Effects of NBP on expression of VEGF and HIF-1α in HUVECs under the condition of oxygen-glucose deprivation

AIM: To investigate the mechanism that dl-3-n-butylphthalide (NBP) protects cells from the induced by oxygen-glucose deprivation (OGD).METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to OGD to induce endothelial damage. Endothelial injury was assessed by measuring the changes of chromatin morphology and MTT method. The protein levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1α) were determined by immunofluorescence and quantitatively analyzed with the software IPP. Western blotting was applied to verify the results.RESULTS: NBP at the concentrations of 0.01 to 100 μmol/L dose-dependently protected against OGD-induced cell damage. Compared with OGD group, NBP enhanced OGD-induced the expression of VEGF and HIF-1α, and the difference was statistically significant. The expression of VEGF and HIF-1α reached to the peak at the time points of 6 h and 8 h after OGD, respectively.CONCLUSION: Under the condition of OGD, NBP enhances the expression of HIF-1α in HUVECs, subsequently promotes the expression of downstream VEGF, and eventually elevates the survival of the cells.     

Oxygen free radical injury of myocardial mitochondria in the experimental type 2 diabetic rats

AIM: To study the mechanism of diabetic cardiomyopathy and abnormality of oxygen free radicals. METHODS: The contents of myocardial cytosolic cytochrome C, mitochondria cytochrome C, mitochondrial calcium, NO, MDA and the activity of SOD and NOS were determined in diabetic rats induced by STZ. The pathological changes were observed under transmission electron microscope. RESULTS: Compared to the normal and ganoderma group, the levels of mitochondrial NO, iNOS, MDA, calcium and plasma Cyt-C in rat myocardium were higher (P<0.05), while mitochondrial Cyt-C and SOD were lowered in model group (P<0.05). The bouncary indistinct, disorganization, a focal loss of muscular fibril, myocardium mitochondria swelling, pulmonary vascular endothelial cellular swelling and obstructed lumen of the capillary were also observed under transmission electronic microscope. CONCLUSION: The findings indicate that oxyradical and lipid peroxidation might be associated with the damage of myocardial mitochondria in NIDDM rats. Cyt-C and mitochondrial calcium is also involved in the process.     

Effects of heptanol preconditioning on structure,function and Cx43 content of mitochondria in rabbit model of myocardial ischemia/reperfusion injury

AIM: To investigate the effects of heptanol preconditioning on the changes of structure, function and connexin 43 (Cx43) content in mitochondria in a rabbit model of myocardial isehemia-reperfusion (IR) injury. METHODS: In anesthetized open-chest rabbits, the left anterior descending artery (LAD) was occluded for 30 min and reperfused for 4 h. Sixty-four rabbits were randomly divided into 4 groups (n=16 in each group): sham operation group (sham group), ischemia-reperfusion group (IR group), ischemic preconditioning group (IP group) and heptanol preconditioning group (HT group). All rabbits in the 4 groups were killed 4 h after reperfusion. Myocardial infarct size was determined at the end of the experiment. Mitochondria was isolated by centrifugations. The ultrastructural changes of the mitochondria were observed under electronic microscope. The mitochondrial membrane potential, Ca2+ concentration, MDA content and SOD activity of myocardial mitochondria were also examined. The content of mitochondria Cx43 was detected by Western blotting. RESULTS: Compared to IR group, the myocardial infarct size was significantly reduced in IP (18.97%±2.80%) and HT (19.97%±3.80%) groups, the damage of mitoehondrial ultrastructure was milder (P<0.05), mitochondrial membrane potential was significantly higher and Ca2+ concentration was much lower (P<0.01) in IP group and HT group. No significant difference of MDA content and SOD activity in myocardial mitochondria between IR group and HT group was found. However, MDA content were much lower and SOD activity was significantly higher in IP group as compared to IR group (P<0.01). Compared to sham group, the mitochondria Cx43 expression was distinctly decreased compared to IR group (P<0.05) and no significant difference was found between IP group and HT group (P>0.05). CONCLUSION: Heptanol preconditioning protects myocardium from ischemia-reperfusion injury. The mechanism may be related to increasing in mitochondrial membrane potential, alleviating Ca2+ overload in myocardial mitochondria and attenuating the decrease in mitochondria Cx43 expression induced by isehemia-reperfusion.     

