蒙古山萝卜酸对肝星状细胞与肝纤维化大鼠超微结构及抗氧化的影响
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摘要
目的探讨蒙古山萝卜酸(SCA)对肝星状细胞(HSC)的基质金属蛋白酶(MMP1)和组织金属蛋白酶抑制酶(TIMP1)的活性,细胞外基质Ⅲ型前胶原(PC-Ⅲ)和Ⅳ型胶原(C-Ⅳ)的合成以及纤维化肝组织细胞外基质(ECM)与HSC体密度、HSC的凋亡及抗氧化作用。方法采用HSC初次传代后,将HSC随机分为4组:空白对照组、SCA高剂量组(15.6μg·mL~(-1))、SCA中剂量组(7.81μg·mL~(-1))和SCA低剂量组(3.9μg·mL~(-1))。加药孵育48h后,采用Elisa法测定HSC中PC-Ⅲ、MMP1与TIMP1活性,Western Blot法检测C-Ⅳ表达,透射电镜扫描SCA对纤维化肝组织ECM与HSC体密度、HSC的凋亡形态及抗氧化测定。结果 SCA可抑制HSC中TIMP1活性与细胞外基质PC-Ⅲ和C-Ⅳ合成,对MMP1活性作用差异无统计学意义,呈抑制趋势。SCA质量浓度在3.9～15.6μg·mL~(-1)范围内呈量效关系;SCA可明显减少大鼠肝脏汇管区纤维化肝组织ECM与HSC体密度、诱导HSC结构转变呈凋亡形态及抑制肝组织氧化应激反应。结论 SCA对体外培养的HSC分泌合成TIMP1活性及PC-Ⅲ、C-Ⅳ合成有抑制作用,可减少纤维化肝组织ECM与HSC体密度、促进HSC的凋亡,抑制丙二醛(MDA)合成、提高超氧化物歧化酶(SOD)和谷胱甘肽氧化酶(GSH)活性,其作用机制与SCA抑制肝组织氧化应激作用有关。
Objective To study the effect of Scabiosa comosa acid(SCA)on matrix metalloproteinases(MMP1)and tissue inhibitor of metalloproteinases(TIMP1),extracellular matrix precollagenⅢ(PC-Ⅲ),Ⅳtype collagen(C-Ⅳ)synthesis in hepatic stellate cells(HSC)and the effect of SCA on extracellular matrix(ECM)and HSC density,HSC apoptosis and antioxidant ability in fibrotic liver tissue.Methods After wedding for the first time,HSC were randomly divided into 4groups:blank control group,SCA high dose group(15.6μg·mL~(-1)),SCA middle dose group(7.81μg·mL~(-1))and SCA low dose group(3.9μg·mL~(-1)),dosed 48h after incubation.Using Elisa method was used to determine PC-Ⅲ,MMP1and TIMP1active in HSC.The C-Ⅳcollagen expression was determined by Western Blot test.The ECM and HSC density were observed by transmission electron microscope.The HSC apoptosis and antioxidant ability were assayed.Results The SCA inhibited the TIMP1enzyme activity in HSC and PC-Ⅲ,C-Ⅳsynthesis in extracellular matrix but showed no significant effect on the MMP1enzyme activity.Within the designed concentration range(3.9-15.6μg·mL~(-1)),SCA showed the concentration-response relationship.SCA can significantly reduce the ECM and HSC density,induce the apoptosis of HSC structure change and inhibit the hepatic tissue oxidative stress in fibrosis hepatic tissue.Conclusion SCA has the inhibitory effect on TIMP1enzyme activity and PC-Ⅲ,C-Ⅳsynthesis in vitro culture of HSC,can reduce the ECM and HSC density,promote the HSC apoptosis,inhibit the malondialdehyde(MDA)synthesis and increase the activity of superoxide dismutase(SOD)and glutathione peroxidase(GSH)in fibrosis liver tissue.The action mechanism is related to the inhibition of oxidative stress.
