Hydrogen alleviates hypoxic-ischaemic brain damage in neonatal rats by inhibiting injury of brain pericytes.

Hypoxia-ischaemia (HI) can induce the death of cerebrovascular constituent cells through oxidative stress. Hydrogen is a powerful antioxidant which can activate the antioxidant system. A hypoxia-ischaemia brain damage (HIBD) model was established in 7-day-old SD rats. Rats were treated with different doses of hydrogen-rich water (HRW), and brain pericyte oxidative stress damage, cerebrovascular function and brain tissue damage were assessed. Meanwhile, in vitro-cultured pericytes were subjected to oxygen-glucose deprivation and treated with different concentrations of HRW. Oxidative injury was measured and the molecular mechanism of how HRW alleviated oxidative injury of pericytes was also examined. The results showed that HRW significantly attenuated HI-induced oxidative stress in the brain pericytes of neonatal rats, partly through the Nrf2-HO-1 pathway, further improving cerebrovascular function and reducing brain injury and dysfunction. Furthermore, HRW is superior to a single-cell death inhibitor for apoptosis, ferroptosis, parthanatos, necroptosis and autophagy and can better inhibit HI-induced pericyte death. The liver and kidney functions of rats were not affected by present used HRW dose. This study elucidates the role and mechanism of hydrogen in treating HIBD from the perspective of pericytes, providing new theoretical evidence and mechanistic references for the clinical application of hydrogen in neonatal HIE.

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.

Atorvastatin inhibits neuronal apoptosis via activating cAMP/PKA/p-CREB/BDNF pathway in hypoxic-ischemic neonatal rats.

Neuronal apoptosis is one of the main pathological processes of hypoxic-ischemic brain damage (HIBD) and is involved in the development of hypoxic-ischemic encephalopathy (HIE) in neonates. Atorvastatin has been found to have neuroprotective effects in some nervous system diseases, but its role in regulating the pathogenesis of neonatal HIBD remains elusive. Thus, this study aimed to explore the effects and related mechanisms of atorvastatin on the regulation of neuronal apoptosis after HIBD in newborn rats. The rat HIBD model and the neuronal oxygen glucose deprivation (OGD) model were established routinely. Atorvastatin, cAMP inhibitor (SQ22536), and BDNF inhibitor (ANA-12) were used to treat HIBD rats and OGD neurons. Cerebral infarction, learning and memory ability, cAMP/PKA/p-CREB/BDNF signaling molecules, and apoptosis-related indicators (TUNEL, cleaved caspase-3, and Bax/Bcl2) were then examined. In vivo, atorvastatin reduced cerebral infarction, improved learning and memory ability, decreased the number of TUNEL-positive neurons, inhibited the expression of cleaved caspase-3 and Bax/Bcl2, and activated the cAMP/PKA/p-CREB/BDNF pathway in the cerebral cortex after HIBD. In vitro, atorvastatin also decreased the apoptosis-related indicators and activated the cAMP/PKA/p-CREB/BDNF pathway in neurons after OGD. Furthermore, inhibition of cAMP or BDNF attenuated the effect of atorvastatin on the reduction of neuronal apoptosis, suggesting that atorvastatin inhibits HIBD-induced neuronal apoptosis and alleviates brain injury in neonatal rats mainly by activating the cAMP/PKA/p-CREB/BDNF pathway. In conclusion, atorvastatin may be developed as a potential drug for the treatment of neonatal HIE.

The time-dependent variations of zebrafish intestine and gill after polyethylene microplastics exposure.