Protective effect of quercetin on L-02 cells by inhibiting DNA damage of INH-induced mitochondrial oxidative stress

AIM: To investigate the role of reactive oxygen species(ROS)-mediated mitochondrial oxidative injury in isonicotinyl hydrazide(INH)-induced DNA damage and the protective effect of quercetin on L-02 cells. METHODS: The injury model of hepatocyte L-02cells in vitro induced by INH was established. The cells were divided into control group, INH group, low-dose quercetin group and high-dose quercetin group. The DNA damage of L-02 cells was evaluated by the comet test. The mitochondrion was prepared, and the level of mitochondrial ROS and the value of mitochondrial membrane potential(ΔΨ_m) were detected by fluorescent probes DCFH-DA and rhodamine 123. The content of MDA was measured by TBA method. The activity of SOD was assessed with the xanthine oxidase method. The protein expression of Bcl-2 and Bax was determined by Western blotting, and the value of Bax/Bcl-2 was calculated. RESULTS: INH induced obvious DNA damage, increased the level of mitochondrial ROS, the content of MDA and the value of Bax/Bcl-2, and markedly reduced the value of ΔΨ_m and the activity of SOD in the L-02 cells. Quercetin attenuated DNA damage, reduced the level of mitochondrial ROS, elevated the value of ΔΨ_m, declined the content of MDA, increased the activity of SOD and decreased the value of Bax/Bcl-2 in the L-02 cells. CONCLUSION: INH induces DNA damage in L-02 cells by generation of mitochondrial oxidative stress. Quercetin has a protective effect on L-02 cells to attenuate the INH-induced DNA damage by inhibiting ROS-mediated mitochondrial oxidative damage.     

Effects of glucagon-like peptide-1 on myocardial ischemia-reperfusion/hypoxia-reoxygenation injury in rats

AIM: To investigate the effects of glucagon-like peptide-1 (GLP-1) on myocardial ischemia-reperfusion (IR)/hypoxia-reoxygenation (HR) injury in rats. METHODS: Sprague-Dawley rats were randomly divided into 5 groups: sham group, IR group and IR+GLP-1 (0.03 nmol/L, 0.16 nmol/L and 0.30 nmol/L) groups. IR group and IR+GLP-1 group were subject to 30 min of ischemia and 3 h of reperfusion. The myocardial infarct size, the ultrastructural changes of the myocardial tissues, the apoptosis of the cardiomyocytes, the activity of superoxide dismutase (SOD) and the concentration of malondialdehyde (MDA) were detected. Primarily cultured cardiomyocytes were divided into 5 groups at random: control group, HR group and HR+GLP-1 (1 μmol/L, 5 μmol/L and 10 μmol/L) groups. The morphology and apoptosis of the cardiomyocytes were observed. The levels of lactate dehydrogenase (LDH),MDA,SOD,reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in different groups were detected. RESULTS: Compared with IR group, the myocardial infarct size and cardiomyocyte apoptosis were remarkably reduced, mitochondrial ultrastructures were improved, the activity of SOD was increased and the concentration of MDA was decreased in IR+GLP-1 (0.03 nmol/L, 0.16 nmol/L and 0.30 nmol/L) groups. Compared with HR group, GLP-1 (1 μmol/L, 5 μmol/L and 10 μmol/L) preconditioning significantly decreased the myocardial injury, increased SOD activity, decreased MDA concentration and ROS production, and heightened MMP in a dose-dependent manner. CONCLUSION: GLP-1 protects cardiomyocytes from IR/HR injury, which may be partially due to the effects of anti-oxidative mechanism and the function of mitochondrial protection.     

