摘要
目的:探究葫芦巴碱通过PTEN诱导性激酶蛋白1(Pink1)/E3泛素连接酶(Parkin)信号通路介导线粒体自噬对氧-糖剥夺/复氧(OGD/R)诱导的神经元凋亡的影响。方法:原代培养大鼠海马神经元,经下调Pink1或葫芦巴碱预处理后通过OGD/R诱导神经元损伤,实验分组包括对照组、OGD/R组、葫芦巴碱组、pLKO空载体组(转染pLKO空载体)、pLKO-Pink1组(转染连接Pink1 shRNA干扰质粒的pLKO载体)、葫芦巴碱+pLKO-Pink1组。Annexin V-FITC/PI双染法测定神经元凋亡率;JC-1法测定线粒体膜电位(MMP);Calcein-AM法测定线粒体膜通透性转换孔(MPTP)开放程度;透射电镜观察自噬小体数量;Western Blot检测线粒体自噬相关蛋白(微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)、Pink1、Parkin)表达。结果:与对照组相比,OGD/R组神经元凋亡率、MPTP开放程度增加,MMP下降,自噬小体数量及LC3-Ⅱ、Pink1、Parkin蛋白表达增加(P<0.05);与OGD/R组相比,葫芦巴碱组神经元凋亡率、MPTP开放程度减少,MMP升高,自噬小体数量及LC3-Ⅱ、Pink1、Parkin蛋白表达增加(P<0.05),pLKO-Pink1组与葫芦巴碱组趋势相反;与葫芦巴碱组相比,葫芦巴碱+pLKO-Pink1组神经元凋亡率、MPTP开放程度增加,MMP下降,自噬小体数量及LC3-Ⅱ、Pink1、Parkin蛋白表达减少(P<0.05)。结论:葫芦巴碱可抑制OGD/R诱导的神经元凋亡,可能通过激活Pink1/Parkin信号通路上调线粒体自噬而实现。
Objective:To investigate the impact of trigonelline on oxygen-glucose deprivation/reoxygenation(OGD/R)-induced neuronal apoptosis mediated by mitophagy via PTEN-inducible kinase protein 1(Pink1)/E3 ubiquitin ligase(Parkin)signaling pathway.Methods:Primary cultured rat hippocampal neurons were induced neuronal damage through OGD/R after pretreatment with down-regulation of Pink1 or trigonelline.There were control group,OGD/R group,trigonelline group,pLKO empty vector group(transfected with pLKO empty vector),pLKO-Pink1 group(transfected with pLKO vector connected with Pink1 shRNA interference plasmid),and trigonelline+pLKO-Pink1 group in the experiment.Annexin V-FITC/PI double staining method was used to determine the apoptosis rate of neurons;the mitochondrial membrane potential(MMP)was measured by JC-1 method;Calcein-AM method was used to measure the opening degree of mitochondrial membrane permeability transition pore(MPTP);the number of autophagosomes was observed by transmission electron microscope;Western Blot was used to determine the expression of mitophagy-related proteins[microtubule-associated protein 1 light chain 3 II(LC3 II),Pink1,Parkin].Results:Compared with the control group,the neuron apoptosis rate and MPTP opening degree in the OGD/R group increased,MMP decreased,the number of autophagosomes and the expression of LC3-II,Pink1 and Parkin proteins increased(P<0.05);compared with the OGD/R group,the neuron apoptosis rate and MPTP opening degree of trigonelline group decreased,MMP increased,the number of autophagosomes and the expression of LC3-II,Pink1 and Parkin proteins increased(P<0.05),the trends of pLKO-Pink1 group were opposite to those of trigonelline group;compared with the trigonelline group,the neuron apoptosis rate and MPTP opening degree in the trigonelline+pLKO-Pink1 group increased,MMP decreased,the number of autophagosomes and the protein expressions of LC3-II,Pink1 and Parkin decreased(P<0.05).Conclusion:Trigonelline can inhibit OGD/R-induced neuronal apoptosis,which may be achieve
作者
黄琦
翟海程
HUANG Qi;ZHAI Haicheng(Hanzhong Central Hospital,Shaanxi Hanzhong 723000,China)
出处
《河北医学》
CAS
2023年第5期716-721,共6页
Hebei Medicine
基金
陕西省汉中市中心医院科研项目,(编号:YK1807)。
