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1.
AIM: To investigate the protective effect of N-acetylcysteine(NAC) on H9c2 cells from injuries induced by methylglyoxal(MG) and the potential mechanism. METHODS: H9c2 cells were divided into control group, MG treatment group, NAC + MG treatment group, SP600125 pretreatment + MG group, NAC group and SP600125 group. The viability of the H9c2 cells was measured by CCK-8 assay. The protein levels of p-JNK and t-JNK were tested by Western blot. The changes of intracellular reactive oxygen species(ROS) were evaluated by 2', 7'-dichlorofluorescein diacetate(DCFH-DA) staining. Mitochondrial membrane potential(MMP) was measured by rhodamine 123(Rh123) staining. The morphological changes in apoptotic cardiomyocytes were detected by Hoechst 33258 staining. RESULTS: Du-ring 100~800μmol/L concentration range, MG caused significantly reduced viability of the H9c2 cells in a dose-dependent manner. NAC had a protective effect on H9c2 cells against the injuries induced by MG during 500~1500μmol/L concentration range through raising cell viability, inhibiting cellular oxidative stress and improving MMP(P<0.01). SP600125, an inhibitor of JNK, showed the protective effect similar to NAC on H9c2 cells against MG-induced injuries, including attenuating oxidative stress, improving MMP and suppressing apoptosis.CONCLUSION: N-acetylcysteine offers obvious protective effect on H9c2 cells against the injuries induced by methylglyoxal. The underlying mechanisms may be associated with decreasing the production of ROS, ameliorating MMP, inhibiting the activation of JNK and suppressing apoptosis.  相似文献   

2.
AIM: To investigate the effect of polysaccharide from Fructus corni(PFC) on cardiomyocytes against hypoxia/reoxygenation (H/R) injury and its possible relationship with ROS/PKC/p38 MAPK pathway.METHODS: Primary cardiomyocytes were isolated from neonatal SD rats and randomly divided into normal group, H/R group, PFC (20 mg/L, 100 mg/L and 200 mg/L) preconditioning+H/R groups, chelerythrine+PFC (100 mg/L)+H/R group and SB203580+PFC (100 mg/L)+H/R group. The cell viability was measured by inverted microscopic observation. Apoptosis in the cardiomyocytes was detected by Hoechst 33258 staining and fluorescence microscopy. The levels of lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in the cell culture supernatants, and the reactive oxygen species (ROS) in the cells were also measured by microplate reader. The protein levels of PKC, p-p38 MAPK and HSP70 in the cells were detected by Western blotting.RESULTS: Compared with normal group, the cell viability and beating frequency were decreased in H/R group. LDH and ROS contents, apoptotic rate and p-p38 MAPK level increased significantly (P<0.01). Compared with H/R group, PFC preconditioning increased beating frequency, SOD activity and the protein level of PKC and HSP70, and decreased ROS production, the protein level of p-p38 MAPK and cell apoptotic rate. However, the effect of PFC was inhibited by chelerythrine or SB203580.CONCLUSION: PFC may protect cardiomyocytes from hypoxia/reoxygenation injury. Its mechanism is possibly involved in the inhibition of ROS via increasing the activity of SOD and the activation of PKC, and suppression of excessive activation of p38 MAPK.  相似文献   

3.
AIM: To explore the production and cytotoxicity of the reactive oxygen species (ROS) induced by diallyl trisulfide (DATS) in HL-60 cells.METHODS: HL-60 cells were either treated with various doses of DATS alone, or DATS combination with apocynin, a specific NADPH oxidase inhibitor, or with antioxidant N-acetyl-L-cysteine (NAC) for 0, 1, 3, 6, 12 and 24 h, respectively. The intracellular ROS level was measured by flow cytometry. The activity of NADPH oxidase was evaluated by NBT reduction experiment. The content of both malondialdehyde (MDA) and the protein carbonyl were analyzed by spectrophotometer. RESULTS: The results from flow cytometry indicated that DATS significantly increased the intracellular ROS level in HL-60 cells (P<0.05), which was dose-and time-dependent. The fluorescence intensities of ROS reached at maximum when HL-60 cells were incubated with 150 μmol/L DATS for 3 h. The NBT reduction experiment showed that DATS activated NADPH oxidase, the highest activity was observed when the cells were exposed to 150 μmol/L DATS for 3 h. DATS induced MDA and protein carbonyl production in HL-60 cells. Furthermore, both MDA and protein carbonyl reached the highest level in the cells exposed to 150 μmol/L DATS for 3 h. Apocynin and NAC attenuated the production of MDA and protein carbonyl, suggesting that ROS induced by DATS was involved in the toxicity to the cells. CONCLUSION: DATS induces ROS production through activating NADPH oxidase in HL-60 cells. ROS increases the oxidation of membrane lipid and protein in HL-60 cells.  相似文献   

