川芎嗪对肝星状细胞CTGF及Smad2/3表达的影响
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摘要
目的:研究川芎嗪(TMP)对大鼠肝星状细胞(HSC-T6)结缔组织生长因子(CTGF)及Smad2/3表达的影响。
方法:
体外培养HSC-T6细胞株,以不同浓度的TMP以及0.005mg/l[1]的转化生长因子-β1(TGF-β1)作用于HSC-T6,采用MTT比色法测定HSC-T6增殖;用免疫细胞化学染色法观察CTGF、Smad2/3在HSC-T6的定位及不同浓度TMP对其表达的影响;用Western印迹法检测CTGF、Smad2/3蛋白表达水平。
结果:
1.一定浓度(100、200、400、600、1000 mg/l) TMP能抑制HSC-T6增殖,且呈剂量依赖性(P<0.05)。
2.免疫细胞化学法结果显示CTGF主要在胞浆表达;Smad2/3蛋白则在胞核及胞浆均有表达。随着TMP浓度的升高,CTGF、Smad2/3表达依次递减,与空白对照组比较,具有显著性差异(P<0.05)。
3. Western印迹法结果亦显示TMP可以明显抑制CTGF及Smad2/3蛋白的表达,两者被抑制程度呈正相关关系(r=0.967,P<0.01)。
结论:
TMP可能通过抑制HSC-T6细胞增殖,下调Smad2/3、CTGF的表达,从而发挥其抗肝纤维化的作用。
Objective: To study the effects of Tetramethylpyrazine(TMP) on expression of connective tissue growth factor(CTGF) and Smad2/3 in hepatic stellate cell-T6 (HSC-T6).
Methods:
HSC-T6 was cultured with varying doses of TMP and 0.005mg/l[1] of transforming growth factor-β1(TGF-β1) in vitro.MTT colorimetric assay was used for detecting the proliferation inhibition ratio of HSC-T6.Immunocytochemistry was used to observe the location of CTGF and Smad2/3 ,to test the effect of TMP on the expression of CTGF and Smad2/3 in HSC-T6.Protein expression level of CTGF and Smad2/3 was analyzed with Western blot.
Results:
1. TMP inhibited the proliferation of HSC-T6 at the concentration of 100、200、400、600 and 1000mg/l in concentration dependent way(P<0.05).
2. The test by immunohistochemical method indicated that the expression of CTGF located in the endochylema of HSC-T6 while Smad2/3 expressed both in the nucleus and endochylema of HSC-T6. When the density of TMP increased, the expression of CTGF and Smad2/3 decreased progressively. The comparision between the control group were remarkably discrepant (P<0.05).
3. The results of Western blot also revealed that TMP could dose- dependently inhibit the protein expression of CTGF and Smad2/3,the inhibition drgree of which showed a positive correlation.(r=0.967,P<0.01).
Conclusion:
Inhibiting proliferation of HSC-T6, down-regulating the protein expression of Smad2/3 and CTGF,might be involved in the anti-fibrosis mechanism of TMP.
方法:
体外培养HSC-T6细胞株,以不同浓度的TMP以及0.005mg/l[1]的转化生长因子-β1(TGF-β1)作用于HSC-T6,采用MTT比色法测定HSC-T6增殖;用免疫细胞化学染色法观察CTGF、Smad2/3在HSC-T6的定位及不同浓度TMP对其表达的影响;用Western印迹法检测CTGF、Smad2/3蛋白表达水平。
结果:
1.一定浓度(100、200、400、600、1000 mg/l) TMP能抑制HSC-T6增殖,且呈剂量依赖性(P<0.05)。
2.免疫细胞化学法结果显示CTGF主要在胞浆表达;Smad2/3蛋白则在胞核及胞浆均有表达。随着TMP浓度的升高,CTGF、Smad2/3表达依次递减,与空白对照组比较,具有显著性差异(P<0.05)。
3. Western印迹法结果亦显示TMP可以明显抑制CTGF及Smad2/3蛋白的表达,两者被抑制程度呈正相关关系(r=0.967,P<0.01)。
结论:
TMP可能通过抑制HSC-T6细胞增殖,下调Smad2/3、CTGF的表达,从而发挥其抗肝纤维化的作用。
Objective: To study the effects of Tetramethylpyrazine(TMP) on expression of connective tissue growth factor(CTGF) and Smad2/3 in hepatic stellate cell-T6 (HSC-T6).
