扶脾柔肝方对肝纤维化大鼠转化生长因子-β1/Smad信号通路的影响
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摘要
目的探讨扶脾柔肝方对肝纤维化(HF)大鼠转化生长因子-β1(TGF-β1)/Smad信号通路的干预作用。方法将SD大鼠随机分为7组:正常对照组,模型组,秋水仙碱组(0.2 mg·kg~(-1)),扶正化瘀组(0.415 g·kg~(-1)),扶脾柔肝方低、中、高剂量组(20,40,80 g·kg~(-1))。采用大鼠背部皮下注射(sc)50%四氯化碳和灌胃(ig)30%乙醇的方法诱导HF,造模10周,成功建立大鼠HF模型,其后给予相应药物10 m L·kg~(-1),每日1次,连续4周,正常对照组、模型组给予等量生理盐水。检测大鼠血清TGF-β1;HE染色和Masson染色观察肝组织病理学变化;免疫组化法检测肝组织中α-平滑肌肌动蛋白(α-SMA)的表达;同时,采用荧光定量PCR方法及免疫印迹法(Western blot)检测肝组织中TGF-β1、Smad 3、Smad 4、Smad7 mRNA及蛋白的表达。结果模型组的HF程度较其余组明显严重,与正常对照组比较,模型组大鼠血清TGF-β1明显升高(P<0.01),肝组织α-SMA蛋白及TGF-β1、Smad3、Smad4 mRNA及蛋白表达升高Smad 7表达降低(P<0.01);与模型组比较,扶脾柔肝方各组大鼠血清TGF-β1明显降低(P<0.05,P<0.01),肝组织α-SMA蛋白及TGF-β1、Smad3、Smad4 mRNA及蛋白表达下调(P<0.01),Smad 7 mRNA及蛋白表达升高(P<0.05,P<0.01)。结论扶脾柔肝方下调HF肝组织α-SMA蛋白及TGF-β1、Smad3、Smad4 mRNA及蛋白表达水平,上调Smad7 mRNA及蛋白表达水平,可能是其抗HF作用机制之一。
Objective To explore the intervention of Fupi Rougan formula on transforming growth factor-β1(TGF-β1)/Smads signal pathway in rats with hepatic fibrosis(HF). Methods SD rats were randomly divided into 7 groups:normal group,model group and colchicine group(0.2 mg·kg~(-1)),the fuzheng huayu group(0.415 g·kg~(-1)),Fupi Rougan formula group low, medium and high dose groups(20,40,80 g·kg~(-1)). Liver fibrosis models were successfully established by subcutaneously injection in back with 50% carbon tetrachloride accompanied with gavage with alcohol for 10 weeks. Then medicines(10 mL·kg~(-1))were given accordingly once a day(qd)for four weeks,the normal group and model group were given normal saline(NS). Rats' serum TGF-β1 was detected by ELISA;liver tissue pathological change was observed by HE staining and Masson staining under microscope;detection of α smooth muscle actin(α-SMA) expression in liver tissue was carried out by immunohistochemical method. Fluorescence quantitative polymerase chain reaction(q PCR) and Western Blot were used to detect mRNA levels and protein expression of TGF-β1,Smad3,Smad4,and Smad7 in liver tissue. Results Degrees of hepatic fibrosis in model group are obviously more serious than that in other groups; serum TGF-β1 in model group increased significantly compared with normal group(P < 0.01), α-SMA protein and TGF-β1, Smad3 and Smad4 mRNA and protein expression in liver tissue increased,too(P < 0.01);while the expression of Smad7 reduced. On the other hand,the TGF-β1 in the Fupi Rougan formula groups were obviously lower than that in model group(P < 0.05), α-SMA protein and TGF-β1,Smad3 and Smad4 mRNA and protein expression in liver tissue were lowered at the same time(P < 0.05), while the expression of Smad7 mRNA and protein increased. Conclusion Fupi Rougan formula down-regulated α-SMA protein and TGF-β1, Smad3, Smad4 mRNA and protein expression levels, meanwhile up-regulated Smad7 mRNA and protein expression level in the liver tissue. This may be involved in its mechanism of the anti-hepatic fibrosis effects.
