The RNA m6A modification might participate in microglial activation during hypoxic-ischemic brain damage in neonatal mice.

BACKGROUND: The RNA m6A modification has been implicated in multiple neurological diseases as well as macrophage activation. However, whether it regulates microglial activation during hypoxic-ischemic brain damage (HIBD) in neonates remains unknown. Here, we aim to examine whether the m6A modification is involved in modulating microglial activation during HIBD. We employed an oxygen and glucose deprivation microglial model for in vitro studies and a neonatal mouse model of HIBD. The brain tissue was subjected to RNA-seq to screen for significant changes in the mRNA m6A regulator. Thereafter, we performed validation and bioinformatics analysis of the major m6A regulators. RESULTS: RNA-seq analysis revealed that, among 141 m6A regulators, 31 exhibited significant differential expression (FC (abs) ≥ 2) in HIBD mice. We then subjected the major m6A regulators Mettl3, Mettl14, Fto, Alkbh5, Ythdf1, and Ythdf2 to further validation, and the results showed that all were significantly downregulated in vitro and in vivo. GO analysis reveals that regulators are mainly involved in the regulation of cellular and metabolic processes. The KEGG results indicate the involvement of the signal transduction pathway. CONCLUSIONS: Our findings demonstrate that m6A modification of mRNA plays a crucial role in the regulation of microglial activation in HIBD, with m6A-associated regulators acting as key modulators of microglial activation.

RP58 knockdown contributes to hypoxia-ischemia-induced pineal dysfunction and circadian rhythm disruption in neonatal rats.

Hypoxia-ischemia (HI) of the brain not only impairs neurodevelopment but also causes pineal gland dysfunction, which leads to circadian rhythm disruption. However, the underlying mechanism of circadian rhythm disruption associated with HI-induced pineal dysfunction remains unknown. The zinc finger protein repressor protein with a predicted molecular mass of 58 kDa (RP58) is involved in the development and differentiation of nerve cells. In this study, we established an HI model in neonatal rats to investigate the expression of RP58 and its role in pineal dysfunction and circadian rhythm disruption induced by HI. We demonstrated that RP58 was highly expressed in the pineal gland under normal conditions and significantly downregulated in the pineal gland and primary pinealocytes following HI. Knockdown of RP58 decreased the expression of enzymes in the melatonin (Mel) synthesis pathway (tryptophan hydroxylase 1 [TPH1], acetylserotonin O-methyltransferase [ASMT], and arylalkylamine N-acetyltransferase [AANAT]) and clock genes (circadian locomotor output cycles kaput [CLOCK] and brain and muscle ARNT-like 1 [BMAL1]), and it also reduced the production of Mel, caused pineal cell injury, and disrupted circadian rhythms in vivo and in vitro. Similarly, HI reduced the expression of Mel synthesis enzymes (TPH1, ASMT, and AANAT) and clock genes (CLOCK and BMAL1), and caused pineal injury and circadian rhythm disruption, which were exacerbated by RP58 knockdown. The detrimental effect of RP58 knockdown on pineal dysfunction and circadian rhythm disruption was reversed by the addition of exogenous Mel. Furthermore, exogenous Mel reversed HI-induced pineal dysfunction and circadian rhythm disruption, as reflected by improvements in Mel production, voluntary activity periods, and activity frequency, as well as a diminished decrease in the expression of Mel synthesis enzymes and clock genes. The present study suggests that RP58 is an endogenous source of protection against pineal dysfunction and circadian rhythm disruption after neonatal HI.

Human bone marrow mesenchymal stem cells-derived exosomes protect against nerve injury via regulating immune microenvironment in neonatal hypoxic-ischemic brain damage model.

Neonatal hypoxic-ischemic (HI) brain injury is a serious injury caused by various perinatal factors, which has become a heavy mental burden to the family. The molecular mechanism underlying neonatal hypoxic-ischemic brain injury remains largely unknown. Human bone marrow mesenchymal stem cells (hBMSCs) have caused wide public concern due to the immunomodulatory properties. Exosomes can polarize human microglia and thus changed it into an anti-inflammatory phenotype to reduce the release of pro-inflammatory factors. However, it is unclear whether hBMSCs-exosomes have effect on neonatal hypoxic-ischemic brain injury. In this study, we aimed at investigating the role of hBMSCs-exosomes in regulating immune response and nerve injury in neonatal hypoxic-ischemic brain damage model. In the research, we identified the exosome secretion of hBMSCs could transferred into human microglia (HMC). Moreover, we determined the importance of hBMSCs-exosomes in regulating HMC polarization and inflammatory response. Our research findings might provide a new insight into slowing the disease progression of neonatal hypoxic-ischemic brain injury.

