调节M2型丙酮酸激酶对TGF-β1诱导人肾小管上皮细胞周期阻滞和细胞外基质分泌的影响  

Functional role of pyruvate kinase M2 in cell cycle arrest and extracellular matrix secretion of renal tubular epithelial cell induced by transforming growth factor-β1

作　　者：吴洪銮 安宁 陈小翠 庞浩文 杨陈 刘华锋 Wu Hong-luan;An Ning;Chen Xiao-cui;Pang Hao-wen;Yang Chen;Liu Hua-feng(Key Laboratory of Prevention&Management of Chronic Kidney Disease of Zhanjiang Municipality,Institute of Nephrology;Department of Emergency Medicine,Affiliated Hospital,Guangdong Medical University,Zhanjiang 524001,China)

机构地区：[1]广东医科大学附属医院肾病研究所暨湛江市慢性肾脏病防治重点实验室,湛江524001 [2]广东医科大学附属医院急诊科,湛江524001

出　　处：《临床肾脏病杂志》2021年第12期1009-1014,共6页Journal Of Clinical Nephrology

基　　金：国家自然科学基金(81700627);广东省自然科学基金(2019A1515010678);湛江市科技发展专项资金竞争性分配项目(2018A01034)。

摘　　要：目的探讨M2型丙酮酸激酶(pyruvate kinase M2,PKM2)在转化生长因子-β1(transforming growth factor-β1,TGF-β1)诱导人肾小管上皮细胞周期阻滞和细胞外基质分泌中的作用。方法利用TGF-β1刺激人肾皮质近曲小管上皮细胞HK-2(human kidney cell 2)细胞构建体外纤维化细胞模型。采用PKM2变构剂TEPP-46和抑制剂紫草素(Shikonin)抑制二聚体PKM2功能。利用蛋白免疫印迹法检测细胞外基质蛋白纤连蛋白和胶原I以及结缔组织生长因子的表达;利用碘化丙啶染色、流式细胞术检测细胞周期分布情况;利用蛋白免疫印迹法检测细胞周期抑制蛋白p21的表达情况。结果蛋白免疫印迹法检测显示,TGF-β1处理显著提高HK-2细胞内二聚体PKM2的表达。加用PKM2变构剂TEPP-46或抑制剂Shikonin,显著抑制TGF-β1诱导的HK-2细胞外基质蛋白纤连蛋白和胶原I以及结缔组织生长因子的表达。此外,TEPP-46或Shikonin均显著抑制TGF-β1诱导的HK-2细胞G1周期阻滞以及周期抑制蛋白p21的表达。结论二聚体PKM2促进TGF-β1诱导人肾小管上皮细胞G1周期阻滞和细胞外基质分泌;抑制二聚体PKM2功能有望成为延缓肾小管-间质纤维化的新策略。Objective To explore the functional role of pyruvate kinase M2(PKM 2) in cell cycle arrest and extracellular matrix secretion of renal tubular epithelial cell induced by transforming growth factor-β1(TGF-β1).Methods HK-2 cells were stimulated by TGF-β1 for establishing an in vitro model of renal tubulointerstitial fibrosis.The function of dimeric PKM2 was suppressed by a PKM2 allosteric agent TEPP-46 or its antagonist shikonin.The expressions of fibronectin, collagen I and connective tissue growth factor(CTGF) were detected by Western blot.The ratio of HK-2 cells in cycle phase was performed by propidium iodide staining and analyzed by flow cytometry.Also the expression of cell cycle inhibitor p21 was detected by Western blot.Results As compared with control-treated cells, a treatment of TGF-β1 significantly boosted the levels of extracellular matrix fibronectin, collagen I,pro-fibrotic CTGF and dimeric PKM2.By contrast, TEPP-46 or shikonin markedly suppressed the elevations of fibronectin, collagen I and CTGF.Moreover, an inhibition of dimeric PKM2 by TEPP-46 or shikonin remarkably suppressed the proportion of TGF-β1-treated HK-2 cells in G1 phase through a down-regulation of p21.Conclusion Dimeric PKM2 promotes cell cycle arrest and extracellular matrix secretion of renal tubular epithelial cell induced by TGF-β1.And targeting dimeric PKM2 may represent a novel therapeutic strategy for delaying the progression of renal tubulointerstitial fibrosis.

关 键 词：肾小管上皮细胞 M2型丙酮酸激酶 TGF-Β1 周期阻滞 细胞外基质 

分 类 号：R692[医药卫生—泌尿科学]