4.
AIM: To investigate the effects of glucagon-like peptide-1 (GLP-1) on myocardial ischemia-reperfusion (IR)/hypoxia-reoxygenation (HR) injury in rats. METHODS: Sprague-Dawley rats were randomly divided into 5 groups: sham group, IR group and IR+GLP-1 (0.03 nmol/L, 0.16 nmol/L and 0.30 nmol/L) groups. IR group and IR+GLP-1 group were subject to 30 min of ischemia and 3 h of reperfusion. The myocardial infarct size, the ultrastructural changes of the myocardial tissues, the apoptosis of the cardiomyocytes, the activity of superoxide dismutase (SOD) and the concentration of malondialdehyde (MDA) were detected. Primarily cultured cardiomyocytes were divided into 5 groups at random: control group, HR group and HR+GLP-1 (1 μmol/L, 5 μmol/L and 10 μmol/L) groups. The morphology and apoptosis of the cardiomyocytes were observed. The levels of lactate dehydrogenase (LDH),MDA,SOD,reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in different groups were detected. RESULTS: Compared with IR group, the myocardial infarct size and cardiomyocyte apoptosis were remarkably reduced, mitochondrial ultrastructures were improved, the activity of SOD was increased and the concentration of MDA was decreased in IR+GLP-1 (0.03 nmol/L, 0.16 nmol/L and 0.30 nmol/L) groups. Compared with HR group, GLP-1 (1 μmol/L, 5 μmol/L and 10 μmol/L) preconditioning significantly decreased the myocardial injury, increased SOD activity, decreased MDA concentration and ROS production, and heightened MMP in a dose-dependent manner. CONCLUSION: GLP-1 protects cardiomyocytes from IR/HR injury, which may be partially due to the effects of anti-oxidative mechanism and the function of mitochondrial protection.  相似文献   

5.
AIM: To investigate the effect of N-acetylcystein (NAC) on oxidant stress, neuron apoptosis in the hippocampal CA1 region of rats exposured to chronic intermittent hypoxia (CIH). METHODS: 30 healthy male Wistar rats were randomly divided into three groups of 10 each, a CIH group, a NAC therapeutic group and a control group. The levels of MDA and SOD were detected by colorimetric method. Immunohistochemistry was used to examine the expression of p-JNK and TUNEL was used to detect the neuron apoptosis in the hippocampal CA1 region. RESULTS: The level of MDA in NAC group were lower than that in CIH group［(1.71±0.43) μmol/g protein vs （1.37±0.26) μmol/g protein, P<0.05)］. The activity of SOD in NAC group was higher than that in CIH group［(44.94±14.01) 103 NU/g protein vs(57.66±14.07) 103 NU/g protein, P<0.05)］. The expression of p-JNK protein and the apoptotic indices ［(0.39±0.16), (0.20±0.11)］ in NAC group were significantly lower than those in CIH group ［(0.53±0.10), (0.32±0.18), all P<0.05］. CONCLUSION: NAC protects hippocampal neuron from apoptosis by suppressing the oxidant stress in the hippocampal CA1 region and inhibiting the activation of JNK signaling pathway.  相似文献   

