Role of heat shock protein 90 and tubulin in oxidative stress preconditioning

AIM:To find the role of heat shock protein 90 （HSP90） and tubulin in oxidative stress preconditioning in HepG2 cells. METHODS:The different doses of H2O2 were used to induce cell injury in HepG2 cells. MTT assay, Western blotting and confocal laser microscopy were also used. RESULTS:MTT colorimetry showed that preconditioning (50 mmo1/L H2O2) provided a temporary resistance against subsequent oxidative stress (500 mmol/L H2O2). Western blotting demonstrated that preconditioning increased the levels of HSP90 and tubulin in HepG2 cells, and lessen the declining of HSP90 and tubulin after stress. Tubulin and HSP90's colocalizations in cells with different doses of H2O2 were also observed under laser scanning confocal microscope. CONCLUSION:Tubulin might play important role in oxidative stress preconditioning in HepG2 cells by combining with HSP90.     

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.     

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.     

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.     

Hesperetin attenuates hypoxia/reoxygenation-induced apoptosis in H9c2 cells via inhibition of calcium overload

AIM: To investigate the effect of hesperetin on hypoxia/reoxygenation (H/R)-induced apoptosis in the H9c2 cells and to clarify the underlying mechanism. METHODS: The H/R model was established and the H9c2 cells were pretreated with hesperetin for 4 h. The cell viability and cell damage were measured by CCK-8 assay and lactate dehydrogenase (LDH) detection. The apoptosis was analyzed by Hoechst 33258 staining and flow cytometry. The intracellular calcium fluorescence intensity was measured by fluorescence microscopy and flow cytometry. The calcium-ATPase activity and the level of adenosine triphosphate (ATP) were measured by ELISA. The mitochondrial membrane potential was measured by JC-1 staining. The protein expression levels of Bcl-2, Bax and cytochrome C (Cyt-C) were determined by Western blot. RESULTS: Hesperetin reduced the apoptosis of the H9c2 cells induced by H/R, decreased intracellular Ca²⁺ fluorescence intensity, elevated Ca²⁺-ATPase activity, inhibited the mitochondrial membrane potential depolarization and increased the level of ATP (P<0.05). In addition, hesperetin significantly reduced the release of Cyt-C protein from mitochondria to cytoplasma and increased the Bcl-2/Bax ratio (P<0.05). After using the calcium ion inhibitor nimodipine, the percentage of the cells with mitochondrial membrane depolarization was decreased, the ATP level was increased and the protein expression of mitochondrion-related apoptosis molecules were decreased (P<0.05). CONCLUSION: Hesperetin reduces the apoptosis of the H9c2 cells induced by H/R, which may be related to inhibition of calcium overload and improvement of mitochondrial function.     

Genipin inhibits oxidative stress and apoptotic damage in high glucose-induced H9c2 cardiomyocytes

AIM:To explore the effects of genipin (GEN) on high glucose (HG)-induced oxidative stress injury and apoptosis in H9c2 cardiomyocytes.METHODS:H9c2 cells were cultured in vitro and HG-induced injury model was established. H9c2 cells were divided into 4 groups:normal control (NC) group (glucose at 5.6 mmol/L), HG group (glucose at 50 mmol/L), NG+GEN group and HG+GEN group. The concentration of genipin was used at 10 μmol/L. The viability of the H9c2 cells was measured by CCK-8 assay. The intracellular malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined by enzyme labeling and WST-1 methods, respectively. The activity of lactate dehydrogenase (LDH) in the cell culture supernatant was detected by microplate method. Fluorescent probe DCF was used to detect intracellular levels of reactive oxygen species (ROS). Nucleosome fragments was measured to evaluate cell apoptosis by ELISA. The intracellular mitochondrial membrane potential was detected by JC-1 method. The protein levels of Mn-SOD, cytochrome C (Cyt C), Bax and cleaved caspase-3 were determined by Western blot. RESULTS:Compared with HG group, the cell viability in HG+GEN group was increased significantly (P<0.05), the levels of MDA and LDH were decreased (P<0.05), SOD activity was increased (P<0.05), the levels of ROS and nucleosome fragments in HG+GEN group were decreased (P<0.05), and the mitochondrial membranes potential was notably increased (P<0.05). Compared with NG group, the activation of Mn-SOD was decreased, but the protein levels of Cyt C, Bax and cleaved caspase-3 were increased in HG group (P<0.05). Compared with HG group, the activation of Mn-SOD was increased, and the protein levels of Cyt C, Bax and cleaved caspase-3 were decreased in HG+GEN group (P<0.05).CONCLUSION:Genipin protects HG-induced H9c2 cardiomyocytes against oxidative stress injury and apoptosis.     

