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1.
AIM: To observe the effect of crocetin on the apoptosis and the changes of its related regulating proteins caspase-3 and Bcl-2 expression induced by hydrogen peroxide (H2O2) in cultured cardiomyocytes. METHODS: Changes of cellular morphology were detected under microscope. Apoptosis rates of the cells were analyzed by PI staining with flow cytometry. Expressions of caspase-3 and Bcl-2 proteins in the cells were determined by immunofluorescence with flow cytometry. RESULTS: In the concentrations used, more severe morphological changes with higher apoptosis rate of the cultured myocardial cells were seen in each H2O2 group than that in control group. When treated with 1×10-4 mol·L－1 H2O2, the caspase-3 was increased and Bcl-2 protein decreased remarkably in the cells. But each dosage of crocetin, especially the highest one (5×10－5 mol·L－1, P<005 compared with 5×10－7 mol·L－1 group), seemed efficient in maintaining the cell morphology, reducing the cell apoptosis rate and improving the changes in caspase-3 and Bcl-2 protein expression in the cells exposed to 1×10－4 mol·L－1 H2O2. CONCLUSION: Crocetin obviously inhibits the apoptosis induced by H2O2 in the cultured myocardial cells. The mechanisms may involve the balance of the functions of the apoptosis-related regulating proteins, caspase-3 and Bcl-2 protein.  相似文献   

2.
AIM:To study the effect of VEGF on extracellular H2O2 production in HUVECs and the role of H2O2 in the VEGF-induced proliferation. METHODS:HUVECs was stimulated with 500 μg/L VEGF. Products of extracellular H2O2 was detected by H2DCFDA staining. MTT method was used to value the influences of 3×106 U/L catalase and 5-20 mmol/L H2O2 to VEGF function. RESULTS:After treatment for 15 min with VEGF, HUVECs appeared fluorescence, and continued to become stronger, peaked at 45 min then decreased. HUVECs, which was treated simultaneity with VEGF and 3×106 U/L catalase, only appeared very faint fluorescence. The proliferation of HUVECs by VEGF was restrained when treated with 3×106 U/L catalase. The extrinsic H2O2 at concentration of 5-10 mmol/L promoted the proliferation of HUVECs but inhibited the proliferation effect of VEGF on HUVECs (P<0.01). CONCLUSION:These findings indicate that VEGF induces HUVECs to produce extracellular H2O2 and plays role in proliferation, but extrinsic H2O2 restrains VEGF function.  相似文献   

3.
AIM: To study the effect of luteolin on endothelial cell injuries induced by H2O2. METHODS: Endothelial cells were cultured in vitro and divided into seven groups: control; solvent; H2O2; quercetin+H2O2; luteolin-L+H2O2; luteolin-M+H2O2; luteolin-H+H2O2 group, respectively. Endothelial cells were incubated with 750 μmol/L H2O2 for 18 h or 14 h in the absence or presence of various concentrations of luteolin and quercetin. The expression of caspase-3, the pro-apoptotic factors, was detected by immunohistochemical technique, and the apoptotic index was detected by flow cytometry. Meanwhile, the amount of malondialdehyde (MDA), nitric oxide (NO), lacate dehydrogenase (LDH) in serum, and cell viability were measured. RESULTS: Incubation of endothelial cells with H2O2 for 18 h elicited the increase in the extent of immunostaining for caspase-3, the rate of apoptosis, the release of LDH, and the number of dead cells accompanied by the decrease in the content of NO in the medium, which were inhibited by pretreatment of luteolin at concentrations of 0.5-50 μmol/L in a concentration-dependent manner (P<0.01). CONCLUSION: Luteolin possesses a protective effect against endothelial cell injuries induced by H2O2, which may be related with anti-oxidation, increasing the concentration of NO and inhibiting the activation of caspase-3.  相似文献   

