Up-regulation of miR-126 via DNA methylation in hypoxia-preconditioned endothelial cells may contribute to hypoxic tolerance of neuronal cells.

BACKGROUND: Endothelial cells (ECs) can confer neuroprotection by secreting molecules. This study aimed to investigate whether DNA methylation contributes to the neuroprotective gene expression induced by hypoxia preconditioning (HPC) in ECs and to clarify that the secretion of molecules from HPC ECs may be one of the molecular mechanisms of neuroprotection. METHODS: Human microvascular endothelial cell-1 (HMEC-1) was cultured under normal conditions (C), hypoxia(H), and hypoxia preconditioning (HPC), followed by the isolation of culture medium (CM). SY5Y cell incubated with the isolated CM from HMEC-1 was exposed to oxygen-glucose deprivation (OGD). The DNA methyltransferases (DNMTs), global methylation level, miR-126 and its promotor DNA methylation level in HMEC-1 were measured. The cell viability and cell injury in SY5Y were detected. RESULTS: HPC decreased DNMTs level and global methylation level as well as increased miR-126 expression in HMEC-1. CM from HPC treated HMEC-1 also relieved SY5Y cell damage, while CM from HMEC-1 which over-expression of miR-126 can reduce injury in SY5Y under OGD condition. CONCLUSIONS: These findings indicate EC may secrete molecules, such as miR-126, to execute neuroprotection induced by HPC through regulating the expression of DNMTs.

Hypoxia preconditioning increases Notch1 activity by regulating DNA methylation in vitro and in vivo.

BACKGROUND: Our previous research has demonstrated that hypoxic preconditioning (HPC) can improve spatial learning and memory abilities in adult mice. Adult hippocampal neurogenesis has been associated with learning and memory. The Neurogenic locus notch homolog protein (Notch) was involved in adult hippocampal neurogenesis, as well as in learning and memory. It is currently unclear whether the Notch pathway regulates hippocampal neuroregeneration by modifying the DNA methylation status of the Notch gene following HPC. METHOD: The HPC animal model and cell model were established through repeated hypoxia exposure using mice and the mouse hippocampal neuronal cell line HT22. Step-down test was conducted on HPC mice. Real-time PCR and Western blot analysis were used to assess the mRNA and protein expression levels of Notch1 and hairy and enhancer of split1 (HES1). The presence of BrdU-positive cells and Notch1 expression in the hippocampal dental gyrus (DG) were examined with confocal microscopy. The methylation status of the Notch1 was analyzed using methylation-specific PCR (MS-PCR). HT22 cells were employed to elucidate the impact of HPC on Notch1 in vitro. RESULTS: HPC significantly improved the step-down test performance of mice with elevated levels of mRNA and protein expression of Notch1 and HES1 (P < 0.05). The intensities of the Notch1 signal in the control group, the H group and the HPC group were 2.62 ± 0.57 × 107, 2.87 ± 0.84 × 107, and 3.32 ± 0.14 × 107, respectively, and the number of BrdU (+) cells in the hippocampal DG were 1.83 ± 0.54, 3.71 ± 0.64, and 7.29 ± 0.68 respectively. Compared with that in C and H group, the intensity of the Notch1 signal and the number of BrdU (+) cells increased significantly in HPC group (P < 0.05). The methylation levels of the Notch1 promoter 0.82 ± 0.03, 0.65 ± 0.03, and 0.60 ± 0.02 in the C, H, and HPC groups, respectively. The methylation levels of Notch1 decreased significantly (P < 0.05). The effect of HPC on HT22 cells exhibited similarities to that observed in the hippocampus. CONCLUSION: HPC may confer neuroprotection by activating the Notch1 signaling pathway and regulating its methylation level, resulting in the regeneration of hippocampal neurons.

Involvement of nerve growth factor in mouse hippocampal neuronal cell line (HT22) differentiation and underlying role of DNA methyltransferases.

