大麻素受体CB2介导脑室出血后米诺环素保护效应的机制研究

Intraventricular hemorrhage (IVH) is often associated with intracerebral hemorrhage (ICH), and is an independent risk factor for poor outcome after ICH. There is currently no effective treatment for IVH.Our previous studies found that minocycline could attenuate IVH-induced brain injury significantly, although the mechanisms remained unknown. Recent studies have shown that the cannabinoid type 2 receptor (CB2R) has potential neuroprotective properties for multiple neurological diseases. We noticed that some of the mechanisms proposed to explain the neuroprotective effects of CB2R activation including modulation of inflammation appeared to be similar with minocycline. Our preliminary results also showed that increased expression of CB2R after rat IVH model and treatment with minocycline, and CB2R antagonist AM630 blocked the protective effect of minocycline against brain edema after IVH. Based on these findings, our central hypothesis is that CB2R activation mediate minocycline-induced neuroprotection after IVH, by upregulation of MKP-1 and inhibition of MAPKs pathways. Following specific aims are proposed to address our hypothesis: ①determine the mechanisms of CB2R activation by minocycline administation following IVH; ②determine whether CB2R activation mediate minocycline-induced neuroprotection following IVH by using CB2R antagonist, and whether CB2R agonist inhibits inflammation and induces neuroprotection following IVH. ③determine whether minocycline-induced and CB2R-induced neuroprotection is mediated by upregulation of MKP-1 and inhibition of MAPKs pathways. Together, the proposed studies will explore the underlying molecular mechanisms of minocycline and CB2R-induced neuroprotection, and provide novel therapeutic strategies.

脑室出血（IVH）后脑损伤尚无有效治疗措施。我们前期研究发现米诺环素可显著减轻IVH后脑损伤，但机制不清。新近研究发现大麻素受体CB2R激活具有神经保护效应，我们注意到米诺环素与CB2R激活后下游效应相似，且预实验结果显示CB2R拮抗剂可阻断米诺环素的脑水肿改善作用。结合近期文献，我们推测：CB2R激活可能是米诺环素保护效应的关键环节，通过上调MKP-1表达，从而抑制IVH后MAPKs信号通路介导的炎症损伤。本项目拟通过体内外研究，阐明下述问题以验证假说：①米诺环素引起CB2R活化的环节及机制？②CB2R激活是否为IVH后米诺环素神经保护效应的关键环节?选择性CB2R激动可否抑制IVH后炎症反应及脑损伤?③MKP-1上调并抑制MAPKs信号通路介导的炎症反应是否为CB2R激活后神经保护效应的关键机制?本项目是首次从CB2R激活为切入点探讨IVH后神经保护策略，有望为脑损伤防治提供新的思路。

脑出血合并脑室出血(intraventricular hemorrhage, IVH)的发生率高达50%以上，目前尚无针对IVH以及继发脑积水的有效策略。本项目立足临床需求，基于课题组前期建立的改良实验性大鼠IVH模型以及新生大鼠ICH/IVH模型（GMH），使用米诺环素、大麻素CB2受体（CB2R）选择性激动剂JWH133及阻断剂AM630等进行干预，并采用神经功能缺损分析和分子生物学技术，阐明了米诺环素通过激活CB2R介导神经保护效应，并明确了CB2R激活在IVH及GMH实验动物神经功能缺损和急性期炎症以及脑室系统纤维化中的作用和分子机制。项目的主要科学发现如下：（1）证实了米诺环素通过上调激活小胶质细胞CB2R介导GMH新生大鼠神经保护效应；（2）明确了CB2R激活通过ERK/p38 MAPK信号通路调控小胶质细胞活化抑制GMH后早期炎症反应；（3）探索发现小胶质细胞细胞极化及其调控的海马区神经回路重塑是CB2R介导的GMH后神经保护效应的重要效应环节；（4）阐明了CB2R激活可抑制大鼠IVH后炎症反应并显著减少蛛网膜下腔区域的纤维化，从而减轻脑积水发挥神经保护效应。上述成果为米诺环素做为ICH/IVH治疗的潜在新策略提供了可靠的理论依据，并为IVH后脑损伤防治研究提供了新的思路。在研究计划外，根据研究结果调整实验方向，获得以下研究成果：初步探索发现阿托伐他汀可能通过上调激活PPARγ促进小胶质细胞对红细胞的吞噬功能，从而加速IVH血肿清除发挥神经保护效应，并在此基础上获得了2017年国家自然科学基金面上项目的资助。总之，本项目完成了预期目标，明确了CB2R激活对IVH后炎症调控和脑损伤的干预效应，及其在米诺环素神经保护作用中的意义，证实了IVH后CB2R通过ERK/p38 MAPK通路调控小胶质细胞活化发挥抗炎保护效应的分子机制，为IVH防治提供了新的思路；在研究计划外，新增研究方向亦取得了显著成果，为ICH/IVH防治提供了可能的潜在治疗靶点。