4.
AIM:To explore the role of extracellular signal-regulated kinases ERK1/2-STAT3 pathway in adaptive cytoprotection induced by H2O2 preconditioning in PC12 cells. METHODS:In PC12 cells, the experimental model of cytoprotection by H2O2 preconditioning against oxidative stress-induced injury was set up. The morphological changes in the apoptotic cells were observed by using of chromatin dye Hoechst 33258. The percent of apoptotic cells was determined by flow cytometry (FCM) with propidium iodide staining. The levels of p-ERK1/2 and p-STAT3 expression were detected by Western blotting assay. RESULTS:Preconditioning with H2O2 at concentration of 100 μmol/L for 90 min obviously inhibited apoptosis induced by 300 μmol/L H2O2, and both ERK1/2 and STAT3 were activated. UO126 (10 μmol/L, an inhibitor of ERK1/2) or AG-490 (10μmol/L, an inhibitor of JAK2) significantly blocked the cytoprotection effect of H2O2 preconditioning. Moreover, UO126 (10 μmol/L) also markedly inhibited the up-regulation of p-STAT3 expression by H2O2 preconditioning. CONCLUSION:H2O2 preconditioning activates ERK1/2-STAT3 signal pathway, which may be one of the mechanisms underlying H2O2 preconditioning-induced cytoprotection.  相似文献   

5.
AIM: To investigate the role of nuclear factor-kappa B (NF-кB) in the adaptive cytoprotection of H2O2 preconditioning and modulation of NF-кB expression by JAK-STAT pathway. METHODS: In PC 12 cells, the experimental model of cytoprotection of H2O2 preconditioning against oxidative stress-induced injury was set up. The apoptotic cells were measured by propidium iodide staining and flow cytometry (FCM). The levels of NF-кB and STAT3 expression were detected by Western blotting. RESULTS: Preconditioning with 100 μmol/L H2O2 for 90 min significantly inhibited apoptosis induced by 300 μmol/L H2O2, and up-regulated expression of NF-кB and STAT3. Both MG-132 (10 μmol/L, an inhibitor of NF-кB) and AG-490 (an inhibitor of JAK2) obviously blocked the expression of NF-кB and cytoprotection induced by H2O2 preconditioning. CONCLUSION: JAK-STAT pathway modulates the cytoprotection of H2O2 preconditioning that is mediated by NF-кB.  相似文献   

6.
AIM: To explore the changes and the possible function of mtCLIC/CLIC4 (mitochondrial chloride intracellular channel 4) proteins in malignant C6 glioma cells treated with hydrogen peroxide (H2O2). METHODS: The viability of C6 cells was measured by MTT assay, LDH release rate was detected by ultraviolet spectrophotometry, CLIC4 mRNA level was determined by RT-PCR and CLIC4 protein level was measured by Western blotting. RESULTS: Compared with the control group, the cell viability was constant, the LDH release rate increased obviously, and the CLIC4 protein level also increased significantly in 500 μmol/L H2O2 treated group (P<0.05, respectively). However, the cell viability decreased, LDH release rate increased significantly (P<0.01, respectively), and the CLIC4 protein level increased obviously in 1 000 μmol/L H2O2 treated group (P<0.05). CONCLUSION: The CLIC4 protein may be involved in the process of C6 injuries induced by H2O2.  相似文献   

7.
AIM:To investigate the mechanisms by which bilirubin inhibits acute lung injury (ALI). METHODS:30 female Wistar rats were divided into normal group, ALI group and bilirubin treatment group. ALI was induced by intravenous injection of LPS. The contents of OH-, H2O2 and O2· in the lung as well as the expression of caspase-3 in the lungs were investigated. RESULTS:(1) The contents of OH-, H2O2 and O2· in the lung homogenate and the expression of caspase-3 in the lungs in ALI group increased compared with those in normal group (P<0.05). (2) The contents of OH-, H2O2 and O2· in the lung homogenate and the expression of caspase-3 in the lungs in bilirubin treatment group increased compared with those in normal group, but decreased compared with those in ALI models (P<0.05). CONCLUSION:(1) Bilirubin was shown to be able to ameliorate apoptosis in ALI rats. (2) The increase in the contents of OH-, H2O2, O2· in ALI group indicated the development of oxidative lung injury, which was ameliorated by bilirubin. (3) Expression of caspase-3 confirmed that the model made by LPS was associated with apoptosis, which was reduced by bilirubin.  相似文献   