Effects of NOD8 on H2O2-induced apoptosis in human hepatocytes

AIM: To observe the effects of NOD8 on H2O2-induced apoptosis in human L02 hepatocytes. METHODS: pEGFP-C2 and pEGFP-NOD8 plasmids were transfected into L02 cells by JetPRIME, respectively. The apoptosis of these transfected cells was induced by H2O2. The cells were divided into pEGFP-C2 group, pEGFP-C2+H2O2 group and pEGFP-NOD8+H2O2 group. MTT assay was used to detect the cell viability. NOD8 protein expression was determined by Western blotting. The cell apoptosis was observed by Hoechst 33342 staining and apoptotic rate was evaluated by flow cytometry. The caspase-3 activity was analyzed by a colorimetric method. RESULTS: L02 cells were stimulated by H2O2 at concentrations of 0.2～2 mmol/L for 6 h, and H2O2 at concentration of 1 mmol/L was chosen to induce apoptosis determined by MTT assay. The protein expression of NOD8 significantly increased in the cells transfected with pEGFP-NOD8 plasmid. More cellular nucleus with strong blue fluorescence by Hoechst 33342 staining in pEGFP-C2+ H2O2 group were observed, indicating that apoptosis was increased, while the apoptosis in pEGFP-NOD8+H2O2 group significantly reduced. The apoptotic rate in pEGFP-C2+H2O2 group was obviously increased, whereas that in pEGFP-NOD8+H2O2 group was significantly decreased. The caspase-3 activity in pEGFP-C2+H2O2 group was remarkably increased. By contrast, the activity of caspase-3 was significantly reduced in pEGFP-NOD8+H2O2 group.CONCLUSION: NOD8 inhibits H2O2-induced apoptosis in L02 cells and the mechanism may be related to inhibition of caspase-3 activity.     

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 endoplasmic reticulum stress induced by increased expression of calcium-sensing receptor in myocardial hypoxia/re-oxygenation injury

AIM: To observe the effects of endoplasmic reticulum stress induced by the increase in expression of calcium-sensing receptor in myocardial hypoxia/re-oxygenation injury. METHODS: The primarily neonatal rat ventricular cardiomyocytes were incubated for 4-5 d, then randomly divided into 5 groups: (1) sham control group; (2) hypoxia/re-oxygenation group; (3) H/Re+ NiCl2, CdCl2-reperfusion group; (4) H/Re+GdCl3, NiCl2, CdCl2-reperfusion group; (5) H/Re+caffeine,GdCl3, NiCl2, CdCl2-reperfusion group. The neonatal cells were in ischemia-mimetic solution for 3 h, and re-incubated cells in normal culture medium for 9 h to establish a model of H/Re. The activity of LDH was determined, the viability and apoptosis of cardiomyocytes were assayed by MTT, the expressions of CaSR and caspase-12 in each group were analyzed using Western blotting, and the concentration of intracellular calcium was measured by laser confocal scanning microscope. RESULTS: The apoptosis index, the activity of LDH, the concentration of intracellular calcium, and quantitative expression of CaSR and caspase-12 in H/Re and activator groups were significantly higher than those in control group, while the viability and apoptosis of cardiomyocytes were lower than those in control group. CONCLUSION: In myocardial hypoxia/re-oxygenation, CaSR induces endoplasmic reticulum stress by altering the intracellular calcium homeostasis. The induction of apoptosis may be due to the increase in the expreesions of caspase-12 and other proapoptotic proteins.     

