Ganoderma lucidum extract protects rat cerebral cortical neurons from hypoxia/reoxygenation injury in vitro

AIM: To investigate the neuroprotective effect of Ganoderma lucidum extract (GLE) in an in vitro model of primary cultured neurons with oxygen and glucose deprivation (OGD). METHODS: Neuronal injury was induced by oxygen and glucose deprivation/reoxygenation (OGD/R). The neuronal injury and viability were determined by LDH leakage and XTT assay at 0 h,3 h,6 h,12 h,24 h,48 h and 72 h after OGD/R. Neuronal apoptosis was detected by flow cytometry (FCM). The expression of apoptosis-related proteins was analyzed by Western blotting.RESULTS: The viability of the neurons increased with exposure to GLE (0.1 mg/L,1 mg/L and 10 mg/L)after OGD/R. The LDH releases were also significantly reduced. GLE significantly inhibited OGD/R-induced apoptosis of cultured rat cortical neurons in a concentration-dependent and time-dependent manner(P<0.05). GLE at concentrations of 0.1 mg/L,1 mg/L and 10 mg/L inhibited the expression of caspase-3 and caspase-8 proenzyme. Additionally,GLE at concentration of 10 mg/L suppressed the expression of caspase-9 proenzyme.CONCLUSION: Our findings provide the evidence that the GLE has neuroprotective effect on cerebral ischemia. The mechanisms are related to the inhibition of caspase-3,-8 and-9 activations. GLE may be a novel and effective reagent for treating ischemic stroke.     

Protective effect of hydrogen sulfide on hypoxia-induced injury of rat cortical neurons

AIM:To explore the role of hydrogen sulfide (H 2S) in cortial neuronal injury induced by hypoxia.METHODS:The SD rat cortical neurons were cultured in hypoxic conditions (2% O 2, 5% CO 2 and 93% N 2 at 37 °C) to establish the hypoxic model. Sodium hydrosulfide (NaHS) was used as the donor of H 2S and neuronal viability was detected by CCK-8 assay. Neuronal content of reactive oxygen species (ROS) was determined by DCFH-DA method, and mitochondrial membrane potential (MMP) was detected using Rh123 staining. Lactate dehydrogenase (LDH) release rate was measured by a commercial kit to reflect the degree of neuronal injury. RESULTS:Hypoxic treatment increased ROS content and the release rate of LDH in the neurons. However, NaHS pretreatment significantly inhibited the hypoxia-induced increases in ROS content and LDH release. Hypoxia decreased MMP and cell viability. Pretreatment with NaHS and N-acetyl-L-cysteine (NAC), a ROS scavenger, significantly inhibited the decreases in MMP and viability of the neurons. CONCLUSION:Hypoxia induces ROS generation in the neurons, thereby decreases MMP and neuronal viability. H 2S significantly attenuates hypoxia-induced neuronal injury by its antioxygenation.     

Effects of Tribulus terrestris L. saponin on apoptosis and changes in cytosolic calcium induced by hypoxia/reoxygenation in rat cortical neurons

AIM: To study the effect of Tribulus terrestris L. saponin (TTLS) on apoptosis and changes in cytosolic calcium concentration induced by hypoxia/re-oxygenation in rat cortical neurons. METHODS: Rat cortical neurons in primary culture were used, and a apoptosis model was induced by hypoxia/reoxygenation. LDH releasing rate was detected by spectrophotometry. The apoptosis rate of cortical neurons was analyzed quantitatively by flow cytometry with Annexin V-FITC and PI staining. Intracellular free Ca2+(［Ca2+］i) was observed with a confocal laser-scanning microscope and determined by mean fluorescent value with Fluo-3 fluometry. RESULTS: Compared to control group, three hours of hypoxia and twelve hours of reoxygenation group induced cortical neuronal apoptosis and significantly increased the intracellular free Ca2+ concentration（P<0.01). Compared with model group, TTLS decreased the percentage of neuronal apoptosis and reduced neuronal ［Ca2+］i（P<0.01).CONCLUSION: TTLS could obviously reduce hypoxia/reoxygenation-induced apoptosis and alleviate the damage degree of rat cortical neurons.The mechanism might be related to inhibiting the calcium overload induced by hypoxia/reoxygenation in rat cortical neurons.     