8.
AIM: To study the mechanisms of salvianolic acid B (Sal B)antagonizing mesangial cell activation and kidney fibrosis through investigating the effect of Sal B on expression of transforming growth factor-β1 (TGF-β1) receptors and Smad2 in TGF-β1-stimulated renal mesangial cell activation. METHODS: Mesangial cells was isolated and purified from rat kidney. TGF-β1 was used to establish rat primary mesangial cell activation model and Smad2,Smad7 protein expression was detected. Sal B (10-6 mol/L and 10-5 mol/L) was employed to treat the cells; α-smooth muscle actin(α-SMA) expression was analyzed by immunofluorescence staining and Western blotting. Mesangial cells were treated with Sal B alone or additional with TGF-β1,and TGF-β1 receptor Ⅰ (TβRⅠ),TGF-β1 receptorⅡ (TβRⅡ),Smad2 phosphorylation and Smad2 protein expression was determined by Western blotting. RESULTS: Cell ular model was established by incubating with 5 μg/L TGF-β1 for 24 h,and in early stage Smad2 was significantly phosphorylated. Sal B (10-6 mol/L and 10-5 mol/L) could inhibit α-SMA expression,which was the biomarker of activated mesangial cells. In addition,in Sal B group,the protein expression of TβRⅠand TβRⅡ was significantly down-regulated while Smad2 phosphorylation in mesangial cells was inhibited. CONCLUSION: Sal B inhibits the TGF-β1-Smad pathway,the protein expression of TβRⅠ,TβRⅡ and Smad2 phosphorylation in mesangial cells,which is probably one of the mechanisms of Sal B alleviating kidney fibrosis.  相似文献   

9.
AIM:To investigate the neuroprotective effect of tubuloside B, one of the phenylethanoids isolated from the stems of Cistanche salsa, on H2O2-induced injury in PC12 cells.METHODS:PC12 cells were exposed to various doses of tubuloside B for 12 h, then treated with H2O2 at concentration of 100 μmol/L for 24 h. The cell viability was observed with MTT assay. Reactive oxygen species and the mitochondrial membrane potential were measured with laser scanning confocal microscopy (LSCM). The DNA content and percentage of apoptosis were assayed by DNA agarose gel electrophoresis and flow cytometry. The activation of caspase-3 was detected with the caspase-3 activity assay kit. RESULTS:Following treatment with H2O2 for 24 h, H2O2 induced a significant decrease in cell viability; DNA ladder was observed and apoptosis percentage was as high as 48.0%. Accumulation of intracellular ROS, increase in caspase-3 activity and the decrease in mitochondrial membrane potential as indicated with the decrease of red/green ratios (from 5.97 to 0.41) were detected. However, pretreatment with tubuloside B (1, 10 or 100 mg·L-1) for 12 h exhibited cytoprotective effects in a dose-dependent manner. Tubuloside B obviously enhanced the cell viability, reduced formation of the DNA ladder, and significantly reduced the number of cells labeled with Annexin-V. The percentage of apoptosis/necrosis neurons was significantly decreased to 30.9%, 18.3% and 6.2%, respectively. LSCM showed that the tubuloside B attenuated the accumulation of ROS and the H2O2-induced collapse of mitochondrial membrane potential in PC12 cells. The significant decrease in caspase-3 activity was detected, compared to the H2O2-treated cells at the same time point. CONCLUSION:Tubuloside B has the neuroprotective capacity to antagonize H2O2-induced apoptosis and injury in PC12 cells, indicating it may be useful for treating some neurodegenerative diseases.  相似文献   

