Changes of mitochondrial membrane potential and mitochondrial mass in camptothecin-induced Jurkat cells

AIM: To study the changes of mitochondrial membrane potential (△ψm) and mitochondrial mass in apoptosis of Jurkat cells induced by camptothecin (CPT). METHODS: Jurkat cells were treated with CPT. Annexin V-FITC/propidium iodine (PI) double stainig was used to detected early stage of apoptosis and PI staining for analyzing the cell cycle. Jurkat cells were stained by annexin V-PE/DiOC6(3) to detect changes of △ψm. The mitochondrial mass was measured by cytometry with NAO staining. RESULTS: 6 h after treated with 10 μmol/L CPT, the rate of early apoptotic cells (22.59±1.04)% had significantly difference compared with control group (3.93±0.73)% (P<0.01). The necrotic rate (2.48±0.53)% had no significant difference to that in control group (2.78±0.63)% (P>0.05). Apoptotic peak appeared obviously after treated with CPT, the percentage of late apoptotic cells (13.58±0.97)% had distinctly difference compared with control group (3.18±0.51)% (P<0.01). The cells in G0/G1 phase (48.14±0.96)% were much higher than that in control group (44.09±0.43)% (P<0.01). Mitochondrial depolarization was very obviously in CPT group. The percentage of annexin V+DiOC6 (3)- cells was (19.47±0.69)%, while in control group, was (4.21±0.40)% (P<0.01). Mitochondrial mass in CPT group was significantly lower than that in control group, the percentage of NAO+ cells (74.77±1.66) % had significantly difference compared with control group (92.24±1.41)% (P<0.01). CONCLUSION: During the process of CPT-induced apoptosis in Jurkat cells, mitochondrial depolarization was very obviously and mitochondrial mass decreased, indicating that the process of apoptosis is nearly related to the mitochondrial pathway.     

Effects of arsenic trioxide on activities of MMPs and expression of TIMP-1 and TGF-β1 in skin fibroblasts and polymorphonuclear neutrophils

AIM: To observe the effects of arsenic trioxide (As₂O₃) on activities of matrix metalloproteinases (MMPs), expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor beta1 (TGF-β₁) in human fibroblast (hFb), and to discuss weather As₂O₃ promotes the healing of chronic skin ulcer through regulating collagen metabolism. METHODS: Zymography was used for testing activity of MMP-9 deriving from rat polymorphonuclear neutrophils (PMNs) and activities of MMP-1, MMP-2 secreted by hFb. Immunocytochemical method was used to determine the expressions of TIMP-1 and TGF-β₁. RESULTS: At the concentration of 50 mg/L, As₂O₃ elevated the activity of MMP-9 (P<0.01). At the concentration of 0.8 mg/L, As₂O₃ increased the activities of MMP-1 and MMP-2 (P<0.01, respectively). After hFb was cultured with As₂O₃ for 6 h, 12 h and 18 h, the expressions of TIMP-1 and TGF-β₁ decreased continuously (P<0.01). CONCLUSION: As₂O₃ elevates the activities of MMP-1, MMP -2 and MMP-9, also inhibits the expressions of TIMP-1 and TGF-β₁, suggesting that arsenic preparation may exert positive effect on healing chronic skin ulcer through regulating collagen metabolism.     

Effect of ERK inhibition on the mitochondrial potential change in dexamethasone-induced thymocyte apoptosis