MiR-17-5p protects neonatal mice from hypoxic-ischemic brain damage by targeting Casp2.

Hypoxia-ischemia brain damage (HIBD) is a leading cause of neonatal death worldwide, which significantly influences the development of newborns; however, effective treatment strategies remain limited. Recent studies have discovered that microRNAs (miRNAs) play essential roles in the progression of HIBD. Our study was designed to explore whether miR-17-5p was involved in the pathological development of HIBD. In our study, HIBD mouse experimental model was established by carotid artery ligation combined with a hypoxic environment. RT-qPCR and western blot analyses found that Casp2 was high expressed while miR-17-5p was poorly expressed in the cerebral cortical tissue of HIBD mice. Knockdown of Casp2 significantly alleviated brain injury and cell apoptosis. Additionally, the luciferase reporter assay confirmed that miR-17-5p targeted the 3' UTR of Casp2 and negatively regulated Casp2 expression. The rescue experiment demonstrated that miR-17-5p mimic significantly relieved brain tissue damage and improved memory ability in the HIBD mouse model, while these functions of miR-17-5p were blocked by overexpression of Casp2. In summary, our results indicated that miR-17-5p exerted protective effects on HIBD by targeting Casp2.

Protective effects of histone deacetylase inhibition by Scriptaid on brain injury in neonatal rat models of cerebral ischemia and hypoxia.

BACKGROUND: Neonatal hypoxia-ischemia brain damage (HBID) can cause a series of neurological sequelae, such as movement and cognitive impairment, and there is currently no clinically effective treatment. Changes in epigenetic processes had been shown to be involved in the development of a series of neurodegenerative diseases, and HDAC inhibition by Scriptaid had been shown to reduce severe traumatic brain injury by suppressing inflammatory responses. This study investigated the protective effect of HDAC inhibition by Scriptaid after HBID. METHODS: We established the neonatal rat HBID model, and used intraperitoneal injection of HDAC inhibitor scriptaid as a treatment. 7 days after HBID, nuclear magnetic resonance imaging (MRI) was used to detect infarct volume. The otarod test, wire hang test and Morris water maze were used to evaluate the HBID model of neurobehavioral dysfunction. Immunoblotting, immunofluorescence, and quantitative real-time PCR (RT-qPCR) were used to detect gene expression. RESULTS: HDAC inhibition by Scriptaid treatment could not only reduce the infarct volume and neuronal degeneration in HBID rats, but also helped to improve their neurobehavioral dysfunction. 7 days after HBID, the expression of HDAC-1, HDAC-2 and HDAC-3 in the infarct volume of HBID + Veh group rats were much more than that in sham group (P<0.05), but Scriptaid could significantly inhibit those expression (P<0.05), and significantly increased the acetylation of H3 and H4 in HBID rats. In vivo and vitro results demonstrated that Scriptaid had no significant effect on oligodendrocyte MBP protein expression after OGD, but Scriptaid -treated microglia cultures had protective effects on OGD-treated OLG, M1 microglia suppressed OLG activity after OGD, and M2 enhanced its activity. In vivo experiments at 7 days after HBIDI injury showed that Scriptaid could promote the polarization of microglia into M2 microglia, reduced the expression of pro-inflammatory factors, and enhance the expression of anti-inflammatory cytokines. CONCLUSION: After HBID, HDAC inhibitor Scriptaid inhibits inflammatory responses and protects the brain by promoting the polarization of microglia in brain tissue to M2 microglia.

Extracellular Vesicle-Derived microRNA-410 From Mesenchymal Stem Cells Protects Against Neonatal Hypoxia-Ischemia Brain Damage Through an HDAC1-Dependent EGR2/Bcl2 Axis.

