Oxygen-glucose Deprivation And Restoration Research Articles

The critical role of KLF4 in regulating the activation of A1/A2 reactive astrocytes following ischemic stroke

BackgroundWe have previously demonstrated that the expression of kruppel-like transcription factor-4 (KLF-4) is upregulated in astrocytes following acute ischemic stroke (AIS) and found that KLF4 confers vascular protection against cerebral ischemic injury. However, the functional role of KLF4 in astrocyte after AIS is far from clear.MethodsThe intrinsic relationship between KLF4 and A1/A2 reactive astrocytes and the impact of astrocytic KLF4 on the activation of A1/A2 subtype astrocytes were evaluated in middle cerebral artery occlusion (MCAO) mice and oxygen–glucose deprivation and restoration (OGD/R) astrocytes.ResultsOur results demonstrated that astrocytic KLF4 expression and complement C3-positive A1 and S100 calcium binding protein A10 (S100A10)-positive A2 astrocytes were induced in the ischemic penumbra following focal cerebral ischemia, and the time course of upregulation of astrocytic KLF4 correlated closely with the activation of A2 astrocytes. The dual immunofluorescent studies displayed that in the ischemic hemisphere, where the high levels of KLF4 were expressed, there were relatively low levels of C3 expressed in the reactive astrocytes and vice versa, but KLF4 was always co-stained well with S100A10. Mechanistic analyses revealed that astrocytic KLF4 inhibited the activation of A1 astrocyte but promoted A2 astrocyte polarization after OGD/R by modulating expressions of nuclear factor-kB.ConclusionsAstrocyte-derived KLF4 has a critical role in regulating the activation of A1/A2 reactive astrocytes following AIS.

Role of GPR35 activation in apoptosis in cortical astrocytes in a rat model of oxygen-glucose deprivation and restoration

Objective To evaluate the role of G protein-coupled receptor 35 (GPR35) activation in apoptosis in cortical astrocytes in a rat model of oxygen-glucose deprivation and restoration (OGD/R). Methods Primary cortical astrocytes of rats cultured in vitro were seeded in 6-well plates or 96-well plates and divided into 4 groups (n=30 each) using a random number table method: control group (group C), OGD/R group (group O), GPR35 inhibitor plus OGD/R group (group GO) and GPR35 inhibitor group (group G). Astrocytes were subjected to 95% N2-5% CO2 in an incubator at 37℃ for 6 h followed by restoration of O2-glucose supply for 24 h. GPR35 inhibitor CID2745687 3 μmol/L was added at 1 h before OGD in GO and G groups.At 24 h after treatment in each group, apoptosis rate was calculated using Annexin V-FITC/GFAP/DAPI immunofluorescent staining, the concentration of calcium in cytoplasm was determined by Fluo3/AM fluorescence, the expression of hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) mRNA was detected by real-time polymerase chain reaction, and the expression of Bax and Bcl-2 was detected by Western blot. Results Compared with group C, the apoptosis rate of cortical astrocytes were significantly increased, the concentration of calcium in cytoplasm was increased, the expression of HCN2 mRNA, cAMP and Bax was up-regulated, and the expression of Bcl-2 was down-regulated in O and GO groups (P 0.05). Conclusion GPR35 activation is involved in the process of apoptosis in cortical astrocytes after OGD/R in rats. Key words: Receptors, G-protein-coupled; Hypoxia, brain; Astrocytes; Apoptosis

Hippocampal neuron-protective mechanism of hydrogen in a rat model of oxygen-glucose deprivation and restoration: promoting mitochondrial autophagy

