右美托咪定预处理减轻脑缺血-再灌注后的脑局部炎症反应

Dexmedetomidine pretreatment attenuates the local inflammation of the cerebral induced by cerebral ischemia-reperfusion

目的利用大鼠大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)模型观察右美托咪定预处理减轻脑炎症反应的机制。方法雄性SD大鼠42只,体重220~250g,随机分为七组,每组6只:假手术组(S组):大鼠不做任何干预,只分离一侧颈动脉;MCAO组(M组):阻断一侧颈内动脉血流,缺血90min;D10组:MCAO前30min腹腔注射右美托咪定10μg/kg;D50组:MCAO前30min腹腔注射右美托咪定50μg/kg;D100组:MCAO前30 min腹腔注射右美托咪定100μg/kg;DY组:腹腔注射右美托咪定50μg/kg前10min给予育亨宾5mg/kg;Y组:MCAO前40min腹腔注射育亨宾5mg/kg。MCAO后24h采用TTC染色法测定脑梗死面积,神经功能评分法评定脑损伤程度。采用TUNEL染色法评估大脑皮层细胞凋亡情况,采用Western blot法检测AMPK和磷酸化AMPK(pAMPK)蛋白含量,并计算pAMPK/AMPK值;采用ELISA法检测脑组织中肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)含量。缺血-再灌注后第1、2、5天评估运动功能。结果与S组比较,M组神经功能评分、脑组织中TNF-α和IL-1β含量明显升高,梗死面积、凋亡细胞数明显增加,运动功能评分明显降低(P<0.01)。与M组比较,D10、D50和D100组神经功能评分、脑组织中TNF-α和IL-1β含量明显降低,梗死面积、凋亡细胞数明显减少,pAMPK/AMPK值、运动功能评分明显升高(P<0.05);D50和D100组上述指标改变较D10组更为明显(P<0.05)。与D50组比较,DY和Y组和YpAMPK/AMPK值明显降低(P<0.01)。结论 MCAO后右美托咪定预处理可以通过激活AMPK减轻脑缺血后炎症反应,保护脑组织,改善脑功能,并且高剂量右美托咪定较低剂量的效应更为明显。采用α2肾上腺素能受体拮抗药育亨宾可阻断右美托咪定的这些效应。

Objective To observe the mechanism of dexmedetomidine pretreatment alleviating inflammation after a middle cerebral artery occlusion（MCAO）in rats.Methods Forty-two male Sprague-Dawley rats,weighing 220-250 g,were assigned into seven experimental groups,6 in each group.Sham-operation group（group S）only received unilateral isolation of the carotid artery,group MCAO（group M）was blocked one side of internal carotid artery for 90 min,group D10（dexmedetomidine 10μg/kg,intraperitoneal injection 30 min before MCAO）,group D50（dexmedetomidine 50μg/kg,intraperitoneal injection 30 min before MCAO）,group D100（dexmedetomidine 100μg/kg,intraperitoneal injection 30 min before MCAO）,group DY（yohimbine 5 mg/kg,10 min before dexmedetomidine 50μg/kg administrated）,group Y（yohimbine 5 mg/kg,intraperitoneal injection40 min before MCAO）.TTC staining was used to determine the area of cerebral infarction and neurological deficit score was used to assess the degree of brain damage 24 hafter MCAO.Apoptosis in cortex was histologically assessed using the TUNEL staining method while Western blotting was used to investigate changes in the contents of AMPK and phospho-AMPK（pAMPK）and the rate of pAMPK to AMPK（pAMPK/AMPK）in ischemia cortex was calculated.In addition,concentrations of tumor necrosis factor-α（TNF-α）and interleukin（IL-1β）were analyzed by ELISA.On the 1 st,2 nd and 5 th day post ischemia reperfusion,the motor function was assessed by a blinded observer.Results The neurological deficit scores,inflammation（pro-inflammatory cytokines including TNF-α,IL-1β）levels,the injury area,the number of apoptotic neurons increased and the score of motor function was significantly reduced in group M compared with group S（P〈0.01）.The neurological deficit scores,levels of TNF-αand IL-1β,the injury area,the number of apoptotic neurons decreased,and the value of pAMPK/AMPK increased in groups D10,D50 and D100 compared with group M（P〈0.01）.Additionally,the effect was remarkable in group D50 and D100 compared with group D10（P〈0.05）.The value of pAMPK/AMPK was decreased in groups DY and Y compared with group D50（P〈0.01）.Conclusion These findings suggest that precondition of dexmedetomidine exerted anti-inflammatory effects after MCAO,and the activation of AMPK may be involved in the mechanism,moreover,these effects are more evident in the high dose dexmedetomidine than in the lower dose.Co-administration with yohimbine abolished these effects of dexmedetomidine.