Hypoxia-ischemia brain damage (HIBD) is a neurological disorder occring in neonates, which is exacerbated by neuronal apoptosis. Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) have been proposed as a promising strategy for treating or preventing ischemia-related diseases. However, their mechanisms in HIBD remain unclear. Thus, we aimed to address the role of EV-derived microRNA (miR)-410 in HIBD. Neonatal HIBD mouse model was constructed using HI insult, from which neurons were isolated, followed by exposure to oxygen glucose deprivation (OGD). EVs were isolated from human umbilical cord (hUC)-derived MSCs. In silico analyses, dual-luciferase reporter gene and chromatin immunoprecipitation assays were adopted to determine relationships among miR-410, histone deacetylase 1 (HDAC1), early growth response protein 2 (EGR2), and B cell lymphoma/leukemia 2 (Bcl2). The functional roles of EV-derived miR-410 were determined using loss- and gain-of functions experiments, and by evaluating neuronal viability, cell-cycle distribution and neuronal apoptosis in vitro as well as modified neurological severity score (mNSS), edema formation, and cerebral infarction volume in vivo. hUC-MSCs-derived EVs protected against HIBD in vivo and inhibited the OGD-induced neuronal apoptosis in vitro. miR-410 was successfully delivered to neurons by hUC-MSCs-EVs and negatively targeted HDAC1, which inversely mediated the expression of EGR2/Bcl2. Upregulation of EV-derived miR-410 promoted the viability but inhibited apoptosis of neurons, which was reversed by HDAC1 overexpression. EV-derived miR-410 elevation reduced mNSS, edema formation, and cerebral infarction volume by increasing EGR2/Bcl2 expression through downregulating HDAC1 expression in vivo. In summary, EV-derived miR-410 impeded neuronal apoptosis by elevating the expression of EGR2/Bcl2 via HDAC1 downregulation, thereby providing a potential strategy for treating or preventing HIBD.

Bumetanide reduce the seizure susceptibility induced by pentylenetetrazol via inhibition of aberrant hippocampal neurogenesis in neonatal rats after hypoxia-ischemia.

Hypoxia-ischemia brain damage (HIBD) is one of prevalent causes of neonatal mortality and morbidity. Our data demonstrated that hypoxia-ischemia (HI) induced Na+-K+-Cl--co-transporter 1 (NKCC1) increasing in hippocampus. Previous studies demonstrated that NKCC1 regulates various stages of neurogenesis. In this study, we studied the role of increased NKCC1 in regulating of HI-induced neurogenesis. HIBD model was established in 7days old Sprague-Dawley rat pup, and the expression of NKCC1 was detected by western blot and qPCR. Brain electrical activity in freely rats was monitored by electroencephalography (EEG) recordings. HI-induced neurogenesis was detected by immunofluorescence staining. Neurobehavioral test was to investigate the neuro-protective role of bumetanide, an inhibitor of NKCC1, on neonatal rats after HI. The results showed that bumetanide treatment significantly reduced brain electrical activity and the seizure stage of epilepsy induced by pentylenetetrazol (PTZ) in vivo after HI. In addition, bumetanide restored aberrant hippocampal neurogenesis and associated cognitive function. Our data demonstrated that bumetanide reduces the susceptibility of epilepsy induced by PTZ in rats suffering from HI injury during neonatal period via restoring the ectopic newborn neurons in dentate gyrus (DG) and cognitive function.

Hyperbaric oxygen preconditioning ameliorates hypoxia-ischemia brain damage by activating Nrf2 expression in vivo and in vitro.

The present study aimed to investigate whether hyperbaric oxygen preconditioning (HBO-PC) could ameliorate hypoxia-ischemia brain damage (HIBD) by an increase of Nrf2 expression. P7 Sprague-Dawley rats (aged 7 d, n = 195) were used in two in vivo experiments, including BO-PC exposure experiments in non-HIBD models and treatment experiments in HIBD models. 2,3,5-triphenyltetrazolium chloride (TTC) staining, Nissl Staining, and TUNEL staining were performed. And expressions of Nrf2, HO-1, and GSTs were measured. For in vitro studies, oxygen-glucose deprivation cells were established. Morphological and apoptotic staining and gene silencing of Nrf2 by siRNA transfection were investigated. For exposure experiments, HBO-PC for longer time increased the expression of Nrf2 significantly. And for treatment experiments, HBO-PC treatment significantly decreased infarction area, lessened neuronal injury, reduced apoptosis, and increased both the expression of Nrf2 and activities of its downstream proteins. Cytology tests confirmed effects of HBO-PC treatments. Besides, Nrf2 siRNA significantly reduced protective effects of HBO-PC. These observations demonstrated that an up-regulation of Nrf2 by HBO-PC might play an important role in the generation of tolerance against HIBD.

[ROLE OF EXTRACELLULAR SIGNAL-RELATED PROTEIN KINASE 1/2 PATHWAY IN GINSENOSIDE Rg1 MEDIATED ANTI-APOPTOTIC EFFECT ON NEURON AFTER HYPOXIA ISCHEMIA BRAIN DAMAGE IN NEONATAL RATS].

