Pepstatin Pr通过YAP-TGFβ-Smad通路发挥体外抗肝纤维化作用
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摘要
肝纤维化是各种慢性因素引起的肝脏损伤后组织修复代偿反应,其进一步恶化会导致肝硬化、肝功能衰竭甚至是肝细胞癌。肝星状细胞的异常活化是肝纤维化发生发展的细胞学基础。化合物pepstatin Pr是从链霉菌CPCC 202950中分离得到的pepstatin A衍生物。本实验目的在于研究pepstatin Pr的抗肝纤维化活性并探索其分子机制。通过给予TGFβ1诱导人肝星状细胞LX-2活化并进行不同浓度的pepstatin Pr处理后,应用磺酰罗丹明B(sulforhodamine B, SRB)比色法检测pepstatin Pr对LX-2细胞的细胞毒性,应用real time PCR和Western blot方法检测pepstatin Pr对COL1A1、α-SMA、组织蛋白酶D (cathepsin D)、TGFβ、Smad以及YAP/TAZ蛋白表达与磷酸化水平的影响。结果显示pepstatin Pr对LX-2细胞无明显细胞毒性,并且可显著降低肝纤维化标志物COL1A1及α-SMA的mRNA和蛋白表达,同时在蛋白水平上抑制cathepsin D、TGFβ1、YAP和TAZ的表达, Smad2的磷酸化水平,以及YAP核转位。综上, pepstatin Pr可通过调控YAP-TGFβ-Smad通路抑制由TGFβ1诱导的肝星状细胞活化,具有良好的体外抗肝纤维化活性。
Liver fibrosis is a tissue repair compensatory response to liver injury caused by various chronic factors,ultimately leading to liver cirrhosis,liver failure and even hepatocellular carcinoma.Abnormal activation of hepatic stellate cells is the cellular basis of liver fibrosis development.Pepstatin Pr,the derivative of pepstatin A was isolated from Streptomyces sp.CPCC 202950.Our purpose was to investigate the anti-fibrotic activity of pepstatin Pr and explore its molecular mechanism.Hepatic stellate cell LX-2 was stimulated by TGFβ1 and subsequently treated with pepstatin Pr.Its cytotoxicity was detected by sulforhodamine B(SRB)assay.The expression of COL1A1,α-SMA and cathepsin D,signaling proteins TGFβ,Smad and YAP/TAZ were detected by Western blot or real-time PCR.The results showed that pepstatin Pr was not cytotoxic to LX-2 cells.And pepstatin Pr signifi-cantly reduced the mRNA and protein expression of COL1A1 andα-SMA,which are important liver fibrosis markers Pepstatin Pr also repressed the protein expression level of cathepsin D,TGFβ1,YAP/TAZ,the phospholation level of Smad2,and YAP nuclear translocation.In conclusion,pepstatin Pr exhibits anti-fibrotic effects in TGFβ1-stimu-laed LX-2 cells by mediating YAP-TGFβ-Smad pathway
Liver fibrosis is a tissue repair compensatory response to liver injury caused by various chronic factors,ultimately leading to liver cirrhosis,liver failure and even hepatocellular carcinoma.Abnormal activation of hepatic stellate cells is the cellular basis of liver fibrosis development.Pepstatin Pr,the derivative of pepstatin A was isolated from Streptomyces sp.CPCC 202950.Our purpose was to investigate the anti-fibrotic activity of pepstatin Pr and explore its molecular mechanism.Hepatic stellate cell LX-2 was stimulated by TGFβ1 and subsequently treated with pepstatin Pr.Its cytotoxicity was detected by sulforhodamine B(SRB)assay.The expression of COL1A1,α-SMA and cathepsin D,signaling proteins TGFβ,Smad and YAP/TAZ were detected by Western blot or real-time PCR.The results showed that pepstatin Pr was not cytotoxic to LX-2 cells.And pepstatin Pr signifi-cantly reduced the mRNA and protein expression of COL1A1 andα-SMA,which are important liver fibrosis markers Pepstatin Pr also repressed the protein expression level of cathepsin D,TGFβ1,YAP/TAZ,the phospholation level of Smad2,and YAP nuclear translocation.In conclusion,pepstatin Pr exhibits anti-fibrotic effects in TGFβ1-stimu-laed LX-2 cells by mediating YAP-TGFβ-Smad pathway
引文
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[15]Yu DK,Zhang CX,Zhao SS,et al.The anti-fibrotic effects of epigallocatechin-3-gallate in bile duct-ligated cholestatic rats and human hepatic stellate LX-2 cells are mediated by the PI3K/Akt/Smad pathway[J].Acta Pharmacol Sin,2015,36:473-482.
[16]Moles A,Tarrats N,Morales A,et al.Acidic sphingomyelinase controls hepatic stellate cell activation and in vivo liver fibro-genesis[J].Am J Pathol,2010,177:1214-1224.
[17]Liu PP,Liu HH,Sun SH,et al.Aspirin alleviates cardiac fibrosis in mice by inhibiting autophagy[J].Acta Pharmacol Sin,2017,38:488-497.
