Effect of asiaticoside on OGD/R induced injury of H9C2 cardiomyocytes based on PI3K/Akt/Beclin-1 signaling pathway / 药学学报

In order to investigate the effects of asiaticoside (Ass) on H9C2 cardiomyocytes, the present study examined the potential intervention of Ass on the proliferation and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/Bcl-2 homology domain protein (Beclin-1) signaling pathway in H9C2 cardiomyocytes following oxygen and glucose deprivation/reperfusion (OGD/R) injury. H9C2 cardiomyocytes were selected as the research objects, and the activity of H9C2 was detected by cell counting kit-8 (CCK-8). H9C2 cells were divided into control group, OGD/R group, Ass low concentration group (10 μmol·L-1), Ass high concentration group (80 μmol·L-1) and Ass high concentration + chloroquine group (80 μmol·L-1 + 50 μmol·L-1). The control group was cultured under normal conditions, and the other groups were treated with oxygen and glucose deprivation for 4 h and reperfusion for 2 h. The activity and content of aspartic aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK) in the supernatant of H9C2 cardiomyocytes were detected by enzyme-linked immunosorbent assay. Autophagy staining assay kit with monodansylcadaverine (MDC) method to observe cellular autophagy; molecular docking technique to identify the molecular targets of Ass. Immunofluorescence was used to observe the effect of the drug on cell number. The expression levels of PI3K, Akt, selective autophagy adaptor protein (P62) and Beclin-1 were detected by Western blot. Compared with OGD/R group, Ass group had a protective effect from 10-80 μmol·L-1, and the activities and contents of AST, LDH and CK were decreased. The protein expression levels of PI3K, Akt, P62 and Beclin-1 were decreased. Compared with the administration group, the activities and contents of AST, LDH and CK in Ass high-concentration + chloroquine group were significantly decreased, and the protein expression levels of PI3K, Akt, Beclin-1 and P62 were significantly decreased. Immunofluorescence showed that the inhibitor group and each administration group had different degrees of protective effect compared with the model group. Asiaticoside can reduce the injury of H9C2 cardiomyocyte induced by OGD/R, reduce the content of AST, LDH and CK, reduce the expression level of P62 protein, and reduce autophagy, which may be closely related to the inhibition of PI3K/Akt/Beclin-1 signaling pathway activation.

Mechanism of Gastrodia elata active ingredient reducing oxygen-glucose deprivation/reoxygenation injury in rat neuron / 中国药房

OBJECTIVE To study the improvement effect and mechanism of Gastrodia elata active ingredient 3，4- dihydroxybenzaldehyde （3，4-DD） on oxygen-glucose deprivation/reoxygenation（OGD/R） injury in rat primary brain microvascular endothelial cells （BMECs）-rat adrenal chromaffin cells PC12 co-culture system. METHODS The co-culture model of BMECs and PC12 cells was replicated in the Transwell chamber， and divided into control group， model group， butylphthalide group （positive control group， 0.1 mmol/L） and 3，4-DD group （0.1 μmol/L）. OGD/R injury model of the co-culture system was induced in those groups except for the control group. After preventively intervention in BMECs with relevant medicine or culture medium for 24 h， cell transendothelial electronic resistance （TEER） value， lactate dehydrogenase （LDH） activity， brain-derived neurotrophic factor （BDNF） level and mRNA expressions of TrkB， Plc-γ， Map-2， GAP-43 in PC12 cells was detected. RESULTS Compared with the control group， TEER of the co-culture model， LDH activity and BDNF level of PC12 cells were decreased significantly in the model group （P＜0.01）， while mRNA expressions of TrkB， Plc-γ， Map-2 and GAP-43 in PC12 cells were increased significantly （P＜0.01）. Compared with the model group， TEER of the co-culture model， LDH activity， BDNF level， and the mRNA expressions of TrkB， Plc-γ， Map-2 and GAP-43 in PC12 cells were increased significantly in the 3，4-DD group and butylphthalide group （P＜0.05 or P＜0.01）. CONCLUSIONS 3，4-DD can relieve the damage of neuronal OGD/R by acting on BMECs， the mechanism of which may be associated with activating the BDNF/TrkB signaling pathway.