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

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

Effect of 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.     

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．     

Protective effect of ecdysterone on H9c2 cells against oxidative stress

AIM: To investigate the effect of ecdysterone (EDS) on H9c2 cardiomyocytes after oxidative stress. METHODS: H9c2 cells were cultured in vitro and divided into control group, high dose (2 μmol/L) of EDS group, middle dose (1.5 μmol/L) of EDS group, low dose (1 μmol/L) of EDS group, and H₂O₂ group. H9c2 cardiomyocytes in H₂O₂ group and high, middle and low doses of EDS groups were exposed to H₂O₂ for 6 h to establish the model of oxidative stress. The viability of the H9c2 cells was detected by CCK-8 assay. The apoptosis of H9c2 cells was analyzed by flow cytometry. The levels of lactate dehydogenase (LDH) and creatine kinase-MB (CK-MB) in the culture medium, and the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in the H9c2 cells were measured by colorimetry. The generation of reactive oxygen species (ROS) and the mitochondrial membrane potential were evaluated by flow cytometry and confocal laser scanning microscopy. The protein levels of Bax, Bcl-2 and cleaved caspase-3 in the H9c2 cells were determined by Western blot. RESULTS: Ecdysterone at the selected concentrations had no effect on the viability of H9c2 cells. Compared with control group, the levels of LDH, CK-MB, ROS and MDA, and the apoptotic rates of the H9c2 cells were significantly increased after treated with H₂O₂, but were decreased by EDS treatment in a dose-dependent manner. The levels of SOD and mitochondrial membrane potential of the H9c2 cells in H₂O₂ group were reduced significantly compared with control group, but high, middle and low doses of EDS treatments up-regulated the levels of SOD and mitochondrial membrane potential in H₂O₂-treated H9c2 cells. The protein levels of Bax and cleaved caspase-3 in the H9c2 cells in H₂O₂ group showed significant elevation in comparison with control group, and the protein expression of Bcl-2 declined in H₂O₂ group compared with control group, but high, middle and low doses of ecdysterone treatments down-regulated the protein levels of Bax, cleaved caspase-3 and up-regulated the expression of Bcl-2 in H₂O₂-treated H9c2 cells. CONCLUSION: Ecdysterone attenuates the effect of H₂O₂-induced oxidative stress on H9c2 cardiomyocytes. The mechanism may be involved in scavenging oxidative stress products, increasing antioxidant enzyme activity and improving mitochondrial function.     

Changes of uncoupling proteins activity and the efficiency of oxidative phosphorylation in hypoxic exposed rat brain mitochondria in vitro

AIM: To observe the effect of GDP on uncoupling proteins(UCPs) activity and the efficiency of oxidative phosphorylation in hypoxic exposed rat brain mitochondria.METHODS: Adult SD rats were randomly divided into three groups (control, acute hypoxia and chronic hypoxia groups). The animals were placed into a hypobaric chamber simulated 5 000 m high altitude for 0, 3 and 30 d, respectively. The mitochondria from rat brain were isolated by centrifugation. The activity of UCPs was detected by the method of ［H3］-GTP binding with UCPs specifically. The maximal binding content (Bmax) and the dissociation constant (Kd) were determined by Scatchard plot. The mitochondrial potential was measured by rhodamine 123 method. Oxidative respiratory consumption was measured by Clark electrode. The experiments were conducted under the conditions with or without GDP (1 mmol/L), respectively. RESULTS: For exposed to hypoxia, Bmax and the oxidative consumption of uncoupling respiration were increased. Kd, MMP and RCR were decreased. UCPs activity was inhibited by GDP in three groups. Kd was increased 61.01%, 83.13% and 71.52% and Bmax was decreased 23.18%, 35.20% and 33.38%, respectively. The values in the acute hypoxic group were changed markedly. The sensitivity of UCPs to GDP was elevated significantly by hypoxia. With the reducing of UCPs activity, oxidative consumption of uncoupling respiration was decreased whereas RCR and MMP were increased. The results elucidated increase in the efficiency of oxidative phosphorylation.CONCLUSION: GDP increases the mitochondrial membrane potential and decreases the oxygen consumption of uncoupling respiration in hypoxic exposed rat brain mitochondria by inhibiting UCPs activity. The results suggest that the change in UCPs activity is one of the factors of mitochondrial dysfunction in oxidative phosphorylation induced by hypoxia.     