10.
ZHAO Ting  LING Wen-hua 《园艺学报》2007,23(10):1950-1954
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11.
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12.
AIM: To investigate the effect of estrogen antagonists on the in vitro growth of human prolactinomas. METHODS: RT-PCR was applied to the detection of estrogen receptor (ER) mRNA expressed in a human prolactinomas CH3 cell strain. Estradiol and 4-hydroxytamoxifen (OHTam) were added respectively at different concentrations into the culture medium. Cell number and levels of ER mRNA were examined. RESULTS: The growth of CH3 cells became slower in estrogen-deprived medium than that in nomal culture and was higher in medium containing estrogen(E2) at concentration of 10-8 mol/L than at concentration of 10-6 mol/L. OHTam (10-6mol/L) inhibited the growth of CH3 cell strain treated with E2. The expression of ER mRNA in CH3 cells was observed, the levels of ER mRNA in the E2 (10-8mol/L) group, higher than those in estrogen deprived group. OHTam (10-6mol/L) obviously inhibited the expression of ER mRNA. CONCLUSION: The growth of CH3 cells depends on estrogen, estrogen antagonists inhibits the growth of CH3 cells and decline the levels of ER mRNA. ER levels in human prolactinomas cell lines can be auto-regulated.  相似文献   

13.
AIM: To study the changes of mitochondria during apoptosis in Jurkat cells induced by arsenic oxide (As2O3). METHODS: By treated with 4×10-6 mol/L As2O3, apoptosis and necrosis of Jurkat cells were assessed by annexin V-FITC/PI double staining flowcytometry. Mitochondrial mass and its membrane potential (△ψm) was measured by NAO/PI and DiOC6 (3)/PI staining, respectively. Free radical formation was detected by DCFDA staining. RESULTS: After 48 h of As2O3 treatment, the rates of early apoptotic Jurkat cells in As2O3 and control groups were (18.98±1.40)% and (5.17±0.80)%, respectively (P<0.01). The necrotic rate in As2O3 group was significantly higher than that in control group (P<0.01), they were (8.56±0.70)% and (1.53±0.55)%, respectively. △ψm decreased Jurkat cells in As2O3 groups and control groups were (23.07±3.62)% and (6.63±1.46)%. The percentages of low mitochondrial mass cells in As2O3 and control groups were (25.90±1.80)% and (6.37±1.04)% (P<0.01). Intracellular free radicals in As2O3 group was increased, compared with control group, their DCFDA-fluorescence mean intensities were (24.41±0.75) and (17.06±0.48) (P<0.01). CONCLUSION: During apoptotsis process in As2O3-induced Jurkat cells, mitochondrial membrane potential and mitochondrial mass loses significantly, with increase in free radicals.  相似文献   

14.
AIM: To study the role of prostaglandin F2α (PGF2α) in cardiac hypertrophy and its relation with calcineurin (CaN) signal transduction pathway in vitro. METHODS: The cultured neonatal rat cardiomyocyte was used to observe the hypertrophic effect of PGF2α, and the hypertrophic response was assayed by measuring the cell diameter, protein content and atrial natriuretic factor (ANF) mRNA expression. For mechanism studies, the intracellular free calcium concentration (［Ca2+］i) in cultured cardiomyocytes was measured by using Fura-2/AM as a fluorescent indicator. ANF and CaN mRNA expressions, and the expressions of CaN and its downstream effectors, NFAT3 and GATA4 proteins were assayed by RT-PCR and Western blotting, respectively.RESULTS: In cultured cardiomyocytes, PGF2α induced profound hypertrophic morphology change, the significant increase in cell diameter and protein content in a concentration-dependent manner compared with those in vehicle control (P<0.01). The same result was found in measuring the ［Ca2+］i in cardiomyocytes (P<0.01). PGF2α at concentration of 10-7 mol/L significantly promoted ANF and CaN mRNA expressions and the protein expressions of CaN/NFAT3/GATA4 compared with those in the vehicle control (P<0.05). Cyclosporin A, a CaN inhibitor, markedly inhibited the myocyte hypertrophy (P<0.01), reduced the increased ［Ca2+］i(P<0.01) and decreased the expressions of CaN mRNA and CaN/NFAT3/GATA4 proteins (P<0.05) compared with those of only PGF2α　10-7 mol/L treatment. CONCLUSION: Cardiomyocyte hypertrophy induced by PGF2α may be, at least in part, mediated by CaN signal transduction pathway activated by increasing ［Ca2+］i.  相似文献   

