脯氨酰寡肽酶抑制剂体外对肝星状细胞凋亡、增殖和肝纤维化相关基因表达的影响
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摘要
目的体外探讨肝星状细胞(HSC)脯氨酰寡肽酶(POP)的生物学功能,以进一步阐述其在肝脏炎症和纤维化发生发展中的作用。方法取HSC-T6细胞,经不同浓度的POP抑制剂(S17092)处理,采用ELISA法检测HSC-T6细胞N-乙酰基-丝氨酸-天冬氨酸-赖氨酸-脯氨酸(Ac-SDKP)水平,使用流式细胞术和CCK8检测HSC-T6凋亡和增殖水平,采用实时定量-PCR法检测相关炎症和肝纤维化基因转化生长因子-β1(TGF-β1)、α-平滑肌肌动蛋白(α-SMA)、单核细胞趋化蛋白-1(MCP-1)、Ⅰ型胶原(Col I)水平,采用Western blot法检测相关蛋白(POP、TGF-β1、α-SMA、Smad7、p-Smad2/3、PPAR-γ)表达。结果与对照组比,POP抑制剂干预组细胞内Ac-SDKP水平显著下降(P<0.05);POP被抑制后对HSC-T6凋亡没有显著的影响(P>0.05),但可抑制其增殖(P<0.05);与对照组比,抑制剂组HSC内α-SMA蛋白表达显著升高(P<0.05),而Smad7和PPAR-γ蛋白表达显著下降(P均<0.05);抑制剂组HSC内MCP-1和α-SMA m RNA水平显著上调(P均<0.05)。结论 POP在肝星状细胞内可能发挥重要的抗炎抗纤维化作用,其机制可能与其调节Ac-SDKP及Smad7和PPAR-γ的表达有关。
Objective To investigate the inhibition of proliferation and regulation of heaptic fibrosis-related genes in HSC-T6 cells by prolyloligopeptidase(POP) inhibitor in vitro. Methods The HSC-T6 cells were cultured,and the POP activity was inhibited by increasing concentrations of POP inhibitor(S17092). The intracellular level of N-acetyl-seryl-aspartyl-lysyl-proline(Ac-SDKP) was determined by ELISA. The cell apoptosis and cell proliferation were detected by flow cytometry and CCK-8 assay,respectively. The m RNA levels of TGF-β1,α-SMA,monocyte chemoattractant protein 1(MCP-1) and collagen I(Col I) were detected by realtimePCR,and the POP,TGF-β1,α-SMA,Smad7,p-Smad2/3 and PPAR-γ expression were detected by Western blot.R esults The intracellular Ac-SDKP level decreased significantly after co-culture with S17092,the POP inhibitor(P<0.05) as compared to that in the control; the proliferation of HSC-T6 cells was significantly inhibited(P<0.05)and the cell apoptosis was not influenced obviously after S17092 intervention(P>0.05);the Smad7(P<0.05) and PPAR-γ(P<0.05) protein levels were significantly down-regulated,and the α-SMA protein level was significantly up-regulated(P<0.05) after S17092 intervention;the MCP-1(P<0.05) and α-SMA(P<0.05) m RNA level were significantly up-regulated. Conclusion POP plays a pivotal role in anti-inflammation and anti-fibrosis in HSC cells,which might be related to the regulation of Smad7 and PPAR-γ expression.
