Neuroprotective effects of miRNA-326 knockout in neonatal hypoxic-ischemic brain damage mice via the δ-opioid receptor.

Hypoxic-ischemic brain damage (HIBD) in the perinatal period is an important cause of cerebral damage and long-term neurological sequelae, and can place much pressure on families and society. Our previous study demonstrated that miRNA-326 reduces neuronal apoptosis by up-regulating the δ-opioid receptor (DOR) under oxygen-glucose deprivation in vitro. In the present study, we aimed to explore the neuroprotective effects of the miRNA-326/DOR axis by inhibiting apoptosis in HIBD using neonatal miRNA-326 knockout mice. Neonatal C57BL/6 mice, neonatal miRNA-326 knockout mice, and neonatal miRNA-326 knockout mice intraperitoneally injected with the DOR inhibitor naltrindole were treated with hypoxic-ischemia (HI). Neurological deficit scores, magnetic resonance imaging, terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling, and Caspase-3, Bax, and B-cell lymphoma 2 (Bcl-2) expression were evaluated on day 2 after HI. Neurobehavioral analyses were performed on days 2 and 28 after HI. Additionally, the Morris water maze test was conducted on days 28. Compared with HI-treated neonatal C57BL/6 mice, HI-treated neonatal miRNA-326 knockout mice had higher neurological deficit scores, smaller cerebral infarction areas, and improved motor function, reaction ability, and long-term spatial learning and memory. These effects were likely the result of inhibiting apoptosis; the DOR inhibitor reversed these neuroprotective effects. Our findings indicate that miRNA-326 knockout plays a neuroprotective effect in neonatal HIBD by inhibiting apoptosis via the target gene DOR.

Effects of HMGB1/RAGE/cathespin B inhibitors on alleviating hippocampal injury by regulating microglial pyroptosis and caspase activation in neonatal hypoxic-ischemic brain damage.

Microglia play a crucial role in regulating neuroinflammation in the pathogenesis of neonatal hypoxic-ischemic brain damage (HIBD). Pyroptosis, an inflammatory form of programmed cell death, has been implicated in HIBD; however, its underlying mechanism remains unclear. We previously demonstrated that high-mobility group box 1 protein (HMGB1) mediates neuroinflammation and microglial damage in HIBD. In this study, we aimed to investigate the association between HMGB1 and microglial pyroptosis and elucidate the mechanism involved in rats with HIBD (both sexes were included) and in BV2 microglia subjected to oxygen-glucose deprivation. Our results showed that HMGB1 inhibition by glycyrrhizin (20 mg/kg) reduced the expression of microglial pyroptosis-related proteins, including caspase-1, the N-terminus fragment of gasdermin D (N-GSDMD), and pyroptosis-related inflammatory factors, such as interleukin (IL) -1ß and IL-18. Moreover, HMGB1 inhibition resulted in reduced neuronal damage in the hippocampus 72 h after HIBD and ultimately improved neurobehavior during adulthood, as evidenced by reduced escape latency and path length, as well as increased time and distance spent in the target quadrant during the Morris water maze test. These results revealed that HIBD-induced pyroptosis is mediated by HMGB1/receptor for advanced glycation end products (RAGE) signaling (inhibition by FPS-ZM1, 1 mg/kg) and the activation of cathespin B (cat B). Notably, cat B inhibition by CA074-Me (5 mg/kg) also reduced hippocampal neuronal damage by suppressing microglial pyroptosis, thereby ameliorating learning and memory impairments caused by HIBD. Lastly, we demonstrated that microglial pyroptosis may contribute to neuronal damage through the HMGB1/RAGE/cat B signaling pathway in vitro. In conclusion, these results suggest that HMGB1/RAGE/cat B inhibitors can alleviate hippocampal injury by regulating microglial pyroptosis and caspase activation in HIBD, thereby reducing the release of proinflammatory mediators that destroy hippocampal neurons and induce spatial memory impairments.

Iron overload facilitates neonatal hypoxic-ischemic brain damage via SLC7A11-mediated ferroptosis.