Microplastics (MPs) are common environmental contaminants that present a growing health concern due to their increasing presence in aquatic and human systems. However, the mechanisms behind MP effects on organisms are unclear. In this study, zebrafish (Danio rerio) were used as an in vivo model to investigate the potential risks and molecular mechanisms of the toxic effects of polyethylene MPs (45-53 µm). In the zebrafish intestine, 6, 5, and 186 genes showed differential expression after MP treatment for 1, 5, and 10 days, respectively. In the gills, 318, 92, and 484 genes showed differential expression after MP treatment for 1, 5, and 10 days, respectively. In both the intestine and the gills, Gene Ontology (GO) annotation showed that the main enriched terms were biological regulation, cellular process, metabolic process, cellular anatomical entity, and binding. KEGG enrichment analysis on DEGs revealed that the dominant pathways were carbohydrate metabolism and lipid metabolism, which were strongly influenced by MPs in the intestine. The dominant pathways in the gills were immune and lipid metabolism. The respiratory rate of gills, the activity of SOD and GSH in the intestine significantly increased after exposure to MPs compared with the control (p < 0.05), while the activity of SOD did not change in the gills. GSH activity was only significantly increased after MP exposure for 5 days. Also, the MDA content was not changed in the intestine but was significantly decreased in the gills after MP exposure. The activity of AChE significantly decreased only after MPs exposure for 5 days. Overall, these results indicated that MPs pollution significantly induced oxidative stress and neurotoxicity, increased respiratory rate, disturbed energy metabolism and stimulated immune function in fish, displaying an environmental risk of MPs to aquatic ecosystems.

Expression and functional analysis of lncRNAs in the hippocampus of immature rats with status epilepticus.

Long non-coding RNAs (lncRNAs) have been implicated in the regulation of gene expression at various levels. However, to date, the expression profile of lncRNAs in status epilepticus (SE) was unclear. In our study, the expression profile of lncRNAs was investigated by high-throughput sequencing based on a lithium/pilocarpine-induced SE model in immature rats. Furthermore, weighted correlation network analysis (WGCNA), gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to construct co-expression networks and establish functions of the identified hub lncRNAs in SE. The functional role of a hub lncRNA (NONRATT010788.2) in SE was investigated in an in vitro model. Our results indicated that 7082 lncRNAs (3522 up-regulated and 3560 down-regulated), which are involved in cell proliferation, inflammatory responses, angiogenesis and autophagy, were dysregulated in the hippocampus of immature rats with SE. Additionally, WGCNA identified 667 up-regulated hub lncRNAs in turquoise module that were involved in apoptosis, inflammatory responses and angiogenesis via regulation of HIF-1, p53 and chemokine signalling pathways and via inflammatory mediator regulation of TRP channels. Knockdown of an identified hub lncRNA (NONRATT010788.2) inhibited neuronal apoptosis in vitro. Taken together, our study is the first to demonstrate the expression profile and potential function of lncRNAs in the hippocampus of immature rats with SE. The defined hub lncRNAs may participate in the pathogenesis of SE via lncRNA-miRNA-mRNA network.

[Expression and role of Pim1 in cultured cortical neurons with oxygen-glucose deprivation/reoxygen injury].

OBJECTIVE: To study the expression and effect of Pim1 in primary cortical neurons after hypoxic-ischemic injury. METHODS: Cortical neurons were isolated from 1-day-old C57BL/6 mice and cultured in neurobasal medium. On the 8th day of neuron culture, cells were subjected to oxygen-glucose deprivation/reoxygen (OGD/R) treatment to mimic in vivo hypoxic injury of neurons. Briefly, medium were changed to DMEM medium, and cells were cultured in 1% O2 for 3 hours and then changed back to normal medium and conditions. Cells were collected at 0 hour, 6 hours, 12 hours and 24 hours after OGD/R. Primary neurons were transfected with Pim1 overexpression plasmid or mock plasmid, and then were exposed to normal conditions or OGD/R treatment. They were named as Pim1 group, control group, OGD/R group and OGD/R+Pim1 group respectively. Real-time PCR was used to detect Pim1 mRNA expression. Western blot was used to detect the protein expression of Pim1 and apoptotic related protein cleaved caspase 3 (CC3). TUNEL staining was used to detect cell apoptosis. RESULTS: Real-time PCR and Western blot results showed that Pim1 mRNA and protein were significantly decreased in neurons after OGD/R. They began to decrease at 0 hour after OGD/R, reached to the lowest at 12 hours after OGD/R, and remained at a lower level at 24 hours after OGD/R (P<0.01). Overexpression of Pim1 significantly upregulated the protein level of Pim1. Under OGD/R conditions, the CC3 expression and the apoptosis rate in cells of the Pim1 group were significantly lower than in un-transfected cells (P<0.01). CONCLUSIONS: Hypoxic-ischemic injury may decrease Pim1 expression in neurons. Overexpressed Pim1 may inhibit apoptosis induced by OGD/R.