OBJECTIVE: To investigate the anti-apoptotic effect of ginsenoside Rg1 in neonatal rats with hypoxia ischemia brain damage (HIBD), and to explore the possible signaling pathway involved in anti-apoptosis. METHODS: Forty-eight 10-day-old Sprague Dawley (SD) rats (weighing 17-21 g, male or female) were randomly allocated into 4 groups (12 rats in each group): sham-operation group (sham group), HIBD group (HI group), HIBD+ginsenoside Rg1 group (HI+Rg1 group), and HIBD+ginsenoside Rg1+U0126 group (HI+Rg1+U0126 group). SD rats in HI group, HI+Rg1 group, and HI+Rg1+U0126 group underwent ligation of the right common carotid artery (CCA) and hypoxic ventilation (8%O2+92%N2) for 2.5 hours to prepare the HIBD model, and rats in sham group underwent only separation of the right CCA. SD rats in HI+Rg1+U0126 group received intraventricular injection of 5 µL phosphate buffer saline (PBS) containing U0126 (25 µg/kg) at 1 hour before HIBD, and rats in the other three groups received intraventricular injection of PBS at the same time. The rats in HI+Rg1 group and HI+Rg1+U0126 group received intraperitoneal injection of 0.1 mL normal saline (NS) containing Rg1 (40 mg/kg) at immediate after HIBD, while rats in HI group and sham group received intraperitoneal injection of 0.1 mL NS at immediate after HIBD. At 4 and 24 hours after HIBD, the right hemisphere and hippocampus were collected to detect the protein expression and distribution of extracellular signal-related protein kinase 1/2 (Erk1/2), phospho-Erk1/2 (p-Erk1/2), hypoxia inducible factor 1α (HIF-1α), and cleaved Caspase-3 (CC3) by Western blot and immunohistochemistry staining. TUNEL staining was used to evaluate neural apoptosis in situ. RESULTS: Western blot results showed that there were expressions of Erk1/2, p-ERK1/2, HIF-1α, and CC3 proteins at 4 and 24 hours after HIBD in each group. The expressions of HIF-1α and CC3 protein at 4 and 24 hours, and expression of p-Erk1/2 protein at 4 hours were significantly increased in HI group when compared with sham group (P<0.05). When compared with HI group, the expressions of p-Erk1/2 and HIF-1α protein in HI+Rg1 group were significantly increased (P<0.05), while the expression of CC3 protein was significantly decreased at 4 and 24 hours (P<0.05). When compared with HI+Rg1 group, the expressions of p-Erk1/2 and HIF-1α proteins in HI+Rg1+U0126 group were significantly decreased (P<0.05), while expression of CC3 protein was significantly increased at 4 and 24 hours (P<0.05). There was no significant difference in Erk1/2 protein expression between groups at different time points (P>0.05). Immunohistochemistry staining displayed that HIF-1α and CC3 proteins mainly distributed in the nucleus and cytoplasma, while Erk1/2 and p-Erk1/2 proteins mainly distributed in the cytoplasma. The expression levels of protein by immunohistochemistry results were similar to the results by Western blot. TUNEL staining showed that the apoptotic neurons were characterized by yellow or brown particle in the nucleus. The apoptotic index (AI) of neurons at 4 and 24 hours was significantly increased in HI group when compared with sham group (P<0.05), and the AI of neurons was significantly decreased in HI+Rg1 group when compared with HI group and HI+Rg1+U0126 group at 24 hours (P<0.05). CONCLUSIONS: Rg1 could enhance HIBD induced HIF-1α expression and inhibit activation of Caspase-3 by Erk1/2 signaling pathway, and play an anti-apoptotic role in neonatal rats with HIBD.

ROLE OF EXTRACELLULAR SIGNAL-RELATED PROTEIN KINASE 1/2 PATHWAY IN GINSENOSIDE Rg1 MEDIATED ANTI-APOPTOTIC EFFECT ON NEURON AFTER HYPOXIA ISCHEMIA BRAIN DAMAGE IN NEONATAL RATS / 中国修复重建外科杂志

OBJECTIVE: To investigate the anti-apoptotic effect of ginsenoside Rg1 in neonatal rats with hypoxia ischemia brain damage (HIBD), and to explore the possible signaling pathway involved in anti-apoptosis.