Stigmasterol protects human brain microvessel endothelial cells against ischemia-reperfusion injury through suppressing EPHA2 phosphorylation / 中国天然药物

Stigmasterol is a plant sterol with anti-apoptotic, anti-oxidative and anti-inflammatory effect through multiple mechanisms. In this study, we further assessed whether it exerts protective effect on human brain microvessel endothelial cells (HBMECs) against ischemia-reperfusion injury and explored the underlying mechanisms. HBMECs were used to establish an in vitro oxygen and glucose deprivation/reperfusion (OGD/R) model, while a middle cerebral artery occlusion (MCAO) model of rats were constructed. The interaction between stigmasterol and EPHA2 was detected by surface plasmon resonance (SPR) and cellular thermal shift assay (CETSA). The results showed that 10 μmol·L-1 stigmasterol significantly protected cell viability, alleviated the loss of tight junction proteins and attenuated the blood-brain barrier (BBB) damage induced by OGD/R in thein vitro model. Subsequent molecular docking showed that stigmasterol might interact with EPHA2 at multiple sites, including T692, a critical gatekeep residue of this receptor. Exogenous ephrin-A1 (an EPHA2 ligand) exacerbated OGD/R-induced EPHA2 phosphorylation at S897, facilitated ZO-1/claudin-5 loss, and promoted BBB leakage in vitro, which were significantly attenuated after stigmasterol treatment. The rat MCAO model confirmed these protective effects in vivo. In summary, these findings suggest that stigmasterol protects HBMECs against ischemia-reperfusion injury by maintaining cell viability, reducing the loss of tight junction proteins, and attenuating the BBB damage. These protective effects are at least meditated by its interaction with EPHA2 and inhibitory effect on EPHA2 phosphorylation.

Mitophagy mediated by ligustilide relieves OGD/R-induced injury in HT22 cells / 中国中药杂志

Mitochondrion, as the main energy-supply organelle, is the key target region that determines neuronal survival and death during ischemia. When an ischemic stroke occurs, timely removal of damaged mitochondria is very important for improving mitochondrial function and repairing nerve damage. This study investigated the effect of ligustilide(LIG), an active ingredient of Chinese medicine, on mitochondrial function and mitophagy based on the oxygen and glucose deprivation/reperfusion(OGD/R)-induced injury model in HT22 cells. By OGD/R-induced injury model was induced in vitro, HT22 cells were pre-treated with LIG for 3 h, and the cell viability was detected by the CCK-8 assay. Immunofluorescence and flow cytometry were used to detect indicators related to mitochondrial function, such as mitochondrial membrane potential, calcium overload, and reactive oxygen species(ROS). Western blot was used to detect the expression of dynamin-related protein 1(Drp1, mitochondrial fission protein) and cleaved caspase-3(apoptotic protein). Immunofluorescence was used to observe the co-localization of the translocase of outer mitochondrial membrane 20(TOMM20, mitochondrial marker) and lysosome-associated membrane protein 2(LAMP2, autophagy marker). The results showed that LIG increased the cell viability of HT22 cells as compared with the conditions in the model group. Furthermore, LIG also inhibited the ROS release, calcium overload, and the decrease in mitochondrial membrane potential in HT22 cells after OGD/R-induced injury, facilitated Drp1 expression, and promoted the co-localization of TOMM20 and LAMP2. The findings indicate that LIG can improve the mitochondrial function after OGD/R-induced injury and promote mitophagy. When mitophagy inhibitor mdivi-1 was administered, the expression of apoptotic protein increased, suggesting that the neuroprotective effect of LIG may be related to the promotion of mitophagy.

Study on Protective Effects of Drug-contained Serum of Xiaoxuming Decoction on Astrocyte of Oxygen and Glucose Deprivation Model Rats / 中国药房

