基于GLP-1R/cAMP/PKA/CREB的泽泻汤促进白色脂肪棕色化/激活棕色脂肪作用机制研究  

Effect and mechanism of Zexie Decoction in promoting white adipose tissue browning/brown adipose tissue activation based on GLP-1R/cAMP/PKA/CREB pathway

作　　者：丁婧 赵洁[1] 王萌萌[1] 苏烜 高改 徐江雁[1] 谢治深 DING Jing;ZHAO Jie;WANG Meng-meng;SU Xuan;GAO Gai;XU Jiang-yan;XIE Zhi-shen(Academy of Chinese Medical Sciences,Henan University of Chinese Medicine,Zhengzhou 450046,China)

机构地区：[1]河南中医药大学中医药科学院,河南郑州450046

出　　处：《中国中药杂志》2023年第21期5851-5862,共12页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82004019,82174267);河南省高校科技创新人才支持计划项目(22HASTIT048);河南省高校科技创新团队支持计划项目(21IRTSTHN026)。

摘　　要：基于GLP-1R/cAMP/PKA/CREB探讨泽泻汤(Zexie Decoction,ZXD)体内外促进白色脂肪棕色化/激活棕色脂肪的作用机制。西式饮食(western diet,WD)诱导高脂血症小鼠模型,设置对照组,WD组(模型组),ZXD低、中、高剂量组;体外诱导3T3-L1细胞成脂分化模型,以毛喉素(forskolin,FSK)作为阳性对照,设置ZXD低、中、高剂量组。免疫组化与免疫荧光结果提示,与WD组相比,泽泻汤促进白色和棕色脂肪组织中UCP1表达;同时泽泻汤上调了细胞中UCP1、CPT1β、PPARα等基因;Western blot检测PGC-1α、UCP1、PPARα的蛋白表达也表现出随泽泻汤剂量依赖性升高,表明泽泻汤促进白色脂肪棕色化/激活棕色脂肪。苏木素-伊红(HE)染色结果显示,相较于WD组,泽泻汤处理后,白色和棕色脂肪细胞明显缩小,ATGL、HSL、MGL、PLIN1的mRNA表达显著上调;油红O染色和生化试剂盒检测细胞TC、TG水平均提示泽泻汤改善脂质蓄积,促进脂肪分解。p-CREB免疫组化和免疫荧光发现,泽泻汤处理逆转了WD导致的p-CREB表达降低;在泽泻汤的体外干预下,CREB、p-CREB、p-PKA substrate的蛋白表达均明显增加,CREB的mRNA水平增加;ELISA检测发现泽泻汤增加细胞内cAMP浓度;分子对接分析结果显示泽泻、白术中多个活性成分可与GLP-1R形成氢键稳定结合。综上所述,泽泻汤在体内外均能促进白色脂肪棕色化/激活棕色脂肪,其作用机制可能与GLP-1R/cAMP/PKA/CREB通路有关。This study investigated the mechanism of Zexie Decoction(ZXD) in promoting white adipose tissue browning/brown adipose tissue activation based on the GLP-1R/cAMP/PKA/CREB pathway.A hyperlipidemia model was induced by a western diet(WD) in mice,and the mice were divided into a control group,a model group(WD),and low-,medium-,and high-dose ZXD groups.An adipogenesis model was induced in 3T3-L1 cells in vitro,and with forskolin(FSK) used as a positive control,low-,medium-,and high-dose ZXD groups were set up.Immunohistochemistry and immunofluorescence results showed that compared with the WD group,ZXD promoted the expression of UCP1 in white and brown adipose tissues,and also upregulated UCP1,CPT1β,PPARα,and other genes in the cells.Western blot analysis showed a dose-dependent increase in the protein expression of PGC-1α,UCP1,and PPARα with ZXD treatment,indicating that ZXD could promote the white adipose tissue browning/brown adipose tissue activation.Hematoxylin-eosin(HE) staining results showed that after ZXD treatment,white and brown adipocytes were significantly reduced in size,and the mRNA expression of ATGL,HSL,MGL,and PLIN1 was significantly upregulated as compared with the results in the WD group.Oil red O staining and biochemical assays indicated that ZXD improved lipid accumulation and promoted lipolysis.Immunohistochemistry and immunofluorescence staining for p-CREB revealed that ZXD reversed the decreased expression of p-CREB caused by WD.In vitro intervention with ZXD increased the protein expression of CREB,p-CREB,and p-PKA substrate,and increased the mRNA level of CREB.ELISA detected an increase in intracellular cAMP concentration with ZXD treatment.Molecular docking analysis showed that multiple active components in Alismatis Rhizoma and Atractylodis Macrocephalae Rhizoma could form stable hydrogen bond interactions with GLP-1R.In conclusion,ZXD promotes white adipose tissue browning/brown adipose tissue activation both in vivo and in vitro,and its mechanism of action may be related to the GLP-

关 键 词：泽泻汤 棕色脂肪 3T3-L1 GLP-1R/cAMP/PKA/CREB 

分 类 号：R285.5[医药卫生—中药学]