Effect of Omega-3 fish oil fat emulsion on the expression of Toll-like receptor 4 and nuclear factor-κB in the neonatal rats hippocampus after hypoxic-ischemic / 中华实用儿科临床杂志

Objective To discuss whether Omega-3 fish oil fat emulsion has the potential protective mechanism for 7-day-old rats with hypoxic-ischemic brain damage (HIBD).Methods One hundred and sixty-eight 7-day-old SD rats were randomly divided into 4 groups:group A (sham group),group B (Omega-3 fish oil fat emulsion group),group C (normal fat emulsion group),group D (model group),and there were 42 cases in each group.Neonatal HIBD was induced by the method of Rice.Rats were sacrificed at 1 d,3 d and 7 d after the surgery.Hippocampus was removed for Real-time PCR and Western blot test to detect Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) mRNA and protein expression.TUNEL staining comparison was done among different groups to observe the number of cellular apoptosis.Results HE staining of hippocampus CA1 area in 3 d showed that brain tissues in group A maintained normal structures;those in group D had much more brain cells with severe edema than other groups;TLR4 and NF-κB mRNA and protein expression levels in group D were higher than those in group A in 1 d (all P ＜0.05);TLR4 and NF-κB expression levels of mRNA and protein in group B (4.89 ± 0.51,9.30 ± 1.53;1.15 ±0.10,1.44 ± 0.14) were lower than those in group C (17.58 ± 2.50,20.13 ± 1.00;2.56 ± 0.10,2.82 ± 0.09) and group D (15.94-± 2.52,26.21 ± 3.00;2.34 ± 0.11,4.51 ± 0.36) in 3 d (all P ＜ 0.05),and compared with group A (6.30 ± 1.52,5.32 ± 1.06;1.32 ± 0.10,2.42 ± 0.14),there was significant difference (all P ＞ 0.05);TLR4 and NF-κB mRNA and protein expression levels in group B were lower than those in group C and group D in 7 d(all P ＜0.05),and compared with group A there was no significant difference (all P ＞ 0.05).The apoptotic cell number of brain tissues in 3 d:group B (13.67 ±2.52) were lower than those in group C (27.67 ±2.52) and group D (41.00 ±3.61) (all P ＜0.05),and compared with the group A (6.00 ±2.00),the difference was not statistically significant (P ＞ 0.05).Conclusions Omega-3 fish oil fat emulsion plays an important role in protecting neonatal rats with HIBD.The mechanisms were likely to reduce TLR4,NF-κB and cell apoptosis levels.

Riboflavin inhibited ischemia brain damage in rats / 中国药理学通报

Aim To investigate the protective effect of riboflavin on ischemia brain damage and the mecha-nism.Methods The in vivo experiments were pro-cessed in male SD rats .Rats were randomly arranged into control group , model group and riboflavin group . The rats in riboflavin group were intraperitoneally in-jected riboflavin at the dose of 1 mg? kg -1 for seven consecutive days .Then the rats in model and riboflavin groups were carried out middle cerebral artery occlu-sion( MCAO) operation.After 24 h, all rats were sacri-ficed and the brain tissue was dissected to observe the infarct area, the edema and the ultrastructure damage . The brain tissue was dyed by triphenyl tetrazolium chloride .The brain edema was observed by the weight of ischemia-side semi-brain.The ultrastructure was ob-served by electron microscope .The in vitro experiments were processed in primary culture neurons by exposed to oxygen and glycose deprivation ( OGD) .The viability of neurons was assayed by MTT method .The enzyme activity of superoxide dismutase ( SOD) , catalase ( CAT)and glutathione peroxidase ( GSH-Px ) was assayed to explore the mechanism .Results Riboflavin signifi-cantly decreased the infarct area ( P<0.01 ) , inhibited the brain edema ( P <0.01 ) and inhibited the ultra-structure damage in rats after MCAO;riboflavin protec-ted the viability ( P <0.01 ) and the ultrastructure of neurons exposed to OGD .The enzyme activity of an-tioxidant SOD1 ( P <0.01 ) , CAT ( P <0.01 ) and GSH-Px ( P <0.01 ) was protected by riboflavin in MCAO model .No difference was found in the activity of SOD2 . Conclusion Riboflavin inhibits ischemia brain damage , and the protection of the activity of an-tioxidants is involved in the mechanism .

Alteration in rectification of potassium channels in perinatal hypoxia ischemia brain damage.