Objective To evaluate the relationship between the hippocampal neuron-protective mechanism of hydrogen in a rat model of oxygen-glucose deprivation and restoration (OGD/R) and mitochondrial autophagy. Methods Hippocampal neurons isolated from healthy Sprague-Dawley rats (24 h after birth) were cultured in vitro, seeded in polylysine-coated 6-well plates at a density of 7×105 cells/well and then divided into 5 groups (n=30 each) using a random number table method: control group (C group), OGD/R group, OGD/R+ H2 group, OGD/R plus 3-methyladenine (3-MA) group (OGD/R+ 3-MA group), and OGD/R plus H2 plus 3-MA group (OGD/R+ H2+ 3-MA group). The cells were cultured for 24 h in normal culture atmosphere (75%N2-20%O2-5%CO2) in group C, and cells were subjected to oxygen-glucose deprivation for 2 h followed by O2-glucose supply for 24 h to establish the model of OGD/R injury in OGD/R, OGD/R+ H2, OGD/R+ 3-MA and OGD/R+ H2+ 3-MA groups.The cells were cultured for 24 h in a hydrogen-rich incubator (60% H2-10% O2-5% CO2-25% N2) after establishing the model in group OGD/R+ H2.Autophagy inhibitor 3-MA 10 mmol/L was added, and then cultured for 24 h in normal culture atmosphere after establishing the model in group OGD/R+ 3-MA.Autophagy inhibitor 3-MA 10 mmol/L was added, and then cultured for 24 h in hydrogen-rich incubator after establishing the model in group OGD/R+ H2+ 3-MA.The cell survival rate was measured using MTT assay.DCFH-DA fluorescent probe was applied for determination of reactive oxygen species (ROS) activity.The mitochondrial membrane potential was measured using a JC-10 assay kit.The neuronal apoptosis was detected by flow cytometry, and apoptosis rate was calculated.The expression of mitophagy-related protein microtubule-associated protein 1 light chain 3 (LC3), PINK1 and Parkin was determined by Western blot, and LC3Ⅱ/LC3Ⅰ ratio was calculated. Results Compared with group C, the cell survival rate and MMP were significantly decreased, the apoptosis rate and ROS activity were increased, and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were increased in OGD/R and OGD/R+ H2 groups (P<0.05). Compared with group OGD/R, the cell survival rate and MMP were significantly increased, the apoptosis rate and ROS activity were decreased, and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were increased in group OGD/R+ H2(P<0.05), and the cell survival rate and MMP were significantly decreased, the apoptosis rate and ROS activity were increased, and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were decreased in group OGD/R+ 3-MA (P<0.05). Compared with group OGD/R+ H2, the cell survival rate and MMP were significantly decreased, the apoptosis rate and ROS activity were increased, and the expression of PINK1 and Parkin and LC3Ⅱ/LC3Ⅰ ratio were decreased in OGD/R+ 3-MA and OGD/R+ H2+ 3-MA groups (P<0.05). Conclusion Hippocampal neuron-protective mechanism of hydrogen against OGDR injury is related to promoting mitochondrial autophagy in rats. Key words: Hydrogen; Hypoxia-ischemia, brain; Reperfusion injury; Hippocampus; Neurons; Mitochondria; Autophagy

Effect of docosahexaenoic acid on microglial activation during oxygen-glucose deprivation and restoration injury

Objective To evaluate the effect of docosahexaenoic acid (DHA) on microglial activation during oxygen-glucose deprivation and restoration (OGD/R) injury. Methods N9 microglia were inoculated in 96-well culture plates at a density of 104 cells/well for 3-5 days and divided into 3 groups (n=18 each) using a random number table method: control group (group C), group OGD/R and DHA+ OGD/R group. The cells were cultured for 24 h in an incubator at 37 ℃ (95% air-5% CO2) in group C. In OGD/R and DHA groups, the culture medium was replaced by glucose-free culture medium, the cells were cultured for 12 h in an incubator at 37 ℃ (95% N2-5% CO2), and then cells were returned to the normal culture medium and cultured for 24 h in an incubator at 37 ℃ (95% air-5% CO2) to establish the OGD/R injury model. The cells in group DHA were incubated with 25 μmol/L DHA at 12 h before OGD/R. The cell viability was detected using the methyl thiazolyl tetrazolium assay, the activity of lactate dehydrogenase (LDH) was measured by using chemical colorimetric method, activated microglia were counted by immunofluorescence staining, the expression of microglia activation marker iba-1 was detected by Western blot, and the concentrations of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and IL-4 and IL-10 in culture medium were determined using enzyme-linked immunosorbent assay. Results Compared with the group C, the cell viability was significantly decreased, the LDH activity was increased, the number of activated microglia was increased, and the expression of iba-1 was up-regulated in OGD/R and DHA+ OGD/R groups, the concentrations of TNF-α and IL-6 were significantly increased in group OGD/R, and the concentrations of TNF-α, IL-6, IL-4 and IL-10 were significantly increased in group DHA+ OGD/R (P<0.05). Compared with group OGD/R, the cell viability was significantly increased, the LDH activity was decreased, the number of activated microglia was decreased, the expression of iba-1 was down-regulated, TNF-α and IL-6 concentrations were decreased, and IL-4 and IL-10 concentrations were increased in group DHA (P<0.05). Conclusion The mechanism by which DHA reduces OGD/R injury may be related to inhibiting activation of microglia and to reducing inflammatory responses. Key words: Docosahexaenoic acids; Reperfusion injury; Microglia

Role of mitochondrial calcium uniporter in mitophagy in SH-SY5Y cells subjected to oxygen-glucose deprivation and restoration