Effects of Homer1a over-expression on apoptosis and AMPK protein expression in mechanically injured neurons

AIM:To investigate the effects of Homer1a over-expression on the apoptosis and AMP-activated protein kinase (AMPK) protein expression in mechanically injured neurons. METHODS:The rat cortical neurons were isolated and cultured in vitro, and then ramdomly divided into control group, model group, empty vector group, and Exp-Homer1a group. Neuron models with mechanical injury were constructed and infected with the Homer1a over-expression vector. The mRNA expression of Homer1a was detected by qPCR. The cell viability in each group was detected by MTT assay. The activity of lactate dehydrogenase (LDH) in the supernatant of each group was measured by LDH test kit. The apoptosis level was analyzed by flow cytometry. The protein levels of Hormer1a, cleaved caspase-3, Bax, Bcl-2, p-AMPKα and AMPKα were determined by Western blot. RESULTS:Compared with control group, the viability of mechanically injured neurons was significantly decreased, the LDH activity in the supernatant and neuronal apoptotic rate were significantly increased (P<0.05), and Homer1a expression at mRNA and protein levels was significantly increased (P<0.05). Compared with model group, the LDH activity in the supernatant and neuronal apoptotic rate in Exp-Homer1a group were significantly decreased, the protein levels of cleaved caspase-3 and Bax were significantly decreased (P<0.05), and the protein levels of Bcl-2 and p-AMPKα were significantly increased (P<0.05). CONCLUSION:Over-expression of Homer1a may increase the viability of mechanically injured neurons and inhibit their apoptosis by promoting the activation of AMPKα phosphorylation.     

Shikonin inhibits neuronal apoptosis induced by OGD through targeting PI3K/Akt signaling pathway

AIM: To explore the effect of shikonin on rat primary cortical neurons in oxygen-glucose deprivation (OGD)-induced injury model.METHODS: The neurons were pretreated with shikonin at different concentrations (0.02, 0.2, 2 and 20 μmol/L) followed by treatment with OGD. Lactate dehydrogenase (LDH) release assay and fluorescein diacetate/propidium iodide (FDA/PI) double staining were used to detect neuronal viability and apoptosis, and then the optimal concentration of shikonin was determined. LY294002 (PI3K/Akt signaling pathway inhibitor, 1 μmol/L) was added before the addition of shikonin, and the protein level of p-Akt (Ser473) in the neurons was determined by Wes-tern blot. LDH release assay and FDA/PI double staining were also used to detect neuronal viability and apoptosis.RESULTS: A certain concentration (0.2~20 μmol/L) of shikonin increased the viability of impaired neurons (P<0.05) and the protein level of p-Akt (Ser473) in the neurons (P<0.05). The effect of shikonin on neuronal p-Akt (Ser473) levels and the cell death were blocked by LY294002 (P<0.05).CONCLUSION: A certain concentration of shikonin reduces OGD-induced apoptosis of rat primary cortical neurons by activating PI3K/Akt signaling pathway.     

Protective effect of procyanidin single active ingredient B2 on human endothelial progenitor cells under high glucose stimulation

AIM: To study the protective effect of procyanidin single active ingredient B2(PC-B2) on human endothelial progenitor cells(EPCs) stimulated with high glucose. METHODS: The human EPCs were isolated from peripheral blood of healthy people and identified. The EPCs were divided into control group(PBS treatment), hypertonic control group(25 mmol/L mannitol treatment), high glucose(30 mmol/L) group, and different concentrations(2, 10 and 50 mg/L) of PC-B2+30 mmol/L glucose groups. The viability of EPCs was detected by CCK-8 assay. The levels of LDH, MDA, SOD and GSH in the EPCs were detected. The changes of NO, ET-1, ICAM-1 and VCAM-1 in the EPCs cultured medium were measured by ELISA. The cell apoptotic rate and reactive oxygen species(ROS) in the EPCs were analyzed by flow cytometry. The expression of VEGF and VEGFR-2 in the EPCs were determined by Western blot. RESULTS: Compared with control group, the viability of human EPCs was decreased significantly in 30 mmol/L glucose group(P<0.05). The LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were induced significantly(P<0.05), but SOD and GSH activity and NO production were decreased significantly(P<0.05). The ROS and cell apoptotic rate were increased significantly(P<0.05). The expression of VEGF and VEGFR-2 in the EPCs were decreased(P<0.05). When human EPCs were treated with different concentrations of PC-B2 and 30 mmol/L glucose, the viability was obviously rebounded(P<0.05), the LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were decreased gradually(P<0.05), the SOD, GSH activity and NO production were increased significantly(P<0.05), the ROS and cell apoptotic rate were decreased significantly(P<0.05), and the expression of VEGF and VEGFR-2 in the EPCs was increased gradually(P<0.05).CONCLUSION: PC-B2 enhances the viability of human EPCs under high glucose condition, reduces high glucose-induced oxidative damage, restores the EPCs normal function, and reduces the releases of inflammatory cytokines and apoptosis, thus playing a protective effect on human EPCs through inducing the expression of VEGF and VEGFR-2.     