Objective To explore the intervention of Fupi Rougan formula on transforming growth factor-β1(TGF-β1)/Smads signal pathway in rats with hepatic fibrosis(HF). Methods SD rats were randomly divided into 7 groups:normal group,model group and colchicine group(0.2 mg·kg~(-1)),the fuzheng huayu group(0.415 g·kg~(-1)),Fupi Rougan formula group low, medium and high dose groups(20,40,80 g·kg~(-1)). Liver fibrosis models were successfully established by subcutaneously injection in back with 50% carbon tetrachloride accompanied with gavage with alcohol for 10 weeks. Then medicines(10 mL·kg~(-1))were given accordingly once a day(qd)for four weeks,the normal group and model group were given normal saline(NS). Rats' serum TGF-β1 was detected by ELISA;liver tissue pathological change was observed by HE staining and Masson staining under microscope;detection of α smooth muscle actin(α-SMA) expression in liver tissue was carried out by immunohistochemical method. Fluorescence quantitative polymerase chain reaction(q PCR) and Western Blot were used to detect mRNA levels and protein expression of TGF-β1,Smad3,Smad4,and Smad7 in liver tissue. Results Degrees of hepatic fibrosis in model group are obviously more serious than that in other groups; serum TGF-β1 in model group increased significantly compared with normal group(P < 0.01), α-SMA protein and TGF-β1, Smad3 and Smad4 mRNA and protein expression in liver tissue increased,too(P < 0.01);while the expression of Smad7 reduced. On the other hand,the TGF-β1 in the Fupi Rougan formula groups were obviously lower than that in model group(P < 0.05), α-SMA protein and TGF-β1,Smad3 and Smad4 mRNA and protein expression in liver tissue were lowered at the same time(P < 0.05), while the expression of Smad7 mRNA and protein increased. Conclusion Fupi Rougan formula down-regulated α-SMA protein and TGF-β1, Smad3, Smad4 mRNA and protein expression levels, meanwhile up-regulated Smad7 mRNA and protein expression level in the liver tissue. This may be involved in its mechanism of the anti-hepatic fibrosis effects.
引文
[1]赵志敏,刘成海.中医药治疗肝纤维化研究进展[J].实用肝脏病杂志,2016,19(1):12-15.
[2]ROBERTS AB,PIEK E,BOTTINGER EP,et al.Is Smad 3 and smad 7 amajor player in signal transducition pathways leading to fibrogenesis?[J].Chest,2013,120(suppl 1):S43-S47.
[3]孙定隆.自拟扶脾柔肝汤治疗肝硬化33例小结[J].贵阳中医学院学报,1998,20(2):21-22.
[4]黄丹,孙定隆,王敏,等.扶脾柔肝汤治疗肝硬化40例临床报道[J].贵阳中医学院学报,2015,37(5):51-53.
[5]安祯祥,何远利,王敏.芒硝外敷联合扶脾柔肝颗粒内服治疗肝硬化腹水的临床研究[J].辽宁中医杂志,2016,43(9):1900-1902.
[6]安祯祥,何远利,王敏.扶脾柔肝方配方颗粒对肝纤维化大鼠肝组织诱导型一氧化氮合酶表达的影响[J].中国实验方剂学杂志,2016,22(18):100-105.
[7]安祯祥,何远利,王敏.四氯化碳不同给药途径复合乙醇诱导大鼠肝纤维化模型的研究[J].贵州医药,2016,40(1):6-8.
[8]中华医学会传染病与寄生虫病学分会、肝病学分会.病毒性肝炎防治方案[J].中华肝脏病杂志,2000,8(6):324-329.
[9]陈晗,杨碧伟,袁满,等.扶正化瘀胶囊对肝纤维化大鼠的防治作用及对结缔组织生长因子表达的影响[J].四川大学学报(医学版),2016,47(2):197-202.
[10]INAGAKI Y,OKAZAKI I.Emerging insights into transforming growth factor beta Smad signal in hepatic fibrogenesis[J].Gut,2007,56:284-292.
[11]DERYNCK R,ZHANG Y E.Smad-dependent and Smadindependent pathways in TGF-beta family signaling[J].Nature,2003,425(6958):577-584.
[12]吴芙蓉,姜玲,何晓丽,等.橙皮苷对肝星状细胞TGF-β1/Smad信号通路的影响[J].中国中药杂志,2015,40(13):2639-2643.
[13]李刚,龚全权.肝纤维化信号传导通路研究进展[J].广东医学,2014,35(3):453-455.
[14]CUTRONEO KR.TGF-beta-induced fibrosis and SMAD signaling:oligo decoys as natural therapeutics for inhibition of tissue fibrosis and scarring[J].Wound Repair Regen,2007,15(Suppl 1):S54-S60.