Objective To investigate the inhibition of proliferation and regulation of heaptic fibrosis-related genes in HSC-T6 cells by prolyloligopeptidase(POP) inhibitor in vitro. Methods The HSC-T6 cells were cultured,and the POP activity was inhibited by increasing concentrations of POP inhibitor(S17092). The intracellular level of N-acetyl-seryl-aspartyl-lysyl-proline(Ac-SDKP) was determined by ELISA. The cell apoptosis and cell proliferation were detected by flow cytometry and CCK-8 assay,respectively. The m RNA levels of TGF-β1,α-SMA,monocyte chemoattractant protein 1(MCP-1) and collagen I(Col I) were detected by realtimePCR,and the POP,TGF-β1,α-SMA,Smad7,p-Smad2/3 and PPAR-γ expression were detected by Western blot.R esults The intracellular Ac-SDKP level decreased significantly after co-culture with S17092,the POP inhibitor(P<0.05) as compared to that in the control; the proliferation of HSC-T6 cells was significantly inhibited(P<0.05)and the cell apoptosis was not influenced obviously after S17092 intervention(P>0.05);the Smad7(P<0.05) and PPAR-γ(P<0.05) protein levels were significantly down-regulated,and the α-SMA protein level was significantly up-regulated(P<0.05) after S17092 intervention;the MCP-1(P<0.05) and α-SMA(P<0.05) m RNA level were significantly up-regulated. Conclusion POP plays a pivotal role in anti-inflammation and anti-fibrosis in HSC cells,which might be related to the regulation of Smad7 and PPAR-γ expression.
引文
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[12]陈源文,董国芳,徐雷鸣,等.内源性Ac SDKP及其前体胸腺素β4在四氯化碳致肝纤维化发生早期的变化.胃肠病学和肝病学杂志,2010,19(1):6-9.
[13]Tenorio-Laranga J,Montoliu C,Urios A,et al.The expression levels of prolyl oligopeptidase responds not only to neuroinflammation but also to systemic inflammation upon liver failure in rat models and cirrhotic patients.J Neuroinflamm,2015,12(1):183.
[14]Zoubek ME,Trautwein C,Strnad P.Reversal of liver fibrosis:From fiction to reality.Best Pract Res Clin Gastroenterol,2017,31(2):129-141.
[15]Shang H,Liu X,Guo H.Knockdown of Fstl1 attenuates hepatic stellate cell activation through the TGFbeta1/Smad3 signaling pathway.Mol Med Rep,2017,[Epub ahead of print].
[16]Kumar N,Nakagawa P,Janic B,et al.The anti-inflammatory peptide Ac-SDKP is released from thymosin-beta4 by renal meprin-alpha and prolyl oligopeptidase.Am J Physiol Renal Physiol,2016,310(10):F1026-1034.
[17]Zhang L,Xu LM,Chen YW,et al.Antifibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline on bile duct ligation induced liver fibrosis in rats.World J Gastroenterol,2012,18(37):5283-5288.
[18]Kanasaki K,Nagai T,Nitta K,et al.N-acetyl-seryl-aspartyl-lysyl-proline:a valuable endogenous anti-fibrotic peptide for combating kidney fibrosis in diabetes.Front Pharmacol,2014,5:70.
[19]Nagai T,Kanasaki M,Srivastava SP,et al.N-acetyl-seryl-aspartyl-lysyl-proline inhibits diabetes-associated kidney fibrosis and endothelial-mesenchymal transition.Biomed Res Int,2014,2014:696475.
[20]Xiaojun W,Yan L,Hong X,et al.Acetylated alpha-tubulin regulated by N-acetyl-seryl-aspartyl-lysyl-proline(Ac-SDKP)exerts the anti-fibrotic effect in rat lung fibrosis induced by silica.Sci Rep,2016,6:32257.
[21]Hrenak J,Paulis L,Simko F.N-acetyl-seryl-aspartyl-lysylproline(Ac-SDKP):Potential target molecule in research of heart,kidney and brain.Curr Pharm Des,2015,21(35):5135-5143.
[22]Myohanen TT,Garcia-Horsman JA,Tenorio-Laranga J,et al.Issues about the physiological functions of prolyl oligopeptidase based on its discordant spatial association with substrates and inconsistencies among m RNA,protein levels,and enzymatic activity.J Histochem Cytochem,2009,57(9):831-848.
[23]Di Daniel E,Glover CP,Grot E,et al.Prolyl oligopeptidase binds to GAP-43 and functions without its peptidase activity.Mol Cell eurosci,2009,41(3):373-382.
[24]Myohanen TT,Venalainen JI,Garcia-Horsman JA,et al.Distribution of prolyl oligopeptidase in the mouse whole-body sections and peripheral tissues.Histochem Cell Biol,2008,130(5):993-1003.
