糖原合成激酶-3β／β-连环蛋白信号通路在贫铀致人肾近曲小管上皮细胞损伤中的作用  

Role of GSK-3β/β-catenin signaling pathway in human renal proximal tubular epithelial cell injury induced by depleted uranium

作　　者：李强 暴一众[1] 张旭霞[1] 高赟[1] 丁德芳[1] 任湘祎 陈红红[1] Li Qiang Bao Yizhong Zhang Xuxia Gao Yun Ding Defang Ren Xiangyi Chen Honghong(Department of Radiation Biology, Institute of Radiation Medicine, Fudan University, Shanghai 200032, Chin)

机构地区：[1]复旦大学放射医学研究所放射生物部,上海200032

出　　处：《中华放射医学与防护杂志》2017年第3期171-176,共6页Chinese Journal of Radiological Medicine and Protection

基　　金：国家科技重大专项子课题（2013ZX09J13101-05B）

摘　　要：目的探索糖原合成激酶-3β（GSK-3β）／β-连环蛋白信号通路对贫铀（depleted uranium，DU）致人肾近曲小管上皮HK-2细胞损伤的作用。方法不同浓度DU染毒HK-2细胞3～24h，采用免疫荧光法检测肾损伤分子-1（KIM-1）和中性粒细胞明胶酶相关脂质运载蛋白（NGAL）表达水平以观察DU诱导HK-2细胞的损伤，免疫荧光法检测β-连环蛋白核转位，Westernblot法检测p-GSK-3β（S9）、GSK-3β、β-连环蛋白及c-mye蛋白表达变化，观察GSK-3β／β-连环蛋白信号通路的时相变化；采用GSK-3β（KD）质粒瞬时转染和GSK-3β特异性抑制剂TDZD-8处理抑制HK-2细胞的GSK-3β活性，采用β-连环蛋白质粒瞬时转染HK-2细胞使其过表达，观察GSK-3β活性和β-连环蛋白表达变化对DU染毒致HK-2细胞损伤的影响。结果300和600μmol／LDU染毒致HK-2细胞KIM-1和NGAL阳性细胞率随染毒时间的延长和染毒剂量的增加而增加，染毒后6、9和24h明显高于空白对照组（KIM-1阳性细胞率：t=11．06、18．97、30．49，P〈0．05；t=6．79、16．02、85．45，P〈0．05；NGAL阳性细胞率：t=11．78、11．37、34．29，P〈0．05；t=7．34、21．63、36．84，P〈0．05）；p—GSK-3β（S9）／GSK-3β比值和核β-连环蛋白阳性细胞率于染毒后3、6、9和24h明显高于空白对照组[p-GSK-313（s9）／GSK-3β比值：t=3．95、4．69、5．40、3．34，P〈0．05]；核β-连环蛋白阳性细胞率：t=4．61、6．52、36．64、14．93，P〈0．05），于染毒后9h达峰值，还激活了β-连环蛋白下游靶基因c-myc的蛋白表达。瞬时转染GSK-3β（KD）质粒明显抑制了HK-2细胞的GSK-3β活性（t=8．07，P〈0．05），降低了DU致染毒HK-2细胞的KIM-1阳性细胞率（t：24．77，P〈0．05）。TDZD-8抑制GSK-3β活性的同时提高了β-连环蛋白核转位，亦能显著降低DU染毒诱导HK-2细胞的KIM-1阳性细胞率（t=6．25、6．73，P〈0．05）。而且，β-连环蛋�Objective To investigate the effects of glycogen synthase kinase-3β （GSK-3β） and β-catenin signaling on the human renal proximal tubular epithelial HK-2 cell injury induced by depleted uranium（DU）, and provide a new enlightenment for the development of DU antidotes. Methods HK-2 ceils were exposed to different concentrations of DU for 3 -24 h, then the protein expressions of kidney injury molecule 1 （KIM-1）, neutrophil gelatinase-associated lipocalin （NGAL） and nuclear β-catenin were detected by immunofluorescence staining. The protein expressions of p-GSK-3β （S9） , GSK-313 and c- myc were detected by Western blot assay. HK-2 cells were transiently transfected by GSK-3β （KD） plasmid or treated by TDZD-8 to inhibit the activity of GSK-3β specifically. Other HK-2 ceils were transiently transfected by β-catenin plasmid to overexpress the β-catenin protein. Results The percentages of KIM-1 and NGAL-positive Cells increased with DU exposure time and concentrations from 300 and 600 μmol/L, and they were significantly higher than those of the blank control at 6 -24 h of DU exposure （KIM-l-positive cells： t = 11.06, 18.97, 30.49, P 〈0.05; t =6.79, 16.02, 85.45, P 〈 0.05; NGAL-positive cells： t=11.78, 11.37, 34.29, P〈0.05; t =7.34, 21.63, 36.84, P〈0.05）. In contrast, the ratio of p-GSK-313 （ S9 ） to GSK-3β and percentage of nuclearβ-catenin-positive cells were significantly higher than that of the blank control at 3 -24 h of DU exposure （p-GSK-313 （S9）/GSK-313： t = 3.95, 4. 69, 5.40, 3.34, P 〈 0. 05 ; nuclear β-catenin-positive cells ： t = 4. 61, 6. 52, 36. 64, 14. 93, P 〈 0. 05） with a maximum response at 9 h of DU exposure accompanied with corresponding increase of protein level of c-myc, a downstream target gene of β-catenin. Transient transfection of HK-2 cells with GSK-3 6 （KD） plasmid significantly inhibited the activity of GSK-3 β（ t = 8.07, P 〈 0.05 ） and reduced the DU-increased percentage of KIM-l-positive cells （ t = 24. 77,

关 键 词：贫铀 糖原合成激酶-3β Β-连环蛋白 肾损伤分子-1 HK-2细胞 

分 类 号：R114[医药卫生—卫生毒理学]