AIM: To study the effect of ERK inhibition on the mitochondrial potential change in dexamethasone (DEX)-induced thymocyte apoptosis. METHODS: ERK activity was inhibited by PD098059 (PD), and 4 experimental groups were set: control, PD only, DEX and PD+DEX. Annexin V-FITC/PI double staining flowcytometry was used to detect apoptotic cells at time points of 3 h, 5 h and 7 h. JC-1 staining flowcytometry was adopted to examine mitochondrial membrane potential (△ψm) at time points of 3 h, 7 h and 11 h. RESULTS: By stimulation with 1 μmol/L DEX, the apoptotic rates of mouse thymocytes at 3 h, 5 h and 7 h were (19.63±0.35)%, (41.84±1.67)% and (67.00±2.43)%, respectively, and had significantly difference from control group ［(4.98±0.39)%, (6.08±0.33)% and (9.31±0.34)%］ (P<0.01). At same time points, the rates in PD only group ［(7.95±0.60)%, (10.69±0.48)% and (22.20±1.24)%］ were higher than that in control group (P<0.01). Apoptotic rates in PD+DEX group at 3 h and 11 h were significantly higher than that in DEX group (P<0.01), and it was of no significance at 7 h (P>0.05). At 3 h, 7 h and 11 h, the rates of low △ψm cells were (21.23±1.43)%, (55.34±1.78)% and (70.88±2.87)%, significantly higher than that in control group (P<0.01). At same time points, the rates in PD group were (11.09±2.00)%, (16.21±2.25)% and (21.15±3.70)%, higher than that in control group (P<0.01). The rates in PD+DEX group at 3 h ［(30.55±2.99)%］ and 7 h ［(65.22±4.32)%］ were significantly higher than that in DEX group (P<0.01), it was of no significance at 11 h (P>0.05). CONCLUSION: DEX induces mouse thymocyte apoptosis at least partly through ERK pathway, and ERK inhibition has an important biological significance during this process.     

Calreticulin is involved in the protection of hypoxic preconditioning against cardiomyoblast H9c2 cell oxidative injury

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.     

COX-2 inhibitor protects rat heart against oxidative stress through a pathway independent of cyclooxygenase

AIM: To investigate whether nimesulide ［a selective cyclooxygenase 2 (COX-2) inhibitor］ and piroxicam (an inhibitor of COX-1) protect the rat hearts against oxidative stress induced by hydrogen peroxide,superoxide anion or hydroxyl free radical.METHODS: Cardiac contractility,lactate dehydrogenase (LDH) and malondialdehyde (MDA) were analyzed by the Langendorff method in isolated rat hearts.Production of 6-Keto-PGF1α,a marker of COX activity,was measured in isolated rat hearts.RESULTS: Rat hearts were exposed to hydrogen peroxide (H₂O₂),pyrogallol (which produced superoxide anion) or Vit C+Fe2+ (which produced hydroxyl free radical) for 10 min followed by reperfusion for 30 min.H₂O₂ decreased cardiac contractility and increased LDH release,which was inhibited by nimesulide (3 mg/kg) ［LVDP 72%±10% vs 61%±11%,LDH （5.5±2.5）U/L vs （8.0±2.1）U/L,P<0.05］.Piroxicam (3 mg/kg) increased systolic function (LVDP 73%±10% vs 61%±11%,P<0.05),but exacerbated diastolic function ［LVEDP （29.00±5.61）mmHg vs （23.16±3.57） mmHg,P<0.01］ in H₂O₂ treated rat hearts.Nimesulide also protected rat hearts against superoxide anion and hydroxyl free radical injury.Nimesulide and piroxicam had no effect on the content of 6-Keto-PGF_1α in rat hearts.Mitochondrial ATP sensitive potassium channel (mitoK_ATP) inhibitor 5-HD blocked the improvement of contractility (LVDP and ±dp/dt_max) induced by nimesulide in H₂O₂ treated rat hearts (53%±12% vs 69%±3%,58%±11% vs 72%±7% and 37%±8% vs 51%±4% respectively,P<0.01).CONCLUSION: The results suggests that COX-2 inhibitor nimesulide can protect rat hearts against oxidative injury.The protection is independent of COX activity.Activation of mitoK_ATP may be involved in nimsulide-induced cardioprotection in rat hearts.     

Advances in experimental research in HCV cell model and anti-HCV effect of As2O3

Hepatitis C virus (HCV) is a human pathogen responsible for liver diseases including acute and chronic hepatitis and hepatocellular carcinoma. However, the high prevalence, the absence of antiviral drugs and vaccines for prevention and treatment are the difficult medical problems. Lacking appropriate culture method and small animal model have severely limited investigation of the HCV infection mechanism and the development of the therapeutic strategy. Recently, the in vitro culture system develops rapidly, and provides a powerful tool for HCV related research. Despite the well known toxicity of the chemical, arsenic trioxide (As₂O₃) is effective for treating the patients with refractory or relapsed acute promyelocytic leukemia and many other cancers. Interestingly, As₂O₃ shows the ability of inhibiting HCV RNA replication and infection. This review describes the different types of in vitro HCV models developed, and many studies of the potent effect of As₂O₃ against HCV and its associated molecular mechanisms.     

