甘参复方对大鼠肝星状细胞增殖和胶原合成的影响
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摘要
目的:观察甘参复方对大鼠肝星状细胞增殖和胶原合成的影响,并分析甘参复方抗肝纤维化的分子机制。方法:采用体外培养的永生化细胞株大鼠肝星状细胞(hepatic sellate cell-T6,HSC-T6)。将甘参复方稀释成6个不同的浓度梯度,采用CCK-8法检测各组细胞的增殖情况,筛选出甘参复方对HSC-T6的有效作用浓度。之后将细胞分为阴性对照组,甘参复方低浓度组、中浓度组、高浓度组。采用ELISA法检测各组细胞上清液中的Ⅰ型胶原蛋白(collagenⅠ,ColⅠ)水平。然后,应用转化生长因子-β1(transforming growth factor-β1,TGF-β1)(1 mg·L~(-1))刺激HSC-T6,观察甘参复方对刺激后细胞的增殖和胶原合成的影响。结果:甘参复方的6个不同浓度对HSC-T6的增殖有明显抑制作用,且浓度为0.5μmol·L~(-1)时抑制作用最明显(P<0.01)。ELISA结果显示,甘参复方能够降低细胞上清中的ColⅠ含量,且与CCK-8结果相吻合,在0.5μmol·L~(-1)时ColⅠ分泌最少。甘参复方能够抑制TGF-β1刺激后的HSC-T6增殖和胶原合成。结论:甘参复方能够显著抑制星状细胞的增殖以及ColⅠ的表达,这是甘参复方抗肝纤维化的作用机制之一。
Objective:To observe the effects of Ganshen Compound on proliferation and collagen synthesis of rat hepatic stellate cells,and to analyze the molecular mechanism of Ganshen Compound on anti-hepatic fibrosis.Methods:The immortalized cell line of hepatic sellate cell-T6(HSC-T6) was cultured in vitro.The Ganshen Compound was diluted into 6 different concentration gradients.The proliferation of cells in each group was detected by CCK-8 method.The effective concentration of Ganshen Compound on HSC-T6 was screened out.After that,the cells were divided into negative control group,Ganshen Compound low concentration group,medium concentration group and high concentration group.The levels of collagenⅠ(Col I) in cell supernatants were detected by ELISA.Then,HSC-T6 was stimulated by transforming growth factor-β1(TGF-β1)(1 mg·L~(-1)),and the effects of Ganshen Compound on cell proliferation and collagen synthesis were observed.Results:The proliferation of HSC-T6 was inhibited by 6 different concentrations of Ganshen Compound,and the inhibition was most obvious when the concentration was 0.5 μmol·L~(-1)(P<0.01).ELISA results showed that Ganshen Compound could reduce the content of collagen type I in cell supernatant,which was consistent with the results of CCK-8.Col I secreted least at 0.5 μmol·L~(-1).Moreover,Ganshen Compound can inhibit the proliferation and collagen synthesis of HSC-T6 stimulated by TGF-β1.Conclusion:Ganshen Compound can significantly inhibit the proliferation of stellate cells and the expression of collagen type I,which is one of the mechanisms of anti-hepatic fibrosis of Ganshen Compound.
Objective:To observe the effects of Ganshen Compound on proliferation and collagen synthesis of rat hepatic stellate cells,and to analyze the molecular mechanism of Ganshen Compound on anti-hepatic fibrosis.Methods:The immortalized cell line of hepatic sellate cell-T6(HSC-T6) was cultured in vitro.The Ganshen Compound was diluted into 6 different concentration gradients.The proliferation of cells in each group was detected by CCK-8 method.The effective concentration of Ganshen Compound on HSC-T6 was screened out.After that,the cells were divided into negative control group,Ganshen Compound low concentration group,medium concentration group and high concentration group.The levels of collagenⅠ(Col I) in cell supernatants were detected by ELISA.Then,HSC-T6 was stimulated by transforming growth factor-β1(TGF-β1)(1 mg·L~(-1)),and the effects of Ganshen Compound on cell proliferation and collagen synthesis were observed.Results:The proliferation of HSC-T6 was inhibited by 6 different concentrations of Ganshen Compound,and the inhibition was most obvious when the concentration was 0.5 μmol·L~(-1)(P<0.01).ELISA results showed that Ganshen Compound could reduce the content of collagen type I in cell supernatant,which was consistent with the results of CCK-8.Col I secreted least at 0.5 μmol·L~(-1).Moreover,Ganshen Compound can inhibit the proliferation and collagen synthesis of HSC-T6 stimulated by TGF-β1.Conclusion:Ganshen Compound can significantly inhibit the proliferation of stellate cells and the expression of collagen type I,which is one of the mechanisms of anti-hepatic fibrosis of Ganshen Compound.
引文
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[14]林如盛.甘利欣联合丹参注射液治疗慢性乙型肝炎的临床观察[J].海峡药学,2007,19(2):81-82.
[15]LI S,WANG L,YAN X,et al.Salvianolic Acid B Attenuates Rat Hepatic Fibrosis via Downregulating Angiotensin Ⅱ Signaling[J].Evid Based Complement Alternat Med,2012(11):160726.