Objective To evaluate the role of mitochondrial calcium uniporter (MCU) in mitophagy in SH-SY5Y cells subjected to oxygen-glucose deprivation and restoration (OGD/R). Methods SH-SY5Y cells were cultured in vitro, seeded in 96-well plates at a density of 2×105 cells/ml, and randomly divided into 4 groups (n=6 each) using a random number table method: control group (group C), group OGD/R, OGD/R plus MCU inhibitor group (group OGD/R + Ru360) and MCU inhibitor group (group Ru360). Cells were cultured in normal culture medium in group C. Cells were subjected to O2-glucose deprivation for 6 h followed by restoration of O2-glucose supply for 24 h in group OGD/R.In group OGD/R+ Ru360, Ru360 at a final concentration of 10 μmol/L was added at 30 min before O2-glucose deprivation, and the other treatments were similar to those previously described in group OGD/R.Ru360 was added at a final concentration of 10 μmol/L, and 30 min later cells were cultured under normoxic conditions in group Ru360.At 24 h of restoration of O2-glucose supply, cell counting kit-8 assay was used to detect the cell survival rate, JC-1 assay was used to detect mitochondrial membrane potential (MMP), the ultrastructure of cells was observed with a transmission electron microscope, and the expression of p62, Tom20 and Beclin-1 was detected by Western blot. Results Compared with group C, no significant change was found in each parameter in group Ru360 (P>0.05), the cell survival rate and MMP were significantly decreased, the expression of Tom20 and p62 was down-regulated, Beclin-1 expression was up-regulated (P<0.01), the mitochondria swelled, and mitochondrial autophagosomes were increased in group OGD/R.Compared with group OGD/R, the cell survival rate and MMP were significantly increased, the expression of Tom20 and p62 was up-regulated, Beclin-1 expression was down-regulated (P<0.01), the mitochondrial morphology kept well, and mitochondrial autophagosomes were decreased. Conclusion MCU is involved in the process of mitophagy in SH-SY5Y cells subjected to OGD/R. Key words: Mitochondria; Ion transport; Cell hypoxia; Autophagy

Effect of carbon monoxide postconditioning on pyroptosis induced by oxygen-glucose deprivation and restoration in rat hippocampal neurons: the relationship with mPTP/ROS signaling pathway

Objective To evaluate the effect of carbon monoxide(CO) postconditioning on pyroptosis induced by oxygen-glucose deprivation and restoration(OGD/R) in rat hippocampal neurons and the relationship with mitochondrial permeability transition pore(mPTP)/reactive oxygen species(ROS) signaling pathway. Methods Primary hippocampal neurons were cultured in vitro, seed in 6-well or 96-well plates, and divided into 5 groups(n=24 each) using a random number table method: control group(C group), OGD/R group, CO postconditioning group(CO group), specific mPTP opener atractyloside plus CO postconditioning group(ACO group), and specific ROS inducer antimycin A plus CO postconditioning group(KCO group). Neurons were subjected to O2-glucose deprivation(OGD) for 16 h followed by restoration of O2 -glucose supply for 24 h to establish the model of OGD/R injury. In group CO, neurons were exposed to 2% CO-5% CO2 for 3 h at 37 ℃ starting from the end of OGD, followed by normal culture for 21 h. In ACO and KCO groups, atractyloside 20 μmol/L and antimycin A 50 μmol/L were added at the end of OGD, respectively, and the other treatments were similar to those previously described in group CO. Neuronal pyroptosis rate was determined using double immunofluorescent staining cleaved caspase-1-AlexaFluor 568/DAPI after the end of treatments in each group. The neuronal survival rate was determined by MTT, opening of mPTP by Calcein-AM fluorescence, ROS content by DCFH-DA, and expression of interleukin-1beta(IL-1β) and IL-18 by Western blot. Results Compared with C group, neuronal pyroptosis rate, ROS content and opening of mPTP were significantly increased, the neuronal survival rate was decreased, and the expression of IL-1β and IL-18 was up-regulated in the other groups(P<0.05). Compared with OGD/R group, neuronal pyroptosis rate, ROS content and opening of mPTP were significantly decreased, the neuronal survival rate was increased, and the expression of IL-1β and IL-18 was down-regulated in CO, ACO and KCO groups(P<0.05). Compared with CO group, neuronal pyroptosis rate and ROS content were significantly increased, the neuronal survival rate was decreased, and the expression of IL-1β and IL-18 was up-regulated in ACO and KCO groups, and opening of mPTP was significantly inctreased in ACO group(P<0.05). Conclusion CO postconditioning can inhibit OGD/R-induced pyroptosis in rat hippocampal neurons, and the mechanism is related to inhibiting mPTP/ROS signaling pathway. Key words: Carbon monoxide; Neurons; Cell death; Reperfusion injury; Reactive oxygen species; Mitochondrial membrane transport proteins

Role of SIRT1/NF-κB signaling pathway in oxygen-glucose deprivation and restoration injury to hippocampal neurons of mice

