大黄酚对局灶性脑缺血再灌注小鼠缺血半暗带区环氧化酶2和基质金属蛋白酶-9表达的影响

摘要

Objective To explore the effects of Chrysophanol (CHR) on the expression levels of cyclooxygenase-2 (COX2) and matrix metalloproteinase-9 (MMP-9) in mice with middle cerebral artery occlusion (MCAO)/reperfusion injury and to investigate the potential anti-inflammatory mechanism of CHR.Methods Eighteen male C57BL mice were divided into 3 groups randomly:Sham group (n =6),MCAO group (n =6),and CHR group [mice received 0.1 mg/kg CHR (i.p.) for 14 days after ischemia/reperfusion].MCAO was induced by using the suture method.The mice underwent 45 min of right MCAO,and then reperfusion by withdrawing filament.Rectal temperature was monitored and kept in normal range during the operation.The mice were sacrificed and the brains were harvested on 14 d after reperfusion.The expressions of COX2 and MMP-9 were detected by immunofluorescent staining.And the cellular location of COX2 and MMP-9 were detected by double immunofluorescence labeled antibody technique.Results 1) In Sham group,little COX2 positive cells and MMP-9 positive cells were observed.Compared with Sham group,the number of COX2 positive cells and MMP-9 positive cells increased significantly in the penumbra of MCAO group (P ＜ 0.05).2) Compared with MCAO group,the number of COX2 positive cells and MMP-9 positive cells decreased significantly in the penumbra of MCAO + CHR group on day 14 after reperfusion (P ＜ 0.05).3) COX2-positive immunoreactive cells were colocalized with the general neuronal marker,NeuN,in the penumbra of ischemia/reperfusion mice.And MMP-9-positive immunoreactive cells were colocalized with NeuN.Conclusion CHR has long-term neuroprotective effect against ischemia/reperfusion injury which might be attributed to its anti-inflammatory actions by decreasing the expression of COX2 and MMP-9.%目的 探究大黄酚(Chrysophanol,CHR)对大脑中动脉梗死(middle cerebral artery occlusion,MCAO)模型小鼠再灌注后脑内环氧化酶2(cyclooxygenase-2,COX2)和基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)表达的影响,探讨CHR保护脑缺血再灌注损伤的抗炎机制.方法 采用数字表法随机将18只健康雄性C57BL小鼠分为3组:假手术(Sham)组、MCAO组、CHR组(小鼠造模当天按0.1 mg/kg腹腔注射CHR,此后每天1次,连续给药14 d),每组6只.使用线栓法制作小鼠右侧大脑中动脉缺血45 min再灌注模型.术中监测小鼠肛温,使其维持在正常范围.于再灌注后14 d处死小鼠,迅速取脑,用免疫荧光染色检测小鼠脑组织冰冻切片缺血半暗带区COX2和MMP-9的表达,并用免疫荧光双标法对COX2和MMP-9在缺血脑组织的表达进行细胞定位.结果 1)Sham组小鼠偶见COX2或MMP-9染色阳性细胞.与Sham组相比,MCAO组小鼠脑缺血半暗带区COX2和MMP-9的表达明显增高(P＜0.05).2)与MCAO组相比,给予CHR治疗后,缺血再灌注小鼠脑缺血半暗带区COX2和MMP-9的表达均明显减少(P＜0.05).3)缺血再灌注小鼠脑缺血半暗带区,COX2或MMP-9免疫荧光染色分别与神经元标志物NeuN免疫荧光染色共定位.结论 CHR可能通过抑制COX2和MMP-9的蛋白表达,减轻炎性反应,从而对脑缺血再灌注损伤发挥长期的神经保护作用.