NMN attenuates brain injury after intracerebral hemorrhage by activating Nrf2/HO-1 signaling pathway

   Replenishment of NAD+ has been shown to protect against brain disorders such as amyotrophic lateral sclerosis and ischemic stroke. However, whether this intervention has therapeutic effects in intracerebral hemorrhage (ICH) is unknown. In this study, we sought to determine the potential therapeutic value of replenishment of NAD+ in ICH. In a collagenase-induced ICH (cICH) mouse model, nicotinamide mononucleotide (NMN), a key intermediate of nicotinamide adenine dinucleotide (NAD+) biosynthesis, was administrated at 30 minutes post cICH from tail vein to replenish NAD+. NMN treatment did not decrease hematoma volume and hemoglobin content. However, NMN treatment significantly reduced brain edema, brain cell death, oxidative stress, neuroinflammation, intercellular adhesion molecule-1 expression, microglia activation and neutrophil infiltration in brain hemorrhagic area. Mechanistically, NMN enhanced the expression of two cytoprotective proteins: heme oxygenase 1 (HO-1) and nuclear factor-like 2 (Nrf2). Moreover, NMN increased the nuclear translocation of Nrf2 for its activation. Finally, a prolonged NMN treatment for 7 days markedly promoted the recovery of body weight and neurological function. These results demonstrate that NMN treats brain injury in ICH by suppressing neuroinflammation/oxidative stress. The activation of Nrf2/HO-1 signaling pathway may contribute to the neuroprotection of NMN in ICH.

   Intracerebral hemorrhage (ICH) is a devastating type of stroke occurring when an abnormal blood vessel within the brain disrupts, allowing blood to leak inside the brain tissue. Although hemorrhagic strokes are less common, accounting less than 15% of all strokes cases, they are responsible for about half of all stroke deaths, and is associated with worse recovery than ischemic stroke both in world. The primary brain injury induced by ICH, which is always considered to be hematoma-caused mechanical damage, takes place within several minutes to hours after the onset of bleeding. Secondary injury is resulted by the subsequent pathophysiological changes and the complex interaction between them. These pathophysiological changes following ICH include, but are not limited to, blood-brain barrier breakdown, hemoglobin-induced iron overload, excitotoxicity, neuroinflammation activation, triggered oxidative stress and neural cell death/apoptosis. Currently, large scale clinical trials have not reached a consensus on the benefit of surgical evacuation in treatment of ICH-induced primary injury. Thus, targeting the secondary injury attracts great attentions for development of novel therapeutic strategies for ICH.

   NAD+ is a well-known ubiquitous pyridine nucleotide that functions as an essential cofactor in mitochondrial oxidative phosphorylation. Classically, NAD+ is considered to be just a coenzyme which is essential for mitochondrial electron transfer chain. However, overwhelming evidence in recent years has demonstrated that NAD+ not only acts as a coenzyme, but also participates in the transduction of numerous important intracellular signaling pathways to critically regulate numerous biological functions including cell death, metabolism, circadian rhythms, aging and immunity through regulating several NAD+-consuming proteins such as sirtuin family proteins and poly(ADP-ribose) polymerases. As NAD+ depletion is a necessary event for neuronal death, supplement of NAD+ is neuroprotective through enhancing NAD+ pool. Our group has provided numerous evidence of the neuroprotection of NAD+ . Nicotinamide mononucleotide (NMN) is a particularly interesting chemical compound used to replenish NAD+. NMN is a key intermediate of nicotinamide adenine dinucleotide (NAD+) biosynthesis from nicotinamide, which is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT) in mammals. Recent evidence suggests that NMN treats obesity, vascular aging and islet damage. Besides, NMN has favorable effects in central nerve system (CNS) by raising brain mitochondrial respiratory deficits, protecting β-amyloid oligomer-induced cognitive impairment, delaying astrocyte-mediated motor neuron death and maintaining neural stem/progenitor cells. We and other group also previously demonstrated that NMN protectes against cerebral ischemia-induced neural apoptosis and promotes neurogenesis after cerebral ischemia. The potential therapeutic values of NMN in cerebral ischemic stroke have been discussed in detail in our previous review. However, the effects of NMN in hemorrhagic stroke have not been examined yet.

  In the present study, we conducted a straightforward study to determine whether replenishment of NAD+ by NMN, the key intermediate of NAD+ biosynthesis, is able to enhance intracerebral NAD+ pool and treat ICH-induced brain injury in animal model and, if so, to further explore the molecular mechanisms underlying the therapeutic action of NMN in ICH.

NMN treatment protects against cICH-induced acute brain injury

   Hematoma volume, brain hemoglobin content, body weight, brain water content and neurological function deficit were evaluated at 24 h after cICH. A single dose of NMN treatment given at 30 minutes (i.v.) post cICH increased intracerebral NAD+ concentrations at 2 and 6 hours post cICH (Fig. 1A). At 12 and 24 hours later, the NAD+ level returned to basal (Fig. 1A). NMN treatment did not affect the hematoma volume (Fig. 1B), decline of body weight (Fig. 1C) and brain hemoglobin content (Fig. 1D). Interestingly, the induction of edema in striatum (water content) by cICH was slightly but significantly reduced by NMN treatment (Fig. 1E). Beam walking test demonstrated that NMN improved the neurological function at 24 hours after cICH (Fig. 1F). These data suggest that NMN is unable to reduce hematoma volume, but it alleviates ICH-induced brain injury.

   Nicotinamide mononucleotide (NMN) treatment protects against cICH-induced brain injury. (A) NAD+ level in brain tissue was measured at 2 hours after injection of NMN from tail vein. *P < 0.05 vs Vehicle; NS, no significance. N = 6 per group. (B) Brain hematoma volume at 24 hours after cICH. n = 8 per group. (C) Body weight at 24 hours after cICH. ***P < 0.001 vs Sham, n = 22, 60 and 63 in Sham, cICH + Vehicle and cICH + NMN groups respectively. (D) Hemoglobin content at 24 hours after cICH. n = 8 per group. (E) Brain water content at 24 hours after cICH in cortex and striatum. **P < 0.01 cICH + Vehicle vs Sham, *P < 0.05 cICH + NMN vs cICH + Vehicle, n = 8. (F) Neurological deficit was evaluated by beam walking test at 24 hours after cICH. **P < 0.01, n = 26, 69 and 73 in Sham, cICH + Vehicle and cICH + NMN groups respectively. NS, no significance.

 Auther:Chun-Chun WeiYuan-Yuan KongGuo-Qiang LiYun-Feng GuanPei Wang & Chao-Yu Miao 

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