基于Calpain与NLRP1炎症小体信号相互作用的局灶性脑缺血固有免疫调节机制研究

NLRP1 inflammasome is one of key factors in innate immune response through mediating the activation of caspase-1subsequently, processing pro-interleukin-1β and pro-interleukin-18, and releasing bioactive interleukin-1β and interleukin-18. It is composed of NLRP1, apoptosis-associated speck-like protein containing a CARD (ASC), pro-caspase-1, pro-caspase-11, P2X7 reseptor, pannexin-1 channel, and X-linked inhibitor of apoptotis (XIAP) in neuron, however, it is composed of NLRP1, ASC, and pro-caspase-1 in macrophages. NLRP1 inflammasome is reported to be involved in ischemic brain injury. Under pathological conditions, calpain, a calcium-dependent neutral protease, promotes the release of high mobility group box-1 protein (HMGB1), and cleaves some important protreins in NLRP1 inflammasome signaling pathway, such as Bcl-2, Bcl-xL, XIAP, and IκB (an endogenous inhibitor of NF-κB), by which, participating in the activation of NLRP1 inflammasome signaling pathway. On the other hand, the activation of NLRP1 inflammasome signaling pathway may be up-regulate the calpain activity through the mechanisms as follows：(1) cleaving calpastatin, an endogenous calpain inhibitor; (2) promoting the release of HMGB1 and IL-1β, subsequently, activating NMDA receptors, and resulting in the intracellular calcium overload. In previous study, we have found that calpain participated in the inflammatory response during focal cerebral ischemia in rats. Therefore, we hypothesized the existence of the positive interaction between calpain signal and NLRP1 inflammasome signaling pathway, which would be contributed to the activation of innate immune response and inflammation during focal cerebral ischemia. This project intends to investigate the interaction between calpain signal and NLRP1 inflammasome signaling pathway and the mechanisms in various cell types (including neuron, astrocyte and microglia) in penumbra and core in a rat model of focal cerebral ischemia by means of the approaches of pharmacology, biochemistry, molecular biology, immunology and morphology, by which, demonstrating our hypothesis, elucidating the new mechanism of penumbra evolution and reversion, and providing new target and strategy for the prevention and therapy of ischemic cerebrovascular diseases.

NLRP1炎症小体参与局灶性脑缺血(FCI)引起的固有免疫应答。体外研究发现蛋白酶Calpain可能通过促进HMGB1释放，裂解NLRP1炎症小体信号通路的一些重要蛋白(如Bcl-xL、XIAP和IκB)，参与NLRP1炎症小体信号的激活。另一方面，NLTP1炎症小体信号的激活能裂解内源性Calpain抑制剂，升高细胞内钙，上调Calpain的活性。前期研究提示Calpain参与FCI后的炎症反应。因此，我们推测在FCI时，Calpain信号与NLTP1炎症小体信号之间可能存在正性的相互作用，共同参与FCI引起的固有免疫应答和炎症反应。本研究拟在前期工作的基础上，利用大鼠FCI模型，研究Calpain信号与NLRP1炎症小体信号之间的相互作用及其机制，阐述FCI后固有免疫应答调节以及半暗带演变和逆转的新机制，为缺血性脑血管病的防治提供新靶点和新策略。

炎症小体(Inflammasome)是固有免疫应答的关键组成部分。研究发现NLRP1炎症小体和钙离子依赖性中性蛋白酶Calpain参与脑缺血引起的炎症应答。本研究本研究利用大鼠局灶性脑缺血再灌注模型，研究了脑缺血不同病变区域(半暗带和核心区)，主要研究局灶性脑缺血再灌注损伤时Calpain信号与NLRP1炎症小体信号之间的相互调控机制。研究发现：研究发现：（1）阻断Calpain的过度活性或抑制NLRP1炎症小体信号（HMGB1、NF-kB或Caspase 1抑制剂以及NLRP1 siRNA）能改善神经功能，减轻细胞的凋亡性死亡，缩小梗死体积；（2）阻断NLRP1炎症小体信号通路（HMGB1、NF-kB或Caspase 1抑制剂以及NLRP1 siRNA）能阻断大鼠局灶性脑缺血再灌注损伤后半暗带和核心区Calpain信号的激活；（3）阻断Calpain活性能降低缺血半暗带和核心区核蛋白HMGB1的释放，下调半暗带和核心区NF-B信号，抑制NLRP1炎症小体的激活；（4）Bcl-xL过表达能能降低缺血半暗带和核心区Calpain信号的激活，下调NF-B信号，抑制NLRP1炎症小体的激活，改善缺血大鼠的神经功能，缩小梗死体积。这些结果证实局灶性脑缺血再灌注损伤时，Calpain的过度活化、Bcl-xL参与NLRP1炎症小体信号的激活，而Bcl-xL和NLRP1炎症小体信号的激活也参与Calpain的活化。结果提示局灶性脑缺血再灌注损伤时，Calpain信号与NLRP1炎症小体信号之间存在相互调制作用，共同参与局灶性脑缺血再灌注引起的NVU损伤，其中Bcl-xL可能在Calpain信号与NLRP1炎症小体信号相互调制中其关键作用。本项目阐述了固有免疫应答参与局灶性脑缺血再灌注损伤的新机制，为缺血性脑血管的防治提供了新的理论基础。