[25]Sakaguchi M,Matsuda T,Matsumura E,et al.Prolyl oligopeptidase participates in cell cycle progression in a human neuroblastoma cell line.Biochem Biophys Res Commun,2011,409(4):693-698.
[26]Lee SJ.Mechanisms of fibrogenesis in liver cirrhosis:The molecular aspects of epithelial-mesenchymal transition.World J Hepatol,2014,6(4):207.
[27]Marra F,Tacke F.Roles for chemokines in liver disease.Gastroenterology,2014,147(3):577-594.
[28]Zardi EM,Navarini L,Sambataro G,et al.Hepatic PPARs:their role in liver physiology,fibrosis and treatment.Curr Med Chem,2013,20(27):3370-3396.
[29]Yu J,Zhang S,Chu ES,et al.Peroxisome proliferator-activated receptors gamma reverses hepatic nutritional fibrosis in mice and suppresses activation of hepatic stellate cells in vitro.Int J Biochem Cell Biol,2010,42(6):948-957.
[30]李兵航,李梦婷,何玲楠,等.Ac SDKP体外抑制脂多糖诱导的小鼠RAW264.7巨噬细胞促炎功能研究.实用肝脏病杂志,2017,20(2):142-147.
[31]王晶,周达,丁永年,等.慢病毒介导脯氨酰寡肽酶过表达抑制硫代乙酰胺诱导的大鼠肝纤维化.实用肝脏病杂志,2015,18(2):168-172.
[2]Penttinen A,Tenorio-Laranga J,Siikanen A,et al.Prolyl oligopeptidase:a rising star on the stage of neuroinflammation research.CNS Neurol Disord Drug Targets,2011,10(3):340-348.
[3]Suzuki K,Sakaguchi M,Tanaka S,et al.Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells.Biochem Biophys Res Commun,2014,443(1):91-96.
[4]Tenorio-Laranga J,Mannisto PT,Storvik M,et al.Four day inhibition of prolyl oligopeptidase causes significant changes in the peptidome of rat brain,liver and kidney.Biochimie,2012,94(9):1849-1859.
[5]Maruyama Y,Matsubara S,Kimura AP.Mouse prolyl oligopeptidase plays a role in trophoblast stem cell differentiation into trophoblast giant cell and spongiotrophoblast.Placenta,2017,53:8-15.
[6]Yamakawa N,Shimeno H,Soeda S,et al.Inhibition of proline endopeptidase activity by acyl-coenzyme A esters.Biochim Biophys Acta,1990,1037(3):302-306.
[7]Zhou D,Li BH,Wang J,et al.Prolyl oligopeptidase inhibition attenuates steatosis in the L02 human liver cell line.Plos One,2016,11(10):e0165224.
[8]Yamakawa N,Shimeno H,Soeda S,et al.Regulation of prolyl oligopeptidase activity in regenerating rat liver.Biochim Biophys Acta,1994,1199(3):279-284.
[9]Matsubara Y,Ono T,Tsubuki S,et al.Transient up-regulation of a prolyl endopeptidase activity in the microsomal fraction of rat liver during postnatal development.Eur J Biochem,1998,252(1):178-183.
[10]Chen YW,Liu BW,Zhang YJ,et al.Preservation of basal Ac SDKP attenuates carbon tetrachloride-induced fibrosis in the rat liver.J Hepatol,2010,53(3):528-536.
[11]周达,陈源文,范建高.脯氨酰寡肽酶的肝内生物学功能研究进展.胃肠病学和肝病学杂志,2015,24(1):12-14.
[12]陈源文,董国芳,徐雷鸣,等.内源性Ac SDKP及其前体胸腺素β4在四氯化碳致肝纤维化发生早期的变化.胃肠病学和肝病学杂志,2010,19(1):6-9.
[13]Tenorio-Laranga J,Montoliu C,Urios A,et al.The expression levels of prolyl oligopeptidase responds not only to neuroinflammation but also to systemic inflammation upon liver failure in rat models and cirrhotic patients.J Neuroinflamm,2015,12(1):183.
[14]Zoubek ME,Trautwein C,Strnad P.Reversal of liver fibrosis:From fiction to reality.Best Pract Res Clin Gastroenterol,2017,31(2):129-141.
