[1,25 -(OH)2-VitD3通过抑制Snail1-SMAD3/SMAD4复合物形成减轻糖尿病肾病肾小管间质纤维化]。

Chengchong Huang, Rong Dong, Zhengsheng Li, Jing Yuan
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The binding of Snail1 with SMAD3/SMAD4 complex to the promoter of Coxsackie-adenovirus receptor (CAR) was detected by chromatin immunoprecipitation. The interaction among Snail1, SMAD3/SMAD4 and E-cadherin were detected by luciferase assay. Small interfering RNA (siRNA) was used to inhibit the expression of Snail1 and SMAD4, and the expression of mRNA of E-cadherin was detected by real-time quantitative PCR. SD rats were randomly divided into control group, DKD group and VitD3-treated group. DKD model was established by injection of streptozotocin (STZ) in DKD group and VitD3-treated group. After DKD modeling, VitD3-treated group was given VitD3 (60 ng/kg) intragastric administration. Control group and DKD group were given normal saline intragastric administration. In the DKD group and VitD3-treated group, insulin (1-2 U/kg) was injected subcutaneously to control blood glucose for 8 weeks. The mRNA and protein levels of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in renal tissues were detected by real-time quantitative PCR and Western blot analysis respectively. Immunohistochemistry was used to detect the expression and localization of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in renal tissue. Results Compared with the control group, the mRNA and protein expressions of Snail1, SMAD3, SMAD4 and α-SMA in NRK-52E cells cultured with high glucose and in DKD renal tissues were up-regulated, while E-cadherin expression was down-regulated. After the intervention of VitD3, the expression levels of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in the DKD model improved to be close to those in the control group. Chromatin immunoprecipitation showed that Snail1 and SMAD3/SMAD4 bound to CAR promoter IV, while VitD3 prevented Snail1 and SMAD3/SMAD4 from binding to CAR promoter IV. Luciferase assay confirmed the interaction among Snail1, SMAD3/SMAD4 and E-cadherin. 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引用次数: 0

摘要

目的探讨1,25 -(OH)2-维生素d3 (VitD3)对糖尿病肾病肾小管间质纤维化的影响。方法将NRK-52E肾小管上皮细胞分为对照组(5.5 mmol/L葡萄糖培养基处理)、高糖组(25 mmol/L葡萄糖培养基处理)和高糖加维生素d3组(25 mmol/L葡萄糖培养基联合10-8 mmol/L VitD3)。采用实时定量PCR和Western blot分别检测NRK-52E细胞中Snail1、SMAD3、SMAD4、α-SMA和E-cadherin的mRNA和蛋白表达。免疫荧光细胞化学染色检测Snail1、SMAD3和SMAD4的表达和定位。采用染色质免疫沉淀法检测Snail1与SMAD3/SMAD4复合物结合柯萨奇腺病毒受体(CAR)启动子的情况。荧光素酶法检测Snail1、SMAD3/SMAD4与E-cadherin的相互作用。采用小干扰RNA (Small interfering RNA, siRNA)抑制Snail1和SMAD4的表达,采用实时定量PCR检测E-cadherin mRNA的表达情况。SD大鼠随机分为对照组、DKD组和vitd3治疗组。DKD组和vitd3组分别注射链脲佐菌素(STZ)建立DKD模型。DKD造模后,VitD3处理组大鼠灌胃维生素d3 (60 ng/kg)。对照组和DKD组给予生理盐水灌胃。DKD组和vitd3治疗组皮下注射胰岛素(1 ~ 2 U/kg)控制血糖,持续8周。采用实时荧光定量PCR和Western blot分别检测肾组织中Snail1、SMAD3、SMAD4、α-SMA和E-cadherin的mRNA和蛋白水平。采用免疫组化方法检测Snail1、SMAD3、SMAD4、α-SMA和E-cadherin在肾组织中的表达和定位。结果与对照组相比,高糖培养的NRK-52E细胞和DKD肾组织中Snail1、SMAD3、SMAD4和α-SMA mRNA和蛋白表达上调,E-cadherin表达下调。VitD3干预后,DKD模型中Snail1、SMAD3、SMAD4、α-SMA、E-cadherin的表达水平提高,与对照组接近。染色质免疫沉淀显示Snail1和SMAD3/SMAD4与CAR启动子IV结合,而VitD3阻止了Snail1和SMAD3/SMAD4与CAR启动子IV的结合。荧光素酶测定证实了Snail1、SMAD3/SMAD4与E-cadherin之间的相互作用。Snail1和SMAD4 mRNA被siRNA抑制后,高糖诱导的E-cadherin表达上调。结论VitD3可抑制Snail1-SMAD3/SMAD4复合物的形成,减轻DKD大鼠肾小管间质纤维化。
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[1, 25-(OH)2-VitD3 attenuates renal tubulointerstitial fibrosis in diabetic kidney disease by inhibiting Snail1-SMAD3/SMAD4 complex formation].

Objective To investigate the effect of 1, 25-(OH)2-VitD3 (VitD3) on renal tubuleinterstitial fibrosis in diabetic kidney disease. Methods NRK-52E renal tubular epithelial cells were divided into control group (5.5 mmol/L glucose medium treatment), high glucose group (25 mmol/L glucose medium treatment) and high glucose with added VitD3 group (25 mmol/L glucose medium combined with 10-8 mmol/L VitD3). The mRNA and protein expression of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in NRK-52E cells were detected by real-time quantitative PCR and Western blot analysis respectively. The expression and localization of Snail1, SMAD3 and SMAD4 were detected by immunofluorescence cytochemical staining. The binding of Snail1 with SMAD3/SMAD4 complex to the promoter of Coxsackie-adenovirus receptor (CAR) was detected by chromatin immunoprecipitation. The interaction among Snail1, SMAD3/SMAD4 and E-cadherin were detected by luciferase assay. Small interfering RNA (siRNA) was used to inhibit the expression of Snail1 and SMAD4, and the expression of mRNA of E-cadherin was detected by real-time quantitative PCR. SD rats were randomly divided into control group, DKD group and VitD3-treated group. DKD model was established by injection of streptozotocin (STZ) in DKD group and VitD3-treated group. After DKD modeling, VitD3-treated group was given VitD3 (60 ng/kg) intragastric administration. Control group and DKD group were given normal saline intragastric administration. In the DKD group and VitD3-treated group, insulin (1-2 U/kg) was injected subcutaneously to control blood glucose for 8 weeks. The mRNA and protein levels of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in renal tissues were detected by real-time quantitative PCR and Western blot analysis respectively. Immunohistochemistry was used to detect the expression and localization of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in renal tissue. Results Compared with the control group, the mRNA and protein expressions of Snail1, SMAD3, SMAD4 and α-SMA in NRK-52E cells cultured with high glucose and in DKD renal tissues were up-regulated, while E-cadherin expression was down-regulated. After the intervention of VitD3, the expression levels of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in the DKD model improved to be close to those in the control group. Chromatin immunoprecipitation showed that Snail1 and SMAD3/SMAD4 bound to CAR promoter IV, while VitD3 prevented Snail1 and SMAD3/SMAD4 from binding to CAR promoter IV. Luciferase assay confirmed the interaction among Snail1, SMAD3/SMAD4 and E-cadherin. After the mRNA of Snail1 and SMAD4 was inhibited by siRNA, the expression of E-cadherin induced by high glucose was up-regulated. Conclusion VitD3 could inhibit the formation of Snail1-SMAD3/SMAD4 complex and alleviate the renal tubulointerstitial fibrosis in DKD.

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