柴竭抑肝纤对硫代乙酰胺诱导的大鼠肝纤维化的影响
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摘要
目的:研究中药复方柴竭抑肝纤(Chaijie Yiganxian,CJYGX)对抗大鼠肝纤维化的作用及其机制。方法:60只SD大鼠随机分为正常组,模型组,阳性组(鳖甲煎丸),CJYGX高、中、低剂量组。除正常组外,其余5组通过腹腔注射硫代乙酰胺(thioacetamide,TAA)诱导建立肝纤维化模型。自造模第4周开始,阳性组给予鳖甲煎丸1 g·kg~(-1)·d-1灌胃,CJYGX高、中、低剂量组分别给予CJYGX 7.96,3.98,1.99 g·kg~(-1)·d-1灌胃,模型组灌服生理盐水,每日灌胃1次,共5周。于末次灌胃24 h后,水合氯醛轻度麻醉大鼠,留取血清及肝组织,称肝脏湿重并计算肝脏指数;全自动分析法检测大鼠肝功能指标丙氨酸氨基转移酶(alanine transaminase,ALT),天门冬氨酸氨基转移酶(aspartate aminotransferase,AST),碱性磷酸酶(alkaline phosphatase,ALP);酶联免疫吸附测定(ELISA)检测肝纤维化血清学标志物层粘连蛋白(laminin,LN),透明质酸酶(hyaluronidase,HA),Ⅳ型胶原(Ⅳcollagen,CⅣ),Ⅲ型前胶原(procollagenⅢ,PCⅢ);马松(Masson)染色观察肝脏纤维化程度;实时荧光定量聚合酶链式反应(Real-time PCR)检测肝脏组织转化生长因子-β1(transforming growth factor-β1,TGF-β1),Smad2,Smad3,Smad4,Smad7 mRNA表达;免疫组化检测肝脏组织血小板源性生长因子(platelet derived growth factor,PDGF)和TGF-β1蛋白表达水平;蛋白免疫印迹法(Western blot)检测肝脏TGF-β1和α-平滑肌肌动蛋白(α-SMA)蛋白表达。结果:与正常组比较,模型组肝脏指数显著升高;肝功能指标ALP,AST,ALT的含量显著升高;肝纤维化指标LN,HA,Ⅳ-C,PCⅢ和HYP显著升高; Masson染色观察到肝纤维化程度显著增加; TGF-β1,Smad2,Smad3,Smad4 mRNA表达显著升高,Smad7 mRNA表达显著降低; TGF-β1,PDGF和α-SMA蛋白表达显著升高(P<0.01)。与模型组比较,各剂量的CJYGX能不同程度的降低肝功能指标ALP,AST,ALT的水平;肝纤维化指标LN,HA,Ⅳ-C,PCⅢ和HYP都明显的降低; Masson组织观察到肝纤维化程度显著减轻; TGF-β1,Smad2,Smad3,Smad4 mRNA表达明显下调,Smad7 mRNA表达明显上调; TGF-β1,PDGF和α-SMA蛋白表达显著下调(P<0.05,P<0.01)。结论:中药复方"柴竭抑肝纤"可能通过干预TGF-β1/Smad信号通路,保护TAA诱导的大鼠肝纤维化损伤。
Objective:To research the effect of traditional Chinese medicine compound Chaijie Yiganxian(CJYGX) on the hepatic fibrosis in rats and explore the mechanism.Method:Totally 60 SD rats were randomly divided into normal group,model group,positive group(Biejia Jianwan),and low,medium and high-dose CJYGX groups.Except for the normal group,the remaining five groups were involved in establishing the hepatic fibrosis model through the intraperitoneal injection of thioacetamide(TAA).Fourth weeks after modeling,the positive group was given Biejiajian pill(1 g·kg~(-1)·d-1),and high,medium and low-dose CJYGX groups were given CJYGX(7.96,3.98,1.99 g·kg~(-1)·d-1),respectively.The model group was given normal saline once a day for 5 weeks.24 h after the last intragastric administration with chloral hydrate,the rats were anesthetized slightly.Serum and liver tissues were collected.The liver wet weight was weighed electronically,and the liver index was calculated.Liver function indexes [alanine transaminase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP) ]and serum markers [laminin(LN),hyaluronidase(HA),Ⅳ collagen(CⅣ),procollagen Ⅲ(PCⅢ) ]of hepatic fibrosis were detected by enzyme linked immunosorbent assay(ELISA).The degree of hepatic fibrosis by Masson stain,mRNA expressions of transforming growth factor-β1(TGF-β1),Smad2,Smad3,Smad4,Smad7 were detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR),and protein expression levels of platelet derived growth factor(PDGF),TGF-β1 and α-smooth muscle actin(α-SMA)were detected by immunohistochemical or Western blot.Result:Compared with the blank group,the model group liver index was significantly increased;the contents of ALP,AST and ALT were significantly increased;the liver fibrosis indexes LN,HA,Ⅳ-C,PC Ⅲ and HYP were significantly increased;the degree of liver fibrosis was significantly increased in Masson tissue;the mRNA expressions of TGF-β1,Smad2,Smad3,Smad4 were significantly increased;and the Smad7 mRNA expression was significantly decreased.The protein expressions of TGF-β1,PDGF and α-SMA increased significantly(P<0.01).Compared with model group,different doses of CJYGX can reduce the concentrations of ALP,AST,ALT,and