DNA methylation is an epigenetic event involved in regulation of gene transcription during cell differentiation. DNA methyltransferases (DNMT) play a role in differentiation of neural stem cells into neurons. The aim of this study was to determine whether nerve growth factor (NGF) was involved in differentiation of mouse hippocampal neuronal cell line (HT22) as assessed by IncuCyte. Quantitative PCR and western blot were used to measure gene and protein expression of DNMT as well as the activity of DNMTs. Treatment with NGF was found to upregulate both gene and protein expressions as well as total activity of DNMTs in differentiating HT22 cells. Compared to undifferentiating cells, the percentage of differentiating cells at S phase increased significantly when incubated with NGF. In undifferentiated cells, NGF failed to induce gene and protein expressions and activity of DNMTs. Data demonstrate that differentiation of HT22 cells by exposure to NGF involve the activation of DNMTs pathway.

5-Aza-2'-deoxycytidine, a DNA methylation inhibitor, induces cytotoxicity, cell cycle dynamics and alters expression of DNA methyltransferase 1 and 3A in mouse hippocampus-derived neuronal HT22 cells.

Epigenetic processes such as DNA methylation are essential for processes of gene expression in normal mammalian development. DNA methyltransferases (DNMT) are responsible for initiating and maintaining DNA methylation. It is known that 5-Aza-CdR, an inhibitor of DNMT induces cytotoxicity by reducing DNMT activity in various tumor cell lines. However, disturbances in neuronal DNA methylation may also play a role in altered brain functions. Thus, it was of interest to determine whether alterations in DNA methylation might be associated with neuronal functions by using 5-Aza-CdR, on mouse hippocampus-derived neuronal HT22 cell line. In particular, the aim of this study was to investigate the effects of 5-Aza-CdR on cell growth inhibition, cell cycle arrest, apoptosis as well as the expression levels of DNMT in HT22 cells. HT22 cells were incubated with 5 or 20 µmol/L 5-Aza-CdR for 24 h. Data showed that 5-Aza-CdR at both concentrations significantly inhibited proliferation of HT22 cells and exacerbated cytoplasmic vacuolization. Flow cytometry analysis demonstrated that 5-Aza-CdR treatment at both concentrations decreased early apoptosis but enhanced late apoptosis. Cell cycle analysis illustrated that 5-Aza-CdR treatment induced S phase arrest. Further, incubation with 5-Aza-CdR produced a down-regulation in expression of mRNA and protein DNMT1 and 3A but no marked changes were noted in DNMT 3B and p21 expression. In addition, DNMT1 activity was significantly decreased at both 5-Aza-CdR concentrations. Evidence indicates that 5-Aza-CdR induced cytotoxicity was associated with altered mRNA and protein expression of DNMT 1 and 3A associated with reduced DNMT1 activity in HT22 cells which might affect brain functions.

Vanadate-induced antiproliferative and apoptotic response in esophageal squamous carcinoma cell line EC109.

Vanadate is a transition element that present in nature and was shown to be a nonspecific inhibitor of protein tyrosine phosphatases. It was reported that vanadium (Vd) compounds exhibit antitumor actions in several cancer cell lines. This study aimed to examine the antiproliferative and apoptotic actions of different concentrations of sodium vanadate (NaVd) (+5) in esophageal squamous carcinoma cell line EC109 by determining the protein expression levels of cyclin D1 and caspase-3 following incubation for various times from 15 min up to 4 h. In addition, cell proliferation of EC109 treated with different concentrations (NaVd) was also measured using the MTT assay at 4, 12, 24, and 48 h. The cell cycle of EC109 cells exposed to different concentrations of NaVd was detected using flow cytometry determination at 24 h. Data showed that NaVd greater than 100 µM significantly increased cyclin D1. In contrast, reduced caspase-3 protein expression levels occurred at 50 µM. Cellular proliferation was significantly decreased at 50uM. The cell cycle was arrested at S phase with 100 µM NaVd. Taken together, data indicate that NaVd produced concentration- and time-dependent antitumor actions in EC109 cell line.

