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

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,80μmol/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。

Objective To detect expression changes of Sfrp1 in NRK-52 E 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 modelingfor 10 weeks;kidney index(KI) was calculated;the expression of Sfrp1 in renal tissue was detected byimmunohistochemistry. NRK-52 E 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,Dnmt3 a, Dnmt3 b, 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 Dnmt3 a, Dnmt3 b, 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 Dnmt3 a, Dnmt3 b, 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 Dnmt3 a, Dnmt3 b, col-Ⅲ and col-Ⅳ in 5-Aza-CdR group were decreased(P<0.05);Sfrp1 was negatively correlated with Dnmt3 a(r=-0.937, P<0.05),Dnmt3 b(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 Dnmt3 a and Dnmt3 b expression and the increase of Sfrp1 expression.