miR-520c-3p对糖尿病肾脏病患者近端肾小管上皮细胞焦亡的影响及机制研究  

Effect of miR-520c-3p on pyroptosis of proximal tubular epithelial cells in diabetic kidney disease and its mechanism

作　　者：宋怡 郭丰[1] 甘甜 邵明玮[1] 范迅捷 徐亚楠 张薇 卫芳祎 秦贵军[1] Song Yi;Guo Feng;Gan Tian;Shao Mingwei;Fan Xunjie;Xu Yanan;Zhang Wei;Wei Fangyi;Qin Guijun(Department of Endocrinology and Metabolism,First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院内分泌与代谢病科,郑州450052

出　　处：《中华糖尿病杂志》2022年第6期592-602,共11页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：国家自然科学基金(81974110,81770812,82170839);河南省教育厅重点项目(20A320013)。

摘　　要：目的探讨miR-520c-3p对糖尿病肾脏病患者近端肾小管上皮细胞焦亡的影响及其分子机制。方法收集2019年6月至2020年12月在郑州大学第一附属医院就诊的糖尿病肾脏病患者(DKD组,4例)和糖耐量正常肾脏肿瘤手术者(正常对照组,4例)的肾脏组织。HE染色、过碘酸-希夫染色(PAS)和透射电镜观察肾脏组织病理学变化。取生长状态良好的人近端肾小管上皮(HK-2)细胞传代至培养板内,并分成如下8组:正常葡萄糖组(NG,5.6 mmol/L葡萄糖)、高糖组(HG,30.0 mmol/L葡萄糖)、抑制物组(正常葡萄糖+转染miR-520c-3p抑制物)、抑制物对照组(正常葡萄糖+转染miR-520c-3p抑制物阴性对照物)、模拟物组(高糖+转染miR-520c-3p模拟物)、模拟物对照组(高糖+转染miR-520c-3p模拟物阴性对照物)、模拟物+TXNIP共转染组(高糖+共转染miR-520c-3p模拟物+TXNIP)、共转染阴性对照组(高糖+共转染miR-520c-3p模拟物+TXNIP阴性对照物)。活细胞动态实时监测细胞活性,碘化丙啶(PI)染色检测细胞焦亡情况;免疫荧光染色、荧光原位杂交、实时定量PCR和Western blotting法检测miR-520c-3p、硫氧还蛋白相互作用蛋白(TXNIP)和细胞焦亡分子NOD样受体蛋白3(NLRP3)、活化半胱氨酸蛋白酶-1(cl-caspase-1)、消皮素D(GSDMD)的mRNA和蛋白表达水平;酶联免疫吸附测定法(ELISA)检测细胞上清液白细胞介素-1β(IL-1β)含量;双荧光素酶报告基因实验明确miR-520c-3p和TXNIP之间的相互作用。两组间比较采用t检验,多组间比较采用单因素方差分析。结果与对照组相比,DKD组肾脏组织肾小管炎性细胞增多,伴随miR-520c-3p表达水平下降,TXNIP表达升高(均P<0.05);肾小管特异性标志物水通道蛋白1(AQP1)与NLRP3和GSDMD焦亡蛋白共定位均增加。在HK-2细胞中,与NG组相比,HG组焦亡细胞增多,伴随miR-520c-3p表达水平下降,TXNIP、NLRP3、cl-caspase-1、GSDMD的蛋白水平和细胞上清液IL-1β浓度升高(均P<0.05);与模�Objective To investigate the protective effect of miR-520c-3p on cell pyroptosis of proximal tubular epithelial cell(PTEC)in diabetic kidney disease(DKD)and its molecular mechanism.Methods Human kidney specimens from patients with DKD(DKD group,n=4)were obtained from the First Affiliated Hospital of Zhengzhou University from June 2019 to December 2020,and the kidney specimens from unaffected portions of the kidney tumor with normal glucose tolerance were served as normal controls(NC group,n=4).Renal histopathological changes were examined by hematoxylin and eosin(HE)staining,periodate-Schiff staining(PAS)and transmission electron microscope(TEM).PTEC(HK-2 cell line)were cultured with normal glucose(NG group,5.6 mmol/L glucose)and high glucose(HG group,30.0 mmol/L glucose),and transfected with miR-520c-3p mimics,miR-520c-3p inhibitor,miR-520c-3p negative control(NC)plasmid,thioredoxin-interacting protein(TXNIP)plasmid and TXNIP negative control(NC)plasmid.Cell pyroptosis was determined by dynamic real-time monitoring of living cells and propidium iodide(PI)staining.The expression of miR-520c-3p,TXNIP,NOD-like receptor thermal protein domain associated protein 3(NLRP3),cleaved caspase-1,Gasdermin D(GSDMD)were detected by immunofluorescence staining,fluorescence in situ hybridization(FISH),real time-PCR and Western blotting.The IL-1βconcentration in cell supernatant was detected by enzyme-linked immunosorbent assay(ELISA)kit.Interaction between miR-520c-3p and TXNIP was analyzed by dual luciferase activity assay.Data were presented as the mean±standard deviation(SD),and the significance was determined using unpaired Student′s t-test for two groups and one-way analysis of variance(ANOVA)for multiple groups.Results Compared with the NC group,inflammatory cells were increased in the DKD group,accompanied with reduction of miR-520c-3p expression level and increased of TXNIP level(P<0.05).The co-localization between renal tubule specific marker aquaporin(AQP1)and NLRP3 and that between AQP1 and GSDMD were both increa

关 键 词：糖尿病 糖尿病肾病 近端肾小管上皮细胞 细胞焦亡 miR-520c-3p 硫氧还蛋白相互作用蛋白 

分 类 号：R587.2[医药卫生—内分泌] R692.9[医药卫生—内科学]