从Sirt6/GCN5/PGC-1α信号通路探讨胡芦巴改善肝糖异生治疗2型糖尿病的作用机制

Hepatic gluconeogenesis disorder is one of the important pathological characteristics of insulin resistance in type 2 diabetes mellitus. It has been found that the activation of Sirt6/GCN5/PGC-1α signaling pathway could inhibit hepatic gluconeogenesis, thus treating type 2 diabetes mellitus. Type 2 diabetes mellitus belongs to the category of diabetes in traditional Chinese medicine. The basic pathogenesis in diabetes is considered as dryness-heat due to yin deficiency, however some physicians in past dynasties also recognized the theory of yang deficiency leading to diabetes. They considered that kidney yang deficiency would result in failure of qi transformation as well as bladder control，which was one of the important pathogenesis in diabetes. Fenugreek is characterized with the function of tonifying kidney yang and dispelling phlegm and dampness. Based on the effects of fenugreek on relieving type 2 diabetes mellitus in our clinical application and improving insulin resistance validated by our preliminary experimental study, we propose that fenugreek may regulate hepatic gluconeogenesis through activating Sirt6/GCN5/PGC-1α signaling pathway to treat type 2 diabetes mellitus. To test our hypothesis, we will use the db/db mice model characterized with spontaneous type 2 diabetes mellitus in vivo, together with in vitro experiment. Several experimental techniques including molecular biology, biochemistry and immunology will be employed to investigate the level of gluconeogenesis, the expression of gluconeogenesis related transcription factors and rate-limiting enzymes and the expression of the related signaling pathway proteins. Our research aims to provid some insights of the mechanism of fenugreek in treating type 2 diabetes mellitus and scientific evidence for its clinical application.

肝糖异生紊乱是2型糖尿病胰岛素抵抗的重要病理特征之一。研究发现激活Sirt6/GCN5/PGC-1α信号通路可抑制肝糖异生，治疗2型糖尿病。2型糖尿病属中医学“消渴病”范畴，虽然阴虚燥热为其基本病机，但是历代也有医家认可“阳虚致消”，认为肾阳不足、气化无权、开合失司乃其重要病机之一。胡芦巴具有温肾助阳、祛痰除湿之功效，本课题组在前期临床应用胡芦巴治疗2型糖尿病有效、实验研究证实胡芦巴可改善胰岛素抵抗的基础上，提出“胡芦巴可能通过激活Sirt6/GCN5/PGC-1α信号通路调控肝糖异生治疗2型糖尿病”的假说，以自发性2型糖尿病db/db小鼠为研究对象，结合离体实验，运用分子生物学、生物化学、免疫学等技术，探讨胡芦巴对肝糖异生水平、糖异生相关转录因子和限速酶的表达、相关信号通路蛋白表达的影响，初步阐明胡芦巴治疗2型糖尿病的机制，为促进其临床应用提供科学依据。

超过70%的2型糖尿病患者同时伴有非酒精性脂肪肝，两者的共存和相互作用增加了非酒精性脂肪肝的难治性。课题组既往研究已经证实，胡芦巴具有降糖和调脂功效，但其具体机制尚不明确。前期实验中，本课题组通过分子对接技术，观察到胡芦巴活性成分薯蓣皂苷元对Sirt6亲和力良好。基于此，本研究以自发性2型糖尿病、非酒精性脂肪肝db/db小鼠为研究对象，用薯蓣皂苷元对其进行干预，结合体外实验，运用ELISA检测、HE染色、油红O染色、DHE染色、BODIPY染色、免疫荧光、免疫组化、转录组学测序、荧光定量PCR等技术，探讨了胡芦巴活性成分薯蓣皂苷元对2型糖尿病伴发的非酒精性脂肪肝的干预作用及其对肝脏Sirt6相关信号通路蛋白表达的影响。结果显示：①薯蓣皂苷元可以显著改善db/db小鼠非酒精性脂肪肝，表现为减轻体重、肝指数、脂肪量，降低血清TG、TC、AST、ALT、IL-6、TNF-α水平，降低肝脏TG水平，改善肝脏脂质堆积和氧化应激；②免疫组学结果显示，薯蓣皂苷元可以上调db/db小鼠肝脏中Sirt6表达；③转录组学结果显示，薯蓣皂苷元对db/db小鼠肝脏组织脂质代谢相关通路有显著影响；结合qPCR结果分析，这种影响主要体现在脂肪酸转运相关基因中，即薯蓣皂苷元可以下调db/db小鼠肝脏中脂肪酸转运体CD36、FATP2、FABP1等基因的表达；④薯蓣皂苷元可以上调棕榈酸诱导的HepG2细胞Sirt6表达，下调脂肪酸转运体CD36、FATP2、FABP1的表达；⑤薯蓣皂苷元可以抑制棕榈酸诱导的HepG2细胞对游离脂肪酸的摄取，改善其脂质堆积；且这种干预作用可以被Sirt6抑制剂OSS_128167阻断，也可以被Sirt6激动剂MDL800重复。上述结果提示：薯蓣皂苷元可以改善db/db小鼠非酒精性脂肪肝，其分子机制可能与调节肝细胞内Sirt6相关脂肪酸转运通路相关。该研究成果为胡芦巴治疗2型糖尿病伴发的非酒精性脂肪肝提供了科学依据。