Hydrogen sulfide regulates NLRP3 to inhibit pyroptosis and protect against hypoxic-ischemic brain injury in neonatal rats

Background: Hypoxic-ischemic encephalopathy (HIE) is a leading cause of neonatal mortality and long-term neurological deficits. This study investigated whether sodium hydrosulfide (NaHS), an exogenous donor of hydrogen sulfide (H2S) could protect against HIE by inhibiting microglial activation and pyroptosis in brain tissue. Methods: A neonatal hypoxic-ischemic encephalopathy (NIE) model was established in rats via hypoxia combined with ligation of the left common carotid artery. Exogenous hydrogen sulfide (NaHS) and the nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain- containing receptor 3 (NLRP3) inhibitor CY-09 were administered post-injury. Cognitive function was assessed using the Morris water maze. Hippocampal injury was examined using Nissl staining. Microglial activation was assessed by immunofluorescence detection of Iba-1 expression. Pyroptosis-related proteins expression levels in brain tissue were measured using Western blotting. Results: Treatment with exogenous hydrogen sulfide significantly reduced neuronal injury, inhibited microglial activation, enhanced memory and spatial learning (as demonstrated by a decrease in escape latency, an increase of platform crossings, and time spent in the target quadrant). Furthermore, it suppressed the expression of NLRP3 inflammasome, Gasdermin D N-terminal domain (GSDMD-N), cleaved caspase-1, Interleukin (IL)-1β, and IL-18 proteins. Conclusions: Exogenous hydrogen sulfide mitigates neuronal damage, improves cognitive outcomes, and suppresses pyroptosis and microglial activation in neonatal rats with hypoxic-ischemic encephalopathy.

Hypoxic-ischemic encephalopathy; Exogenous hydrogen sulfide; Cognitive dysfunction’ microglial activation; NLRP3

[1] Lu L, Pang M, Chen T, Hu Y, Chen L, Tao X, et al. Protopine exerts neuroprotective effects on neonatal hypoxic-ischemic brain damage in rats via activation of the AMPK/PGC1α pathway. Drug Design, Development and Therapy. 2024; 18: 4975–4992.

[2] Zen R, Tsuji S, Maeda T, Urushitani M, Murakami T, Terashima T. Pro-inflammatory microglia-targeted peptide therapy ameliorates neonatal hypoxic-ischemic encephalopathy in mice. Molecular Therapy. 2025; 33: 3177–3194.

[3] Shao R, Sun D, Hu Y, Cui D. White matter injury in the neonatal hypoxic-ischemic brain and potential therapies targeting microglia. Journal of Neuroscience Research. 2021; 99: 991–1008.

[4] Sha S, Jin N, Zhou R, Ruan Y, Ouyang Y. The activation of PKM2 induces pyroptosis in hippocampal neurons via the NLRP3/Caspase-1/GSDMD pathway in neonatal rats with hypoxic-ischemic brain injury. Brain and Behavior. 2024; 14: e70108.

[5] Li T, Li J, Li T, Zhao Y, Ke H, Wang S, et al. L-cysteine provides neuroprotection of hypoxia-ischemia injury in neonatal mice via a PI3K/Akt-dependent mechanism. Drug Design, Development and Therapy. 2021; 15: 517–529.

[6] Cebeci B, Yalinbas EE, Akcilar R, Metineren H. Evaluation of the neuroprotective efficiency of sodium hydrosulfide in neonatal rats with the induced hypoxic-ischemic encephalopathy model. Bratislava Medical Journal. 2020; 121: 789–795.

[7] Liu M, Zeng C, Zhang Y, Xin Y, Deng S, Hu X. Protective role of hydrogen sulfide against diabetic cardiomyopathy by inhibiting pyroptosis and myocardial fibrosis. Biomedicine & Pharmacotherapy. 2024; 175: 116613.

[8] Chen A, Teng C, Wei J, Wu X, Zhang H, Chen P, et al. Gut microbial dysbiosis exacerbates long-term cognitive impairments by promoting intestinal dysfunction and neuroinflammation following neonatal hypoxia-ischemia. Gut Microbes. 2025; 17: 2471015.

[9] Fang M, Yu Q, Ou J, Lou J, Zhu J, Lin Z. The neuroprotective mechanisms of PPAR-γ: inhibition of microglia-mediated neuroinflammation and oxidative stress in a neonatal mouse model of hypoxic-ischemic white matter injury. CNS Neuroscience & Therapeutics. 2024; 30: e70081.

[10] Hu Y, Nan Y, Lin H, Zhao Q, Chen T, Tao X, et al. Celastrol ameliorates hypoxic-ischemic brain injury in neonatal rats by reducing oxidative stress and inflammation. Pediatric Research. 2024; 96: 1681–1692.

[11] Chen L, Xiong S, Zhou X, Fu Q. Aloesin ameliorates hypoxic-ischemic brain damage in neonatal mice by suppressing TLR4-mediated neuroinflammation. Immunity, Inflammation and Disease. 2024; 12: e1320.

[12] Fang M, Lu L, Lou J, Ou J, Yu Q, Tao X, et al. FGF21 alleviates hypoxic-ischemic white matter injury in neonatal mice by mediating inflammation and oxidative stress through PPAR-γ signaling pathway. Molecular Neurobiology. 2025; 62: 4743–4768.

[13] Shi H, Shi J, Wang Z, Zuo H, Guo T, Zheng H, et al. GAS reduced inflammatory responses in activated microglia by regulating the Ccr2/Akt/Gsk-3β pathway. Molecular Brain. 2025; 18: 40.

[14] Zhang Z, Shimizu T. Recent advances in structural studies of NLRP3 and NLRP1 inflammasome regulation. Current Opinion in Structural Biology. 2025; 92: 103057.

[15] Dou JY, Wu YN, Gao C, Zheng S, Wang CY, Dai X, et al. Ginseng and platycodon grandiflorum ameliorated pulmonary fibrosis and inflammation targeting TLR4-P2X7r/NLRP3 signaling pathway. Journal of Ethnopharmacology. 2025; 348: 119913.

[16] Jung H, Kim B, Jang G, Kim H, Lee AR, Yoon SH, et al. The NLRP3 inflammasome in microglia regulates repetitive behavior by modulating NMDA glutamate receptor functions. Cell Reports. 2025; 44: 115656.

[17] Barnett KC, Li S, Liang K, Ting JP. A 360° view of the inflammasome: mechanisms of activation, cell death, and diseases. Cell. 2023; 186: 2288–2312.

[18] Zhu JJ, Yu BY, Huang XK, He MZ, Chen BW, Chen TT, et al. Neferine protects against hypoxic-ischemic brain damage in neonatal rats by suppressing NLRP3-mediated inflammasome activation. Oxidative Medicine and Cellular Longevity. 2021; 2021: 6654954.

[19] Sha S, Jin N, Xie X, Zhou R, Ruan Y, Ouyang Y. Ethyl pyruvate alleviates NLRP3/Caspase-1/GSDMD-mediated neuronal pyroptosis in neonatal rats with hypoxic-ischemic brain damage. International Journal of Developmental Neuroscience. 2024; 84: 594–604.