Protective effect of progesterone on PC12 cells injured by oxygen-glucose deprivation

AIM: To investigate the effects of progesterone on the cell viability and expression of glucose transporter type 3(GLUT3) in PC12 cells injured by oxygen-glucose deprivation (OGD) in attempt to prove the neuroprotection of progesterone (PROG) against the hypoxic-ischemic injury in cultured cells in vitro. METHODS: Well-differentiated PC12 cells induced by nerve growth factor were randomly divided into 3 groups. In normal group, the cells were cultured without OGD treatment. In OGD group, the culture medium was replaced by glucose-free medium and the cells were transferred to a humidified incubation chamber flushed by a gas mixture of 95% N₂ and 5% CO₂ for 30 min. After that, the cells were fed with glucose-supplemented medium and cultured under normoxic condition for 24 h. In PROG+OGD group, the cells were given the same treatments as those in OGD group except that the medium contained progesterone at concentration of 10 nmol/L. Cellular morphological changes were observed after OGD for 30 min. The cell viability was assessed by WST-8 assay. The degree of the cell damage was evaluated by determining lactate dehydrogenase (LDH) leakage. The expression of GLUT3 at mRNA and protein levels was examined by RT-PCR and Western blotting, respectively. RESULTS: Progesterone attenuated the cellular swelling, decreased the leakage of LDH and improved the viability of PC12 cells injured by OGD (P<0.01). The expression of GLUT3 at mRNA and protein levels in PC12 cells in PROG+OGD group was significantly higher than that in OGD group (P<0.05). CONCLUSION: Progesterone has protective effect on in vitro cultured PC12 cells injured by OGD. The mechanism may be related to the up-regulation of GLUT3 protein.     

Antagonistic action of Jiunaoning injection aganist oxygen/glucose- deprived and reperfusion injury-induced cultured rat cortical neurons

AIM: To determine whether Jiunaoning injection has protective effects on oxygen/glucose-deprived and reperfusion injury-induced neurons. METHODS: Various concentrations of Jiunaoning injection (0.5-5 mL/L) were used to observe their effects on cultured rat cortical neurons induced by oxygen/glucose-deprived and reperfusion injury in various time points. The neuronal metabolic rate and viability were assessed by using 3-(4,5-dimethylthiazol)-2, 5-diphenyl-tetra zoliumbromide (MTT) and lactate dehydrogenase (LDH) assay. RESULTS: Jiunaoning injection enhanced the neuronal metabolic rate in a dose-dependent manner in the range from 0.5 to 5 mL/L, and Jiunaoning injection (1.5-2.5 mL/L) enhanced the neuronal metabolic rate, decreased the cell death rate and depressed LDH leak rate significantly. CONCLUSION: Jiunaoning injection has an affirmative protective effect on oxygen/glucose-deprived and reperfusion-induced neuronal injury.     

17β-estradiol protects cortical neurons from propofol-induced apoptosis by activating PI3K-Akt signaling pathway

AIM: To investigate the protective effects and the mechanisms of 17β-estradiol on the propofol-induced neuroapoptosis in primary cultured cortical neurons. METHODS: The neurons were cultured for 7 d and treated with different concentrations of propofol and/or 17β-estradiol, respectively. The neuron viability, neuroapoptosis and the protein level of p-Akt was determined by MTT assay, Hoechst 33258 staining and Western blot 12 h after different treatments, respectively. RESULTS: Compared with vehicle-control group, propfol inhibited neuron viability in a dose-dependent manner (P<0.05). Compared with propofol treatment group, 17β-estradiol increased the neuron viability in a dose-dependent manner (P<0.05), and IGF increased the neuron viability greatly (P<0.01). Compared with vehicle-control group, the number of apoptotic neurons which was significantly decreased by treatment of 17β-estradiol was markedly increased by propofol (P<0.01). Compared with the 17β-estradiol+propofol group, LY294002 increased the number of apoptotic neurons (P<0.01). Compared with vehicle-control group, propfol decreased the protein level of p-Akt in a dose-dependent manner (P<0.05). Compared with propofol treatment group, 17β-estradiol increased the protein level of p-Akt in a dose-dependent manner (P<0.05). Compared with 17β-estradiol+propofol group, LY294002 significantly decreased the protein level of p-Akt (P<0.01). CONCLUSION: 17β-estradiol exerts the neuroprotective effects against propofol-induced neuroapoptosis by activating the PI3K-Akt signaling pathway.     

