NLRP3炎性小体在脑缺血再灌注损伤中的作用及机制

Nucleotide-binding oligomerization domain like receptors (NLRs) play an important role in innate immunity-associated inflammatory responses. As the key member of NLRs family, NLR family, pyrin domain-containing 3 (NLRP3), assembles with adapter protein apoptosis-associated Speck-like protein (ASC), and procaspase-1 to compose NLRP3-inflammasome which is a hot topic in scientific research. It is confirmed that innate immunity is involved in the occurance and development of cerebral ischemia-reperfusion injury (CIRI), however, the role and mechanism of NLRP3-inflammasome in cerebral ischemia-reperfusion injury is unknown. Our recent studies demonstrated that the expression of NLRP3 mRNA and protein was significantly increased in the ischemic penumbra of mice after 24h reperfusion which is subsequent to 2h middle cerebral artery occlusion. Moreover, compared with wild-type mice, the neurological scores were decreased, the number of survival neurons in hippocampus CA1 region were increased, and the infarct volumes were obviously reduced in NLRP3-deficient middle cerebral artery occlusion (MCAO) mice model. It indicated that NLRP3-inflammasome is important for CIRI. We will establish the CIRI model both in vitro (primary microglia cells and BV-2 cells) and in vivo (NLRP3-/-, ASC-/- and wild-type mice) and use multiple assays/techniques, such as siRNA, transfection and co-immunoprecipitation (Co-IP), to verify the role and make clear the mechanism of NLRP3-inflammsome in CIRI. Our study will provide solid scientific and theoretic basis for targeting therapy in NLRP3-inflammasome signaling pathway. It will also provide new strategy for the prevention and treatment of cerebral ischemic disease.

NLRs家族在固有免疫炎症反应中发挥重要作用，NLRP3作为家族重要成员，与ASC，procaspase-1构成NLRP3炎性小体，成为目前研究热点。固有免疫参与了脑缺血再灌注损伤(CIRI)中炎症反应的发生发展，但NLRP3炎性小体在CIRI中的作用及机制在国内外研究领域尚无报道。本课题组已证实，脑缺血2h再灌注24h后缺血半暗区NLRP3表达上调，且与野生型小鼠模型相比，NLRP3-/-小鼠神经功能学评分及海马锥体存活细胞数目明显改善，脑梗死面积明显减少，提示NLRP3在CIRI中发挥重要作用。我们拟建立NLRP3、ASC敲除小鼠和野生型小鼠的体内外CIRI模型，应用siRNA，转染，免疫共沉淀等技术，首次从整体-脑组织-细胞多角度证明NLRP3炎性小体在CIRI中的作用，阐述其相关机制，为针对NLRP3炎性小体通路进行靶向治疗提供坚实的理论基础，为缺血性脑血管疾病的防治提供新的策略。

缺血性脑卒中是中风的主要类型，也是目前继心血管疾病之后的第二大死亡原因，目前尚无有效治疗措施。固有免疫参与了缺血性脑卒中炎症的发生发展，是造成脑缺血继发性脑损伤的关键因素之一。NLRs家族在固有免疫炎症反应中发挥重要作用，NLRP3及NLRP2作为家族重要成员，与ASC，caspase-1构成NLRP3及NLRP2炎性小体，成为目前研究热点，尤其是NLRP2炎性小体在脑缺血损伤中的作用及机制未见报道。本项目进行了系统的细胞及动物实验，从整体-脑组织-细胞多角度证明了NLRP炎性小体在大脑中动脉栓塞及糖氧剥夺造成的动物及细胞的脑缺血模型中的作用，并对其作用机制进行了探讨。研究表明，NLRP3, NLRP2, caspase-1 及ASC 在C57BL/6 小鼠脑缺血损伤后脑组织内表达明显升高。NLRP3基因敲除或者shRNA 基因沉默NLRP2均可通过改善神经病学评分及减少脑梗死体积而减轻脑缺血损伤。接下来我们发现NLRP3及NLRP2可以通过促进白介素-1β (IL-1β)及白介素-18 (IL-18)的自分泌/旁分泌而加重星形胶质细胞凋亡。研究进一步表明NLRP2通过促进炎症反应而加重糖氧剥夺诱导的星形胶质细胞的损伤。NLRP2通过NF-κB信号通路参与了缺血性脑损伤。研究首次揭示，NLRP2作为重要的靶点分子通过NF-κB介导的神经血管损伤进而参与了脑缺血损伤过程，为此提供了理论依据。研究结果进一步补充了脑缺血发病的病理生理机制。药物靶向NLRP3 或者NLRP2-介导的炎症反应可以为缺血性脑血管疾病的治疗提供新策略。