MSCs激活巨噬细胞TFEB介导的线粒体溶酶体自噬改善糖尿病脂代谢紊乱

Dyslipidemia is one of the common complications that seen in most diabetic patients, and is linked to a state of chronic low-grade inflammation, which result in infiltration of macrophage into several organs including liver. Upon high glucose, impaired macrophage autophagy produces inflammatory cytokines, and alter the liver lipid metabolism via the effects on insulin signaling. MSCs have been reported to play an important role in immune regulation. In our previous study, we found that MSC infusion is able to significantly improve the dyslipidemia..Based on the immunological mechanism involved in lipid metabolism disorders and the new finding of MSCs-mediated regulation of blood lipid, 1) we hypothesized that lipid metabolism modulatory properties of MSCs may dependent on the regulation of liver inflammatory microenvironment via the restored macrophage autophagy..In vitro: To further study the role of autophagy in high glucose-induced inflammation, autophagy agonists and inhibitors were administrated into the culture system. .1)Macrophage was treated with autophagy agonists or inhibitors in the presence of high glucose to further study the role of autophagy in high glucose-induced inflammation;.2)After the treatment with high glucose, macrophage co-culture with MSCs in transwell system to explore the role of MSCs in impaired macrophage autophagy;.3)Macrophage isolated from that trasnwell system co-culture with hepatocyte to study the specific mechanism involved in the modulation of liver lipid metabolism via inflammation..In vivo: Applying metabonomics to study the change in lipid metabolism..1)Construct of Atg7 knock-out or Mφ-depleted diabetic recipient mice;.2)GFP-MSCs were infused intravenously into recipient mice;.3)Isolate primary hepatocyte or liver tissue, to evaluate the role of inflammation microenvironment in liver lipid metabolism.

糖尿病患者多存在脂代谢紊乱(LMD)，机体处于慢性低度炎症状态，引起肝脏等器官Mφ浸润。Mφ在高糖环境下功能活化，可分泌炎症因子调控胰岛素信号干扰肝脏脂质代谢。MSCs有重要免疫调节功能，前期研究发现糖尿病大鼠Mφ自噬受损，经MSCs治疗可诱导TFEB核转位，提高Mφ溶酶体自噬活性、促进M2型Mφ分化、纠正血脂紊乱.根据糖尿病LMD发生的免疫学机制和MSCs调节血脂的新发现，提出MSCs修复Mφ自噬调控肝脏炎性微环境改善LMD的假说。本课题通过干预自噬通路，探讨自噬在调控高糖诱导Mφ炎症表型中的作用；用MSCs混培方法，探讨混培前后Mφ对肝细胞脂质代谢影响的分子机制。体内 用Mφ自噬相关蛋白7特异性敲除和Mφ清除小鼠建立糖尿病模型，代谢组学技术研究MSCs治疗对小鼠脂质代谢的转归，阐明Mφ及其自噬功能在MSCs调控炎症微环境和治疗糖尿病LMD中的作用，为MSCs治疗糖尿病LMD提供科学依据

本项目按计划执行，取得以下研究成果：.1.通过建立2型糖尿病小鼠模型和非酒精性脂肪肝炎大鼠及小鼠模型，多方位验证了MSCs静脉输注对糖尿病及脂代谢紊乱的调控作用。血生化、组织病理学及分子生物学检测发现：.1)MSCs干预可降低动物外周胰岛素水平，改善糖耐量及胰岛素敏感性，并通过缓解肝细胞内质网应激，降低肝脏组织TG及TC的含量，减轻肝脏脂质沉积，改善糖、脂代谢紊乱，抑制肝脏纤维化进程。.2)MSCs干预降低高脂饲养大鼠的血清TG、TC和LDL，提高HDL浓度，改善高脂血症，同时减轻肝脏脂肪堆积和缓解血管内膜增厚和内皮细胞损伤。.2.采用饱和脂肪酸（PA）刺激HepG2及大鼠原代肝细胞建立肝细胞脂代谢紊乱模型，通过Transwell体系研究发现：.1）MSCs可改善PA诱导的肝细胞活性降低、细胞凋亡、脂质沉积、ATP含量降低、葡萄糖摄取受损、胰岛素信号通路受损；其机制可能是通过修复肝细胞内质网钙离子泵SERCA2的表达及活性，保护肝细胞内钙离子稳态，减轻内质网应激，进而对肝细胞起到保护作用。.2）MSCs还可降低肝细胞IL-1β、IL-18、NRLP3、GSDMD和Caspase-1表达，抑制胞内炎性反应，减少肝细胞焦亡，进而提高细胞活力。.3.利用RAW264.7细胞株和原代腹腔Mφ建立糖脂损伤模型，经与MSCs混培或通过Transwell培养体系，研究发现MSCs可以通过旁分泌和线粒体转移，激活巨噬细胞TFEB介导的溶酶体-自噬途径改善Mφ线粒体功能，调控其极化过程（抑制Mφ向M1型分化，促进向M2型转化），进而抑制机体炎性反应，改善糖尿病状态下肝、肾等组织的糖脂毒性损伤。 .4.糖尿病动物组织器官均存在支链氨基酸（BCAA）代谢紊乱现象。我们利用BCAA代谢激动剂（BT2）干预糖尿病小鼠，观察发现：db/db小鼠血中BCAA及其产物支链氨基酸酮酸（BCKA）减少的同时，也降低了其肝肾等组织的炎性反应；收集原代Mφ并用BCAA/BCKA进行处理，结果显示BCKA可降低Mφ的mtDNA拷贝数，并诱导Mφ线粒体碎片化、mtROS生成和细胞因子产生。提示：以Mφ代谢调控为出发点可能阐释MSCs干预脂代谢紊乱的新机制。