冬凌草甲素诱导TLR4-NLRP3炎症小体-自噬途径吞噬清除白血病细胞凋亡小体调控机制研究

Induced apoptosis of tumor cells is the main way for tumor therapy, however, if the large number of apoptotic corpse (apoptotic bodies) not be engulfed and removed rapidly, toxic and inflammatory contents will be released to cause internal-environment disturbance, gene mutation, inflammatory injury and immune deficiency disorder, which may further lead to tumor recurrence. The medicine which targeting endocytosis and clearance of tumor apoptotic bodies as well as the mechanism are rarely reported and remains unclear. In our previous study, we found that oridonin could enhance endocytosis of U937 leukemia cells apoptotic bodies by phagocyte through PI3K, Ras, inflammation and autophagy pathways. Nevertheless, the starting targets and key pathways are required to further investigate. In this study, human and murine macrophages will be incubated with UV-induced apoptotic bodies of leukemia cells in vitro, and the regulatory mechanism of Toll-like receptor 4（TLR4）-NLRP3 inflammasome-autophagy pathway in oridonin enhanced endocytosis of apoptotic bodies will be investigated by fluorescent staining, flow cytometry, siRNA and plasmid transfection. The relationship and interaction between TLR4-NLRP3 and autophagy will be revealed. The hypothesis will also be verified in vivo through detecting the endocytosis function of murine peritoneal and peripheral blood macrophage and some immune indices. The original results of promoted effect of oridonin on endocytosis of leukemia apoptotic cells as well as its targets and mechanism will provide the new ideas for leukemia or other tumor adjuvant therapy. It may assist in prevent recurrence, immune regulation and improve the tumor microenvironment.

肿瘤治疗后，体内大量细胞凋亡残骸（凋亡小体）若不及时清除，毒性、炎性物质外泄将引起内环境紊乱、基因突变等，导致肿瘤复发。调控肿瘤细胞凋亡小体吞噬清除的药物及机制尚不明确。我们前期研究发现冬凌草甲素可促进人巨噬细胞吞噬白血病U937细胞凋亡小体，并通过激活PI3K、Ras、炎症通路诱导自噬协同吞噬，然而其调控吞噬的启动性靶点和关键性通路有待更深入揭示。本研究通过体外培养人和小鼠巨噬细胞与白血病细胞凋亡小体共同孵育，及体内腹腔巨噬细胞、外周血巨噬细胞吞噬白血病细胞凋亡小体等模型，采用荧光染色、流式细胞术、siRNA、质粒转染等技术，考察冬凌草甲素促进吞噬作用中，Toll样受体4（TLR4）及其可能激活的NLRP3炎症小体的调控作用，并探索此通路与自噬的相互作用，阐明自噬协同吞噬机制。揭示冬凌草甲素促吞噬作用及关键靶点，将为肿瘤凋亡小体的吞噬清除、防止复发、改善微环境、免疫调节等治疗提供依据。

本研究采用紫外线诱导白血病系肿瘤细胞凋亡，与巨噬细胞共同孵育，旨在模拟机体大量凋亡细胞聚集产生的生理病理过程，尤其在肿瘤放化疗后，机体大量凋亡细胞存在的情况，考察天然药物冬凌草甲素对巨噬细胞吞噬凋亡小体的作用及机制。研究在体外人单核U937巨噬细胞，小鼠Raw264.7巨噬细胞及体内腹腔巨噬细胞，血液单核细胞等巨噬细胞中，通过细胞生物学、药理学、生化免疫等多种技术手段，揭示了低剂量冬凌草甲素能够促进巨噬细胞吞噬凋亡小体，其作用机制涉及激活膜上吞噬受体TLR4，促进其向核内转位，并调控下游MyD88-TRAF通路，激活NLRP3炎症小体；TLR4-NLRP3炎症小体的活化，促进巨噬细胞自噬，溶酶体数量、活力显著升高，炎症因子适度释放，促进巨噬细胞向凋亡细胞迁移，从而调控吞噬活力。同时，冬凌草甲素增强巨噬细胞对凋亡小体的廓清能力，对机体有一定免疫促进作用。课题基本按照计划，完成了相应实验内容，达到了预计目标，得到初步结论，现已发表SCI论文1篇，国际会议报告1次，培养本科毕业生6名，大学生创新项目3项。部分实验数据在整理中，预计再发表SCI论文1-2篇，申请专利保护1项。研究对冬凌草甲素促进吞噬作用及关键靶点的揭示，将为肿瘤凋亡小体的吞噬清除、防止肿瘤复发、改善微环境、免疫调节等研究治疗提供参考依据。