Objective To evaluate the role of sirtuin 1 (SIRT1)/nuclear factor kappa B (NF-κB) signaling pathway in oxygen-glucose deprivation and restoration (OGD/R) injury to hippocampal neurons of mice. Methods The HT22 hippocampal neurons were seeded in a culture plate (96-well plate, 100 μl/well; 6-well plate, 2 ml/well) at the density of 5×104 cells/ml or in a culture dish (6 cm in diameter), and then divided into 4 groups (n=24 each) using a random number table method: control group (group C), OGD/R group (group OGD), SIRT1 inhibitor EX-527 preconditioning group (group EX) and SIRT1 agonist SRT1720 preconditioning group (group SRT). Neurons were cultured in normal culture atmosphere at 37 ℃ in group C. In OGD, EX and SRT groups, the culture medium was replaced with oxygen-poor fluid, and neurons were exposed to 5% CO2-95% N2 for 12 h in an incubator at 37 ℃, oxygen-poor fluid was replaced with the culture medium, and neurons were cultured for 24 h in normal culture atmosphere at 37 ℃.SIRT1 inhibitor EX-527 1 μmol/L and SIRT1 agonist SRT1720 10 μmol/L were added at 12 h before OGD in EX and SRT groups, respectively.The cell viability was measured by CCK8 assay, the activity of LDH was detected by chemical colorimetry, cell apoptosis rate was determined by flow cytometry, the expression of SIRT1, NF-κB, IκBα, Bcl-2 and Bax was detected by Western blot, and the Bcl-2/Bax ratio was calculated. Results Compared with group C, the cell viability was significantly decreased, LDH activity and cell apoptosis rate were increased, the expression of SIRT1, IκBα and Bcl-2 was down-regulated, the expression of NF-κB and Bax was up-regulated, and the Bcl-2/Bax ratio was decreased in OGD, EX and SRT groups (P<0.05). Compared with group OGD, the cell viability was significantly increased, the LDH activity and cell apoptosis rate were decreased, the expression of SIRT1, IκBα and Bcl-2 was up-regulated, the expression of NF-κB and Bax was down-regulated, and the Bcl-2/Bax ratio was increased in group SRT, and the cell viability was significantly decreased, LDH activity and cell apoptosis rate were increased, the expression of SIRT1, IκBα and Bcl-2 was down-regulated, the expression of NF-κB and Bax was up-regulated, and the Bcl-2/Bax ratio was decreased in group EX (P<0.05). Conclusion SIRT1/NF-κB signaling pathway inhibition is involved in OGD/R injury to hippocampal neurons of mice. Key words: SIRT1; NF-κB; Reperfusion injury; Cell hypoxia; Hippocampal neurons

Role of mito-KATP channels in sevoflurane postconditioning-induced inhibition of oxygen-glucose deprivation and restoration-induced pyroptosis in primary rat cardiomyocytes

Objective To evaluate the role of mitochondrial ATP-sensitive potassium (mito-KATP) channels in sevoflurane postconditioning-induced inhibition of oxygen-glucose deprivation and restoration (OGD/R)-induced pyroptosis in primary rat cardiomyocytes. Methods Cardiomyocytes of newborn Sprague-Dawley rats (<48 h after birth) were cultured in vitro and seeded in 6-well dishes (2 cm in diameter) or in 96-well plates.The cells were divided into 6 groups (n=15 each) using a random number table: control group (group C), OGD/R group (group O), sevoflurane postconditioning group (group Sev), sevoflurane postconditioning plus 5-hydroxydecanoate (5-HD) group (group SH), 5-HD group (group H) and OGD/R plus 5-HD group (group HO). The cardiomyocytes were subjected to oxygen-glucose deprivation for 4 h followed by restoration of oxygen-glucose supply for 24 h. After oxygen-glucose restoration, the cardiomyocytes in the culture media were exposed to 2% sevoflurane for 1 h to perform sevoflurane postconditioning.At 1 h before oxygen-glucose deprivation, a specific mito-KATP channel blocker 5-HD 100 μmol/L was added to the culture media.Cardiomyocytes were cultured in normal culture atmosphere in group C. Cardiomyocytes were collected at 24 h of oxygen-glucose restoration.Cell pyroptosis was detected by double flow cytometry AlexaFour488 (caspase-1 FLICA staining) and TMR red (DNA staining) staining.The pyroptosis rate was calculated.The cell survival rate was measured by methyl thiazolyl tetrazolium assay.The content of reactive oxygen species (ROS) in mitochondria was determined by 2′, 7′-dichlorofluorescin diacetate assay.The mitochondrial membrane potential (MMP) was measured by using JC-1 fluorescent probe.The expression of interleukin-1beta (IL-1β) was determined by Western blot. Results Compared with group C, the pyroptosis rate and ROS content were significantly increased, the cell survival rate and MMP were decreased, and the expression of IL-1β was up-regulated in group O (P<0.05). Compared with group O, the pyroptosis rate and ROS content were significantly decreased, the cell survival rate and MMP were increased, and the expression of IL-1β was down-regulated in group Sev (P<0.05). Compared with group Sev, the pyroptosis rate and ROS content were significantly increased, the cell survival rate and MMP were decreased, and the expression of IL-1β was up-regulated in group SH (P<0.05). Compared with group SH, the pyroptosis rate and ROS content were significantly increased, the cell survival rate and MMP were decreased, and the expression of IL-1β was up-regulated in group HO (P<0.05). Conclusion The mechanism by which sevoflurane postconditioning inhibits OGD/R-induced pyroptosis in primary rat cardiomyocytes is probably associated with increasing mito-KATP channel opening. Key words: Anesthetics, inhalation; Anoxia; Myocytes, cardiac; Cell death; Postconditioning

