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.     

Preliminary mechanism of senegenin against H/R-induced apoptosis of primary cortical neurons

AIM：To explore the preliminary mechanism of senegenin (Sen) on inhibiting hypoxia/reoxygenation(H/R)-induced apoptosis of primary cortical neurons. METHODS：The cultured cortical neurons were randomly divided into normal group (control group), model group (H/R group), Sen+H/R group and Sen group. Flow cytometry was used to evaluate the effect of Sen on H/R-induced cell apoptosis. The protein levels of JNK, p-JNK, c-Jun, p-c-Jun, Bcl-2 and Bax were assessed by Western blotting. RESULTS：The apoptotic rate in H/R group was obviously higher than that in control group (P<0.05), while the apoptotic rate in Sen+H/R group was obviously lower than that in H/R group (P<0.05), suggesting that the model of apoptosis was established successfully. The results of Western blotting showed that Sen increased the expression of JNK and c-Jun, inhibited the phosphorylation of JNK and c-Jun (P<0.05), increased the protein level of Bcl-2 and inhibited the protein level of Bax in H/R treated primary cortical neurons (P<0.05). CONCLUSION：Sen has a protective effect against H/R-induced neuronal apoptosis by increasing the expression of JNK and c-Jun, inhibiting the phosphorylation of JNK and c-Jun, increasing the protein level of Bcl-2 and decreasing the protein level of Bax.     

Influence of Bcl-2 inhibitor on Astragalus injection-reduced expression of caspase-3 in rat hippocampal neurons with oxygen-glucose deprivation and reoxygenation

AIM: To observe the influence of Bcl-2 inhibitor on the expression of caspase-3 reduced by Astra-galus injection in rat hippocampal neurons with oxygen-glucose deprivation and reoxygenation (OGD/R). METHODS: The primary rat hippocampal neurons cultured in vitro for 8 d were chosen and randomly divided into 6 groups: normal control group, model group (OGD/R group), Astragalus injection group, Astragalus injection solvent (sterile deionized water)group, Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group. The cells in all groups were tested 24 h after they were treated with reoxygenation after deprived of oxygen and glucose for 30 min except normal control group. The cell type and rate of positive cells were observed by immunohistochemistry. The protein levels of Bcl-2 and cleaved caspase-3 in the hippocampal neurons were measured by Western blotting. The mRNA expression of caspase-3 was detected by RT-PCR. RESULTS: Compared with normal control group, the caspase-3 positive rate of the cells, the protein levels of Bcl-2 and cleaved caspase-3, and the mRNA expression of caspase-3 in model group enhanced significantly (P < 0.05). Compared with model group, the expression of Bcl-2 in Astragalus injection group obviously enhanced, while the caspase-3 positive rate of the cells, the protein level of cleaved caspase-3 and the mRNA expression of caspase-3 in the Astragalus injection group decreased significantly (P < 0.05). No significant difference in injection solvent group, Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group was observed (P > 0.05). The expression of Bcl-2 was decreased sharply in Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group. CONCLUSION: Bcl-2 inhibitor antagonizes the inhibitory effect of Astragalus injection on caspase-3 expression in rat hippocamal neurons with OGD/R, which may be one of the possible target for the inhibitory action of Astragalus injection on the apoptosis of rat hippocampal neurons induced by OGD/R.     

Inhibition of Toll-like receptor 9 activation in microglia after oxygen-glucose deprivation and reoxygenation protects neurons from damage

AIM: To observe the Toll-like receptor 9 (TLR9) activation in microglia BV-2 cells after oxygen-glucose deprivation and reoxygenation (OGDR), and its effects on neuronal apoptosis. METHODS: The BV-2 cell supernatants were collected after the corresponding treatment and added to mouse primary cortical neurons after OGDR for 4 h, followed by normal culture for 24 h. The cells were divided into normal BV-2 group, NC-siRNA group, TLR9-siRNA group, OGDR group, OGDR+NC-siRNA group, OGDR+TLR9-siRNA group and control group (without adding BV-2 cell supernatant). The changes of the neuronal morphology were observed under an inverted phase- contrast microscope, and the neuronal apoptosis was detected by TUNEL. The protein expression of cleaved caspase-3 was detected by Western blotting. RESULTS: After OGDR, the axon turned thin, twisted and broken, and neuronal swelling, decrease in refraction and vacuolar degeneration were observed. The green-stained apoptotic bodies in the neurons in all groups were positive. Compared with control group, the caspase-3 protein levels in other groups were increased. Compared with the normal BV-2 group, the caspase-3 protein in OGDR group and TLR9-siRNA group was increased. Compared with OGDR+TLR9-siRNA group, the caspase-3 protein in TLR9-siRNA group and OGDR group was decreased. CONCLUSION: After OGDR, TLR9 activation in BV-2 cells induces neuronal apoptosis with the increase in caspase-3 protein level. Inhibition of TLR9 expression reduces neuronal damage.     

