RasGRP1/MAPK在肠道免疫-慢性炎症-肠道胰岛素抵抗中的作用及中药健脾清化方干预研究

Intestinal insulin resistance (IR), which can affect the sugar and lipid absorption, and then cause systemic glucose and lipid metabolic disorders, is important pathophysiological mechanisms in occurrence and development of type 2 diabetes mellitus (T2DM). The latest studies have confirmed that the intestinal Th1 and Th17 play important roles in promoting the intestinal inflammation and causing intestinal IR. The abnormal activation of MAPK pathway in Th1 and Th17 cells can make themselves activation. RasGRP1 can activate MAPK pathway and was related with the onset of diabetes.Jianpi Qinghua Recipe is effective in the treatment of T2DM, which can regulate the glucose and lipid metabolism and alleviate IR in T2DM patients. Previous studies have found that Jianpi Qinghua Recipe can activate intestinal insulin signaling pathway, reduce the concentration of the proinflammatory cytokines in intestinal Th1/Th17 and inhibit the mRNA level of intestinal RasGRP1 in diabetic rats.Therefore, we speculate that Jianpi Qinghua Recipe regulates the intestinal Th1/Th17 cells function by inhibiting RasGRP1 / MAPK pathways to relieve intestinal inflammation and then alleviate intestinal IR.We will carry out experiments in db/db mice and use in vivo transfection and in vitro culture technology by the forward and reverse intervention to explicit the role of RasGRP1 / MAPK pathway in the intestinal Th1/Th17 cells—chronic inflammation—intestinal IR and the interventional mechanism of Jianpi Qinghua Recipe and clarify the key target in regulating intestinal immunity, reducing inflammation and alleviating intestinal IR.

肠道胰岛素抵抗（IR）影响肠糖脂吸收，导致全身糖脂代谢紊乱，是2型糖尿病（T2DM）发生发展的重要病理生理机制。研究证实肠道Th1和Th17是促发肠道炎症、引起肠IR的关键免疫细胞亚群，其胞内MAPK通路的异常激活可使其活化。RasGRP1可激活MAPK，并被证实与糖尿病发病密切相关。健脾清化方是治疗T2DM的验方，明确改善患者糖脂代谢及IR。前期研究发现该方能活化糖尿病大鼠肠道胰岛素信号通路；下调肠Th1/Th17细胞型因子浓度；抑制肠RasGRP1表达。由此推测：健脾清化方通过抑制RasGRP1/MAPK通路，调控肠Th1/Th17细胞功能，缓解肠道慢性炎症，从而改善肠IR。拟采用db/db小鼠，体内转染和体外共培养相结合，正反向干预，阐明RasGRP1/MAPK在调控肠Th1/Th17—慢性炎症—肠IR中的作用及健脾清化方干预机制，阐明其调节肠道免疫、减轻炎症、改善肠IR的关键靶点。

能量摄入过多是肥胖、T2DM、代谢综合征的重要原因。高脂饮食可诱导肠 道免疫炎症，导致肠道上皮细胞胰岛素信号通路钝化，肠道发生 IR，进而增加糖脂吸收；亦可引起肠道通透性增加，大量促炎物质渗透入血。全身糖脂代谢紊乱及慢性低反应性炎症，最终引发、加重机体 IR。前期研究发现肠道Th1和Th17是促发肠道炎症、引起肠IR的关键免疫细胞亚群，其胞内MAPK通路的异常激活可使其活化。RasGRP1可激活MAPK，并被证实与糖尿病发病密切相关。前期研究发现该方能活化糖尿病大鼠肠道胰岛素信号通路；下调肠Th1/Th17细胞型因子浓度；抑制肠RasGRP 1表达。由此提出“健脾清化方通过抑制 RasGRP1/ MAPK，调控肠Th1/Th17 细胞功能，缓解慢性炎症，从而改善肠IR”的假说。本课题采用T2DM大鼠模型，随机分为正常对照组、模型对照组和健脾清化方组，灌胃给药4周，观察健脾清化方对T2DM大鼠肠道胰岛素抵抗和肠道炎症的影响；同时采用DIO小鼠模型，随机分为正常对照组、模型对照组、健脾清化方组、二甲双胍组、RasGRP1干扰组、RasGRP1干扰对照组、RasGRP1干扰+健脾清化方组，观察健脾清化方及RasGRP1对肠道胰岛素抵抗、肠道炎症及肠道屏障功能的影响。.实验结果表明，健脾清化方显著降低T2DM大鼠小肠组织apoB48表达、促进GLUT2 向肠上皮细胞刷状缘和基底侧转位、激活胰岛素信号通路及降低Th1/Th17细胞比例、转录因子、细胞因子浓度。同时，健脾清化方组及干扰RasGRP1表达均可显著抑制DIO小鼠小肠组织RasGRP1/MAPK信号通路，降低apoB48浓度、促进GLUT2转位、激活胰岛素信号通路、降低FD4浓度、恢复受损上皮紧密连接超微结构、提高肠道紧密连接蛋白表达并降低血清中LPS的浓度。.健脾清化方显著改善肠道IR，减少糖脂吸收，治疗全身IR，可能与其抑制RasGRP1/MAPK，调控肠Th1/Th17 细胞功能，缓解慢性炎症有关。