[18]Uemura M,Swenson ES,Gaca MD,et al.Smad2 and Smad3play different roles in rat hepatic stellate cell function and alphasmooth muscle actin organization[J].Mol Biol Cell,2005,16:4214-4224.
[19]Leto G,Tumminello FM,Pizzolanti G,et al.Cathepsin D serum mass concentrations in patients with hepatocellular carcinoma and/or liver cirrhosis[J].Eur J Clin Chem Clin Biochem,1996,34:555-560.
[20]Kristensen DB,Kawada N,Imamura K,et al.Proteome analysis of rat hepatic stellate cells[J].Hepatology,2000,32:268-277.
[21]Moles A,Tarrats N,Fernandez-Checa JC,et al.Cathepsins Band D drive hepatic stellate cell proliferation and promote their fibrogenic potential[J].Hepatology,2009,49:1297-1307.
[2]Trappoliere M,Caligiuri A,Schmid M,et al.Silybin,a component of sylimarin,exerts anti-inflammatory and anti-fibrogenic effects on human hepatic stellate cells[J].J Hepatol,2009,50:1102-1111.
[3]Gressner AM,Weiskirchen R.Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets[J].J Cell Mol Med,2006,10:76-99.
[4]Dooley S,ten Dijke P.TGF-βin progression of liver disease[J].Cell Tissue Res,2012,347:245-256.
[5]Dooley S,Hamzavi J,Breitkopf K,et al.Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats[J].Gastroenterology,2003,125:178-191.
[6]Mannaerts I,Leite SB,Verhulst S,et al.The Hippo pathway effector YAP controls mouse hepatic stellate cell activation[J].JHepatol,2015,63:679-688.
[7]Ge M,Liu H,Zhang Y,et al.The anti-hepatic fibrosis effects of dihydrotanshinone I are mediated by disrupting the Yes-associated protein and transcriptional enhancer factor D2 complex and stimu-lating autophagy[J].Br J Pharmacol,2017,174:1147-1160.
[8]Zhang H,von Gise A,Liu Q,et al.Yap1 is required for endothelial to mesenchymal transition of the atrioventricular cushion[J].JBiol Chem,2014,289:18681-18692.
[9]Narimatsu M,Labibi B,Wrana JL,et al.Analysis of Hippo and TGFβsignaling in polarizing epithelial cells and mouse embryos[J].Differentiation,2016,91:109-118.
[10]Wang C,Zhang L,He Q,et al.Differences in Yes-associated protein and mRNA levels in regenerating liver and hepatocellular carcinoma[J].Mol Med Rep,2012,5:410-414.
[11]Chen MH,Chang SS,Dong B,et al.Ahmpatinin iBu,a new HIV-1protease inhibitor,from Streptomyces sp.CPCC 202950[J].RSCAdv,2018,8:5138-5144.
[12]Cao W,Li Y,Li M,et al.Txn1,Ctsd and Cdk4 are key proteins of combination therapy with taurine,epigallocatechin gallate and genistein against liver fibrosis in rats[J].Biomed Pharmacother,2017,85:611-619.
[13]Gohda E,Nagahama J,Nakamura O,et al.Increased activities of liver cathepsins T and D in carbon tetrachloride-treated rats[J].Biochim Biophys Acta,1984,802:362-371.
[14]Zhao SS,Wang JX,Wang YC,et al.Establishment and application of a high-throughput drug screening model based on COL1A1promoter for anti-liver fibrosis[J].Acta Pharm Sin(药学学报),2015,50:169-173.
[15]Yu DK,Zhang CX,Zhao SS,et al.The anti-fibrotic effects of epigallocatechin-3-gallate in bile duct-ligated cholestatic rats and human hepatic stellate LX-2 cells are mediated by the PI3K/Akt/Smad pathway[J].Acta Pharmacol Sin,2015,36:473-482.
[16]Moles A,Tarrats N,Morales A,et al.Acidic sphingomyelinase controls hepatic stellate cell activation and in vivo liver fibro-genesis[J].Am J Pathol,2010,177:1214-1224.
[17]Liu PP,Liu HH,Sun SH,et al.Aspirin alleviates cardiac fibrosis in mice by inhibiting autophagy[J].Acta Pharmacol Sin,2017,38:488-497.
[18]Uemura M,Swenson ES,Gaca MD,et al.Smad2 and Smad3play different roles in rat hepatic stellate cell function and alphasmooth muscle actin organization[J].Mol Biol Cell,2005,16:4214-4224.
[19]Leto G,Tumminello FM,Pizzolanti G,et al.Cathepsin D serum mass concentrations in patients with hepatocellular carcinoma and/or liver cirrhosis[J].Eur J Clin Chem Clin Biochem,1996,34:555-560.
[20]Kristensen DB,Kawada N,Imamura K,et al.Proteome analysis of rat hepatic stellate cells[J].Hepatology,2000,32:268-277.
[21]Moles A,Tarrats N,Fernandez-Checa JC,et al.Cathepsins Band D drive hepatic stellate cell proliferation and promote their fibrogenic potential[J].Hepatology,2009,49:1297-1307.