基于TLR4/NF-κB通路黄芪多糖调控miR-223对树突状细胞免疫活性的影响及机制研究

Immature and functional defect of dendritic cell (DC) leading to inefficient activation of cytotoxic T cell which results in important manifestation of Qi deficiency in cancer patients. Astragalus Polysaccharide (APS), as the main component of Astragalus which is one of the typical tonic traditional Chinese medicines, could promote immunocompetence of DC; however, the mechanism of APS immunocompetence of DC is still not clear. Researches show that miR-223 can regulate maturation, activation of DC. The expression of miR-223 is controlled by TLR4 pathway, and regulates the immunocompetence of DC through promoting nuclear translocation of NF-κB. Our previous work showed that APS could kill tumor cells through TLR4 pathway and decreased the expression of miR-223.Based on the theory of Traditional Chinese Medicine and previous work, we raise the hypothesis of our study is that: APS could improve the immunocompetence of DC through activating TLR4 pathway, which lead to the decreasing expression of miR-223, then promoting nuclear translocation of NF-κB. In this project, inhibitors of key factors on TLR4 pathway are used, and miR-223 is being over expressed also miR-223 target genes are blocked to understand DC immunocompetence effect of APS; This study will provide scientific support for the theory of“strengthening body resistance and eliminating evil”of traditional Chinese Medicine.

树突状细胞（DC）成熟障碍及功能缺陷，将不能有效活化T细胞杀伤肿瘤细胞。现代研究揭示，肿瘤患者成熟DC数量不足及功能缺陷是机体正气亏虚的微观体现。黄芪多糖(APS)是扶正中药黄芪的主要成分，可提高DC免疫活性，但作用机制尚不明确。研究表明miR-223对DC成熟、活化等功能有重要的调控作用，其表达受TLR4信号通路的影响，并通过调节TLR4下游NF-κB核移位影响DC免疫活性。我们前期工作证实，APS可通过TLR4信号通路发挥肿瘤细胞的杀伤作用，并可显著下调miR-223表达。据此我们提出：“APS通过抑制miR-223的表达调节TLR4信号通路下游NF-κB核转位，提高DC免疫活性”的假说。课题通过阻断TLR4及其下游关键因子、过表达miR-223、阻断miR-223靶基因等方案，考察APS对DC免疫活性的影响及作用机制，为APS抗肿瘤治疗及中医“扶正祛邪”理论奠定分子生物学基础。

现代研究揭示，成熟DC数量不足及功能缺陷是机体正气亏虚的微观体现。黄芪多糖(APS)是扶正中药黄芪的主要成分，可提高DC免疫活性，但作用机制尚不明确。在前期工作基础上提出：“APS通过抑制miR-223的表达调节TLR4信号通路下游NF-κB核转位，提高DC免疫活性”的假说。实验结果：①APS可有效激活DC的TLR4通路关键基因表达，在加入TLR4和MyD88抑制剂后，APS不能有效的激活TLR4通路；进一步实验得出APS可下调miR-223表达的影响，在加入TLR4和MyD88抑制剂后，APS不能显著下调miR-223的表达。②黄芪多糖可显著促进DC成熟、细胞因子的分泌；促进共培养T细胞增殖及活化；miR-223 过表达+黄芪多糖组及miR-223 过表达组，均无显著差异。初步证明APS可能是通过下调miR-223影响DC免疫活性。③黄芪多糖可促进C/EBPβ表达及NF-κB活化，通过过表达miR-223的DC进一步明确黄芪多糖通过miR-223促进C/EBPβ表达及NF-κB活化；抑制C/EBPβ表达后，APS不能促进DC 表面分子表达的变化、细胞因子水平的变化以及对DC共培养T细胞增殖及活化，明确黄芪多糖通过miR-223调节C/EBPβ影响DC免疫活性。④黄芪多糖对荷瘤小鼠实体瘤生长均具有一定的抑制作用，可显著提高脾指数及胸腺指数、外周血中IL-6、IL-12、TNF-α分泌；免疫荧光法检肿瘤组织中DC成熟的情况可发现，黄芪多糖高剂量组可显著增加肿瘤组织中浸润DC的成熟。⑤抗纤丸含药血清可显著促进DC成熟，提高IL-6分泌；抗纤丸（不含黄芪）组不能有效促进DC成熟及IL-6分泌，由此得出扶正祛邪方抗纤丸可影响DC免疫活性，其促进DC成熟及IL-6分泌作用与方中黄芪密不可分。结论：黄芪多糖通过TLR4通路抑制miR-223表达促进树突状细胞免疫活性的分子作用机制；进一步通过内体实验证明黄芪多糖对DC的免疫调节作用；并通过黄芪为君药的扶正祛邪方，证明黄芪对DC的免疫调节作用。为补益中药作用机制提供新的研究方案与思路，并为“扶正祛邪”理论注入新的科学内涵。