Protective effect of astragalus polysaccharide on mitochondria and lysosome in H2O2-stressed skin fibroblasts

AIM: To verify the protection of astragalus polysaccharide (APS) on H2O2-stressed skin fibroblasts. METHODS: A model of acute H2O2 stress in primary skin fibroblast was used at concentration of 0.5 mmol/L by 30 min incubation. Dose responses of APS on cell survival was measured by MTT, cell death was evaluated by DAPI, and effect of APS on mitochondria, mitochondrial membrane potential and lysosome stabilization were measured by confocal microscopy. RESULTS: APS improved cell survival in a dose-dependent manner, starting at 0.5 mg/L and with a maximum at 1 mg/L. Moreover, APS inhibited mitochondrial membrane potential collapse, protected mitochondrial morphology and stablized lysosomal membrane. CONCLUSION: The results suggest the existence, at the mitochondria-lysosome level, of a new pathway of apoptotic regulation by APS. This might constitute a new therapeutic target where oxidative stress and lysosomal impairment are involved.     

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.     

Protective effects of nmhaFGF on NRK52E cell apoptosis induced by H2O2

AIM: The present study was designed to establish H2O2-induced NRK52E cell apoptotic model and to investigate the effects and the mechanism of nmhaFGF on the NRK52E cell apoptosis induced by H2O2. METHODS: In vitro experiment, a apoptotic model of normal rat kidney epithelium (NRK52E) was made by MTT method, Hoechst33342 dyeing and flow cytosorting (FCR). NRK52E cells were cultured with different concentrations of nmhaFGF and haFGF for 24 h before H2O2 was added. The apoptotic rate was detected with FCR method. RESULTS: H2O2 at concentration of 0.4 mmol/L, the optimal condition to establish the apoptotic model, was used to treat NRK52E cells for 18h. Different doses of nmhaFGF (0.01, 0.03, 0.10, 0.30, 1.00 mg/L) reduced the apoptotic rates with the dose rising. However, the decreasing tends of apoptotic rates with dose increasing for haFGF was not so obvious. CONCLUSION: nmhaFGF protects the NRK52E cells against apoptosis. The mechanism might be connected with its non-mitogenic property.     