the hepatic fibrosis indexes(LN and HA,Ⅳ-C,PCⅢ) and HYP also were significantly lower.Masson staining show that CJYGX can significantly reduce the degree of hepatic fibrosis.Real-time PCR show that CJYGX can significantly down-regulate mRNA expressions of TGF-β1,Smad2,Smad3,Smad4 and up-regulate mRNA expression of Smad7.Immunohistochemical and Western blot analysis show that,compared with model group,CJYGX could significantly reduce the protein expressions of TGF-β1,PDGF and α-SMA(P<0.05,P<0.01).Conclusion:Traditional Chinese medicine compound CJYGX may protect TAA-induced hepatic fibrosis injury by interfering with TGF-β1/Smad signaling pathway.
Objective:To research the effect of traditional Chinese medicine compound Chaijie Yiganxian(CJYGX) on the hepatic fibrosis in rats and explore the mechanism.Method:Totally 60 SD rats were randomly divided into normal group,model group,positive group(Biejia Jianwan),and low,medium and high-dose CJYGX groups.Except for the normal group,the remaining five groups were involved in establishing the hepatic fibrosis model through the intraperitoneal injection of thioacetamide(TAA).Fourth weeks after modeling,the positive group was given Biejiajian pill(1 g·kg~(-1)·d-1),and high,medium and low-dose CJYGX groups were given CJYGX(7.96,3.98,1.99 g·kg~(-1)·d-1),respectively.The model group was given normal saline once a day for 5 weeks.24 h after the last intragastric administration with chloral hydrate,the rats were anesthetized slightly.Serum and liver tissues were collected.The liver wet weight was weighed electronically,and the liver index was calculated.Liver function indexes [alanine transaminase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP) ]and serum markers [laminin(LN),hyaluronidase(HA),Ⅳ collagen(CⅣ),procollagen Ⅲ(PCⅢ) ]of hepatic fibrosis were detected by enzyme linked immunosorbent assay(ELISA).The degree of hepatic fibrosis by Masson stain,mRNA expressions of transforming growth factor-β1(TGF-β1),Smad2,Smad3,Smad4,Smad7 were detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR),and protein expression levels of platelet derived growth factor(PDGF),TGF-β1 and α-smooth muscle actin(α-SMA)were detected by immunohistochemical or Western blot.Result:Compared with the blank group,the model group liver index was significantly increased;the contents of ALP,AST and ALT were significantly increased;the liver fibrosis indexes LN,HA,Ⅳ-C,PC Ⅲ and HYP were significantly increased;the degree of liver fibrosis was significantly increased in Masson tissue;the mRNA expressions of TGF-β1,Smad2,Smad3,Smad4 were significantly increased;and the Smad7 mRNA expression was significantly decreased.The protein expressions of TGF-β1,PDGF and α-SMA increased significantly(P<0.01).Compared with model group,different doses of CJYGX can reduce the concentrations of ALP,AST,ALT,and the hepatic fibrosis indexes(LN and HA,Ⅳ-C,PCⅢ) and HYP also were significantly lower.Masson staining show that CJYGX can significantly reduce the degree of hepatic fibrosis.Real-time PCR show that CJYGX can significantly down-regulate mRNA expressions of TGF-β1,Smad2,Smad3,Smad4 and up-regulate mRNA expression of Smad7.Immunohistochemical and Western blot analysis show that,compared with model group,CJYGX could significantly reduce the protein expressions of TGF-β1,PDGF and α-SMA(P<0.05,P<0.01).Conclusion:Traditional Chinese medicine compound CJYGX may protect TAA-induced hepatic fibrosis injury by interfering with TGF-β1/Smad signaling pathway.
