香紫苏醇通过调控TGF-β/Smad信号通路抑制肝星状细胞活化及肝纤维化  

Sclareol Inhibits Hepatic Stellate Cell Activation and Liver Fibrosis by Regulating the TGF-β/Smad Signaling Pathway

作　　者：宋安宁 张甜甜 郑珊珊 宋杨璐 郑志勇 舒广文[1] 黎炎梅[2] 邓旭坤[1] SONG Anning;ZHANG Tiantian;ZHENG Shanshan;SONG Yanglu;ZHENG Zhiyong;SHU Guangwen;LI Yanmei;DENG Xukun(National Experimental Teaching Demonstration Center of Ethnic Pharmacy,School of Pharmacy,South Central Minzu University,Wuhan 430074,China;Hubei Provincial Center for Disease Control and Prevention/Hubei Provincial Key Laboratory of Applied Toxicology Wuhan 430070,China)

机构地区：[1]中南民族大学药学院/民族药学国家级实验教学示范中心,武汉430074 [2]湖北省疾病预防控制中心/应用毒理湖北省重点实验室,武汉430070

出　　处：《世界科学技术-中医药现代化》2024年第12期3136-3144,共9页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：江西赣江海智人才计划(GHZ22018):负责人:邓旭坤;江西省“双千计划”科技创新高端人才(青年)项目(JXSQ2019201105):熊胆产品基础研究与产业升级研究,负责人:任永申;中南民族大学2019中央科研业务费(自然科学)(CZD19007):巴东县中医药产业发展调研与精准扶贫,负责人:邓旭坤。

摘　　要：目的观察香紫苏醇(Sclareol,SCL)对LX-2肝星状细胞活化及CCl_(4)诱导的小鼠肝纤维化的影响,并进一步探索其作用机制。方法取KM小鼠40只,随机分为正常组、模型组(10%CCl_(4))和SCL给药组以及水飞蓟宾阳性对照组(10%CCl_(4)+100 mg·kg^(-1)Silybin),SCL给药组分为SCL低(10%CCl_(4)+20 mg·kg^(-1)SCL)、高剂量组(10%CCl_(4)+40 mg·kg^(-1)SCL)。除正常组以外,其余各组小鼠每周3次腹腔注射10%橄榄油稀释的CCl_(4),持续4周。从第3周开始,给药组每天灌胃给予不同剂量的SCL,阳性对照组每天灌胃水飞蓟宾,4周结束后处死小鼠并采集血清和肝脏组织。整体动物实验采用生化试剂盒检测肝纤维化小鼠血清中谷氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)水平的变化;利用苏木精-伊红(HE)、天狼星红(Sirius red)和Masson染色法检测显微结构变化和肝脏组织胶原沉积情况;采取免疫组化法检测肝脏组织中纤维化标志蛋白α-平滑肌肌动蛋白(α-SMA)和纤维状胶原蛋白Ⅰ(CollagenⅠ)的表达情况。体外实验采用LX-2人肝星状细胞,正常组正常培养,模型组采用转化生长因子-β1(TGF-β1)诱导LX-2肝星状细胞活化,SCL给药组分为SCL低剂量组(5 ng·mL^(-1)TGF-β1+10μmol·L^(-1)SCL)、高剂量组(5 ng·mL^(-1)TGF-β1+20μmol·L^(-1)SCL)。随后以Transwell和EdU法检测SCL对LX-2细胞的迁移与增殖能力变化的影响;通过免疫荧光法检测SCL影响细胞中纤维化标志蛋白α-SMA和CollagenⅠ的表达情况;采用免疫印迹法检测细胞中TGF-β/Smad通路中相关蛋白的表达情况。结果动物实验中,与模型组相比,SCL能显著改善肝纤维化模型小鼠的各项肝功能指标与肝脏组织病理学变化。体外细胞实验中,与模型组相比,SCL能有效抑制肝星状细胞的迁移与增殖从而抑制其活化,进一步研究发现,与模型组相比,SCL显著上调Smad7蛋白表达,同时显著下调了Smad2、Smad3蛋白的磷酸化水平。结论SCLObjective To investigate the effect of Sclareol(SCL)on LX-2 hepatic stellate cell activation and CCl_(4)-induced liver fibrosis in mice,and to further explore its mechanism.Methods A total of 40 Kunming mice were randomly divided into healthy group,model group(10%CCl_(4))and SCL administration group,and silybin positive control group(10%CCl_(4)+100 mg·kg^(-1)Silybin),and SCL administration group was divided into low SCL(10%CCl_(4)+20 mg·kg^(-1)SCL)and high dose group(10%CCl_(4)+40 mg·kg^(-1)SCL).Mice in all groups were intraperitoneally injected with 10% olive oil-diluted CCl_(4) three times a week for four weeks,except for the healthy group.Starting from the third week,the dosing group was given different doses of SCL by gavage daily,and the positive control group was given silybin daily,and the mice were sacrificed and serum and liver tissue were collected after four weeks.In whole animal experiments,biochemical kits were used to detect the changes in the serum levels of glutamate aminotransferase(ALT)and aspartate aminotransferase(AST)in mice with liver fibrosis.Hematoxylin-eosin(HE),Sirius Red and Masson staining were used to detect microstructural changes and collagen deposition in liver tissues.Immunohistochemistry was used to detect the expression of fibrosis marker proteins α-smooth muscle actin(α-SMA)and fibrous collagen I.in liver tissues.In vitro,LX-2 human hepatic stellate cells were used for normal culture in the blank group,and the activation of LX-2 hepatic stellate cells was induced by transforming growth factor-β1(TGF-β1)in the model group,and the SCL administration group was divided into SCL low-dose group(5 ng·mL^(-1)TGF-β1+10μmol·L^(-1)SCL)and high-dose group(5 ng·mL^(-1)TGF-β1+20μmol·L^(-1)SCL).Subsequently,Transwell and EdU assays were used to detect the effects of SCL on the migration and proliferation of LX-2 cells.The expression of fibrosis marker proteinsα-SMA and Collagen I.affected by SCL was detected by immunofluorescence.Western blot was used to detect the expression

关 键 词：香紫苏醇 肝纤维化 LX-2肝星状细胞活化 TGF-β/Smad通路 

分 类 号：R285.5[医药卫生—中药学]