银杏二萜内酯葡胺对脑缺血再灌注损伤的保护作用

The protective effect of ginkgo diterpene lactone meglumine on cerebral ischemia-reperfusion

目的探讨银杏二萜内酯葡胺(GDLM)对脑缺血再灌注(I-R)损伤和氧糖剥夺后海马神经元的保护作用及其可能机制。方法体内实验:SD大鼠30只按随机数字表法均分为假手术(Sham)组、I-R组和GDLM组。GDLM组在I-R建模后,给予GDLM 10 mg/kg尾静脉注射,Sham组和I-R组同时给予等剂量生理盐水。脑组织行TTC染色。海马组织行尼氏染色和TUNEL染色,Bradford法定量检测3组海马氧化应激指标丙二醛(MDA)含量、谷胱甘肽过氧化物酶(GSH-PX)和超氧化物歧化酶(SOD)活性的变化。体外实验:胎鼠原代海马神经元培养后分为对照(Con)组、氧糖剥夺复氧模型(OGD/R)组及GDLM组。对OGD/R组和GDLM组进行氧糖剥夺复氧复糖处理,Con组不处理。MTT法观察并计算神经元细胞存活率,TUNEL和Annexin V-FITC/PI流式细胞仪检测并计算神经元凋亡率,Western blot检测磷酯酰肌醇-3激酶(PI3K)、磷酸化蛋白激酶B(pAKt)、雷帕霉素靶蛋白(mTOR)的蛋白表达水平。结果体内实验:与I-R组比较,GDLM组大鼠脑梗死体积减小,海马神经细胞凋亡率降低,形态改善显著,MDA表达水平降低,SOD和GSH-PX活性升高(P<0.01)。体外实验:与OGD/R组比较,GDLM组细胞存活率增高,凋亡率降低,PI3K、pAKt和mTOR表达水平升高(P<0.01)。结论GDLM能够抑制脑I-R和氧糖剥夺复氧后海马神经元的凋亡,具有神经保护作用,其作用机制可能与其可调控PI3K/AKt/mTOR信号通路和氧化应激有关。

Objective To investigate the protective effect and possible mechanism of ginkgo diterpene lactone meglumine(GDLM)on cerebral ischemia-reperfusion(I-R)injury and oxyglucose deprived(OGD)hippocampal neurons.Methods In vivo experiments:30 SD rats were divided into sham operation group(Sham),I-R group and GDLM group by random number table method.GDLM group was injected with GDLM 10 mg/kg by tail vein after I-R modeling,and Sham and I-R groups were given equal dose of normal saline.The brain tissue was stained with TTC.Hippocampus tissues were stained by Nissl and TUNEL,and Bradford method was used to quantitatively detect the changes in MDA content,gSH-PX and SOD activity of hippocampus oxidative stress indexes in 3 groups.In vitro experiments:primary fetal hippocampal neurons were cultured and divided into control group(Con),OGD/reoxygenation(R)model group and GDLM group.OGD/R and GDLM groups were treated with oxyglucose deprivation and reoxygenation,while the control group was not treated.The survival rate of neurons was calculated by MTT assay,apoptosis rate of neurons was measured by TUNEL and Annexin VFITC/PI flow cytometry,and PI3K,pAKt,mTOR protein expression levels were measured by Western blot assay.Results In vivo experiment:compared with Sham group and I-R group,cerebral infarction volume and apoptosis rate of hippocampal neurons were significantly reduced in GDLM group,and morphological improvement was significant.MDA expression level was significantly decreased in GDLM group,while SOD and GSH-PX activities were significantly increased(P<0.01).In vitro experiment:compared with OGD/R group,the cell survival rate was increased and apoptosis rate was decreased in GDLM group,and the expressions of PI3K,pAKt and mTORwere significantly increased(P<0.01).Conclusion GDLM can inhibit the neuron apoptosis of cerebral ischemia-reperfusion and oxygen-deprived reoxygenated hippocampal neurons,and has a neuroprotective effect,which may be related to its regulation of PI3K/AKt/mTOR signaling pathway and oxidative stress.