Methods:
HSC-T6 was cultured with varying doses of TMP and 0.005mg/l[1] of transforming growth factor-β1(TGF-β1) in vitro.MTT colorimetric assay was used for detecting the proliferation inhibition ratio of HSC-T6.Immunocytochemistry was used to observe the location of CTGF and Smad2/3 ,to test the effect of TMP on the expression of CTGF and Smad2/3 in HSC-T6.Protein expression level of CTGF and Smad2/3 was analyzed with Western blot.
Results:
1. TMP inhibited the proliferation of HSC-T6 at the concentration of 100、200、400、600 and 1000mg/l in concentration dependent way(P<0.05).
2. The test by immunohistochemical method indicated that the expression of CTGF located in the endochylema of HSC-T6 while Smad2/3 expressed both in the nucleus and endochylema of HSC-T6. When the density of TMP increased, the expression of CTGF and Smad2/3 decreased progressively. The comparision between the control group were remarkably discrepant (P<0.05).
3. The results of Western blot also revealed that TMP could dose- dependently inhibit the protein expression of CTGF and Smad2/3,the inhibition drgree of which showed a positive correlation.(r=0.967,P<0.01).
Conclusion:
Inhibiting proliferation of HSC-T6, down-regulating the protein expression of Smad2/3 and CTGF,might be involved in the anti-fibrosis mechanism of TMP.
引文
[1] Runyan CE,Schnaper HW ,Poncelet AC.The role of internalization in transforming growth factor betal-induced Smad2 association with Smad anchor for receptor activation(SARA)and Smad2-dependent signaling in human mesangial cells[J]. J Biol Chem,2005,280:8300-8308.
[2] Ramon B,David A.Liver fibrosis[J].J. Clin. Invest. 2005,115:209–218.
[3] Dubuisson L , Desmouliere A , Decou NB, et al . Inhibition of rat liver fibrogensis through noradrenergic antagonism[J]. Hepatology , 2002,35: 325-331.
[4] Nakata E,Nakanishi T,Kawai A,et al.Expression of connective tissue factor/hypertrophic chondrocyte-specific gene product 24(CTGF/Hcs)during fracture healing [J]. Bone,2002,31: 441-447.
[5] Riser BL, Denichilo M, Cortes P, et al. Regulation of connetive tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomeralosclerosis[J].JM Soc Nephrol , 2000,11:25-28.
[6] Hirasaki S, Koide N, Ujike K, et al. Expression of Nov, CYR61 and CTGF genes in human hepatocellular carcinoma [J]. Hepatol Res.,2001 Mar 26,19(3):294-305.
[7] Lucia W, Nicolas C, Karin K, et al. Glucocorticoids activate TGF-βinduced PAI-1 and CTGF expression in rat hepatocytes[J]. Comp Hepatol., 2007, 6:5926-5931.
[8] Runyan CE,Schnaper HW,Poncelet AC.The role of internalization in transforming growth factor betal-induced Smad2 association with Smad anchor for receptor activation fSARA1 and Smad2 dependent signaling in human mesangial cells[J]. J Biol Chem,2005,280:8300-8308.
[9] Safadi R,Friedman SL.Hepatic fibrosis-role of hepatic stelate cell activation[J].Med Gen Med,2002,4(3):27.
[10] Less KS.Hepatic fibrogenesis[J].Korean J Gastroenterol,2006,97(12):741-750.
[11] Solis-Herruzo JA,Dela-Torre P,Minoz-Yague MT,et a1.Hepatic stelate cells (HSC): architects of hepatic fibrosis[J].Rev Esp Enferm Dig,2003,95:438-439.
[12] Marra F.Hepatic stellate cells and the regulation of liver inflammation[J].J Hepatol1999,31:1120-1130.