引文
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[10]陈冠新,文彬,孙海涛,等.鳖甲煎丸对CCl_4致大鼠肝纤维化模型中NF-κB信号通路的影响[J].中国实验方剂学杂志,2018,24(10):161-167.
[11]高强,李旭光,樊莉,等.肝纤维化发病机制的研究进展[J].现代生物医学进展,2017,17(14):2780-2785.
[12]Thieringer F,Maass T,Czochra P,et al.Spontaneous hepatic fibrosis in transgenic mice over expressing PDGF-A[J].Gene,2008,423(1):23-28.
[13]Brew K,Nagase H.The tissue inhibitors of metalloproteinases(TIMPs):an ancient family with structural and functional diversity[J].BBA-Mol Cell Res,2010,1803(1):55-71.
[14]Wynn T A.Cellular and molecular mechanisms of fibrosis[J].J Pathol,2010,214(2):199-210.
[15]张家富,姜辉,高家荣,等.肝乐颗粒对肝纤维化大鼠TGF-β1/Smad信号通路的调控作用[J].中国实验方剂学杂志,2017,23(6):169-174.
[16]黄进,张晨,詹菲,等.黄芪多糖对肝纤维化大鼠TGF-β1/Smads信号通路的影响[J].中华中医药杂志,2015,30(6):2184-2186.
[17]魏珂,赵慧玲,田年秀,等.中医药治疗肝纤维化的研究进展[J].国际中医中药杂志,2014,36(11):1049-1053.
[2]Asrani S K,Larson J J,Yawn B,et al.Underestimation of liver-related mortality in the United States[J].Gastroenterology,2013,145(2):375-382.
[3]陈宵瑜,杨长青.肝纤维化发病机制研究新进展[J].实用肝脏病杂志,2016,19(1):121-124.
[4]Lieber C S.Prevention and treatment of liver fibrosis based on pathogenesis[J].Alcohol Clin Exp Res,2010,23(5):944-949.
[5]李光全,黄奕雪,尹萍,等.中药复方抗肝纤维化药理作用研究进展[J].中药与临床,2016,7(2):95-98.
[6]郑玉,姜莉云,李垚,等.自拟柴竭抑肝纤汤对慢性肝病患者肝纤维化指标的影响[J].云南中医中药杂志,2018,39(3):102.
[7]郭会君,张志毕,余晓玲,等.柴竭抑肝纤对大鼠肝星状细胞增殖和TGF-β1/Smad信号通路的影响[J].中国实验方剂学杂志,2018,24(13):100-104.
[8]刘煜,吕若芸,万博,等.硫代乙酰胺诱导肝纤维化模型及其血清学诊断数学模型的构建[J].药物生物技术,2009,16(6):554-558.
[9]张再康,邓国兴,郑玉光.鳖甲煎丸的临床和实验研究进展[J].中国中药杂志,2008,33(8):965-967.
[10]陈冠新,文彬,孙海涛,等.鳖甲煎丸对CCl_4致大鼠肝纤维化模型中NF-κB信号通路的影响[J].中国实验方剂学杂志,2018,24(10):161-167.
[11]高强,李旭光,樊莉,等.肝纤维化发病机制的研究进展[J].现代生物医学进展,2017,17(14):2780-2785.
[12]Thieringer F,Maass T,Czochra P,et al.Spontaneous hepatic fibrosis in transgenic mice over expressing PDGF-A[J].Gene,2008,423(1):23-28.
[13]Brew K,Nagase H.The tissue inhibitors of metalloproteinases(TIMPs):an ancient family with structural and functional diversity[J].BBA-Mol Cell Res,2010,1803(1):55-71.
[14]Wynn T A.Cellular and molecular mechanisms of fibrosis[J].J Pathol,2010,214(2):199-210.
[15]张家富,姜辉,高家荣,等.肝乐颗粒对肝纤维化大鼠TGF-β1/Smad信号通路的调控作用[J].中国实验方剂学杂志,2017,23(6):169-174.
[16]黄进,张晨,詹菲,等.黄芪多糖对肝纤维化大鼠TGF-β1/Smads信号通路的影响[J].中华中医药杂志,2015,30(6):2184-2186.
[17]魏珂,赵慧玲,田年秀,等.中医药治疗肝纤维化的研究进展[J].国际中医中药杂志,2014,36(11):1049-1053.