Oligodendrocyte progenitor cells (OPCs) are susceptible to perinatal hypoxia ischemia brain damage (HIBD), which results in infant cerebral palsy due to the effects on myelination. The origin of OPC vulnerability in HIBD, however, remains controversial. In this study, we defined the HIBD punctate lesions by MRI diffuse excessive high signal intensity (DEHSI) in postnatal 7-day-old rats. The electrophysiological functional properties of OPCs in HIBD were recorded by patch-clamp in acute cerebral cortex slices. The slices were intracellularly injected with Lucifer yellow and immunohistochemically labeled with NG2 antibody to identify local OPCs. Passive membrane properties and K(+) channel functions in OPCs were analyzed to estimate the onset of vulnerability in HIBD. The resting membrane potential, membrane resistance, and membrane capacitance of OPCs were increased in both the gray and white matter of the cerebral cortex. OPCs in both the gray and white matter exhibited voltage-dependent K(+) currents, which consisted of the initiated rectified potassium currents (IA) and the sustained rectified currents (IK). The significant alternation in membrane resistance was influenced by the diversity of potassium channel kinetics. These findings suggest that the rectification of IA and IK channels may play a significant role in OPC vulnerability in HIBD.

Role of melatonin for repairing the periventricular white matter damage due to hypoxia-ischemia in the developing brain of rat / 中华实用儿科临床杂志

Objective To investigate the protective effect of melatonin and its possible mechanism for repairing in the immature white matter damage due to brain hypoxia-ischemia (HI).Methods Forty-eight three-day SD rats after birth were randomly divided into 3 groups:sham-operated(SHAM) group,HI group and melatonin treatment(MT) group.Periventricular white matter damage (PWMD) to animal models were estabished according to Rice modeling.MT group was treated with melatonin pre-operatively,immediately postoperation,1 hour postoperation and 24 hours postoperation via intraperitoneal injection,and the other groups were injected with the same volume of dissolvent.The rats were executed by decollation after 2 days and 14 days.The histological changes in periventricular white matter were observed by HE staining and immunohistochemistry.Results For the 3 groups,the structure in ope-ration side of the white matter in the peripheral ventricles of the brain 2 days postoperation were significant different (P ＜0.05).The O4 positive cells decreased one by one/greatest in the SHAM group[(75.548 ± 7.333)/hpf] followed by MT group [(59.971 ± 3.635)/hpf],and HI group [(40.511 ± 2.848)/hpf] (P ＜ 0.05).The expression of Casepase-3 increased in the SHAM group (107.724 ± 10.266),MT group (132.289 ± 8.537),and HI group (202.168 ± 14.367),and the difference was statically significant (P ＜ 0.05).Ventricular index was greater in operation side of the white matter in the peripheral ventricles of the 14-day-brain in the SHAM group(0.928 ±0.063),MT group (1.813 ± 0.110),HI group (2.752 ± 0.201),increasingly,while absorbance value of myelin basic protein decreased one by one in sequence(39.504 ± 1.673,21.729 ± 1.614,11.344 ± 1.118).Conclusions MT plays a role in protecting the periventricular white matter via inhibiting the apoptosis of oligodendrocyte progenitor cell,and thus benefits the PWMD.

MiR-139-5p inhibits HGTD-P and regulates neuronal apoptosis induced by hypoxia-ischemia in neonatal rats.

Human growth transformation dependent protein (HGTD-P) is a newly identified protein that promotes neuronal apoptosis in hypoxia-ischemia brain damage (HIBD) in neonatal rats. However, the mechanisms regulating HGTD-P expression are not clear. Here we describe microRNAs targeted to HGTD-P and examine their effects on regulating neuronal apoptosis in HIBD. We use samples from cultured neurons after oxygen-glucose deprivation (OGD) and postnatal day 10 rat brains after hypoxia-ischemia (HI). RT-PCR, Western blotting, and immunostaining are used to detect the expression of HGTD-P and cleaved caspase 3, as well as real-time PCR detects microRNA expression. MicroRNA agomir is used to inhibit the expression of HGTD-P, and DAPI, TUNEL, and TTC staining are employed to detect cell apoptosis and brain damage. Moreover, in vitro processing assay is used to examine the mechanism by which HI down-regulates miR-139-5p expression. We found that miR-139-5p is down-regulated in neurons and rat brains after HI treatment. The expression pattern of miR-139-5p correlates inversely with that of HGTD-P. Furthermore, miR-139-5p agomir inhibits neuronal apoptosis and attenuates HIBD, which is concurrent with down-regulation of HGTD-P. Moreover, pre-miR-139 processing activity decreases in extracts from OGD neurons, and OGD neuronal extracts attenuates the processing of pre-miR-139 by Dicer. In conclusion, HI induces inhibitors which block the processing step of pre-miR-139, resulting in the down-regulation of mature miR-139-5p. The down-regulation of miR-139-5p plays a critical role in the up-regulation of HGTD-P expression. MiR-139-5p agomir attenuates brain damage when used 12h after HI, providing a longer therapeutic window than anti-apoptosis compounds currently available.

