基于MiR21/RasGRP1/MAPK通路探讨健脾清化方调控NK细胞-巨噬细胞轴改善2型糖尿病肠道胰岛素抵抗的作用

Low-grade chronic inflammation is primary cause of insulin resistance(IR). NK cell-macrophagocyte axis plays an important role in IR induced by chronic inflammation. Recent evidence has demonstrated that abnormity of RasGRP1／MAPK pathway activates NK cell, and then regulates the macrophage polarization. MiR21 could regulate the function of RasGRP1.Our previous studies showed that Jianpi Qinghua Recipe can regulate the glucose and lipid metabolism and alleviate IR in T2DM patients. And our animal studies showed that Jianpi Qinghua Recipe can activate insulin signaling pathway in intestinal tissue in diabetic rats, downregulate E4bp4 mRNA (NK cells transcription factor) from intestinal tissue and the protein expression of intestinal RasGRP1, and upregulate MiR21 mRNA from intestinal tissue. Therefore, we speculate scientific hypothesis "Jianpi Qinghua Recipe could regulate NK cell-macrophagocyte axis to alleviate intestinal insulin resistance in T2DM through MiR21/RasGRP1/MAPK pathway". We carry out experiments by using overexpression in vivo, flow cytometry, RNAi and other techniques to explore mechanism of MiR21/RasGRP1/MAPK on regulation of intestinal NK cell-macrophagocyte axis in T2DM and effect of Jianpi Qinghua recipe, clarify key target of Jianpi Qinghua recipe on alleviating intestinal insulin resistance in T2DM, and then enrich the theory of "treating T2DM from spleen".

慢性炎症是肠道胰岛素抵抗（Insulin Resistance，IR）的重要原因，NK细胞-巨噬细胞轴在慢性炎症引起IR中发挥重要作用。研究发现RasGRP1／MAPK异常可使NK细胞活化，进而调节巨噬细胞极性。而MiR21对RasGRP1有调控作用。我们前期研究发现：健脾清化方明确改善患者糖脂代谢及IR；活化糖尿病大鼠肠胰岛素信号通路；下调肠NK细胞转录因子E4bp4水平；抑制肠RasGRP1表达；上调肠MiR21水平。故提出“健脾清化方通过MiR21/RasGRP1/MAPK通路调控NK细胞-巨噬细胞轴功能，改善2型糖尿病肠道胰岛素抵抗”科学假说。本研究拟采用体内过表达、细胞流式、RNAi等技术，研究MiR21/RasGRP1/MAPK调节糖尿病肠道NK细胞-巨噬细胞轴的机制及健脾清化方的干预作用，阐明健脾清化方改善2型糖尿病肠道胰岛素抵抗的关键靶点，丰富中医从脾论治糖尿病理论内涵。

NK细胞-巨噬细胞轴在慢性炎症引起肠道胰岛素抵抗(IR)中发挥重要作用。课题组前期研究证实RasGRP1与T2DM、IR发病密切相关，健脾清化方具有改善糖尿病患者和动物IR的作用，但其机制不明确。首先，我们通过db/db小鼠和高脂饮食诱导肥胖、胰岛素抵抗小鼠（DIO）的比较，发现DIO小鼠更适合RasGRP1基因对肠道胰岛素抵抗的研究。第二，我们使用DIO小鼠模型，并予以健脾清化方干预，结果显示健脾清化方可改善DIO小鼠代谢表型及肠道胰岛素抵抗、恢复受损肠道屏障功能，降低DIO小鼠肠道NK细胞比例、提高M2/M1型巨噬细胞比值、减少促炎性细胞因子分泌，从而调节肠道免疫，这些改变可能与RasGRP1/MAPK通路有关，但不受miR 21调控。第三，体外细胞实验运用LPS+PA或/和健脾清化方冻干粉干预巨噬细胞，结果显示LPS+PA可诱导巨噬细胞M1型活化，而健脾清化方能通过RasGRP1/MAPK通路逆转LPS+PA诱导的M1型活化，使其向M2型活化。第四，我们建立DIO小鼠小肠组织RasGRP1干扰模型，并予以健脾清化方干预，结果显示健脾清化方抑制RasGRP1/MAPK通路，缓解肠道免疫炎症，改善肠道IR。综上所述，我们的工作阐明了健脾清化方通过调控NK细胞-巨噬细胞轴，改善肠道IR的作用及其机制，从而为健脾清化方治疗T2DM提供提供理论依据。总之，本研究基本上圆满完成了任务计划书中的主要研究内容，达到了预期目标。