OBJECTIVE：To investigate the protective effects of drug-contained serum of Xiaoxuming decoction （XXM）on astrocyte of oxygen and glucose deprivation model rats ，and to explore its mechanisms. METHODS ：The astrocytes of rats were randomly divided into control group ，model group and XXM low-dose ，middle-dose，high-dose groups. The cells in the control group were not treated ；after 2.5 h of OGD ，model group and XXM low-dose ，middle-dose，high-dose groups were reoxygenated for 0，3，6，12 h in 0（i.e. the model group was not added with drugs ），2.5％，5％，10％ of XXM ，respectively. The content of lactate dehydrogenase （LDH）was detected by colorimetry. The reactive oxygen species （ROS）level was detected by fluorescence probe method ，and the expression of Manganese superoxide dismutase （MnSOD）was determined by immunofluorescence double staining method in control group ，model group and XXM high-dose group after 12 h of reoxygenation following OGD. RESULTS ： The content of LDH in the control group was always kept at a low level ；LDH content in the model group gradually increased from （110.99±17.06）U/L to （436.64±55.29）U/L after 0-12 h of reoxygenation following OGD ，which was significantly higher than that in the control group （P＜0.05）. Compared with model group at the same time point after reoxygenation following OGD ，the contents of LDH in the cells of XXM low-dose ，medium-dose and high-dose groups were decreased to different extents ，and showed a time-and dose-dependent trend. The contents of LDH in XXM groups at 6 and 12 h after reoxygenation following OGD were significantly lower than that of the model group （P＜0.05）. At 12 h after reoxygenation following OGD ，the levels of ROS in model group were significantly higher than control group ， while the level of MnSOD was significantly lower than control group（P＜0.05）. The level of ROS in XXM high-dose group hospital.sh.cn was significantly lower than model group ，while the level of MnSOD was significantly higher than model group （P＜0.05）.. CONCLUSIONS：XXM can protect astrocyte by up-regulating sh.cn levels of MnSOD ，scavenging excessive oxygen free radicals ， to relieve the OGD induced astrocytic injury ，with protective effect.

Effect of silencing beclinl gene on apoptosis of HT22 cells after oxygen and glucose deprivation/reoxygenation / 解剖学报

Objective To investigate the effect of Beclinl gene on apoptosis of HT22 of mouse hippocampus neuron treated with oxygen and glucose deprivation/reoxygenation ( OGD/R). Methods HT22 cells in logarithmic growth phase were randomly divided into 4 groups; normal, model, beclin1 -/-and control. All groups were reoxygenated after 6 hours of oxygen and glucose deprivation except for normal group. The beclinl interference sequence was designed for mouse cDNA sequence using RNAi technology, and was transfected into HT22 cells by liposome Lipo2000. The transfection efficiency was observed under fluorescence microscope and Western blotting after 48 hours of transfection. Cell viability was detected by CCK-8 method. Cell damage was detected by lactate dehydrogenase(LDH) method , Bax and Bcl-2 were detected by immunofluorescence staining and the expression of LC3, P62 and Caspase-3 were tested by Western blotting after 24 hours of reoxygenation. SPSS 19. 0 statistical software was used for data analysis. Results Compared with the normal group, the cell viability and P62 expression decreased significantly (P<0. 01) , the LDH leakage rate, LC3 II/LC3 I , Caspase-3 expression and Bax/Bcl-2 increased significantly in model group (P<0. 01). Compared with the model group, the cell viability and LC3 II /LC3 I decreased significantly (P<0. 01) , the LDH leakage rate, Bax/Bcl-2,P62 and Caspase-3 expression increased significantly in beclin1-/-group (P<0.01). There was no difference between the control group and the model group. Conclusion Silencing beclinl inhibites autophagy, which aggravates the damage of OGD/R HT22 cells and further increases apoptosis.

Effect of ligustilide on oxygen and glucose deprivation/reperfusion-induced mitochondria fission in PC12 cells / 中国中药杂志

This study aimed to investigate the effect and mechanism of ligustilide, the main active ingredient in Ligusticum wallichii, on mitochondria fission after PC12 cell injury induced by oxygen and glucose deprivation/reperfusion(OGD/R). In the experiment, an OGD/R model was established in vitro, and PC12 cells were pre-treated with ligustilide for 3 h, and then the cell viability was detected by CCK-8 method. The effect of different concentrations of ligustilide on the morphology of PC12 cells after OGD/R injury was observed under an inverted microscope. Transmission electron microscopy was used to observe the mitochondrial fission of PC12 cells after OGD/R injury. DCFH-DA immunofluorescence staining method was used to detect intracellular reactive oxygen species(ROS) changes. Changes in mitochondria membrane potential(MMP) were detected by flow cytometry. Hochest 33258 was used to observe the apoptosis of PC12 cells. Western blot was used to detect changes in cytochrome C(Cyt C) content in mitochondria and cytoplasm, and mitochondrial fission-related proteins Drp 1 and Fis 1. All results showed that compared with the model group, ligustilide significantly increased the survival rate of PC12 cells and the number of cells. Further experiments showed that ligustilide inhibited the release of ROS and decline of mitochondrial membrane potential in PC12 cells after OGD/R injury. Moreover, ligustilide reduced the release of Cyt C and promoted the expressions of Drp1 and Fis1 in mitochondrial fission proteins. Verification experiments showed that mitochondrial fission inhibitor mdivi-1 decreased cell survival rate and inhibited fission. The results indicated that ligustilide exerted neuro-protective effects by promoting mitochondrial fission and reducing cell damage. It preliminary proves that the mechanism of ligustilide on ischemic brain injury may be related to the promotion of mitochondrial fission and the maintenance of cell homeostasis.