Oxidative damage and cell death are involved in the pathogenesis of hypoxic-ischemic brain damage (HIBD). Ferroptosis is a newly identified mode of cell death that results from the oxidative damage induced by excessive iron. In HIBD, iron accumulates in brain tissues due to the massive destruction of red blood cells and increased permeability of the blood brain barrier vasculature, which can trigger ferroptosis. Ferroptosis is implicated in various diseases involving neuronal injury; however, the roles of iron and ferroptosis in HIBD have not been identified. In the present study, we investigated the role of iron overload in neuronal ferroptosis both in HIBD rat models and in oxygen- and glucose-deprived (OGD) SH-SY5Y cells. We observed that iron deposition in the cerebral cortex was significantly increased in HIBD rats. Features of ferroptosis such as shrunken mitochondria, increased MDA (malondialdehyde) levels, and reduced solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) expression were observed in the cerebral cortex of HIBD rats. Administration of an iron chelator in HIBD rats upregulated SLC7A11 expression and alleviated neuronal ferroptosis in cerebral cortex tissue. Additionally, overexpression of SLC7A11 in SH-SY5Y cells increased cell viability and attenuated OGD-induced ferroptosis. Our results demonstrate that iron overload induces neuronal ferroptosis by inhibiting SLC7A11 expression in HIBD. Inhibition of neuronal ferroptosis may be a promising strategy to alleviate brain damage in HIBD.

A Review of Plant Extracts and Plant-Derived Natural Compounds in the Prevention/Treatment of Neonatal Hypoxic-Ischemic Brain Injury.

Neonatal hypoxic-ischemic (HI) brain injury is one of the major drawbacks of mortality and causes significant short/long-term neurological dysfunction in newborn infants worldwide. To date, due to multifunctional complex mechanisms of brain injury, there is no well-established effective strategy to completely provide neuroprotection. Although therapeutic hypothermia is the proven treatment for hypoxic-ischemic encephalopathy (HIE), it does not completely chang outcomes in severe forms of HIE. Therefore, there is a critical need for reviewing the effective therapeutic strategies to explore the protective agents and methods. In recent years, it is widely believed that there are neuroprotective possibilities of natural compounds extracted from plants against HIE. These natural agents with the anti-inflammatory, anti-oxidative, anti-apoptotic, and neurofunctional regulatory properties exhibit preventive or therapeutic effects against experimental neonatal HI brain damage. In this study, it was aimed to review the literature in scientific databases that investigate the neuroprotective effects of plant extracts/plant-derived compounds in experimental animal models of neonatal HI brain damage and their possible underlying molecular mechanisms of action.

Neuroprotective strategies for neonatal hypoxic-ischemic brain damage: Current status and challenges.

Neonatal hypoxic-ischemic brain damage (HIBD) is a prominent contributor to both immediate mortality and long-term impairment in newborns. The elusive nature of the underlying mechanisms responsible for neonatal HIBD presents a significant obstacle in the effective clinical application of numerous pharmaceutical interventions. This comprehensive review aims to concentrate on the potential neuroprotective agents that have demonstrated efficacy in addressing various pathogenic factors associated with neonatal HIBD, encompassing oxidative stress, calcium overload, mitochondrial dysfunction, endoplasmic reticulum stress, inflammatory response, and apoptosis. In this review, we conducted an analysis of the precise molecular pathways by which these drugs elicit neuroprotective effects in animal models of neonatal hypoxic-ischemic brain injury (HIBD). Our objective was to provide a comprehensive overview of potential neuroprotective agents for the treatment of neonatal HIBD in animal experiments, with the ultimate goal of enhancing the feasibility of clinical translation and establishing a solid theoretical foundation for the clinical management of neonatal HIBD.

RNA-seq Analysis Reveals Potential Molecular Mechanisms of ZNF580/ZFP580 Promoting Neuronal Survival and Inhibiting Apoptosis after Hypoxic-ischemic Brain damage.

