摘要
目的:探讨维生素D3上调蛋白1(VDUP-1)对高糖诱导的肾小管上皮细胞凋亡的影响及机制。方法:人近端肾小管上皮HK-2细胞用高糖处理后,real-time PCR和Western blot检测细胞中VDUP-1的水平。HK-2细胞转染VDUP-1小干扰RNA,real-time PCR和Western blot检测抑制效果。高糖条件培养VDUP-1表达下调的HK-2细胞,流式细胞术检测细胞凋亡,试剂盒检测细胞中caspase-3和caspase-9的活性,ELISA法测定培养上清液中肿瘤坏死因子α(TNF-α)含量,Western blot检测细胞中音猬因子(Shh)信号通路关键蛋白Patched 1 (Ptch1)、Smoothened (Smo)、锌指蛋白Gli2和Shh的水平。用外源性Shh处理HK-2细胞,Western blot检测Ptch1、Smo和Gli2的水平。用外源性Shh处理VDUP-1表达下调的HK-2细胞,高糖处理后,流式细胞术检测细胞凋亡,试剂盒检测细胞中caspase-3和caspase-9的活性,ELISA法测定培养液上清中TNF-α含量。结果:高糖处理后,HK-2细胞中VDUP-1的mRNA和蛋白水平升高(P <0. 05)。转染VDUP-1小干扰RNA后,HK-2细胞中VDUP-1的mRNA和蛋白水平下降(P <0. 05)。与正常培养的细胞相比,高糖处理后HK-2细胞凋亡率显著升高,细胞中caspase-3和caspase-9活性明显升高,TNF-α含量亦明显升高(P <0. 05);下调VDUP-1表达后的HK-2细胞经高糖处理后细胞凋亡率显著降低,细胞中caspase-3和caspase-9活性也明显降低(P <0. 05)。高糖培养后细胞中Ptch1、Smo、Gli2和Shh的蛋白水平下降,而下调VDUP-1表达部分拮抗高糖对HK-2细胞中Ptch1、Smo、Gli2和Shh表达的影响。外源性Shh可以促进细胞中Ptch1、Smo和Gli2的表达,抑制高糖诱导的HK-2细胞凋亡和分泌TNF-α,与下调VDUP-1共同抑制高糖诱导的肾小管上皮细胞凋亡。结论:干扰VDUP-1表达可能通过激活Shh信号通路抑制高糖诱导的肾小管上皮细胞凋亡和分泌TNF-α。
AIM:To investigate the effect of vitamin D3 up-regulated protein 1(VDUP-1)on apoptosis of renal tubular epithelial cells induced by high glucose and its mechanism.METHODS:Human renal proximal tubular epithelial cell line HK-2 was treated with high glucose.The mRNA and protein levels of VDUP-1 in HK-2 cells were detected by real-time PCR and Western blot.HK-2 cells were transfected with VDUP-1 small interfering RNA(siRNA).Real-time PCR and Western blot were used to detect the inhibitory effect.The HK-2 cells were treated with high glucose,and the change of VDUP-1 expression was detected.The apoptosis was analyzed by flow cytometry.The activities of caspase-3 and caspase-9 in the cells were measured.The tumor necrosis factor-α(TNF-α)content in the culture supernatant was examined by ELISA.The key proteins of Sonic hedgehog(Shh)signaling pathway,Patched 1(Ptch1),Smoothened(Smo),zinc finger protein Gli2 and Shh,were determined by Western blot.The HK-2 cells were treated with exogenous Shh,and the levels of Ptch1,Smo and Gli2 were detected by Western blot.After the HK-2 cells with VDUP-1 silencing were treated with exogenous Shh and high glucose,the apoptosis was analyzed by flow cytometry,the activities of caspase-3 and caspase-9 in the cells were examined,and the TNF-αcontent in culture supernatant was measured by ELISA.RESULTS:High levels of VDUP-1 mRNA and protein were observed in the HK-2 cells treated with high glucose.The mRNA and protein levels of VDUP-1 were decreased in the HK-2 cells transfected with VDUP-1 siRNA(P<0.05).Compared with the normally cultured cells,the apoptotic rate of HK-2 cells was increased after high glucose treatment,and the activities of caspase-3 and caspase-9 and the content of TNF-αwere also significantly increased(P<0.05).After down-regulation of VDUP-1 expression by siRNA transfection,the apoptotic rate of HK-2 cells decreased after high glucose treatment,and the activities of caspase-3 and caspase-9,and the content of TNF-αwere also significantly decreased(P<0.05).The protei
作者
张明霞
刘伦志
向海燕
谭千林
ZHANG Ming-xia;LIU Lun-zhi;XIANG Hai-yan;TAN Qian-lin(Department of Nephrology,University Hospital of Hubei University for Nationalities,Enshi 445000,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2018年第11期1968-1975,共8页
Chinese Journal of Pathophysiology
基金
恩施州科技局计划项目(No.D20170017)
关键词
维生素D3上调蛋白1
肾小管上皮细胞
糖尿病肾病
凋亡
SHH信号通路
Vitamin D3 up-regulated protein 1
Renal tubular epithelial cells
Diabetic nephropathy
Apoptosis
Shh signaling pathway