Astragaloside IV inhibits apoptosis of HT22 after oxygen and glucose deprivation/reoxygenation by regulating autophagy / 中国药理学通报

Aim: To investigate the effect of astragaloside IV on apoptosis of HT22 after oxygen and glucose deprivation/reoxygenation by regulating autophagy. Methods: HT22 cells were randomly divided into control, model, DMSO, AS-IV, AS-IV + N, AS-IV + 3-MA, 3-MA and Rapa group. Except for control group, cells in other groups were reoxygenated after 6 h of oxygen and glucose deprivation. Invertded microscope was employed to observe cell morphology. CCK-8 method was used to test cell survival rate. LDH method was applied to detect cell damage, and Bax, Bcl-2 immunofluorescence was used to detect apoptosis. Results: Compared with control, the synapses of cell bodies decreased, the cells shrank, the number of intercellular connections decreased, cell viability was significantly reduced, and LDH leakage and Bax/Bcl-2 significantly increased in model group (P <0. 01). Compared with model group, cell viability in AS-IV group and Rapa group markedly increased, and LDH leakage and Bax/ Bcl-2 significantly decreased (P < 0. 01); cell viability was significantly down-regulated, and Bax/Bcl-2 was markedly up-regulated in 3-MA group (P < 0. 01). Astragaloside IV +3-MA group had no significant difference compared with model group. Conclusion: Astragaloside IV protects OGD/R HT22 by inhibiting apoptosis via activating autophagy.

Effects of Xuesaitong Injection on expression of Lingo-1 in SH-SY5Y cells after oxygen and glucose deprivation / 中草药

Objective: To establish an oxygen and glucose deprivation model (OGD) of SH-SY5Y cells, and investigate the effects of Xuesaitong Injection on the cell survival, apoptosis rate of SH-SY5Y cells and the mRNA and protein expressions of Lingo-1. Methods: The model was established using 1640 sugar-free medium and three-gas incubator. The cell survival rate was determined by CCK8 method to determine the optimal time for hypoxia and the optimal concentration of Xuesaitong Injection. The apoptosis rate of SH-SY5Y cells was detected by Annexin V-FITC/PI double staining. The mRNA and protein expressions of Lingo-1 were determined by qRT-PCR and Western blotting, respectively, and the effect of Xuesaitong on Lingo-1 expression was finally determined. Results: In this study, the optimal hypoxia time for the establishment of the OGD model of SH-SY5Y cells was 16 h and the optimum concentration of Xuesaitong Injection was 640 mg/L. The apoptosis rate of SH-SY5Y cells was significantly reduced, and the expression levels of lingo-1 mRNA and protein were decreased in the Xuesaitong group compared with the model group under the condition of this concentration. Conclusion: The apoptosis rate was significantly increased and Lingo-1 was highly expressed when SH-SY5Y cells were damaged by oxygen and glucose deprivation. Xuesaitong can significantly reduce the apoptosis of SH-SY5Y cells induced by oxygen and glucose deprivation, and inhibit the high expression of lingo-1, which has the anti-apoptosis and significant neuroprotective effect.

Effects of puerarin on the expressions of CaM, CaMKⅡ, MECP2, BDNF and Akt in oxygen and glucose deprivation cell models / 西安交通大学学报(医学版)

