TLR2对急性脑缺血微血管内皮细胞MMP-2/9上调机制研究

Toll-like receptors（TLRs）are important recognition receptrors of the innate immune system and have the ability bo bind endogenous danger-associated molecular pattern ligands. The engagement of TLRs by different ligands triggers the activation of signaling cascades and ultimately induce the activation of mitogen-activated protein kinases (MAPKs),such as extracellular signal-regulated kinase1/2(ERK1/2) which lead to transcription factor activation and generation of cytokines and chemokines.Recent evidence suggests that TLR2 may play a key role in the evolution of brain damage following cerebral ischemia through ERK1/2 signal pathway.But few have been known.Evidence suggests that matrix metalloproteinase-2/9 (MMP-2/9) have been implicated to play a deleterious role on blood brain barrier(BBB) by degrading the tight junction proteins and basal lamina proteins, thereby leading to BBB leakage, leukocyte infiltration, brain edema, and hemorrhage during acute stroke. TLRs are located on brain microvascular endothelium cells(BMECs). MMP-2/9 can be also expressed on BMECs.This study is intended to investigate whether Toll-like receptor 2(TLR2) upregulate MMP-2/9 activity during acute phase after cerebral ischemia. In vitro BMECs will be cultured. The expression of TLR2 and MMP-2/9 on BMECs are observed by fluorecent immunohistochemical staining. TLR2，mitogen- activated protein kinase ERK1/2, MMP-2/9 and tight junction proteins(occludin, claudin-5 and ZO-1) expression on BMECs will be measured by western blot before and after oxygen-glucose deprivation，TLR2 ligand Pam3CSK4,TLR2-blocking anti-TLR2 antibody (T2.5)，and ERK1/2 inhibitor U0126 respectively.The change of TLR2, ERK1/2 and MMP-2/9 mRNA will be examined by RT-PCR.Middle cerebral artery occlusion(MCAo) in the rat will be used as the ischemia model.The anatomical location of TLR2 and MMP-2/9 on BMECs will be determined by triple fluorescent immunohistochemical staining.At different time points after ischemia /reperfusion, the combination of laser capture microdissection and RT-PCR allows the quntification of the TLR2,ERK1/2 and MMP-2/9 mRNA on BMECs.The protein expression of tight junction proteins will be determined by using western blot.To examine the potential benefit of TLR2 inhibition, rats will be treated intraarterially with T2.5.After T2.5 treatment tight junction proteins will be measured respectively by western blot. TLR2,ERK1/2 and MMP-2/9 mRNA on BMECs will be obtained by the combination of laser capture microdissection and RT-PCR. BBB permeability,brain edema and infarction volume will also be determined.This study will elucidate the molecular mechanism of TLR2 on regualtion of MMP-2/9 through ERK1/2 signal pathway on BMECs after cerebral ischemia.It will also investigate the potential treatment of TLR2 inhibitor on acute cerebral ischemia by BBB protection.

近年发现Toll样受体2（TLR2）可能通过细胞外信号调节激酶（ERK1/2）参与炎症反应。急性脑缺血后，基质金属蛋白酶-2/9（MMP-2/9）上调可致血脑屏障（BBB）破坏，但机制不全清楚。TLR2是否介导脑微血管内皮细胞（BMEC）MMP-2/9上调致BBB破坏，尚无报道。故培养BMEC，通过激活与抑制TLR2及氧-葡萄糖剥夺，用免疫荧光法定位TLR2与MMP-2/9，用RT-PCR与western blot法分别测定TLR2、ERK1/2、MMP-2/9及BBB紧密连接蛋白的mRNA与蛋白表达。大鼠大脑中动脉闭塞后，用免疫荧光法观察BMEC上TLR2与MMP-2/9表达；用激光显微切割与RT-PCR技术测定二者mRNA含量。最后予抗TLR2抗体治疗，观察BBB破坏、脑水肿及脑梗死变化。揭示急性脑缺血，TLR2对BMEC MMP-2/9上调机制及应用TLR2抗体对脑缺血治疗的意义。

探讨急性脑缺血后，Toll样受体2（TLR2）是否介导脑微血管内皮细胞（BMEC）MMP-2/9上调，并致血脑屏障（BBB）破坏。.体外培养BMEC给氧糖剥夺复氧实验（OGDR），观察MMP-2/9、TLR2和紧密连接蛋白（TJs）的蛋白表达。给予TLR2激动剂-Pam3CSK4，研究MAPKs信号通路对MMP-2/9表达的影响。建立大脑中动脉闭塞2 h再灌注模型，给TLR2抗体T2.5。再灌注24h时测定脑梗死体积、脑水肿程度、血脑屏障通透性，神经功能缺损评分以及缺血皮质TLR2、MyD88和MMP-9表达。在MCAO1h后，在脑缺血/再灌注前30min侧脑室注射JNK的抑制剂SP600125，再灌注48h评估神经功能缺损，脑梗死体积，脑水肿及p-JNK/JNK，MMP-9蛋白表达。.①OGDR模型BMECs上MMP-9的表达明显增高,TLR2表达亦明显增高。TJs蛋白表达在复氧培养不同时间亦不同程度下调。②抑制TLR2能下调MMP-9的表达和上调BMECs上TJs蛋白的表达。加入T2.5后，复氧培养6小时组和24小时组MMP-9蛋白表达均明显减低；TJs蛋白表达有上调趋势。③用Pam3CSK4刺激BMECs，能明显上调MMP-9的表达，而MMP-2 表达却明显下调；claudin 5，ZO-1和collagen Ⅳ蛋白表达明显下调。④用Pam3CSK4刺激BMECs，pERK1/2、pJNK、pP38和pNF-κB 四种磷酸化蛋白表达明显增高。⑤研究发现 ERK1/2 和 JNK 抑制剂能明显下调Pam3CSK4引起的MMP-9增高。⑥大鼠脑缺血再灌注后6 h，TLR2和MyD88蛋白表达显著升高；而MMP-9在缺血再灌注后24 h才显著升高。缺血再灌注后24 h时，BBB通透性、脑水肿、脑梗死体积和神经功能缺损评分均较假手术组增高；给与T2.5后，上述结果显著降低。⑦SP600125可下调p-JNK/JNK，MMP-9的表达，减轻缺血脑损伤。.细胞实验示T2.5能下调BMECs经OGDR后MMP-9的表达；TLR2受体激动剂Pam3CSK4激活TLR2，通过ERK1/2与JNK信号途径调节MMP-9的表达，且能通过NF-κB信号通路负性调节MMP-2/9的表达。T2.5可能通过TLR2-MyD88信号通路，抑制MMP-9表达，减轻BBB破坏与缺血再灌注损伤。