[13] Lamireau T,Desmoyliere A,Bioulac-Sage P,et a1.Mechanisms of hepatic fibmgenesis[J].Arch Pediatr, 2002,9(4):392-405.
[14] Hui AY,Friedman SL.Molecular basis of hepatic fibrosis[J].Expert Rev Mol Med, 2003,2003:1-23.
[15] Friedman SL.Stellate cells:a moving target in hepaticfibrogenesis[J]. Hepatology, 2004,40(5):1041-1043.
[16] Pan Q,Li DG,Lu HM,et a1.Establishment and identification of a novel immortalized rat hepatic stellate cell line[J].China Journal of Modem Medicine, 2005,15(10):1441-1446.
[17] Friedman SL,Lalazar A,Crong L,et a1.HSC-T6 cells,an immortalized rat hepatic stelate cell line[j].Hepatology,1997,26(4):338-342.
[18] Gressner AM, Weiskirchen R, Breitkopf K, et al. Roles of TGF-beta in hepatic fibrosis[ J ]. FrontBiosci, 2002,7 (4):793 - 807.
[19] 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.
[20] Parsonsp CJ,Takashima M,Rippe RA.Molecular mechanisms of hepatic fibrogenesis[J].J Gastroenterol Hepatol, 2007, 22(1):79-84.
[21] Philippe B, Kjetil A,Kathy F, et al.TGF-βand Smad3 Signaling Link Inflammation to Chronic Fibrogenesis[J].The Journal of Immunology, 2005,175:5390–5395.
[22] Xavier LL,Viola GG,Ferraz AC,et al.A simple and fast densitometric method for the analysis of tyrosine hydroxylase immunoreactivity in the substantia nigra parscompacta and in the ventral tegmental area[J].Brain Res Brain Res Protoc,2005,16(1-3):58-64.
[23] Arnott JA,Zhang X,Sanjay A, et al. Molecular Requirements for Induction of CTGF Expression by TGF-β1 in Primary Osteoblasts[J].Bone,2008, 42(5): 871–885.
[24] Yokoi H,Mukoyama H,Sugawara A,et a1.Role of connective tissue growth factor in fibronectin expression and tubulointerstial fibrosis[J].Am J Physiol,2002,282(5):933–942.
[25] Chen Y,Blom IE,Sa S,et a1.CTGF expression in mesangial cells:Involvement of SMADs,MAPK and PKC[J].Kidney Int,2002,62(4):l149-l152.
[26] Blom IE,Goldsohmeding R, Leusk A.Gene regulation of connective tissue growth factor:new targets for antifibrotic therapy[J].Matrix Bial,2002,21:473-479.
[27] Leask A,Abraham DJ.The role of connective tissue growth factor,a multifunctional matricellular protein,in fibroblast biology[J].Biochem Cell Biol,2003,81:355-379.
[28] Rchfal AW,Brigstock DR.Connective tissue growth factor(CTGF/CCN2) in hepatic fibrosis[J].Hepatol Res,2003,26(1):1-9.
[29] Gao R,Brigstock DR.Low density lipoprotein receptor-related protein(LRP) is a heparin-dependent adhesion receptor for connective tissue growth factor(CTGF) in rat activated hepatic stellate cells[J].Hepatol Res,2003,27(3):214-220.
[30] Ball DK,Wilson AW,Kemper SA,et al.The heparinbinding 10kDa fragment of connective tissue growth factor(CTGF) containing module 4 alone stimulate cell adhesion[J].J Endocrinol,2003,176(2):1-7.
[1] Friedman SL,Bansal MB.Reversal of hepatic fibrosis-Fact or fantasy[J]? Hepatology,2006,43:82-88.
[2] Nakata E,Nakanishi T,Kawai A,et al.Expression of connective tissue factor/hypertrophic chondrocyte-specific gene product 24(CTGF/Hcs)during fracture healing[J].Bone,2002,31:441-447.
[3] Lewindon PJ,Pereira IN,Hoskins AC,et a1.The role of hepatic satellite cells and transforming growth factor-β1 in cystic fibro6is liver disease[J].Am J Pathol, 2002,160(5):1705-1715.