Objective: To observe the effects of puerarin on the expressions of CaM, CaMKⅡ, BDNF and Akt in vascular dementia cell models induced by oxygen and glucose deprivation (OGD). Methods: The passaged well-differentiated PC12 cells were randomly divided into control group, model group, and low-dose, medium-dose and high-dose intervention groups. Vascular dementia cell model was established by OGD. Suitable OGD time and concentration of puererin were obtained from the cell viability measured by MTT assay. The release of LDH was measured to assess the extent of cell damage and identify cell models. The expressions of CaM, CaMKⅡ, MECP2, BDNF and Akt were detected by Western blot. Results: PC12 cells with OGD prolonged viability decreased in a time-dependent manner, with increased concentrations of puerarin increased in a concentration-dependent manner. Effective intervention of puerarin was 0.1-10 μmol/L and optimal time of OGD was 6 h. Compared with control group, the release of LDH in model group was significantly increased (P0.05). Puerarin could down-regulate the level of CaM protein, increase the expressions of MECP2 and BDNF and the phosphorylation of CaMKⅡ, and also increase the phosphorylation of Akt in addition to the low-dose group (P<0.05). Conclusion: The neuroprotective effect of puerarin may be related to the increase of the autophosphorylation of CaMKⅡ mediated by Ca2+-CaM complex, induce the phosphorylation of MECP2, up-regulate the expression of BDNF and activate the PI3K-Akt pathway to inhibit the expression of apoptotic genes and proteins.

Inhibitory effect of calyeosin on apoptosis of PC12 cells under oxygen and glucose deprivation/reoxygenation / 中国药理学通报

Aim To study the effect of calycosin on ap-optosis of PC 12 cells under oxygen and glucose deprivation/reoxygenation. Methods PC 12 cells in logarithmic phase were randomly divided into four groups: Normal control group, model group, calycosin group (0.07 (xmol • L"1) and nimodipine group (5.00 mi-cromol • L"1, positive control group). CCK-8 assay was used to detect cell survival rate; flow cytometry and TUNEL staining were used to detect apoptotic rate and apoptotic index; immunofluorescence staining was used to detect Bax/Bcl-2 ratio; Western blot was used to detect the expression of caspase-3 apoptotic protein. Results Compared with control group, the cell survival rate significantly declined (P <0. 05) , the apoptotic rate and apoptotic index significantly rose (P < 0. 05), and Bax/Bcl-2 ratio and caspase-3 protein ex-pression were significantly up-regulated (P < 0. 05) ; compared with model group, the cell survival rate significantly increased in calycosin group and nimodipine group (P < 0. 05) , the mortality and apoptotic index significantly decreased (P <0. 05) , the Bax/Bcl-2 ratio and caspase-3 protein expression significantly decreased (P <0. 05) , and the difference was statistically significant. Conclusions Calycosin can significantly improve the survival rate of oxygen-glucose depriva-tion/reoxygenation PC 12 cells and inhibit cell apopto-sis. Its mechanism is related to the regulation of expression of apoptotic proteins Bax, Bcl-2 and caspase-3 by calycosin.

Optimization for extraction of an oil recipe consisting of white pepper, long pepper, cinnamon, saffron, and myrrh by supercritical carbon dioxide and the protective effects against oxygen-glucose deprivation in PC12 cells

ABSTRACT This study is to investigate the most efficient extractives of extracting oil recipe for stroke treatment and the protective effects on an oxygen and glucose deprivation model in PC12 cells. An orthogonal experimental design L9 (34) was carried out for oil recipe's optimization with supercritical CO2 fluid extraction. 2-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and enzyme-linked immunosorbent assay were conducted to evaluate cell activity and indexes in the cell lysate. The result showed that the optimum extraction condition was 30 Mpa, 50 ºC, 100 min, the extracts were analyzed by gas chromatography-mass spectrometry and among forty detected compounds 27 were identified, representing 80.86% of the total oil content. trans-Cinnamaldehyde (14.14%), piperine (9.32%), β-amyrin (6.79%), lupenone (6.28%), longifolene (6.07%), β-caryophyllene (5.21%), α-bisabolol (4.11%), and β-bisabolene (2.56%) were high mass fraction. Oil recipe could significantly attenuate PC12 cell damage, the lactate dehydrogenase release and decreased the malondialdehyde levels, glutathione peroxidase and nicotinamide adenine dinucleotide phosphate oxidase activity, glutathione and nitric oxide content (p < 0.01) and increased the level of superoxide dismutase after oxygen and glucose deprivation. The protective mechanism may be related to oil recipe's antioxidant effect by scavenging free radicals.