碱胁迫下杜鹃花抗氧化体系的响应及亚细胞分布

为探讨杜鹃花属（Rhododendron）植物的耐碱性机理，以3个杜鹃花园艺栽培品种‘胭脂蜜’（R. obtusum‘Yanzhimi’）、‘红珊瑚’（R. obtusum‘Hongshanhu’）及‘红月’（R. obtusum‘Hongyue’）为试验材料，对植株进行为期2个月的NaHCO3和Na2CO3混合碱胁迫处理，观察植株在碱胁迫下的形态变化并测定叶片叶绿体、线粒体和细胞溶质3个亚细胞部位中活性氧及抗氧化体系的活性。结果表明：根据碱害指数，3个杜鹃花品种耐碱性从高到低依次为‘胭脂蜜’>‘红珊瑚’>‘红月’。在碱胁迫下，3个品种的3个亚细胞部位中H2O2和超氧阴离子水平显著提高，导致MDA含量增加，抗氧化酶的变化存在品种间差异。‘胭脂蜜’的3个亚细胞部位中SOD、POD、CAT和GR酶活性显著提高，APX在叶绿体和细胞溶质中的活性显著上升；而‘红珊瑚’仅叶绿体中的CAT和APX、线粒体中的SOD、POD和CAT以及细胞溶质中的SOD、GR活性显著提高；‘红月’的SOD活性在线粒体和细胞溶质中显著提高，CAT活性在叶绿体和线粒体中显著提高，POD及APX活性显著下降，GR活性无显著变化。碱胁迫下，3个品种的非酶促抗氧化剂AsA和GSH含量在叶绿体中降低，在线粒体和细胞溶质中增加。抗碱性强的‘胭脂蜜’在碱胁迫后ROS水平较稳定，MDA含量较低，SOD、POD、APX和GR活性较高。3个亚细胞部位比较发现，细胞溶质中H2O2和超氧阴离子的水平显著高于线粒体和叶绿体，抗氧化酶SOD、POD和GR及抗氧化剂AsA和GSH也主要分布在细胞溶质中，活性氧与抗氧化系统的亚细胞分布具有较高的一致性；APX主要分布在叶绿体中；CAT在线粒体活性最高。碱胁迫下不同亚细胞部位活性氧积累和过氧化损伤程度不同，相应的抗氧化防御体系在各亚细胞的响应也不同，细胞溶质是杜鹃花活性氧产生和清除的主要场所。     

Effects of maternal limb ischemic preconditioning on structural and functional changes of mitochondria in fetal hippocampal neurons induced by intrauterine distress-reoxygenation in rats

AIM: To investigate the effects of maternal limb ischemic preconditioning (LIP) on the mitochondrial structures and functions of the hippocampal neurons induced by reoxygenation in the intrauterine distress fetal rats. METHODS: Pregnant rats (n=40) were randomly divided into 4 groups: sham (S) group, LIP group, fetal distress (FD) group and LIP+FD group. Intrauterine ischemia model was established through the experimental design. The ultrastructure of the mitochondria in CA1 area of the hippocampus was observed. The mitochondrial membrane potential and reactive oxygen species (ROS) were measured. The content of ATP and MDA in the hippocampus tissue was detected. The activity of Mn-SOD was observed. RESULTS: Compared with sham group, the ultrastructure of mitochondria in CA1 area of the hippocampus was damaged in FD group and LIP+FD group. The mitochondrial membrane potential, the content of ATP and the activity of Mn-SOD were decreased. However, the content of ROS and MDA was increased. Compared with FD group, the ultrastructure of mitochondria in CA1 area of the hippocampus was intact in LIP+FD group. Furthermore, the reduced mitochondrial membrane potential and ATP content were inhibited. The activity of Mn-SOD was increased, but the content of ROS and MDA was decreased in LIP+FD group. CONCLUSION: Limb ischemia preconditioning inhibits the damage the mitochondria of fetal hippocampal neurons induced by reoxygenation in the intrauterine distress fetal rats.     

