异氟醚预处理对局灶性脑缺血再灌注损伤大鼠缺血半暗带TLR4-MyD88信号通路的影响

Effect of isoflurane preconditioning on TLR4-MyD88 signaling pathway in ischemic penumbra following focal cerebral ischemia-reperfusion in rats

目的探讨异氟醚预处理对局灶性脑缺血再灌注损伤大鼠缺血半暗带TLR4-MyD88信号通路的影响。方法成年雄性SD大鼠54只，体重250～280g，采用随机数字表法，将其随机分为3组（n=18）：假手术组（S组）、脑缺血再灌注组（I／R组）和异氟醚预处理组（1P组）。S组仅分离血管不留置线栓；I／R组采用线栓法制备右侧局灶性脑缺血再灌注损伤模型，缺血2h，再灌注24h；IP组吸入2．0％异氟醚2h，预处理结束后24h时制备右侧局灶性脑缺血再灌注损伤模型。于再灌注24h时行神经功能缺陷评分，随后处死大鼠，每组随机抽取5只大鼠，取脑组织，测定脑梗死体积，采用Westernblot法和RT—PCR法检测大鼠右侧脑缺血半暗带区HSP60、TLR4、MyD88蛋白及mRNA的表达情况；每组剩余的3只大鼠，采用TUNEL法检测大鼠右侧脑缺血半暗带区细胞凋亡情况。结果与S组比较，I／R组和IP组神经功能缺陷评分升高，脑梗死体积增大，右侧脑缺血半暗带区凋亡指数升高，HSP60、TLR4、MyD88蛋白及mRNA表达均上调（P〈0．05）；与I／R组比较，IP组神经功能缺陷评分降低，脑梗死体积减小，右侧脑缺血半暗带区凋亡指数降低，HSP60、TLR4、MyD88蛋白及mRNA表达均下调（P〈0．05）。结论异氟醚预处理可保护脑缺血再灌注大鼠缺血半暗带，其机制可能与抑制大鼠脑缺血半暗带TLR4-MyDg8信号通路有关。

Objective To investigate the effect of isoflurane preconditioning on Toll-like receptor 4 （TLR4）-myeloid differentiation factor 88 （MyD88） signaling pathway in ischemic penumbra following focal cerebral ischemia-reperfusion （I/R） in rats. Methods Fifty-four healthy male SD rats, aged 3 months, weighing 250- 280 g, were randomly divided into 3 groups （n = 18 each）： sham operation group （group S）, I/R group and isofiurane preconditioning group （group IP）. Focal cerebral I/R was induced by middle cerebral artery occlusion. In groups I/R and IP, a nylon thread with rounded tip was inserted into the fight internal jugular vein and threaded cranially until resistance was met. The middle cerebral artery was occluded for 2 h, followed by 24 h reperfusion. In group IP, the animals inhaled 2.0 % isoflurane for 2 h, and middle cerebral artery occlusion was performed at 24 h after the end of preconditioning. Neurological deficit was scored at 24 h of reperfusion and then the rats were sacrificed. Five rats in each group were chosen and the brains removed for measurement of the cerebral infarct vol- ume. The right cerebral ischemic penumbra was removed for detection of the expression of HSP60, TLR4, MyD88 protein and mRNA by Western blot analysis and real time-PCR. Apoptosis was detected in the ischemic penumbra in the left 3 rats in each group using TUNEL. Apoptosis index （AI） was calculated. Results Neurological deficit scores and AI were significantly increased, the cerebral infarct volume was significantly enlarged, and the expression of HSP60, TLR4, MyD88 protein and mRNA was upregulated in groups I/R and IP as compared with group S （ P 〈 0.05） . Isoflurane preconditioning significantly reduced the cerebral infarct volume and decreased neurological deficit scores and AI, and down-regulated the expression of HSP60, TLR4, MyD88 protein and mRNA （ P 〈 0.05）. Conclusion The mechanism by which isoflurane preconditioning protects ischemic penumbra following focal cerebral I/R may be related to inhibition of TLR4-MyD88 signaling pathway.