CB2受体激动剂对小鼠肝纤维化影响的机制

Liver fibrosis has always been a hotspot in liver disease research field，there is no effective treatments for liver fibrosis currently. Oxidative stress is considered critital in the pathogenesis of the liver diseases especially liver fibrosis. Previous studies showed that endocannabinoids system (ECS) played a very important role in regulating oxidative stress related liver injury. The dysregulation of ECS is associated with hepatitis, alcoholic liver disease and many other liver diseases. Activation of cannabinoid receptors-2 (CB2) by selective CB2 receptor agonist protects against hepatic ischemia/reperfusion damage by decreasing oxidative stress without addiction. CB2 receptor agonist appears to be an attractive target in managing liver diseases. Nuclear factor erythroid 2-related factor 2(Nrf2) is an important transcription factor regulating antioxidative stress reaction. In our previous study,we found CB2 receptor agonist protects against chronic-binge ethanol-induced liver injury by attenuating oxidative stress and liver inflammation. Our preliminary data also showed that CB2 receptors agonist can prevent liver fibrosis by attenuating liver oxidative stress. Therefore, we plan to examine the benificial effects of CB2 receptor agonist in mice hepatic fibrosis and explore the mechanism is related to increased antioxidative capability of liver by stimulating phase II enzymes expression through the Nrf2 pathway. The study will provide the experimental basis and theoretical foundation for the discovery of novel the anti-fibrosis drugs.

肝纤维化一直是肝病领域研究的热点，目前尚无有效的治疗方法。氧化应激是肝纤维化的重要发病机制。内源性大麻素系统(ECS)在炎症反应、氧化应激损伤中发挥重要的调节作用，ECS失调和肝炎、酒精性肝病等许多肝病有关系。选择性大麻素受体-2（CB2）激动剂能特异性结合CB2受体，从而减轻肝缺血再灌注损伤等疾病的氧化应激损伤，而无成瘾性，因此CB2受体激动剂可能为保肝药物研究的新亮点。核转录相关因子2 （Nrf2） 是细胞调节抗氧化应激反应的重要转录因子。申请人在国外前期实验发现，CB2受体激动剂能通过减轻氧化应激和炎症反应防治酒精性肝损伤。预实验也发现，CB2受体激动剂能减轻肝纤维化的程度及氧化应激损伤。因此，本课题拟研究选择性CB2受体激动剂对小鼠肝纤维化的作用，并探讨其机制是否与激活肝脏Nrf2及调控Ⅱ相酶的表达，增加机体的抗氧化能力有关。预期结果为抗纤维化药物开发提供一定的实验和科学基础。

肝纤维化的治疗目前尚无有效的方法,因此寻找预防和逆转肝纤维化的物质具有重要意义。内源性大麻素系统(ECS)在炎症反应、氧化应激损伤中发挥十分重要的调节作用，ECS失调和肝炎、酒精性肝病等许多肝脏疾病有关系。选择性CB2受体激动剂能特异性结合CB2受体，从而减轻肝缺血再灌注损伤等疾病的氧化应激损伤，而无成瘾性，因此CB2受体激动剂可能会成为保肝药物研究的新亮点。我们以CCl4诱导WT小鼠和CB2-/-小鼠肝纤维化模型4个月，同时用CB2受体激动剂AM1241及拮抗剂AM630干预。采用ELISA、Real-time PCR、Western blot等方法检测各相关指标的变化。另外用葡萄糖氧化酶诱导HSC-T6成氧化应激状态，分别用AM1241和拮抗剂AM630干预，采用细胞免疫化学染色法、ELISA、Western blot等方法观察各组HSC-T6胞浆或培养上清液各指标的变化。结果发现1）CCl4诱导CB2-/-小鼠发生更严重的肝纤维化表明CB2受体在CCl4诱导的小鼠肝纤维化中具有抗肝纤维化的作用；2）CB2受体激动剂可缓解肝损伤和肝纤维化发展； 3）CB2受体激动剂可缓解CCl4诱导的小鼠肝纤维化可能与其下调p-ERK1/2-p-CREB-Bcl-2信号通路，促进活化HSC凋亡有关。 4）大麻素CB2受体激动剂AM1241可抑制HSC-T6的增殖与活化，其机制可能与AM1241经大麻素CB2受体通道，激活HSC-T6细胞内Nrf2，使Nrf2发生核内转移，导致Nrf2和HO-1的蛋白表达量上调，增加HSC-T6的抗氧化作用有关。结果期望为抗纤维化药物开发提供一定的实验基础和科学依据。