Mechanism underlying docosahexaenoic acid-induced regulation of angiopoietin expression in human brain vascular pericytes subjected to oxygen-glucose deprivation and restoration: the relationship with SSeCKS

Objective To evaluate the relationship between the mechanism underlying docosahexaenoic acid (DHA)-induced regulation of angiopoietin expression and Src-suppressed C kinase substrate (SSeCKS) in human brain vascular pericytes (HBVPs) subjected to oxygen-glucose deprivation and restoration (OGD/R). Methods HBVPs were seeded in 96-well or in 6-well plates at a density of 2×105 cells/ml and divided into 5 groups (n=18 each) using a random number table: control group (group C), OGD/R group, DHA group (group D), SSeCKS gene silencing group (group S) and SSeCKS gene silencing plus DHA group (group SD). The model of OGD/R injury was established as follows: the cells were subjected to O2-glucose deprivation for 24 h in glucose- and serum-free culture medium aerated with 94%N2-5%CO2-1%O2 followed by restoration of O2-glucose supply for 6 h in high-glucose DMEM culture medium in normal atmosphere.DHA was added at 1 h before hypoxia with the final concentration of 40 μmol/L in group D. Small interfering RNA induced SSeCKS gene silencing in S and SD groups.Subsequently, DHA with the final concentration of 40 μmol/L was added at 1 h before hypoxia in group SD.At 6 h of reoxygenation, the cell survival rate was determined by CCK-8 assay, the amount of LDH released was detected using ELISA, and the expression of SSeCKS, angiopoietin-1 (Ang-1) and Ang-2 was detected by Western blot. Results Compared with group C, the cell survival rate was significantly decreased, the amount of LDH released was increased, the expression of SSeCKS and Ang-1 was down-regulated, the expression of Ang-2 was up-regulated, and Ang-1/Ang-2 ratio was decreased in group OGD/R, and the expression of SSeCKS was down-regulated in group S (P 0.05). Compared with group D, the cell survival rate was significantly decreased, the amount of LDH released was increased, the expression of SSeCKS and Ang-1 was down-regulated, the expression of Ang-2 was up-regulated, and Ang-1/Ang-2 ratio was decreased in group SD (P<0.05). Conclusion The mechanism by which DHA increases the ratio of Ang-1/Ang-2 may be totally related to up-regulation of SSeCKS expression in HBVPs subjected to OGD/R. Key words: Docosahexaenoic acids; A kinase anchor proteins; Angiopoietins; Pericytes; Brain; Reperfusion injury

Relationship between TDAG8 and endogenous neuron-protective mechanism against oxygen-glucose deprivation and restoration-induced apoptosis in rat neurons

Objective To investigate the relationship between T-cell death-associated gene 8 (TDAG8) and endogenous neuron-protective mechanism against oxygen-glucose deprivation and restoration (OGD/R)-induced apoptosis in rat neurons. Methods The primary cortical neurons obtained from fetal rats were seeded in 6-well plates at a density of 1×105 cells/ml and divided into 5 groups using a random number table: control group (group C, n=24), group OGD/R (n=48), TDAG8 agonist BTB09089 group (group BTB, n=24), TDAG8-siRNA group (group siRNA, n=24), and blank vehicle group (group V, n=24). The medium was replaced with glucose- and serum-free Locke′s buffer, and the neurons were exposed to 95% N2-5% CO2 in an air-tight incubator at 37 ℃ for 60 min followed by routine culture to establish the model of OGD/R.In BTB, siRNA and V groups, 20 μmol/L TDAG8 agonist BTB09089, 200 pmol/L TDAG8-siRNA, and 6 μl/200 μl transfection reagent were added, respectively, at 24 h before oxygen-glucose restoration.At 6 h of oxygen-glucose restoration, the neuronal viability and amount of lactic dehydrogenase (LDH) released were measured, and the expression of TDAG8 and caspase-3 mRNA in neurons was detected by fluorescent quantitative real-time polymerase chain reaction.In group OGD/R, the expression of TDAG8 and caspase-3 was measured by Western blot at 0, 3, 6, 12 and 24 h of oxygen-glucose restoration.In C, OGD/R, BTB, siRNA and V groups, the expression of TDAG8, caspase-3 and p-Akt was detected at 6 h of oxygen-glucose restoration. Results In group OGD/R, the expression of TDAG8 was gradually up-regulated after oxygen-glucose restoration, and the expression of caspase-3 peaked at 6 h of oxygen-glucose restoration.Compared with group C, the neuronal viability was significantly decreased, the amount of LDH released was significantly increased, and the expression of TDAG8 and caspase-3 protein and mRNA and p-Akt was significantly up-regulated in OGD/R, V and siRNA groups (P 0.05). Conclusion TDAG8 is partially involved in the endogenous neuron-protective mechanism against OGD/R-induced apoptosis in rat neurons, which may be related to activation of Akt signaling pathway. Key words: Receptors, G-protein-coupled; Apoptosis; Neurons; Reperfusion injury