Minocycline inhibits neurogenic inflammation by blocking the effects of tumor necrosis factor-α.

It has been well established that neurogenic inflammation is one of the major pathological processes underlying inflammatory pain, but there are few effective anti-inflammatory drugs to alleviate such pain. The present study shows that minocycline, a widely used glial activation inhibitor, is effective in reducing neurogenic inflammation. Patch-clamp recordings showed that small sized dorsal root ganglion (DRG) neurons were dramatically excited following intradermal capsaicin injection in the rat hind paw, evidenced by decreased rheobase and membrane threshold. Pretreatment with minocycline (30 mg/kg for 1 day, intraperitoneal injection) blocked the increased neuronal excitability. Western blot and immunostaining of DRG revealed the activation of satellite glial cells (SGCs) following capsaicin injection. The up-regulation of glial fibrillary acidic protein (GFAP) was significantly inhibited by minocycline pre-administration. Measurement of tumor necrosis factor α (TNF-α) and its receptor, TNF-α receptor 1 (TNFR1), showed that minocycline mainly blocked the up-regulation of TNF-α in SGCs and TNFR1s in neurons following capsaicin injection. The pivotal role of TNF-α in neurogenic inflammation was further supported by the findings that incubation DRG with TNF-α mimicked the increased excitability of DRG neurons induced by capsaicin injection, and that TNF-α application enhanced cutaneous vasodilation in the hind paws induced by antidromic electrical stimulation of dorsal roots. Based on these results, we propose that minocycline is a potential therapeutic drug that can reduce neuronal excitability and neurogenic inflammation by working on SGCs to inhibit the expression of TNF-α.

Growth of the skull in young children in Baotou, China.

BACKGROUND: There are some controversies about the optimal time to perform skull repair in very young Chinese children because of the rapid skull growth in this stage of life. The purpose of this current study is to describe the characteristics of skull growth and to discuss the optimal time for skull repair in young Chinese children with skull defects. METHODS: A total of 112 children born in the First Affiliated Hospital of Baotou Medical College were measured for six consecutive years starting in 2006. Cranial length (CL, linear distance between the eyebrows to the pillow tuberosity), cranial width (CW, double-sided linear distance of connection of external auditory canal), ear over the top line (EOTL), the eyebrows-the posterior tuberosity line (EPTL), and head circumference (HC) were measured to describe the skull growth. RESULTS: The most rapid period of skull growth occurs during the first year of life. The second and third most rapid periods are the second and third years, respectively. Then, the skull growth slowed and the values of the skull growth index of 6-year-old children were close to those of adults. CONCLUSION: Children 0-1 years old should not receive skull repair due to their rapid skull growth. The indexes of children 3 years old or older were close to those of the adult; therefore, 3 years old or older may receive skull repair.

The effects of CoCl2 on HIF-1α protein under experimental conditions of autoprogressive hypoxia using mouse models.

It is well known that cobalt chloride (CoCl2) can enhance the stability of hypoxia-inducible factor (HIF)-1α. The aim of this study is to detect the effect of CoCl2 on the hypoxia tolerance of mice which were repeatedly exposed to autoprogressive hypoxia. Balb/c mice were randomly divided into groups of chemical pretreatment and normal saline (NS), respectively injected with CoCl2 and NS 3 h before exposure to hypoxia for 0 run (H0), 1 run (H1), and 4 runs (H4). Western Blot, electrophoretic mobility shift assay (EMSA), extracellular recordings population spikes in area cornus ammonis I (CA 1) of mouse hippocampal slices and real-time were used in this study. Our results demonstrated that the tolerance of mice to hypoxia, the changes of HIF-1α protein level and HIF-1 DNA binding activity in mice hippocampus, the mRNA level of erythropoietin (EPO) and vascular endothelial growth factor (VEGF), and the disappearance time of population spikes of hippocampal slices were substantially different between the control group and the CoCl2 group. Over-induction of HIF-1α by pretreatment with CoCl2 before hypoxia did not increase the hypoxia tolerance.