Over-expression of PGC-1α reverses mitochondrial function reduction and apoptosis in OGD/R-induced neurons

AIM: To investigate the effect of over-expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) on mitochondrial morphology and cell apoptosis in the cortical neurons with oxygen glucose deprivation/reoxygenation (OGD/R). METHODS: The whole gene sequence of PGC-1α was obtained from the cerebral cortex of C57BL/6 mice by RT-PCR and cloned into the eukaryotic expression vector pEGFP-N1. The pEGFP-N1-PGC-1α was identified by PCR, and transfected into cortical neurons. The level of PGC-1α expression was identified by Western blot. The cortical neurons transfected with pEGFP-N1 and pEGFP-N1-PGC-1α vectors were treated with OGD/R. The mitochondrial mass, reactive oxygen species (ROS) and ATP production, cell apoptosis and changes of cleaved caspase-3 were detected by MitoTracker Red staining, flow cytometry, ATP metabolic assay kit and TUNEL. RESULTS: Over-expression of PGC-1α inhibited the decrease in mitochondrial biogenesis capacity and the ROS formation of OGD/R neurons (P<0.05), enhanced the ability of ATP synthesis (P<0.01), inhibited neuronal apoptosis (P<0.01) and decreased the activation of caspase-3 (P<0.01). CONCLUSION: PGC-1α over-expression inhibits neuronal apoptosis with OGD/R treatment by promoting mitochondrial biogenesis, inhibiting the production of ROS and maintaining mitochondrial function. PGC-1α may be used as a target for the development of cerebral ischemia/reperfusion injury drugs.     

Significance and change of SCF/KIT following cerebral ischemia reperfusion

AIM: To study the expressions and significance of stem cell factor (SCF) and tyrosine kinase receptor (KIT) in neurons after cerebral ischemia reperfusion injury. METHODS: Western blotting was used to examine the expression of SCF and KIT in the cortex after cerebral ischemia and oxygen glucose deprivation (OGD) neurons, then the OGD neurons preincubated with SCF and SCF-Ab were assessed the cell viability by MTT to determine its significance. RESULTS: ①Compared to control group, the expressions of M-SCF and S-SCF in cortex increased markedly while KIT decreased significantly in ipsilateral hemisphere. However, no obvious change in the contraleteral hemisphere was observed. ②Compared to control group, the levels of S-SCF and M-SCF were increased significantly in the OGD neurons while KIT was not obvious changed. ③The cell viability of OGD neurons decreased significantly. Preincubation with SCF inhibited cell damage while SCF-Ab deteriorated OGD injury. CONCLUSION: The expression of SCF may be induced by cerebral ischemia reperfusion, which can protect the neurons from ischemia injury.     

Protective effects of adenosine on cultured rat hippocampal neurons after oxygen-glucose deprivation

AIM:To investigate the protective effects of adenosine on cultured rat hippocampal neurons after oxygen-glucose deprivation.METHODS:The control and adenosine-treated hippocampal neurons cultured for 12 d were exposed to oxygen-glucose deprivation environment for 0.5-4 h and then cultured with original medium in normoxia for 24 h. The soma area, survival rate, effluxes of lactate dehydrogenase (LDH)and apoptosis of neurons were observed.RESULTS:The soma area, effluxes of lactate dehydrogenase from neurons and apoptosis were increased while survival rate of neurons was decreased after oxygen-glucose deprivation compared with those pre-oxygen-glucose deprivation. Compared with the control, after oxygen-glucose deprivation the soma area, effluxes of lactate dehydrogenase from neurons and apoptosis were decreased, however, the survival rate of neurons was increased in the adenosine group.CONCLUSION:Oxygen-glucose deprivation can lead to the severe damage of cultured hippocampal neurons, and adenosine can reduce neuronal injury induced by oxygen-glucose deprivation.     

