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

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

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 NS-398 on the proliferation and apoptosis of HepG2 cells

AIM: To evaluate the effects of NS-398, a cyclooxygenase-2(COX-2) inhibitor, on the proliferation and apoptosis in HepG2 cells. METHODS: The effects of NS-398 on the proliferation of HepG2 cells was evaluated by MTT. DNA fragmentation gel analysis was used to analyze the apoptotic cells; DNA ploidy and apoptotic cell percentage were examined by flow cytometry. Furthermore, the expression of COX-2 and Bcl-2 mRNA was identified by competitive RT-PCR. RESULTS: NS-398 inhibited cell proliferation and induced apoptosis in HepG2 in a concentration-dependent manner. DNA ploidy analysis showed that S phase cells were significantly decreased with NS-398 concentration increasing. The quiescent G₀/G₁ phase was accumulated with decreasing of Bcl-2 mRNA. Whereas NS-398 had no effect on the expression of COX-2 mRNA, no correlations were found between COX-2 mRNA and the HepG2 cell proliferation and apoptosis induced by NS-398 (r=0.056 and r=0.119, respectively). CONCLUSION: NS-398 significantly inhibits the proliferation and induces apoptosis in HepG2. Mechanisms may be involved in accumulation of quiescent G₀/G₁ phase and decrease in Bcl-2 mRNA expression, but independent to COX-2 mRNA expression.     

Inhibitory effects of a cyclooxygenase-2 inhibitor,NS398,on cancer cells

AIM:To study the mechanism of growth inhibitory effect of a selective cyclooxygenase-2 (COX-2) inhibitor,NS-398,on cancer cells.METHODS:The esophageal cancer cell line (EC9706),which expresses COX-2 constitutively,and hepatocellular carcinoma cell line (SMMC7721),which expresses no COX-2,were studied.The cell lines were incubated with NS-398 at doses of 10,20,50,100 μmol/L for 24 h,48 h and 72 h.Antiproliferation effect was measured by ［3H］-TdR incorporation.The cell apoptosis were determined by flow cytometry (FCM) and DNA fragmentation analysis.Survivin was detected by immunocytochemical technique.RESULTS:The growth inhibition was induced by NS398 in a dose- and time-dependent manners in both cell lines.FCM analysis revealed a high sub-G1 cell peak in EC9706 group and agarose electrophoresis showed marked apoptosis ladder pattern.However,no apoptosis was observed in SMMC7721 cells treated with NS-398.The difference of apoptosis percentage in EC9706 and SMMC7721 was (45.23±1.08)% and (3.05±0.15)% (P<0.01).After 24 h incubation with NS-398 at concentration of 100 μmol/L,the expression of survivin was markedly reduced in EC9706,no change was observed in SMMC7721.CONCLUSION:NS-398 suppresses cell growth in cancer cell lines by different mechanism.NS-398 suppresses cell growth and increases apoptosis in the cancer cells that expresses COX-2.     

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.     

Influence of normal conditioned medium of astrocytes on the damaged neurons of cerebral hypoxia and reoxygenation in vitro

AIM: To study the nutritional role of the normal conditioned medium of astrocytes (NCMA) on the injured cerebral cortex neurons induced by cerebral hypoxia and reoxygenation in vitro. METHODS: The normal and damaged neurons induced by hypoxia and reoxygenation were cultured with normal conditioned medium of astrocytes (NCMA) after the cerebral cortical astrocytes and neurons of rats were isolated. The viability and survival rate of cultured neurons were investigated by MTT method. RESULTS: NCMA increased the viability and survival rate of cultured neurons. CONCLUSION: NCMA has nutritional and supporting roles on cultured neurons.     

Tert-butyl hydroperoxide damages mitochrondria and induces apoptosis in cortical neurons

AIM: To explore the possible mechanism of tert-butyl hydroperoxide (t-BHP)-induced apoptosis in rat cortical neurons. METHODS: Primary cultured rat cortical neurons were performed in vitro and cell viability was measured by MTT assay. DNA fragmentation was used to evaluate cell apoptosis and mitochondrial transmembrane potential (ΔΨm) was determined by flow cytometric assay. Cellular glutathione (GSH) content was measured by spectrophotometer. Bcl-2 and Bax protein, cytosolic cytochrome c, cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were detected by Western blotting. RESULTS: After exposure of cortical neurons to tBHP (25-400 μmol/L), the cell viability was reduced. ΔΨm and cellular GSH content were also decreased significantly. The level of Bcl-2 protein was reduced and Bax was elevated. Meanwhile, tBHP exposure resulted in cytochrome c release, caspase-3 and PARP proteolysis, DNA fragmentation and eventually neuron apoptosis. CONCLUSION: Mitochondrial damage may mediate tBHP- induced apoptosis in cortical neurons.     