Role of ERK signaling pathway in inhibition of OGD/R-induced apoptosis in rat cortical neurons by sevoflurane: the relationship with mPTP

Objective To evaluate the role of extracellular signal-regulated kinase(ERK)signaling pathway in inhibition of oxygen-glucose deprivation and restoration(OGD/R)-induced apoptosis in rat cortical neurons by sevoflurane and the relationship with mitochondrial permeability transition pore(mPTP). Methods The rat cortical neurons were cultured in vitro and seeded in 6-well or 12-well culture plates.The neurons were randomly divided into 4 groups(n=18 each)using a random number table: control group(group C); OGD/R group(group O); sevoflurane group(group OS); sevoflurane+ ERK1/2 inhibitor PD98059 group(group OSP). The neurons were subjected to O2-glucose deprivation for 90 min followed by restoration of O2-glucose supply for 24 h in group O. The neurons were incubated with 2% sevoflurane for 2 h after OGD/R in group OS.OGD/R was performed at 1 h after ERK1/2 inhibitor PD98059 30 μmol/L was added, and the neurons were incubated with 2% sevoflurane for 2 h after OGD/R in group OSP.At 24 h of restoration of O2-glucose supply, the expression of phosphorylated ERK1/2(p-ERK1/2)in neurons was measured by Western blot, the neuronal apoptosis was detected using Annexin V-FITC/PI double staining combined with flow cytometry, and the opening of mPTP was determined through measuring the optical density at 540 nm.The apoptosis rate was calculated. Results Compared with group C, the expression of p-ERK1/2 in neurons was significantly up-regulated, and the apoptosis rate and mPTP opening were significantly increased in group O(P<0.05). Compared with group O, the expression of p-ERK1/2 in neurons was significantly up-regulated, and the apoptosis rate and mPTP opening were significantly decreased in group OS(P<0.05). Compared with group OS, the expression of p-ERK1/2 in neurons was significantly down-regulated, and the apoptosis rate and mPTP opening were significantly increased in group OSP(P<0.05). Conclusion The mechanism by which sevoflurane inhibits OGD/R-induced apoptosis in rat cortical neurons is related to inhibition of mPTP opening after activation of ERK signaling pathway. Key words: Extracellular signal-regulated MAP kinases; Mitochondrial membrane transport proteins; Anesthetics, inhalation; Neurons; Apoptosis

Efficacy of HET0016 for improving delayed mild hypothermia-induced protection of rat neurons subjected to oxygen-glucose deprivation and restoration in vitro

Objective To evaluate the efficacy of HET0016 for improving the protection of rat neurons subjected to oxygen-glucose deprivation and restoration (OGD/R) by delayed mild hypothermia. Methods The hippocampal neurons were isolated from healthy neonatal Sprague-Dawley rats and then cultured.The neurons were seeded in 6-well plates at a density of (2-3)×105 cells/ml, and randomly divided into 4 groups (n=12 each) using a random number table: control group (group C), group OGD, OGD/R-mild hypothermia group (group OGD/R-MH), and OGD/R-mild hypothermia-HET0016 group (group OGD/R-MH-HET). In group C, the neurons were cultured in the normal culture medium.In group OGD, the neurons were subjected to OGD for 2 h. In group OGD/R-MH, the neurons were subjected to OGD for 2 h followed by restoration of oxygen-glucose for 4 h, and then mild hypothermia was applied for 24 h. In group OGD/R-MH-HET, HET0016 with the final concentration of 1 μmol/L was added immediately after OGD (before restoration of oxygen-glucose), and the procedures else were same as those in group OGD/R-MH.The neurons were then continuously cultured for 24 h. After 24 h of culture, the viability of neurons was measured using methyl thiazolyl tetrazolium assay, and the survival rate was measured using PI/FDA double staining. Results Compared with group C, the viability of neurons was significantly weakened, and the survival rate was significantly decreased in group OGD (P<0.05). Compared with group OGD, the viability of neurons was significantly enhanced, and the survival rate was significantly increased in OGD/R-MH and OGD/R-MH-HET groups, especially in group OGD/R-MH-HET (P<0.05). Conclusion HET0016 improves delayed mild hypothermia-induced protection of isolated rat neurons subjected to OGD/R to some extent. Key words: Hypothermia, induced; Hypoxia-ischemia, brain; Infant, newborn; Neurons; HET0016

Effect of etomidate post-conditioning on mPTP in rat cortical neurons subjected to oxygen-glucose deprivation and restoration: the relationship with Robo receptors