Genipin inhibits oxidative stress and apoptotic damage in high glucose-induced H9c2 cardiomyocytes

AIM:To explore the effects of genipin (GEN) on high glucose (HG)-induced oxidative stress injury and apoptosis in H9c2 cardiomyocytes.METHODS:H9c2 cells were cultured in vitro and HG-induced injury model was established. H9c2 cells were divided into 4 groups:normal control (NC) group (glucose at 5.6 mmol/L), HG group (glucose at 50 mmol/L), NG+GEN group and HG+GEN group. The concentration of genipin was used at 10 μmol/L. The viability of the H9c2 cells was measured by CCK-8 assay. The intracellular malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined by enzyme labeling and WST-1 methods, respectively. The activity of lactate dehydrogenase (LDH) in the cell culture supernatant was detected by microplate method. Fluorescent probe DCF was used to detect intracellular levels of reactive oxygen species (ROS). Nucleosome fragments was measured to evaluate cell apoptosis by ELISA. The intracellular mitochondrial membrane potential was detected by JC-1 method. The protein levels of Mn-SOD, cytochrome C (Cyt C), Bax and cleaved caspase-3 were determined by Western blot. RESULTS:Compared with HG group, the cell viability in HG+GEN group was increased significantly (P<0.05), the levels of MDA and LDH were decreased (P<0.05), SOD activity was increased (P<0.05), the levels of ROS and nucleosome fragments in HG+GEN group were decreased (P<0.05), and the mitochondrial membranes potential was notably increased (P<0.05). Compared with NG group, the activation of Mn-SOD was decreased, but the protein levels of Cyt C, Bax and cleaved caspase-3 were increased in HG group (P<0.05). Compared with HG group, the activation of Mn-SOD was increased, and the protein levels of Cyt C, Bax and cleaved caspase-3 were decreased in HG+GEN group (P<0.05).CONCLUSION:Genipin protects HG-induced H9c2 cardiomyocytes against oxidative stress injury and apoptosis.     

miR-422a regulates SOX6 to inhibit injury of PC12 cells induced by hydrogen peroxide

AIM: To investigate the effects of microRNA-422a (miR-422a) on the damage of rat adrenal gland pheochromocytoma PC12 cells induced by hydrogen peroxide (H₂O₂). METHODS: The expression of miR-422a in the PC12 cells treated with H₂O₂ was detected by real-time PCR. After miR-422a mimics were transfected into PC12 cells, the cell viability was measured by MTT assay, the lactate dehydrogenase (LDH) leakage rate was detected, and the apoptosis was analyzed by flow cytometry. Target gene prediction software was used to predict that sex-determining region Y box 6 (SOX6) may be the target gene of miR-422a. Luciferase reporter assay was used to identify the targeting relationship. miR-422a mimics and SOX6 over-expression vector were co-transfected into the PC12 cells. The effects of SOX6 over-expression on the viability, LDH leakage rate and apoptosis of PC12 cells treated with H₂O₂ and transfected with miR-422a mimics were evaluated. RESULTS: The expression of miR-422a in the PC12 cells was decreased after treatment with H₂O₂ (P<0.05). The viability of PC12 cells treated with H₂O₂ was decreased, and the LDH leakage rate and apoptotic rate were increased. Transfection with miR-422a mimics enhanced the viability of PC12 cells treated with H₂O₂, and the leakage rate of LDH and apoptotic rate of the PC12 cells were reduced. The expression of SOX6 was negatively regulated by miR-422a. SOX6 over-expression reversed the effects of miR-422a on PC12 cell viability, LDH leakage and apoptosis. CONCLUSION: miR-422a reduces the damage of PC12 cells induced by H₂O₂ via targeting SOX6.     