Vitamin D prevents high glucose-induced human umbilical vein endothe-lial cell apoptosis by inhibition of prolyl isomerase 1-mediated mitochon-drial oxidative stress

AIM: To observe the effects of vitamin D on the apoptosis, prolyl isomerase 1 (Pin1) protein expression and activity, mitochondrial translocation of p66Shc, and reactive oxygen species (ROS) production in high glucose-cultured human umbilical vein endothelial cells (HUVECs), and to explore the role of vitamin D receptor (VDR) in these processes. METHODS: HUVECs were treated with high glucose (33 mmol/L) in the presence or absence of vitamin D or Pin1 inhibitor juglone. The cell apoptosis was measured by flow cytometry and TUNEL staining. Intracellular ROS levels were examined by flow cytometry and fluorescence microscopy. The protein levels of Pin1, p66Shc, p-p66Shc, mitochondria to cytoplasm ratio of p66Shc, and caspase-3 in HUVECs were measured by Western blot. Pin1 activity in HUVECs lysate was assessed by a commercial kit. Knockdown of VDR by siRNA was conducted to evaluate the role of VDR in the regulatory effects of vitamin D on Pin1 protein expression and activity in HUVECs under high-glucose condition. RESULTS: Vitamin D suppressed the apoptosis and intracellular ROS generation of HUVECs induced by high glucose (P<0.05). Vitamin D inhibited high glucose-induced upregulation of Pin1 protein expression and activity (P<0.05). Vitamin D inhibited the phosphorylation and mitochondrial translocation of p66Shc and caspase-3 protein expression induced by high glucose (P<0.05). Knockdown of VDR by siRNA abolished the inhibitory effects of vitamin D on high glucose-induced upregulation of Pin1 protein expression and activity. CONCLUSION: Vitamin D alleviates high glucose-induced endothelial cell apoptosis by inhibition of Pin1 protein expression and activity, and attenuation of p66Shc-mediated mitochondrial oxidative stress, which are dependent on VDR activation.     

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.     

Hypoxic preconditioning alleviates oxygen-glucose deprivation in PC12 cells:the involvement of autophagy

AIM: To examined the effects of hypoxic preconditioning(HPC) on oxygen-glucose deprivation(OGD)-induced PC12 cells, and to investigate its possible mechanisms of autophagy.METHODS: Cultured PC12 cells were randomly divided into control group, HPC group, 3-methyladenine(3-MA) group, HPC+OGD group, 3-MA+HPC+OGD group and OGD group. CCK-8 assay was used to detect the cell viability. The caspase-3 activity was also tested. TUNEL staining and flow cytometry were used to detect the cell apoptosis. The protein levels of apoptosis-related protein caspase-3 and autophagy-marked protein LC3-2 and beclin-1 were determined by Western blot.RESULTS: Compared with control group, the viability of PC12 cells was significantly reduced, and the activity of caspase-3 was significantly increased in OGD group. Compared with 3-MA+ HPC+OGD group and OGD group, the viability of PC12 cells was significantly increased, and the activity of caspase-3 was significantly reduced in HPC+OGD group(P<0.05). The PC12 cell injury was apparent after OGD with a great increase in the apoptotic rate(P<0.05). Compared with OGD group, the apoptotic rate significantly decreased in HPC+OGD group(P<0.05). Compared with control group, the protein level of cleaved caspase-3 was significantly increased in OGD group(P<0.05). Compared with OGD group, the protein level of cleaved caspase-3 was significantly decreased, and the levels of LC3-2 and beclin-1 were significantly increased in HPC+OGD group(P<0.05).CONCLUSION: OGD decreases cell survival and induces apoptosis.Activation of cell autophagy may be the mechanism by which hypoxic preconditioning protects the PC12 cells from OGD induced injury.     

