巨噬细胞CB2R激活介导的Bcl-2降解在抑制巨噬细胞HMGB1释放中的作用与机制研究

Controlling inflammation is an important strategy in the treatment of inflammation-related diseases. As a late-acting inflammatory mediator, extracellular HMGB1 (High-mobility group box 1) could boost inflammation leading to tissue injury and organ dysfunction. Meanwhile, HMGB1 in cytoplasm could compete with Bcl-2 to bind with Beclin-1, leading to enhancement of autophagy which could in turn inhibit inflammation. This suggestes the important role of Bcl-2 in this process. Our previous researches showed that activation of cannabinoid receptor Ⅱ (CB2R) could inhibit the HMGB1level in plasma of animal with sepsis and supernatant of macrophages stimulated by lipopolysaccharide (LPS), thus, protect against inflammation, but the mechanism is not clear. Here we propose a hypothesis that activation of CB2R could enhance autophagy by promoting Bcl-2 degradation to inhibit inflammation and HMGB1secretion in macrophage. The project will focus on autophagy and HMGB1 of macrophage, is being designed to study the role of CB2R-regulated Bcl-2 degradation on its promotion of autophagy and inhibition of HMGB1 secretion in macrophage, furthermore, to reveal the mechanisms involved in the CB2R-mediated anti-inflammatory function. The work is meaningful for the treatment of inflammatory diseases.

控制炎症是炎症相关性疾病治疗的重要策略。高迁移率蛋白1（high mobility group box 1，HMGB1）在胞外可扩大炎症反应，同时胞内HMGB1能与Bcl-2竞争结合Beclin-1，促进细胞自噬并抑制炎症，反映Bcl-2在这一过程中作用关键。我们前期研究发现：Ⅱ型大麻素受体 (cannabinoid receptor Ⅱ，CB2R)激活能有效抑制动物血清和巨噬细胞培养上清中HMGB1的产生，发挥保护作用，但其机制并不清楚。由此提出假说：CB2R通过促进Bcl-2降解，从HMGB1-Beclin-1途径促进巨噬细胞自噬并抑制HMGB1释放，并发挥抗炎保护作用。本项目拟以巨噬细胞自噬和HMGB1为聚焦点，利用基因改造、药理学和分子生物学等方法系统地研究Bcl-2降解在CB2R抗HMGB1释放和促进细胞自噬中的作用和机制，阐明CB2R的抗炎机制，对炎症相关性疾病的治疗意义重大。

本项目着眼于如何通过有效途径抑制HMGB1从细胞中分泌到胞外，防止其产生扩大化的病理作用，从而达到控制病理损伤、延缓疾病进程的目的。.受资助项目《巨噬细胞CB2R激活介导的Bcl-2降解在抑制巨噬细胞HMGB1释放中的作用与机制研究(81501364)》，取得了如下研究成果：.1）CB2R激动剂GW405833（以下简称GW）能够抑制LPS诱导的巨噬细胞HMGB1释放，但不影响HMGB1 mRNA表达水平；ELISA结果显示，与对照组相比，GW并不影响HMGB1胞外释放；运用WB方法，我们观察到抗炎有效剂量的GW单独处理巨噬细胞能时间依赖性促进胞内HMGB1蛋白的降解，提示CB2R激动剂对HMGB1的抑制作用发生在细胞内。.2）我们进一步探索CB2R激动剂致HMGB1降解的途径，利用自噬阻断剂3-MA及泛素化蛋白酶体抑制剂MG-132，我们发现3-MA能阻断GW诱导的HMGB1降解，但MG-132不能，提示GW可能通过自噬溶酶体途径促进胞内HMGB1降解；同时，我们发现GW能时间依赖性和剂量依赖性促进巨噬细胞自噬，并促进HMGB1核转位，3-MA能抑制GW诱导的HMGB1核转位，说明CB2R激动剂可能通过促进巨噬细胞自噬进而促进胞内HMGB1分子的核转位。3）接下来我们发现Cathepsin B特异性抑制剂CA-74Me能阻断GW诱导的HMGB1降解，提示胞内HMGB1可能是Cathepsin B底物。.总之，受基金资助，本项目以巨噬细胞自噬和HMGB1为聚焦点，深入系统地研究了CB2R激动后是如何通过促进自噬来影响胞内HMGB1的行为并促进其降解，最终达到抑制其胞外分泌的目的。