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Activation of the TLR4/NF-κB signaling pathway and autophagy, and release of inflammatory cytokines in the hippocampus during brain damage after HIBD. (A) Representative western blot bands of TLR4, p-NF-κB p65, Beclin-1, and LC3 at different time points after HIBD. (B-E) The protein expression levels of TLR4 (B), p-NF-κB p65 (C), Beclin-1 (D), and LC3 (E) were significantly increased and reached peak levels at 48 hours, and had gradually decreased by 72 hours. Protein expression levels of TLR4, p-NF-κB p65, and Beclin-1 were assessed according to the ratio of gray values between the target protein bands and β-actin bands. (F, G) An enzyme-linked immunosorbent assay showed that the expression levels of TNF-α (F) and IL-1β (G) were markedly elevated after HIBD such that they reached peak levels at 48 hours and had gradually decreased by 72 hours. Data are expressed as the mean ± SD (n = 6 per group) and were analyzed via one-way analysis of variance followed by the Student-Newman-Keuls test. *P < 0.05, **P < 0.01, vs. sham-operated group. HIBD: Hypoxic-ischemic brain damage; IL-1β: interleukin-1β; LC3: microtubule-associated protein l light chain 3; NF-κB: nuclear factor kappa-B; p-NF-κB: phos-pho-NF-κB; TLR4: Toll-like receptor 4; TNF-α: tumor necrosis factor-α.
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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 mechani...
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... found that compared with the sham-operated group, rats that underwent HIBD exhibited significantly increased levels of TLR4, p-NF-κB p65, Beclin-1, LC3, TNF-α, and IL-1β in the left hippocampus after HIBD. These increases reached peak levels at 48 hours, and had gradually decreased by 72 hours after HIBD (P < 0.01, vs. sham-operated group; Figure 1). ...
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... present, it is believed that neuronal autophagy can be divided into neuronal basal autophagy and neuronal inducible autophagy. Neuronal basal autophagy protects neurons, while neuronal inducible autophagy damages neurons because its intensity is extremely strong (Hou et al., 2019). Many experiments have ...
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... Neurodegenerative and neuropsychiatric disorders may be caused by dysfunctional autophagy [10][11][12]. Accumulating evidence indicates that autophagy activation is crucial in the pathogenesis of brain damage following neonatal HI [13,14]. Further, aggravated autophagy accumulation eventually results in synaptic and cognitive impairment [15]. ...
Hypoxic-ischemic brain damage (HIBD) can result in significant global rates of neonatal death or permanent neurological disability. N6-methyladenosine (m6A) modification of RNA influences fundamental aspects of RNA metabolism, and m6A dysregulation is implicated in various neurological diseases. However, the biological roles and clinical significance of m6A in HIBD remain unclear. We currently evaluated the effect of HIBD on cerebral m6A methylation in RNAs in neonatal rats. The m6A dot blot assay showed a global augmentation in RNA m6A methylation post-HI. Herein, we also report on demethylase FTO, which is markedly downregulated in the hippocampus and is the main factor involved with aberrant m6A modification following HI. By conducting a comprehensive analysis of RNA-seq data and m6A microarray results, we found that transcripts with m6A modifications were more highly expressed overall than transcripts without m6A modifications. The overexpression of FTO resulted in the promotion of Akt/mTOR pathway hyperactivation, while simultaneously inhibiting autophagic function. This is carried out by the demethylation activity of FTO, which selectively demethylates transcripts of phosphatase and tensin homolog (PTEN), thus promoting its degradation and reduced protein expression after HI. Moreover, the synaptic and neurocognitive disorders induced by HI were effectively reversed through the overexpression of FTO in the hippocampus. Cumulatively, these findings demonstrate the functional importance of FTO-dependent hippocampal m6A methylome in cognitive function and provides novel mechanistic insights into the therapeutic potentials of FTO in neonatal HIBD.
... A possible intervention to address bone pathology and simultaneously test the hypothesis that bone manifestations of MPS IIIB are partially driven by TLR4 overactivation would be a simple small-molecule TLR4 or TLR pathway inhibitor. Resatorvid is a TLR-4 antagonist [27][28][29] that crosses the blood-brain barrier (BBB) and has shown activity in hypoxic and traumatic brain injury models [30]. Additionally, Resatorvid (TAK-242) has demonstrated disease modification in mouse models of arthritis, indicating our ability to affect bone and joint physiology [31]. ...
