5-氮杂-2’-脱氧胞苷对高糖培养的NRK-52E细胞中分泌型卷曲相关蛋白1表达的影响

doi:10.16352/j.issn.1001-6325.2022.08.1194

基础医学与临床 ›› 2022, Vol. 42 ›› Issue (8): 1194-1199.doi: 10.16352/j.issn.1001-6325.2022.08.1194

5-氮杂-2’-脱氧胞苷对高糖培养的NRK-52E细胞中分泌型卷曲相关蛋白1表达的影响

田平平¹, 宁洁¹, 邹琴^2,3, 卢雨微², 郭兵^2,3, 石明隽^2,3*

1.新乡医学院三全学院扩理学院,河南新乡 453003;
2.贵州医科大学病理生理学教研室,贵州贵阳 550025;
3.贵州省常见慢性疾病发病机制及药物研究重点实验室,贵州贵阳 550025

收稿日期:2021-09-15 修回日期:2022-04-26 出版日期:2022-08-05 发布日期:2022-08-01
通讯作者: ^*smjtyf@126.com
基金资助:
国家自然科学基金(82060142,81860135)

Effect of 5-Aza-2'-deoxycytidine on the expression of Sfrp1 in NRK-52E cells cultured with high glucose

TIAN Ping-ping¹, NING Jie¹, ZOU Qin^2,3, LU Yu-wei², GUO Bing^2,3, SHI Ming-jun^2,3*

1. School of Nursing, Sanquan College of Xinxiang Medical University, Xinxiang 453003;
2. Department of Pathophysiology, Guizhou Medical University,Guiyang 550025;
3. Guizhou Provincial Key Laboratory of Pathogenesis ＆ Drug Research on Common Chronic Diseases, Guiyang 550025, China

Received:2021-09-15 Revised:2022-04-26 Online:2022-08-05 Published:2022-08-01
Contact: ^*smjtyf@126.com

摘要/Abstract

摘要： 目的检测DNA甲基转移酶抑制剂5-氮杂-2’-脱氧胞苷(5-Aza-CdR)干预肾小管上皮细胞(NRK-52E)后对分泌型卷曲相关蛋白1(Sfrp1)表达变化的影响。方法将SD大鼠随机分为正常组(NC组)和糖尿病肾病组(DN组),DN组建模^[1-2]10周后检测相关生化指标、计算肾脏指数(KI);免疫组化法检测肾组织中Sfrp1的表达;将NRK-52E细胞分为正常糖组(NG,葡萄糖5.5 mmol/L)、高糖组(HG,葡萄糖30 mmol/L)和高糖加5-Aza-CdR组(5-Aza-CdR组,80 μmol/L),用Western blot检测Sfrp1、DNA甲基转移酶(Dnmt)3a、Dnmt3b、col-Ⅲ和col-Ⅳ蛋白的表达;RT-qPCR检测 Sfrp1 mRNA的表达;Pearson检验分析Sfrp1与Dnmt3a、Dnmt3b、col-Ⅲ及col-Ⅳ的相关性。结果 DN组大鼠KI、血糖(BG)、24 h尿蛋白(24 h UP)、肌酐水平均高于NC组。与NC组比,DN组肾组织中Sfrp1表达明显降低。与NG组比,HG组Sfrp1 mRNA 和蛋白表达减少(P＜0.05),Dnmt3a、Dnmt3b、col-Ⅲ和col-Ⅳ蛋白表达增加(P＜0.05);与HG组相比,5-Aza-CdR组Sfrp1 mRNA 和蛋白表达增多(P＜0.05);Dnmt3a、Dnmt3b、col-Ⅲ和col-Ⅳ蛋白表达减少(P＜0.05);Sfrp1与Dnmt3a(r=-0.937,P＜0.05)、Dnmt3b(r=-0.965,P＜0.05)、col-Ⅲ(r=-0.694,P＜0.05)和col-Ⅳ(r=-0.888,P＜0.05)均呈负相关。结论 5-氮杂-2’-脱氧胞苷可能通过降低Dnmt3a和Dnmt3b表达,进而使Sfrp1表达升高。

