胡黄连苷Ⅰ、Ⅱ通过调节C/EBP-PPARγ通路抑制3T3-L1脂肪前体细胞的分化和脂肪合成  

Picroside Ⅰ and Ⅱ inhibit differentiation and lipid synthesis of 3T3-L1 preadipocytes by regulating C/EBP-PPARγpathway

作　　者：常华杰 勾文峰 郭江红 许飞飞 侯文彬 李祎亮[2] CHANG Huajie;GOU Wenfeng;GUO Jianghong;XU Feifei;HOU Wenbin;LI Yiliang(Tianjin University of Traditional Chinese Medicine,Tianjin 301617,China;Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine,Institute of Radiation Medicine,Chinese Academy of Medical Sciences,Tianjin 300192,China)

机构地区：[1]天津中医药大学,天津301617 [2]中国医学科学院放射医学研究所,天津放射医学与分子核医学重点实验室,天津300192

出　　处：《药物评价研究》2023年第6期1193-1200,共8页Drug Evaluation Research

基　　金：中央高校基本科研业务费专项资金资助项目(3332022063);国家自然科学基金资助项目(82202950);国家自然科学基金资助项目(82104012);中国医学科学院医学与健康科技创新工程重大协同创新项目资助(2021-I2M-1-042)。

摘　　要：目的探讨胡黄连主要有效成分胡黄连苷I、II对3T3-L1前脂肪细胞成脂分化的影响。方法采用MTT法检测胡黄连苷Ⅰ、Ⅱ对3T3-L1细胞活力的影响,确定给药浓度;使用诱导分化培养基诱导3T3-L1细胞分化为成熟脂肪细胞,通过油红O染色法观察胡黄连苷I、II(20、40μmol·L^(-1))对细胞脂肪蓄积的影响;实时荧光定量PCR(qRT-PCR)法检测3T3-L1细胞脂肪合成相关的乙酰辅酶A羧化酶1(ACACA)、脂肪酸合酶(FASN)、硬脂酰辅酶A去饱和酶(SCD1)、脂肪酸结合蛋白(FABP4)和调节脂肪合成的转录因子过氧化物酶体增殖物激活受体γ(PPARγ)、固醇调节元件结合蛋白1(SREBP1)的mRNA表达水平;Western blotting实验检测CCAAT/增强子结合蛋白β(C/EBPβ)、SCD1和PPARγ的蛋白表达。结果浓度低于50μmol·L^(-1)的胡黄连苷Ⅰ、Ⅱ处理不会影响细胞的存活率;与对照组比较,模型组在诱导分化后细胞形态变圆,油红O染色显示细胞中有大量脂肪蓄积(P<0.01);与模型组比较,胡黄连苷Ⅰ、Ⅱ显著减少了脂肪蓄积(P<0.01)。与对照组比较,模型组ACACA、FASN、SCD1、FABP4、PPARγ和SREBP1的mRNA表达均显著上调(P<0.05、0.01);与模型组比较,胡黄连苷Ⅰ、Ⅱ20、40μmol·L^(-1)均显著降低了ACACA、SCD1、FASN、FABP4、SREBP1 mRNA的表达(P<0.05、0.01),胡黄连苷Ⅰ、Ⅱ仅在40μmol·L^(-1)时显著抑制SREBP1 mRNA的表达(P<0.05、0.01)。Western blotting结果显示,与对照组比较,模型组PPARγ、C/EBPβ和SCD1的蛋白表达显著上调(P<0.01);与模型组比较,胡黄连苷I、II各浓度均显著降低了SCD1蛋白的表达(P<0.01),胡黄连苷I 40μmol·L^(-1)和胡黄连苷II 20、40μmol·L^(-1)组PPARγ、C/EBPβ蛋白的表达显著降低(P<0.05、0.01)。结论胡黄连苷Ⅰ、Ⅱ均可以显著抑制3T3-L1细胞的脂肪分化,并减少脂肪蓄积,胡黄连苷I表现出更好的剂量相关性,其作用机制可能与抑制C/EBPβ-PPARγ通路有关。Objective To investigate the effects of picrosideⅠandⅡ,the main active components of Picrorhiza scrophulariiflora,on adipogenic differentiation of 3T3-L1 preadipocytes.Methods MTT assay was used to detect the effect of picrosideⅠandⅡon the viability of 3T3-L1 preadipocytes,and to determine the concentration of drug administered.3T3-L1 preadipocytes were induced to differentiate into mature adipocytes by differentiation medium.The effects of picrosideⅠand picrosideⅡ(20 and 40μmol·L^(-1))on lipid accumulation were observed by oil red O staining.Real-time quantitative PCR(qRT-PCR)was used to detect the expression of Acetyl-CoA carboxylase 1(ACACA),fatty acid synthase(FASN),Stearoyl-CoA desaturase(SCD1),fatty acid binding protein(FABP4),peroxisome proliferator-activated receptor gamma(PPARγ),and sterol regulatory element binding protein 1(SREBP1)mRNA expression levels in 3T3-L1 preadipocytes.Western blotting assay was used to detected the protein expression of CCAAT/enhancer binding proteinβ(C/EBPβ),SCD1,and PPARγ.Results The treatment of picrosideⅠandⅡwith concentration below 50μmol·L^(-1)does not affect the cell survival rate.Compared with control group,the vast majority of 3T3-L1 preadipocytes in the model group became circular after induced differentiation,and oil red O staining showed that there was a large amount of lipid accumulation in the cells(P<0.01).Compared with model group,lipid accumulation was significantly reduced in the picrosideⅠandⅡadministration groups.Compared with the control group,the mRNA expression of ACACA,FASN,SCD1,FABP4,PPARγand SREBP1 in the model group was significantly up-regulated(P<0.05).Compared with model group,picroside I and II 20,40μmol·L^(-1)significantly reduced the expression of ACACA,SCD1,FASN,FABP4,and SREBP1 mRNA(P<0.05,0.01),while picrosideⅠandⅡ40μmol·L^(-1)significantly reduced the expression of SREBP1 mRNA(P<0.05,0.01).Western blotting results showed that,compared with control group,the protein levels of PPARγ,C/EBPβ,and SCD1 in th

关 键 词：胡黄连苷Ⅰ 胡黄连苷Ⅱ 3T3-L1细胞 成脂分化 脂肪生成 过氧化物酶体增殖物激活受体γ(PPARγ) CCAAT/增强子结合蛋白β(C/EBPβ) 

分 类 号：R285.5[医药卫生—中药学]