Sanfilippo syndrome Type-B, also known as mucopolysaccharidosis IIIB (MPS IIIB), accounts for approximately one-third of all Sanfilippo syndrome patients and is characterized by a similar natural history as Type-A. Patients suffer from developmental regression, bone malformation, organomegaly, GI distress, and profound neurological deficits. Despite human trials of enzyme replacement therapy (ERT) (SBC-103, AX250) in MPS IIIB, there is currently no FDA approved treatment and a few palliative options. The major concerns of ERT and gene therapy for the treatment of bone malformation are the inadequate biodistribution of the missing enzyme, N-acetyl-α-glucosaminidase (NAGLU), and that the skeleton is a poorly hit target tissue in ERT and gene therapy. Each of the four known human types of MPS III (A, B, C, and D) is usually regarded as having mild bone manifestations, yet it remains poorly characterized. This study aimed to determine bone mineral content (BMC), volumetric bone mineral density (vBMD), and biomechanical properties in femurs MPS IIIB C57BL/6 mice compared to phenotypic control C57BL/6 mice. Significant differences were observed in MPS IIIB mice within various cortical and cancellous bone parameters for both males and females (p < 0.05). Here, we establish some osteogenic manifestations of MPS IIIB within the mouse model by radiographic and biomechanical tests, which are also differentially affected by age and sex. This suggests that some skeletal features of the MPS IIIB mouse model may be used as biomarkers of peripheral disease correction for preclinical treatment of MPS IIIB.
... Previous studies have also reported that TAK-242 alleviates brain edema and neurological deficits following TBI [11], and therefore, we did not conduct repeated verification in this study. Studies on the effects of TAK-242 on hypoxic-ischemic encephalopathy (HIE) showed that TAK-242 alleviates neurological deficits and improves neurobehavioral function in a neonatal HIE rat model [37]. In addition, TAK-242 was similarly confirmed to improve the neurobehavior of intraventricular hemorrhage rats and attenuate their learning and spatial memory deficits, suggesting early TAK-242 intervention is beneficial for blocking the progression of neurocognitive deficits induced by hydrocephalus [22]. ...
Inhibition of Toll-like receptor 4 (TLR4)-mediated inflammatory pathways exerts a critical effect on neuronal death; therefore, it is a possible new therapeutic approach for traumatic brain injury (TBI). Resatorvid (TAK-242) is a novel small-molecule compound widely used to inhibit TLR4-mediated pathways, but the protective mechanism of TAK-242 in TBI remains unclear. Herein, we analyzed the neuroprotective effects of TAK-242 in rats after TBI. The rat model of brain injury was established using a modified Free-fall device, and the rats were injected with TAK-242 (0.5 mg/kg) through the caudal vein before TBI. The rats were allocated into four groups: a sham group, a TBI group, a TBI + vehicle group, and a TBI + TAK-242 group. The brain tissue was extracted for histology and determination of the expression of autophagy-related proteins and inflammatory mediators. TAK-242 pretreatment significantly reduced the damage to hippocampal neurons. Neuronal autophagy increased after brain injury, whereas TAK-242 significantly reduced autophagy marker protein LC3-II in the hippocampus. In addition, TAK-242 pretreatment significantly downregulated NF-κB p65, TNF-α, and IL-1β in the hippocampus. In conclusion, TAK-242 significantly reduced hippocampal neuronal damage by inhibiting autophagy and neuroinflammatory activity, possibly via the NF-κB signaling pathway.
... The changes in circulating iron levels may cause iron accumulation in the nervous system [9], and excessive iron accumulation causes neurodegenerative changes that contribute to damage to neurons, astrocytes, and cerebrovascular endothelial cells [10]. Clinical studies have shown that obvious bleeding points appear in the periventricular white matter after WMI in newborns [11], and abnormal accumulation of iron content appears in the basal ganglia [12] while non-protein bound iron levels in the cerebrospinal fluid and serum are significantly increased [13]. Our previous study results also indicate that the iron content in the brains of neonatal rats increased significantly after HI [14]. ...