Protective effects of high-density lipoprotein on mice cardiomyocytes induced by oxygen and glucose deprivation through Akt signaling pathway / 中华危重病急救医学

Objective To investigate the protective effect of high-density lipoprotein (HDL) on the mice cardiac myocytes induced by oxygen and glucose deprivation (OGD).Methods Cardiac cells of primary scavenger receptor-B1 knockout mice (SR-B1-/-) and normal C57 mice (SR-B1+/+) were obtained by protease digestion and differential adhesion method. ① The two kinds of cells were divided into normal control group (Con group), OGD group, OGD+HDL group. Propidium iodide (PI) staining were used to determine the necrosis of cardiac myocytes. ② SR-B1+/+cardiac cells were divided into Con group, OGD group, OGD+HDL group, and phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) inhibitor LY294002 group. PI staining were used to determine the necrosis of cardiac myocytes. TUNEL staining was used to determine the cell apoptosis. The kit was used to determine the contents of MB isoenzyme of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) in the culture medium supernatant. The expressions of SR-B1 and Akt protein were determined by Western Blot.Results ① In SR-B1+/+ cardiomyocytes, HDL could inhibit cell necrosis induced by OGD. There was no protective effect of HDL on OGD in the SR-B1-/- cardiomyocytes.② The study of SR-B1+/+ cells was showed that compared with Con group, necrotic cells were significantly increased and cell activity were significantly decreased, the cell viability were significantly decreased, the contents of LDH and CK-MB in supernatant were significantly increased, the expressions of phosphorylated Akt (p-Akt) and SR-B1 were significantly decreased in OGD group. Compared with OGD group, the number of necrotic cells in the OGD+HDL group was significantly decreased [PI positive cells rate: (26.71±5.94)％ vs. (64.24±18.34)％], the cell activity was significantly increased [(63.84±6.95)％ vs. (26.71±5.13)％], the contents of LDH and CK-MB in supernatant were significantly decreased [LDH (U/L): 896.3±161.5 vs. 1568.3±243.5, CK-MB (U/L): 304.3±72.9 vs. 583.6±81.6], the expressions of p-Akt and SR-B1 were significantly increased (p-Akt/t-Akt: 0.84±0.13 vs. 0.18±0.06, SR-B1/β-actin: 1.23±0.19 vs. 0.09±0.02), with statistically significant differences (allP < 0.05). Compared with OGD+HDL group, necrotic cells in LY294002 group were increased, cell activity was decreased, LDH and CK-MB contents in supernatant were increased, p-Akt and SR-B1 expressions were decreased; there was no statistical difference between LY294002 group and OGD group. There was no significant difference in cell apoptosis among the 4 groups.Conclusions HDL has protective effect on the mice myocardial cells. The mechanism may be related with the up regulation of the expression of SR-B1 protein by the activation of PI3K/Akt pathway.

Preconditioning of ulinastatin alleviates GES-1 cell injury induced by oxygen and glucose deprivation / 实用医学杂志

Objective To observe the effects of the preconditioning of ulinastatin on GES-1 cell injury induced by oxygen and glucose deprivation (OGD). Methods GES-1 cells were cultured in vitro and divided into three groups: normal control group (group N), oxygen and glucose deprivation group (group O), and ulinastatin preconditioning group (group U). The OGD model of GES-1 cells were established by glucose-free medium and three-gas incubator for 6h. Ulinastatin was added to group U 12h before the deprivation of oxygen and glucose. The cell viability and apoptosis were determined by cck-8 and flow cytometry respectively. Western Blot was used to examine the protein expression of Caspase-3 and Cleaved Caspase-3. The TRPV1 mRNA expression was measured by quantitative real-time PCR. Results As compared with group N, the viability of GES-1 was decreased, the apoptotic rate and the expression of Caspase-3 and Cleaved Caspase-3 were increased, and the TRPV1 mRNA expression decreased greatly in group O (P < 0.05). As compared with group O, the aforementioned changes were significantly inhibited in group U. Conclusions Ulinastatin preconditioning could effectively inhibit GES-1 cell injury induced by OGD, which may be related to the inhibition of apoptosis and the upregulation of TRPV1 mRNA expression.

Effect of resveratrol on oxidative stress of rat primary cortical neurons during different time windows of oxygen-glucose deprivation/reperfusion injury / 第二军医大学学报

