葫芦巴碱通过Pink1/Parkin信号通路介导线粒体自噬对OGD/R诱导的神经元凋亡的影响

doi:10.3969/j.issn.1006-6233.2023.05.03

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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3 Ⅱ(LC3 Ⅱ)、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。

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	黄琦
	翟海程

关键词 ：葫芦巴碱, 氧-糖剥夺/复氧损伤, Pink1/Parkin信号通路, 线粒体自噬, 神经元, 凋亡

Abstract：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 achieved by activating Pink1/Parkin signaling pathway and up-regulating mitophagy.

Key words： Trigonelline Oxygen-glucose deprivation/reoxygenation injury Pink1/Parkin signaling pathway Mitophagy Neurons Apoptosis

基金资助:陕西省汉中市中心医院科研项目,(编号:YK1807)

通讯作者: 翟海程

引用本文:

黄琦, 翟海程. 葫芦巴碱通过Pink1/Parkin信号通路介导线粒体自噬对OGD/R诱导的神经元凋亡的影响[J]. 河北医学, 2023, 29(5): 716-721.
HUANG Qi, ZHAI Haicheng. Impact of Trigonelline on OGD/R-Induced Neuronal Apoptosis Mediated by Mitophagy via Pink1/Parkin Signaling Pathway. HeBei Med, 2023, 29(5): 716-721.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2023.05.03 或 http://www.hbyxzzs.cn/CN/Y2023/V29/I5/716

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