Relationship between morphological changes of autophagy and apoptosis in PC12 cells induced by oxygen-glucose deprivation and reoxygenation

AIM: To investigate the relationship between morphological changes of autophagy and apoptosis in the PC12 cells induced by oxygen-glucose deprivation and reoxygenation. METHODS: The PC12 cells were randomly divided into normal control group, oxygen-glucose deprivation and reoxygenation group, autophagy inhibitor group and autophagy activator group. The cells in oxygen-glucose deprivation and reoxygenation group, autophagy inhibitor group and autophagy activator group were exposed to reoxygenation (12 h) after 3 h of oxygen-glucose deprivation, and autophagy inhibitor 3-methyladenine and autophagy activator rapamycin were added into the cells at the same time. Using transmission electron microscope and monodansylcadaverine fluorescence staining, the morphological changes of autophagosome were observed. The apoptosis of the PC12 cells were analyzed by flow cytometry with Annexin V-FITC/PI staining and TUNEL method. RESULTS: Compared with normal control group, the numbers of autophagosomes and the apoptotic rates increased in oxygen-glucose deprivation and reoxygenation group (P<0.05). Compared with oxygen-glucose deprivation and reoxygenation group, the numbers of autophagosomes decreased obviously (P<0.05) and the apoptotic rates increased markedly in autophagy inhibitor group (P<0.05). The numbers of autophagosomes increased obviously (P<0.05), the apoptotic rates decreased markedly (P<0.05), the autophagosomes became bigger in size, and autolysosomes was also found in autophagy activator group. CONCLUSION: Oxygen-glucose deprivation and reoxygenation induce autophagy in PC12 cells, and autophagy inhibits cell apoptosis to play a protective role.     

Effect of rosuvastatin on cerebral microvascular endothelial cell injury induced by oxygen-glucose deprivation/reoxygenation

AIM: To explore the effect of rosuvastatin on the oxygen-glucose deprivation (OGD)/reoxygenation induced injury of cerebral microvascular endothelial cells (BMECs). METHODS: BMECs derived from BALB/c mice were isolated and cultured. BMECs were pretreated with rosuvastatin, followed by OGD for 3 h or 6 h and reoxygenation for 24 h. The morphological changes of BMECs were observed under light microscope. MTT assay was used to measured the cell viability, and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining was used to assess the proliferation of BMECs. The protein levels of cleaved caspase-3 was observed by immunofluorescence staining. The protein levels of Bcl-2, Bax, matrix metalloproteinase (MMP) 2, MMP9, phosphorylated nuclear factor kappa B (p-NF-κB), phosphorylated P38 mitogen-activated protein kinase (p-P38) and phosphorylated c-Jun N-terminal kinase (p-JNK) were determined by Western blot. RESULTS: Rosuvastatin at 10 μmol/L improved the viability of the BMECs with OGD/reoxygenation-induced damage, and maintained the structure of BMECs. Moreover, rosuvastatin significantly prohibited the protein levels of cleaved caspase-3, MMP2, MMP9, p-NF-κB, p-P38 and p-JNK, and up-regulated the ratio of Bcl-2/Bax (P<0.05). CONCLUSION: Rosuvastatin reduces OGD/reoxygenation-induced injury of BMECs by inhibiting the expression of apoptosis-related proteins and MMPs, suggesting that rosuvastatin has potential value for the maintenance of blood-brain barrier.     

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.     