Neonatal hypoxic-ischemic brain damage (HIBD) is one of the main causes of neonatal acute death and chronic nervous system impairment, but still lacks effective treatments. ZNF580/ZFP580, reported in our previous studies, may be a newly identified member of the Krüppel-like factor (KLF) family, and has anti-apoptotic effects during ischemic myocardial injury. In the present study, we showed that the expression levels of both ZFP580/ZNF580 mRNA and protein increased significantly in neonatal HIBD rats and oxygen-glucose deprivation (OGD) SH-SY5Y cell models. ZNF580 overexpression promoted neuron survival and suppressed neuron apoptosis after OGD in neuron-like SH-SY5Y cells, while interference with ZNF580 resulted in the opposite results. RNA-seq analysis identified 248 differentially-expressed genes (DEGs) between ZNF580 overexpression SH-SY5Y cells and interference-expressed SH-SY5Y cells. Gene Ontology functional enrichment analysis showed that these DEGs played significant roles in the growth, development, and regeneration of axons, DNA biosynthetic processes, DNA replication, and apoptosis. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that these DEGs were found in some pathways, including ferroptosis, glutamatergic synapses, protein processing in the endoplasmic reticulum, estrogen signaling pathways, the TGF-beta signaling pathway, and the longevity regulating pathway. The qRT-PCR validation results were consistent with RNA-seq results, which showed that HSPA5, IGFBP3, NTN4, and KLF9 increased in ZNF580-overexpressed SH-SY5Y cells and decreased in interference-expressed SH-SY5Y cells, when compared with normal cells. Together, the results suggested that ZNF580 targeted these genes to inhibit neuronal apoptosis.

Macamide B Pretreatment Attenuates Neonatal Hypoxic-Ischemic Brain Damage of Mice Induced Apoptosis and Regulates Autophagy via the PI3K/AKT Signaling Pathway.

Lepidium meyenii (maca) is an annual or biennial herb from South America that is a member of the genus Lepidium L. in the family Cruciferae. This herb possesses antioxidant and antiapoptotic activities, enhances autophagy functions, prevents cell death, and protects neurons from ischemic damage. Macamide B, an effective active ingredient of maca, exerts a neuroprotective effect on neonatal hypoxic-ischemic brain damage (HIBD), but the mechanism underlying its neuroprotective effect is not yet known. The purpose of this study was to explore the effect of macamide B on HIBD-induced autophagy and apoptosis and its potential neuroprotective mechanism. The modified Rice-Vannucci method was used to induce HIBD in 7-day-old (P7) macamide B- and vehicle-pretreated pups. TTC staining was performed to evaluate the cerebral infarct volume in pups, the brain water content was measured to evaluate the neurological function of pups, neurobehavioural testing was conducted to assess functional recovery after HIBD, TUNEL and FJC staining was performed to detect cellular autophagy and apoptosis, and Western blot analysis was used to detect the levels of proteins in the pro-survival phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway and autophagy and apoptosis-related proteins. Macamide B pretreatment significantly decreases brain damage and improves the recovery of neural function after HIBD. At the same time, macamide B pretreatment activates the PI3K/AKT signaling pathway after HIBD, enhances autophagy, and reduces hypoxic-ischemic (HI)-induced apoptosis. In addition, 3-methyladenine (3-MA), an inhibitor of the PI3K/AKT signaling pathway, significantly inhibits the increase in autophagy levels, aggravates HI-induced apoptosis, and reverses the neuroprotective effect of macamide B on HIBD. Our data indicate that a macamide B pretreatment might regulate autophagy through the PI3K/AKT signaling pathway, thereby reducing HIBD-induced apoptosis and exerting neuroprotective effects on neonatal HIBD. Macamide B may become a new drug for the prevention and treatment of HIBD.

Asiaticoside attenuates neonatal hypoxic-ischemic brain damage through inhibiting TLR4/NF-κB/STAT3 pathway.