[Effect of irisin on hypoxic-ischemic brain damage in neonatal rats].

OBJECTIVE: To study the effect and mechanism of action of irisin on hypoxic-ischemic brain damage in neonatal rats. METHODS: A total of 248 7-day-old Sprague-Dawley rats were randomly divided into a sham-operation group, a model group, and low- and high-dose irisin intervention groups (n=62 each). The rats in the model and irisin intervention groups were given hypoxic treatment after right common carotid artery ligation to establish a model of hypoxic-ischemic brain damage. Those in the sham-operation group were given the separation of the right common carotid artery without ligation or hypoxic treatment. The rats in the high- and low-dose irisin intervention groups were given intracerebroventricular injection of recombinant irisin polypeptide at a dose of 0.30 µg and 0.15 µg respectively. Those in the model and sham-operation groups were given the injection of an equal volume of PBS. The water maze test was used to compare neurological behaviors between groups. TTC staining, hematoxylin-eosin staining and TUNEL staining were used to observe histopathological changes of the brain. Western blot was used to measure the expression of the apoptosis-related molecules cleaved-caspase-3 (CC3), BCL-2 and BAX. RESULTS: Compared with the sham-operation group, the model group had a significant increase in latency time and a significant reduction in the number of platform crossings (P<0.05). Compared with the model group, the high-dose irisin intervention group had a significant reduction in latency time and a significant increase in the number of platform crossings (P<0.05). Compared with the sham-operation group, the model group had massive infarction in the right hemisphere, with significant increases in karyopyknosis and karyorrhexis. Compared with the model group, the high-dose irisin intervention group had a smaller infarct area of the right hemisphere, with reductions in karyopyknosis and karyorrhexis. The model group had a significantly higher apoptosis rate of cells in the right cerebral cortex and the hippocampus than the sham-operation group. The high-dose irisin intervention group had a significantly lower apoptosis rate than the model group (P<0.05). At 24 and 48 hours after modeling, the sham-operation group had a significantly lower level of CC3 than the model group (P<0.05). Compared with the model group, the high-dose irisin intervention group had a significantly lower level of CC3 and a significantly higher BCL-2/BAX ratio (P<0.05). The low-dose irisin intervention group had similar laboratory markers and histopathological changes of the brain to the model group. CONCLUSIONS: Irisin can alleviate hypoxic-ischemic brain damage in neonatal rats in a dose-dependent manner, possibly by reducing cell apoptosis in the cerebral cortex and the hippocampus.

Excessively tilted fiber grating based Fe3O4 saturable absorber for passively mode-locked fiber laser.

A novel approach to saturable absorber (SA) formation is presented by taking advantage of the mode coupling property of excessively tilted fiber grating (Ex-TFG). Stable mode-locked operation can be conveniently achieved based on the interaction between Ex-TFG coupled light and deposited ferroferric-oxide (Fe3O4) nanoparticles. The central wavelength, bandwidth and single pulse duration of the output are 1595 nm, 4.05 nm, and 912 fs, respectively. The fiber laser exhibits good long-term stability with signal-to-noise ratio (SNR) of 67 dB. For the first time, to the best of our knowledge, Ex-TFG based Fe3O4 SA for mode-locked fiber laser is demonstrated.

High temperature resistant ultra-short DBR Yb-doped fiber laser.