[16]于首元,于兆安.脱氧核苷酸钠注射液联合甘草酸二铵胶囊对人血白蛋白所致免疫性肝纤维化大鼠的影响[J].中国医药指南,2014,12(8):37-38.
[17]XU F,LIU C,ZHOU D,et al.TGF-beta/SMAD Pathway and Its Regulation in Hepatic Fibrosis[J].J Histochem Cytochem,2016,64(3):157-167.
[18]CARGNELLO M,ROUX P P.Activation and Function of the MAPKs and Their Substrates,the MAPK-Activated Protein Kinases[J].Microbiol Mol Biol Rev,2012,76(2):496.
[19]LV Z,SONG Y,XUE D,et al.Effect of Salvianolic-acid B on inhibiting MAPK signaling induced by transforming growth factor-β1 in activated rat hepatic stellate cells[J].J Ethnopharmacol,2010,132(2):384-392.
[20]张英博,孟凡谋,张英慧,等.甘草酸二铵对肝纤维化实验大鼠TGF-β/Smad信号转导通路相关因子的影响[J].齐齐哈尔医学院学报,2016,37(12):1509-1511.
[2]SHANG L,HOSSEINI M,LIU X,et al.Human hepatic stellate cell isolation and characterization[J].J Gastroenterol.2018,53(1):6-17.
[3]SEYER J M,HUTCHESON E T,KANG A H.Collagen polymorphism in normal and cirrhotic human liver[J].J Clin Invest,1977,59(2):241-248.
[4]肝纤维化中西医结合诊疗指南[J].中华肝脏病杂志,2006,14(11):866-870.
[5]庄灿皇.基于近二十年文献中医药治疗肝纤维化的方药规律研究[D].广州:广州中医药大学,2016.
[6]刘树锋,蔡大勇,李澎涛,等.优化甘参复方抗肝纤维化组分配伍的研究[J].中华中医药杂志,2005,20(6):373-375.
[7]DU Q H,HAN L,JIANG J J,et al.Glytan decreases portal pressure via mesentery vasoconstriction in portal hypertensive rats[J].World J Gastroenterol,2014,20(44):16674-16682.
[8]杜庆红,韩琳,姜俊杰,等.肝络通对门静脉高压症大鼠肝组织纤维化的影响[J].世界科学技术——中医药现代化,2012,14(3):1631-1635.
[9]SONG Y F,LU Z G,XU L M.Salvianolic acid B inhibits ERK signal transduction pathway activated by transforming growth factor-β1 in rat hepatic stellate cells[J].Zhong Xi Yi Jie He Xue Bao,2012,10(4):454-461.
[10]刘洪亮,吕靖,赵志敏,等.丹参水溶性成分抑制肝窦内皮细胞功能及血管新生的活性评价[J].中草药,2016,47(6):938-943.
[11]ZHAO X,DENG B,XU X Y,et al.Glycyrrhizinate reduces portal hypertension in isolated perfused rat livers with chronic hepatitis[J].World J Gastroenterol,2013,19(36):6069-6076.
[12]徐列明,刘成,刘平.丹参酚性酸B对大鼠传代肝贮脂细胞增殖、形态和合成细胞外基质的影响[J].中华肝脏病杂志,1996,4(2):86-89.
[13]LIU P,LIU C H,WANG H N,et al.Effect of salvianolic acid B on collagen production and mitogen-activated protein kinase activity in rat hepatic stellate cells[J].Acta Pharmacol Sin,2002,23(8):733-738.
[14]林如盛.甘利欣联合丹参注射液治疗慢性乙型肝炎的临床观察[J].海峡药学,2007,19(2):81-82.
[15]LI S,WANG L,YAN X,et al.Salvianolic Acid B Attenuates Rat Hepatic Fibrosis via Downregulating Angiotensin Ⅱ Signaling[J].Evid Based Complement Alternat Med,2012(11):160726.
[16]于首元,于兆安.脱氧核苷酸钠注射液联合甘草酸二铵胶囊对人血白蛋白所致免疫性肝纤维化大鼠的影响[J].中国医药指南,2014,12(8):37-38.
[17]XU F,LIU C,ZHOU D,et al.TGF-beta/SMAD Pathway and Its Regulation in Hepatic Fibrosis[J].J Histochem Cytochem,2016,64(3):157-167.
[18]CARGNELLO M,ROUX P P.Activation and Function of the MAPKs and Their Substrates,the MAPK-Activated Protein Kinases[J].Microbiol Mol Biol Rev,2012,76(2):496.
[19]LV Z,SONG Y,XUE D,et al.Effect of Salvianolic-acid B on inhibiting MAPK signaling induced by transforming growth factor-β1 in activated rat hepatic stellate cells[J].J Ethnopharmacol,2010,132(2):384-392.
[20]张英博,孟凡谋,张英慧,等.甘草酸二铵对肝纤维化实验大鼠TGF-β/Smad信号转导通路相关因子的影响[J].齐齐哈尔医学院学报,2016,37(12):1509-1511.