Objective To study the effect of Scabiosa comosa acid(SCA)on matrix metalloproteinases(MMP1)and tissue inhibitor of metalloproteinases(TIMP1),extracellular matrix precollagenⅢ(PC-Ⅲ),Ⅳtype collagen(C-Ⅳ)synthesis in hepatic stellate cells(HSC)and the effect of SCA on extracellular matrix(ECM)and HSC density,HSC apoptosis and antioxidant ability in fibrotic liver tissue.Methods After wedding for the first time,HSC were randomly divided into 4groups:blank control group,SCA high dose group(15.6μg·mL~(-1)),SCA middle dose group(7.81μg·mL~(-1))and SCA low dose group(3.9μg·mL~(-1)),dosed 48h after incubation.Using Elisa method was used to determine PC-Ⅲ,MMP1and TIMP1active in HSC.The C-Ⅳcollagen expression was determined by Western Blot test.The ECM and HSC density were observed by transmission electron microscope.The HSC apoptosis and antioxidant ability were assayed.Results The SCA inhibited the TIMP1enzyme activity in HSC and PC-Ⅲ,C-Ⅳsynthesis in extracellular matrix but showed no significant effect on the MMP1enzyme activity.Within the designed concentration range(3.9-15.6μg·mL~(-1)),SCA showed the concentration-response relationship.SCA can significantly reduce the ECM and HSC density,induce the apoptosis of HSC structure change and inhibit the hepatic tissue oxidative stress in fibrosis hepatic tissue.Conclusion SCA has the inhibitory effect on TIMP1enzyme activity and PC-Ⅲ,C-Ⅳsynthesis in vitro culture of HSC,can reduce the ECM and HSC density,promote the HSC apoptosis,inhibit the malondialdehyde(MDA)synthesis and increase the activity of superoxide dismutase(SOD)and glutathione peroxidase(GSH)in fibrosis liver tissue.The action mechanism is related to the inhibition of oxidative stress.
引文
[1]姜慧卿,姚希贤.肝星状细胞的活化与肝纤维化[J].胃肠病学和肝病学杂志,2007,16(1):86-89.
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[7]田国才,刘朝,沈敬华,等.蒙古山萝卜总黄酮激活小鼠腹腔巨噬细胞的动态研究[J].中华微生物学和免疫学杂志,1986,6(2):107-111.
[8]白音夫,李瑞峰,顾维彰.蒙古山萝卜花总黄酮镇痛、镇静、消炎、解热作用的研究[J].内蒙古药学,1987,6(1)43-45.
[9]杨静华,常亮,齐明玉,等.山萝卜酸对不同组织匀浆中MDA生成的影响[J].中国民族医药杂志,2011,17(1):39-40.
[10]欧阳辉,黎田儿,何明珍,等.超高效液相色谱与飞行时间质谱联用技术鉴定蒙药蓝盆花在大鼠体内效应成分及其代谢物[J].中国药学杂志,2016,51(14):1197-1203.
[11]白音夫,周雪梅,周长凤,等.蒙古山萝卜有效成分的分离、鉴定与含量测定[J].西北药学杂志,2014,29(6):564-566.
[12]董珉翔.蒙古山萝卜酸对肝纤维化大鼠血清PC-Ⅲ、C-Ⅳ、HA、LN及肝组织HSCs、胶原纤维的影响[D].呼和浩特:内蒙古医科大学,2014.
[13]杨宏昕,白音夫,米裕青,等.蒙古山萝卜酸对实验性肝纤维化大鼠MMP-1、TIMP-1、TGF-β1和PDGF表达的影响[J].西北药学杂志,2017,32(4):477-480.
[14]胡丹华,禄保平.刺蒺藜总皂苷对雷公藤所致急性肝损伤小鼠肝细胞超微结构的立体计量学分析[J].中医学报,2012,27(5):588-589.
[15]郑富盛.细胞体密度特征参数的测算方法[J].细胞生物学杂志,1984,6(2):80-82.
[16]Zhou X,Murphy F R,Gshdu N,et al.Engagement of alphavbeta3integrin regulates proliferation and apoptosis of hepatic stellate cells[J].J Biol Chem,2004,279(23):23996-24006.
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[18]徐新保,何振平,梁志清.阻断转化生长因子-β1信号传导治疗大鼠实验性肝纤维化[J].中华肝脏病杂志,2004,12(5):263-266.
[19]钟晓琴,沈薇.肝细胞生长因子对大鼠原代肝星状细胞凋亡的影响及其机制[J].中华肝脏病杂志,2008,16(9):665-668.
[20]常亮,马忠良.肝纤维化的机制、临床诊断与蒙医药研究概述[J].中国民族医药杂志,2015,(3):66-68.
[21]Parola M,Robino G.Oxidative stress-related molecules and liver fibrosis[J].J Hepatol,2001,35(2):297-306.
[22]Pinto C,Duque A L,Rodríguez-Galdón B,et al.Xanthohumol prevents carbon tetrachloride-induced acute liver injury in rats[J].Food Chem Toxicol,2012,50(10):3405-3412.