Effect of glycine liposomes on mitochondrial membrane potential and apoptosis in cultured cardiomyocytes

AIM: To observe the effect of glycine liposomes on the mitochondrial membrane potential and the apoptosis rate in cardiomyocytes induced by hypoxia/reoxygenation injury. METHODS: A cardiomyocyte injury model was established by using hypoxia/reoxygenation. DiOC6(3) as fluorescence molecular probe was used to detect the mitochondrial membrane potential in each group. The method of Annexin V associated with PI was used to detect the apoptosis ratio in each group. RESULTS: (1) The result of flow cytometry showed that the mitochondrial membrane potential of cardiomyocytes in H/R group was obviously lower than that in control group (P<0.01). The decrease in mitochondrial membrane potential in Gly-liposome group was the lowest, the percentage of cells about the part of hypofluorescence was (9.61±0.76)%, which was lower than that in glycine group (P<0.01). (2) The apoptosis rate of cardiomyocytes in H/R group was higher than that in control group (20.78±1.58)%,P<0.01. After the treatment of Gly-liposome, the apoptosis rate of cardiomyocytes was lower than that in glycine group (P<0.01). No difference in the apoptosis ratios between blank-liposome group and H/R group was observed(P>0.05).CONCLUSION: Glycine liposomes protect cultured cardiomyocytes against hypoxia/reoxygenation injury. Glycine liposomes produce the better protective effects than glycine.     

Effect of Ginsenoside Rb1 on apoptosis induced by hypoxia/hypoglycemia and reoxygenation in cultured rat hippocampal neurons

AIM: The aim of this work is to investigate the protective effects of Ginsenoside Rb₁ (Rb₁) on apoptosis induced by hypoxia/hypoglycemia and reoxygenation in cultured rat hippocampal neurons. METHODS: Apoptosis were measured by flow cytometry; Morphological changes and neuronal necrosis were examined under microscope; The leakage of lactic dehydrogenase (LDH) and the product of nitric oxide (NO) were measured. RESULTS: hypoxia/hypoglycemia cultures for 5 h and reoxygenation induced neuronal apoptosis and necrosis,and significantly increased the leakage of LDH and the product of NO. The effects were enhanced with the extending time of reoxygenation. Rb₁ could significantly decrease the percentage of neuronal apoptosis and necrosis, and reduce the leakage of LDH and the product of NO. CONCLUSION: Rb₁ had an effect of anti-neuronal apoptosis. This effect might be related to the inhibition of the activity of NO synthase and NO production.     

Role of Nrf2-ARE signaling pathway in protective effect of hypoxia or pinacidil postconditioning against hypoxia-reoxygenation injury in adult rat cardiomyocytes

AIM：To investigate whether nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is involved in the protective effect of hypoxia or pinacidil postconditioning against hypoxia-reoxygenation injury in adult rat cardiomyocytes. METHODS：Cardiomyocytes were isolated from the left ventricle of male adult Sprague-Dawley rats (250~300 g) by Langendorff perfusion and collagenase II digestion. The cells were randomly divided into six groups (n=8 in each group). The cells in N group were cultured for 22 h without any treatment. The cells in the other five groups were cultured for 20 h and then underwent 45 min of hypoxia followed by 60 min of reoxygenation (M group), three 5-min cycles of reoxygenation/hypoxia plus 60 min of reoxygenation (IPO group), or treatment with pinacidil (25, 50 and 100 μmol/L) for 5 min plus 60 min of reoxygenation (P25, P50 and P100 groups, respectively). The expression of Nrf2, NAD(P)H:quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1) and superoxide dismutase 1 (SOD1) at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. RESULTS：The mRNA and protein levels of Nrf2, HO-1, NQO1 and SOD1 in M, IPO, P25, P50 and P100 groups were significantly decreased compared with N group (P＜0.05), and those in IPO, P25, P50 and P100 groups were obviously higher than those in M group (P＜0.05). The mRNA levels of Nrf2, HO-1, NQO1 and SOD1 in IPO and P50 groups were obviously higher than those in P25 and P100 groups (P＜0.05). The mRNA expression of SOD1 in P25 group was obviously higher than that in P100 group (P＜0.05). The protein levels of Nrf2, NQO1 and SOD1 in IPO and P50 groups were obviously higher than those in P25 and P100 groups (P＜0.05). The protein expression of HO-1 in P50 group was obviously higher than that in IPO, P25 and P100 groups (P＜0.05). The protein levels of HO-1 and NQO1 in P25 group were obviously higher than those in P100 group (P<0.05). CONCLUSION：Hypoxia or pinacidil postconditioning may attenuate hypoxia-reoxygenation injury in adult rat cardiomyocytes via activation of Nrf2-ARE signaling pathway.     