Effects of luteolin on endothelial cell injury induced by H2O2

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.     

Effects of crocetin on the apoptosis and the changes of its related regulating proteins caspase-3 and Bcl-2 induced by H2O2 in myocardial cells

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.     

The mitochondrial and structural protein changes in dexamethasone-induced mouse thymocyte apoptosis

AIM: To study mitochondrial mass and structural protein changes in dexamethasone (DEX)-mediated mouse thymocyte apoptosis process. METHODS: DEX-induced mouse thymocyte apoptosis model was established. Annexin V-FITC/PI double staining was used to identify apoptotic and necrotic cells by flowcytometry, JC-1 staining was adopted to test mitochondrial membrane potential (△Ψm), and cellular structural protein changes were studied with CFDA-SE staining. RESULTS: By 1×10-6 mol/L DEX stimulation, the apoptotic rate was 51.25%±5.51% and had significantly difference from control group (12.03%±2.00%); the necrotic rate in DEX group was 30.25%±3.67% and also had significantly difference from control group (10.11%±1.11%, P<0.01). Mitochondrial mass in DEX group ［FL1 (561.62±54.27)］ was significantly lower than that in control (900.25±38.80, P<0.01). DEX also caused significant down-regulation of △Ψm (P<0.01), FL2 in control group was 267.51±26.48, and in DEX group was 133.17±12.29. Mature T cells cultured for 48 h showed only one peak by CFDA-SE staining; while by Con A stimulation, there were three offspring peaks. Low FL1 cell cluster (5.25%±1.15％) exited in control group, while by DEX stimulation, this cluster significantly was larger (47.39%±9.76％, P<0.01). CONCLUSIONS: In mouse thymocytes, mitochondrial mass and cellular structural protein amount are reduced by DEX; CFDA-SE staining flowcytometry can served as an apoptosis detection method based on cellular structural protein amount change.     

Upregulatory effect of isopsoralen on expression of estrogen receptor in human lens epithelial cells

AIM: To investigate the effect of isopsoralen(ISR) on the expression of estrogen receptor (ER) in human lens epithelial cells (HLECs). METHODS: HLECs were cultured and sub-cultured in vitro. The cultured HLECs pretreated with E₂ or ISR were exposed to H₂O₂ at the concentration of 300 μmol/L. The expression of ERα and ERβ in HLECs was analyzed by flow cytometry. RESULTS: The expression of ERα and ERβ in H₂O₂ group was obviously decreased as compared to control group (P<0.01). The expression of ERα and ERβ in the cells treated with E₂ and with ISR at the concentration of 10^-5 mol/L, 10^-6 mol/L or 10^-7 mol/L plus H₂O₂ was obviously increased as compared to the cells treated with H₂O₂ only (P<0.01). A concentration-dependent effect of ISR was observed. CONCLUSION: H₂O₂ decreases the expression of ERα and ERβ in HLECs.E₂ and ISR increase the expression of ERα and ERβ in HLECs treated with H₂O₂ in a concentration-dependent manner, which may account for their antioxidative effect.     

Effect of diazoxide on change of H2O2 in rat pulmonary artery smooth muscle cells and proliferation of hypoxic rat pulmonary artery smooth muscle cells