关键词: 5-氮杂-2’-脱氧胞苷, DNA甲基转移酶3a, DNA甲基转移酶3b, 分泌型卷曲相关蛋白1, 肾脏纤维化

Abstract: Objective To detect expression changes of Sfrp1 in NRK-52E cells after 5-Aza-2'deoxycytidine (5-Aza-CdR) intervention. Methods Sprague-Dawley (SD) rats were divided into normal group (NC group) and diabetic nephropathy group (DN group) randomly. The related biochemical indexes were detected after successfully modeling^[1-2] for 10 weeks;kidney index (KI) was calculated; the expression of Sfrp1 in renal tissue was detected by immunohistochemistry. NRK-52E cells were divided into normal glucose group (NG, 5.5 mmol/L), high glucose group (HG, 30 mmol/L) and high glucose +5-Aza-CdR group(5-Aza-CdR group);the expressions of Sfrp1,Dnmt3a,Dnmt3b,col-Ⅲ and col-Ⅳ were detected by Western blot;the mRNA expression of Sfrp1 was detected by RT-qPCR;pearson test was used to analyze the correlation between Sfrp1 and Dnmt3a,Dnmt3b,col-Ⅲ or col-Ⅳ. Results The level of KI, blood glucose, 24 h urinary protein and creatinine in DN group were all higher than those in NC group.The expression of Sfrp1 in renal tissue of DN group was significantly lower than that of NC group.The results of in vitro experiments showed as follows:the mRNA and protein expression of Sfrp1 were decreased in HG group(P＜0.05),while the protein expressions of Dnmt3a,Dnmt3b,col-Ⅲ and col-Ⅳ in HG group were increased (P＜0.05);compared with HG group,the mRNA and protein expression of Sfrp1 was increased in 5-Aza-CdR group(P＜0.05), the protein expressions of Dnmt3a,Dnmt3b,col-Ⅲ and col-Ⅳ in 5-Aza-CdR group were decreased (P＜0.05); Sfrp1 was negatively correlated with Dnmt3a (r=-0.937, P＜0.05),Dnmt3b (r=-0.965, P＜0.05),col-Ⅲ(r=-0.694, P＜0.05) and col-Ⅳ(r=-0.888, P＜0.05). Conclusions The mechanism through which 5-Aza-2′-deoxycytidine inhibits renal fibrosis is potentially related to the reduction of Dnmt3a and Dnmt3b expression and the increase of Sfrp1 expression.

Key words: 5-Aza-2′ deoxycytidine (5-Aza-CDR), DNA methyltransferase 3a(Dnmt3a), DNA methyltransferase 3b(Dnmt3b), secreted frizzled-related protein 1(Sfrp1), renal fibrosis

中图分类号:

田平平, 宁洁, 邹琴, 卢雨微, 郭兵, 石明隽. 5-氮杂-2’-脱氧胞苷对高糖培养的NRK-52E细胞中分泌型卷曲相关蛋白1表达的影响[J]. 基础医学与临床, 2022, 42(8): 1194-1199.

TIAN Ping-ping, NING Jie, ZOU Qin, LU Yu-wei, GUO Bing, SHI Ming-jun. Effect of 5-Aza-2'-deoxycytidine on the expression of Sfrp1 in NRK-52E cells cultured with high glucose[J]. Basic & Clinical Medicine, 2022, 42(8): 1194-1199.

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5-氮杂-2’-脱氧胞苷对高糖培养的NRK-52E细胞中分泌型卷曲相关蛋白1表达的影响

Effect of 5-Aza-2'-deoxycytidine on the expression of Sfrp1 in NRK-52E cells cultured with high glucose

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