MALAT1 and NEAT1 Are Neuroprotective During Hypoxic Preconditioning in the Mouse Hippocampus Possibly by Regulation of NR2B.

Wang L, Fu G, Han R, Fan P, Yang J, Gong K, Zhao Z, Zhang C, Sun K, Shao GMALAT1 and NEAT1 Are Neuroprotective during Hypoxic Preconditioning in the Mouse Hippocampus Possibly by Regulation of NR2B High Alt Med Biol. 00:000-000, 2024. Background: The regulation of noncoding ribonucleic acid (ncRNA) has been shown to be involved in cellular and molecular responses to hypoxic preconditioning (HPC), a situation created by the induction of sublethal hypoxia in the brain. The ncRNAs metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and nuclear paraspeckle assembly transcript 1 (NEAT1) are abundantly expressed in the brain, where they regulate the expression of various genes in nerve cells. However, the exact roles of MALAT1 and NEAT1 in HPC are not fully understood. Methods: A mouse model of acute repeated hypoxia was used as a model of HPC, and MALAT1 and NEAT1 levels in the hippocampus were measured using real-time polymerase chain reaction (PCR). The mRNA and protein levels of N-methyl-d-aspartate receptor subunit 2 B (NR2B) in the mouse hippocampus were measured using real-time PCR and western blotting, respectively. HT22 cells knocked-down for MALAT1 and NEAT1 were used for in vitro testing. Expression of NR2B, which is involved in nerve cell injury under ischemic and hypoxic conditions, was also evaluated. The levels of spectrin and cleaved caspase-3 in MALAT1 and NEAT1 knockdown HT22 cells under oxygen glucose deprivation/reperfusion (OGD/R) were determined by western blotting. Results: HPC increased the expression of MALAT1 and NEAT1 and decreased the expression of NR2B mRNA in the mouse hippocampus (p < 0.05). Knockdown of MALAT1 and NEAT1 increased both NR2B mRNA and protein levels nearly twofold and caused damage under OGD/R conditions in HT22 cells (p < 0.05). Conclusion: MALAT1 and NEAT1 exert neuroprotective effects by influencing the expression of NR2B.

Vascular Endothelial Growth Factor and Erythropoietin Show Different Expression Patterns in the Early and Late Hypoxia Preconditioning Phases and May Correlate with DNA Methylation Status in the Mouse Hippocampus.

Liu, Na, Yanbo Zhang, Pu Zhang, Kerui Gong, Chunyang Zhang, Kai Sun, and Guo Shao. Vascular endothelial growth factor and erythropoietin show different expression patterns in the early and late hypoxia preconditioning phases and may correlate with DNA methylation status in the mouse hippocampus. High Alt Med Biol. 23:361-368, 2022. Background: Vascular endothelial growth factor (VEGF) and erythropoietin (EPO) have been proven to participate in neuroprotection induced by hypoxia preconditioning (HPC), and they can be regulated by hypoxia-inducible factor 1 (HIF-1). It has been reported that DNA methylation can affect VEGF and EPO expression. This study aimed to explore the expression of VEGF and EPO in the early phase and late phase of HPC and whether their expression was affected by DNA methylation. Method: Acute repeated HPC mice were used as the animal model, and detection of molecular changes was performed immediately (early phase) and 1 day (late phase) after HPC treatment. The mRNA and protein expression levels of VEGF, EPO, and DNA methyltransferases (DNMTs) in the hippocampi were measured by real-time polymerase chain reaction and western blotting, respectively. The activity of DNMTs and global methylation levels were analyzed by enzyme-linked immunosorbent assay. DNA methylation levels of VEGF and EPO promoters, which were catalyzed by DNMTs, were determined by bisulfite-modified DNA sequencing. Results: The expression of VEGF was increased in the early phase and late phase of HPC (p < 0.05), whereas the expression of EPO was unchanged in the early phase (p > 0.05) of HPC and was increased in the late phase (p < 0.05). VEGF and EPO expression were negatively correlated with the DNA methylation levels of their promoters. DNMT3A and DNMT3B were decreased in the early phase and late phase (p < 0.05), whereas DNMT1 was unchanged in the early phase and late phase (p > 0.05). Conclusions: Our data demonstrated that DNMTs affect VEGF and EPO expression by regulating the DNA methylation levels of the promoters of VEGF and EPO.

