大鼠脑缺血再灌注损伤后HMG-COA还原酶抑制剂下调Caveolin-1表达对血脑屏障的影响

HMG-COA reductase inhibitor induced blood-brain barrier permeability decrease is mediated by downregulation of caveolin-1 protein after middle cerebral artery occlusion and reperfusion injury rats

目的采用线栓法构建大鼠的中动脉缺血再灌注(middle cerebral artery occlusion,MCAO)模型,应用不同剂量辛伐他汀(HMG-COA reductase inhibitor)下调Caveolin-1表达,研究被不同程度抑制的Caceolin-1对BBB的作用。方法 160只雄性SD大鼠分成高、中、低剂量的辛伐他汀预处理组,同等剂量生理盐水缺血组及假手术组。预处理及生理盐水组连续灌胃给药7 d,最后一次灌胃3 h后建立MCAO,缺血2 h后抽出线栓再灌,假手术组仅结扎颈外动脉。手术24 h后进行神经功能评分,取脑分别进行TTC染色检测脑梗死体积、干湿质量法测量脑含水量、分光光度计测OD值测量伊文思蓝渗出、免疫组化法及蛋白印记杂交检测Caveolin-1的表达。结果神经功能评分结果显示,全部辛伐他汀组明显优于生理盐水组(P<0.05);TTC染色示,低剂量及中剂量预处理组梗死体积百分比明显小于生理盐水缺血组(P<0.01),高剂量组未见优势,各预处理组两两比较,低及中剂量组梗死体积小于高剂量组(P<0.05);干湿质量百分率测定预处理组湿质量明显小于生理盐水缺血组(P<0.05),且各预处理组间两两比较发现中剂量组湿质量小于高剂量组(P<0.05);分光光度计测伊文思蓝渗透OD值结果,各给药组伊文思蓝渗透水平均低于生理盐水缺血组(P<0.05),各预处理组两两比较,低剂量组优于中及高剂量组且中剂量组优于高剂量组(P<0.05);Western Blot定量分析结果显示,各预处理组Caceolin-1表达水平均低于生理盐水缺血组(P<0.05),各给药组两两比较发现,随剂量增加Caveolin-1被降低水平增加即低剂量组<中剂量组<高剂量组(P<0.05);免疫组化示,Caveolin-1蛋白在各组大鼠脑组织中均沿微血管阳性表达,免疫组化染色半定量测量分布于血管内皮的Caveolin-1表达,除中与高剂量间差异无统计学意义(P>0.05)外,与Western Blot分析结果大概一致。结论大鼠MCAO再灌注模型中Caveolin-1蛋白的表达量会增加。辛伐他汀的预处理可以有效抑制Caveolin-1的表达,适当剂量辛伐他汀可以通过抑制Caveolin-1表达保护血脑屏障完整,但过高或过低水平Caveolin-1蛋白都会破坏血脑屏障增大梗死的体积。

Objective To investigate the relationship between the effects of HMG COA reductase inhibitor downregulates the expression of caveolin 1 and degree of permeability of BBB increased in the rats that accept surgery for middle cerebral artery occlusion. Methods Adult male SD rats (270-320 g) were purchased from the center for experimental animals,China Medieal University.Animals were randomly assigned to control,sham operated group and simvastatin groups.Different degree of simvastatin (100 mg/(kg·d),60 mg/(kg·d) and 30 mg/(kg·d)) were given orally to the rats onece a day for 7 days before operation.Saline was given in the same way in the eontrol group.All groups above were performed surgery for middle cerebral artery occlusion (MCAO) with sham operated group excepted.The following Procedures should be taken:Observation of behavioral testing;Evaluation of infarct volume by TTC testing;Weigh the dry/wet weight ratio;Measurement the permeability of BBB by tseting extravasation of Evans blue dye using spectrophotometer,Immunohistochemical assessment,Westen blot assessment. Results All groups accept treatment of simvastatin revealed that the score of behavioral testing were superior to control group ( P <0.05).All groups accept treatment of simvastatin showed less infarct volume than control group by TTC testing ( P <0.05).In addition,both low and middle dose group reveal less infarct volume than high dose group ( P <0.05).The results of ratio of the dry/wet weight revealed that all groups accept treatment of simvastatin show less ratio than control group ( P <0.05).Meanwhile,the middle dose group reveal less ratio than high dose group ( P <0.05).Measuring the extravasation of Evans blue dye by spectrophotometer revealed that all groups accept treatment of simvastatin show less OD than control group ( P <0.05).Western blot analysis on expression of cav -1 showed that ischemia reperfusion induced the up regulation of cav-1 ( P <0.05) by calculating the gray level ratio. Thus,the level of Caveolin-1 expression decreased with the increase of dose (high<middle<low dose group)( P <0.05).Effects on the expression of caveolin-1 by immunohistochemisitry showed that the expression of caveolin-1 protein was investigated in the microvessel fragnlents of ischemic brain tissue.Caveolin-1 expression was in common with the result of western blot analysis except that there hasn’t statistically significant between middle dose and high dose group ( P >0.05). Conclusion The advent of cerebral I/R injury may evoke the overexpression of Caveolin-1 in rats.Pretreatment of statins exert direct down regulation effects on Caveolin-1.A suitable dose of statins may induce BBB permeability decrease by downregulation of Caveolin-1 protein,so important as that extreme high or low dose induced larger infarction volumes.