陆倩,张金娟,蒋先慧,国小利,廖尚高
LU Qian,ZHANG Jinjuan,JIANG Xianhui,GUO Xiaoli,LIAO Shanggao

• 1:贵州医科大学药学院
• 2:贵州医科大学基础医学院机能实验室
• 3:贵州医科大学民族药与中药开发应用教育部工程研究中心/贵州省药物制剂重点实验室

摘要(Abstract)：

目的 探讨木犀草素(Lut)、欧当归内酯A(Lev)和黄芪皂苷Ⅰ(Ast)单用及联用对人肝星状细胞株LX-2增殖、活化的影响。方法 取对数生长期的LX-2细胞，采用噻唑蓝(MTT)法考察Lut、Lev和Ast对转化生长因子-β1(TGF-β1)诱导的LX-2细胞增殖的影响、联用比例确定及Lut、Lev和Ast联用对TGF-β1诱导LX-2细胞增殖的影响；应用CalcuSyn软件计算Lut、Lev及Ast联用抑制LX-2细胞增殖的联合指数(CI),分析3者联用类型；通过蛋白免疫印迹法(Western blot)检测3者对LX-2细胞中α-平滑肌肌动蛋白(α-SMA)和TGF-β1蛋白表达的影响。结果 MTT结果显示，Lut、Lev和Ast联用摩尔比为1∶1∶10,3者单用及联用对TGF-β1诱导的LX-2细胞增殖均具有抑制作用(P<0.05),并呈浓度依赖性；CalcuSyn软件分析结果显示，3者联用对LX-2细胞的增殖抑制率>15%时CI<1,说明3者联用具有协同增效的作用；Western blot结果显示，Lut、Lev及Ast联用能够显著降低TGF-β1诱导的LX-2细胞中α-SMA蛋白的表达(P<0.01),降低LX-2细胞中TGF-β1蛋白的表达(P<0.05)。结论 Lut、Lev和Ast单用及联用均能抑制LX-2细胞的增殖、活化，3者联用作用更强、具有协同增效的作用，该作用可能与下调肝星状细胞TGF-β1蛋白表达有关。
Objective To investigate the synergistic effect of luteolin(Lut),levistilide A(Lev), and astragalosideⅠ(Ast) on the proliferation and activation of human hepatic stellate cell line LX-2 by single and combined administvation. Methods LX-2 cells in the logarithmic growth phase were evaluated by methyl thiazolyl tetrazolium(MTT) method for the effects of Lut, Lev, and Ast on the proliferation of LX-2 cells induced by transforming growth factor-β1(TGF-β1), determination of combination ratio, and combination of Lut, Lev, and Ast on the proliferation of LX-2 cells induced by TGF-β1. The combination indexes(CI) of Lut, Lev, and Ast to inhibit the proliferation of LX-2 cells were calculated by CalcuSyn software to determine the types of combined use of the three.The effects of them on the protein expression of α-smooth actin(α-SMA) and TGF-β1 in LX-2 cells were evaluated by Western blot. Results The results of MTT experiments showed that the molar ratio of Lut, Lev, and Ast in combination was 1 ∶1 ∶10 and the single use of the and in combination of Lut, Lev, and Ast had inhibitory effects on the proliferation of LX-2 cells induced by TGF-β1(P<0.05) in a concentration-dependent manner; CalcuSyn software analysis results displayed CI<1 when the cell proliferation inhibition rate of LX-2 cells induced by TGF-β1 was more than 15%, indicating that combination of the three compounds was more synergistic and effective. Western blot results showed that the combined use of Lut, Lev, and Ast significantly reduced the expression of α-SMA protein in LX-2 cells induced by TGF-β1(P<0.01) and the expression of TGF-β1 protein in LX-2 cells(P<0.05). Conclusion Although the single use of each of Lut, Lev, and Ast can inhibit the proliferation and activation of LX-2 cells, the combination use of the three has stronger and more synergistic clinical result. It may be related to the down-regulation of TGF-β1 protein expressions in hepatic stellate cells.

关键词(KeyWords)： 木犀草素;欧当归内酯A;黄芪皂苷Ⅰ;LX-2细胞;联合指数;协同作用
luteolin;levistilide A;astragaloside Ⅰ;LX-2 cells;combination index;synergistic effect

Abstract:

Keywords:

基金项目(Foundation): 贵州省高层次创新型人才百层次人才项目(黔科合平台人才[2020]6011);; 贵州省中药、民族药活性物质筛选新技术开发应用创新人才团队项目(黔科合平台人才[2020]5006)

作者(Author): 陆倩,张金娟,蒋先慧,国小利,廖尚高
LU Qian,ZHANG Jinjuan,JIANG Xianhui,GUO Xiaoli,LIAO Shanggao

