载脂蛋白A-Ⅳ对大鼠脑缺血/再灌注损伤神经功能的影响

Effect of apolipoprotein A-Ⅳ on neurological function in rats with cerebral ischemia-reperfusion injury

目的研究载脂蛋白(ApoA-Ⅳ)对大鼠缺血/再灌注损伤神经功能的影响。方法成年雄性SD大鼠120只,随机原则分为4组:假手术(MS)组、脑缺血再灌注(IR)组、ApoA-Ⅳ小剂量(LD,50μg/kg体重)组和ApoA-Ⅳ大剂量(HD,75μg/kg体重)组,每组30只,每组按缺血2 h再灌注后12 h、24 h、48 h分为3个时间点,每个时间点各10只。IR组、LD组及HD组采用改良后的Longa线栓法,制备右侧大脑中动脉阻塞再灌注大鼠动物模型,MS组只切开皮肤,分离动脉。LD组及HD组在缺血2 h后再灌注时阴茎背静脉注射相应剂量ApoA-Ⅳ一次。在大鼠缺血再灌注后相应时间点,用Longa评分标准评价神经功能;用TTC染色法检测脑梗死体积;用qPCR及Western检查脑组织中TNF-αmRNA、Lp-PLA2mRNA和MMP-9mRNA的表达。结果IR组、LD组和HD组大鼠神经功能评分在缺血再灌注24 h最差,ApoA-Ⅳ治疗后大鼠的Longa评分明显改善,LD组在48 h,HD组在12 h、24 h、48 h显著低于IR组(P<0.05)。IR组、LD组和HD组大鼠脑梗死体积在48 h达最大;ApoA-Ⅳ治疗后脑梗死体积明显缩小,LD组在24 h、48 h,HD组在12 h、24 h、48 h与IR组比较有显著性差异(P<0.05,P<0.01)。在IR组、LD组和HD组中,大鼠脑组织MMP-9mRNA的表达明显增高,在48 h最高;大鼠脑组织TNF-αmRNA的表达明显增加,在24 h达高峰;大鼠脑组织Lp-PLA2mRNA的表达于12 h最高,以后逐渐下降,仍高于MS组;三组各时间点与MS组各相应时间点比较有显著性差异(P<0.01)。ApoA-Ⅳ治疗后各时间点脑组织MMP-9mRNA、TNF-αmRNA和Lp-PLA2mRNA的表达明显减少,与IR组比较有显著性差异(P<0.01)。结论ApoA-Ⅳ通过减少大鼠缺血再灌注后MMP-9、TNF-α和Lp-PLA2的生成,减少梗死体积,改善大鼠缺血后的神经功能。

Objective To study the effects of ApoA-Ⅳon the neurological function of rats with ischemia/reperfusion injury.Methods A total of 120 adult male Sprague-Dawley rats were randomly divided into 4 groups:mock surgical(MS)group,cerebral ischemia-reperfusion(IR)group,ApoA-Ⅳlow-dose(LD,50μg/kg)group and ApoA-Ⅳ high-dose(HD,75μg/kg)group,30 rats in each group,each group was divided into three subgroups according to 3 time points(12h,24h and 48h)after reperfusion behind 2 hours of ischemia,and 10 rats in each group.In the IR group,LD group and HD group,the modified Longa suture method was used to prepare the animal model of right middle cerebral artery occlusion and reperfusion.Only have the skin cut was performed in the MS group and separated the artery.In the LD group and the HD group,the corresponding dose of ApoA-Ⅳ was injected once in the dorsal vein of the penis after reperfusion for 2 hours behind ischemia.At the corresponding time points after ischemia-reperfusion in rats,the neurological function was evaluated by Longa scoring standard.The volume of cerebral infarction was detected by TTC staining.The expressions of TNFα mRNA,Lp-PLA2 mRNA and MMP-9 mRNA in brain group were detected by qPCR and Western blot.Results The neurological scores of rats in IR group,LD group and HD group were the worst at 24h after ischemia-reperfusion.The Longa score of rats was significantly improved after ApoA-Ⅳ treatment.The Longa score of rats in LD group at 48h and the Longa scores of rats in HD group at 12h,24h,and 48h after ischemia-reperfusion were significantly lower than those in IR group(P<0.05).The cerebral infarction volume of rats in IR group,LD group and HD group reached the maximum at 48h after ischemia-reperfusion.The volume of cerebral infarction was significantly reduced after ApoA-Ⅳ treatment.The volume of cerebral infarction of LD group at 24h and 48h,and the volume of cerebral infarction in HD group at 12h,24h and 48h were significantly different from that in the IR group(P<0.05,P<0.01).In the IR group,LD group and HD group,the expression of MMP-9 mRNA in rat brain tissue was significantly increased,which was the highest at 48h;The expression of TNF-α mRNA in rat brain tissue increased significantly and reached a peak at 24h;The expression of Lp-PLA2 mRNA in rat brain was the highest at 12h,and then decreased gradually,however,it was still higher than that in MS group.There were significant differences between the three groups at each time point and the corresponding time points of the MS group(P<0.01).The expressions of MMP-9 mRNA,TNF-α mRNA and Lp-PLA2 mRNA in brain tissue were significantly decreased at each time point after ApoA-Ⅳ treatment,and there was significant difference compared with IR group(P<0.01).Conclusions ApoA-Ⅳ could reduce the infarct volume and improve the neurological function after ischemia in rats by reducing the formation of MMP-9,TNF-α and Lp-PLA2 after ischemia-reperfusion in rats.