Effect of CsA on mitochondria stress after microperfusing glutamate and calcium into tree shrews’ hippocampus

AIM: To observe the changes of glutamate and calcium within the hippocampal microenvironment in mitochondrial stress.METHODS: A lateral hippocampus was microperfused with glutamate and calcium chloride solution by a kind of single-pumped push-pull perfusion system in Tree Shrews. At 24 h, the expression of cytochrome C (Cyt C)was observed by immunochemistry. Also, the hippocampus was removed, then mitochondria and cytoplasmic fragment were divided by low temperature centrifugation and the distribution of cytochrome C was assessed through Western blotting. The relative amounts of caspase-3 and caspase-9 mRNA were evaluated by real time fluorescence polymerase chain reaction. In the treated group, cyclosporin A (CsA，40 mg/kg) was intravascularly injected at 6 h after perfusion of glutamate-calcium chloride solutions into the hippocampus and inspected the above-mentioned items at 24 h. RESULTS: In the glutamate-calcium group, compared with the control group, cytochrome C immunoreactivity increased and the content of hippocampal mitochondrial cytochrome C decreased. Also, the cytochrome C was detected in cytosol. Cyclosporin A treatment at 6 h after microperfusion, the cytochrome C expression weakened and no Cyt C in cytosol fraction was observed. By real time PCR, in relation to the control group, the caspase-3 and caspase-9 mRNA was higher in the glutamate-calcium group. Cyclosporin A treatment cut down both caspase-3 and caspase-9 mRNA contents. CONCLUSION: The accumulation of glutamate and calcium may promote Cyt C release, caspase cascade activation and the mitochondrial stress. The neuroprotection of CsA may results from uniquely inhibiting the mitochondrial permeability transition pore, and preventing Cyt C release and caspase activation.     

Et-DHA induces apoptosis of HepG2 cells through activation of mitochondrial pathway and induction of granzyme B expression in T cells

AIM：To investigate the effect of ethyl docosahexaenoate (Et-DHA) on the apoptosis of human hepatocarcinoma HepG2 cells. METHODS：HepG2 cells were used to test the anticarcinogenicity of Et-DHA. The direct inhibition of HepG2 cells by Et-DHA was detected by MTT. Nuclear morphological features of the HepG2 cells were observed under fluorescence microscope after staining with Hochest 33258. The levels of Bax, Bak, Bid, Bcl-2, Smac and cytochrome C (Cyt C) in mitochondria and cytosol, the cleaved caspase-8, cleaved caspase-9, and cleaved caspase-3 in cytosol, as well as the release of reactive oxygen species (ROS), total superoxide dismutase (SOD) and caspase-9 activity in the Et-DHA-treated HepG2 cells were determined by Western blotting and ELISA. Furthermore, by co-culturing the HepG2 cells with T cells, the effects of proliferation of Et-DHA-treated T cells on the activity of HepG2 cells were observed, and the level of granzyme B was detected. RESULTS：Et-DHA significantly inhibited the growth of HepG2 cells in a concentration- and time-dependent manner. The ROS release and caspase-9 activity increased markedly in Et-DHA-treated HepG2 cells, and no significant change of the total SOD activity was observed. The levels of the pro-apoptotic proteins Bax, Bak and Bid in mitochondria increased, the anti-apoptotic protein Bcl-2 as well as mitochondrial Cyt C and Smac levels decreased, and the cytoplasmic Cyt C, Smac, cleaved caspase-8, cleaved caspase-9, cleaved caspase-3 and cleaved Bid levels showed dose-dependent increases. Additionally, the degree of Et-DHA-induced apoptosis in HepG2 cells in the co-culture group (T cells+HepG2 cells) showed a further increase as compared with the HepG2 cells treated with Et-DHA alone. Due to Et-DHA inducing elevation of granzyme B level in the T cells, the granzyme B released into HepG2 cells was significantly increased. CONCLUSION:Et-DHA might induce the apoptosis of HepG2 cells through activation of caspase-3 mainly via a mitochondrial intrinsic pathway and a caspase-8 pathway, and promote the increase in granzyme B indirectly by activating T cells, thus enhancing the cytotoxic effect on HepG2 cells.     