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.     

Effect of L-carnitine on apoptosis and oxidant damage of cardiomyocytes induced by hypoxia/reoxygenation in vitro

AIM: To examine the effects of L-carnitine on apoptosis and oxidant injury in cultured neonatal rat cardiomyocytes induced by hypoxia/reoxygenation and its possible mechanism. METHODS: The cultured cardiomyocytes were divided into three groups， control， A/R group （anoxia for 120 min, reoxygenation for 240 min） and L-carnitine treatment group, in which cells were exposed to 20 mg/L, 50 mg/L, 100 mg/L, 200 mg/L L-carnitine respectively at 2 h before anoxia. The superoxide dismutase (SOD), succinate dehydrogenase (SDH) activities and malondialdehyde (MDA) content were examined, and the apoptosis was determined by flow of cytometry (FCM). In addition, the ultrastructure was observed by transmission electron microscopy. RESULTS: In A/R group, SOD and SDH activities were lower, the apoptosis rate and MDA content were higher than those in control group (P<0.05). In L-carnitine treatment group, SOD and SDH activities were higher, the apoptosis rate and MDA content were lower than those in A/R group, a L-carnitine concentration-dependent effect was found. Moreover, impairment of myocardial ultrastructure was more severe in A/R group than L-carnitine treatment group. CONCLUSION: L-carnitine can protect cardiomyocytes against hypoxia/reoxygenation-induced injury in a dose-dependent manner.     

ZFP580, a novel transcription factor,is involved in cardioprotection of hypoxic preconditioning against hypoxia-reoxygenation injury in myocardial cells

AIM：To elucidate whether ZFP580 is involved in the cardioprotective effects of hypoxic preconditioning (HPC) against hypoxia-reoxygenation (H/R) injury in H9c2 myocardial cells. METHODS：Rat heart-derived H9c2 cells were cultured in DMEM. H/R was induced by incubation under ischemic hypoxia for 3 h and reoxygenation for 2 h. HPC was induced by exposing the H9c2 cells to 10 min of hypoxia and 20 min of reoxygenation for 3 cycles before H/R treatment. MTT staining and LDH leakage detection were used to evaluate the effects of HPC. Western blotting was used to detect the protein levels of ZFP580, phosphorylated ERK1/2 and cleaved caspased-3. The effects of ZFP580 overexpre-ssion or knockdown on H/R induced apoptosis were determined. RESULTS：The results of MTT staining and LDH leakage detection showed evidence of HPC cytoprotection against H/R-induced cell death in H9c2 cells. ZFP580 protein level and ERK1/2 phosphorylation were significantly increased in the HPC group compared with control group and H/R group. PD98059, an inhibitor of ERK1/2 phosphorylation, significantly suppressed the HPC-induced up-regulation of ZFP580 protein expression. ZFP580 overexpression significantly inhibited apoptosis and caspase-3 activation in H9c2 cells. CONCLUSION：HPC exhibits cytoprotection against H/R and leads to high level of ZFP580 protein in H9c2 cells. ZFP580 is regulated by ERK1/2 activation and mediates the anti-apoptotic effect of the ERK1/2 signaling pathway in HPC cytoprotection.     

Astragalus injection inhibits the expression of JNK3 mRNA after hypoxia/hypoglycemia and reoxygenation in hippocampal neurons of rats

