小檗碱通过调控胰岛素抵抗肝源性外泌体miR-383-5p促进星形胶质细胞AQP4表达的作用研究  

Berberine promotes expression of AQP4 in astrocytes by regulating production of miR-383-5p in HepG2 cell-derived exosomes under insulin resistance

作　　者：林雪玲 李英[1,2] 郭梦情 张艳军[1,2,3,4] 尹清晟 庄朋伟 LIN Xue-ling;LI Ying;GUO Meng-qing;ZHANG Yan-jun;YIN Qing-sheng;ZHUANG Peng-wei(National Key Laboratory of Chinese Medicine Modernization,Tianjin University of Traditional Chinese Medicine,Tianjin 301617,China;Haihe Laboratory of Modern Chinese Medicine,Tianjin University of Traditional Chinese Medicine,Tianjin 301617,China;First Teaching Hospital of Tianjin University of Traditional Chinese Medicine,Tianjin 300193,China;National Clinical Research Center for Chinese Medicine Acupuncture and Moxibustion,Tianjin 300193,China)

机构地区：[1]天津中医药大学现代中药创制全国重点实验室,天津301617 [2]天津中医药大学现代中医药海河实验室,天津301617 [3]天津中医药大学第一附属医院,天津300193 [4]国家中医针灸临床医学研究中心,天津300193

出　　处：《中国中药杂志》2025年第3期768-775,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82174112);现代中医药海河实验室科技项目(22HHZYSS00015)。

摘　　要：探究小檗碱调控胰岛素抵抗(insulin resistance,IR)状态下肝源性外泌体miR-383-5p促进星形胶质细胞水通道蛋白4(aquaporin 4,AQP4)表达的作用及机制。采用1×10^(-6)mol·L^(-1)胰岛素建立IR-HepG2细胞模型;以二甲双胍为阳性对照,采用细胞活力法(cell counting kit-8,CCK-8)、乳酸脱氢酶(lactate dehydrogenase,LDH)法筛选小檗碱和二甲双胍安全浓度,采用葡萄糖消耗量评价小檗碱对HepG2细胞胰岛素抵抗的影响;NanoSight鉴定各组细胞分泌外泌体的粒径和浓度;将各组HepG2细胞外泌体加入星形胶质细胞共孵育24 h,Western blot和qRT-PCR分别检测HA1800细胞AQP4蛋白和mRNA表达;利用qRT-PCR检测各组HepG2细胞外泌体及共孵育后HA1800细胞中miR-383-5p mRNA表达;Western blot检测HepG2细胞中miRNAs及外泌体生成释放相关蛋白表达水平。结果显示小檗碱和二甲双胍给药浓度分别为10μmol·L^(-1)、1 mmol·L^(-1),可显著改善HepG2细胞胰岛素抵抗,同时可降低HepG2细胞外泌体浓度;小檗碱和二甲双胍干预的HepG2细胞外泌体明显增加了HA1800细胞AQP4蛋白和mRNA表达;小檗碱及二甲双胍干预后的HepG2细胞外泌体及其共孵育后的HA1800细胞中miR-383-5p mRNA显著降低;小檗碱和二甲双胍干预可显著降低胰岛素抵抗状态下HepG2细胞miRNAs生成(Dicer、Drosha)及外泌体生成(Alix、Vps4A)和释放(Rab35、VAMP3)蛋白表达。因此,小檗碱可通过抑制胰岛素抵抗状态下肝源性外泌体中miR-383-5p的生成和释放,增加了星形胶质细胞AQP4蛋白表达。This study aims to explore the role and mechanism of berberine in promoting the expression of aquaporin 4(AQP4)in astrocytes by regulating the expression of miR-383-5p in HepG2 cell-derived exosomes under insulin resistance(IR).The IR-HepG2 cell model was established with 1×10^(-6)mol·L^(-1)insulin.With metformin as the positive control,the safe concentrations of berberine and metformin were screened by cell counting kit-8(CCK-8)and lactate dehydrogenase(LDH)leakage assays,and the effect of berberine on the IR of HepG2 cells was evaluated by glucose consumption.NanoSight was used to measure the particle size and concentration of exosomes secreted by HepG2 cells in each group.HepG2 cell-derived exosomes in each group were incubated with astrocytes for 24 h,and the protein and mRNA levels of AQP4 in HA1800 cells were determined by Western blot and qRT-PCR,respectively.qRT-PCR was performed to determine the expression of miR-383-5p in HepG2 cell-derived exosomes and HA1800 cells after co-incubation.Western blotting was employed to determine the expression levels of miRNAs and proteins associated with exosome production and release in HepG2 cells.The results showed that 10μmol·L^(-1)berberine and 1 mmol·L^(-1)metformin significantly alleviated the IR of HepG2 cells and reduced the concentration of exosomes in HepG2 cells.The exosomes of HepG2 cells treated with berberine and metformin significantly up-regulated the protein and mRNA levels of AQP4 in HA1800 cells.The mRNA level of miR-383-5p in HepG2 cell exosomes and HA1800 cells co-incubated with berberine and metformin decreased significantly.The intervention with berberine and metformin significantly down-regulated the expression of proteins associated with the production of miRNAs(Dicer,Drosha)as well as the production(Alix,Vps4A)and release(Rab35,VAMP3)of exosomes in IR-HepG2 cells.In conclusion,berberine can promote the expression of AQP4 in astrocytes by inhibiting the production and release of miR-383-5p in HepG2-derived exosomes under IR.

关 键 词：小檗碱 外泌体 AQP4 miR-383-5p HEPG2细胞 HA1800细胞 

分 类 号：R285[医药卫生—中药学]