DOI: 10.19367/j.cnki.2096-8388.2024.02.006

参考文献(References)：

• [1] ZHU J,ZHANG Z Q,ZHANG Y T,et al.MicroRNA-212 activates hepatic stellate cells and promotes liver fibrosis via targeting SMAD7[J].Biochemical and Biophysical Research Communications,2018,496(1):176-183.
• [2] ARROYO N,VILLAMAYOR L ,DíAZ I,et al.GATA4 induces liver fibrosis regression by deactivating hepatic stellate cells[J].The American Society for Clinical Investigation,2021,6(23):174-192.
• [3] LEE Y A,WALLACE M C,FRIEDMAN S L.Pathobiology of liver fibrosis:a translational success story [J].Gut,2015,64(8):830-841.
• [4] 徐诗雨，郭惠婕，刘揭，等.肝纤维化治疗靶标研究进展[J].医学综述，2020,26(10):1930-1934.
• [5] LI H.Advances in anti hepatic fibrotic therapy with traditional chinese medicine herbal formula[J].Journal of Ethnopharmacology,2020,251(8):1124-1142.
• [6] AYAH M H,GOWIFE L,MONA G,et al.Combination of pomegranateextract and curcumin ameliorates thioacetamide-induced liver fibrosis in rats:impact on TGF-β/Smad3 and NF-κB signaling pathways[J].Toxicology Mechanisms and Methods,2020,30(8):620-633.
• [7] 韦凌霞，丁茂鹏，王志旺，等.基于数据挖掘及网络药理学方法探讨含当归中药复方抗肝纤维化的用药规律及作用机制[J].中药新药与临床药理，2020,31(11):1323-1331.
• [8] 符燕青，农小欣，苏晓文，等.中医药抗肝纤维化机制研究进展[J].河南中医，2021,41(8):1256-1261.
• [9] 范月莹，张洪，冯豆，等.苦豆碱对活化的LX-2细胞增殖及凋亡的影响[J].中南药学，2021,19(3):438-443.
• [10]GUO T,LIU Z L,ZHAO Q,et al.A combination of astragaloside I,levistilide A and calycosin exerts anti-liver fibrosis effects in vitro and in vivo[J].Acta Pharmacologica Sinica,2018,39(1):1483-1492.
• [11]ROBERTO G,STASI C.Recent advancements in diagnosis and therapy of liver cirrhosis[J].Current Drug Targets,2016,17(15):1804-1817.
• [12]COLLABORATORS G.Global,regional,and national comparative risk assessment of 84 behavioural,environmental and occupational,and metabolic risks or clusters of risks for 195 countries and territories,1990-2017:a systematic analysis for the Global Burden of Disease Study 2017[J].Lancet,2018,392(10):1923-1994.
• [13]SHAYJESSICA E S,HAMILTONJAMES P.Hepatic fibrosis:avenues of investigation and clinical implications[J].Clinics in Liver Disease,2018,11(5):111-114.
• [14]莫婵.保肝宁和人参皂苷Rg1对肝纤维化小鼠IDO1及树突状细胞的作用机制研究[D].广州：南方医科大学，2021.
• [15]范月莹，张洪，等.苦豆碱对活化的LX-2细胞增殖及凋亡的影响[J].中南药学，2021,19(3):438-443.
• [16]XIANG D J,ZOU J,ZHU X Y,et al.Physalin D attenuates hepatic stellate cell activation and liver fibrosis by blocking TGF-β/Smad and YAP signaling[J].Phytomedicine,2020,78:153-154.
• [17]HASSOUNI E,MANTINI B,PETRI G,et al.To combine or not combine:drug interactions and tools for their analysis.Reflections from the eortc-pamm course on preclinical and earlyphase clinical pharmacology[J].Anticancer Research,2019,39:3303-3309.
• [18]朱正文，蒋晴，王平，等.基于中效方程的黄芩苷与汉黄芩苷调控NF-κB信号通路的协同作用[J].中国实验方剂学杂志，2020,26(21):84-89.
• [19]王文斌，任飞桦，胡少洋，等.二甲双胍与二氯乙酸盐对MYCN扩增神经母细胞瘤BE-2C细胞的协同抗肿瘤作用[J].中国药理学通报，2019,35(9):1256-1261.
• [20]黄甫静，张金娟，董莉，等.黄水枝醇提物对CCl4致小鼠肝纤维化的改善作用及其机制初探[J].中国药房，2021,32(14):1685-1691.
• [21]NAKANO Y,KAMIYA A,SUMIYOSHI H,et al.A deactivation factor of fibrogenic hepatic stellate cells induces regression of liver fibrosis in mice[J].Hepatology,2020,71(4):1437-1452.
• [22]ZHONG F,JIANG Y,XIANG C,et al.Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis[J].Theanostics,2020,11(7):361-378.

文章评论(Comment)：