6.
AIM: To investigate the protective effect of astragaloside IV (ASIV) on angiotensin II (Ang II)- induced apoptosis of H9c2 cardiomyocytes. METHODS: H9c2 cardiomyocytes were treated with different concentrations of Ang II and ASIV. The effects of Ang II and ASIV on the viability of H9c2 cells was measured by CCK-8 assay. The optimum concentration of Ang II was 1 μmol/L and the concentrations of ASIV were 25, 50 and 100 μmol/L. The H9c2 cells was divided into 6 groups:control group, ASIV group, Ang II group, Ang II+ASIV (25 μmol/L) group, Ang II+ASIV 50 (μmol/L) group and Ang II+ASIV (100 μmol/L) group. The morphological changes of the H9c2 cells were observed under inverted phase-contrast microscope. Apoptosis was detected by TUNEL assay. The generation of reactive oxygen species (ROS) was detected by DCFH-DA staining. The protein expression of Bax, Bcl-2, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was determined by Western blot. H9c2 cardiomyocytes were transfected with negative control shRNA (NC) or Nrf2-shRNA (shRNA), and the cells were divided into 8 groups:NC+control group, NC+AngⅡgroup, NC+ASIV group, NC+AngⅡ+ASIV group, shRNA+control group, shRNA+AngⅡgroup, shRNA+ASIV group and shRNA+AngⅡ+ASIV group. ROS level was detected by ROS detection kit. The protein expression of Nrf2 and HO-1 was determined by Western blot. RESULTS: Ang II decreased the viability of H9c2 cells in a concentration-dependent manner (P<0.05). ASIV reversed the effect of Ang II on the viability of H9c2 cells in a concentration-dependent manner (P<0.05). Compared with control group, the apoptotic rate, the level of ROS and the protein expression of Bax in Ang II group were increased significantly, while the protein expression of Bcl-2, Nrf2 and HO-1 was decreased significantly (P<0.05). Compared with Ang II group, ASIV reversed the increase in apoptotic rate of H9c2 cells induced by Ang II in a concentration-dependent manner, reduced ROS level, down-regulated the protein expression of Bax and up-regulated the protein expression of Bcl-2, Nrf2 and HO-1 (P<0.05). After shRNA transfection, the effects of ASIV decreasing ROS production induced by Ang II and up-regulating the expression of Nrf2 and HO-1 were eliminated. CONCLUSION: ASIV protects H9c2 cardiomyocytes from apoptosis induced by Ang II, which may be related to reducing ROS generation and mediating the Nrf2/HO-1 signaling pathway.  相似文献   

7.
AIM: To investigate the effects of CR6-interacting factor 1 (CRIF1) on the apoptosis of mouse cardiomyocytes (MCMs) induced by palmitic acid. METHODS: The MCMs cultured with medium containing palmitic acid at 0 and 300 μmol/L for 24 h were divided into control group and palmitic acid group, respectively. In order to explore the effects of CRIF1 on MCMs injuries induced by high fat, MCM exposed to palmitic acid at 300 μmol/L for 24 h were divided into vehicle group, scrambled (Scra) siRNA group and CRIF1 siRNA group. The cells in vehicle group were only treated with transfection reagent, the cells in Scra siRNA group were given a treatment with transfection reagent and scrambled RNA, and the cells in CRIF1 siRNA group were given a treatment with transfection reagent and CRIF1 specific siRNA. In order to further confirm the specific mechanism of CRIF1 in high fat-induced MCM injuries, MCMs in CRIF1 siRNA group were divided into DMSO group and N-acetyl cysteine (NAC) group, and were given the same intervention of palmitic acid. RT-qPCR, Western blot and immunofluorescence staining were used to detect the mRNA and protein expression of CRIF1. The apoptotic rate was analyzed by flow cytometry, and the level of intracellular reactive oxygen species (ROS) was tested by DHE staining and ELISA. RESULTS: The expression of CRIF1 was significantly increased after exposure to palmitic acid (P<0.05). Compared with control group, the apoptotic rate was increased significantly in vehicle group and Scra siRNA group (P<0.05). The apoptotic rate was significantly increased in CRIF1 siRNA group (P<0.05). Compared with control group, the intracellular ROS content was significantly increased in vehicle group and Scra siRNA group (P<0.05). Compared with vehicle group and Scra siRNA group, the intracellular ROS content was significantly increased in CRIF1 siRNA group (P<0.05). Compared with DMSO group, the intracellular ROS content and the apoptotic rate were remarkably decreased in NAC group (P<0.05). CONCLUSION: With the inhibition of oxidative stress, CRIF1 may reduce the apoptosis of MCMs induced by high fat.  相似文献   