BACKGROUND: Neonatal hypoxic ischemic encephalopathy (HIE) is currently a leading cause of neonatal death. Asiaticoside (AT), a bioactive constituent isolated from Centella asiatica, possesses numerous biological properties. For instance, previous studies showed that AT could protect ischemia hypoxia neurons by mediating BCL-2 protein. However, the roles and underlying mechanisms of AT in neonatal HIE have not been clarified. METHODS: Rice-Vannucci was applied to construct a hypoxic-ischemic brain damage (HIBD) model. Pathological damage of brain neuron tissue was determined by hematoxylin-eosin (HE) staining, while apoptosis was evaluated by terminal-deoxynucleoitidyl transferase nick end labeling (TUNEL) staining. Western blot and immunohistochemistry were applied to monitor related proteins levels. Enzyme-linked immunosorbent assay (ELISA) was conducted to measure the expression levels of inflammatory cytokines. RESULTS: The present study indicated that AT dose-dependently ameliorated histologic damage and inhibited apoptosis induced by hypoxic ischemia (HI) (P<0.01). AT also dose-dependently alleviated oxidative damage and reduced the levels of proinflammatory cytokines (ICAM-1, IL-18, and IL-1ß) and TLR4 level. In terms of mechanism, decrease of TLR and IL-18 suppressed NF-κB phosphorylation and reduced the levels of TNFα, IL-6, and p-STAT3, leading to the inactivation of NF-κB/STAT3 pathway. Interestingly, with the addition of lipopolysaccharide (LPS), the increase of TLR4 activated NF-κB/STAT3 pathway again. CONCLUSIONS: Collectively, the data provide insight into a novel mechanism by which AT may be an effective agent for HIE via the TLR4/NF-κB/STAT3 pathway.

bFGF promotes neurological recovery from neonatal hypoxic-ischemic encephalopathy by IL-1ß signaling pathway-mediated axon regeneration.

INTRODUCTION: Neonatal hypoxia-ischemic brain damage (HIBD) can lead to serious neuron damage and dysfunction, causing a significant worldwide health problem. bFGF as a protective reagent promotes neuron repair under hypoxia/ischemia (HI). However, how bFGF and downstream molecules were regulated in HI remains elusive. METHODS: We established an in vitro HI model by culturing primary cortical neurons and treated with oxygen-glucose deprivation (OGD). We suppressed the expression of bFGF by using siRNA (small interfering RNA) interference to detect the neuronal morphological changes by immunofluorescence staining. To determine the potential mechanisms regulated by bFGF, the change of downstream molecular including IL-1ß was examined in bFGF knockdown condition. IL-1ß knockout (KO) rats were generated using CRISPR/Cas9-mediated technologies. We used an accepted rat model of HI, to assess the effect of IL-1ß deletion on disease outcomes and carried out analysis on the behavior, histological, cellular, and molecular level. RESULTS: We identified that OGD can induce endogenous expression of bFGF. Both OGD and knockdown of bFGF resulted in reduction of neuron numbers, enlarged cell body and shortened axon length. We found molecules closely related to bFGF, such as interleukin-1ß (IL-1ß). IL-1ß was up-regulated after bFGF interference under OGD conditions, suggesting complex signaling between bFGF and OGD-mediated pathways. We found HI resulted in up-regulation of IL-1ß mRNA in cortex and hippocampus. IL-1ß KO rats markedly attenuated the impairment of long-term learning and memory induced by HI. Meanwhile, IL-1ß-/- (KO, homozygous) group showed better neurite growth and less apoptosis in OGD model. Furthermore, serine/threonine protein kinase (AKT1) mRNA and protein expression was significantly up-regulated in IL-1ß KO rats. CONCLUSIONS: We showed that IL-1ß-mediated axon regeneration underlie the mechanism of bFGF for the treatment of HIBD in neonatal rats. Results from this study would provide insights and molecular basis for future therapeutics in treating HIBD.

Resatorvid protects against hypoxic-ischemic brain damage in neonatal rats.

