樟芝提取物Fr.3抑制TGF-β1诱导的肝纤维化机制
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摘要
探讨樟芝菌丝体正己烷提取物Fr.3组分对TGF-β1诱导的肝星状细胞(CFSC-8B)活化的影响及作用机制。采用TGF-β1诱导CFSC-8B活化,通过MTT、WST-1、小室迁移、qRT-PCR及western blotting法检测Fr.3组分对TGF-β1诱导后的CFSC-8B细胞增值、迁移及细胞外基质的变化,并研究了其对TGF-β1介导的Smad、PI3K、MAPK信号通路中相关蛋白质表达的影响。Fr.3组分的IC_(50)值为54.56μg/mL;在细胞存活率80%以上范围内,Fr.3组分(<20μg/mL)可显著抑制TGF-β1诱导的CFSC-8B细胞增值及迁移、明显下调ECM中的相关基因的表达水平及降低α-SMA、Col1、p-Smad2、p-ERK、p-P38及p-AKT的蛋白质表达水平。樟芝菌体正己烷提取物Fr.3可通过调控TGF-β1介导的Smad、PI3K、MAPK信号通路来抑制肝纤维化。
To investigate the effects of hexane extract fraction 3(Fr.3) from Antrodia camphorata on hepatic stellate cells(HSCs) activation induced by TGF-β1,as well as mechanisms of its anti-fibrotic action in vitro. We established a cell model of liver fibrosis by using CFSC-8 B stimulated by TGF-β1. Cell proliferation,cell migration,extracellular matrix(ECM) production and protein expression in the TGF-β1 signaling pathways were detected by the MTT,WST-1 assay,cell migration assay,q RT-PCR and western blotting analyses. The IC_(50) value of Fr.3 was 54.56 μg/mL and we found that less than 20 μg/mL of Fr.3 might be safe for CSFC-8 B cells. Fr.3(< 20 μg/mL)significantly inhibited CFSC-8 B cell proliferation and migration stimulated by TGF-β1. Fr.3 also downregulated α-SMA,Col1,Col3 and Fn m RNA expression. Mechanistically,Fr.3 blocked TGF-β1 induced protein expression of α-SMA,Col1,p-Smad2,p-ERK,p-P38 and p-AKT. Fr.3 from A.camphorata exhibits anti-hepatic fibrosis activity through negatively modulating TGF-β1/Smad/PI3 K/MAPK signaling pathways in CFSC-8 B cells.
To investigate the effects of hexane extract fraction 3(Fr.3) from Antrodia camphorata on hepatic stellate cells(HSCs) activation induced by TGF-β1,as well as mechanisms of its anti-fibrotic action in vitro. We established a cell model of liver fibrosis by using CFSC-8 B stimulated by TGF-β1. Cell proliferation,cell migration,extracellular matrix(ECM) production and protein expression in the TGF-β1 signaling pathways were detected by the MTT,WST-1 assay,cell migration assay,q RT-PCR and western blotting analyses. The IC_(50) value of Fr.3 was 54.56 μg/mL and we found that less than 20 μg/mL of Fr.3 might be safe for CSFC-8 B cells. Fr.3(< 20 μg/mL)significantly inhibited CFSC-8 B cell proliferation and migration stimulated by TGF-β1. Fr.3 also downregulated α-SMA,Col1,Col3 and Fn m RNA expression. Mechanistically,Fr.3 blocked TGF-β1 induced protein expression of α-SMA,Col1,p-Smad2,p-ERK,p-P38 and p-AKT. Fr.3 from A.camphorata exhibits anti-hepatic fibrosis activity through negatively modulating TGF-β1/Smad/PI3 K/MAPK signaling pathways in CFSC-8 B cells.
引文
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[2]HUANG T H,CHIU Y H,CHAN YL,et al.Antrodia cinnamomea alleviates cisplatin-induced hepatotoxicity and enhances chemo-sensitivity of line-1 lung carcinoma xenografted in BALB/cByJ mice[J].Oncotarget,2015,6(28):25741-25754.
[3]LI M H,WU H C,YAO H J,et al.Antrodia cinnamomea extract inhibits Th17 cell differentiation and ameliorates imiquimodinduced psoriasiform skin inflammation[J].The American Journal of Chinese Medicine,2015,43(7):1401-1417.