We present a distributed Bragg reflector (DBR) Yb-doped fiber laser based on a pair of type IA fiber Bragg gratings (FBGs). The high temperature resistant gratings are fabricated in high absorption hydrogen loaded Yb-doped silica fiber by use of a 244 nm argon laser and phase mask method. The DBR laser, with only 10 mm cavity length, exhibits high signal-noise ratio (SNR) of over 50 dB and can survive at 450°C in a long term with stable output power and central wavelength. Besides, the laser has a relatively low temperature sensitivity of 8.9 pm/°C, which contributes to cross-sensitization of stress and temperature.

Stable clinical course in three siblings with late-onset isolated sulfite oxidase deficiency: a case series and literature review.

BACKGROUND: Isolated sulfite oxidase deficiency (ISOD) is an autosomal recessive disorder caused by a deficiency of sulfite oxidase, which is encoded by the sulfite oxidase gene (SUOX). Clinically, the disorder is classified as one of two forms: the late-onset mild form or the classic early-onset form. The latter is life-threatening and always leads to death during early childhood. Mild ISOD cases are rare and may benefit from dietary therapy. To date, no cases of ISOD have been reported to recover spontaneously. Here, we present three mild ISOD cases in one family, each with a stable clinical course and spontaneous recovery. CASE PRESENTATION: All three siblings had two novel compound heterozygous mutations in the SUOX gene (NM_000456; c.1096C > T [p.R366C] and c.1376G > A [p.R459Q]). The siblings included two males and one female with late ages of onset (12-16 months) and presented with specific neuroimaging abnormalities limited to the bilateral globus pallidus and substantia nigra. The three patients had decreased plasma homocysteine levels. They exhibited a monophasic clinical course continuing up to 8.5 years even without dietary therapy. CONCLUSION: This is the first report of mild ISOD cases with a stable clinical course and spontaneous recovery without dietary therapy. Our study provides an expansion for the clinical spectrum of ISOD. Furthermore, we highlight the importance of including ISOD in the differential diagnosis for patients presenting with late-onset symptoms, bilaterally symmetric regions of abnormal intensities in the basal ganglia, and decreased plasma homocysteine levels.

Cumulative evidence for relationships between multiple variants of HNF1B and the risk of prostate and endometrial cancers.

BACKGROUND: To provide a synopsis of the current understanding of the association between variants of HNF1B and cancer susceptibility, we conducted a comprehensive research synopsis and meta-analysis to evaluate associations between HNF1B variants and prostate and endometrial cancers. RESULTS: Eighteen studies totaling 34,937 patients and 55,969 controls were eligible for this meta-analysis. Four variants showed a significant association with the risk of individual cancer. Strong significant associations were found between rs4430796 A and the risk of both prostate cancer (OR = 1.247, p = 2.21 × 10- 77) and endometrial cancer (OR = 1.217, p = 8.98 × 10- 16); the AA, AG genotypes also showed strong significant associations with the risk of prostate cancer (OR1 = 1.517, p = 4.46 × 10- 22; OR2 = 1.180, p = 0.002). There was a strong significant association between rs7501939 G and the risk of prostate cancer (OR = 1.201, p = 9.31 × 10- 31). Strong significant association was found between rs11649743 G (OR = 1.138, p = 1.08 × 10- 12), rs3760511 C (OR = 1.214, p = 1.57 × 10- 19) and the prostate cancer risk;the GG, AG genotypes of rs11649743 also showed strong significant associations with the risk of prostate cancer (OR1 = 1.496, p = 3.32 × 10- 6; OR2 = 1.276, p = 7.82 × 10- 6). All the cumulative epidemiological evidence of associations was graded as strong. CONCLUSIONS: Our study summarizes the evidence and helps to reveal that common variants of HNF1B are associated with risk of prostate and endometrial cancer.

[Investigation of Lentiviral Vectors Based Integrin ß8 RNAi System in Neonatal Rats' Brain].

