大麻素受体2靶向调控代谢重编程对急性肝衰竭保护作用的机制研究

Inflammation is critical for the development of acute liver failure (ALF). The proliferation of hepatic macrophages plays a key role in the inflammatory process of ALF. Cannabinoid receptor 2 (CB2R) is mainly expressed on immune cells, and up-regulated in liver macrophages in injured liver.Our previous experiments have shown that activation of cannabinoid receptor 2 (CB2R) in mice protects ALF as well as reducing the level of pyruvate kinase M2, lactate dehydrogenase and lactate, which are key indicators of metabolic reprogramming.In consist with literature that the proliferation and activation of macrophages requires metabolic reprogramming, we hypothesized that CB2R regulates the inflammatory response by regulating metabolic reprogramming. This project designed to focus on the PI3K/AKT/mTOR/HIF-1 alpha signal pathways related to metabolic reprogramming. And we are planning to prove that CB2R inhibits those pathways to suppress macrophages metabolic reprogramming and proliferation using multiple molecular manipulations such as siRNA interference. We expect to elucidate the effects and mechanisms of CB2R regulated metabolic reprogramming pathways in triggering inflammation of hepatocytes by means of co-culture. Finally, our theory will be validated in the CB2R-/- mouse model of ALF. This project will provide new insights of pathological roles of CB2R in ALF and provide new therapeutic targets for ALF treatment.

炎症反应是急性肝衰竭（ALF）的核心环节。肝巨噬细胞增殖活化在ALF炎症过程中起关键作用。大麻素受体2（CB2R）主要表达于免疫细胞，其在受损肝脏的巨噬细胞上表达明显上调。我们近期实验显示：特异性激活CB2R在保护小鼠ALF的同时，还降低肝组织代谢重编程关键指标丙酮酸激酶M2、乳酸脱氢酶A和乳酸水平。结合文献报道，我们推测：ALF炎症反应涉及巨噬细胞代谢重编程过程，CB2R靶向调控这一过程保护ALF。本项目拟围绕代谢重编程的关键信号通路PI3K/AKT/mTOR/HIF-1α为核心轴，结合CB2R-/-的ALF小鼠和巨噬细胞炎症模型，用生物分子学和siRNA干扰等技术，捕获ALF炎症过程涉及代谢重编程的关键证据；通过共培养等方法，揭示CB2R调控代谢重编程，抑制巨噬细胞增值活化和减轻肝脏炎症的分子机制。本项目从代谢重编程角度切入，将为ALF炎症机制及治疗靶点提供新思路和新依据。

炎症反应是急性肝衰竭（ALF）的核心环节。肝巨噬细胞增殖活化在ALF炎症过程中起关键作用。大麻素受体2（CB2R）主要表达于免疫细胞，在受损肝脏的巨噬细胞上表达明显上调。前期研究发现CB2R激动剂GW405833可保护ALF小鼠，降低肝组织糖代谢关键酶丙酮酸激酶M2与乳酸脱氢酶水平。故本研究从糖代谢角度切入，以阐明CB2R调控糖代谢、抑制巨噬细胞增殖活化减轻肝脏炎症的分子机制。首先，我们利用小鼠急性肝衰竭模型，以血生化结果、肝脏病理、肝脏细胞凋亡多角度验证了大麻素受体2激动剂GW405833对急性肝衰竭的保护作用，并发现大麻素受体2激动剂GW405833可减少急性肝衰竭过程中的肝脏巨噬细胞浸润，抑制炎症因子释放。随后，我们利用巨噬细胞RAW264.7构建了体外炎症模型，验证了大麻素受体2激动剂GW405833可抑制LPS诱导的巨噬细胞增殖、炎症因子释放，并改变其糖代谢过程（增加LPS诱导下的有氧磷酸化，抑制其糖酵解），并通过构建干扰CB2R表达的稳转细胞株再次验证前述结果，同时发现GW405833的作用可能与HIF-1α有关。进而，我们建立了干扰HIF-1α表达的稳转细胞株，验证了GW405833靶向HIF-1α发挥减少巨噬细胞炎症因子释放及抑制糖酵解的作用。最后，使用CB2R-/-小鼠再次验证激活大麻素受体2对急性肝衰竭的保护作用及GW405833通过激活大麻素受体2从而减少HIF-1α，进而发挥调节巨噬细胞糖代谢、增殖及炎症因子释放的效应。综上所述，我们阐明了大麻素受体2激动剂可通过靶向HIF-1α调节肝脏巨噬细胞的糖代谢（糖酵解/有氧磷酸化），抑制其增殖及炎症因子释放，进而发挥保护急性肝功能衰竭的作用。

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