[15]Shang H,Liu X,Guo H.Knockdown of Fstl1 attenuates hepatic stellate cell activation through the TGFbeta1/Smad3 signaling pathway.Mol Med Rep,2017,[Epub ahead of print].
[16]Kumar N,Nakagawa P,Janic B,et al.The anti-inflammatory peptide Ac-SDKP is released from thymosin-beta4 by renal meprin-alpha and prolyl oligopeptidase.Am J Physiol Renal Physiol,2016,310(10):F1026-1034.
[17]Zhang L,Xu LM,Chen YW,et al.Antifibrotic effect of N-acetyl-seryl-aspartyl-lysyl-proline on bile duct ligation induced liver fibrosis in rats.World J Gastroenterol,2012,18(37):5283-5288.
[18]Kanasaki K,Nagai T,Nitta K,et al.N-acetyl-seryl-aspartyl-lysyl-proline:a valuable endogenous anti-fibrotic peptide for combating kidney fibrosis in diabetes.Front Pharmacol,2014,5:70.
[19]Nagai T,Kanasaki M,Srivastava SP,et al.N-acetyl-seryl-aspartyl-lysyl-proline inhibits diabetes-associated kidney fibrosis and endothelial-mesenchymal transition.Biomed Res Int,2014,2014:696475.
[20]Xiaojun W,Yan L,Hong X,et al.Acetylated alpha-tubulin regulated by N-acetyl-seryl-aspartyl-lysyl-proline(Ac-SDKP)exerts the anti-fibrotic effect in rat lung fibrosis induced by silica.Sci Rep,2016,6:32257.
[21]Hrenak J,Paulis L,Simko F.N-acetyl-seryl-aspartyl-lysylproline(Ac-SDKP):Potential target molecule in research of heart,kidney and brain.Curr Pharm Des,2015,21(35):5135-5143.
[22]Myohanen TT,Garcia-Horsman JA,Tenorio-Laranga J,et al.Issues about the physiological functions of prolyl oligopeptidase based on its discordant spatial association with substrates and inconsistencies among m RNA,protein levels,and enzymatic activity.J Histochem Cytochem,2009,57(9):831-848.
[23]Di Daniel E,Glover CP,Grot E,et al.Prolyl oligopeptidase binds to GAP-43 and functions without its peptidase activity.Mol Cell eurosci,2009,41(3):373-382.
[24]Myohanen TT,Venalainen JI,Garcia-Horsman JA,et al.Distribution of prolyl oligopeptidase in the mouse whole-body sections and peripheral tissues.Histochem Cell Biol,2008,130(5):993-1003.
[25]Sakaguchi M,Matsuda T,Matsumura E,et al.Prolyl oligopeptidase participates in cell cycle progression in a human neuroblastoma cell line.Biochem Biophys Res Commun,2011,409(4):693-698.
[26]Lee SJ.Mechanisms of fibrogenesis in liver cirrhosis:The molecular aspects of epithelial-mesenchymal transition.World J Hepatol,2014,6(4):207.
[27]Marra F,Tacke F.Roles for chemokines in liver disease.Gastroenterology,2014,147(3):577-594.
[28]Zardi EM,Navarini L,Sambataro G,et al.Hepatic PPARs:their role in liver physiology,fibrosis and treatment.Curr Med Chem,2013,20(27):3370-3396.
[29]Yu J,Zhang S,Chu ES,et al.Peroxisome proliferator-activated receptors gamma reverses hepatic nutritional fibrosis in mice and suppresses activation of hepatic stellate cells in vitro.Int J Biochem Cell Biol,2010,42(6):948-957.
[30]李兵航,李梦婷,何玲楠,等.Ac SDKP体外抑制脂多糖诱导的小鼠RAW264.7巨噬细胞促炎功能研究.实用肝脏病杂志,2017,20(2):142-147.
[31]王晶,周达,丁永年,等.慢病毒介导脯氨酰寡肽酶过表达抑制硫代乙酰胺诱导的大鼠肝纤维化.实用肝脏病杂志,2015,18(2):168-172.