OBJECTIVE: To construct lentiviral vectors expressing pSicoR-ß8 shRNA and evaluate its efficiency of RNA interference in neonatal rats' brain. METHODS: Plasmid vectors pSicoR-ß8 shRNA and pSicoR-control,as well as lentiviral packaging system pDM2G,g/p RRE and pRSV Rev were amplified respectively and plasmid DNA was identified by restriction enzyme digestion. Lentiviral packaging system and expressing vector pSicoR-ß8 shRNA/pSicoR-control were co-transfected into packaging cell line 293T. Lentiviral particles expressing ß8-shRNA or control sequence packaged and secreted by 293T were collected,concentrated by PEG-it,and viral titers were assayed by 50% tissue culture infective dose (TCID50). RNAi for integrin ß8 in neonatal rats' brain was performed by intraventricular injection of lentivirus expressing ß8-shRNA and rats received lentivirus expressing ß8-shRNA were served as control. Green fluorescent protein (GFP) expression after intraventricular injection of GFP-Lentivirus was observed under fluorescence microscope,ß8 mRNA and ß8 protein expression were detected by RT-PCR and Western blot respectively,all of which were performed to evaluate the RNAi efficiency and to choose the optimal time for intervention. RESULTS: Restrictive endonuclease digestion and agarose gel electrophoresis showed plasmids as same as the expected size. Lentiviral titers for LV-control after concentration was 1.0×108 PFU/mL,and for LV-ß8 shRNA 5.0×108 PFU/mL.One day after intraventricular injection of lentiviral vectors containing GFP sequence,lenticivirus genome was integrated into host cells and emitted green fluorescence. A relatively strong green fluorescence could be observed in brain slides 2 d,3 d and 5 d after intraventricular injection. Western blot and RT-PCR demonstrated a maximum inhibition happened 3 d after intraventricular injection of LV-ß8 shRNA,the inhibitory rate for ß8 mRNA and ß8 protein were 56% and 51%,respectively. CONCLUSION: Lentiviral vectors expressing ß8-shRNA are successfully constructed and lentiviral mediated ß8-RNAi is successfully applied for in vivo use.

T1 polymorphism in a disintegrin and metalloproteinase 33 (ADAM33) gene may contribute to the risk of childhood asthma in Asians.

OBJECTIVE: Polymorphisms in ADAM33 gene have been implicated in susceptibility to the risk of childhood asthma. However, the results remain controversial. We performed meta-analyses to clarify the relationship between them. METHODS: Relevant articles were searched in PubMed, Embase, Wanfang, and China National Knowledge Infrastructure. The Odds ratio (OR) with 95% confidence interval (CI) was used to assess the strength of the associations. RESULTS: Fourteen studies with five ADAM33 polymorphisms (F + 1, T1, T2, S2, and V4) were identified, involving 2687 cases and 2996 controls. ADAM33 F + 1, T2, and T1 polymorphisms showed significant associations with asthma risks in the overall and Caucasian children, Asian children, and Caucasian and Chinese children, respectively; however, these significant results were unstable in sensitivity analysis. T1 revealed significant and stable associations with asthma risks among Asian children in the dominant (OR = 2.00, 95% CI = 1.40-2.87, P = 0.0002) and codominant (OR = 3.06, 95% CI = 1.71-5.50, P = 0.0002) models; in cumulative meta-analyses, these significant results were robust. Concerning S2 or V4 polymorphism, no significant associations were observed. CONCLUSION: These findings demonstrate that ADAM33 T1 polymorphism might be a potential susceptible predictor of asthma for Asian children. Further functional studies between this polymorphism and asthma risks are warranted.

[Protective effect of histone acetylation against cortical injury in neonatal rats].