矮化中间砧‘宫藤富士’苹果栽植密度对树体生长、冠层光照和果实产量的影响

2011 年春季定植的矮化中间砧苹果成品苗（3 年根 1 年干的‘宫藤富士’/SH6/平邑甜茶）为试材，设置 7 种不同的栽植密度（株行距分别为 1 m × 3 m、1.5 m × 3 m、2 m × 3 m、0.75 m × 4 m、1 m × 4 m、1.25 m × 4 m 和 1.5 m × 4 m），细纺锤形整枝修剪，自栽植第 2 年，连续 7 年调查 7 种栽植密度对树体生长、冠层光照分布、果实产量和品质的影响。随着树龄的增长，不同栽植密度下树干粗度和总枝量逐年增加，不同处理间树干粗度无显著差异，第 7 年 1 m × 3 m 和 0.75 m × 4 m 两个栽植密度下树体总枝量超过 140 万条 · hm-2，第 8 年均超过 140 万条 · hm-2。栽植前期（第 2 ~ 4 年）各栽植密度树体短枝比例不断增加，长枝比例不断减少，第 5 年各栽植密度枝类组成趋于稳定；综合稳产 3 年（第 6 ~ 8 年）树体的枝类组成数据，4 m 行距的短枝比例明显高于 3 m 行距，长枝比例略低。树体冠层平均相对光照强度由高到低的株行距处理依次为 1.5 m × 4 m（63.87%）、1.25 m × 4 m（61.44%）、2 m × 3 m（61.27%）、1 m × 4 m（59.19%）、0.75 m × 4 m（55.79%）、1.5 m × 3 m（53.67%）和 1 m × 3 m（49.37%）；相同栽植株数下，4 m 行距处理低光效（相对光照强度小于 40%）的区域比例显著小于 3 m 行距。比较前 5 年的累计产量，以行距 4 m 和 1 m × 3 m 的最高。综合稳产 3 年的结果情况，大果率（单果质量 > 200 g 的果实产量占总产量的比例）以 4 m 行距和 2 m × 3 m 的最高。各栽植密度下的果实的可溶性固形物含量、固酸比、果形指数和果实硬度均无显著差异。综上，采用 4 m 行距，1 ~ 1.25 m 株距，树体成形快，稳产后树体结构合理，冠层光照充足，低效光区比例少，前期产量高。     

The effect of Shenmai injection on cardiac myocyte apoptosis after hypoxia

AIM: To observe the effect of Shenmai injection, a chinese medicine, on apoptosis of cardiac myocytes after hypoxia. METHODS: Cardiac myocytes were separated from neonate rat heart and cultivated in vitro. Hypoxia condition was induced by mixture of 95%N2 and 5%CO2. Cells were exposed to hypoxia for 6 h or 12 h and treated with Shenmai injection (5 mL/L) from 24 h before hypoxia until the end of hypoxia. First, apoptosis was detected with Annexin V-FITC and PI staining by flowcytometry. Then, the activity of cardiac myocyte mitochondria was observed by MTT method. Mitochondria membrane potential and the activity of caspase 3,7 were also measured by laser scan microscopy and multi-detection microplate reader, respectively. RESULTS: The apoptotic cells became more and more with prolonged hypoxia. Shenmai injection enhanced mitochondria activity, kept membrane potential, inhibited the activation of caspase3,7 and then decreased apoptotic cells (P<0.01). CONCLUSION: Hypoxia induces apoptosis in cardiac myocytes by mitochondria pathway. Shenmai injection can decrease cardiomyocyte apoptosis after hypoxia, the mechanism is related to mitochondria membrane potential stabilization and caspase inhibition.     

Quercetin regulates Fas expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To investigate the role of 1, 4, 5- trisphosphate inositol (IP3) and Fas gene expression in apoptosis of HepG2 cells induced by quercetin. METHODS: HepG2 cells were treated with quercetin at different concentrations (including 20, 40, 60, 80 μmol/ L) for 72 h and treated with 60 μmol/ L quercetin for 6 h, 12 h, 24 h, 48 h and 72 h. IP3, Fas mRNA, Fas protein and apoptosis rate were assayed by IP3 - ［3H］ Birtrak assay, RT-PCR, Western blotting and flow cytometry, respectively. RESULTS: When HepG2 cells were incubated with different concentrations of quercetin for 72 h, the IP3 content was lower than those in control. Fas mRNA expression, Fas protein expression and the apoptosis rate were higher than those in control. When HepG2 cells were incubated with quercetin for 6 h, 12 h, 24 h, 48 h, 72 h, the IP3 contents were lower than those in control incubated with 60 μmol/L quercetin for 12 h. Fas mRNA expression was higher than that in control incubated with 60 μmol/L quercetin for 12 h . Fas protein expression was higher than that in control. The apoptosis rate was significantly higher than that in control incubated with 60 μmol/L quercetin for 24 h (P<0.01). CONCLUSION: Quercetin induces apoptosis of HepG2 cells by reducing IP3 production and upregulating Fas gene expression.