The expression and role of integrin-linked kinase in neonatal rats after hypoxia-ischemia brain damage / 临床儿科杂志

Objective To investigate the possible function of integrin-linked kinase (ILK)/protein kinase B (PKB/Akt) signaling in repair of neonatal rat hypoxia-ischemia brain damage (HIBD). Methods Postnatal day 10 SD rats were randomly divided into hypoxia ischemia (HI) group and sham control group. Rat brains were collected at 0 h, 4 h, 6 h, 12 h, 24 h, 48 h and 72 h after hypoxia ischemia damage. Immunolfuorescence staining was used to observe the distribution and expression of ILK. Western blot was used to detect the expression of ILK, Akt, phosphorylated Akt (p-Akt) and vascular endothelial growth factor (VEGF). Lentiviral vectors expressing ILK shRNA were constructed to inhibit the expression of ILK in neonatal rats. After intracerebroventricular injections of LV-ILK shRNA lentivirus and LV-control respectively, HIBD model was established. Rat brains were collected at 4 h and 24 h after HIBD. Western blot was used to detect the expression of ILK, p-Akt, and VEGF. TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining was used to detect cell apoptosis. Results Immunolfuorescence staining showed that ILK was widely distributed in cortex and hippocampus both in HI group and sham control group. ILK located at cell membrane and cytoplasm. Western blot results demonstrated that ILK protein increased after HI, with a peak at 24 h, and maintained higher level than those in sham control group. The p-Akt protein signiifcantly increased at 4 h after HI, and signiifcantly decreased in the following 24 h, and then increased again, with a peak at 48 h, but the level of p-Akt protein was higher than that of sham control group. The VEGF protein increased at 4 h after HI, with a peak at 12 h, higher than that of sham control group. The expression of Akt protein showed no signiifcant difference between HI group and sham control group. Lentiviral vectors containing RNAi targeting ILK was applied successfully in vivo. At 4 h and 24 h after HIBD model, the expression of ILK, p-Akt, and VEGF proteins in right side brain received LV-ILK shRNA signiifcantly decreased compared with those of right side brain received LV-control at the same time point. And cell apoptosis signiifcantly increased in LV-ILK shRNA group. Conclusions The expression of ILK, p-Akt, VEGF proteins increased after HI. By inhibiting the expression of ILK, the expression of p-Akt and VEGF proteins can be reduced, and cell apoptosis could increase in newborn rats after HIBD. The results suggest that ILK may induce the expression of VEGF through activating the PI3K/Akt signaling pathway, and promote cell survival and angiogenesis after HIBD.

Role and mechanism of L-thyroxine on brain tissue hypoxia-inducible factor 1α expression in neonatal rats with hypoxia-ischemia brain damage / 中华实用儿科临床杂志

Objective To observe the expression of hypoxia-inducible factor 1 α (HIF-1α) in rat brain after hypoxia-ischemia(HI),and to explore the possible mechanism of L-thyroxine (L-T4) on HIF-1α expression.Methods Sixty-four postnatal 7-day Sprague-Dawley rats were randomly divided into 4 groups:the sham operation group,HI group,menstruum-treated group and L-T4-treated group.HIBD models were generated according to Rice model method.The rats in menstruum-treated group and L-T4-treated group were respectively administrated of intraperitoneal injection of menstruum with the equal volume and 2 μg/100g L-T4,once a day,for 5 days.The expressions of HIF-1α and phospho-protein kinase B(p-Akt) protein were detected by means of immunohistochemistry.Reverse transcription-polymerase chain reaction was used to detect the level of HIF-1α mRNA.Results The levels of p-Akt protein(50.168 ±4.259),HIF-1α protein (72.795 ±6.121) and HIF-1α mRNA (0.448 ± 0.035) were upregulated compared with those in the sham operation group (8.080 ±0.369,38.581 ± 2.846,0.174 ± 0.015),and the differences were significant (all P ＜ 0.05).The levels of p-Akt protein (82.765 ± 6.271),HIF-1 α protein (117.350 ± 9.374) and HIF-1 α mRNA (0.618 ± 0.042) in L-T4-treated group were higher than those in HI group,and the differences were significant (all P ＜ 0.05).The level of HIF-1 α protein was positively correlated with p-Akt protein in HI group and L-T4-treated group [r(HI) =0.635,P=0.048;r(L-T4) =0.694,P=0.026].Conclusions L-T4 can upregulate HIF-1α mRNA and protein expression in neonatal rats with hypoxia-ischemia brain damage.Phosphatidylinositol-3-kinase/protein kinase B signaling pathway may be involved in L-T4 upregulating HIF-1α mRNA and protein expression.

