高温通过下调密封蛋白-1表达加重局灶性脑缺血再灌注大鼠脑损伤

Hyperthermia aggravates focal cerebral ischemia-reperfusion injury in rats by down-regulating claudin-1 expression

目的 探讨高温对局灶性脑缺血大鼠密封蛋白-1表达的影响及其在缺血性脑损伤中的机制.方法 健康雄性Sprague-Dawley大鼠100只,随机分为假手术组、常温组和高温组,常温组和高温组再分为缺血（1 h）再灌注3h、6h、24h3个时间点.采用线栓法制作大鼠大脑中动脉闭塞模型.再灌注24 h时,采用干湿重法检测脑含水量,2,3,5-氯化三苯基四氮唑染色法检测脑梗死体积.再灌注3h、6h和24 h时分别应用蛋白质印迹法和免疫组化染色法检测缺血脑组织密封蛋白-1表达.结果 再灌注24h时,常温组平均神经功能评分显著性低于高温组[（2.3±0.48）分对（3.2±0.63）分；=3.576,P=0.002）],假手术组、常温组和高温组手术侧脑组织含水量分别为（79.31±0.60）％、（81.13 ±0.12）％和（84.4±0.55）％,存在显著性差异（F=147.115,P=0.000）.蛋白质印迹分析显示,再灌注3h、6h和24 h时,常温组和高温组密封蛋白-1表达水平均较假手术组显著性降低（P均=0.000）,而且密封蛋白-1表达水平均随着缺血再灌注时间的延长进行性降低（P均＜0.05）.在相同时间点,高温组密封蛋白-1表达水平均显著性低于常温组（P均＜0.01）.假手术组、常温组再灌注3h和6h以及高温组再灌注3h和6h均可见密封蛋白-1在脑血管内皮细胞间呈阳性表达,而在再灌注24h时均未见密封蛋白-1阳性细胞.与假手术组相比,再灌注3h时,常温组和高温组密封蛋白-1阳性细胞数量和密封蛋白-1 IOD/Area（累计光密度/阳性细胞累计面积）均开始下降,6h时显著性降低,24h时消失（P=0.000）.再灌注3h和6h时,高温组密封蛋白-1 IOD/Area均显著低于常温组（P均＜0.01）.结论 脑缺血再灌注时,高温可能通过下调血脑屏障密封蛋白-1表达加重缺血性脑水肿和脑损伤.

Objective To investigate the impact of hyperthermia on the expression of claudin-1 in focal cerebral ischemia-reperfusion injury in rats and its mechanism in ischemic cerebral injury.Methods A total of 100 male Sprague-Dawley rats were randomly divided into 3 groups：sham operation,normothermia and hyperthermia groups.Both the normothermia and hyperthermia groups were redivided into 3 time points：Ischemia （1 hour） and reperfusion for 3,6,and 24 h.A model of middle cerebral artery occlusion was induced by the intraluminal suture method.At 24 h after reperfusion,brain water content was measured by the wet and dry weight method.The volume of cerebral infarction was assessed by 2,3,5 triphenyl tetrazolium chloride staining.At 3,6,and 24 h after reperfusion,the claudin-1 expression in ischemic brain tissue was measured by Western blot and immunohistochemical staining Results At 24 h after reperfusion,the mean neurological function score in the normothermia group was significantly lower than that in the hyperthermia group （2.3 ± 0.48 vs.3.2 ± 0.63; t =3.576,P =0.002）.The brain water content on the operated sides in the sham operation,normothermia and hyperthermia groups was 79.31％ ± 0.60％,81.13％ ± 0.12％,and 84.4％ ± 0.55％,respectively.There were significant differences （F=147.115,P=0.000）.Western blot analysis showed that at 3,6,and 24 h after reperfusion,the expression levels of claudin-1 in the normothermia and hyperthermia groups were significantly lower than the sham operation group （all P =0.000）,and the expression levels of claudin-1 progressively decreased with the extension of ischemia-reperfusion time （all P ＜ 0.05）.At the same time point,the expression level of claudin-1 in the hyperthermia group was significantly lower than that in the normothermia group （all P ＜ 0.01）.At 3 and 6 h after reperfusion,the positive expression of claudin-1 among the cerebrovascular endothelial cells was observed in the sham operation,normothermia and hyperthermia groups,while at 24 h after reperfusion,no claudin-1 positive cells were observed.Compared to the sham operation group,at 3 h after reperfusion,the numbers of claudin-1 positive cell and claudin-1 IOD/area （integrated optical density/accumulated positive cell area） in the normothermia and hyperthermia groups begin to decrease,they decreased significantly at 6 h and disappeared at 24 h （P=0.000）.At 3 and 6 h after reperfusion,claudin-1 IOD/area in the hyperthermia group was significantly lower than that in the normothermia group （all P ＜ 0.01）.Conclusions During cerebral ischemia-reperfusion,hyperthermia may aggravate ischemic brain edema and brain injury by down-regulating the expression of claudin-1 in blood-brain barrier.