[4] Sun KA, Wang Q, Huang XH.PPAR gamma inhibits growth of rat hepatic stellate cells and TGF beta–induced connective tissue growth factor expression[J].Acta Pharmacologica Sinica,2006,27 (6):715–723.
[5] Shek FW, Benyon RC.How can transforming growth factor beta be targeted usefully to combat liver fibrosis[J]? Eur.J.GastroenterolHepatol,2004,16 :123–126.
[6] Li JH.Inhibition of p38 Mitogen-Activated Protein Kinase and Transforming Growth Factor-β1/Smad Signaling Pathways Modulates the Development of Fibrosis in Adriamycin-Induced Nephropathy [J].Am J Pathol,2006,169(5): 1527–1540.
[7] Lucia W.Glucocorticoids activate TGF-βinduced PAI-1 and CTGF expression in rat hepatocytes[J].Comp Hepatol,2007,6:5926-5931.
[8] Wu XY. The role of connective tissue growth factor, transforming growth factor-β1 and Smad signaling pathway in cornea wound healing[J].Chinese MedicalJournal, 2006,119(1):57-62.
[9] Rachfal AW,Brigstock DR.Connective tissue growth factor(CTGF / CCN2)in hepatic fibrosis[J].Hepatol Res,2003,26(1):1- 9.
[10] Paradis V,Dargere D,Bonvoust F,et al.Effects and regulation of connective tissue growth factor on hepatic stellate cells[J].Lab invest,2002,82:767-774.
[11] Liu FY.Inhibition effect of small interfering RNA of connective tissue growth factoron the expression of vascular endothelial growth factor and connective tissue growth factor in cultured human peritoneal mesothelial cells[J].Chinese Medical Journal,2007,120 (3):231-236.
[12] Leask A, Abraham DJ.The role of connective tissue growth factor,a multifunctional matricellular protein,in fibroblast biology[J].Biochem Cell Biol,2003,81:355-379.
[13] Kuiper EJ,Witmer AN,Klaassen,et al.Differential expression of connective tissue growth factor in microglia and pencytes in the human diabetic[J].Ophthalmology, 2004,88:1082-1087.
[14] Natalia DL, Heras, et al.Role of connective tissue growth factor in vascular and renal damage associated with hypertension in rats[J].Interactions with angiotensin II jraas,2006,7:192–200.
[15] Umezono T.Glomerular expression of CTGF, TGF-beta1 and type IV collagen in diabetic nephropathy[J].Jnephrol,2006,19:751-757.
[16] Lam S.Connective tissue growth factor and IGF-1 are produced by human renal fibroblasts and cooperate in the induction of collagen production by high glucose[J].Diabetes,2003,52:2975-2983.
[17] Zhou G.Advanced glycation end-products induce connective tissue growth factor-mediated renal fibrosis predominantly through transforming growth factor beta-independent pathway[J].Am J Pathol,2004,165:2033-2043.
[18] Yokoi H.Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis[J].J Am Soc Nephrol,2004,15:1430- 1440.
[2] Ramon B,David A.Liver fibrosis[J].J. Clin. Invest. 2005,115:209–218.
[3] Dubuisson L , Desmouliere A , Decou NB, et al . Inhibition of rat liver fibrogensis through noradrenergic antagonism[J]. Hepatology , 2002,35: 325-331.
[4] Nakata E,Nakanishi T,Kawai A,et al.Expression of connective tissue factor/hypertrophic chondrocyte-specific gene product 24(CTGF/Hcs)during fracture healing [J]. Bone,2002,31: 441-447.
[5] Riser BL, Denichilo M, Cortes P, et al. Regulation of connetive tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomeralosclerosis[J].JM Soc Nephrol , 2000,11:25-28.
[6] Hirasaki S, Koide N, Ujike K, et al. Expression of Nov, CYR61 and CTGF genes in human hepatocellular carcinoma [J]. Hepatol Res.,2001 Mar 26,19(3):294-305.
[7] Lucia W, Nicolas C, Karin K, et al. Glucocorticoids activate TGF-βinduced PAI-1 and CTGF expression in rat hepatocytes[J]. Comp Hepatol., 2007, 6:5926-5931.