The Role of the lncRNA MALAT1 in Neuroprotection against Hypoxic/Ischemic Injury.

Hypoxic and ischemic brain injury can cause neurological disability and mortality, and has become a serious public health problem worldwide. Long-chain non-coding RNAs are involved in the regulation of many diseases. Metastasis-related lung adenocarcinoma transcript 1 (MALAT1) is a type of long non-coding RNA (lncRNA), known as long intergenic non-coding RNA (lincRNA), and is highly abundant in the nervous system. The enrichment of MALAT1 in the brain indicates that it may be associated with important functions in pathophysiological processes. Accordingly, the role of MALAT1 in neuronal cell hypoxic/ischemic injury has been gradually discovered over recent years. In this article, we summarize recent research regarding the neuroprotective molecular mechanism of MALAT1 and its regulation of pathophysiological processes of brain hypoxic/ischemic injury. MALAT1 may function as a regulator through interaction with proteins or RNAs to perform its role, and may therefore serve as a therapeutic target in cerebral hypoxia/ischemia.

Overview of Traditional Mongolian Medical Warm Acupuncture.

Mongolian medical warm acupuncture is a traditional therapy of Mongolian medicine and was developed by people living on the Mongolian Plateau. This kind of traditional oriental medicine has a long history. The main characteristics of Mongolian medical warm acupuncture are the acupoints and the needles used. Its theory is based on the human anatomical structure and the distinct local culture. Mongolian medical warm acupuncture has been practiced for centuries and proved to be very effective in the treatment of age-related diseases, including the musculoskeletal and nervous diseases. This paper aims to briefly introduce the history and scope of Mongolian medical warm acupuncture, with a particular focus on age-related diseases, where Mongolian medical warm acupuncture has shown significant beneficial effects.

Effects of 5-Aza on neurogenesis contribute to learning and memory in the mouse hippocampus.

BACKGROUND: 5-Aza-2'-deoxycytidine (5-Aza-CdR) is a demethylating agent that has various biological effects related to DNA methylation. DNA methylation plays important roles in learning and memory. We have reported that 5-Aza-CdR improved the performance of mice in the water maze and step-down tests. Some behaviours have been well recognized to be mediated by neurogenesis in the hippocampus. The Notch signalling pathway plays a key role in adult hippocampal neurogenesis. In this study, we examined whether 5-Aza-CdR (DNA methyltransferase inhibitor) affects neurogenesis and Notch1 expression. METHODS: The learning and memory behaviour of mice was evaluated by a conditioned avoidance learning 24 h after 5-Aza-CdR treatment. The mRNA and protein expression levels of Notch1 and HES1 were measured by real-time PCR and Western blotting. The 5-bromo-2'-deoxyuridine (BrdU)-positive cells and the expression of Notch1 in the hippocampal DG were observed through laser confocal microscopy. To further clarify whether 5-Aza-CdR affects behaviour through neurogenesis, the expression level of Notch1, cell viability and cell cycle were analysed using the HT22 cell line. RESULTS: The behaviour in conditioned avoidance learning was improved, while neurogenesis and the Notch1 pathway were increased in the hippocampus of mice that were injected with 5-Aza-CdR. In vitro experiments showed that 5-Aza-CdR increased the expression of the Notch1 pathway and upregulated S-phase in the cell cycle and cell viability. CONCLUSIONS: Our results suggest that the effect of 5-Aza-CdR on behaviour may be related to an increase in neurogenesis with upregulation of the Notch1 pathway in the hippocampus.