Effect of miR-25 on apoptosis of H9c2 cells induced by hypoxia/reoxygenation

AIM:To investigate the role and mechanism of microRNA-25 (miR-25) in apoptosis of H9c2 cells induced by hypoxia/reoxygenation. METHODS:The H9c2 cells with over-expression of miR-25 were treated with hypo-xia/reoxygenation. Real-time PCR was used to detect the expression of miR-25 and high mobility group box-1 (HMGB1) mRNA. Western blot was performed to examine the protein expression levels of HMGB1, Bcl-2 and cleaved caspase-3. Flow cytometry was used to analyze the proportion of apoptotic cells and the cell cycle. Dual-luciferase reporter assay was used to confirm that HMGB1 was the target gene of miR-25 in the H9c2 cells. Moreover, the H9c2 cells transfected with HMGB1-shRNA were subjected to hypoxia/reoxygenation to verify whether HMGB1 participated in the regulation of apoptosis of H9c2 cells. RESULTS:Over-expression of miR-25 significantly reduced the protein expression levels of HMGB1 and cleaved caspase-3, and increased the expression of Bcl-2 and the entrance into S phase in H9c2 cells induced by hypoxia/reoxygenation (P<0.01). The result of dual-luciferase reporter assay showed that compared with the control group, transfection with HMGB1-3' UTR-psi-CHECK2+miR-25 mimic strongly inhibited the luciferase activity (P<0.05). After the H9c2 cells transfected with HMGB1-shRNA was treated with hypoxia/reoxygenation, the expression of Bcl-2 was up-regulated, the expression of cleaved caspase-3 was down-regulated, and the cells in S phase were increased (P<0.05). CONCLUSION:miR-25 reduces apoptosis of H9c2 cells induced by hypoxia/reoxygenation, and its mechanism may be related with the inhibition of HMGB1 expression via interacting with its 3'-UTR.     

Effect of Astragalus injection on expression of calmodulin after hypoxia/hypoglycemia and reoxygenation in rat hippocampal neurons

AIM: To investigate the effect of Astragalus injection on the expression of calmodulin(CaM) after hypoxia/ hypoglycemia and reoxygenation in rat hippocampal neurons.METHODS: The hippocampal neurons were cultured for 8 days and divided into 4 groups: normal control group (normal control), hypoxia/hypoglycemia and reoxygenation group (model), Astragalus injection solution group (solution control) and Astragalus injection group ( Astragalus ).The cells in all groups were treated with reoxygenation and normal medium after deprived of oxygen and glucose for 30 min except normal control group.The method of immunohistochemistry was used to measure the number of caspase-3 positive neurons.The expression of CaM at mRNA and protein levels was measured at time points of 0 h, 0.5 h, 2 h, 6 h, 24 h, 48 h, 72 h and 120 h after hypoxia/hypoglycemia and reoxygenation by RT-PCR and Western blotting, respectively.RESULTS: No difference of the parameters at all time points between model group and solution control group was found.Compared with normal control group, the numbers and the percentages of caspase-3 positive cells at all time points obviously increased in model group except at 0 h and 0.5 h (P<0.05).Compared with model group, the numbers and the percentages of caspase-3 positive cells were decreased in Astragalus injection group except at 0 h and 0.5 h (P<0.05).Compared with normal control group, the protein expression of CaM in rat hippocampal neurons at all time points obviously increased in model group (P<0.05).However, the protein expression of CaM in rat hippocampal neurons at all time points obviously decreased in Astragalus injection group as compared with model group (P<0.05).Compared with normal control group, the mRNA expression of CaM in rat hippocampal neurons at all time points obviously decreased in model group (P<0.05).The mRNA expression of CaM in rat hippocampal neurons at all time points obviously increased in Astragalus injection group as compared with model group (P<0.05).CONCLUSION: Astragalus injection inhibits the protein expression of CaM, the calcium overload and the expression of caspase-3 after hypoxia/hypoglycemia and reoxygenation, thus inhibiting hippocampal neuronal apoptosis.     