Angiotensin 1-7 attenuates angiotensin Ⅱ-induced human glomerular endothelial cell injury via Mas receptor

AIM:To investigate the effect of angiotensin 1-7 (Ang1-7) on the human glomerular endothelial cells (HGECs) injury induced by angiotensin Ⅱ (Ang Ⅱ) and its possible mechanism. METHODS:Cultured HGECs were divided into 6 groups randomly:control group, Ang Ⅱ group, Ang1-7 group, Ang Ⅱ +Ang1-7 group, Ang Ⅱ +Ang1-7+A779 (an inhibitor of Mas receptor) group and A779 group. The apoptotic rate and reactive oxygen species (ROS) of HGECs were analyzed by flow cytometry and photographed by fluorescence microscopy. The levels of lactate dehydrogenase (LDH), nitric oxide (NO), endothelin-1 (ET-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) in the supernatant of cell cultures were measured. RESULTS:Compared with the control group, the apoptotic rate and the average fluorescence intensity of ROS were increased in the Ang Ⅱ group, IL-6, TNF-α, TGF-β, ICAM-1 and MCP-1 in cell supernatants were also increased in the Ang Ⅱ group (P<0.05). Compared with the Ang Ⅱ group, the apoptotic rate, ROS level, and the above inflammatory factors were decreased in Ang Ⅱ +Ang1-7 group (P<0.05). Compared with the Ang Ⅱ +Ang1-7 group, adding A779 increased the cell apoptotic rate, ROS production and the releases of the above inflammatory factors in cell supernatants (P<0.05). Compared with the Ang Ⅱ group, adding Ang1-7 inhibited the LDH leakage, ET-1 secretion and promoted the release of NO in a dose-dependent manner (P<0.05). CONCLUSION:Ang1-7 attenuates the HGECs injury induced by Ang Ⅱ by inhibiting the Mas receptor.     

Effect of nicotine against apoptosis of rat cortical neurons induced by colchicines

AIM:To explore the mechanism of nicotine against the apoptosis induced by colchicines in rat cortical neurons. METHODS:Cortical neurons were cultured from newborn Sprague-Dawley (SD) rats (less than 12 h). The rate of apoptosis was measured by Hoechst33258 fluorescence staining in the neurons, and the activity of Akt473 was analyzed by assay kit Akt473. RESULTS:The apoptosis of cortical neurons can be induced by 0.1 μmol/L colchicine. The phosphorlation of Akt 473 decreased significantly (1/3 times of the control group, P<0.01). However, when cortical neurons pretreated with 10 μmol/L nicotine for 2 h were cultured with 0.1 μmol/L colchicine for 24 h, the rate of apoptosis decreased from 62% to 38%. The phosphorlation of Akt473 increased significantly in a bell-shape time-dependent manner, which was respectively 1.3, 3.7, 2.4, 2.1 and 1.9 times compared with the control group (P<0.01). CONCLUSION:By activating the signal pathway of Akt473, nicotine may attenuate the apoptosis of cortical neurons induced by colchicines.     

Effects of intermittent high glucose on apoptosis and PTEN expression in islet cells

AIM: To investigate whether the increase in PTEN expression is related to apoptosis, and whether it is regulated by reactive oxygen species(ROS). METHODS: The rat islet cells were divided into constant low glucose group (group L), constant high glucose group (group H), glucose fluctuation group (group F), low glucose after high glucose group (group HL) and low glucose after fluctuation group (group FL). The ROS level, apoptotic rate, intracellular calcium, insulin release and PTEN protein expression were analyzed. RESULTS: Compared with groups H and L, the insulin secretion decreased, and intracellular calcium, ROS level, PTEN protein expression and apoptotic rate increased in group F (P<0.05). Compared with group H, the intracellular calcium, ROS level, PTEN protein expression and apoptotic rate in group HL decreased, but were still higher than those in group L (P<0.05). Compared with group F, the intracellular calcium, ROS level, PTEN protein expression and apoptotic rate in group FL decreased, but were still higher than those in group L (P<0.05). CONCLUSION: Glucose fluctuation can cause the apoptosis of islet cells more easily than constant high glucose. This may be related to the change of intracellular calcium and increase in oxidative stress which promotes PTEN expression. The recovery of glucose level to some extent relieves oxidative stress, decrease PTEN expression and reduce cell damage.     