[8] Runyan CE,Schnaper HW,Poncelet AC.The role of internalization in transforming growth factor betal-induced Smad2 association with Smad anchor for receptor activation fSARA1 and Smad2 dependent signaling in human mesangial cells[J]. J Biol Chem,2005,280:8300-8308.
[9] Safadi R,Friedman SL.Hepatic fibrosis-role of hepatic stelate cell activation[J].Med Gen Med,2002,4(3):27.
[10] Less KS.Hepatic fibrogenesis[J].Korean J Gastroenterol,2006,97(12):741-750.
[11] Solis-Herruzo JA,Dela-Torre P,Minoz-Yague MT,et a1.Hepatic stelate cells (HSC): architects of hepatic fibrosis[J].Rev Esp Enferm Dig,2003,95:438-439.
[12] Marra F.Hepatic stellate cells and the regulation of liver inflammation[J].J Hepatol1999,31:1120-1130.
[13] Lamireau T,Desmoyliere A,Bioulac-Sage P,et a1.Mechanisms of hepatic fibmgenesis[J].Arch Pediatr, 2002,9(4):392-405.
[14] Hui AY,Friedman SL.Molecular basis of hepatic fibrosis[J].Expert Rev Mol Med, 2003,2003:1-23.
[15] Friedman SL.Stellate cells:a moving target in hepaticfibrogenesis[J]. Hepatology, 2004,40(5):1041-1043.
[16] Pan Q,Li DG,Lu HM,et a1.Establishment and identification of a novel immortalized rat hepatic stellate cell line[J].China Journal of Modem Medicine, 2005,15(10):1441-1446.
[17] Friedman SL,Lalazar A,Crong L,et a1.HSC-T6 cells,an immortalized rat hepatic stelate cell line[j].Hepatology,1997,26(4):338-342.
[18] Gressner AM, Weiskirchen R, Breitkopf K, et al. Roles of TGF-beta in hepatic fibrosis[ J ]. FrontBiosci, 2002,7 (4):793 - 807.
[19] 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.
[20] Parsonsp CJ,Takashima M,Rippe RA.Molecular mechanisms of hepatic fibrogenesis[J].J Gastroenterol Hepatol, 2007, 22(1):79-84.
[21] Philippe B, Kjetil A,Kathy F, et al.TGF-βand Smad3 Signaling Link Inflammation to Chronic Fibrogenesis[J].The Journal of Immunology, 2005,175:5390–5395.
[22] Xavier LL,Viola GG,Ferraz AC,et al.A simple and fast densitometric method for the analysis of tyrosine hydroxylase immunoreactivity in the substantia nigra parscompacta and in the ventral tegmental area[J].Brain Res Brain Res Protoc,2005,16(1-3):58-64.
[23] Arnott JA,Zhang X,Sanjay A, et al. Molecular Requirements for Induction of CTGF Expression by TGF-β1 in Primary Osteoblasts[J].Bone,2008, 42(5): 871–885.
[24] Yokoi H,Mukoyama H,Sugawara A,et a1.Role of connective tissue growth factor in fibronectin expression and tubulointerstial fibrosis[J].Am J Physiol,2002,282(5):933–942.
[25] Chen Y,Blom IE,Sa S,et a1.CTGF expression in mesangial cells:Involvement of SMADs,MAPK and PKC[J].Kidney Int,2002,62(4):l149-l152.
[26] Blom IE,Goldsohmeding R, Leusk A.Gene regulation of connective tissue growth factor:new targets for antifibrotic therapy[J].Matrix Bial,2002,21:473-479.
[27] Leask A,Abraham DJ.The role of connective tissue growth factor,a multifunctional matricellular protein,in fibroblast biology[J].Biochem Cell Biol,2003,81:355-379.
[28] Rchfal AW,Brigstock DR.Connective tissue growth factor(CTGF/CCN2) in hepatic fibrosis[J].Hepatol Res,2003,26(1):1-9.