AIM: To investigate the effect of astragalus injection on the expression of c-jun N terminal kinase (JNK3) mRNA interrelated with apoptosis after hypoxia/hypoglycemia and reoxygenation in hippocampal neurons of rats. METHODS: The hippocampal neurons cultured for eight days were divided into 4 groups: normal control group, original astragalus injection group, hypoxia/hypoglycemia and reoxygenation group, astragalus injection group. Hypoxia/ hypoglycemia and reoxygenation group, astragalus injection group and original astragalus injection group were treated with hypoglycemia and reoxygenation after deprived of oxygen and glucose for 30 min. Methods of in situ hybridization and RT-PCR were used respectively to measure the expression of JNK3 mRNA after hypoxia/hypoglycemia and reoxygenation 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. RESULTS: In normal control group the volume of hippocampal neuronal nucleolus was accretion, cellular tuber was distinct and cytokinesis was dyed by yellow a lot. In hypoxia/hypoglycemia and reoxygenation group the hippocampal neuronal nucleolus was crimple, cellular tuber was shrinked, large number of cytokinesis was dyed by yellow and yellow granule was observed. Compared with normal control group, the numbers of JNK3 mRNA positive neuronal cells at each time point increased obviously in the hypoxia/hypoglycemia and reoxygenation group (P<0.05). The change of neuronal configuration and the numbers of JNK3 mRNA positive neuronal cells in original astragalus injection group accorded with hypoxia/ hypoglycemia and reoxygenation group (P>0.05). In astragalus injection group the hippocampal neuronal nucleolus was crimple slightly and segmental cytokinesis was dyed by yellow.Compared to hypoxia/hypoglycemia and reoxygenation group, the numbers of JNK3 mRNA positive neuronal cells at each time point were less obviously in the astragalus injection group besides 120 h (P<0.05). Compared to normal control group, the mean optic density of expression of JNK3 mRNA in hippocampal neurons of rats at each time point increased obviously in hypoxia/ hypoglycemia and reoxygenation group (P<0.05). Compared to hypoxia/hypoglycemia and reoxygenation group, the mean optic density of JNK3 mRNA expression at each time point in original astragalus injection group had no obvious change (P>0.05), however the mean optic density of JNK3 mRNA expression in hippocampal neurons of rats at each time point decreased obviously in the astragalus injection group besides 120 h (P<0.05). CONCLUSION: Astragalus injection inhibits the expression of JNK3 mRNA after hypoxia/hypoglycemia and reoxygenation, accordingly inhibits hippocampal neuronal apoptosis.     

Effects of adenosine A1 receptor antagonist DPCPX on cerebral neurons after hypoxia and reoxygenation

AIM: To investigate the effects of adenosine A₁ receptor antagonist DPCPX on the release of cerebral neuronal lactate dehydrogenase (LDH), the activity of calcineurin (CaN) and acetylcholinesterase (AChE), and the level of extracellular amino acid after hypoxia and reoxygenation (H/R).METHODS: Primary cultured rat cerebral cortical neurons were used to establish an H/R injury model. Different concentrations of DPCPX (the final concentrations were as follows: 0, 25, 50 and 100 nmol/L) were added at the same time of hypoxia treatment for 8 h, 12 h or 24 h,followed by reoxygenation for 24 h. The LDH release from the neurons was measured. The effects of DPCPX (100 nmol/L) on the activity of CaN and AChE, and the level of extracellular amino acid in neurons treated with hypoxia for 12 h followed by reoxygenation were observed. RESULTS: Compared to the cells in control groups, the neurons treated with 100 nmol/L DPCPX and exposed to hypoxia for 12 h followed by reoxygenation, showed significantly higher LDH release, higher activity of CaN and AChE, and lower level of extracellular γ-aminobutyric acid.CONCLUSION: These results suggest that DPCPX increases the LDH release and the activity of CaN and AChE, decreases the level of extracellular γ-aminobutyric acid in neurons with H/R.     

Ectopic hCG in endometrial carcinoma and its effect on COX-2 expression

AIM: To investigate the effect of human chorionic gonadotropin(hCG) on cyclooxygenase-2(COX-2) expression in endometrial carcinoma so as to study the function of ectopic hCG.METHODS: The ectopic β-hCG in JEC endometrial carcinoma cell lines was quantified by radioimmunoassay. By MTT assay, the effect of hCG on the cell viability of JEC cell lines and the inhibition of selective COX-2 inhibitor NS398 on JEC cell lines were examined. The effect of hCG on COX-2 expression was detected by Western blotting. RESULTS: The ectopic β-hCG release from cultured JEC cell lines were observed. HCG promoted the cell viability, upregulated the expression of COX-2 protein and increased the inhibition of selective COX-2 inhibitor NS398 in JEC cell lines. CONCLUSION: The ectopic hCG in JEC endometrial carcinoma cell lines increases cell proliferation, which may be mediated by upregulating the expression of COX-2 protein.     

