黄芪甲苷调控Akt信号通路阻抑人腹膜间皮细胞间充质转化的实验研究
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摘要
目的观察黄芪甲苷(AS-Ⅳ)对TGF-β1诱导人腹膜间皮细胞HMrSV5EMT及β-catenin的影响,探讨Akt信号通路的作用及AS-Ⅳ干预机制。方法采用TGF-β1诱导建立HMrSV5EMT模型,Western blot检测不同浓度TGF-β1对EMT标记蛋白、β-catenin及Akt信号蛋白的影响;予不同浓度AS-Ⅳ干预HMrSV5EMT模型,Real-time PCR检测EMT相关基因及β-catenin mRNA水平,Western blot检测Akt信号蛋白表达;与Akt通路抑制剂MK2206、雷帕霉素及激动剂Insulin作对照,观察AS-Ⅳ干预HMrSV5EMT指标及β-catenin蛋白的变化。结果 (1)TGF-β1诱导HMrSV5细胞发生EMT、上调β-catenin水平,激活Akt信号通路;(2)AS-Ⅳ能不同程度改善TGF-β1诱导的HMrSV5EMT、降解β-catenin,并在一定程度上抑制Akt信号通路活化;(3)Akt通路参与调控HMrSV5EMT及β-catenin,其抑制剂MK2206、雷帕霉素的调控作用与AS-Ⅳ类似;(4)Akt通路激动剂Insulin明显减弱AS-Ⅳ对EMT及β-catenin的抑制效果。结论 TGF-β1可诱导HMrSV5EMT,上调β-catenin,AS-Ⅳ可能通过抑制Akt信号通路活化,阻抑HMrSV5EMT,降解β-catenin。
OBJECTIVE To observe the effect of AstragalosideⅣ(AS-Ⅳ)on EMT of human peritoneal mesothelial cells(HPMCs)HMrSV5,exploring the function of Akt signaling and regulatory mechanism of AS-Ⅳ.METHODS We used TGF-β1 to establish EMT model of HMrSV5.EMT marker proteins,β-catenin and Akt signaling-related proteins were detected by Western blotting analysis.β-catenin and EMT-related mRNA were expressed by Real-time PCR analysis.Compared with MK2206,Rapamycin and Insulin,the effect of AS-Ⅳon EMT andβ-catenin was observed by Western blotting.RESULTS(1)The EMT model of HMrSV5 was established by TGF-β1.TGF-β1 increasedβ-catenin expression and activated Akt signaling.(2)AS-Ⅳcould alleviate TGF-β1-treated EMT of HMrSV5,decreaseβ-catenin expression,as well as inhibit activation of Akt signaling.(3)EMT of HMrSV5 andβ-catenin were regulated by Akt signaling,MK2206 and Rapamycin had the similar effect with AS-Ⅳ.(4)Insulin could reduce obviously inhibitory effect on EMT andβ-catenin of AS-Ⅳ.CONCLUSION TGF-β1 can induce EMT of HMrSV5 and upregulateβ-catenin,AS-Ⅳ has inhibitory effect on EMT andβ-catenin of HMrSV5 by regulating Akt signaling.
OBJECTIVE To observe the effect of AstragalosideⅣ(AS-Ⅳ)on EMT of human peritoneal mesothelial cells(HPMCs)HMrSV5,exploring the function of Akt signaling and regulatory mechanism of AS-Ⅳ.METHODS We used TGF-β1 to establish EMT model of HMrSV5.EMT marker proteins,β-catenin and Akt signaling-related proteins were detected by Western blotting analysis.β-catenin and EMT-related mRNA were expressed by Real-time PCR analysis.Compared with MK2206,Rapamycin and Insulin,the effect of AS-Ⅳon EMT andβ-catenin was observed by Western blotting.RESULTS(1)The EMT model of HMrSV5 was established by TGF-β1.TGF-β1 increasedβ-catenin expression and activated Akt signaling.(2)AS-Ⅳcould alleviate TGF-β1-treated EMT of HMrSV5,decreaseβ-catenin expression,as well as inhibit activation of Akt signaling.(3)EMT of HMrSV5 andβ-catenin were regulated by Akt signaling,MK2206 and Rapamycin had the similar effect with AS-Ⅳ.(4)Insulin could reduce obviously inhibitory effect on EMT andβ-catenin of AS-Ⅳ.CONCLUSION TGF-β1 can induce EMT of HMrSV5 and upregulateβ-catenin,AS-Ⅳ has inhibitory effect on EMT andβ-catenin of HMrSV5 by regulating Akt signaling.