Objective To investigate the effect of resveratrol on oxidative stress of rat primary cortical neurons during different time windows of oxygen-glucose deprivation/reperfusion (0GD/RP) injury. Methods Rat cortical neurons were cultured under oxygen and glucose deprivation for 150 min and returned to normal culture for 24 h. The experiment was divided into 6 groups, including the normal group, model group, pre-treatment group (treated with resveratrol for 24 h prior to OGD), OGD-treatment group (treated with resveratrol during 150 min of 0GD and 24 h of reperfusion), post-treatment group(treated with resveratrol during 24 h of reperfusion), and thewhole processing group (treated with resveratrol for 24 h prior to 0GD, during 150 min of OGD, and 24 h ofreperfusion). Invert microscope was used to observe cell morphology. Chemical colorimetry was used to detect the activity of superoxide dismutase (SOD) and the content of nitric oxide (NO). Immunofluorescencewas used to detect the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Western blotting analysis was used reveal the the proteinexpressions of Nrf2, heme oxygenase-1 (HO-1) and NADP (H); quinone oxidoreductase-1 (NQO-1). Results The stereoscopic effect and refraction ofneurons were enhanced at the sixth day of culture. Compared with the model group, resveratrol of various concentrations (10, 20, 40, 60 and 80 μmol/L) significantly elevated the activity of SOD and decreased N0 content in the whole processing group (P<0.05) with the effect being the besttranslocation of Nrf2 from the cytoplasm into the nuclei and upregulated Nrf2, HO-1 and NQO-1 protein expression at all stages of OGD/RP injury, with thebest effect found in the whole processing group, followed by the pre-treatment group. Conclusion Resveratrol has a dose-dependent anti-oxidative stress effect on rat cortical neurons during OGD/RP injury, with the best effect seen in the whole processing group, followed by the pre-treatment group. The mechanism might be associated with activation of Nrf2/antioxidant response element (ARE) signaling pathway and the sbusequent upregulation of antioxidant protein expression.

Dynamic signal pathway changes of signal transducer and activator of transcription factor-3 in hippocampal neurons induced by oxygen and glucose deprivation in vitro / 中华创伤杂志

Objective To observe the phosphorylation level and nuclear translocation of signal transducer and activator of transcription factor-3 (STAT3) in hippocampal neurons induced by oxygen and glucose deprivation in vitro and discuss the dynamic changes of STAT3 signal pathway in an in vitro cell model of brain hypoxia and ischemia.Methods Hippocampal neurons from newly born SD rats (within 24 hours from birth) were cultured with DMEM/F12 for nine days,and then were transferred to oxygen and glucose deprivation environment for four hours to establish experimental cell models.The distribution of phosphorylated STAT3 (p-STAT3) in the hippocampal neurons in different groups was observed under laser scanning confocal microscope after immunofluorescence staining.Expression intensity of p-STAT3 at different time points after oxygen and glucose deprivation in the hippocampal neurons was detected by Western blotting.Results Expression of p-STAT3 was unobvious in the nucleus of the control group,but it was observed in the nucleus of the model group one hour after modeling,and peaked at three hour.Expression levels of p-STAT3 in the hippocampal neurons at each time point between the two groups showed significant difference (P ＜ 0.05).Conclusion Oxygen and glucose deprivation induces noticeable up-regulation of p-STAT3 in the hippocampal neuronal nucleus,which indicates the overactivation of signal transduction pathway of STAT3.

The expression of cylindromatosis in oxygen and glucose deprivation and reperfusion induced primary cortical neuron necroptosis / 中华行为医学与脑科学杂志

Objective To investigate the location of CYLD in the neurons and explore the expression of CYLD in OGD/reperfusion-induced neuronal necroptosis.Methods Primary cortical neurons were cultured for 6days and neuronal purity was observed by double staining immunofluorescence of β3-tubulin and DAPI.The location of CYLD was identified by double staining immunofluorescence of NeuN,DAPI and CYLD using primary cortical neurons cultured for 14 days.Then,primary cortical neurons were divided into 8 groups:Control,EBSS,DMSO,OGD/reperfusion(0 h,2 h,6 h,8 h,12 h).Neurons were pretreated with zVAD-fmk for 30 min,OGD for 2 h and the levels of CYLD were evaluated after reoxygenation at different time points.The peak value(8 h) was chosen as reoxygenation time point.Neurons were divided into two groups as Control and OGD.The levels of CYLD were determined in both cytoplasm and nucleus after OGD 2 h and reoxygenation 8 h.Results The double staining immunofluorescence showed that neuronal cultured purity was about 70％ and the CYLD strongly expressed in nucleus but weakly in cytoplasm.The levels of CYLD increased gradually with different reoxygenation time and arrived at peak value after reoxygenation for 8 h (P ＜ 0.05),which was in accordance with the change of LDH (P ＜0.05) (Control (1.00±0.00),EBSS (1.07 ±0.03),DMSO (1.09 ±0.03),0h (1.40±0.12),2 h (1.74±0.08),6 h (2.25 ± 0.12),8 h (2.97 ± 0.15),12 h (3.01 ± 0.08)).The level of cytoplasm CYLD increased significantly in the OGD group (reoxygenation for 8 h)than that in control group (P＜0.05).But the level of nucleus CYLD had no difference between OGD and control group (P ＞ 0.05),which was in accordance with the results of immunofluorescence.Conclusion The CYLD in neurons cytoplasm is involved in necroptosis induced by OGD/deprivation and downregulating of CYLD has a protective effect on the brain injury resulted from ischemia/ reperfusion.