Possible Involvement of DNA Methylation in TSC1 Gene Expression in Neuroprotection Induced by Hypoxic Preconditioning.

Background: It has been reported that ischemia and ischemic preconditioning (IPC) have different effects on the expression of tuberous sclerosis complex 1 (TSC1), which may contribute to the tolerance to ischemia/hypoxia with the increase of autophagy. The mechanisms of TSC1 differential expression are still unclear under ischemia/IPC conditions in hippocampal Cornu Ammon 1 (CA1) and Cornu Ammon 3 (CA3) area neuronal cells. While we have shown that 5-Aza-CdR, a DNA methyltransferase inhibitor, can upregulate TSC1 and increase hypoxic tolerance by autophagy in vivo and in vitro, in this study, we examined whether DNA methylation was involved in the differential expression of TSC1 in the CA1 and CA3 regions induced by hypoxic preconditioning (HPC). Methods: Level of rapamycin (mTOR) autophagy, a downstream molecular pathway of TSC1/TSC2 complex, was detected in HPC mouse hippocampal CA1 and CA3 areas as well as in the HPC model of mouse hippocampal HT22 cells. DNA methylation level of TSC1 promoter (-720 bp~ -360 bp) was determined in CA1 and CA3 areas by bisulfite-modified DNA sequencing (BMDS). At the same time, autophagy was detected in HT22 cells transfected with GFP-LC3 plasmid. The role of TSC1 in neuroprotection was measured by cell viability and apoptosis, and the role of TSC1 in metabolism was checked by ATP assay and ROS assay in HT22 cells that overexpressed/knocked down TSC1. Results: HPC upregulated the expression of TSC1, downregulated the level of P-mTOR (Ser2448) and P-p70S6K (Thr389), and enhanced the activity of autophagy in both in vivo and in vitro. The increased expression of TSC1 in HPC may depend on its DNA hypomethylation in the promoter region in vivo. HPC also could reduce energy consumption in HT22 cells. Overexpression and knockdown of TSC1 can affect cell viability, cell apoptosis, and metabolism in HT22 cells exposed to hypoxia. Conclusion: TSC1 expression induced by HPC may relate to the downregulation of its DNA methylation level with the increase of autophagy and the decrease of energy demand.

Involvement of subtypes γ and ε of protein kinase C in colon pain induced by formalin injection.

Protein kinase C (PKC) has been widely reported to participate in somatic pain; however, its role in visceral pain remains largely unclear. Using a colon inflammatory pain model by intracolonic injection of formalin in rats, the present study was to examine the role of PKC in visceral pain and determine which subtypes may be involved. The colon pain behavior induced by formalin injection could be enhanced by intrathecal pretreatment with a PKC activator (PMA), and alleviated by a PKC inhibitor (H-7). Wide dynamic range (WDR) neurons in the L6-S1 spinal dorsal horn that were responsive to colorectal distension were recorded extracellularly. It was found that neuronal activity was greatly increased following formalin injection. Microdialysis of PMA near the recorded neuron in the spinal dorsal horn facilitated the enhanced responsive activity induced by formalin injection, while H-7 inhibited significantly the enhanced response induced by formalin injection. Western blot analysis revealed that membrane translocation of PKC-γ and PKC-ε, but not other subtypes, in the spinal cord was obviously increased following formalin injection. Therefore, our findings suggest that PKC is actively involved in the colon pain induced by intracolonic injection of formalin. PKC-γ and PKC-ε subtypes seem to significantly contribute to this process.

DNMT3B Expression Might Contribute to Abnormal Methylation of RASSF1A in Lager Colorectal Adenomatous Polyps.