OBJECTIVE: To investigate the protective effect of histone acetylation against hypoxic-ischemic cortical injury in neonatal rats. METHODS: A total of 90 neonatal rats aged 3 days were divided into three groups: sham-operation, cortical injury model, and sodium butyrate (a histone deacetylase inhibitor) treatment. The rats in the model and the sodium butyrate treatment groups were intraperitoneally injected with lipopolysaccharide (0.05 mg/kg), and then right common carotid artery ligation was performed 2 hours later and the rats were put in a hypoxic chamber (oxygen concentration 6.5%) for 90 minutes. The rats in the sham-operation group were intraperitoneally injected with normal saline and the right common carotid artery was only separated and exposed without ligation or hypoxic treatment. The rats in the sodium butyrate treatment group were intraperitoneally injected with sodium butyrate (300 mg/kg) immediately after establishment of the cortical injury model once a day for 7 days. Those in the sham-operation and the model groups were injected with the same volume of normal saline. At 7 days after establishment of the model, Western blot was used to measure the protein expression of histone H3 (HH3), acetylated histone H3 (AH3), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (BAX), cleaved caspase-3 (CC3), and brain-derived neurotrophic factor (BDNF). Immunofluorescence assay was used to measure the expression of 5-bromo-2'-deoxyuridine (BrdU) as the cortex cell proliferation index. RESULTS: The sodium butyrate treatment group had a significantly lower HH3/AH3 ratio than the model group (P<0.05), which suggested that the sodium butyrate treatment group had increased acetylation of HH3. Compared with the model group, the sodium butyrate treatment group had a significant increase in Bcl-2/Bax ratio, a significant reduction in CC3 expression, and a significant increase in BDNF expression (P<0.05). The sodium butyrate treatment group had a significant increase in the number of BrdU-positive cells in the cortex compared with the model group (P<0.05), and BrdU was mainly expressed in the neurons. CONCLUSIONS: Increased histone acetylation may protect neonatal rats against cortical injury by reducing apoptosis and promoting regeneration of neurons. The mechanism may be associated with increased expression of BDNF.

[Effect of telomerase activation on biological behaviors of neural stem cells in rats with hypoxic-ischemic insults].

OBJECTIVE: To investigate the effect of telomerase activation on biological behaviors of neural stem cells after hypoxic-ischemic insults. METHODS: The neural stem cells passaged in vitro were divided into four groups: control, oxygen-glucose deprivation (OGD), OGD+cycloastragenol (CAG) high concentration (final concentration of 25 µM), and OGD+CAG low concentration (final concentration of 10 µM). The latter three groups were subjected to OGD. Telomerase reverse transcriptase (TERT) expression level was evaluated by Western blot. Telomerase activity was detected by telomerase repeat amplification protocol (TRAP). Cell number and neural sphere diameter were measured under a microscope. The activity of lactate dehydrogenase (LDH) was examined by chemiluminescence. Cell proliferation rate and apoptosis were detected by flow cytometry. RESULTS: After OGD insults, obvious injury of neural stem cells was observed, including less cell number, smaller neural sphere, more dead cells, lower proliferation rate and decreased survival rate. In CAG-treated groups, there were higher TERT expression level and telomerase activity compared with the control group (P<0.05). In comparison with the OGD group, CAG treatment attenuated cell loss (P<0.05) and neural sphere diameter decrease (P<0.05), promoted cell proliferation (P<0.05), and increased cell survival rate (P<0.05). Low and high concentrations of CAG had similar effects on proliferation and survival of neural stem cells (P>0.05). In the normal cultural condition, CAG treatment also enhanced TERT expression (P<0.05) and increased cell numbers (P<0.05) and neural sphere diameter (P<0.05) compared with the control group. CONCLUSIONS: Telomerase activation can promote the proliferation and improve survival of neural stem cells under the state of hypoxic-ischemic insults, suggesting telomerase activators might be potential agents for the therapy of hypoxic-ischemic brain injury.

[Expression rhythm of autophagic gene in neurons of neonatal rats with hypoxia/ischemia and its regulatory mechanism].