Effects of PDCD5 on hypoxia/reoxygenation-induced autophagy and apoptosis of cardiomyocytes

AIM: To investigate the influence of programmed cell death 5 (PDCD5) on apoptosis and autophagy in the cardiomyocytes exposed to hypoxia/reoxygenation (H/R) and its potential mechanism.METHODS: H9c2 cells were exposed to H/R. PDCD5 was downregulated by RNA interference. The cell viability was measured by MTT assay. TUNEL assay was used to detect cell apoptosis. The mRNA and protein levels were determined by RT-qPCR and Western blot.RESULTS: The expression of PDCD5 was upregulated in the cardiomyocytes after H/R injury. Furthermore, H/R injury obviously reduced the cell viability and enhanced the apoptosis and autophagy of the cardiomyocytes. However, knockdown of PDCD5 increased the cell viability, and attenuated H/R-induced apoptosis, accompany with reduction of Bax and augment of Bcl-2 expression. Additionally, silencing PDCD5 markedly inhibited H/R-induced autophagy by regulating the expression of LC3-II/LC3-I and beclin-1. Moreover, downregulation of PDCD5 suppressed NF-κB signaling by redu-cing the protein level of p-P65.CONCLUSION: Silencing PDCD5 suppresses H/R-induced H9c2 cells apoptosis and autophagy by blocking NF-κB signaling pathway. The result indicates a new strategy for the prevention and treatment of myocardial I/R injury.     

Effect of IL-8 on mitochondrial fusion and fission gene expression in human lung adenocarcinoma cell line A549 during cell migration

AIM: To investigate the changes of migration of lung adenocarcinoma cells promoted by IL-8 and the inner and outer mitochondrial membrane dynamic changes during this process.METHODS: Human lung adenocarcinoma cell line A549 was divided into control group and IL-8 group. Cell migration was analyzed by scratch detection and Transwell assay. The secretion of endogenous IL-8 was detected by ELISA. The protein levels of mitochondrial cytochrome C (Cyt C) and mitochondrial outer membrane protein Tom20 was detected by Western blot. The mRNA expression of mitochondrial fusion genes Mfn1, Mfn2 and OPA1 and fission genes Fis1, Drp1 and MTP18 was detected by RT-PCR. The morphological changes of mitochondria were observed by MitoTracker Red CMXRos dye staining and confocal microscopy.RESULTS: The migratory rate of A549 cells and endogenous secretion of IL-8 in A549 cells were higher than those in SPC-A-1 cells. The migratory rate of A549 cells was improved by IL-8 in a time-dependent manner. Compared with control group, the Tom20 protein expression was increased (P<0.05), and the Cyt C protein expression was decreased (P<0.05). The expression of mitochondrial outer membrane fusion genes Mfn1 and Mfn2 was increased (P<0.05), and the expression of mitochondrial inner membrane fusion gene OPA1 was decreased (P<0.05). The expression of fission genes Drp1 and MTP18 were decreased (P<0.05), while the expression of Fis1 was no change (P>0.05). Under confocal microscope, the punctate aggregates in the mitochondria of the A549 cells treated with IL-8 were observed.CONCLUSION: The migratory rate of A549 cells is increased by IL-8, which is related to the changes of mitochondrial fusion genes and the fission genes.     

Inhibitory effect of butylphthalide on apoptosis of hippocampal neurons in Alzheimer disease rats via SIRT1/NF-κB signaling pathway

AIM: To investigate the effect of butylphthalide on apoptosis of hippocampal neurons in Alzheimer disease (AD) rats via SIRT1/NF-κB signaling pathway and its mechanism. METHODS: AD rat model was established by intragastric administration of AlCl₃ and intraperitoneal injection of D-galactose. After treated with butylphthalide at 25 mg/kg (low dose), 50 mg/kg (medium dose) and 100 mg/kg (high dose), the effects of butylphthalide on the morphology of hippocampal neurons, apoptosis rate, and the protein levels of Bcl-2, Bax, cleaved caspase-3 and the SIRT1/NF-κB signaling pathway associated proteins were determined by HE staining, flow cytometry and Western blot, respectively. After treated with SIRT1 agonist SRT1720 and inhibitor sirtinol, the role of SIRT1/NF-κB signaling pathway in hippocampal neuronal apoptosis was observed. On the basis of giving 50 mg/kg butylphthalide, sirtinol was administered, and the effect of butylphthalide on neuronal apoptosis regulated by SIRT1/NF-κB signaling pathway was evaluated. RESULTS: The morphology of hippocampal neurons in the AD rats were improved, the apoptosis rate of hippocampal neurons and the protein levels of Bax and cleaved caspase-3 were inhibited, and the protein levels of Bcl-2 and the activation of SIRT1/NF-κB signaling pathway were promoted by butylphthalide significantly (P<0.05). The protein expression of Bcl-2 and the activation of SIRT1/NF-κB signaling pathway were promoted, and the apoptosis of hippocampal neurons and the protein levels of Bax and cleaved caspase-3 were inhibited by SRT1720 remarkably (P<0.05), whereas the effect of sirtinol was contrary to that of SRT1720. After sirtinol treatment, the inhibitory effect of butylphthalide on apoptosis of hippocampal neurons, the protein levels of Bax and cleaved caspase-3, and the promotion of Bcl-2 protein expression in hippocampal neurons were markedly weakened (P<0.05). CONCLUSION: Butylphthalide inhibits the apoptosis of hippocampal neurons in the AD rats by down-regulating the protein expression of Bax and cleaved caspase-3, and up-regulating the protein expression of Bcl-2 through activating SIRT1/NF-κB signaling pathway.     

