Hydrogen Gas Attenuates Hypoxic-Ischemic Brain Injury via Regulation of the MAPK/HO-1/PGC-1a Pathway in Neonatal Rats

Abstract:

Neonatal hypoxic-ischemic encephalopathy (HIE) is a leading cause of death in neonates with no effective treatments. Recent advancements in hydrogen (H2) gas offer a promising therapeutic approach for ischemia reperfusion injury; however, the impact of this approach for HIE remains a subject of debate. We assessed the therapeutic effects of H2 gas on HIE and the underlying molecular mechanisms in a rat model of neonatal hypoxic-ischemic brain injury (HIBI). H2 inhalation significantly attenuated neuronal injury and effectively improved early neurological outcomes in neonatal HIBI rats as well as learning and memory in adults. This protective effect was associated with initiation time and duration of sustained H2 inhalation. Furthermore, H2 inhalation reduced the expression of Bcl-2-associated X protein (BAX) and caspase-3 while promoting the expression of Bcl-2, nuclear factor erythroid-2-related factor 2, and heme oxygenase-1 (HO-1). H2 activated extracellular signal-regulated kinase and c-Jun N-terminal protein kinase and dephosphorylated p38 mitogen-activated protein kinase (MAPK) in oxygen-glucose deprivation/reperfusion (OGD/R) nerve growth factor-differentiated PC12 cells. Inhibitors of MAPKs blocked H2-induced HO-1 expression. HO-1 small interfering RNA decreased the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and sirtuin 1 (SIRT1) and reversed the protectivity of H2 against OGD/R-induced cell death. These findings suggest that H2 augments cellular antioxidant defense capacity through activation of MAPK signaling pathways, leading to HO-1 expression and subsequent upregulation of PGC-1α and SIRT-1 expression. Thus, upregulation protects NGF-differentiated PC12 cells from OGD/R-induced oxidative cytotoxicity. In conclusion, H2 inhalation exerted protective effects on neonatal rats with HIBI. Early initiation and prolonged H2 inhalation had better protective effects on HIBI. These effects of H2 may be related to antioxidant, antiapoptotic, and anti-inflammatory responses. HO-1 plays an important role in H2-mediated protection through the MAPK/HO-1/PGC-1α pathway. Our results support further assessment of H2 as a potential therapeutic for neurological conditions in which oxidative stress and apoptosis are implicated. 1. Background Neonatal hypoxic-ischemic encephalopathy (HIE) is a severe disease with high neonatal morbidity and mortality; 25% of HIE survivors have permanent neurological defects [1]. Before irreversible brain injury occurs, the specific pathological process of hypoxia-ischemia (HI) occurs through a combination of multiple mechanisms [2, 3]. The severity and duration of these mechanisms determine the extent of brain injury in HIE [4]. Molecular hydrogen (H2) easily penetrates the blood-brain barrier and is a novel antioxidant [5]. Previous studies have shown that H2 mitigates ischemia/reperfusion- (I/R-) induced injury to different organs [6–8]. However, the neuroprotective effects of H2 treatment on hypoxic-ischemic brain injury (HIBI) are controversial. For example, a recent study showed that H2-enriched water exerts neuroprotective effects on brain tissue after induction of HIBI [9]. However, another study found no protective effects [10]. Oxidative stress is a well-recognized consequence of HIE and is considered an important contributor to early brain injury after HI [11] working in combination with inflammation [12]. Growing evidence suggests that oxidative stress and neuroinflammation underpin a diverse range of central nervous system (CNS) diseases including stroke, traumatic brain injury, multiple sclerosis, Alzheimer’s, Parkinson’s, and other neurodegenerative diseases [13–15]. Heme oxygenase-1 (HO-1) is an important component of the cellular defense enzyme that is induced by and acts against oxidant-induced I/R injury [16]. HO-1 overexpression may also affect the regulation of apoptotic pathway genes such as B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (BAS), and caspases [17]. HO-1 has antineuroinflammatory and neuroprotective properties in the CNS [18, 19]. Thus, therapies targeting HO-1 may be potential treatments for protection against inflammation, oxidative stress, and apoptosis after HI. Multiple signaling kinases related to cell survival and proliferation reportedly regulate the nuclear translocation of HO-1. Mitogen-activated protein kinases (MAPKs) are some of the most common signaling pathways, which serve to coordinate the cellular response to a variety of extracellular stimuli [20]. These are well characterized in mammals and include c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK) [21, 22]. Activation of MAPKs modulates HO-1 expression [23]. In this study, we evaluated the neuroprotective effects of H2 on neonatal HIBI rats through behavioral tests, immunofluorescence, and western blot analysis to determine the appropriate therapeutic window for of H2. In vivo, we also tested the expression of high-mobility group protein B1 (HMGB1) and toll-like receptor 4 (TLR-4), the apoptosis-related factors Bcl-2/BAX-caspase3, and the oxidative stress signaling molecules p38 MAPK/Nrf2/HO-1. Then, we investigated the potential MAPK/HO-1/peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) signaling pathway in nerve growth factor- (NGF-) differentiated PC12 cells after oxygen-glucose deprivation/reperfusion (OGD/R). 2. Methods 2.1. Experimental Design In Vivo A total of 320 neonatal rats were randomly divided into the following eight groups (): HIBI, HIBI+H2-30 min, HIBI+H2-60 min, HIBI+H2-90 min, HIBI+H2-90 min (12 h), HIBI+H2-90 min (24 h), sham surgery, and control. Animals in the HIBI and all HIBI+H2 treatment groups were rats with induced HIBI. After establishing the HIBI model, neonatal rats in the HIBI+H2-30 min, HIBI+H2-60 min, and HIBI+H2-90 min groups were immediately placed in a box for 30, 60, or 90 min of H2 inhalation, respectively. This was followed by twice daily H2 inhalation (30, 60, or 90 min each time) over the next 3 days (7 : 00–9 : 00 am and 7 : 00–9 : 00 pm). Animals in the HIBI+H2-90 min (12 h) and HIBI+H2-90 min (24 h) groups underwent 3% H2 inhalation for 90 min at 12 and 24 h, respectively, after induction of HI. This was followed by twice daily H2 inhalation for 90 min for 3 days. Animals in the sham surgery group underwent left carotid artery isolation without ligation and hypoxia induction. Animals in the control group were healthy neonatal rats without induction of HIBI. Detection of early neurological reflexes was performed ~24 h after HIBI induction, and then some neonatal rats were subjected to triphenyltetrazolium chloride (TTC) staining (). At 96 h after inducing HIBI, brain samples from neonatal rats were collected for western blotting, paraffin-embedded, and sectioned for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and immunofluorescence staining. In addition, some of the neonatal rats () in adulthood (postnatal days 70–74) underwent testing in a Morris water maze (Figure 1). The study was performed in a blinded and randomized matter with respect to treatment administration and histological and functional assessments.