引文
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[2]YAEZ-MO M,LARA-PEZZI E,SELGAS R,et al.Peritoneal dialysis and epithelial-to-mesenchymal transition of mesothelial cells[J].N Engl J Med,2003,348(5):403-413.
[3]史俊,俞曼殊,盛梅笑,等.黄芪甲苷抑制高糖腹透液诱导HMrSV5氧化应激与EMT的实验研究[J].南京中医药大学学报,2016,32(4):337-341.
[4]YOSHIZAWA H,MORISHITA Y,WATANABE M,et al.TGF-β1-siRNA delivery with nanoparticles inhibits peritoneal fibrosis[J].Gene Ther,2015,22(4):333.
[5]HUANG TS,LI L,MOALIM‐NOUR L,et al.A regulatory network involvingβ‐Catenin,E-cadherin,PI3k/Akt,and Slug balances self‐renewal and differentiation of human pluripotent stem cells in response to wnt signaling[J].Stem Cell,2015,33(5):1419-1433.
[6]ZHOU D,TAN RJ,FU H,et al.Wnt/β-catenin signaling in kidney injury and repair:a double-edged sword[J].Lab Invest,2016,96(2):156-167.
[7]BAO Z,XU X,LIU Y,et al.CBX7negatively regulates migration and invasion in glioma via Wnt/β-catenin pathway inactivation[J].Oncotarget,2017,8(24):39048-39063.
[8]GUO Y,GUPTE M,UMBARKAR P,et al.Entanglement of GSK-3β,β-catenin and TGF-β1signaling network to regulate myocardial fibrosis[J].J Mol Cell Cardiol,2017,110:109.
[9]LIEN EC,LYSSIOTIS CA,CANTLEY LC.Metabolic reprogramming by the PI3K-Akt-mTOR pathway in cancer[J].Metab Cancer,2016:39-72.
[10]MATSUMOTO T,YOKOI A,HASHIMURA M,et al.TGF-β-mediated LEFTY/Akt/GSK-3β/Snail axis modulates epithelial-mesenchymal transition and cancer stem cell properties in ovarian clear cell carcinomas[J].Mol Carcinogen,2018,57(8):957-967.
[11]YU D,LEI JQ,GUO SL,et al.The CNPY2enhances epithelial-mesenchymal transition via activating the AKT/GSK3βpathway in non-small cell lung cancer[J].Cell Biol Int,2018,42(8):959-964.
[12]BACHELDER RE,YOON SO,FRANCI C,et al.Glycogen synthase kinase-3is an endogenous inhibitor of Snail transcription:implications for the epithelial mesenchymal transition[J].J Cell Biol,2005,168(1):29-33.
[13]WANG H,ZHANG G,ZHANG H,et al.Acquisition of epithelial–mesenchymal transition phenotype and cancer stem cell-like properties in cisplatin-resistant lung cancer cells through AKT/β-catenin/Snail signaling pathway[J].Eur J Pharmacol,2014,723:156-166.
[14]WU K,FAN J,ZHANG L,et al.PI3K/Akt to GSK3β/β-catenin signaling cascade coordinates cell colonization for bladder cancer bone metastasis through regulating ZEB1transcription[J].Cell Signal,2012,24(12):2273-2282.
[15]WALKER NM,BELLOLI EA,STUCKEY L,et al.Mechanistic target of rapamycin complex 1(mTORC1)and mTORC2as key signaling intermediates in mesenchymal cell activation[J].J Biol Chem,2016,291(12):6262-6271.
[16]LAMOUILLE S,CONNOLLY E,SMYTH JW,et al.TGF-β-induced activation of mTOR complex 2drives epithelial-mesenchymal transition and cell invasion[J].J Cell Sci,2012,125(5):1259-1273.
[17]HAN Q,LIN L,ZHAO B,et al.Inhibition of mTOR ameliorates bleomycin-induced pulmonary fibrosis by regulating epithelial-mesenchymal transition[J].Biochem Bioph Res Comm,2018,500(4):839-845.
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