Effect of low molecular weight heparin on hypoxia-induced apoptosis in primary cultured neonate rat cerebral cortical neurons

AIM:To investigate the mechanism by which low molecular weight heparin (LMWH) inhibits apoptosis of primary cultured cerebral cortical neurons caused by hypoxia. METHODS:The anti-apoptosis effect of LMWH on primary cultured neurons was observed by methods of the primary culture of cerebral neurons of postnatal rats in free-serum with neurobasal medium supplied with 2% B27 supplement, hypoxic culture of neurons, Hoechst 33342 staining and immunocytochemistry. RESULTS:At concentrations of 250-500 U/L, LMWH reduced apoptosis rate of cerebral cortical neurons induced by hypoxia (P<0.05) and apoptosis rate was lower in LMWH groups than that in BDNF (50 μg/L) group (P<0.05). LMWH (500 U/L) increased Bcl-2 protein expression (P<0.05) and decreased Bax protein expression (P<0.01) in the cerebral cortical neurons induced by hypoxia, the ratio of Bcl-2/Bax was improved (P<0.01). The ratio of Bcl-2/Bax in LMWH (500 U/L) group was higher than that in normal control group, BDNF group and apoptosis positive group (P<0.05). CONCLUSION:LMWH at concentrations of 250-500 U/L is able to prevent cerebral cortical neurons from apoptosis in primary culture under hypoxia. The effect of anti-apoptosis may be related to up-regulation of Bcl-2 protein expression, down-regulation of Bax-2 protein expression, and increase in the ratio of Bcl-2/Bax.     

Effects of Panax quinquefolium saponin on mitochondrial membrane potential and cardiomyocyte apoptosis in ischemia/reperfusion rats

AIM：To investigate whether mitochondrial membrane potential (ΔΨm) and the mitochondrial apoptotic pathway are involved in the protective mechanism of Panax quinquefolium saponin (PQS) against cardiomyocyte apoptosis after ischemia/reperfusion (I/R) injury in rat myocardium. METHODS：Ninety healthy male SD rats were randomly divided into sham group, I/R group, PQS (200 mg·kg-1·d-1) +I/R group, cyclosporine A (CsA) group, CsA (10 mg·kg-1) +I/R group and PQS +CsA +I/R group. The model of myocardial I/R injury in vivo was established by ligating the left anterior descending artery (LAD) for 30 min followed by 120 min of reperfusion in the rats. The serum activity of lactate dehydrogenase (LDH) was measured by automatic chemistry analyzer. The myocardial infarct size was measured by Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining. Cardiomyocyte apoptosis was detected by in situ TDT-mediated dUTP nick end labeling (TUNEL). The protein levels of Bcl-2, Bax, cleaved caspase-3 and cytosolic cytochrome C were determined by Western blotting. ΔΨm was measured by laser scanning confocal microscopy and fluorescence microplate reader. RESULTS：Compared with I/R group, the serum content of LDH,the infarction size in PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group and the myocardial apoptotic index were decreased. Compared with I/R group, the fluorescence intensity of mitochondria after JC-1 staining was enhanced in PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group, and the relative fluorescence units (RFU) of ΔΨm were improved in those 3 groups. In PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group, the protein expression of Bcl-2 was increased, and that of Bax was decreased compared with I/R group. Moreover, in those 3 groups, the protein levels of cleaved-caspase-3 and cytosolic cytochrome C were decreased compared to I/R group, respectively. CONCLUSION：PQS attenuates myocardial injury and cardiomyocyte apoptosis during I/R, and the protective mechanisms of PQS were associated with the modulation of ΔΨm and the inhibition of mitochondrial apoptosis pathway.     

