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Background: Microglia-mediated neuroinflammation plays a crucial role in the pathogenesis of hypoxic-ischemic (HI)-induced brain injury. Activation of melanocortin-1 receptor (MC1R) has been shown to exert anti-inflammatory and neuroprotective effects in several neurological diseases. In the present study, we have explored the role of MC1R activation on neuroinflammation and the potential underlying mechanisms after neonatal hypoxic-ischemic brain injury in rats., Methods: A total of 169 post-natal day 10 unsexed rat pups were used. HI was induced by right common carotid artery ligation followed by 2.5 h of hypoxia. BMS-470539, a specific selective MC1R agonist, was administered intranasally at 1 h after HI induction. To elucidate the potential underlying mechanism, MC1R CRISPR KO plasmid or Nurr1 CRISPR KO plasmid was administered via intracerebroventricular injection at 48 h before HI induction. Percent brain infarct area, short- and long-term neurobehavioral tests, Nissl staining, immunofluorescence staining, and Western blot were conducted., Results: The expression levels of MC1R and Nurr1 increased over time post-HI. MC1R and Nurr1 were expressed on microglia at 48 h post-HI. Activation of MC1R with BMS-470539 significantly reduced the percent infarct area, brain atrophy, and inflammation, and improved short- and long-term neurological deficits at 48 h and 28 days post-HI. MC1R activation increased the expression of CD206 (a microglial M2 marker) and reduced the expression of MPO. Moreover, activation of MC1R with BMS-470539 significantly increased the expression levels of MC1R, cAMP, p-PKA, and Nurr1, while downregulating the expression of pro-inflammatory cytokines (TNFα, IL-6, and IL-1β) at 48 h post-HI. However, knockout of MC1R or Nurr1 by specific CRISPR reversed the neuroprotective effects of MC1R activation post-HI., Conclusions: Our study demonstrated that activation of MC1R with BMS-470539 attenuated neuroinflammation, and improved neurological deficits after neonatal hypoxic-ischemic brain injury in rats. Such anti-inflammatory and neuroprotective effects were mediated, at least in part, via the cAMP/PKA/Nurr1 signaling pathway. Therefore, MC1R activation might be a promising therapeutic target for infants with hypoxic-ischemic encephalopathy (HIE).

Background: Inhibition of inositol-requiring enzyme-1 alpha (IRE1α), one of the sensor signaling proteins associated with endoplasmic reticulum (ER) stress, has been shown to alleviate brain injury and improve neurological behavior in a neonatal hypoxic-ischemic encephalopathy (HIE) rat model. However, there is no information about the role of IRE1α inhibitor as well as its molecular mechanisms in preventing neuronal pyroptosis induced by NLRP1 (NOD-, LRR- and pyrin domain-containing 1) inflammasome. In the present study, we hypothesized that IRE1α can degrade microRNA-125-b-2-3p (miR-125-b-2-3p) and activate NLRP1/caspased-1 pathway, and subsequently promote neuronal pyroptosis in HIE rat model., Methods: Ten-day old unsexed rat pups were subjected to hypoxia-ischemia (HI) injury, and the inhibitor of IRE1α, STF083010, was administered intranasally at 1 h after HI induction. AntimiR-125 or NLRP1 activation CRISPR was administered by intracerebroventricular (i.c.v) injection at 24 h before HI induction. Immunofluorescence staining, western blot analysis, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), brain infarct volume measurement, neurological function tests, and Fluoro-Jade C staining were performed., Results: Endogenous phosphorylated IRE1α (p-IRE1α), NLRP1, cleaved caspase-1, interleukin-1β (IL-1β), and interleukin-18 (IL-18) were increased and miR-125-b-2-3p was decreased in HIE rat model. STF083010 administration significantly upregulated the expression of miR-125-b-2-3p, reduced the infarct volume, improved neurobehavioral outcomes and downregulated the protein expression of NLRP1, cleaved caspase-1, IL-1β and IL-18. The protective effects of STF083010 were reversed by antimiR-125 or NLRP1 activation CRISPR., Conclusions: IRE1α inhibitor, STF083010, reduced neuronal pyroptosis at least in part via miR-125/NLRP1/caspase-1 signaling pathway after HI.

Background: Cardiac arrest survivors suffer from neurological dysfunction including cognitive impairment. Cerebral mast cells, the key regulators of neuroinflammation contribute to neuroinflammation-associated cognitive dysfunction. Mast cell tryptase was demonstrated to have a proinflammatory effect on microglia via the activation of microglial protease-activated receptor-2 (PAR-2). This study investigated the potential anti-neuroinflammatory effect of mast cell tryptase inhibition and the underlying mechanism of PAR-2/p-p38/NFκB signaling following asphyxia-induced cardiac arrest in rats., Methods: Adult male Sprague-Dawley rats resuscitated from 10 min of asphyxia-induced cardiac arrest were randomized to four separate experiments including time-course, short-term outcomes, long-term outcomes and mechanism studies. The effect of mast cell tryptase inhibition on asphyxial cardiac arrest outcomes was examined after intranasal administration of selective mast cell tryptase inhibitor (APC366; 50 μg/rat or 150 μg/rat). AC55541 (selective PAR-2 activator; 30 μg/rat) and SB203580 (selective p38 inhibitor; 300 μg/rat) were used for intervention. Short-term neurocognitive functions were evaluated using the neurological deficit score, number of seizures, adhesive tape removal test, and T-maze test, while long-term cognitive functions were evaluated using the Morris water maze test. Hippocampal neuronal degeneration was evaluated by Fluoro-Jade C staining., Results: Mast cell tryptase and PAR-2 were dramatically increased in the brain following asphyxia-induced cardiac arrest. The inhibition of mast cell tryptase by APC366 improved both short- and long-term neurological outcomes in resuscitated rats. Such behavioral benefits were associated with reduced expressions of PAR-2, p-p38, NFκB, TNF-α, and IL-6 in the brain as well as less hippocampal neuronal degeneration. The anti-neuroinflammatory effect of APC366 was abolished by AC55541, which when used alone, indeed further exacerbated neuroinflammation, hippocampal neuronal degeneration, and neurologic deficits following cardiac arrest. The deleterious effects aggregated by AC55541 were minimized by p38 inhibitor., Conclusions: The inhibition of mast cell tryptase attenuated neuroinflammation, led to less hippocampal neuronal death and improved neurological deficits following cardiac arrest. This effect was at least partly mediated via inhibiting the PAR-2/p-p38/NFκB signaling pathway. Thus, mast cell tryptase might be a novel therapeutic target in the management of neurological impairment following cardiac arrest.