15.
AIM: To determine the differences in phosphoproteome between LPS stimulated THP-1 cells with and without previous oxidative stress for screening of more potential regulators.METHODS: Differentiation of THP-1 cells into macrophages was induced by treatment with 100 μg/L PMA for 36 h. Differentiated cells were rested for additional 36 h without PMA treatment, then treated with 100 μmol/L H2O2 or medium for 1 h followed by LPS or medium treatment for 30 min. After desalted, phosphoproteins were enriched by phosphoprotein metal affinity column, and were run on 2-D electrophoresis, then the spots were analyzed to show the difference between LPS group (cells treated with LPS alone) and H2O2+LPS group (LPS stimulated cells also pretreated with H2O2). Finally, some of these spots were identified by MS and subsequent bioinformatic analysis was also conducted. RESULTS: Compared to LPS group, 29 reproducibly changed spots on the 2-D map in H2O2+LPS group were visualized and selected for MS analysis. Among these, 12 down-regulated spots (include those disappeared), 17 up-regulated spots (include those newly emerged) were selected. Up to now, 5 of these were identified, which were shown to be involved in various cellular processes such as proteolysis, signal transduction and protein folding. Among these, proteasome beta-4 subunit, which was dramatically down-regulated in H2O2+LPS group, was a major component of the proteasome complex and might participate in LPS signalling through various ways.CONCLUSION: With comparative phosphoprotein-affinity profiling, the interference brought by highly abundant house-keeping proteins is minimized, rendering us to detect less abundant signalling molecules. Aforementioned 5 proteins, especially proteasome beta-4 subunit, might be involved in LPS pathway reprogrammed by oxidative stress.  相似文献   

16.
AIM: To observe the effect of 5-nitro-2- (3-phenylpropylamino) benzoate acid (NPPB), niflumic acid (NFA) (chloride channel blockers) on malignant glioma C6 cells injured by hydrogen peroxide (H2O2). METHODS: The viability of C6 cells treated with NPPB, NFA and H2O2 was measured by MTT assay. LDH release rate and GSH contents were detected by ultraviolet spectrophotometry. mRNA levels of GCLC, GCLM and CLIC4 were determined by RT-PCR. CLIC4 protein level was detected by Western blotting. RESULTS: Compared to the control group, H2O2 treatment induced the decrease in cell viability and GSH contents, the increase in LDH release rate, the decrease in the expression of GCLC, GCLM and CLIC4 mRNA and the increase in CLIC4 protein level (P<0.05 ), respectively. Compared with the H2O2 group, H2O2 combined with NPPB or NFA treatment did not change the cell viability, the GSH contents and the GCLC, GCLM mRNA expression. However, the LDH release rate and CLIC4 protein level decreased (P<0.05). CONCLUSION: The chloride channel blockers NPPB or NFA lessen the oxidative injury of C6 cells through modulating the function of membrane and down-regulating the protein expression of CLIC4.  相似文献   

17.
AIM:To observe the STEAP expression and its function in human prostate tissues.METHODS:The expressions of STEAP mRNA and protein were detected by RT-PCR and Western blotting. H2O2 and SOD levels were detected by molybdic acid reduction and xanthine oxidation methods respectively.RESULTS:STEAP mRNA and protein were highly expressed in prostate cancer tissues. H2O2 and SOD levels in prostate cancer were obviously higher than those in normal prostate tissue.CONCLUSION:The function of STEAP gene is possibly to induce intracellular H2O2 and promote cell growth.  相似文献   