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．     

Etoposide induces apoptosis via mitochondrial signaling pathway with cytochrome c release in Jurkat leukemia cells

AIM:To investigate the molecular mechanisms of apoptosis and to elucidate the apoptosis signaling pathway triggered by etoposide in Jurkat human leukemia cells. METHODS:Apoptosis was detected using annexin V-FITC and propidium iodide (PI) staining, respectively, and annexin V-FITC positive cells and hypodiploid cells were analyzed by flow cytometry. Mitochondrial membrane potential (△Ψm) was detected using 3, 3-dihexyloxycarbocyanine iodide ［DiOC6(3)］ staining and △Ψm low cells were analyzed by flow cytometry. Preparation of cytosolic extracts and isolation of mitochondria were completed by centrifugation. Western blotting analysis was used to evaluate the level of cytochrome c, caspase-3, and poly (ADP-ribose) polymerase (PARP) expression. RESULTS:Etoposide induced apoptosis showing phosphatidylserine externalization and DNA fragmentation in a time-dependent manner and the apoptosis could be inhibited by a broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk). Collapse of △Ψm induced by etoposide preceded DNA fragmentation and phosphatidylserine externalization. In contrast, it was not blocked by zVAD.fmk. Etoposide caused cytochrome c release from mitochondria into cytosol, subsequent activation of caspase-3 (32 kD) presented with an intermediate (20 kD) and its active product (17 kD), and cleavage of full-length PARP (116 kD) into the so-called apoptotic 85 kD fragment. CONCLUSION:Etoposide-induced Jurkat cell apoptosis is initiated through mitochondria signaling pathway with cytochrome c release into cytoplasm and caspase is the ultimate executioner of cell apoptosis.     

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.     

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

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

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

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

Oxidative stress induces apoptosis in HepG2 cells

AIM: Direct exposure of cells to reactive oxygen species can induce apoptosis. In this study we investigate how oxidative stress induces cell death in HepG2 cells and characterize the molecular events involved.METHODS: Oxidative stress was created by exposing HepG2 cells to 2 mmol/L H2O2. Apoptosis was determined by analysis of DNA fragmentation by agarose gel electorphoresis. The mitochondrial membrane potential was analyzed using DePsipher fluorescent staining and the expression of cytochrome c in the cytosolic fraction was measured by Western blotting analysis. The caspase activity was detected using fluorometric assay kit by a fluorescence microplate reader.RESULTS: When HepG2 cells were treated with 2 mmol/L H2O2, the cells displayed DNA fragmentation, a typical feature of apoptosis, after 12 h. The mitochondrial membrane potential appeared different in two group of cells. H2O2-treated cells appeared green fluorescence as early as 4 h, which represents de-energized mitochondria, the untreated cells appeared red fluorescence, a feature of mitochondria with intact membrane potential. In treated cells, the expression of cytochrome c increased and accumulated in cytosolic fraction with treatment time, caspase-3 activity increased by 6.7-fold (P<0.01) at 8 h and caspase-9 activity increased by 3.6-fold (P<0.01) at 12 h, respectively, however, the activity of caspase-8 remained unchanged.CONCLUSION: These findings suggest that oxidative stress can induce apoptotic cell death in HepG2 cells, and the mechanism is related to mitochondrial pathway, which activates caspase-9 and-3, but not caspase-8.     

Effect of lentinan on apoptosis and PI3K/AKT signaling pathway in leukemic HL-60 cells in vitro

AIM:To study of the regulatory effect of lentinan on human leukemic HL-60 cell apoptosis and its effect on PI3K/AKT signaling pathway in HL-60 cells in vitro.METHODS:Lentinan at concentrations of 0 mg/L, 15 mg/L, 30 mg/L and 45 mg/L was applied to HL-60 cells cultured to the logarithmic phase in vitro, and the inhibitory effect of lentinan on the viability of HL-60 cells was measured by MTT assay after 24 h, 48 h and 72 h. The apoptosis induced by lentinan was analyzed by flow cytometry. The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3, cleaved caspase-8, cytochrome C, PI3K, AKT and p-AKT were determined by Western blot. After treatment with PI3K inhibitor LY294002 at 5 mg/L for 72 h, the apoptosis of HL-60 cells was analyzed by flow cytometry. RESULTS:The viability of HL-60 cells was inhibited after treatment with lentinan at concentrations of 15 mg/L, 30 mg/L and 45 mg/L for 24 h, 48 h and 72 h in concentration-dependent and time-dependent manners (P<0.05). The apoptosis of HL-60 cells was promoted after treatment with lentinan (15 mg/L, 30 mg/L and 45 mg/L) for 72 h in a concentration-dependent manner (P<0.05). The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3 and cytoplasmic cytochrome C in the HL-60 cells induced by 30 mg/L lentinan were increased significantly with the increase in the treatment time (P<0.05), but caspase-8 did not show any change. The protein levels of PI3K, AKT and p-AKT were decreased obviously with the increase in the lentinan concentration (P<0.05). Treatment of HL-60 cells with LY294002, a PI3K pathway inhibitor, produced apoptosis-inducing effect similar to lentinan (P<0.05). CONCLUSION:Lentinan induces HL-60 cell apoptosis by inhibiting PI3K/AKT signaling pathway.     

