黄芪建中汤靶向BMAL1调控cAMP/PKA/CREB信号通路对3T3-L1细胞脂解及棕色化模型影响的研究  

Study on the effect of Huangqi Jianzhong Decoction on BMAL1 targeted regulation of cAMP/PKA/CREB signaling pathway in 3T3⁃L1 adipose lipolysis and browning model

作　　者：吴涛 曲一玮 徐圣洁 李晓[3] 王咏 马度芳 WU Tao;QU Yiwei;XU Shengjie;LI Xiao;WANG Yong;MA Dufang(College of Traditional Chinese Medicine,Shandong University of Traditional Chinese Medicine,Jinan 250013,China;The First Clinical Medical College of Shandong University of Traditional Chinese Medicine,Jinan 250199,China;Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan 250014,China)

机构地区：[1]山东中医药大学中医学院,济南250013 [2]山东中医药大学第一临床医学院 [3]山东中医药大学附属医院

出　　处：《北京中医药大学学报》2023年第10期1400-1410,共11页Journal of Beijing University of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(No.82004280)。

摘　　要：目的探索黄芪建中汤含药血清对异丙肾上腺素(ISO)诱导的3T3-L1脂肪细胞脂解及棕色化模型的抑制作用及与脑和肌肉芳烃受体核转录因子样蛋白-1(BMAL1)调控环磷酸腺苷(cAMP)/蛋白激酶A(PKA)/环磷酸腺苷反应成分结合蛋白(CREB)信号通路的关系。方法制备空白及黄芪建中汤含药血清。体外培养3T3-L1脂肪细胞,诱导其分化成熟后分为对照组、空白组、2.5%含药血清组、5%含药血清组、10%含药血清组。除对照组外,其余各组加入10 mmol/L ISO以构建脂肪细胞过度脂解及棕色化模型。采用油红O染色法和酶联免疫吸附剂测定(ELISA)法检测各组脂肪细胞内脂滴阳性面积比及其水解后产物的含量,采用实时荧光PCR法和蛋白质印迹法检测各组脂肪细胞脂解及棕色化相关基因及蛋白的表达,以筛选出最优体积分数的含药血清进行下一步慢病毒转染实验。取分化成熟的3T3-L1脂肪细胞,分别加入BMAL1小干扰RNA、空载体小干扰RNA,构建沉默BMAL1的3T3-L1脂肪细胞。将细胞分为空载体组、含药血清空载体组、BMAL1沉默组和含药血清BMAL1沉默组。各组加入ISO孵育后,油红O染色法检测各组脂肪细胞内脂滴阳性面积比;ELISA法检测各组脂肪细胞内甘油三酯(TG)、cAMP及培养基中游离脂肪酸(FFA)的含量;蛋白质印迹法检测PKA、CREB的蛋白表达;实时荧光PCR法检测激素敏感性脂肪酶(HSL)、甘油三酯脂肪酶(ATGL)、线粒体解偶联蛋白1(UCP1)和T-box转录因子1(TBX1)的mRNA表达。结果与2.5%及5%含药血清组比较,10%含药血清组细胞内cAMP含量及培养基中FFA含量降低(均P<0.05)、TG含量升高(P<0.05)、PKA蛋白表达降低(P<0.05)、HSL及ATGL mRNA表达降低(均P<0.05)。慢病毒转染3T3-L1脂肪细胞后,与空载体组比较,BMAL1沉默组中BMAL1蛋白表达降低(P<0.05),表明BMAL1沉默的脂肪细胞成功构建。与空载体组比较,BMAL1沉默组细胞内脂滴阳性面积比及TG含量降低Objective We aimed to investigate the inhibitory effect of serum containing Huangqi Jianzhong Decoction on isoproterenol(ISO)induced lipolysis and browning in 3T3⁃L1 adipocytes and its relationship with the regulation of the cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)/cyclic adenosine monophosphate response element binding protein(CREB)signaling pathway by brain and muscle aromatic hydrocarbon receptor nuclear transcription factor like protein⁃1(BMAL1).Methods Blank serum and Huangqi Jianzhong Decoction drug⁃containing serum were prepared.3T3⁃L1 adipocytes were cultured in vitro and induced to differentiate and mature.They were divided into the control group,the blank group,the 2.5%drug⁃containing serum group,the 5%drug⁃containing serum group,and the 10%drug⁃containing serum group.10 mmol/L ISO was given to all groups except for the control group to establish the hypertipolysis and browning model.Oil red O staining and enzyme⁃linked immunosorbent assay(ELISA)were used to detect the lipid droplet positive area ratio and the content of hydrolyzed products.Real⁃time PCR and Western blotting were conducted to detect the expression of lipolysis⁃and browning⁃related genes and proteins,respectively,to select the optimal volume fraction of drug⁃containing serum for the lentivirus transfection experiment.Mature adipocytes were transfected with si⁃BMAL1 to silence BMAL1.Control cells were transfected with si⁃NC.Then the cells were divided into the si⁃NC group,the containing serum si⁃NC group,the si⁃BMAL1 group,and the drug⁃containing serum si⁃BMAL1 group.After incubation with ISO,the lipid droplet positive area ratio in adipocytes was detected by oil red O staining.The contents of triglyceride(TG)and cAMP in adipocytes and free fatty acid(FFA)in medium were detected by ELISA.The protein expression levels of PKA and CREB were determined by Western blotting.The mRNA expression levels of hormone⁃sensitive lipase(HSL),triglyceride lipase(ATGL),uncoupling protein 1(UCP1),an

关 键 词：黄芪建中汤 恶病质 脂肪消耗 脑和肌肉芳烃受体核转录因子样蛋白-1 环磷酸腺苷/蛋白激酶A/环磷酸腺苷反应成分结合蛋白信号通路 大鼠 

分 类 号：R285.5[医药卫生—中药学]