Autophagy attenuates injury of human pulmonary microvascular endothelial cells under mimic ischemia/reperfusion microenvironment

AIM: To detect the autophagic changes of human pulmonary microvascular endothelial cells (HPMVECs) under ischemia/reperfusion (I/R) microenvironment, and to clarify the effects of autophagy on the HPMVECs survival and endothelial barrier integrity under I/R condition. METHODS: Rapamycin (RAP) was applied to promote autophagy of HPMVECs. These cells were then incubated under the condition of oxygen-glucose deprivation/oxygen-glucose restoration (OGD). After exposure to OGD, the changes of autophagy, cellular death and permeability of the cells were determined by transmission electron microscopy, flow cytometry and transwell assay, respectively. RESULTS: Compared with the control cells, OGD-challenged cells had a much higher level of autophagy. The apoptotic rate was much higher and endothelial permeability was more serious in OGD group than those in control group. Preconditioning with RAP effectively improved OGD induced autophagy, it did not affect the cell survival and endothelial permeability under normal living condition, but obviously decreased the cells apoptotic rate, and remarkably lowered OGD-induced high permeability of the cells. CONCLUSION: Autophagy protects HPMVECs against I/R-induced injury. Promotion of autophagy is helpful for attenuating I/R-induced cell death and sustaining the endothelial barrier integrity.     

Effect of rapamycin on apoptosis of mouse astrocytes in vitro

AIM: To observe the effect of rapamycin on the apoptosis of mouse astrocytes in vitro.ME-THODS:The astrocytes from C57BL/6J newborn mouse pups were isolated and primarily cultured. The effect of rapamycin on the viability of astrocytes was assessed by MTT assay. The mean fluorescence intensity of SYTOX^® Green stain in the astrocytes was detected by fluorescence microplate reader in order to analyze the effects of rapamycin on the cell death induced by H₂O₂, ionomycin and/or deferorxamin. DiOC₆(3) staining was used to analyze the mitochondrial membrane potential of the astrocytes induced by H₂O₂. Flow cytometry analysis was used to determine the production of ROS in the astrocytes and mitochondria by staining with H₂DCFDA and MitoSOX^TM Red reagent, respectively.RESULTS: Rapamycin at concentration of 0.5 μmol/L protected the astrocytes against cell death induced by H₂O₂ or deferoxamine plus ionomycin. Rapamycin protected the mitochondrial membrane potential of astrocytes from the injury of H₂O₂. It also reduced the production of ROS in the astrocytes and decreased the level of ROS in the mitochondria.CONCLUSION: Rapamycin reduces the ROS overload in the mitochondria, keeps mitochondrial membrane potential safety and protects the astrocytes against apoptosis in vitro.     

Expression and role of TRPM2 in H9N2 swine influenza virus infection-induced mouse pulmonary microvascular endothelial cell damage

AIM: To investigate the expression and role of transient receptor potential cation channel M2 (TRPM2) in H9N2 swine influenza virus (H9N2-SIV) infection-induced mouse pulmonary microvascular endothelial cell (PMEVC) damage. METHODS: At 24 h and 48 h after PMEVC (3 parallel test in each group) was infected by H9N2-SIV, the superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity, reactive oxygen species (ROS) level and nitric oxide synthase (NOS) activity were measured according to the manufacturer's instructions. The ultrastructural changes of PMVEC were observed under electron microscope. Transendothelial electrical resistance (TEER) and the percentage of horse radish peroxidase (HRP) influx was measure by Transwell culture. The expression of TRPM2 at mRNA and protein levels was determined by real-time PCR and Western blot. The apoptosis of PMVEC was observed with Annexin V/PI staining. RESULTS: The SOD and GSH levels were decreased significantly in H9N2-SIV-infected PMEVC as compared with control group(P<0.05). However, NOS activity and ROS level were increased dramatically (P<0.01). The organelles of PMVEC infected by H9N2-SIV were decreased sharply, and vacuolization in mitochondria and apoptosis were also observed under electron microscope. The value of TEER was decreased significantly and the percentage of HRP influx was increased dramatically with the H9N2-SIV infection (P<0.01). The expression of TRPM2 at mRNA and protein levels in H9N2-SIV-infected PMVEC was increased dramatically at 24 h and 48 h as compared with control group (P<0.01). Annexin V/PI double staining showed that the cytomembrane was stained by great green fluorescence and some nuclei were stained by red fluorescence in H9N2-SIV-infected PMVEC. However, less fluorescence stained PMVEC were found in control group. CONCLUSION: The expression of TRPM2 at mRNA and protein levels is increased significantly, which is involved in cell damage and apoptosis induced by oxidative stress of H9N2-SIV infection.     