Silencing of FoxM1 induces apoptosis of oral squamous-cell carcinoma cells by promoting mitochondrial release of cytochrome C

AIM: To study the effect of forkhead box protein M1 (FoxM1) silencing on apoptosis of oral squamous-cell carcinoma. METHODS: Oral squamous-cell carcinoma SCC9 cells were infected with FoxM1-shRNA lentivirus or negative control lentivirus. The silencing effect was measured by RT-qPCR and Western blot. The changes of cell viability effect was measured by MTT saay. The cell colony formation ability was measured by plate experiment. Flow cytometry was used to analyze the changes of apoptotic rate. Western blot was used to measure the protein levels of cleaved caspase-3 and cleaved caspase-9 in the cells. The changes of mitochondrial membrane potential were measured by JC-1 method. Western blot was used to measure the protein level of cytochrome C in the mitochondria and cytoplasm. RESULTS: Infection with FoxM1-shRNA lentivirus successfully reduced the expression of FoxM1 in oral squamous-cell carcinoma cells (P<0.05). Negative control lentivirus had no effect on the expression level of FoxM1 in the cells. The cell viability was reduced by FoxM1 silencing, and the ability of cell colony formation was also decreased. The apoptotic rate and the protein levels of cleaved caspase-3 and cleaved caspase-9 were all increased (P<0.05), and the mitochondrial membrane potential was decreased. The protein level of cytochrome C in the cytoplasm was increased, while the protein level of cytochrome C in the mitochondria was decreased (P<0.05). CONCLUSION: Silencing of FoxM1 induces the apoptosis of oral squamous-cell carcinoma cells by decreasing the mitochondrial membrane potential and promoting the release of cytochrome C from mitochondria.     

JNK pathway promotes apoptosis of rat hippocampal neurons after cerebral ischemia and reperfusion

AIM:To investigate the effect of c-Jun N-terminal kinase(JNK) pathway on the apoptosis of hippocampal neurons after cerebral ischemia-reperfusion(IR) in SD rats. METHODS:Ninety rats were randomly divided into 5 groups:sham group, cerebral IR group,cerebral IR+JNK inhibitor(SP600125) group,cerebral IR+JNK agonist(anisomycin) group and cerebral IR+vehicle group. The brain samples were collected 24 h after reperfusion. The protein level of caspase-3 in hippocampal neurons was measured by immunohistochemical and Western blotting techniques. The mRNA expression of caspase-3 in the hippocampus was determined by real-time fluorescence quantitative PCR. The apoptosis of hippocampal neurons was detected by TUNEL staining. RESULTS:Compared with sham group, the expression of caspase-3 at mRNA and protein levels in cerebral IR group increased obviously(P<0.05). Compared with cerebral IR group, the expression of caspase-3 at mRNA and protein levels in cerebral IR+JNK inhibitor group decreased obviously(P<0.05), and those in cerebral group increased obviously(P<0.05). However, the expression of caspase-3 at mRNA and protein levels in cerebral IR+vehicle group had no obvious change(P>0.05).The apoptosis of hippocampal neurons in each group was consistent with the changes of caspase-3 at mRNA and protein levels. CONCLUSION:Activation of JNK pathway enhances caspase-3 expression in rat hippocampal neurons after cerebral IR,thus promoting the apoptosis of the neurons.     

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 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.     

Osthole enhances doxorubicin-induced apoptosis in p53-wildtype prostate cancer cells by down-regulating SIRT1 expression

AIM: To investigate the effect and mechanism of osthole on increasing the cytotoxicity of doxorubicin (DOX) to prostate cancer cells. METHODS: MTT assay was performed to evaluate the viability of LNCaP cells treated with osthole and DOX. The protein expression of silent information regulator 1 (SIRT1), p53, acetylated p53 and Puma, as well as release of cytochrome C and activation of caspase-9 and caspase-3 in the LNCaP cells treated with osthole and DOX were determined by Western blot. The apoptosis of the LNCaP cells treated with osthole and DOX was analyzed by flow cytometry. RESULTS: Osthole significantly increased the cytotoxicity of DOX against p53-wildtype prostate cancer cell line LNCaP. Osthole significantly inhibited the expression of SIRT1 in the LNCaP cells. Transfection with SIRT1 plasmid decreased the cytotoxicity of osthole and DOX co-treatment against LNCaP cells. Combination with osthole and DOX significantly induced the over-expression and acetylation of p53. Transfection with p53 siRNA significantly decreased the synergistic effect of osthole on cytotoxicity of DOX-treated LNCaP cells. Combination with osthole and DOX significantly induced the release of cytochrome C into the cytoplasm from mitochondria, followed by activation of caspase-9 and its downstream molecule caspase-3, thus leading to cell apoptosis in the LNCaP cells. CONCLUSION: Osthole promotes the p53-dependent apoptosis in DOX-treated prostate cancer LNCaP cells by down-regulating the expression of SIRT1.     