Effect of cyclooxygenase 2 on hippocampal neuronal damage induced by aluminum in rats

AIM:To study the relationship between cyclooxygenase 2(COX-2) and the damage of hippocampal neurons by aluminum overload in rats. METHODS:Newborn SD rats(less than 24 h) were used to establish the model of primary culture of hippocampal neurons. The neurons were treated with aluminum at concentration of 200 μmol/L. The techniques of RNA interference(RNAi) and cell transfection were used to study the role of COX-2 in hippocampal neuron. Following RNAi by cell transfection, Western blotting analysis was used to determine the protein expression of COX-2. Cell growth was assayed by the method of MTT. The pathological changes of the neurons were observed by fluorescence labeling. The activity of superoxide dismutase(SOD) and lactate dehydrogenase(LDH), and the content of malondialdehyde(MDA) were detected for evaluating cell damage. RESULTS:COX-2 RNAi by cell transfection significantly decreased the protein expression of COX-2 without changing the neuronal pathomorphology, cell viability, SOD activity and MDA content. However, it obviously improved livability and SOD activity of the hippocampal neurons, which were aluminum-overloaded. Inhibition of COX-2 expression also reduced the leakage of LDH and the content of MDA, and ameliorated the pathological changes in neurons. CONCLUSION:Moderate silence of COX-2 expression not only significantly affects the morphological changes and physiological functions of hippocampal neurons, but also prevents the neurons from aluminum-induced damages.     

Protective role and mechanism of peroxysome proliferator activated receptor-γ in injury of cultured rat cortical neurons induced by hypoxia/ reoxygenation

AIM: To observe the role of peroxysome proliferator activated receptor-γ (PPAR-γ) and the relationship of cyclooxygenase-2 (COX-2) and PPAR-γ in injury of cultured rat cortical neurons induced by hypoxia/reoxygenation. METHODS: Primary rat cortical neurons were cultured. Experiments include control group, hypoxia/ reoxygenation group and hypoxia/ reoxygenation with PPAR-γ agonist group. Cell viability was surveyed by MTT assay. COX-2 protein expression was measured by Western blotting.RESULTS: Neuron viability raised dramatically in hypoxia/reoxygenation with PPAR-γ agonist group, compared with hypoxia/reoxygenation group (P<0.05). The COX-2 protein expression in hypoxia/ reoxygenation with PPAR-γ agonist group decreased significantly compared with hypoxia/ reoxygenation group (P<0.05). CONCLUSION: PPAR-γ agonist inhibits the expression of COX-2 and reduces obviously cortical neuron injury induced by hypoxia/ reoxygenation. It may protect cortical neurons by down-regulating the expression of COX-2.     

Effect of progesterone on SH-SY5Y cells injured by ATP

AIM: To investigate the neuroprotective effect of progesterone against adenosine triphosphate (ATP)-injured human neuroblastoma SH-SY5Y cells.METHODS: The SH-SY5Y cells in the logarithmic phase were divided into different groups according to the progesterone and ATP concentrations. The cell viability was measured by CCK-8 assay. The membrane permeability was detected using fluorescent dye YO-PRO-1. Cytosolic Ca²⁺ concentration was measured with fluorescent dye Fluo-3/AM. The expression of purinergic P2X₇ receptor was assessed by Western blot.RESULTS: The viability of the SH-SY5Y cells was significantly decreased (P<0.05) and YO-PRO-1 uptake was obviously increased (P<0.05) in a concentration-dependent manner compared with control group when SH-SY5Y cells were treated with ATP at 1, 3, 5 and 7 mmol/L for 2 h. The viability reduction of the SH-SY5Y cells induced by ATP was obviously counteracted by treatment with progesterone at 3, 10 and 30 nmol/L for 30 min (P<0.05) as compared with ATP group. YO-PRO-1 fluorescence enhancement induced by ATP in SH-SY5Y cells was significantly reduced (P<0.05) by progesterone (30 nmol/L) or P2X₇ receptor antagonist KN-62 (500 nmol/L) pretreatment for 30 min, and no obvious difference between treatments with progesterone and KN-62 was observed. Cytosolic Ca²⁺ fluorescence intensity in normal group was a little, but that in ATP group was increased (P<0.05). Progesterone or KN-62 pretreatment significantly decreased the cytosolic fluorescence intensity of Ca²⁺ induced by ATP (P<0.05). However, no obvious difference between treatments with progesterone and KN-62 was found. The expression of P2X₇ receptor in ATP group was significantly higher than that in control group (P<0.05), and progesterone inhibited ATP-induced P2X₇ receptor expression (P<0.05).CONCLUSION: Progesterone inhibits P2X₇ receptor expression, membrane pore formation, intracellular Ca²⁺ increase and cell death induced by ATP, so progesterone may protect SH-SY5Y cells against ATP-induced injuries.