Hypoxic-ischemic white matter injury (WMI) pathogenesis in preterm infants is not well established, and iron-related proteins in the brain may play an important role in imbalanced iron metabolism. We aimed to investigate the iron-related protein changes in neonatal rats after hypoxia-ischemia (HI), clarify the role of iron-related proteins in hypoxic-ischemic WMI, and potentially provide a new target for the clinical treatment of hypoxic-ischemic WMI in preterm infants. We adopted a WMI animal model of bilateral common carotid artery electrocoagulation combined with hypoxia in neonatal 3-day-old Sprague-Dawley rats. We observed basic myelin protein (MBP) and iron-related protein expression in the brain (ferritin, transferrin receptor [TfR], and membrane iron transporter 1 [FPN1]) via Western blot and double immunofluorescence staining. The expression of MBP in the WMI group was significantly downregulated on postoperative days (PODs) 14, 28, and 56. Ferritin levels were significantly increased on PODs 3, 7, 14, and 28 and were most significant on POD 28, returning to the sham group level on POD 56. FPN1 levels were significantly increased on PODs 7, 28, and 56 and were still higher than those in the sham group on POD 56. TfR expression was significantly upregulated on PODs 1, 7, and 28 and returned to the sham group level on POD 56. Immunofluorescence staining showed that ferritin, TfR, and FPN1 were expressed in neurons, blood vessels, and oligodendrocytes in the cortex and corpus callosum on POD 28. Compared with the sham group, the immune-positive markers of three proteins in the WMI group were significantly increased. The expression of iron-related proteins in the brain (ferritin, FPN1, and TfR) showed spatiotemporal dynamic changes and may play an important role in hypoxic-ischemic WMI.
... Hypoxic-ischemic (HI) brain damage (HIBD) occurs when cerebral blood flow is decreased or suspended, resulting in partial or complete hypoxia of brain tissues [1]. In neonates and adults, brain injury produced by HI is recognized as a devastating incident that frequently leads to death or profound long-term neurological morbidity [2]. ...
Previous studies have shown that mfat-1 transgenic mice have protective effects against some central nervous system (CNS) disorders, owing to the high docosahexaenoic acid (DHA) content enriched in their brains. However, whether this protective effect is connected to the blood–brain barrier (BBB) remains unclear. This study aims to investigate the mechanisms of the protective effect against hypoxic-ischemic brain damage (HIBD) of mfat-1 transgenic mice. mfat-1 mice not only demonstrated a significant amelioration of neurological dysfunction and neuronal damage but also partly maintained the physiological permeability of the BBB after HIBD. We initially showed this was associated with elevated major facilitator superfamily domain-containing 2a (Mfsd2a) expression on the BBB, resulting from more lysophosphatidylcholine (LPC)-DHA entering the brain. Wild-type (WT) mice showed a similar Mfsd2a expression trend after long-term feeding with an LPC-DHA-rich diet. Knockdown of Mfsd2a by siRNA intra-cerebroventricular (ICV) injection neutralized the protective effect against HIBD-induced BBB disruption in mfat-1 mice, further validating the protective function of Mfsd2a on BBB. HIBD-induced BBB high permeability was attenuated by Mfsd2a, primarily through a transcellular pathway to decrease caveolae-like vesicle-mediated transcytosis. Taken together, these findings not only reveal that mfat-1 transgenic mice have higher expression of Mfsd2a on the BBB, which partly sustains BBB permeability via vesicular transcytosis to alleviate the severity of HIBD, but also suggest that dietary intake of LPC-DHA may upregulate Mfsd2a expression as a novel therapeutic strategy for BBB dysfunction and survival in HIBD patients.
... The NF-κB pathway is the main signaling pathway that activates LCN2 transcription. Secondary brain injury resulting from neuronal ferroptosis and over-activation of the inflammatory response plays an important role in hypoxic ischemic brain injury [51]. Although previous studies have shown that NF-κB is involved in the neuroinflammatory response induced by brain injury, the role and mechanism of NF-κB-mediated transcriptional activation of LCN2 in neonatal HIBD is unclear. ...