Mitochondrial mechanism of hyperglycemia-induced apoptosis in primary mouse hepatocytes with steatosis

AIM: To investigate the role of high glucose in primary hepatocytes of mice fed with a high fat diet.METHODS: Male C57BL/6J mice were fed a high fat (45% of calories) diet ad libitum for 6 weeks to induce hepatic steatosis. Primary hepatocytes were isolated from the mouse liver by the 2 step collagenase perfusion method. The cells were incubated in low glucose (5 mmol/L), low glucose plus mannitol (30 mmol/L), or high glucose (35 mmol/L) DMEM medium for 12 h. The cell viability, apoptosis, mitochondrial membrane potential, and caspase enzymatic activities were measured. Furthermore, proteins related to the stress-sensitive signaling pathway of regulating high glucose-induced apoptosis in primary hepatocytes were determined by Western blotting.RESULTS: Incubation with 35 mmol/L glucose resulted in a significant decrease in cell viability and an increase in apoptosis, whereas mannitol had no significant effect on the cell viability or apoptosis. A progressive depolarization of the mitochondria, an increase in cytosol cytochrome C and a dramatic decrease in mitochondrial cytochrome C in high-glucose stressed hepatocytes were observed. The enzymatic activities of caspase-9 and caspase-3, but not caspase-8, were significantly increased in high glucose-stressed hepatocytes (P<0.05). High glucose treatment suppressed the expression of Bcl-2 and Bcl-xL, while it increased the expression of the pro-apoptotic factor Bax.CONCLUSION: High glucose stress reduces mitochondrial membrane potential, initiates mitochondria-mediated apoptotic pathways and promotes apoptosis of hepatocytes with steatosis. This may be an important pathological mechanism of hyperglycemia-induced progression of nonalcoholic fatty liver disease.     

Effects of ginkgolide B on apoptosis of cultured rat retina neurons in vitro

AIM: To observe the effect of ginkgolide B (GB) on glutamate-induced apoptosis in the cultured neurons of rat retina. METHODS: Neurons of rat retina were cultured and apoptosis was induced by glutamate. The neurons were cultured with different concentration of GB and the survival rate was monitored by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The apoptosis in the cultured neurons and the expression of Bcl-2 and Bax were observed by flow cytometry. RESULTS: After exposed to glutamate, the survival rate and the number of Bcl-2 positive cells obviously decreased. At the same time, the number of Bax positive cells obviously increased, and the number of the apoptotic cells also obviously increased. Such phenomena were relieved by the treatment of ginkgolide B, with raise of survival rate and the expression of Bcl-2. Meanwhile, the expression of Bax and the apoptosis of neurocytes obviously decreased. CONCLUSIONS: Ginkgolide B protects retinal neurons from the virulence induced by glutamate. Such effects of GB might be brought about by increasing the expression of Bcl-2 while decreasing Bax, resulting in increasing the ratio of Bcl-2 to Bax and so reducing the apoptosis in the cultured neurons of rat retina.     

Celastrol induces apoptosis of multiple myeloma H929 cells

AIM: To investigate the effect of celastrol on the apoptosis of human multiple myeloma H929 cells and its molecular mechanism. METHODS: The H929 cells were cultured in vitro and treated with celastrol at different concentrations (0.5, 1, 5 and 10 mg/L). The viability of H929 cells was analyzed by CCK8 assay. Annexin V-PE/7-AAD staining was used to analyzed the effect of celastrol on apoptosis of H929 cells, and mitochondrial membrane potential was observed by flow cytometry. The effect of celastrol on DNA damage was detected by comet assay. The protein levels of apoptosis-related molecules P53, XIAP, cleaved PARP-1 and cleaved caspase-3, and the release of mitochondrial cytochrome C in the H929 cells treated with celastrol were determined by Western blot. RESULTS: The viability of H929 cells was significantly inhibited by different concentrations of celastrol in a concentration-dependent and time-dependent manner. Apoptosis and decreased mitochondrial membrane potential of H929 cells in a concentration-dependent manner were observed after treatment with celastrol (P<0.05). The results of comet assay showed that celastrol induced DNA damage in the H929 cells. The protein levels of apoptotic molecules P53, cleaved PARP-1 and cleaved caspase-3 were significantly increased and the expression level of anti-apoptotic protein XIAP was significantly decreased in the H929 cells treated with celastrol (P<0.05). Celastrol promoted the release of cytochrome C in mitochondria, and activated caspase-3 in dependence on caspase-9. CONCLUSION: Celastrol has an apoptosis-inducing effect on multiple myeloma H929 cells. Its mechamism may be related to activation of mitochondrial apoptosis pathway by inducing DNA damage.     

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

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