[15]MAS N,TASCI I,COMERT B,et al.Ursodeoxycholic acid treatment improves hepatocyte ultrastructure in rat liver fibrosis[J].World J Gastroenterol,2008,14(7):1108.
[16]秦致中,冷希圣,李涛,等.白细胞介素-10对与枯否细胞共培养的肝星状细胞表达转移生长因子-β和血小板源生长因子的影响[J].中华实验外科杂志,2004,21:666-668.
[17]CARPINO G,MORINI S,GINANNI S C,et al.Alpha-SMA expression in he-patic stellate cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis after liver transplantation[J].Dig Liver Dis,2005,35:349-356.
[18]郑亚江,周振华,任朦,等.补肾柔肝方对肝纤维化大鼠肝组织病理学以及α-SMA蛋白表达的影响[J].现代中西医结合杂志,2014,23(34):3770-3773.
[19]慕永平,刘平.中西医结合抗肝纤维化的研究思路与方法[J].上海医药,2016,37(13):8-12.
[2]ROBERTS AB,PIEK E,BOTTINGER EP,et al.Is Smad 3 and smad 7 amajor player in signal transducition pathways leading to fibrogenesis?[J].Chest,2013,120(suppl 1):S43-S47.
[3]孙定隆.自拟扶脾柔肝汤治疗肝硬化33例小结[J].贵阳中医学院学报,1998,20(2):21-22.
[4]黄丹,孙定隆,王敏,等.扶脾柔肝汤治疗肝硬化40例临床报道[J].贵阳中医学院学报,2015,37(5):51-53.
[5]安祯祥,何远利,王敏.芒硝外敷联合扶脾柔肝颗粒内服治疗肝硬化腹水的临床研究[J].辽宁中医杂志,2016,43(9):1900-1902.
[6]安祯祥,何远利,王敏.扶脾柔肝方配方颗粒对肝纤维化大鼠肝组织诱导型一氧化氮合酶表达的影响[J].中国实验方剂学杂志,2016,22(18):100-105.
[7]安祯祥,何远利,王敏.四氯化碳不同给药途径复合乙醇诱导大鼠肝纤维化模型的研究[J].贵州医药,2016,40(1):6-8.
[8]中华医学会传染病与寄生虫病学分会、肝病学分会.病毒性肝炎防治方案[J].中华肝脏病杂志,2000,8(6):324-329.
[9]陈晗,杨碧伟,袁满,等.扶正化瘀胶囊对肝纤维化大鼠的防治作用及对结缔组织生长因子表达的影响[J].四川大学学报(医学版),2016,47(2):197-202.
[10]INAGAKI Y,OKAZAKI I.Emerging insights into transforming growth factor beta Smad signal in hepatic fibrogenesis[J].Gut,2007,56:284-292.
[11]DERYNCK R,ZHANG Y E.Smad-dependent and Smadindependent pathways in TGF-beta family signaling[J].Nature,2003,425(6958):577-584.
[12]吴芙蓉,姜玲,何晓丽,等.橙皮苷对肝星状细胞TGF-β1/Smad信号通路的影响[J].中国中药杂志,2015,40(13):2639-2643.
[13]李刚,龚全权.肝纤维化信号传导通路研究进展[J].广东医学,2014,35(3):453-455.
[14]CUTRONEO KR.TGF-beta-induced fibrosis and SMAD signaling:oligo decoys as natural therapeutics for inhibition of tissue fibrosis and scarring[J].Wound Repair Regen,2007,15(Suppl 1):S54-S60.
[15]MAS N,TASCI I,COMERT B,et al.Ursodeoxycholic acid treatment improves hepatocyte ultrastructure in rat liver fibrosis[J].World J Gastroenterol,2008,14(7):1108.
[16]秦致中,冷希圣,李涛,等.白细胞介素-10对与枯否细胞共培养的肝星状细胞表达转移生长因子-β和血小板源生长因子的影响[J].中华实验外科杂志,2004,21:666-668.
[17]CARPINO G,MORINI S,GINANNI S C,et al.Alpha-SMA expression in he-patic stellate cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis after liver transplantation[J].Dig Liver Dis,2005,35:349-356.
[18]郑亚江,周振华,任朦,等.补肾柔肝方对肝纤维化大鼠肝组织病理学以及α-SMA蛋白表达的影响[J].现代中西医结合杂志,2014,23(34):3770-3773.
[19]慕永平,刘平.中西医结合抗肝纤维化的研究思路与方法[J].上海医药,2016,37(13):8-12.
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