Hypoxic-ischemic brain damage (HIBD) is a common cause of death or mental retardation in newborns. Ferroptosis is a novel form of iron-dependent cell death driven by lipid peroxidation, and recent studies have confirmed that ferroptosis plays an important role in the development of HIBD. However, HIBD ferroptosis-related biomarkers remain to be discovered. An artificial neural network (ANN) was established base on differentially expressed genes (DEGs) related to HIBD and ferroptosis and validated by external dataset. The protein–protein interaction (PPI) network, support vector machine-recursive feature elimination (SVM-RFE) algorithms, and random forest (RF) algorithm were utilized to identify core genes of HIBD. An in vitro model of glutamate-stimulated HT22 cell HIBD was constructed, and glutamate-induced ferroptosis and mitochondrial structure and function in HT22 cells were examined by propidium iodide (PI) staining, flow cytometry, Fe2+ assay, Western blot, JC-1 kit, and transmission electron microscopy (TEM). In addition, Western blot and immunofluorescence assays were used to detect the NF-κB/STAT3 pathway. An HIBD classification model was constructed and presented excellent performance. The PPI network and two machine learning algorithms indicated two hub genes in HIBD. Lipocalin 2 (LCN2) was the core gene correlated with the risk of HIBD according to the results of differential expression analysis and logistic regression diagnostics. Subsequently, we verified in an in vitro model that LCN2 is highly expressed in glutamate-induced ferroptosis in HT22 cells. More importantly, LCN2 silencing significantly inhibited glutamate-stimulated ferroptosis in HT22 cells. We also found that glutamate-stimulated HT22 cells produced mitochondrial dysfunction. Furthermore, in vitro experiments confirmed that NF-κB and STAT3 were activated and that silencing LCN2 could have the effect of inhibiting their activation. In short, our findings reveal a molecular mechanism by which LCN2 may promote ferroptosis in HIBD through activation of the NF-κB/STAT3 pathway, providing new and unique insights into LCN2 as a biomarker for HIBD and suggesting new preventive and therapeutic strategies for HIBD.
... NEURAL REGENERATION RESEARCH www.nrronline.org Research Article crucial role in pathological features of diabetes and ischemic stroke (Liu et al., 2013;Zhang et al., 2013;Han et al., 2016;Jiang et al., 2020;Lang et al., 2021). This pathway was first discovered in the context of tumor immunity and subsequently found to play essential roles in the pathophysiology of many diseases (Muzio et al., 1998;Yang et al., 2015;Dong et al., 2018;Wang et al., 2018). ...
Diabetes mellitus is an independent risk factor for ischemic stroke. Both diabetes mellitus and stroke are linked to systemic inflammation that aggravates patient outcomes. Stellate ganglion block can effectively regulate the inflammatory response. Therefore, it is hypothesized that stellate ganglion block could be a potential therapy for ischemic stroke in diabetic subjects. In this study, we induced diabetes mellitus in rats by feeding them a high-fat diet for 4 successive weeks. The left middle cerebral artery was occluded to establish models of ischemic stroke in diabetic rats. Subsequently, we performed left stellate ganglion block with 1% lidocaine using the percutaneous posterior approach 15 minutes before reperfusion and again 20 and 44 hours after reperfusion. Our results showed that stellate ganglion block did not decrease the blood glucose level in diabetic rats with diabetes mellitus but did reduce the cerebral infarct volume and the cerebral water content. It also improved the recovery of neurological function, increased 28-day survival rate, inhibited Toll like receptor 4/nuclear factor kappa B signaling pathway and reduced inflammatory response in the plasma of rats. However, injection of Toll like receptor 4 agonist lipopolysaccharide 5 minutes before stellate ganglion block inhibited the effect of stellate ganglion block, whereas injection of Toll like receptor 4 inhibitor TAK242 had no such effect. We also found that stellate ganglion block performed at night had no positive effect on diabetic ischemic stroke. These findings suggest that stellate ganglion block is a potential therapy for diabetic ischemic stroke and that it may be mediated through the Toll like receptor 4/nuclear factor kappa B signaling pathway. We also found that the therapeutic effect of stellate ganglion block is affected by circadian rhythm.
... Pups in each group were subjected to 3 short-term neurobehavioral tests [21][22][23] 3-day post-HI: (1) cliff avoidance reaction for evaluating the capacity of rodents to react to adverse environments; (2) geotaxis reflex for assessing the function of vestibular and proprioception; and (3) the grip test for estimating the grip force and fatigability. All experiments were observed and evaluated by no less than two naïve lab workers, and all animal groups kept the same testing methods [14]. ...