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 soybean isflavones on mitochondrial damage and neuronal apoptosis induced by cerebral ischemia/reperfusion

AIM: To study the effects of soybean isoflavones on mitochondrial ultrastructure, neuronal apoptosis and expression of cytochrome C, caspase-9 and caspase-3 in the rats with cerebral ischemia/reperfusion.METHODS: Adult healthy SD rats (n=60) were randomly divided into 3 groups: sham group, ischemia/reperfusion injury (I/R) group and soybean isoflavone (SI) pretreatment group. Soybean isoflavones (120 mg·kg-1·d-1) were fed by gastric lavage for 21 d. The global ischemia/reperfusion model of the rats was established by blocking 3 vessels, and then reperfused for 1 h after 1 h of ischemia. The morphological change of the cerebral cortex cells was observed under light microscope. The mitochondrial ultrastructure of the cerebral cortex cells was determined by transmission electron microscope. The apoptotic rate of the cerebral cortex cells was detected by flow cytometry. The expression of cytochrome C, caspase-9 and caspase-3 in the cerebral cortex cells was determined by semi-quantitative RT-PCR and immunohistochemical techniques.RESULTS: Disintegration of mitochondria membrane and disappearance of the mitochondrial cristae were seen in I/R group. Compared with I/R group, the change of ultrastructure of mitochondria was significantly improved by soybean isoflavone pretreatment, and the neuronal apoptotic rate was also significantly decreased (P<0.01). The mRNA expression and protein content of cytochrome C, caspase-9 and caspase-3 in I/R group were obviously higher than those in sham group (P<0.01). Compared with I/R group, the mRNA expression and protein content of cytochrome C, caspase-9 and caspase-3 in SI group were significantly decreased (P<0.01).CONCLUSION: Soybean isoflavones attenuate cerebral ischemia/reperfusion injury by stabilizing the structure of mitochondria, preventing cytochrome C release to the cytoplasm, inhibiting the activation of caspase-9 and caspase-3 and decreasing cell apoptosis．     

Effect of salidroside on mitochondrial membrane potential during injury induced by hypoxia/hypoglycemia in cultured SH-SY5Y cells

AIM: To investigate the effects of salidroside on intracellular free calcium concentration [Ca²⁺]i, apoptosis, mitochondrial membrane potential (MMP) and activity during injury induced by hypoxia/hypoglycemia in cultured SH-SY5Y cells. METHODS: Mitochondrial activity was measured by methylthiazolyl tetrazolium test. MMP,[Ca²⁺]i and apoptosis were measured by flow cytometry. RESULTS: SH-SY5Y cells were cultured in a hypoxia/hypoglycemia condition for 2, 4, 6 and 12 h,[Ca²⁺]i and apoptosis rate significantly increased compared with control group (P<0.01). After hypoxia /hypoglycemia cultures, MMP and mitochondrial activity declined 29.17% (P<0.01) and 38.80% (P<0.01) at 2 h, 56.72% (P<0.01) and 63.58% (P<0.01) at 12 h, were lower than that in control group (P<0.01). Salidroside significantly decreased [Ca²⁺]i and apoptosis rate, and increased MMP and mitochondrial activity in hypoxia /hypoglycemia-treated SH-SY5Y cells. CONCLUSIONS: Salidroside might inhibit the decline in MMP and mitochondrial activity induced by hypoxia /hypoglycemia, and has an inhibitory effects on neuronal apoptosis. The mechanism might be related to inhibiting intracellular calcium overload.     

