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中国应用生理学杂志 ›› 2020, Vol. 36 ›› Issue (6): 576-581.doi: 10.12047/j.cjap.6010.2020.121

• 研究论文 • 上一篇    下一篇

柔木丹改善小鼠肝纤维化的TGF-β1/Smad4信号通路机制*

李倩1, 闫曙光2, 惠毅2, 李京涛3, 魏海梁3, 张红1,2△   

  1. 1.陕西中医药大学医学科研实验中心, 咸阳 712046;
    2.陕西中医药大学基础医学院, 咸阳 712046;
    3.陕西中医药大学附属医院, 咸阳 712000
  • 收稿日期:2020-01-19 修回日期:2020-11-25 出版日期:2020-11-28 发布日期:2021-03-15
  • 通讯作者: Tel: (029)38183453; E-mail: zhangh1227@163.com
  • 基金资助:
    *陕西省自然科学基础研究计划(2020JQ-864);陕西省教育厅科学研究项目(17JK0216);陕西省中药基础与新药研究重点实验室开放基金项目(2017KF07);陕西中医药大学学科创新团队建设项目(2019-YL05);陕西中医药大学科研基金项目(2016QN30)

Roumudan formulation attenuated liver fibrosis by inhibiting TGF-β1/Smad4 pathway in mice

LI Qian1, YAN Shu-guang2, HUI Yi2, LI Jing-tao3, WEI Hai-liang3, ZHANG Hong1,2△   

  1. 1. Medical Experiment Center, Shaanxi University of Chinese Medicine, Xianyang 712046;
    2. Department of Pathophysiology, Shaanxi University of Chinese Medicine, Xianyang 712046;
    3. Affiliated Hospital of Shaanxi University of Chinese Medicine, Xianyang 712000, China
  • Received:2020-01-19 Revised:2020-11-25 Online:2020-11-28 Published:2021-03-15

摘要: 目的:探讨柔木丹(RMD)对改善CCl4诱导的小鼠肝纤维化的TGF-β1/果蝇抗生物皮肤生长因子蛋白家族4号因子(Smad4)信号通路机制。方法:雄性BALB/c小鼠随机分为空白对照组、模型组、RMD治疗组(n=11)。腹腔注射CCl4诱导小鼠肝纤维化模型,模型及RMD治疗组小鼠腹腔注射20 % CCl4(CCl4∶橄榄油=1∶4),注射量为2.5 ml/kg,空白对照组以同样方法注射等量橄榄油,每周2次;第2周起调整模型及RMD治疗组小鼠CCl4腹腔注射量为5 ml/kg(空白对照组注射等量橄榄油),每周2次。成模后,RMD治疗组小鼠使用RMD灌胃给药(6.2 g/(kg·d);空白对照组、模型组使用等量的水灌胃),模型及RMD治疗组小鼠继续腹腔注射20 % CCl4,注射量为1.5 ml/kg(空白对照组注射等量橄榄油),每周1次,持续3 周。采取各组小鼠血清样本检测谷丙转氨酶(ALT)、谷草转氨酶(AST)活性;采取各组小鼠肝组织样本使用HE、Masson、原位杂交、免疫组织化学染色、Western blot、Q-PCR等方法进行检测。结果:与正常组相比,CCl4造模5 周后,模型组小鼠肝脏纤维化病理特征明显。与模型组相比,RMD治疗3 周,治疗组小鼠肝组织病理学改变减轻,小鼠的肝脏指数(P<0.01)、血清中的ALT(P< 0.01)、AST(P<0.01)活性、肝组织中羟脯氨酸的含量(P<0.05)均降低;Ⅰ型胶原(Collagen Ⅰ,P<0.01)、Ⅲ型胶原(Collagen Ⅲ,P<0.01)表达减少,胶原沉积减少;肝组织中TGF-β1(P<0.05)和α-SMA(P<0.05)表达均降低;肝组织中Smad4阳性表达区域缩小、表达强度降低。结论:RMD通过抑制TGF-β1/Smad4通路信号转导,减少胶原沉积,进而发挥抗小鼠肝纤维化的作用。

关键词: 纤维化, 肝, 柔木丹, 四氯化碳, 小鼠

Abstract: Objective: To investigate underlying mechanism involving Roumudan(RMD) formulation (Z20160012) suppressed liver fibrosis induced by CCl4 injection in mice by inhibiting TGF-β1/Smad4 pathway. Methods: Male BALB/c mice were randomly divided into control group, liver fibrosis model group and RMD-treated group(n=11). Mice in liver fibrosis model and RMD-treaded groups were injected intraperitoneally with CCl4 (20% in olive oil) at the dose of 2.5 mL/kg two times for one week and 5 mL/kg two times for 4 weeks. Mice in control group were treated intraperitoneally with the same volume of olive oil at the same time intervals. From sixth week, Mice in liver fibrosis model group were administrated with CCl4 (20% in olive oil, 1.5 ml/kg once per week) intraperitoneally and given distilled water by intragastric gavage. Mice in the RMD-treated group were administrated with CCl4 (20% in olive oil, 1.5 ml/kg/mouse once per week) intraperitoneally and given RMD(6.2 g/kg everyday) by intragastric gavage. Mice in the control group were administrated with olive oil (1.5 ml/kg/mouse once per week) intraperitoneally and given distilled water by intragastric gavage. The serum AST and ALT levels were estimated for assessment of liver function. The pathologic changes of mice' livers were examined by the HE, Masson, immunohistochemical staining, Western Blot, Q-PCR and so on. Results: After intraperitoneally injected with CCl4 in mice, the pathological characteristics of liver fibrosis were observed compared with the control group at the sixth week. Compared with the liver fibrosis model group, RMD improved the liver function significantly through reducing liver index(P<0.01) and the levels of ALT(P<0.01), AST(P<0.01) and HYP(P<0.05). The expression of TGF-β1(P<0.05), α-SMA(P<0.05), COL1A1(P<0.01) and COL3A1(P<0.01) were decreased by RMD. The positive expression area of Smad4 mRNA in RMD treated group was lower than that in liver fibrosis model group. Conclusion: The RMD formulation could attenuate liver fibrosis by inhibiting TGF-β1/Smad4 pathway and extracellular matrix (ECM) production in mice.

Key words: fibrosis, liver, roumudan, carbon tetrachloride, mice

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