18.
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19.
AIM: In order to investigate the mechanisms involved in the vascular hyporeactivity after hemorrhagic shock, the changes of Ca2+ release from calcium store in vascular smooth muscle cells (VSMCs) with hypoxia were observed and the role of Ca2+ release from calcium store in the occurrence of vascular hyporeactivity to norepinephrine (NE) after hemorrhagic shock in rats was further explored.METHODS: A hemorrhagic shock model (40 mmHg for 2 h) in rats and a VSMCs hypoxic model were established. The changes of intracellular Ca2+ concentration in VSMCs were evaluated by fura3-AM and the role of IP3R and RyR mediated Ca2+ release from calcium store was further observed. The role of IP3R and RyR mediated Ca2+ release from Ca2+ store in the development of vascular hyporeactivity was measured with an isolated organ perfusion system. RESULTS: In the absence of extracellular Ca2+, NE upregulated by mobilizing Ca2+ release through calcium store. Compared to the normal control, the VSMCs had a slight increase when treated with hypoxia and NE-induced intracellular down-regulated, both without significant difference. Compared to the normal control cells, there was a significant change of Ca2+ release from calcium store in hypoxia-treated VSMCs, characterized by the significant increase in triggered by RyR-sensitive Ca2+ releasing activator caffeine. However, the increase in triggered by IP3R-mediated Ca2+ release agonist adenophostin A (10-5 mol/L) and ATP-Na2 (10-4 mol/L) had no significant difference in hypoxic VSMCs. Furthermore, the vascular reactivity to NE decreased in abdominal aorta in hemorrhagic shock (40 mmHg, 2 h) rats. The activation of IP3R mediated Ca2+ release with ATP-Na2 (10-4 mol/L) did not improve the vascular reactivity to NE, while inhibition of IP3R mediated Ca2+ release with heparin (104 U/L) significantly antagonized the vascular reactivity to NE in hemorrhagic shock rats. In addition, in normal K-H solution (with about 2.2 mmol/L) and Ca2+-free K-H solution, RyR antagonist ryanodine (10-5 mol/L) partly restored the vascular reactivity to NE in hemorrhagic shock rats, while RyR agonist caffeine(10-3 mol/L) further decreased the vascular reactivity. CONCLUSION: The over-activation of RyR-mediated Ca2+ release from calcium store is partly involved in the development of vascular hyporeactivity after hemorrhagic shock in rats.  相似文献   

20.
AIM: To investigate whether hypoxic preconditioning (HPC) protects cardiomyoblast H9c2 cells against oxidative injury, and to discuss whether calreticulin (CRT) contribute to this protection through p38 MAPK signaling pathway. METHODS: Cardiomyoblast H9c2 cells were randomly divided into eight groups as follows: hydrogen peroxide stress (H2O2); brief hypoxic exposure of 20 min to simulate hypoxic preconditioning (HPC); 20 min of hypoxic exposure followed by 24 h of normoxic reoxygenation before hydrogen peroxide stress (HPC+H2O2), SB203580 (the specific inhibitors of p38 MAPK)+HPC+H2O2, antisense oligonucleotides transfection of calreticulin (AS), AS+H2O2, AS+HPC+H2O2 and control. Morphological studies, estimation of lactate dehydrogenase (LDH) leakage and flow cytometry were employed to assess the cell apoptosis and necrosis. RT-PCR and Western blotting analysis was used to detect calreticulin expression and phosphorylation of p38 MAPK. RESULTS: The results obtained are as follows: (1) HPC relieved cell injury caused by H2O2. Compared with those in H2O2 group, apoptosis rate and LDH leakage in culture medium in HPC + H2O2 group decreased 13.4% and 44.0%, respectively (P<0.05), and cell survive rate increased 12.7% (P<0.05). SB203580, a selective p38 MAPK inhibitor presented before HPC, eliminated the cytoprotection of HPC. Compared with HPC+H2O2 group, apoptosis rate and LDH leakage increased 5.4% and 45.0%, respectively (P<0.05), and cell survive rate decreased 5.0%(P<0.05). (2) Brief hypoxia intimating HPC resulted in mild CRT up-regulation (1.4-fold increased vs control group, P<0.05), but this up-regulation was lower than that of 3.6-fold increase induced by oxidative stress. HPC relieved the over-expression of CRT induced by H2O2 (26% decreased vs H2O2 group, P<0.05). (3) Transfection of antisense oligonucleotides of CRT before HPC reduced cytoprotection against oxidative stress. Correlative analysis indicated that mild up-regulation of CRT induced by HPC was positively correlated with survive rate (r=0.8573, P<0.05). (4) SB203580 suppressed CRT up-regulation (the expression of CRT decreased 38% or 23%, vs HPC+H2O2 group or HPC group, respectively). CONCLUSION: These results suggest that hypoxic preconditioning up-regulates calreticulin expression through p38 MAPK signaling pathway and protects cardiomyoblast H9c2 cells against oxidative injury.  相似文献   

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