| 李强,暴一众,张旭霞,等.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[J].中华放射医学与防护杂志,2017,37(3):171-176.Li Qiang,Bao Yizhong,Zhang Xuxia,et al.Role of GSK-3β/β-catenin signaling pathway in human renal proximal tubular epithelial cell injury induced by depleted uranium[J].Chin J Radiol Med Prot,2017,37(3):171-176 |
| 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 |
| 投稿时间：2016-11-07 |
| DOI：10.3760/cma.j.issn.0254-5098.2017.03.002 |
| 中文关键词: 贫铀 糖原合成激酶-3β β-连环蛋白 肾损伤分子-1 HK-2细胞 |
| 英文关键词:Depleted uranium GSK-3β β-catenin KIM-1 HK-2 cell |
| 基金项目:国家科技重大专项子课题（2013ZX09J13101-05B） |
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| 中文摘要: |
| 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3~24 h，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，Western blot法检测p-GSK-3β（S9）、GSK-3β、β-连环蛋白及c-myc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600 μmol/L DU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24 h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24 h明显高于空白对照组；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9 h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-连环蛋白过表达显著减轻了DU诱导的HK-2细胞损伤（t=7.48，P<0.05）。结论 GSK-3β/β-连环蛋白信号通路是调控DU致HK-2细胞毒性的关键通路，抑制GSK-3β活性及β-连环蛋白表达能有效拮抗DU诱导的HK-2细胞损伤。 |
| 英文摘要: |
| 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 cells 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-3β 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 cells 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-1-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-3β(S9) to GSK-3β and percentage of nuclear β-catenin-positive cells were significantly higher than that of the blank control at 3-24 h of DU exposure (p-GSK-3β(S9)/GSK-3β: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-3β(KD) plasmid significantly inhibited the activity of GSK-3β (t=8.07, P<0.05) and reduced the DU-increased percentage of KIM-1-positive cells (t=24.77, P<0.05). Treatment cells with TDZD-8 inhibited the activity of GSK-3β and enhanced the percentage of nuclear β-catenin-positive cells, and it also significantly reduced the percentage of KIM-1-positive cells in HK-2 cells exposed to DU (t=6.25, 6.73, P<0.05). Moreover, overexpression of β-catenin significantly reduced DU-induced cell injury (t=7.48, P<0.05). Conclusions GSK-3β/β-catenin signaling plays a key role in regulating the DU-induced cytotoxicity of HK-2 cells. Inhibition of GSK-3β activity and overexpression of β-catenin can protect the HK-2 cells from DU-induced damage. |
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