[4]LU Z M,TAO W Y,XU H Y,et al.Further studies on the hepatoprotective effect of Antrodia camphorata in submerged culture on ethanol-induced acute liver injury in rats[J].Natural Product Research,2011,25(7):684-695.
[5]HUANG G J,DENG J S,HUANG S S,Hepatoprotective effects of eburicoic acid and dehydroeburicoic acid from Antrodia camphorata in a mouse model of acute hepatic injury[J].Food Chemistry,2013,141(3):3020-3027.
[6]HUANG Y L,CHU Y L,HO C T,et al.Antcin K,an active triterpenoid from the fruiting bodies of basswood-cultivated Antrodia cinnamomea,inhibits metastasis via suppression of integrin-mediated adhesion,migration,and invasion in human hepatoma cells[J].Journal of Agricultural and Food Chemistry,2015,63(18):4561-4569.
[7]WANG Jing,GENG Yan,SUN Qing,et al.Inhibitory effects of extract fraction 2 from Antrodia cinnamomea on the activation of CFSC-8B cells induced by PDGF-BB[J].Journal of Food Science and Biotechnology,2014,33(9):917-923.(in Chinese)
[8]GENG Y,WANG J,SUM Q,et al.Identification of antrodin B from Antrodia camphorata as a new anti-hepatofibrotic compound using a rapid cell screening method and biological evaluation[J].Hepatology Research,2016,46(3):E15-25.
[9]KUMAR K J,VANI M G,CHUEH P J,et al.Antrodin C inhibits epithelial-to-mesenchymal transition and metastasis of breast cancer cells via suppression of Smad2/3 andβ-catenin signaling pathways[J].Plos One,2015,10(2):e0117111.
[10]BATALLER R,BRENNER D A.Liver fibrosis[J].Journal of Clinical Investigation,2005,115(2):209-218.
[11]GRESSNER A M,WEISKIRCHEN R,BREITKOPF K,et al.Roles of TGF-beta in hepatic fibrosis[J].Frontiers in Bioscience,2002,7(1-3):793-807.
[12]DE MINICIS S,SEKI E,UCHINAMI H,et al.Gene expression profiles during hepatic stellate cell activation in culture and in vivo[J].Gastroenterology,2007,132(5):1937-1946.
[13]SEKI E,DE MINICIS S,OSTERREICHER C H,et al.TLR4 enhances TGF-beta signaling and hepatic fibrosis[J].Nature medicine,2007,13(11):1324-1332.
[14]MENG X M,TANG P M,LI J,et al.TGF-beta/Smad signaling in renal fibrosis[J].Frontiers in Physiology,2015,6:82.
[15]WANG Y.Phosphatidylinositol 3-kinase/Akt pathway regulates hepatic stellate cell apoptosis[J].World Journal of Gastroenterology,2008,14(33):5186-5191.
[16]GABELE E,REIF S,TSUKADA S,et al.The role of p70S6K in hepatic stellate cell collagen gene expression and cell proliferation[J].The Journal of Biological Chemistry,2005,280(14):13374-13382.
[17]CHENG K,YANG N,MAHATO R I.TGF-beta1 gene silencing for treating liver fibrosis[J].Molecular Pharmaceutics,2009,6(3):772-779.
[18]UENO Y,FRANCIS H,GLASER S,et al.Taurocholic acid feeding prevents tumor necrosis factor-alpha-induced damage of cholangiocytes by a PI3K-mediated pathway[J].Experimental Biology and Medicine,2007,232(7):942-949.
[19]POULOS J E,WEBER J D,BELLEZZO J M,et al.Fibronectin and cytokines increase JNK,ERK,AP-1 activity and transin gene expression in rat hepatic stellate cells[J].The American Journal of Physiology,1997,273(4):G804-811.
[20]LU Z M,TAO W Y,ZOU X L,et al.Protective effects of mycelia of Antrodia camphorata and Armillariella tabescens in submerged culture against ethanol-induced hepatic toxicity in rats[J].Journal of Ethnopharmacology,2007,110(1):160-164.
[21]SONG T Y,YEN G C.Protective effects of fermented filtrate from Antrodia camphorata in submerged culture against CCl4-induced hepatic toxicity in rats[J].Journal of Agricultural and Food Chemistry,2003,51(6):1571-1577.
[22]BENYON R C,IREDALE J P.Is liver fibrosis reversible?[J].Gut,2000,46(4):443-446.