中国中药杂志2025，Vol.50Issue(15)：4293-4303,11.DOI:10.19540/j.cnki.cjcmm.20250410.401
基于BMAL1:CLOCK复合体探讨小檗碱调控肝糖酵解、糖氧化和糖异生改善能量代谢的体外降糖作用机制
Hypoglycemic effect and mechanism of berberine in vitro based on regulation of BMAL1:CLOCK complex involved in hepatic glycolysis,glucose oxidation and gluconeogenesis to improve energy metabolism
摘要
Abstract
This paper aims to investigate the hypoglycemic effect and mechanism of berberine in improving energy metabolism based on the multi-pathway regulation of brain and muscle aromatic hydrocarbon receptor nuclear translocal protein 1(BMAL1):cyclin kaput complex of day-night spontaneous output cyclin kaput(CLOCK).The dexamethasone-induced hepatic insulin resistance(IR)HepG2 cell model was used;0.5,1,5,10,20 μmol·L-1 berberine were administered at 15,18,21,24,30,36 h.The time-dose effect of glucose content in extracellular fluid was detected by glucose oxidase method.The optimal dosage and time of berberine were determined for the follow-up study.Glucose oxidase method and chemiluminescence method were respectively performed to detect hepatic glucose output and relative content of ATP in cells;Ca2+,reactive oxygen species(ROS),mitochondrial structure and membrane potential were detected by fluorescent probes.Moreover,ultraviolet colorimetry method was used to detect the liver type of pyruvate kinase(L-PK)and phosphoenol pyruvate carboxykinase(PEPCK).In addition,pyruvate dehydrogenase E1 subunit α1(PDHA1),phosphate fructocrine-liver type(PFKL),forkhead box protein O1(FoxO1),peroxisome proliferator-activated receptor gamma co-activator 1α(PGC1α),glucose-6-phosphatase(G6Pase),glucagon,phosphorylated nuclear factor-red blood cell 2-related factor 2(p-Nrf2)(Ser40),heme oxygenase 1(HO-1),NAD(P)H quinone oxidoreductase 1(NQO1),fibroblast growth factor 21(FGF21),uncoupled protein(UCP)1 and UCP2 were detected by Western blot.BMAL1:CLOCK complex was detected by immunofluorescence double-staining method,combined with small molecule inhibitor CLK8.Western blot was used to detect PDHA1,PFKL,FoxO1,PGC1α,G6Pase,glucagon,Nrf2,HO-1,NQO1,FGF21,UCP1 and UCP2 in the CLK8 group.The results showed that berberine downregulated the glucose content in extracellular fluid in IR-HepG2 cells in a time-and dose-dependent manner.Moreover,berberine inhibited hepatic glucose output and reduced intracellular Ca2+and ROS whereas elevated JC-1 membrane potential and improved mitochondrial structure to enhance ATP production.In addition,berberine upregulated the rate-limiting enzymes such as PFKL,L-PK and PDHA1 to promote glycolysis and aerobic oxidation but also downregulated PGC1α,FoxO1,G6Pase,PEPCK and glucagon to inhibit hepatic gluconeogenesis.Berberine not only upregulated p-Nrf2(Ser40),HO-1 and NQO1 to enhance antioxidant capacity but also upregulated FGF21,UCP1 and UCP2 to promote energy metabolism.Moreover,berberine increased BMAL1,CLOCK and nuclear BMAL1:CLOCK complex whereas CLK8 reduced the nuclear BMAL1:CLOCK complex.Finally,CLK8 decreased PDHA1,PFKL,Nrf2,HO-1,NQO1,FGF21,UCP1,UCP2 and increased FoxO1,PGC1α,G6Pase and glucagon compared with the 20 μmol·L-1 berberine group.BMAL1:CLOCK complex inhibited gluconeogenesis,promoted glycolysis and glucose aerobic oxidation pathways,improved the reduction status within mitochondria,protected mitochondrial structure and function,increased ATP energy storage and promoted energy consumption in IR-HepG2 cells.These results suggested that berberine mediated BMAL1:CLOCK complex to coordinate the regulation of hepatic IR cells to improve energy metabolism in vitro.关键词
胰岛素抵抗/糖代谢/能量代谢/BMAL1:CLOCK复合体/小檗碱Key words
insulin resistance/glucose metabolism/energy metabolism/BMAL1:CLOCK complex/berberine引用本文复制引用
徐中华,延李科,刘伟华,崔灿,肖汉月,李慧平,涂珺..基于BMAL1:CLOCK复合体探讨小檗碱调控肝糖酵解、糖氧化和糖异生改善能量代谢的体外降糖作用机制[J].中国中药杂志,2025,50(15):4293-4303,11.基金项目
国家自然科学基金项目(82160838,82460859) （82160838,82460859）
江西中医药大学校级科技创新团队发展计划项目(CXTD22007) （CXTD22007）
江西中医药大学中西医结合一级学科(江西省双一流学科)项目(zxyylxk20220103) （江西省双一流学科）