Protective effects of IGF-1 on neurons under condition of hypoxia and the role of PI3K signal pathway / 中南大学学报(医学版)

Objective To investigate the protective effects of insulin like growth factor 1(IGF-1) on cortical neurons under condition of hypoxia and the possible mechanism. Methods Cerebral cortical neurons from newborn rats were cultured under the condition of oxygen and glucose deprivation (OGD) . On day 7, neurons were treated with IGF-1 or IGF-1 plus LY294002 or PD98059 under condition of OGD or normal condition. MTT assay was used to analyze the viability of neurons in each group. The expression of total Akt and p-Akt were analyzed by Western blot. Results Compared with the control, the neuron viability was significantly higher in IGF-1 treated group under normal or OGD condition (P＜0.05). The protective effects of IGF-1 were attenuated in the presence of LY294002 but not PD98059. The result of Western blot showed IGF-1 upregulated the expression of p-Akt, which was inhibited by LY294002. Conclusion PI3K pathway may play an important role in neuroprotection afforded by IGF-1.

Study of the effects of mild hypothermia on improvement of cardiomyocyte contractility after ischemia-reperfusion in rats / 中华急诊医学杂志

Objective To study the effects of mild hypothermia on cardiomyocyte contractility improvement after ischemia-repeffusion injury and on the preservation of well-functioning mitochondrial respiratory capability.Methods A total of 50 newborn SD rats 1 ～ 2 days after delivery were sacrificed and their hearts taken to preserved in 4 ℃ cold D-hanks buffer solution with 0.12％ pancreatic proteinase and collagenase and then processed with 37 ℃ water bath to collect the cardiomyocytes cultured in DMEM medium with 10％ FBS for 5 days.The cardiomyocytes of rats were subjected to ischemia/reperfusion,in vitro,by oxygen and glucose deprivation(OGD)/oxygen and glucose restoration(OGR).The cardiomyocytes of rats after ischemia/reperfusion were divided into three groups:control group,hypothemia group and normothermia group.Contractile frequency and velosity were determined before OGD and 0 h,0.5h,1 h,1.5 h and 2 h after OGR.Ultrastructure changes of cardiomyocytes and mitochondrion were observed under transmission electron microscope(TEM)0 h and 2 h after OGR as well as assessment ot respiratory rate and respiratory control rate(RCR)with Clark oxygen electrode in each group.All data were analyzed with statistical software of SPSS 13.0.Results Contractile function of cardiomyocytes in hypothermia group and normothermia group declined to nadir at 0 h after OGR(P =0.000)and the contractile function of cardiomyocytes in hypothermia group was improved one hour later,compared with the normothermia group(P =0.000).Obvious swelling of mitochondrion was observed under TEM in normothermia group with little alteration after OGR.The RCR assessments indicated respiratory function in normothermia group was impaired after OGR(P =0.000)and this may be responsible for contractility dysfunction.Conclusions Mild hypothemia used after ischaemia can optimize the contractility of cardiomyocytes after a normothermia OGR,and the well-functioning respiratory capability of mitochondrion may be preserved in this process.

Antioxidant and protective effects of LWMH-SOD on neurocytes / 中国药理学通报

Aim To investigate the protective effects of LMWH-SOD(Low molecular weight heparin- Superoxide dismutase Conjugate)on the injuries induced by oxygen and glucose deprivation in cultured neurons. Methods The cortical neurons of fetal rat were cultured in vitro. The antioxidant and protective effects of LMWH-SOD were observed by treating neurons with oxygen and glucose deprivation. Results LMWH-SOD reduced the number of cell death and the efflux of LDH and the content of NO,MDA and increased the membrane fluidity after the injuries of cells. Conclusion LMWH-SOD has protective effects on cerebral cortical neurons through its action of scavenging free radicals.