白藜芦醇对大鼠脑缺血再灌注损伤的作用

Role of cell autophagy in protection of resveratrol against cerebral ischemia-reperfusion injury in rats

目的探讨白藜芦醇(Res)预处理对脑缺血再灌注大鼠神经功能损伤的作用及其机制。方法将80只成年雄性SD大鼠随机分为假手术组、模型组、Res组、Res+PI3K抑制剂组,每组20只。以线栓法建立大脑中动脉阻塞后缺血再灌注模型。造模前6 d,Res组和Res+PI3K抑制剂组腹腔注射Res(15μg/g,1次/d),假手术组和模型组腹腔注射等体积生理盐水;造模前30 min,Res组和Res+PI3K抑制剂组先腹腔注射Res(15μg/g),然后,Res+PI3K组用微量注射器向右侧脑室注射PI3K抑制剂3-MA 5μl(50μg),假手术组和模型组右侧脑室注射等体积生理盐水。造模后24 h处死大鼠采用TTC染色法评估大鼠脑梗死面积,取材前行神经功能评分;以尼氏染色以及透射电镜观察神经细胞尼氏体和自噬体数量;通过Q-PCR法检测脑组织自噬基因mRNA水平,免疫印迹法检测脑组织PI3K/Akt/mTOR信号通路相关蛋白及磷酸化表达水平。结果与假手术组相比,模型组神经功能评分明显增高(P<0.05),脑梗死体积明显增大(P<0.05),损伤脑组织尼氏体明显减少(P<0.05),自噬相关基因Beclin1 mRNA水平明显升高(P<0.05),PI3K、mTOR和Akt蛋白表达水平无显著变化(P>0.05),而各自蛋白磷酸化水平均显著降低(P<0.05)。相比于模型组,Res组上述变化明显改善(P<0.05),而PI3K抑制剂明显抑制Res的作用(P<0.05)。结论Res能有效减轻缺血再灌注模型大鼠脑组织造成的神经功能损伤,可能与抑制PI3K/AKT/mTOR信号通路促进细胞自噬有关。

Objective To explore the effect and mechanism of resveratrol(Res)pretreatment on neurological damage in rats after cerebral ischemia-reperfusion injury.Methods Eighty adult male SD rats were randomly divided into four groups,i.e.,sham operation group,model group,Res group,Res+PI3K inhibitor group,with 20 rats in each group.The model of ischemia-reperfusion injury was established by tread embolization of middle cerebral artery.Six days before injury,the rats in Res group and Res+PI3K inhibitor group were intraperitoneally injected with Res(15μg/g,once a day),and the rats in sham operation group and model group were intraperitoneally injected with equal volume of saline.Thirty minutes before injury,the rats in Res group and Res+PI3K inhibitor group were injected with Res(15μg/g)intraperitoneally.Then,the rats in Res+PI3K group was injected with PI3K inhibitor 3-MA(5μl,50μg)into the right ventricle using a microsyringe.An equal volume of saline was injected into the right ventricle of rats in the model group.Twenty-four hours after injury,nerve function scores were assessed in all the rats and then were sacrificed.TTC staining method was used to evaluate the cerebral infarction area of rats.Nissl staining and transmission electron microscopy were used to observe the number of Nissl bodies and autophagosomes.The mRNA level of autophagy gene in brain tissue was detected by Q-PCR method.The protein and phosphorylation expression levels of PI3K/Akt/mTOR signaling pathway were detected by immunoblotting.Results Compared with the sham operation group,the neurological function score was significantly increased(P<0.05),the volume of cerebral infarction was significantly increased(P<0.05),the damaged brain tissue Nissl body was significantly reduced(P<0.05),autophagy gene Beclin1 mRNA level was significantly increased(P<0.05),and the phosphorylation level of PI3K/Akt/mTOR protein was significantly reduced(P<0.05)in the model group.Compared with the model group,the above changes in the Res group were significantly improved(P<0.05),while PI3K inhibitors significantly inhibited the effect of Res(P<0.05).Conclusions Res can effectively reduce the neurological damage caused by ischemia-reperfusion injury in rats,which may be related to the promotion of cell autophagy by inhibition of PI3K/Akt/mTOR signaling pathway.