AIM: To investigate the contribution of diazoxide,an opener of mitochondrial ATP-sensitive K+ channel (MitoK_ATP),and mitochondrial membrane potential (ΔΨm) to change of H₂O₂ in rat pulmonary artery smooth muscle cells (PASMCs) and to unbalance between cell proliferation and apoptosis of PASMCs induced by hypoxia.METHODS: The rat PASMCs were isolated from fresh normal lung tissues and cultured,which were divided into 6 groups,as follows: ① control group;② diazoxide group;③ 5-HD group;④chronic hypoxia group;⑤ chronic hypoxia+diazoxide group;⑥ chronic hypoxia +5-HD group.The relative change in mitochondrial potential was detected with rhodamine fluorescence (R-123) technique.The level of H₂O₂ in rat PASMCs was detected with chemiluminescence method.The proliferation of rat PASMCs was examined by cell cycle analysis and MTT colorimetric assay.RESULTS: After exposed to diazoxide for 24 h,the intensity of R-123 fluorescence,the level of H₂O₂ and the A value in normoxic rat PASMCs were significantly increased,and the apoptosis of rat PASMCs was significantly decreased as compared with control group (P<0.05).However,there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24 h.Chronic hypoxia or chronic hypoxia+diazoxide markedly increased the intensity of R-123 fluorescence,the level of H₂O₂ and the A value in rat PASMCs,and also markedly decreased the apoptosis of rat PASMCs as compared with control group (P<0.05),and these changes were more significant in chronic hypoxia +diazoxide group than those in chronic hypoxia group (P<0.05).5-HD partly weakened the effect of hypoxia on the intensity of R-123 fluorescence,the level of H₂O₂,the A value and the apoptosis of rat PASMCs (P<0.05).Significant and positive correlations were found between the intracellular H₂O₂ and the R-123 fluorescence or the A value.Significant and negative correlation was found between the intracellular H₂O₂ and the apoptosis of rat PASMCs.CONCLUSION: The results suggest that the opening of MitoK_ATP followed by a depolarization of ΔΨm can contribute to the increase in the level of H₂O₂ in rat PASMCs and to the proliferation of rat PASMCs induced by hypoxia.This might be a mechanism of the development of hypoxic pulmonary hypertension.     

Role of mtCLIC/CLIC4 in injured C6 glioma cells induced by hydrogen peroxide

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.     

VEGF induces HUVECs to produce extracellular H2O2 and its proliferation role

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.     

Role of ERK1/2-STAT3 pathway in adaptive cytoprotection induced by H2O2 preconditioning

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.     

Role of preconditioning to oxidative stress in HepG2 cells

AIM:To induce preconditioning and oxidativ e stress by H₂O₂ in HepG2 cells.METHODS:The different doses of H₂O₂ were used to induce apo ptosis in HepG2 cells,which was estimated by AO/EB staining,MTT assay and flow cytometry.RESULTS:The different group of HepG2 cells stained with AO/EB s howed different staining state.The high dose of H₂O₂ resulted in the increa se in apoptosis rate of HepG2 cells and made MTT activity decreased.However,af ter pretreated with low dose of H₂O₂,the apoptosis rate was decreased and M TT activity was increased.CONCLUSION:The high dose H₂O₂ induced apoptosis in HepG2 ce lls and the low dose H₂O₂ protected HepG2 cells against the oxidative stress .     

Tubuloside B rescues the PC12 neuronal cells from H2O2-induced apoptosis

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.     

JAK-STAT pathway modulates NF-кB-mediated cytoprotection of H2O2 preconditioning

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.     

Effect of NPPB and niflumic acid on expression of GCL and CLIC4 in H2O2 injured C6 glioma cells

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.     

Adiponectin activates AKT and protects rat cardiomyocytes against H2O2-induced apoptosis

AIM: To study the effect of adiponectin on H₂O₂-induced apoptosis in rat cardiomyocytes (H9c2 cells). METHODS: Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) was used to determine H₂O₂-induced apoptosis of H9c2 cells in the absence or presence of adiponectin. The content of caspase-3 and Akt (protein kinase B, PKB) was examined by Western blotting. RESULTS: Adiponectin significantly inhibited H₂O₂-induced apoptosis in H9c2 cells (P<0.01). Adiponectin increased basal and H₂O₂-induced Akt activity and significantly inhibited H₂O₂-induced activation of caspase-3 (P<0.01). Pretreatment with LY294002, a specific inhibitor of Akt upstream kinase PI3K (phosphatidylinositol 3-kinase), not only increased H₂O₂-induced H9c2 cell apoptosis, but also abrogated the anti-apoptotic effect of adiponectin. CONCLUSION: Adiponectin protects H9c2 cells against H₂O₂-induced apoptosis by activating PI3K/Akt signaling pathway.