Cathepsin S对小鼠脑缺血/再灌注损伤的作用及其机制

Immunity and inflammation are key elements of the pathobiology of stroke. Recent developments have revealed that stroke engages both innate and adaptive immunity.Anti-inflammatory and immunomodulation are promising therapeutic approaches. Cystein protease Cathepsin (Cat S) can liberate a kind of chemokine, fractalkine (FKN), from the membrane of neurons, and the free soluble FKN can aggravate inflammation during acute stroke by recruitment of inflammtory cells via chemotaxis. Cat S also plays a key role in antigen presentation and T cell adaptive immunity and could be engaged in chronic autoimmune brain damage after ischemia and influence long-term neurological recovery. Our preliminary experiments showed that Cat S gene knock out could protect against functional and tissue damage due to transient focal brain ischemia at 24 h after MCAO/reperfusion, and could protect against functional damage during 7 d after MCAO/reperfusion; Cat S inhibitor (LHVS, i.c.v) could also protect against acute brain ischemic damage at 24h. Therefore we hypothesizes that Cat S can aggravate acute brain ischemic damage via FKN pathway, and perniciously influence long-term neurological recovery via T cell adaptive immunity after stroke. In this study, we will use various research methods like gene knock-out to investigate the effects of Cat S on the acute and long-term brain ischemic damage in vivo and in vitro ischemic models (including acute and long term animal models), and try to carify the protective effect of Cat S inhibition,as a kinde of anti-inflammatory or immunomodulation agent,against brain ischemia.

固有免疫和适应性免疫分别参与急性和慢性脑缺血损伤，脑缺血抗炎治疗研究方兴未艾。半胱氨酸蛋白酶（Cathepsin S，Cat S)可以裂解神经细胞膜上的趋化因子Fractalkine（FKN），可能通过FKN加剧急性脑缺血的固有免疫反应；另外，Cat S是适应性免疫中的关键因子，可能参与缺血后的慢性自身免疫性脑损伤。我们的预实验发现，Cat S基因敲除可以保护小鼠局灶脑缺血后24 h神经功能或者组织学的损害，也可以保护局灶脑缺血后7 d之内的神经功能损害；Cat S抑制剂也可以保护急性脑缺血。由此，我们推测，Cat S可通过FKN途径参与急性缺血损伤；也可通过适应性免疫影响缺血后长期脑功能恢复。本研究中，我们将利用体、内外脑缺血模型（包括急性和长期模型）、采用基因敲除等手段，深入阐明Cat S在急性和长期脑缺血损伤中的作用和机制，探索Cat S抑制作为免疫调节在脑缺血治疗中的作用。

目的：本研究旨在研究Cathepsin S对小鼠脑缺血/再灌注损伤的作用及其机制，以及Cathepsin K对急性脑梗死后出血转化患者和rt-PA诱导的小鼠脑梗死后出血转化的影响。.方法：建立稳定的小鼠脑线栓栓塞再灌注（缺血时间为90 min，再灌注24h）模型及rt-PA诱导的脑梗死后出血转化（缺血时间为60 min，股静脉注射rt-PA 30 min）小鼠模型，进行神经行为学评分、出血转化评分、血红蛋白浓度检测、伊文思蓝血脑屏障通透性检测、western-blot紧密连接蛋白检测，并采用Image J软件计算脑梗死体积。.结果：成功建立了小鼠脑线栓栓塞再灌注模型；根据麻醉方式不同，Cathepsin S对小鼠脑缺血/再灌注损伤的作用不同。腹腔注射水合氯醛麻醉的小鼠梗死体积大，神经行为学损害严重，Cathepsin S对小鼠脑缺血/再灌注损伤有一定的保护作用；而气体麻醉的小鼠梗死体积小，神经行为学损害较轻，未见到Cathepsin S对小鼠脑缺血/再灌注损伤的保护作用。在急性缺血性脑卒中静脉溶栓后发生出血转化的患者血清中Cathepsin K浓度较未发生出血转化的患者有明显的下降趋势；且在出血转化模型小鼠的脑组织中Cathepsin K的表达水平较Sham组小鼠明显下调。Cathepsin K基因敲除后会加重小鼠急性缺血性脑损伤后rt-PA诱导的出血转化，血脑屏障的通透性明显增加。该模型小鼠的神经功能学损伤加重，脑梗死体积及脑水肿体积增加。.结论：Cathepsin S对小鼠脑缺血/再灌注损伤的作用与脑梗死严重程度有关，脑梗死严重时Cathepsin S对其有一定的保护作用。Cathepsin K通过对神经血管单元产生影响，从而在急性脑梗死后rt-PA诱导出血转化的产生中发挥着重要的作用。在今后的研究中我们将增加各组小鼠的样本量，另外，Cathepsin S抑制剂LHVS对小鼠脑缺血/再灌注损伤的作用研究正在进行中，在稳定各方面实验条件时，我们也在进行Cathepsin S基因敲除对小鼠局灶脑缺血/再灌注后慢性（4 周后）脑功能损害的作用研究。