A list of microRNAs (miRs) has been referred to involve in the development of hypoxic–ischemic brain damage (HIBD). Based on that, we probed the concrete role of miR-214-3p regulating thioredoxin-interacting protein (TXNIP) in the illness. A neonatal HIBD mouse model was established using the Rice–Vannucci method, followed by measurements of miR-214-3p and TXNIP levels in brain tissues. After modeling, mice were given brain injection of the compounds that could alter miR-214-3p and TXNIP expression. Afterward, neurological function, neuronal inflammation, neuronal apoptosis, neuron morphology, and the number of Nissl body were assessed in HIBD mice. The binding of miR-214-3p to TXNIP was analyzed. Lower miR-214-3p and higher TXNIP were analyzed in brain tissues of mice with HIBD. Up-regulating miR-214-3p or depleting TXNIP improved neurological function, reduced neuronal inflammation and neuronal apoptosis, attenuated morphological damage of neurons, and increased the number of Nissl bodies in mice with HIBD. TXNIP was targeted by miR-214-3p and overexpressing TXNIP reversed the therapeutic effect of miR-214-3p on HIBD mice. It is noted that promotion of miR-214-3p relieves HIBD in mice through inhibiting TXNIP expression.
... Церебральная ишемия является самой частой формой патологии мозга у детей раннего возраста, перенесших перинатальное поражение ЦНС (ПП ЦНС) и регистрируется с частотой 1-3 на 1000 доношенных новорожденных в развитых странах и в 5-10 раз чаще в развивающихся [1, 2,3]. При этом около 40 % детей погибают в раннем неонатальном периоде, у 30 % -формируются отсроченные неврологические осложнения, такие как детский церебральный паралич, церебральная эпилепсия и когнитивные нарушения [1,2]. ...
... Активация врожденной иммунной системы является важной частью воспалительного ответа и ключевым событием в неблагоприятном исходе ишемии головного мозга [4]. Известно, что нейроглиальная активация сопровождается увеличением синтеза про-и противовоспалительных цитокинов, что приводит к индукции нейроповреждающих медиаторов и лейкоцитарной инфильтрации ткани мозга [2,3,4]. Установлена нейродеструктивная роль IL1, IL6, TNFα и нейропротективная -IL-10 и TGFβ [2]. ...
... Neonatal hypoxic-ischemic brain damage (HIBD) is one of the major neurological disorders in neonates caused by perinatal asphyxia, leading to permanent neurological deficits, including cerebral palsy, mental retardation, and epilepsy and even neonatal death [1,2]. In spite of emerging therapeutic strategies, such as therapeutic hypothermia and stem cell therapy, there are no specialized treatment measures for neonatal HIBD [3,4]. ...
Long noncoding RNA (LncRNA) Peg13 has been demonstrated to protect against neurological diseases. However, its underlying mechanism in the progression of hypoxic-ischemic brain damage (HIBD) has not been well investigated. The expression of target genes was determined in neonatal mice with HIBD and in mouse hippocampal neurons during oxygen–glucose deprivation (OGD) using quantitative real-time PCR (qRT-PCR) and immunoblotting. Functional assays, including CCK-8 cell viability and apoptotic cell detection using TdT mediated dUTP nick ending labeling (TUNEL) assay were used to examine the neuroprotective role of Peg13 in mouse hippocampal neurons. Luciferase assays were performed to determine the regulatory mechanism of Peg13 in OGD-induced neuronal apoptosis. Peg13 was reduced in HIBD mice and OGD-treated mouse hippocampal neurons. Altered Peg13 expression relieved OGD-induced neuronal apoptosis. Mechanistically, Peg13 may serve as a sponge for miR-20a-5p to increase the expression of X chromosome-linked inhibitor of apoptosis (XIAP), a downstream target of miR-20a-5p. Our study showed that Peg13 fulfilled its anti-apoptotic function in neurons through suppressing XIAP expression by sponging miR-20a-5p. Together, Peg13 binds to miR-20a-5p to upregulate XIAP and alleviate HIBD in neonatal mice. The Peg13/miR-20a-5p/XIAP competing endogenous RNA (ceRNA) axis could be a potential therapeutic target for HIBD.
