枸杞多糖经TLR/NF-κB通路对2型糖尿病炎症因子生成的抑制

Recent studies suggest that type 2 diabetes (T2DM) may be an inflammatory processes mediated by cytokines. Inflammation acts as an intermediary in the pathogenesis of T2DM. Intervention of inflammatory processes may reduce insulin resistance as well as islet beta cell apoptosis. Lycium barbarum polysaccharide is the main active constituent of famous traditional Chinese medicinal herb - Chinese wolfberry. In previous study, we found that Lycium barbarum polysaccharide could inhibit the serum TNF-α level of T2DM and thereby alleviating insulin resistance in T2DM patients. But its mechanism was not clear. TLRs is a kind of ligand to recognize potential pathogens. TLR2/4 has been shown to play an important role in the development of T2DM. TLR2/4 can identify polysaccharides and is involved in the regulation of immune system through the TLR/NF-κB signal transduction pathway for polysaccharides. Thus, it is hypothesized that TLR/NF-κB signal transduction pathway is inhibited to relieve insulin resistance by Lycium barbarum polysaccharide in T2DM. Therefore, this article studies the inhibited mechanism of inflammatory cytokines by Lycium barbarum polysaccharide through TLR/NF-κB signal transduction pathway in T2DM, in ordor to provide a theoretical basis for the application of Lycium barbarum polysaccharide on T2DM treatment.

近年的研究认为2型糖尿病（T2DM）可能是细胞因子介导的炎症反应，炎症在T2DM的发病中起媒介作用。干预炎症过程同时具有减轻胰岛素抵抗，减少胰岛β细胞凋亡的作用。枸杞多糖是我国传统名贵中药材--枸杞的主要活性成分。在前期的研究中我们发现枸杞多糖能够抑制T2DM患者血清炎症因子TNF-α的水平，进而缓解T2DM患者的胰岛素抵抗，但其机制尚不清楚。TLRs是一类能够识别潜在病原体结构的配体。其中TLR2/4已被证实在T2DM的发展中占有重要作用。TLR2/4能识别多糖，并通过TLR/NF-κB信号传导通路参与多糖对免疫系统的调节。由此我们猜测枸杞多糖是否通过TLR/NF-κB信传导号通路对炎症因子产生了抑制，并进而缓解了T2DM的胰岛素抵抗。因此，本文拟从TLR/NF-κB信号传导通路入手，研究枸杞多糖抑制T2DM炎症因子产生的机制，以期为枸杞多糖应用于T2DM治疗提供理论基础。

为了明确枸杞多糖是否通过TLR/NF-κB信号通路对炎症因子产生抑制，项目设计了两部分内容“枸杞多糖对NF-κB转导的MyD88依赖性途径的调节”和“枸杞多糖对MyD88非依赖性途径的调节”。项目采用总糖含量为77.38%的枸杞多糖，依照低剂量组（20mg/kg）、中（40mg/kg）、高（80mg/kg）分别干预KKAy（MyD88依赖性途径模型）和MyD88.KO（MyD88非依赖性途径模型）高糖小鼠，ELISA法检测小鼠的血清炎症因子水平，RP-PCR及Western blot分别测定小鼠腹腔巨噬细胞中的关键点的基因和蛋白的表达量（MyD88依赖性途径：TLR4、MyD88、TRAF6、IKKβ；MyD88非依赖性途径：TRIF、TRAM、RIP1、TRAF6）。并采用脂多糖（LPS）诱导小鼠巨噬细胞RAW264.7细胞，利用不同浓度（25、50、100ug/mL）LBP干预，Western blot检测各组细胞NF-κB的核转位数量及P-IκB、IκB的蛋白表达量。结果显示LBP能够激活MyD88依赖性通路关键点Tlr4、Myd88、TRAF6和IKKβ的基因及蛋白表达，进而影响其血清中IL-10、TNF-α和IL-1β的水平，并降低KKAy小鼠的血糖；但LBP虽然能够抑制MyD88非依赖性通路上TRIF、TRAM、RIP1、TRAF6基因表达，但对其蛋白表达作用不明显，高糖的MyD88.KO小鼠血清中各炎性因子水平变化紊乱，血糖未发生明显改变。本课题的结果表明MyD88依赖性通路在LBP调节炎症因子生成的过程中占据主导地位，同时LBP还能够抑制NF-κB核转位的发生，进而降低体内促炎症因子水平，增强抗炎症因子水平，发挥降糖作用；敲除MyD88基因后，LBP对2型糖尿病小鼠的血糖无影响。