[23]戚晓渊,史秀灵,高银辉,等.绿原酸抗肝纤维化作用的研究[J].中国实验方剂学杂志,2011,17(15):139-143.
[24]白清云.中国医学百科全书:蒙医学[M].上海:上海科学技术出版社,1992:204.
[2]Benyon R C,Arther M J.Mechanisms of hepatic fibrosis[J].J Pediatr Gastroenterol Nutr,1998,27(1):75-85.
[3]姜宪辉,叶华,孙雷.MMP-1、TIMP-1在肝纤维化组织中的表达及其病理意义[J].医学信息,2008,21(12):2291-2294.
[4]Friedman S L.Mechanisms of hepatic fibrogenesis[J].Gastroenterology,2008,134(6):1655-1669.
[5]中华人民共和国卫生部药典委员会.中华人民共和国卫生部药品标准:蒙药分册[S].北京:人民卫生出版社,1998:52-194.
[6]内蒙古自治区卫生厅.内蒙古蒙药材标准[S].呼和浩特:内蒙古科学技术出版社,1987:502.
[7]田国才,刘朝,沈敬华,等.蒙古山萝卜总黄酮激活小鼠腹腔巨噬细胞的动态研究[J].中华微生物学和免疫学杂志,1986,6(2):107-111.
[8]白音夫,李瑞峰,顾维彰.蒙古山萝卜花总黄酮镇痛、镇静、消炎、解热作用的研究[J].内蒙古药学,1987,6(1)43-45.
[9]杨静华,常亮,齐明玉,等.山萝卜酸对不同组织匀浆中MDA生成的影响[J].中国民族医药杂志,2011,17(1):39-40.
[10]欧阳辉,黎田儿,何明珍,等.超高效液相色谱与飞行时间质谱联用技术鉴定蒙药蓝盆花在大鼠体内效应成分及其代谢物[J].中国药学杂志,2016,51(14):1197-1203.
[11]白音夫,周雪梅,周长凤,等.蒙古山萝卜有效成分的分离、鉴定与含量测定[J].西北药学杂志,2014,29(6):564-566.
[12]董珉翔.蒙古山萝卜酸对肝纤维化大鼠血清PC-Ⅲ、C-Ⅳ、HA、LN及肝组织HSCs、胶原纤维的影响[D].呼和浩特:内蒙古医科大学,2014.
[13]杨宏昕,白音夫,米裕青,等.蒙古山萝卜酸对实验性肝纤维化大鼠MMP-1、TIMP-1、TGF-β1和PDGF表达的影响[J].西北药学杂志,2017,32(4):477-480.
[14]胡丹华,禄保平.刺蒺藜总皂苷对雷公藤所致急性肝损伤小鼠肝细胞超微结构的立体计量学分析[J].中医学报,2012,27(5):588-589.
[15]郑富盛.细胞体密度特征参数的测算方法[J].细胞生物学杂志,1984,6(2):80-82.
[16]Zhou X,Murphy F R,Gshdu N,et al.Engagement of alphavbeta3integrin regulates proliferation and apoptosis of hepatic stellate cells[J].J Biol Chem,2004,279(23):23996-24006.
[17]F Murphy,J Waung,J Collins,et al.N-Cadherin cleavage during activated hepatic stellate cell apoptosis is inhibited by tissue inhibitor of metalloproteinase-1[J].Comp Hepatol,2004,3(Suppl 1):S8.
[18]徐新保,何振平,梁志清.阻断转化生长因子-β1信号传导治疗大鼠实验性肝纤维化[J].中华肝脏病杂志,2004,12(5):263-266.
[19]钟晓琴,沈薇.肝细胞生长因子对大鼠原代肝星状细胞凋亡的影响及其机制[J].中华肝脏病杂志,2008,16(9):665-668.
[20]常亮,马忠良.肝纤维化的机制、临床诊断与蒙医药研究概述[J].中国民族医药杂志,2015,(3):66-68.
[21]Parola M,Robino G.Oxidative stress-related molecules and liver fibrosis[J].J Hepatol,2001,35(2):297-306.
[22]Pinto C,Duque A L,Rodríguez-Galdón B,et al.Xanthohumol prevents carbon tetrachloride-induced acute liver injury in rats[J].Food Chem Toxicol,2012,50(10):3405-3412.
[23]戚晓渊,史秀灵,高银辉,等.绿原酸抗肝纤维化作用的研究[J].中国实验方剂学杂志,2011,17(15):139-143.
[24]白清云.中国医学百科全书:蒙医学[M].上海:上海科学技术出版社,1992:204.