小檗碱对小鼠肝缺血再灌注致脑损伤的预防效应:与GSK-3β活性的关系

Efficacy of berberine in preventing brain injury induced by hepatic ischemia-reperfusion in mice:the relationship with GSK-3βactivity

目的评价小檗碱对小鼠肝缺血再灌注致脑损伤的预防效应及其与糖原合成酶激酶3β(GSK-3β)活性的关系.方法健康清洁级雄性C57BL∕6小鼠48只,体重20~25g,采用随机数字表法分为3组(n=16):假手术组(S组)、肝缺血再灌注组(I∕R组)和小檗碱组(B组).B组于术前7d经胃灌注小檗碱50mg∕kg,1次∕d,连续7d.采用夹闭肝左、中叶脉管的共干制备70%肝缺血再灌注损伤模型.再灌注后6h处死小鼠,取海马组织,光镜下观察病理学结果,TUNEL法确定凋亡指数;采用黄嘌呤氧化酶法测定SOD活性,硫代巴比妥酸比色法测定MDA含量;采用Westernblot法测定磷酸化GSK-3β(p-GSK-3β)和GSK-3β表达,计算p-GSK-3β∕GSK-3β比值;采用分光光度法测定线粒体通透性转换孔(mPTP)开放程度.结果与S组比较,I∕R组和B组凋亡指数和MDA含量升高,SOD活性和p-GSK-3β∕GSK-3β比值降低,mPTP开放程度增加(P<0.05);与I∕R组比较,B组凋亡指数和MDA含量降低,SOD活性和p-GSK-3β∕GSK-3β比值升高,mPTP开放程度降低(P<0.05),海马组织病理损伤减轻.结论小檗碱对小鼠肝缺血再灌注所致的脑损伤有预防效应,机制可能与抑制GSK-3β活性,从而抑制mPTP开放有关.

Objective To evaluate the efficacy of berberine in preventing brain injury induced by hepatic ischemia-reperfusion(I/R)and the relationship with glycogen synthase kinase 3 beta(GSK-3β)activity in mice.Methods Forty-eight healthy clean-grade male C57BL/6 mice,weighing 20-25 g,were randomized into 3 groups(n=16 each)using a random number table method:sham operation group(S group),hepatic I/R group(I/R group)and berberine group(B group).The model of 70%liver I/R injury was established by clamping the portal vein and hepatic artery supplying left and middle lobes of the liver in mice anesthetized with 2%pentobarbital sodium 40 mg/kg.In B group,berberine 50 mg/kg was administered through a gastric tube once a day for 7 consecutive days starting from 7 days before operation.The equal volume of normal saline was given instead in S group and I/R group.The mice were sacrificed at 6 h after reperfusion,brains were removed and hippocampal tissues were harvested for microscopic examination of pathological changes(with a light microscope)and for determination of cell apoptosis(by TUNEL),superoxide dismutase(SOD)activity(by xanthine oxidase method),malondialdehyde(MDA)content(by thiobarbituric acid colorimetric method),expression of phosphorylated GSK-3β(p-GSK-3β)and GSK-3β(by Western blot),and opening of mitochondrial permeability transition pore(mPTP).The apoptosis index and p-GSK-3β/GSK-3βratio were calculated.Results Compared with S group,the apoptosis index and MDA content were significantly increased,SOD activity and p-GSK-3β/GSK-3βratio were decreased,and mPTP opening was increased in I/R and B groups(P<0.05).Compared with group I/R,the apoptosis index and MDA content were significantly decreased,SOD activity and p-GSK-3β/GSK-3βratio were increased,mPTP opening was decreased(P<0.05),and the pathological changes of hippocampal tissues were significantly attenuated in B group.Conclusion Berberine can prevent the brain injury induced by hepatic I/R,and the mechanism may be related to inhibiting GSK-3βactivity and thus inhibiting mPTP opening in mice.