参苓白术散调控miR223介导单纯性肥胖巨噬细胞极化的机制研究

M1/M2 macrophage polarization in adipose tissue is considered to be an important pathogenesis of obesity-induced insulin resistance (IR).Recent study shows that miRNA223 is an important dual-directional regulatory factor of M1/M2 macrophage polarization. Traditional Chinese Medicine considers that the Spleen-deficicency and Phlegm Stagnation is the basic pathogenesis of obesity. Shenling Baizhu San is the classical TCM prescriptions for Strengthening Spleen and Removing Dampness and had good effect on weight reduction, but its mechanism has not been reported. Our previous work found that the patients with Spleen-deficicency and Phlegm Stagnation syndrome have an obvious imbalance of Th1/Th2 ratio in peripheral blood and is closely related to glucose and lipid metabolism. Th1/Th2 influence M1/M2 polarization directly. Shenling Baizhu San had been found can regulated the cell immunity by changed T cell subsets. Further research shows that there is differentially expressed of miRNA in the patients with Spleen-deficicency and Phlegm Stagnation syndrome and chinese medicine had regulated effects on it. Thus, we suppose that Shenling Baizhu San can regulate the M1/M2 macrophage polarization in adipose tissue and improve IR. Its effect target was miR223 probably. This study use the IR obesity mice model to observe the intervention effect of Shenling Baizhu San in miRNA223-mediated macrophage polarization ; use the co-culture system of U937 cells and SW872 cells, over expressed or inhibeted miR223, to study the regulation mechanism of Shenling Baizhu San in miRNA223-mediated macrophage polarization. This study aims at provide the experimental basis for prevention and treatment of obesity with TCM.

脂肪组织巨噬细胞极化失衡是肥胖致胰岛素抵抗（IR）的重要机制。新近研究发现miR223是双向调节巨噬细胞极化的重要调节因子。脾虚痰湿是肥胖的基本中医病机。参苓白术散是健脾祛湿经典方，临床发现它减重效佳,但其作用机制研究尚未见报道。前期研究发现脾虚痰湿者Th1/Th2比例明显失衡且与糖脂代谢紊乱相关，Th1/Th2可直接影响巨噬细胞分化。研究提示参苓白术散可调控细胞免疫，因此，我们推测参苓白术散具有调控巨噬细胞极化的作用。研究还发现脾虚痰湿者存在miRNA差异表达且党参等参苓白术散成分可调节miRNA，故推测其作用靶点可能与某种miRNA有关。本项目采用肥胖IR小鼠模型，观察参苓白术散对miR223介导的巨噬细胞极化的干预作用；采用U937巨噬细胞与SW872脂肪细胞共培养体系，过表达或抑制miR223，研究参苓白术散调控miR223介导的巨噬细胞极化的机制，为中医药防治肥胖提供实验基础。

有效防治单纯性肥胖及其并发症是一个严重的公共卫生问题，也是目前国内外研究热点。本研究以C57BL/6J肥胖小鼠模型、Raw246.7巨噬细胞及3T3L-1脂肪细胞为研究对象，通过实验研究探讨Pknox1在miR223介导肥胖脂肪组织M1/M2极化中的调控机制及健脾祛湿法的干预作用：1、观察miR223对Raw246.7巨噬细胞及3T3L-1脂肪细胞共培养体系Pknox1、PPARs表达情况及JAK/STAT6通路的影响。2、观察健脾祛湿法对C57BL/6J肥胖小鼠miRNA223水平及腹部脂肪组织中M1/M2极化的影响。3、进一步观察健脾祛湿法对C57BL/6J肥胖小鼠腹部脂肪中的Pknox1、PPARs表达及JAK/STAT6通路的影响。结果发现参苓白术散可明显改善C57BL/6J肥胖小鼠的糖脂代谢相关指标，增加胰岛素及C肽分泌，并且可使脂肪组织的巨噬细胞向M1分化减少，向M2分化增多，从而导致血清及脂肪组织中的M1相关炎症因子TNF-a水平减少，M2抗炎因子Arg1水平增加。进一步机制研究发现参苓白术散主要是通过增加miR223水平，并进一步通过影响其下游通路Pknox1-PPARs-JAK/STAT6的磷酸化水平来实现对M1/M2极化的调控。细胞实验证实，给予miR223过表达或抑制miR223表达均可出现上述通路的磷酸化水平的改变，给予参苓白术含药血清处理后，可逆转这些改变。中医药在防治单纯性肥胖及其所致的IR取得了较好的疗效，但其作用机制不清，基础研究不深入，导致在临床应用中无法更有效地更有针对性进行加减辨证，从而极大地影响了中医药临床应用和发展。本项目在前期的临床与实验研究基础上，选择确有疗效的经典方参苓白术散，以机体免疫状态（M1/M2活化）、表观遗传（miR223）和信号通路（JAK/STAT）变化为切入点，结合现代生命科学技术，探讨参苓白术散调控M1/M2极化、改善胰岛素敏感性的分子机制，为中医药防治单纯性肥胖提供理论依据，有非常重要的探索意义和科学价值。