普罗布考对高脂血症大鼠脑缺血再灌注的脑保护作用及机制研究

Study on cerebral protective effect and mechanism of probucol on cerebral ischemia-reperfusion in hyperlipidemia rats

目的探讨普罗布考对高脂血症大鼠脑缺血再灌注的脑保护作用及可能机制。方法140只SD大鼠随机分为普通组(20只)和高脂组(120只),分别给予普通饲料、高脂饲料喂养8周,全自动生化分析仪检测其血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白-胆固醇(LDL-C)、高密度脂蛋白-胆固醇(HDL-C)水平以判断高脂血症大鼠造模成功与否。100只造模成功的高脂血症大鼠采用随机数字表法分为假手术组、缺血再灌注组、低剂量普罗布考组、高剂量普罗布考组,每组各25只。普罗布考低剂量组、普罗布考高剂量组分别给予0.5%羧甲基纤维素(CMC)配制的普罗布考混悬液100 mg/kg、500 mg/kg灌胃,假手术组、缺血再灌注组同时给予相同剂量0.5%CMC灌胃,1次/天,连续4周。缺血再灌注组、普罗布考低剂量组、普罗布考高剂量组采用改良Longa法建立大脑中动脉阻断模型,假手术组大鼠除不插鱼线外,其余手术步骤同前。造模成功大鼠缺血2 h后,于不同再灌注时间点(6、12、24、48、72 h)对大鼠进行神经功能缺损评分(mNSS);再灌注24 h后2,3,5-氯化三苯基四氮唑(TTC)染色法检测脑梗死体积;ELISA检测缺血脑组织肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)水平;蛋白免疫印迹(Western blot)法检测缺血脑组织核转录因子-κB(NF-κB)通路相关蛋白表达水平。结果与普通组比较,高脂组大鼠血清TC、TG、LDL-C水平升高,HDL-C水平降低(P<0.05)。与假手术组比较,缺血再灌注组大鼠再灌注不同时间点mNSS降低,脑梗死体积增大,缺血脑组织TNF-α、IL-1β、p-p65水平升高(P<0.05);与缺血再灌注组比较,普罗布考低剂量组、普罗布考高剂量组大鼠再灌注不同时间点mNSS升高,脑梗死体积减小,缺血脑组织TNF-α、IL-1β、p-p65蛋白水平降低(P<0.05);与普罗布考低剂量组比较,普罗布考高剂量组大鼠再灌注6、48、72 h mNSS升高,脑梗死体积减小,缺血脑组织TNF-α、IL-1β、p-p65水平降低(P<0.05)。结论普罗布考对高脂血症大鼠脑缺血再灌注具有脑保护作用,机制可能与调控NF-κB通路有关。

Objective To investigate the cerebral protective effect and possible mechanism of probucol on cerebral ischemia-reperfusion in hyperlipidemia rats.Methods A total of 110 SD rats were randomly divided into the normal group(10 rats)and the high-fat group(100 rats),and the rats were fed with normal diet and high-fat diet for 8 weeks,respectively.The levels of serum total cholesterol(TC),triglyceride(TG),low-density lipid cholesterol(LDL-C)and high-density lipid cholesterol(HDL-C)were detected by automatic biochemical analyzer,to judge whether the hyperlipidemia rat models were successfully established or not.A total of 100 successfully hyperlipidemia rats-models were randomly divided into the sham operation group,the ischemia-reperfusion group,the low-dose probucol group and the high-dose probucol group,with 25 rats in eachgroup.The probucol low-dose group and high-dose group were given probucol suspension mad with 0.5%carboxymethyl cellulose(CMC)at the dose of 100 mg/kg,500 mg/kg gavage,respectively;the sham operation group and the ischemia-reperfusion group were given the same dose of 0.5%CMC gavage,1 time/d,for 4 weeks.The middle cerebral artery occlusion model was established by the modified Longa method in the ischemia-reperfusion group,the low-dose probucol group and the high-dose probucol group.The rats in the sham operation group were treated with the same procedure as those in the operation group before except that no fish line was inserted.After 2 h of ischemia,neurological deficit scores(mNSS)were evaluated at different reperfusion time points(6,12,24,48,72 h).Cerebral infarction volume was detected by 3,5-triphenyltetrazole chloride(TTC)staining after 24 h reperfusion.The levels of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in ischemic brain tissue were detected by ELISA.Western blot was used to detect the expression levels of nuclear transcription factor-κB(NF-κB)pathway-related proteins in the ischemic brain tissue.Results Compared with the normal group,the serum levels of TC,TG and LDL-C of rats in the the high-fat group were increased,while HDL-C serum levels was decreased(P<0.05).Compared with the sham operation group,the mNSS of rats in the model group at different time points after reperfusion decreased,the volume of cerebral infarction increased,and the levels of TNF-α,IL-1βand p-p65 protein in the ischemic brain tissue increased(P<0.05).Compared with the model group,the mNSS of rats in the low-dose probucol group and the high-dose probucol group at different time points of reperfusion increased,the volume of cerebral infarction decreased,and the levels of TNF-α,IL-1βand p-p65 protein in the ischemic brain tissue decreased(P<0.05).Compared with the low-dose probucol group,the mNSS of rats in the high-dose probucol group at 6,48,72 h after reperfusion were increased,the volume of cerebral infarction was decreased,and the levels of TNF-α,IL-1βand p-p65 protein in ischemic brain tissue were decreased(P<0.05).Conclusion Probucol has a cerebral protective effect on cerebral ischemia-reperfusion in hyperlipidemia rats,and the mechanism may be related to the regulation of NF-κB pathway.