BACKGROUND: It is pretty well known that DNA methyltransferases (DNMTs) are actively involved in abnormal cell growth. The goal of the current study is to explore the correlation between DNMT expression and colorectal adenomatous polyps (CAPs). METHOD: Twenty pairs of CAP samples with a diameter ≥ 10 mm and corresponding normal colorectal mucosa (NCM) tissues from patients were used in the present study. The expression levels and activity of DNA methyltransferases (DNMTs) were measured in the CAP tissues. The global methylation and the promoter methylation level of 3 kinds of tumour suppressor gene were detected. RESULTS: mRNA and protein levels of DNMT3B were found to be elevated in the CAP tissues compared with the control tissue. Additionally, the methylation of long interspersed nuclear elements-1 (LINE-1/L1) was decreased in the CAP tissue. Furthermore, methylation of the promoter of a tumour suppressor gene Ras association domain family 1A (RASSF1A) was increased in the CAP tissues, while the mRNA levels of RASSF1A were decreased. CONCLUSIONS: These results suggest that the overexpression of DNMT3B may contribute to a role in the genesis of CAPs through the hypomethylation of chromosomes in the whole cell and promoter hypermethylation of RASSF1A.

Neuroprotective mechanisms of DNA methyltransferase in a mouse hippocampal neuronal cell line after hypoxic preconditioning.

Hypoxic preconditioning has been shown to improve hypoxic tolerance in mice, accompanied by the downregulation of DNA methyltransferases (DNMTs) in the brain. However, the roles played by DNMTs in the multiple neuroprotective mechanisms associated with hypoxic preconditioning remain poorly understood. This study aimed to establish an in vitro model of hypoxic preconditioning, using a cultured mouse hippocampal neuronal cell line (HT22 cells), to examine the effects of DNMTs on the endogenous neuroprotective mechanisms that occur during hypoxic preconditioning. HT22 cells were divided into a control group, which received no exposure to hypoxia, a hypoxia group, which was exposed to hypoxia once, and a hypoxic preconditioning group, which was exposed to four cycles of hypoxia. To test the ability of hypoxic preadaptation to induce hypoxic tolerance, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay. Cell viability improved in the hypoxic preconditioning group compared with that in the hypoxia group. The effects of hypoxic preconditioning on the cell cycle and apoptosis in HT22 cells were examined by western blot assay and flow cytometry. Compared with the hypoxia group, the expression levels of caspase-3 and spectrin, which are markers of early apoptosis and S-phase arrest, respectively, noticeably reduced in the hypoxic preconditioning group. Finally, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and western blot assay were used to investigate the changes in DNMT expression and activity during hypoxic preconditioning. The results showed that compared with the control group, hypoxic preconditioning downregulated the expression levels of DNMT3A and DNMT3B mRNA and protein in HT22 cells and decreased the activities of total DNMTs and DNMT3B. In conclusion, hypoxic preconditioning may exert anti-hypoxic neuroprotective effects, maintaining HT22 cell viability and inhibiting cell apoptosis. These neuroprotective mechanisms may be associated with the inhibition of DNMT3A and DNMT3B.

Exosomal MicroRNA-126 from RIPC Serum Is Involved in Hypoxia Tolerance in SH-SY5Y Cells by Downregulating DNMT3B.

