天然产物研究与开发 ›› 2024, Vol. 36 ›› Issue (2): 260-267.doi: 10.16333/j.1001-6880.2024.2.009

• 研究简报 • 上一篇    下一篇

β-烟酰胺单核苷酸对氧糖剥夺PC12细胞自噬的影响

田梦芝1，龙佳欣1，陈笑一1，陈惠媚1,2，金泽龙1，李思特1，谢明霞1，杜   可1*   

  1. 1湖南中医药大学；2中医药防治眼耳鼻咽喉疾病湖南省重点实验室，长沙410208
  • 出版日期:2024-02-23 发布日期:2024-02-23
  • 基金资助:
    国家重点实验室开放基金（GTZK201711）；研究生创新创业训练计划（CX20220794），大学生创新创业训练计划（S202210541074）；2022年度湖南省普通高等学校教学改革研究项目（HNJG-2022-0134 ）

Effect of β-nicotinamide mononucleotide on autophagy in oxyglucose-deprived PC12 cells

TIAN Meng-zhi1,LONG Jia-xin1,CHEN Xiao-yi1,CHEN Hui-mei1,2,JIN Ze-long1,LI Si-te1,XIE Ming-xia1,DU Ke1*   

  1. 1Hunan University of Traditional Chinese Medicine;2Hunan Provincial Key Laboratory for the Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Traditional Chinese Medicine,Changsha 410208,China
  • Online:2024-02-23 Published:2024-02-23

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摘要：

为了探讨β-烟酰胺单核苷酸（β-nicotinamide mononucleotide，NMN）对氧糖剥夺（oxygen glucose deprivation，OGD）诱导的PC12细胞自噬的影响及其可能机制。通过体外建立OGD PC12细胞自噬损伤模型，MTT法检测各组细胞存活率。透射电镜观察NMN处理后的自噬小体及溶酶体，MDC荧光染色观测NMN对自噬小体的影响。Western blot检测LC3-II/LC3-I、Beclin1、p62、P-mTOR/mTOR等自噬相关蛋白表达水平。结果显示，与Con比，OGD组细胞存活率显著降低（P<0.01），自噬小体及自噬溶酶体增多（P<0.01），MDC荧光斑点及强度增强（P<0.01），Beclin1、LC3-II/LC3-I表达量显著上调（P<0.01），p62、P-mTOR/mTOR表达量显著下调（P<0.01）。与OGD组比，NMN组细胞存活率显著提高（P<0.01），NMN组和3-甲基腺嘌呤（3-methyladenine，3-MA）组自噬小体及自噬溶酶体减少（P<0.01），MDC荧光斑点及强度减弱（P<0.01），Beclin1、LC3-II/LC3-I表达量下调（P<0.01），p62、P-mTOR/mTOR表达量上调（P<0.01），而雷帕霉素（rapamycin，RAPA）组则与之相反。与RAPA组相比，RAPA+NMN组自噬小体及自噬溶酶体显著减少（P<0.01），MDC荧光斑点及强度减弱（P<0.01），Beclin1、LC3-II/LC3-I表达量显著下调（P<0.01），p62、P-mTOR/mTOR表达量显著上调（P<0.01）。因此，NMN可抑制OGD诱导的PC12细胞自噬损伤，发挥抗神经元自噬损伤的保护作用，并且这种保护作用可能与 mTOR相关通路有关。本研究可为NMN防治OGD诱导的细胞自噬损伤提供一定的靶标参考，为天然化合物的开发累积一定实验室数据。

关键词: β-烟酰胺单核苷酸；氧糖剥夺；哺乳动物雷帕霉素靶蛋白；自噬损伤 

Abstract:

This study aims to explore the impact of β-nicotinamide mononucleotide(NMN) on oxygen glucose deprivation(OGD)-induced autophagy in PC12 cells and its potential mechanisms.An in vitro model of OGD-induced autophagic injury in PC12 cells was established,and cell viability in each group was measured using the MTT assay.The autophagosomes and autolysosomes after NMN treatment were observed by transmission electron microscopy,and the effect of NMN on autophagosomes was observed by MDC fluorescence staining.Western blot analysis was conducted to evaluate the expression levels of autophagy-related proteins,including LC3-II/LC3-I,Beclin1,p62,P-mTOR/mTOR,and others.The results showed that compared with control group,the survival rate of OGD group was significantly decreased (P<0.01),the number of autophagosomes and autolysosomes increased (P<0.01),and the fluorescence spots and intensity of MDC were enhanced (P<0.01).The expressions of Beclin1 and LC3-II/LC3-I were significantly up-regulated (P<0.01),while the expressions of p62 and P-mTOR /mTOR were significantly down-regulated (P<0.01).Compared to the OGD group,the survival rate of NMN group was significantly increased (P<0.01),the autophagosome and autolysomes were decreased in the NMN and 3-methyladenine(3-MA) group (P<0.01),and the fluorescence spots and intensity of MDC were decreased (P<0.01).the expressions of Beclin1 and LC3-II/LC3-I were down-regulated (P<0.01),and the expressions of p62 and P-mTOR /mTOR were up-regulated (P<0.01),but the opposite was true in RAPA group.Compared with rapamycin(RAPA) group,the autophagosomes and autolysosomes in RAPA+NMN group were significantly decreased (P<0.01),the fluorescence spots and intensity of MDC were decreased (P<0.01),and the expressions of Beclin1 and LC3-II/LC3-I were significantly down-regulated (P<0.01).The expressions of p62 and P-mTOR /mTOR were significantly up-regulated (P<0.01).Therefore,NMN can inhibit OGD-induced autophagy injury in PC12 cells and play a protective role against neuronal autophagy injury,and this protective effect may be related to mTOR pathways.This study can provide a certain target reference for NMN prevention and treatment of OGD-induced autophagy injury,and accumulate some laboratory data for the development of natural compounds.

Key words: β-nicotinamide mononucleotide, oxygen glucose deprivation, mammalian target of rapamycin, autophagic injury

中图分类号:  R932

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