital,Chengdu 610072,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2020年第24期2960-2965,共6页
Chinese Journal of Immunology
基金
四川省干部保健科研课题(川干研2018-204)。