OBJECTIVE: To investigate the expression of autophagic gene and circadian gene in the neurons of neonatal rats after hypoxic-ischemic brain damage and the mechanism of nerve injury induced by hypoxia/ischemia. METHODS: Twelve Sprague-Dawley (SD) rats were randomly divided into hypoxic-ischemic (HI) group and sham-operation group, with 6 rats in each group. Ligation of the right common carotid artery and hypoxic treatment were performed to establish a model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of the circadian protein Clock in the cortex and hippocampus. The neurons of the rats were cultured in vitro and randomly divided into oxygen glucose deprivation (OGD) group and control group. The neurons in the OGD group were treated with DMEM medium without glucose or serum to simulate ischemic state, and hypoxic treatment was performed to establish an in vitro model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of autophagy-related proteins Beclin1 and LC3 and Clock protein at different time points. The changes in the expression of Beclin1 and LC3 were measured after the expression of Clock protein in neurons was inhibited by small interfering RNA technique. RESULTS: The expression of autophagy-related proteins Beclin1 and LC3Ⅱ in neurons cultured in vitro displayed a rhythmic fluctuation; after OGD treatment, the expression of Beclin1 and LC3Ⅱ gradually increased over the time of treatment and no longer had a rhythmic fluctuation. Compared with the sham-operation group, the HI group had a significant reduction in the expression of Clock protein in the cortex and hippocampus (P<0.05). After OGD treatment, the neurons cultured in vitro had a significant reduction in the expression of Clock protein (P<0.05). Compared with the negative control group, the Clock gene inhibition group had significant reductions in the expression of Beclin1 and LC3Ⅱ (P<0.05). CONCLUSIONS: Hypoxia/ischemia induces the disorder in the expression rhythm of autophagy-related proteins Beclin1 and LC3, and the mechanism may be associated with the fact that the circadian protein Clock participates in the regulation of the expression of Beclin1 and LC3.

[Long non-coding RNAs and hypoxic-ischemic brain damage].

Long non-coding RNAs (lncRNAs) are transcripts with a complex structure and a length of >200 nt which are unable to encode proteins. The lncRNAs interact with DNA, mRNA, and proteins and regulate gene expression through various mechanisms, thus participating in the regulation of various biological processes. Studies have shown that lncRNAs play important roles in neural development and the pathogenesis of diseases. This article reviews the roles of lncRNAs in hypoxic-ischemic brain damage.

[Role of STAT3 signaling pathway in hypoxic-ischemic brain damage of neonatal rats].

OBJECTIVE: To study the role and mechanisms of STAT3 signaling pathway in hypoxic-ischemic brain damage (HIBD) of neonatal rats. METHODS: Eighty 7-day-old Sprague-Dawley rats were randomly divided into two groups: HI and sham-operated (n=40 each). The rats in the HI group were subjected to right carotid artery ligation and subsequent hypoxia exposure (8% O2) for 2.5 hours, and the rats in the sham-operated group underwent the right carotid artery dissection without subsequent ligation or hypoxia treatment. Brain tissue samples were collected at 4, 6, 8, 12 and 24 hours after operation and hypoxic exposure. Immunohistochemistry and Western blot were used to detect the expression of STAT3, phosphorylated STAT3 (p-STAT3) and vascular endothelial growth factor (VEGF) proteins. TUNEL staining was used to detect apoptotic cells. RESULTS: No significant difference in STAT3 expression was observed at all time points between the HI and sham-operated groups (P>0.05). Compared with the sham-operated group, the expression of p-STAT3 protein in the HI group was significantly upregulated at 4, 6, 8, 12 hours after operation and hypoxic exposure, and peaked at 6 hours (P<0.01). The VEGF expression in the HI group was higher than that in the sham-operated group at all time points, which peaked at 8 hours (P<0.05). TUNEL staining showed that the apoptotic cells increased significantly in a time-dependent manner compared with the sham-operated group (P<0.01). CONCLUSIONS: HI may lead to phosphorylation of STAT3 which probably induces the VEGF expression in the brain of neonatal rats. The activated STAT3 signaling pathway may be involved in the apoptosis regulation of nerve cells, and related to apoptosis inhibition of nerve cells.