Objective To evaluate the role of etomidate post-conditioning on mitochondrial permeability transition pore (mPTP) in the rat cortical neurons subjected to oxygen-glucose deprivation and restoration (OGD/R) and the relationship with Robo receptors. Methods The cortical neurons obtained from Sprague-Dawley rats (< 24 h after birth) were cultured in vitro and seeded in 6-well plates (2 ml/well). The neurons were divided into 4 groups (n=24 each) using a random number table: control group (group C), OGD/R group, etomidate post-conditioning group (group E), and etomidate post-conditioning + Robo receptor blocker group (group ER). The neurons were subjected to O2-glucose deprivation for 90 min followed by restoration of O2-glucose supply for 24 h. In E and ER groups, etomidate was added to the culture medium with the final concentration of 6 μmol/L immediately after onset of O2-glucose supply.In group ER, Robo blocker RoboN was added to the culture medium with the final concentration of 1 μg/ml at 6 h before O2-glucose deprivation.The neuronal apoptosis was detected using Hoechst/PI double staining, the viability of neurons was measured by MTT assay, and the amount of lactic dehydrogenase (LDH) released was measured using colorimetric method.The mitochondria were extracted, and mitochondrial permeability transition pore (mPTP) opening was detected. Results Compared with group C, the apoptosis rate, amount of LDH released, and mPTP opening were significantly increased, and the cell survival rate was decreased in OGD/R, E and ER groups (P<0.05). Compared with group OGD/R, the apoptosis rate, amount of LDH released, and mPTP opening were significantly decreased, and the cell survival rate was increased in group E, and the apoptosis and amount of LDH released were significantly decreased, and the cell survival rate was increased in group ER (P<0.05). Compared with group E, the apoptosis rate, amount of LDH released, and mPTP opening were significantly increased, and the cell survival rate was decreased in group ER (P<0.05). Conclusion Etomidate post-conditioning mitigates OGD/R-induced damage to the cortical neurons through activating Robo receptors and inhibiting mPTP opening in rats. Key words: Etomidate; Ischemic postconditioning; Cell hypoxia; Oxygen; Cerebral cortex; Neurons; Mitochondrial membrane transport proteins; Nerve tissue proteins; Receptors, immunologic

Role of mitochondrial permeability transition pore in sevoflurane-induced inhibition of apoptosis induced by oxygen-glucose deprivation and restoration in rat neurons

Objective To evaluate the role of mitochondrial permeability transition pore (mPTP) in sevoflurane-induced inhibition of apoptosis induced by oxygen-glucose deprivation and restoration (OGD/R) in rat neurons. Methods Cortical neurons isolated from neonatal Sprague-Dawley rats born within 24 h, were cultured primarily and seeded in 6-well plates (2 ml/well) at a density of 1×106 cells/ml.The neurons were randomly divided into 5 groups (n=72 each) using a random number table: control group (group C), OGD/R group (group O), mPTP opener group (group A), mPTP opener + sevoflurane group (group AS), and sevoflurane group (group Sev). The cells were cultured in normal culture medium in group C, and the cells were subjected to OGD for 90 min followed by restoration of O2-glucose supply for 24 h in the other groups.In A and AS groups, mPTP opener atractyloside 20 μmol/L was added immediately after OGD.In Sev and AS groups, the cells were post-conditioned with 2% sevoflurane for 1 h immediately after OGD.The neurons were collected at 24 h of OGD in O and A groups, or at 23 h of OGD in Sev and AS groups.Annexin Ⅴ-FITC/PI double staining was performed to count apoptotic neurons, apoptotic rate was calculated, and cell survival rate was measured using MTT assay.The mitochondrial membrane potential (MMP) was measured by using JC-1 fluorescence probe as indicator.The opening of mPTP was determined through assessing the changes of mitochondrial optical density (ΔOD540 of mPTP). The expression of pro-apoptotic factors Bid, Bim, Puma protein and mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction and Western blot. Results Compared with group C, apoptotic rate was significantly increased, cell survival rate and MMP were decreased, the opening of mPTP was increased, and the expression of Bid, Bim, Puma protein and mRNA was up-regulated in the other groups.Compared with group O, apoptotic rate was significantly decreased, cell survival rate and MMP were increased, the opening of mPTP was decreased, and the expression of Bid, Bim, Puma protein and mRNA was down-regulated in Sev group.Compared with group Sev, apoptotic rate was significantly increased, cell survival rate and MMP were decreased, the opening of mPTP was increased, and the expression of Bid, Bim, Puma protein and mRNA was up-regulated in A and As groups.Compared with group A, apoptotic rate was significantly decreased, cell survival rate and MMP were increased, the opening of mPTP was decreased, and the expression of Bid, Bim, Puma protein and mRNA was down-regulated in group AS. Conclusion Sevoflurane mitigates OGD/R-induced apoptosis in rat neurons through inhibiting mPTP opening. Key words: Mitochondrial membrane transport proteins; Anesthetics, inhalation; Reperfusion injury; Neurons; Apoptosis

The effects of exogenous hydrogen sulfide on injury of rat hippocampus neurons induced by oxygen-glucose deprivation and restoration