[29] Gao R,Brigstock DR.Low density lipoprotein receptor-related protein(LRP) is a heparin-dependent adhesion receptor for connective tissue growth factor(CTGF) in rat activated hepatic stellate cells[J].Hepatol Res,2003,27(3):214-220.
[30] Ball DK,Wilson AW,Kemper SA,et al.The heparinbinding 10kDa fragment of connective tissue growth factor(CTGF) containing module 4 alone stimulate cell adhesion[J].J Endocrinol,2003,176(2):1-7.
[1] Friedman SL,Bansal MB.Reversal of hepatic fibrosis-Fact or fantasy[J]? Hepatology,2006,43:82-88.
[2] Nakata E,Nakanishi T,Kawai A,et al.Expression of connective tissue factor/hypertrophic chondrocyte-specific gene product 24(CTGF/Hcs)during fracture healing[J].Bone,2002,31:441-447.
[3] Lewindon PJ,Pereira IN,Hoskins AC,et a1.The role of hepatic satellite cells and transforming growth factor-β1 in cystic fibro6is liver disease[J].Am J Pathol, 2002,160(5):1705-1715.
[4] Sun KA, Wang Q, Huang XH.PPAR gamma inhibits growth of rat hepatic stellate cells and TGF beta–induced connective tissue growth factor expression[J].Acta Pharmacologica Sinica,2006,27 (6):715–723.
[5] Shek FW, Benyon RC.How can transforming growth factor beta be targeted usefully to combat liver fibrosis[J]? Eur.J.GastroenterolHepatol,2004,16 :123–126.
[6] Li JH.Inhibition of p38 Mitogen-Activated Protein Kinase and Transforming Growth Factor-β1/Smad Signaling Pathways Modulates the Development of Fibrosis in Adriamycin-Induced Nephropathy [J].Am J Pathol,2006,169(5): 1527–1540.
[7] Lucia W.Glucocorticoids activate TGF-βinduced PAI-1 and CTGF expression in rat hepatocytes[J].Comp Hepatol,2007,6:5926-5931.
[8] Wu XY. The role of connective tissue growth factor, transforming growth factor-β1 and Smad signaling pathway in cornea wound healing[J].Chinese MedicalJournal, 2006,119(1):57-62.
[9] Rachfal AW,Brigstock DR.Connective tissue growth factor(CTGF / CCN2)in hepatic fibrosis[J].Hepatol Res,2003,26(1):1- 9.
[10] Paradis V,Dargere D,Bonvoust F,et al.Effects and regulation of connective tissue growth factor on hepatic stellate cells[J].Lab invest,2002,82:767-774.
[11] Liu FY.Inhibition effect of small interfering RNA of connective tissue growth factoron the expression of vascular endothelial growth factor and connective tissue growth factor in cultured human peritoneal mesothelial cells[J].Chinese Medical Journal,2007,120 (3):231-236.
[12] Leask A, Abraham DJ.The role of connective tissue growth factor,a multifunctional matricellular protein,in fibroblast biology[J].Biochem Cell Biol,2003,81:355-379.
[13] Kuiper EJ,Witmer AN,Klaassen,et al.Differential expression of connective tissue growth factor in microglia and pencytes in the human diabetic[J].Ophthalmology, 2004,88:1082-1087.
[14] Natalia DL, Heras, et al.Role of connective tissue growth factor in vascular and renal damage associated with hypertension in rats[J].Interactions with angiotensin II jraas,2006,7:192–200.
[15] Umezono T.Glomerular expression of CTGF, TGF-beta1 and type IV collagen in diabetic nephropathy[J].Jnephrol,2006,19:751-757.
[16] Lam S.Connective tissue growth factor and IGF-1 are produced by human renal fibroblasts and cooperate in the induction of collagen production by high glucose[J].Diabetes,2003,52:2975-2983.
[17] Zhou G.Advanced glycation end-products induce connective tissue growth factor-mediated renal fibrosis predominantly through transforming growth factor beta-independent pathway[J].Am J Pathol,2004,165:2033-2043.
[18] Yokoi H.Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis[J].J Am Soc Nephrol,2004,15:1430- 1440.