摘要
探讨小檗碱介导脑和肌肉芳香烃受体核转运样蛋白1基因(brain and muscle arnt-like 1,BMAL1)为中心的钟控基因调节代谢网络改善肝细胞胰岛素抵抗(insulin resistance, IR)的分子机制。优化建立地塞米松诱导肝IR-HepG2模型,5、10、20μmol·L^(-1)小檗碱干预24 h。葡萄糖氧化酶法和细胞活力(cell counting kit-8,CCK-8)法分别检测细胞葡萄糖消耗量和细胞活力;过碘酸-雪夫(periodic acid-Schiff stain, PAS)染色法和脂质荧光法分别检测糖原和脂质;免疫荧光检测BMAL1和昼夜自发输出周期蛋白kaput(circadian locomotor output cycles kaput, CLOCK);蛋白免疫印迹法检测BMAL1、CLOCK、昼夜节律调节因子2(period circadian clock 2,PER2)、隐花色素昼夜节律调节因子1(cryptochrome circadian regulator 1,CRY1)、Rev-Erbα、碳水化合物应答元件结合蛋白(carbohydrate response element-binding protein, ChREBP)、过氧化物酶体增殖物激活受体α/γ(peroxisome proliferators-activated receptors α/γ,PPARα/γ)、固醇调节元件结合蛋白1C(sterol regulatory element-binding protein 1C,SREBP-1C)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)、蛋白激酶B(protein kinase B,Akt)、糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK3β)、乙酰辅酶A羧化酶1(acetyl coenzyme A carboxylase 1,ACC1)、脂肪酸合成酶(fatty acid synthase, FASN)、肉碱棕榈酰转移酶1α(carnitine palmitoyltransferase 1α,CPT1α)、烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyltransferase, NAMPT)、沉默信息调节因子1(silent information regulator 1,SIRT1)、脂联素(adiponectin, ADPN)、胰岛素受体底物2(insulin receptor substrate 2,IRS2)、磷脂酰肌醇3-激酶p85调控亚基(phosphatidylinositol 3-kinase regulatory subunit p85,PI3Kp85)。此外,检测cAMP依赖蛋白激酶α(adenosine monophosphate-activated protein kinase, AMPKα)、Akt、mTOR、GSK3β和BMAL1磷酸化水平。添加20μmol·L^(-1)CLK8抑制剂检测葡萄糖消耗量及mTOR、ChREBP和PPARα蛋白。结果显示,小檗碱可增加IR-HepG2细胞葡萄糖消耗量和糖原含量且降低胞内脂质;增加IR-HepG2细胞时钟蛋白BMAL1和CLOCK表达及核定位,上调BMAL1。小檗碱可上调ADPN、IRS2、PI3Kp85、p-Akt(Ser473)/Akt、p-mTOR(Ser2448)/mTOR、PPARα和CPT1α水平,下调p-GSK3β(Ser9)/GSK3β、ChREBP、SREBP-1C、ACC1和FASN水平;CLK8反证研究提示抑制BMAL1:CLOCK相互作用可减弱小檗碱降糖效应,上调ChREBP且下调mTOR和PPARα水平。因而推测小檗碱介导BMAL1为中心的时钟调控代谢网络改善肝细胞IR。
To investigate the mechanism of berberine on the orchestrated regulation of metabolism based on clock-controlled genes by brain and muscle arnt-like 1(BMAL1)regulation to improve hepatic insulin resistance(IR)in vitro.An optimized dexamethasone-induced hepatic IR-HepG2 model was established,which was administrated with berberine at concentrations of 5,10 and 20μmol·L^(-1) for 24 hours.Glucose oxidase method and cell counting kit-8(CCK-8)assay were employed to measure extracellular glucose concentration and cell viability respectively.Periodic acid-schiff(PAS)staining and BOPIDY 493/503 fluorescence probe were used to detect glycogen and lipid content.Moreover,immunofluorescence(IF)was performed to detect the nuclear localization of BMAL1,circadian locomotor output cycles kaput(CLOCK)in IR-HepG2 cells.In addition,western blot was used to detect BMAL1/p-BMAL1,CLOCK,period circadian clock 2(PER2),cryptochrome circadian regulator 1(CRY1),Rev-Erbα,carbohydrate response element binding protein(ChREBP),peroxisome proliferator-activated receptor alpha and gamma(PPARα/γ),sterol regulatory element-binding protein 1c(SREBP-1C),mammalian target of rapamycin(mTORC),acetyl coenzyme A carboxylase 1(ACC1),fatty acid synthase(FASN),carnitine palmitoyltransferase 1α(CPT1α),nicotinamide phosphoribosyltransferase(NAMPT),silent information regulator 1(SIRT1),adiponectin(ADPN),insulin receptor substrate 2(IRS2),phosphatidylinositol 3-kinase regulatory subunit p85(PI3Kp85),adenosine monophosphate-activated protein kinase(AMPKα),protein kinase B(Akt)/p-Akt,glycogen synthase kinase 3β(GSK3β)/p-GSK3β,mTORC/p-mTORC.Except for,20μmol·L^(-1) CLK8 was added to detect glucose consumption as well as ChREBP,PPARαand mTORC in IR-HepG2 cells.The results showed that berberine reduced extracel lular glucose content and did not change cell viability in IR-HepG2 cells whereas increase hepatic glycogen synthesis and decreased intracellular lipid.Moreover,berberine increased the expression and nuclear localization of BMAL1 and CLOCK whereas upregu lated p-BMAL1(Ser42)in IR-HepG2 cells.Frutherly,berberine upregulated ADPN,IRS2,PI3Kp85,p-Akt(Ser473)/Akt,p-mTORC(Ser2448)/mTORC,PPARαand CPT1α,downregulated p-GSK3β(Ser9)/GSK3β,ChREBP,SREBP-1C,ACC1 and FASN.In addition,the addition of CLK8 to 20μmol·L^(-1) berberine group reduced glucose consumption in IR-HepG2 cells,which upregulated ChREBP but downregulated PPARαand mTORC.In summary,berberine orchestratelly regulated glucose and lipid metabolism based on clock-controlled genes by BMAL1 mediation,which maybe improve hepatic IR.It would provide a theoretical basis for the clinical application of berberine.
作者
延李科
崔灿
王颖
朱水兰
徐中华
肖汉月
刘伟华
涂珺
YAN Li-ke;CUI Can;WANG Ying;ZHU Shui-lan;XU Zhong-hua;XIAO Han-yue;LIU Wei-hua;TU Jun(Jiangxi Province Key Laboratory of Traditional Chinese Medicine Etiopathogenisis&Research Center for Differentiation and Development of Traditional Chinese Medicine Basic Theory,Jiangxi University of Chinese Medicine,Nanchang 330004,China;Key Laboratory of Traditional Chinese Medicine Pharmacology of Jiangxi Province,Nanchang 330004,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2024年第23期6368-6377,共10页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(82160838,81960809)
江西中医药大学校级科技创新团队发展计划项目(CXTD22007)。