Effect of senegenin on H2O2-induced damage in hippocampal neurons of SD rats

AIM: To observe the effect of senegenin (Sen) on hippocampal neuron injuries induced by H₂O₂.METHODS: Hippocampal neurons were isolated from neonatal SD rats. The primarily cultured neurons were divided into control group, H₂O₂ group, Sen group and Sen+H₂O₂ group. The cell viability, the content of malondialdehyde(MDA) and the activity of superoxide dismutase(SOD) in the neurons were detected after treated with Sen. The morphological changes of nucleus of the neurons were observed by Hoechst 33258 staining. The mRNA expression of bcl-2 and bax was quantified by real-time PCR. The protein levels of Bcl-2 and bax were measured by Western blotting. The activity of caspase-3 was also assayed.RESULTS: Compared with H₂O₂ group, the levels of antioxidative enzyme were increased in Sen+H₂O₂ group (P<0.05). In addition, mRNA expression of bcl-2 increased and that of bax decreased (P<0.05) in Sen+H₂O₂ group. Moreover, Sen increased the protein level of Bcl-2, and reduced the protein level of Bax and the activity of caspase-3 in the neurons exposed to H₂O₂ (P<0.05).CONCLUSION: The protective effect of Sen on hippocampal neurons with H₂O₂ -induced injury may be involved in the mechanisms of     

Effect of SAPK/JNK signal pathway on neuronal apoptosis in cerebral infarction rats

AIM: To investigate the molecular mechanism of neuronal apoptosis by observing the changes of key proteins in SAPK/JNK and Bcl-2/Bax signal pathways after brain infarction. METHODS: The cortical infarction was induced by photochemistry, namely photothrombotic cortical injury (PCI). Thirty-six Sprague-Dawley rats were randomly divided into 2 groups: PCI group and sham-operated group. The ipsilesional cortex was harvested for histomorphometry and transmission electron microscopy 7 days after PCI. Some key proteins including p-JNK1, p-JNK2, p-c-Jun, p-ATF-2, total JNK1, total JNK2, Bcl-2 and Bax were detected by Western blotting analysis.RESULTS: The cortical infarction in rats was successfully induced by photochemistry. The apoptosis of neurons in cortex was more obvious in PCI group than that in sham-operated group 7 days after PCI. The levels of p-JNK1, p-JNK2, p-c-Jun and p-ATF-2 in PCI group were significantly higher than those in sham-operated group, whereas the ratio of Bcl-2/Bax was significantly lower(P<0.05). CONCLUSION: Apoptosis is a major contributor to neuronal loss induced by cerebral hypoxia-ischemia for a long period after cortical infarction. The process is related to some apoptotic proteins such as Bcl-2/Bax and the SAPK/JNK signal pathways activated by ischemic injury.     

Effect of AMPKα2 gene silencing by shRNA recombinant plasmid on chloride-mediated anoxia/reoxygenation injury in rat cardiomyocytes

AIM: To explore the role of AMP-activated protein kinase α2 subunit (AMPKα2) gene in chloride-mediated anoxia/reoxygenation (A/R) injury by transfection of short-hairpin RNA (shRNA) expression vector targeting to AMPKα2 gene into H9c2 cardiomyocytes. METHODS: Recombinant shRNA expression vector pSuper-AMPKα2 targeting to AMPKα2 gene was constructed and transfected into H9c2 cardiomyocytes. The protein expression of AMPKα2 was determined by Western blotting. The cells were divided into 5 groups: control group, A/R group, Cl^--free A/R group, pSuper+Cl^--free A/R group and pSuper-AMPKα2 shRNA+Cl^--free A/R group. After treatment, the cell viability was detected by MTT assay. LDH activity was analyzed with an automatic biochemical analyzer. The apoptotic rate and the level of intracellular ROS was measured by flow cytometry. The activity of SOD and GSH-Px was analyzed by a colorimetric method. RESULTS: The result of sequencing proved that the recombinant plasmid pSuper-AMPKα2 shRNA was correctly constructed. The protein level of AMPKα2 significantly decreased after the plasmid was transfected into the cardiomyocytes. Compared with A/R group, the cell viability and the activity of SOD and GSH-Px were significantly increased, while the activity of LDH, apoptotic rate and ROS production were significantly decreased in Cl^--free A/R group. The protective effect of Cl^--free solution on the A/R-induced injury of cardiomyocytes was abolished, and the ROS production was increased and the activity of SOD and GSH-Px was decreased after the cells were transfected with pSuper-AMPKα2 shRNA. CONCLUSION: Recombinant plasmid pSuper-AMPKα2 shRNA is successfully constructed, and silencing of AMPKα2 gene abolishes the protective effect of Cl^--free solution on A/R injury.