Role of prostaglandin E2 receptor 1 in neuronal cell death induced by hypoxia on rat cortical neurocytes

AIM: To observe the effects of prostaglandin E2 receptor1 (EP1) in neuronal cell death induced by hypoxia/re-oxygenation and ischemia/reperfusion. METHODS: The cortical neurocytes of neonatal Wistar rats were cultured for 12 days and exposed to hypoxia/re-oxygenation to establish a hypoxia/re-oxygenation model. Another set of cultured primary neonatal cortical neurocytes of rats were pre-treated with 17-pt (an antagonist of EP1), then underwent hypoxia for 3 h, re-oxygenated for 21 h. MTT reagent was added 1 h before measuring the cell viability. Neuron apoptosis was determined by flow cytometry. The protein expression was examined by Western blotting. RESULTS: Compared to the control cells (only underwent hypoxia /re-oxygenation and without any pretreatment), the neurons pretreated with 17-pt and then underwent hypoxia/re-oxygenation showed significantly lower survival rate (P<0.05) and increased expression level of caspase-3 and neuron apoptosis. CONCLUSION: EP1 is involved in the pathogenesis of neuron death induced by hypoxia/ischemia and may be a new target for treating ischemic stroke.     

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

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

Injury pathways of hypoxia/reoxygenation in AC16 cardiomyocyte

AIM:To investigate the effects of hypoxia/reoxygenation (H/R) for different reoxygenation times on cardiomyocyte injury. METHODS:Human cardiomyocyte AC16 was cultured in glucose-free and serum-free DMEM with 1% O₂ for 24 h, 10% fetal bovine serum and low glucose DMEM combined with 21% O₂ were used to establish reoxygenation for 2 h, 6 h and 12 h, respectively. The cell viability was measured by CCK-8 assay. The protein levels of different cell injury pathway related molecules, such as LC3-Ⅱ/-I (autophagy), caspase-1 and gasdermin D (pyroptosis) and caspase-3 and Bax/Bcl2 (apoptosis), were determined by Western blot. RESULTS:Compared with blank control group, the cell viability in each H/R group was continuously decreased with the extension of reoxygenation time (P<0.05). The expression of LC3-Ⅱ/-I was up-regulated in hypoxia group and H/R group compared with blank control group (P<0.05). In addition, the protein levels of cleaved caspase-1 and cleaved gasdermin D were increased in H/R groups for 6 h and 12 h, respectively (P<0.05). Cleaved caspase-3 and Bax/Bcl2 were increased after reoxygenation for 12 h (P<0.05). CONCLUSION:Autophagy in hypoxia-induced AC16 cells is up-regulated, and then decreased by reoxygenation. The cell pyroptosis is activated earlier than the apoptosis during reoxygenation.     

The characteristics of hypertrophic cardiomyocyte injury and the effect of intervention on energy metabolism after different time of hypoxia/reoxygenation

AIM:To investigate the characteristics of pathological injury and its relationship with the transformation of energy metabolism of hypertrophic cardiomyocytes after hypoxia-reoxygenation. METHODS:Cultured rat cardiomyocytes were induced to be hypertrophy by angiotensin Ⅱ (Ang Ⅱ) and norepinephrine (NE). Glucose oxidation rate (GOR), glucolysis rate (GLR) and fatty acid oxidation rate (FOR) were determined by liquid scintillation counting, and cell apoptosis was detected by TUNEL. RESULTS:(1) Compared with the normal cardiomyocytes (NC), the GOR and GLR were slightly higher and the FOR was slightly lower in the group of hypertrophic cardiac cells (HC) than that in the group of normal cardiomyocytes cultured under the normal oxygen partial pressure. The apoptosis rate had no difference between the two groups. (2) The apoptosis rate of hypertrophic cardiomyocytes after hypoxia was significantly higher than that of hypertrophic cardiomyocytes in normal culture. It was higher and moreover, some necrosis cardiomyocytes appeared after reoxygenation. (3) GOR and FOR in both group (NC and HC) were slightly lower in a time-dependent manner after hypoxia than that in each group in normal culture condition. GLR had no difference in both group. The GOR was more lower in both NC and HC group when reoxygenation than that at the point of hypoxia for 2 hours, but the GLR and FOR were significantly higher in HC than that in NC when reoxygenation. (4) The GOR was significantly higher and the GLR and FOR were significantly lower in the hypertrophic cardiomyocytes group (HC) with dichloroacetate (DCA, 1 000 μmol/L) or trimetazidine (TMZ, 1 μmol/L) treated respectively than that in the responded hypertrophic cardiomyocytes after stimulation by hypoxia-reoxygenation. In the meanwhile, the apoptosis rate also was markedly lower in the treated hypertrophic cardiomyocytes group. CONCLUSION:The transformation of energetic metabolism pathway plays an important role in the pathogenesis (mainly the apoptosis) of the hypertrophic cardiomyocytes after hypoxia-reoxygenation.