Role of calreticulin-induced mitochondrial damage in high glucose-induced apoptosis of myocardial cells

AIM: To observe the effect of high glucose on the protein expression of calreticulin (CRT) and its association with cell apoptosis and mitochondrial dysfunction in the cardiomyocytes. METHODS: AC-16 cardiomyocytes were randomly divided into normal glucose group, high glucose group, high glucose+ CRT siRNA group and isotonic control group. The cell apoptotic rate, reactive oxygen species (ROS), mitochondrial membrane potential level, respiratory enzyme activity, and protein expression of CRT were observed. RESULTS: Compared with the cardiomyocytes in normal glucose group, the apoptotic rate and ROS production of cardiomyocytes increased in high glucose group, accompanying with the decreases in the mitochondrial membrane potential level and enzyme activitiy of the respiratory chain. The protein expression of CRT was significantly increased in high glucose group. However, compared with high glucose group, high glucose+ CRT siRNA decreased the expression of CRT and attenuated the damage of mitochondria, but CRT siRNA did not reduce the ROS level in cardiomyocytes. CONCLUSION: High glucose brings about CRT over-expression to induce mitochondrial injury, thus increasing myocardial apoptosis.     

Oxidative stress plays an important role in acetaldehyde-induced cardiomyocytes apoptosis

AIM: To elucidate the mechanism of alcoholic myocardiopathy (AHMD) by exploring the role of ROS mediated oxidative stress in acetaldehyde-induced cardiomyocytes apoptosis. METHODS: Cultured rat cardiomyocytes were stimulated with acetaldehyde (100 μmol/L) for 24 h, then the cell apoptotic index were examined and compared to that with alcohol (100 μmol/L) stimulation. The changes of ROS levels and SOD activities were explored by a time-dependent model in acetaldehyde-induced cardiomyocytes. Meanwhile, the phosphorylation changes of ROS mediated MAPK signaling pathway correlated proteins were also detected, with which compared that changes both in pre-adding NAC acetaldehyde-induced cardiomyocytes, and in H2O2 (100 μmol/L) induced cardiomyocytes, respectively. RESULTS: Acetaldehyde had more obvious apoptotic effect than alcohol through caspase 3 activating (P<0.05, vs control), both cellular ROS level and SOD activity increased in a time-dependent way, and reached the peak value of (78.84%±4.33%) for ROS and (0.55±0.02) for SOD among 18 to 24 h, respectively. Meanwhile, JNK and ERK protein phosphorylation upregulated in acetaldehyde-induced cardiomyocytes, and was reversed by NAC anti-oxidative effect. However all the phosphorylation levels were weaker than that in H2O2-induced group. CONCLUSION: Acetaldehyde causes oxidative damage in cardiomyocytes through enhancing cellular ROS level, and induces cardiocytes apoptosis by activating ROS mediated JNK pathway. The novel way of depressing cellular ROS level or blocking the special apoptotic pathway may have effects on AHMD preservation and therapy.     

