小檗碱介导BMAL1:CLOCK复合体调控糖脂代谢改善脂肪胰岛素抵抗的效应机制  

Mechanism of berberine in improving adipocytic IR by mediating BMAL1:CLOCK complex and regulating glucose and lipid metabolism

作　　者：王颖[1,2] 徐中华 延李科 崔灿 刘伟华 肖汉月[1] 涂珺 WANG Ying;XU Zhong-hua;YAN Li-ke;CUI Can;LIU Wei-hua;XIAO Han-yue;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)

机构地区：[1]江西中医药大学江西省中医病因学重点实验室&中医基础理论分化发展研究中心,江西南昌330004 [2]江西省中药药理重点实验室,江西南昌330004

出　　处：《中国中药杂志》2024年第17期4586-4596,共11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)。

摘　　要：探究小檗碱介导脑和肌肉芳香烃受体核转位样蛋白1(brain and muscle arnt-like 1,BMAL1):昼夜自发输出周期蛋白kaput(circadian locomotor output cycles kaput,CLOCK)复合体,调控糖脂代谢改善脂肪胰岛素抵抗(insulin resistance,IR)的作用机制。地塞米松诱导96 h建立IR-3T3-L1脂肪细胞模型,0.5、1、5、10、20μmol·L^(-1)小檗碱给药24 h。葡萄糖氧化酶法和细胞活性(cell counting kit-8,CCK-8)试剂盒分别检测胞外葡萄糖含量和细胞活力;酶比色法检测甘油三酯(triglyceride,TG)和甘油含量;油红O染色检测脂滴;荧光染色检测Ca2+、线粒体结构及活性氧(reactive oxygen species,ROS);蛋白免疫印迹(Western blot,WB)检测脂联素(adiponectin,ADPN)、BMAL1、CLOCK、激素敏感性脂肪酶(hormone-sensitive triglyceride lipase,HSL)、碳水化合物反应元件结合蛋白(carbohydrate-responsive element-binding protein,ChREBP)、固醇调节元件结合蛋白1C(sterol regulatory element-binding protein 1C,SREBP-1C)、过氧化物酶体增殖物激活受体γ共激活剂1α(peroxisome proliferator activated receptorγcoactivator 1α,PGC1α)、肉碱棕榈酰基转移酶1α(choline phosphotransferase 1α,CPT1α)、过氧化物酶体增殖物激活受体α(peroxisome proliferators-activated receptorsα,PPARα),免疫荧光检测BMAL1核定位。此外,添加20μmol·L^(-1)CLK8(CLOCK抑制剂)检测葡萄糖消耗量及BMAL1/ChREBP/PPARα蛋白。研究结果显示小檗碱增加IR-3T3-L1脂肪细胞葡萄糖消耗量,且不影响细胞活力;小檗碱降低IR-3T3-L1脂肪细胞胞内TG,5μmol·L^(-1)小檗碱升高甘油含量,减少脂滴积累,提示其加强脂解,而10μmol·L^(-1)小檗碱不影响甘油含量且脂滴更少,提示其可能同时加强脂解和甘油利用。此外,小檗碱降低IR-3T3-L1脂肪细胞胞内Ca^(2+)和ROS改善线粒体功能,上调PGC1α有助于维护线粒体结构。结果还显示小檗碱上调ADPN,提示其增加IR-3T3-L1脂肪细胞胰岛素敏感性,上调外周节律调�To explore the action mechanism of berberine in improving adipocytic insulin resistance(IR)by mediating brain and muscle arnt-like 1(BMAL1):circadian locomotor output cycles kaput(CLOCK)complex and regulating glucose and lipid metabolism.After the IR-3T3-L1 adipocyte model was established by dexamethasone induction for 96 h,0.5,1,5,10,and 20μmol·L-1 berberine was administered for 24 h.The glucose oxidase method and cell counting kit-8(CCK-8)were used to detect extracellular glucose content and cell viability,respectively.The triglyceride(TG)and glycerol contents were detected by enzyme colorimetry.Oil red O staining was used to detect lipid droplets,and fluorescence staining was used to detect Ca2+,mitochondrial structure,and reactive oxygen species(ROS).Adiponectin(ADPN),BMAL1,CLOCK,hormone-sensitive triglyceride lipase(HSL),carbohydrate-response element-binding protein(ChREBP),sterol regulatory element-binding protein 1C(SREBP-1C),peroxisome proliferator-activated receptorγcoactivator 1α(PGC1α),carnitine palmitoyl transferase 1α(CPT1α),and peroxisome proliferator-activated receptorα(PPARα)were detected by Western blot(WB).Moreover,the nuclear localization of BMAL1 was detected by immunofluorescence.In addition,20μmol·L-1 CLK8 inhibitor was added to detect glucose consumption and BMAL1/ChREBP/PPARαprotein.The results showed that berberine increased glucose consumption in IR-3T3-L1 adipocytes without affecting cell viability and reduced TG content.In addition,5μmol·L-1 berberine increased glycerol content and reduced lipid droplet accumulation due to enhanced lipolysis,while 10μmol·L-1 berberine did not affect glycerol content,and fewer lipid droplets were observed due to enhanced lipolysis and glycerol utilization.Berberine improved mitochondrial function by reducing intracellular Ca2+and ROS in IR-3T3-L1 adipocytes and upregulated PGC1αto improve the mitochondrial structure.The results also showed that berberine elevated ADPN to increase the insulin sensitivity of IR-3T3-L1 adipocytes,upregulat

关 键 词：胰岛素抵抗 脂代谢 糖代谢 BMAL1:CLOCK复合体 小檗碱 

分 类 号：R285[医药卫生—中药学]