The regulation of deferoxamine on HIF-1α expression after hypoxia-ischemia brain damage in neonatal rats / 临床儿科杂志

Objective To study the role of deferoxamine(DFO)on regulating hypoxia-inducible factor 1α(HIF-1α)expression after hypoxia-ischemia brain damage(HIBD)in neonatal rats,to explore the therapeutic strategy for HIBD. Methods Postnatal day 10 SD rats were divided into four groups: hypoxia-ischemia(HI)group,DFO-treated group,normal saline(NS)-treated group,and sham operation group. HIBD model was established by the ligation of right common carotid artery following the inhalation of nitrogen-oxygen mixtures containing 92% nitrogen and 8% oxygen. DFO-treated group and NS-treated group were treated by intraperitoneal injection of DFO or NS respectively. The brains were collected at 4 h,8 h,and 24 h after hypoxia. HIF-1α protein expression was detected by Western blot analysis,and HIF-1α mRNA expression was detected by using RT-PCR at each time point. Results The synthetic level of HIF-1α protein increased significantly at 4 h,peaked at 8 h,and decreased at 24 h after HI in HI group,as well as NS-treated group. However,in DFO-treated group HIF-1α protein was peaked at 4 h,maintained higher level at 8 h and 24 h after HI. The level of HIF-1α protein was much higher in HI and DFO-treated groups than those in sham controls(P ＜ 0.05). The synthetic level of HIF-1α protein were higher in DFO-treated groups than those in HI groups at each time point(P ＜ 0.05). HIF-1α mRNA expression was higher in DFO-treated groups than those in HI groups at each time point. Conclusions DFO upregulate HIF-1α protein and mRNA expression in neonatal rats with HIBD. The peak of HIF-1α protein expression are also more advanced and lasted longer after DFO-treatment.

Corticotropin-releasing factor and hypoxic-ischemic brain damage / 中国小儿急救医学

Corticotropin-releasing factor(CRF)is one neuroendocrine peptide which is closely related with the stress.It has been proved that any stress can lead to the person and animal's blood CRF level increasing,and high-level CRF urges the hypothalamic neuron calcium ion inflow.Newborn pediatricians pay more attention to whether blood CRF level can act as one director of assessing severity of newborn hypoxic-schemic brain damage.This article reviews the CRF and it's acceptors,CRF secretion,CRF physiological action and it's adjustment,and hypoxia-ischemia stress and CRF,in order to provide the theory basis for reviewing the severity of hypoxic-schemic brain damage in neonates.

Effect of Progesterone on Progesterone Receptor in Brain Tissue with Hypoxia-Ischemia Brain Damage in Neonatal Rats / 实用儿科临床杂志

ObjectiveTo observe the effect of progesterone on progesterone receptor in brain tissue with hypoxia-ischemia brain da-mage(HIBD) in neonatal rats,and discuss the protective mechanism of progesterone on HIBD of neonatal rats.MethodsTwenty-four 7-day-old neonatal rats were randomly divided into 3 groups:sham-operated group,hypoxia-ischemia group and pretreatment group.Rats in hypoxia-ischemia group and pretreatment group were subjected to left common carotid artery ligation,then were exposed to 80 mL/L oxygen and 920 mL/L nitrogen gas in 37 ℃ closed container for up to 2.5 h to establish hypoxia-ischemia encephalopathy(HIE) model.Progestero-ne was injected intraperitoneally into pretreatment group rats respectively at 30 minutes before hypoxia,solution was injected into the first 2 groups.All rats were killed at the 24 hour after operation,immunohistochemistry staining was used to examine the expression of progesterone receptor in brain.ResultsTotally 24 neonatal rats entered the result analysis without loss.Progesterone receptor was expressed in both endochylema and nucleus in every group rat.The amount of the positive cell of progesterone receptor in hypoxia-ischemia group significantly decreased compared with that in sham-operated group(P