Ischemic tolerance in the brain can be induced by transient limb ischemia, and this phenomenon is termed remote ischemic preconditioning (RIPC). It still remains elusive how this transfer of tolerance occurs. Exosomes can cross the blood-brain barrier, and some molecules may transfer neuroprotective signals from the periphery to the brain. Serum miRNA-126 is associated with ischemic stroke, and exosomal miRNA-126 has shown protective effects against acute myocardial infarction. Therefore, this study aims to explore whether exosomal miRNA-126 from RIPC serum can play a similar neuroprotective role. Exosomes were isolated from the venous serum of four healthy young male subjects, both before and after RIPC. Exosomal miRNA-126 was measured by real-time PCR. The miRNA-126 target sequence was predicted by bioinformatics software. SH-SY5Y neuronal cells were incubated with exosomes, and the cell cycle was analyzed by flow cytometry. The expression and activity of DNA methyltransferase (DNMT) 3B, a potential target gene of miRNA-126, were examined in SH-SY5Y cells. The cell viability of SH-SY5Y cells exposed to oxygen-glucose deprivation (OGD) was also investigated. To confirm the association between miRNA-126 and DNMT3B, we overexpressed miRNA-126 in SH-SY5Y cells using lentiviral transfection. miRNA-126 expression was upregulated in RIPC exosomes, and bioinformatics prediction showed that miRNA-126 could bind with DNMT3B. DNMT levels and DNMT3B activity were downregulated in SH-SY5Y cells incubated with RIPC exosomes. After overexpression of miRNA-126 in SH-SY5Y cells, global methylation levels and DNMT3B gene expression were downregulated in these cells, consistent with the bioinformatics predictions. RIPC exosomes can affect the cell cycle and increase OGD tolerance in SH-SY5Y cells. RIPC seems to have neuroprotective effects by downregulating the expression of DNMTs in neural cells through the upregulation of serum exosomal miRNA-126.

Antihypoxic effects of neuroglobin in hypoxia-preconditioned mice and SH-SY5Y cells.

This work aims at investigating the neuroprotective effects of neuroglobin (Ngb) in vivo and in vitro. RT-PCR and Western blotting were used to examine Ngb mRNA and protein levels in the mouse cortex after acute and repeated exposure to hypoxia. The cDNAs of mouse Ngb were cloned and transfected into SH-SY5Y cells to examine Ngb function in vitro. Expression of Ngb and mRNA was upregulated in the cortex of mice preconditioned by repetitive exposure to hypoxia. Tolerance to hypoxia of Ngb-transformed SH-SY5Y cells was enhanced. These results suggest that Ngb might be involved in hypoxic preconditioning which protects neurons from hypoxic injury.

Imbalance Between Excitatory and Inhibitory Synaptic Transmission in the Primary Somatosensory Cortex Caused by Persistent Nociception in Rats.

There is substantial evidence supporting the notion that the primary somatosensory (S1) cortex is an important structure involved in the perceptional component of pain. However, investigations have mainly focused on other pain-related formations, and few reports have been provided to investigate the synaptic plasticity in the S1 cortex in response to persistent pain. In the present study, we report that bee venom (BV) injection triggered an imbalance between excitatory and inhibitory synaptic transmission in the S1 cortex in rats. Using a multi-electrode array recording, we found that BV-induced persistent inflammatory pain led to temporal and spatial enhancement of synaptic plasticity. Moreover, slice patch clamp recordings on identified pyramidal neurons demonstrated that BV injection increased presynaptic and postsynaptic transmission in excitatory synapses and decreased postsynaptic transmission in inhibitory synapses in the layer II/III neurons within the S1 cortex. In immunohistochemistry and Western blot sections, the distribution and expression of total AMPA receptor subunits and gamma-amino butyric acid-A (GABAA) were unaffected, although the membrane fractions of GluR2 and GABAA were decreased, and their cytosolic fractions were increased in contrast. The change of GluR1 was opposite to that of GluR2, and GluR3 did not change significantly. Our studies, therefore, provide direct evidence for both presynaptic and postsynaptic changes in synapses within the S1 cortex in persistent nociception, which are probably related to the membrane trafficking of GluR1, GluR2, and GABAA. Perspective: Increased synaptic plasticity was detected in S1 after peripheral nociception, with enhanced excitatory and decreased inhibitory synaptic transmissions. Increased GluR1, and decreased GABAAα1 and GluR2 membrane trafficking were detected. Therefore, the disrupted excitatory/inhibitory balance in transmissions is involved in nociception processing, and S1 can be a potential antinociceptive site.