Ischemic cerebrovascular disease includes two types: transient ischemic stroke and cerebral infarction. Among them, cerebral infarction is named as ischemic stroke (IS) in traditional Chinese medicine diagnosis, which is a disease caused by various reasons leading to local blood supply disorders in brain tissue regions, resulting in ischemic and hypoxic lesions and necrosis of brain tissue, and subsequently producing a series of neurological functional disorders. Its high mortality and disability rates, as well as complex pathological mechanisms, impose a significant economic burden on the world every year. Oxygen glucose deprivation (OGD) in brain cells is the most commonly used in vitro model of ischemic stroke, widely used in basic and preclinical stroke research.
Autophagy is widely present in eukaryotic cells and plays an important role in regulating cell survival and death processes. A large number of studies suggest that autophagy is closely related to the occurrence and development of ischemic brain injury. Some studies have shown that neuronal autophagy can alleviate ischemic brain injury, while others have reported that neuronal autophagy can exacerbate ischemic brain injury, but the specific mechanism is still unclear.
β – nicotinamide mononucleotide (NMN) is present in various foods and is crucial for regulating cellular aging and maintaining normal bodily functions. It participates in the transduction of many important intracellular signaling pathways. There are research reports that administering NMN in vitro can rapidly convert it into NAD+to regulate cell aging and maintain normal bodily functions. NMN can compensate for the decrease in NAD+caused by IS and improve neuronal damage in cerebral ischemia. This suggests that NMN plays a certain role in IS. Studies have shown that NMN can promote neurovascular regeneration, improve brain microvascular endothelial function, anti-inflammatory, and anti apoptotic effects. There are also a few studies suggesting that NMN can exert anti ischemic brain injury effects by regulating autophagy, but the specific mechanism by which NMN regulates autophagy and anti IS is not yet clear.
The aim of this study is to construct an oxygen glucose deprivation PC12 cell model. Cell survival rate was measured by MTT assay in vitro, autophagosomes and autolysosomes were detected by transmission electron microscopy, autophagosome fluorescence intensity was observed by MDC, and autophagy related protein expression of LC3-II/LC3-I, Beclin1, p62, and P-mTOR/mTOR were detected by Western blot. The aim is to clarify the effect of NMN on OGD induced autophagic damage in PC12 cells.

The 2017 Lancet study showed that stroke is the main disease causing years of life loss in China, with rapid disease progression, high mortality and disability rates. Among them, IS accounts for 60% to 70% of all strokes, which is caused by vascular obstruction leading to a decrease in blood supply to a certain area of the brain. After cerebral ischemia and hypoxia, it can trigger a series of pathological and physiological reactions such as autophagy, apoptosis, oxidative stress, and intracellular calcium overload. The long recovery cycle of IS survivors has increased the global economic burden. At present, there are many contraindications to drugs used in the market for the prevention and treatment of IS. Therefore, it is necessary to continuously explore drugs for the prevention and treatment of IS. OGD is a classic model for studying IS in vitro. The PC12 cell line is one of the most commonly used cell lines for studying neuronal injury, commonly used in research on ischemia and hypoxia injury. Therefore, this experiment used the OGD PC12 cell model for in vitro research.
NMN is a naturally occurring substance in the human body and also present in many foods, with a molecular weight of 334.22. It is a product of the nicotinamide phosphoribosyltransferase reaction and also one of the key precursors of NAD+. Studies have found that regulating the levels of NMN in organisms has good therapeutic and reparative effects on cardiovascular and cerebrovascular diseases, neurodegenerative diseases, and age-related degenerative diseases. There are also research reports that administering NMN can reduce the infarct size and neurological damage in rat MCAO models. In this experiment, MTT assay was used to demonstrate that OGD can reduce the survival rate of PC12 cells. NMN can increase the survival rate of OGD PC12 cells at concentrations of 400, 800, and 1600 μ mol/L, with the highest survival rate observed at a concentration of 800 μ mol/L; However, there was no significant difference in cell survival rate between NMN200 and 3200 μ mol/L concentrations, indicating that 200 μ mol/L concentration has not yet reached the effective concentration for improving OGD induced PC12 cell survival rate, while 3200 μ mol/L concentration may have caused certain damage to the cells due to excessive concentration.
Autophagy, also known as type II programmed cell death, refers to the process in which cells, under the regulation of autophagy related genes, use lysosomes to degrade damaged, denatured, or aging macromolecules and organelles to maintain their survival, differentiation, growth, and stability. Research shows that autophagy is induced after IS, and autophagy also occurs and develops with the pathological process of IS, playing different regulatory roles in the acute, subacute, recovery, and sequelae stages of IS.
MTOR is an atypical serine/threonine protein kinase with a relative molecular weight of 289 kDa. The binding of different proteins can form two different complexes, namely mTORCl and mTORC2. MTORCl is sensitive to rapamycin and is responsible for integrating growth factors and nutrient signals, mainly regulating cellular autophagy, ribosome biogenesis, protein translation, and lipid synthesis. MTORT is considered a valve for autophagy. Research has shown that phosphorylated mTOR can alleviate oxidative glucose deprivation damage and play a protective role in cells. 3-MA is a commonly used autophagy inhibitor. RAPA is an mTOR inhibitor that can induce autophagy by inhibiting mTORC1, also known as an autophagy activator. This experiment set up 3-MA and RAPA groups to intervene and regulate OGD induced autophagy in PC12 cells; At the same time, 3-MA, RAPA, and drug combination groups were also set up to observe whether NMN could counteract the effects of 3-MA or RAPA on OGD induced autophagy in PC12 cells.
The levels of Beclin-1, LC3, and p62 proteins can serve as important detection indicators for autophagy. Beclin1 is a mature autophagy regulator that is positively correlated with autophagy. Beclin1 interacts with proteins such as VPS15, VPS34, ATG14 to perform autophagy and membrane transport functions. LC3 is a homolog of the ubiquitin like modifier ATG8 in yeast, believed to play a role in autophagy. After treatment with ATG4, LC3 loses its C-terminal residue and transforms into LC3-I. LC3-I undergoes a cascade of ubiquitination like enzymatic reactions, covalently linked to the lipid molecule phosphatidylethanolamine on the autophagosome membrane, transforming into LC3-II. An increase in the LC3-II/LC3-I ratio indicates an increase in autophagy levels. P62 is a marker protein that reflects autophagy activity, and its protein level is negatively correlated with autophagy. That is, when autophagy occurs, p62 protein is continuously degraded in the cytoplasm; When autophagy activity is weakened and autophagy function is impaired, p62 protein will continuously accumulate in the cytoplasm. This experiment confirmed through Western blot technology that NMN can downregulate the relative expression levels of Beclin1 and LC3-II/LC3-I proteins, and upregulate the expression levels of P-mTOR/mTOR and p62 proteins. In addition, transmission electron microscopy and MDC method were used to confirm that NMN can reduce the number and intensity of autophagosomes and autolysosomes in OGD PC12 cells. The above indicates that NMN can inhibit OGD induced autophagy in PC12 cells.
In summary, it suggests that a certain dose of NMN can counteract OGD induced autophagic damage in PC12 cells, thereby exerting a cellular protective effect, and this protective effect may be related to mTOR related pathways. This study can provide a certain target reference for the prevention and treatment of OGD induced autophagic damage by NMN, and accumulate laboratory data for the development of natural compound NMN.