Effects of silymarin on homocysteine-induced apoptosis in human umbilical vein endothelial cells

AIM:To investigate the effect of silymarin on homocysteine-induced cell viability and apoptosis in human umbilical vein endothelial cells (HUVECs). METHODS:Cell viability was analyzed by using MTT and LDH assay. Apoptotic cells were detected by using DNA fragmentation and flow cytometric analysis. The level of intracellular reactive oxygen species (ROS) and the potential of mitochondrial membrane were determined by flow cytometric assay. The activity of caspase-3, -6 and -9 were measured with microplate spectrofluorometer. Protein levels were examined by Western blotting. RESULTS:Treatment of cultured HUVECs with HCY for 48 h induced a significant decrease in cell viability, and the percentage of apoptosis increased to 76.8%. The level of intracellular ROS and activity of caspase-3, -6 and -9 enhanced, and the red/green ratios of mitochondrial membrane decreased. However, simultaneous treatment with silymarin exhibited cytoprotective effects, reduced formation of the DNA ladder, prevented the levels of Bax and Bcl-2 proteins and the accumulation of ROS as well as caspase-3, -6 and -9 activation, reconverted the potential of mitochondrial membrane, and the percentage of apoptosis/necrosis was significantly decreased to 12.7% in a dose-dependent manner. CONCLUSION:These results demonstrate that silymarin has the protective capacity to antagonize HCY-induced apoptosis in HUVECs. The antiapoptotic action of silymarin may be partially dependent on an anti-oxidative stress effects, inhibition of caspases activity, and maintenance of mitochondria function.     

Effect of miRNA-21 on protection of PC12 cells from oxygen-glucose deprivation damage

AIM: To investigate the effect of microRNA (miRNA)-21 on the PC12 cells with hypoxic-ischemic damage.METHODS: The PC12 cells were cultured in vitro, and the cell model of oxygen-glucose deprivation (OGD) was established. In accordance with the following requirements, the cells were randomly divided into control group, OGD group, negative control sequence+OGD group, miRNA-21 inhibitor+OGD group and miRNA-21 mimic+OGD group. The effects and mechanism of miRNA-21 on the protection of PC12 cells from OGD damage were determined by CCK-8 assay, real-time PCR and Western blot.RESULTS: Decrease in the expression of miRNA-21 by transfection with miRNA-21 inhibitor inhibited the viavility of the PC12 cells subjected to OGD damage. Increase in the expression of miRNA-21 by transfection with miRNA-21 mimic promoted the viability of the PC12 cells subjected to OGD damage. It was further confirmed that miRNA-21 promoted the AKT phosphorylation in OGD-damaged PC12 cells.CONCLUSION: miRNA-21 significantly increases the viability of PC12 cells subjected to OGD damage, which may be related to the activation of PI3K/AKT signaling pathway.     

Mild hypothermia protects against injury of rat hippocampal neurons induced by oxygen-glucose deprivation

AIM：To study the protective effect of mild hypothermia (31~32 °C) on rat hippocampal neurons against oxygen-glucose deprivation (OGD)-induced injury and its possible mechanisms. METHODS：An OGD experimental model of rat hippocampal neurons in vitro was established to simulate cerebral ischemic-hypoxic injury. The rat hippocampal neurons were randomly divided into 4 groups：control group, mild hypothermia group, OGD group and mild hypothermia+OGD group. The cell morphology was observed under light and electron microscopes. The neuronal apoptosis was detected by flow cytometry. The activity of caspase-3 in the cytoplasm was measured by colorimetry. RESULTS：The neuronal injury was apparent after OGD, with a great increase in apoptotic rate (P<0.01). Compared with OGD group, the morphology of neuronal injury in mild hypothermia+OGD group was attenuated, and the neuronal apoptotic rate and the activity of caspase-3 in the cytoplasm decreased. The activity of caspase-3 in the cytoplasm increased after OGD, and was positively correlated with the neuronal apoptotic rate (r=0.823, P<0.05). The activity of caspase-3 in the cytoplasm also increased after mild hypothermia and OGD, and was also positively correlated with the neuronal apoptotic rate (r=0.841, P<0.05). CONCLUSION：OGD can increase caspase-3 activity in the neuronal cytoplasm and induce neuronal apoptosis. Restraint on caspase-3 activity in the neuronal cytoplasm may be the mechanism by which mild hypothermia protects against neuronal injury induced by OGD.