Secondary brain damage caused by hyperactivation of autophagy and inflammatory responses in neurons plays an important role in hypoxic-ischemic brain damage (HIBD). Although previous studies have implicated Toll-like receptor 4 (TLR4) and nuclear factor kappa-B (NF-κB) in the neuroinflammatory response elicited by brain injury, the role and mechanisms of the TLR4-mediated autophagy signaling pathway in neonatal HIBD are still unclear. We hypothesized that this pathway can regulate brain damage by modulating neuron autophagy and neuroinflammation in neonatal rats with HIBD. Hence, we established a neonatal HIBD rat model using the Rice-Vannucci method, and injected 0.75, 1.5, or 3 mg/kg of the TLR4 inhibitor resatorvid (TAK-242) 30 minutes after hypoxic ischemia. Our results indicate that administering TAK-242 to neonatal rats after HIBD could significantly reduce the infarct volume and the extent of cerebral edema, alleviate neuronal damage and neurobehavioral impairment, and decrease the expression levels of TLR4, phospho-NF-κB p65, Beclin-1, microtubule-associated protein l light chain 3, tumor necrosis factor-α, and interleukin-1ß in the hippocampus. Thus, TAK-242 appears to exert a neuroprotective effect after HIBD by inhibiting activation of autophagy and the release of inflammatory cytokines via inhibition of the TLR4/NF-κB signaling pathway. This study was approved by the Laboratory Animal Ethics Committee of Affiliated Hospital of Yangzhou University, China (approval No. 20180114-15) on January 14, 2018.

miRNA-7a-2-3p Inhibits Neuronal Apoptosis in Oxygen-Glucose Deprivation (OGD) Model.

Neuronal apoptosis is a major pathological hallmark of the neonatal hypoxic-ischemic brain damage (HIBD); however, the role of miR-7a-2-3p in the regulation of HIBD remains unknown. The purpose of this study was to explore the possible roles of miR-7a-2-3p in brain injury using a hypoxia-ischemia model in rats and oxygen-glucose deprivation (OGD) model in vitro. Firstly, we established the hypoxia-ischemia (HI) model and verified the model using Zea Longa scores and MRI in rats. Next, the changes of miR-7a-2-3p were screened in the ischemic cortex of neonatal rats by qRT-PCR at 12, 48, and 96 h after HIBD. We have found that the expression of miR-7a-2-3p in the HI rats decreased significantly, compared with the sham group (P < 0.01). Then, we established the OGD model in PC12 cells, SH-SY5Y cells and primary cortical neurons in vitro and qRT-PCR was used to confirm the changes of miR-7a-2-3p in these cells after the OGD. In order to determine the function of miR-7a-2-3p, PC12 cells, SH-SY5Y cells and rat primary cortical neurons were randomly divided into normal, OGD, mimic negative control (mimic-NC) and miR-7a-2-3p groups. Then, Tuj1+ (neuronal marker) staining, TUNEL assay (to detect apoptotic cells) and MTT assay (to investigate cell viability) were performed. We have found that the number of PC12 cells, SH-SY5Y cells and cortical neurons in the miR-7a-2-3p groups increased significantly (P < 0.01) in comparison to the OGD groups. The survival of cortical neurons in the miR-7a-2-3p group was improved markedly (P < 0.01), while the apoptosis of neurons in the miR-7a-2-3p group was significantly decreased (P < 0.01), compared with the normal group. Lastly, we investigated the target genes of miR-7a-2-3p by using the prediction databases (miRDB, TargetScan, miRWalk, and miRmap) and verified the target genes with qRT-PCR in the HI rats. Bioinformatics prediction showed that Vimentin (VIM), pleiomorphic adenoma gene 1(PLAG1), dual specificity phosphatase 10 (DUSP10), NAD(P)H dehydrogenase, quinone 1 (NQO1) and tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) might be the targets of miR-7a-2-3p and the qRT-PCR confirmed that VIM increased in the HI rats (P < 0.01). In conclusion, miR-7a-2-3p plays a crucial role in the hypoxic-ischemic injury, and is associated with regulation of VIM.