高温通过上调组织蛋白酶L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 up-regulating cathespin L expression

目的研究高温对局灶性脑缺血组织蛋白酶L（CTSL）表达的影响，探讨高温加重缺血性脑损伤的机制。方法100只雄性SD大鼠按照随机数字表法分为假手术组（n=20）、常温组（n=40）和高温组（n=40）。常温组和高温组参照Longa等方法制作大鼠大脑中动脉闭塞（MCAo）缺血1h再灌注模型。高温组保持肛温39．0℃（瑞沃德恒温加热装置），共3h。假手术组和常温缺血组保持肛温37．5℃。假手术组只分离血管，不结扎，不插线。测定再灌注24h后3组大鼠神经功能缺损评分、脑梗死体积及脑含水量。Western blotting和免疫组化法检测再灌注3h、6h和24h后大鼠缺血脑组织CTSL表达。结果（1）高温组大鼠再灌注24h后神经功能缺损评分、脑梗死体积百分比及手术侧脑含水量均高于常温组大鼠再灌注24h后结果（3．37±0．48 vs．2．20±0．42、59．08％±0．98％vs．37．96％±0．16％、84．82％±1．79％强82．18％±0．45％），差异均有统计学意义（P〈0.05）。（2）Western blotting检测结果显示,常温组大鼠各时间点CTSL表达无明显变化；而高温组大鼠CTSL表达于再灌注后3h即开始增加，24h仍未恢复正常，各时间点均高于常温组，差异有统计学意义（P〈0．05）。（3）免疫组化法显示神经元与神经胶质细胞均可表达CTSL，以神经胶质细胞为主。再灌注后3h、6h、24h。高温组大鼠CTSL阳性表达神经元中CTSL平均密度值均明显高于常温组及假手术组大鼠。差异均有统计学意义（P〈0．05）。结论脑缺血再灌注时。高温可能通过上调CTSL的表达加重缺血性脑损伤。

Objective To investigate the effect of hyperthermia on expression of cathespin L （CTSL） in focal cerebral ischemia-reperfusion injury of rats and its mechanism in ischemia injury. Methods A total of 100 male Sprague-Dawley rats were randomly divided into sham-operated group （n=20）, normothermia group （n=40） and hyperthermia group （giving constant temperature heating for 39 ℃, n=40）; rats in the sham-operated group and normothermia group were given constant temperature heating for 37.5 ℃. Models of middle cerebral occlusion reperfusion in the later two groups were induced by intraluminal suture method. Both the normothermia and hyperthermia groups were sub-divided into 3 time point groups： ischemia for one h and reperfusion for 3, 6 and 24 h （n=10, 10 and 20）. At 24 h after reperfusion, the neurological deficit scores, volume of infarction and brain water content were assessed. At 3, 6, and 24 h after reperfusion, the expression of CTSL in ischemic brain tissues was measured by Western blotting and immunohistochemical staining. Results At 24 h after reperfusion, the mean neurological deficit scores, volume of infarction and brain water content in the hyperthermia groups were significantly higher than those in the normothermia groups （3 h： 3.37±0.48 vs. 2.20±0.42, 6 h： 59.08%±0.98% vs. 24 h： 37.96%±0.16% and 84.82%±1.79% vs. 82.18%±0.45%, P〈 0.05）. Western blotting showed that CTSL expression did not change at each time point in the normothermia group, while the CTSL expression in the hyperthermia group was significantly higher than that in the normothermia group at each time point, respectively （P〈0.05）. CTSL expressed in neuron and glia of sham-operated group delected by immunohistochemistry, especially in glia. As compared with that in the sham-operated group, glia CTSL integrated optical density/accumulated positive cell area （IOD/area） began to decrease in the normothermia group at 24 h after reperfusion; neuron CTSL IOD/area began to increase at 3 h, reached to the summit at 6 h, and then, decreased at 24 h （P〈0.05）. At each time point, neuron CTSL IOD/area in the hyperthermia group was significantly higher than that in the normothermia group （P〈0.05）. Conclusion Hyperthermia can probably worsen the brain edema and damage by up-regulating CTSL expression after ischemia/reperfusion.