Objective To investigate the effects of exogenous hydrogen sulfide (H2S) on injury of rat hippocampus neurons induced by oxygen-glucose deprivation and restoration (OGD/R) and explore its mechanism.Methods Hippocampus neurons were isolated from embryonic day 16-18 (E16-18) rat embryos.Hippocampus was immediately removed and digested with 0.25％ trypsin.The neurons were isolated and cultured at 37 ℃ for 7 days and neuron-specific enolase (NSE) was detected by immunohistochemical staining method to identify neurons.At 8th day,the neurons were placed in deoxygenated glucose-free medium and exposed to 95％ N2-5％ CO2 in an air tight chamber for 1 hour,and then replaced the glucose-free medium with original medium,and returned the cultures to a standard incubator in 5％ CO2 at 37 ℃ and incubated for another 24 h.The neurons were divided into 3 groups:group Ⅰ control; group Ⅱ OGD/R,and group ⅢOGD + NaHS,the latter was further divided into 5 subgroups:groups Ⅲ1-5 with 25,50,100,200,400 μmol/L NaHS added,respectively.Then cell viability was quantified by MTT method,the level of lactate dehydrogenase (LDH) were detected,apoptosis was measured by Annexin V FITC/PI Apoptosis Kits,and RT-PCR was used to assay HIF-1 α mRNA in neurons in all groups.Results Compared with control group,the LDH level,apoptosis and expression of HIF-1α mRNA in group Ⅱ were significantly increased,the cell viability was significantly decreased (P ＜ 0.01).There were no significant differences in the LDH level,apoptosis and expression of HIF-1 α mRNA and the cell viability between group Ⅱ and group Ⅲ1 (P ＞ 0.05).Compared with group Ⅱ,the LDH level,apoptosis and expression of HIF-1α mRNA in group Ⅲ2-4 were significantly increased,the cell viability was significantly increased (P ＜ 0.01).Compared with group Ⅱ,the LDH level,apoptosis and expression of HIF-1 α mRNA in the group Ⅲ 5 were significantly decreased,the cell viability was significantly decreased (P ＜ 0.01).Conclusions H2S of low concentration has no significant effects on injury of rat hippocampus neurons induced by OGD/R.H2S of moderate doses can protect rat hippocampus neurons from OGD/R injury and H2S of high concentration can aggravate injury.The expression of HIF-1α mRNA in rat hippocampus neurons was increased after OGD/R,and the protective role of H2S is associated with increase in the expression of HIF-1α mRNA. Key words: Hydrogen sulfide; Oxygen-glucose deprivation and recovery ; Hippocampal neurons ; Apoptosis; Hypoxia-induced factor-1α

Role of mTOR in reduction of oxygen-glucose deprivation and restoration-induced injury to isolated cardiomyocytes by hypoxic preconditioning in neonatal rats

Objective To evaluate the role of mammalian target of rapamycin （mTOR ） in reduction of oxygen-glucose deprivation and restoration （OGD/R ）-induced injury to isolated cardiomyocytes by hypoxic preconditioning （HPC） in neonatal rats .Methods Primary cardiomyocytes were obtained from neonatal Sprague-Dawley rats （1-3 days after birth ） ,and seeded in 96-well plates at the density of 1 × 105 cells/ml （200μl/well ） . The cells were randomly divided into 6 groups （ n=6 each ） using a random number table：control group （group C） ,rapamycin group （group R） ,group OGD/R ,rapamycin＋OGD/R group （group R＋OGD/R）;group HPC＋OGD/R ,and rapamycin＋HPC＋OGD/R group （group R＋HPC＋OGD/R） .Rapamycin （final concentration 20 nmol/L） was added to the culture media and the cells were then incubated for 1 h in group R .In R＋OGD/R group ,rapamycin （final concentration 20 nmol/L ） was added to the culture media ,the cells were then incubated for 1 h ,and then OGD/R was performed .In HPC＋OGD/R group ,OGD/R was performed immediately after HPC was completed .In R＋HPC＋OGD/R group ,rapamycin （final concentration 20 nmol/L） was added to the culture media ,the cells were then incubated for 1 h , and then HPC and OGD/R were performed . All the cells were subjected to OGD for 2 h followed by restoration of O2-glucose supply for 3 h to induce OGD/R .HPC was induced by 3 cycles of 10 min hypoxia followed by 30 min reoxygenation .The cell viability （using MTT assay ） and lactic＆nbsp;dehydrogenase （LDH） activity in the culture medium were determined .Results Compared with group C ,the cell viability was significantly decreased , and LDH activities in the culture medium were increased in OGD/R , R＋OGD/R ,HPC＋OGD/R and R＋ HPC＋OGD/R groups （ P0.05） .The cell viability was significantly higher ,and LDH activity in the culture medium were lower in HPC＋OGD/R group than in OGD/R group ,and in R＋HPC＋OGD/R group than in HPC ＋ OGD/R group （ P〈 0.05 ） .Conclusion mTOR is involved in HPC-induced attenuation of OGD/R injury to isolated cardiomyocytes in neonatal rats . Key words: Anoxia; Ischemic preconditioning; Myocardial reperfusion injury; Mammalian target of rapamycin