8.
AIM: To investigate the changes of reactive oxygen species (ROS) in apoptosis of LoVo cells induced by 23-hydroxybetulinic acid.METHODS: LoVo cells were treated with 23-hydroxybetulinic acid. The apoptotic morphological change was observed under the light microscope. Intracellular ROS production and the rate of apoptosis were detected by flow cytometry.RESULTS: LoVo cells improved apoptotic morphological changes treated with 23-hydroxybetulinic acid for 48 h. At concentrations of 25, 50, 100, 200 μmol/L of 23-hydroxybetulinic acid, the apoptotic rates of LoVo cells were (7.17±2.31)%, (15.60±4.02)%, (32.47±5.25)% and (52.71±5.93)%, respectively. The results indicated a certain concentration-dependent relationship. 23-hydroxybetulinic acid caused an increase in the ROS production, and the ROS levels were 2.83±0.80, 5.97±1.72, 12.53±2.57 and 16.73±4.58. Compared with the control group (2.13±0.32), the increase in ROS production in LoVo cells at the concentration of 100, 200 μmol/L of 23-hydroxybetulinic acid treatment was significant (P<0.05). CONCLUSION: 23-hydroxybetulinic acid induces LoVo cell apoptosis. The production of ROS may play a crucial role in the process of the LoVo cell apoptosis.  相似文献   

9.
AIM: To study whether the angiotensin-(1-7)[Ang-(1-7)]/Mas receptor axis protects cardiomyocytes against high glucose(HG)-induced injury by inhibiting nuclear factor-κB(NF-κB) pathway. METHODS: The cell viability was measured by CCK-8 assay. The intracellular levels of reactive oxygen species(ROS) were detected by DCFH-DA staining. The number of apoptotic cells was tested by Hoechst 33258 nuclear staining. Mitochondrial membrane potential(MMP) was examined by JC-1 staining. The levels of NF-κB p65 subunit and cleaved caspase-3 protein were determined by Western blotting. RESULTS: Treatment of H9c2 cardiac cells with 35 mmol/L glucose(HG) for 30, 60, 90, 120 and 150 min significantly enhanced the levels of phosphorated(p) NF-κB p65, peaking at 60 min. Co-treatment of the cells with 1 μmol/L Ang-(1-7) and HG for 60 min attenuated the up-regulation of p-NF-κB p65 induced by HG. Co-treatment of the cells with Ang-(1-7) at concentrations of 0.1~30 μmol/L and HG for 24 h inhibited HG-induced cytotoxicity, evidenced by an increase in cell viability. On the other hand, 1 μmol/L Ang-(1-7) ameliorated HG-induced apoptosis, oxidative stress and mitochondrial damage, indicated by decreases in the number of apoptotic cells, cleaved caspase-3 level, ROS generation and MMP loss. However, the above cardioprotective effects of Ang-(1-7) were markedly blocked by A-779, an antagonist of Ang-(1-7) receptor(Mas receptor). Similarly, co-treatment of H9c2 cardiac cells with 100 μmol/L PDTC(an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries induced by HG. CONCLUSION: Ang-(1-7)/Mas receptor axis prevents the cardiomyocytes from the HG-induced injury by inhibiting NF-κB pathway.  相似文献   

10.
AIM: To investigate the role of mitogen-activated protein kinases (MAPKs) pathways and the molecular mechanism by which the proto-oncogene Pim-3 protects cardiomyocyte against anoxia/reoxygenation (A/R) injury. METHODS: The primarily cultured neonatal rat ventricular cardiomyocytes were randomly divided into 4 groups: control group; A/R group; APC+A/R group; SB203850, U0126 or SP600125+APC+A/R group. The cells were pre-incubated with U0126 (ERK1/2 inhibitor), SP600125 (SAPK/JNK inhibitor), or SB203850 (p38 MAPK inhibitor) at concentration of 10 μmol/L for 30 min before the APC. The activities of p38 MAPK, JNK and ERK1/2 were detected by Western blotting. The viability of cardiomyocytes was assayed by MTT and the apoptosis of cardiomyocyte was detected by TUNEL. RESULTS: U0126, SB203850, and SP600125 abolished the increased expression of ERK1/2, p38-MAPK, and JNK proteins induced by APC+A/R or A/R, respectively. The expression level of Pim-3 protein significantly decreased when the p38 MAPK signal pathway was inhibited. Meanwhile, the activity of LDH and the apoptosis index increased, and the viability of cardiomyocytes decreased. CONCLUSION: Pim-3 expression through a p38 MAPK signaling pathway may protect cardiomyocytes from A/R injury.  相似文献   

11.
AIM: To study the effects of exogenous spermine on human umbilical veins endotheliocytes (HUVECs) and to explore its possible mechanism. METHODS: The serial subculture of HUVECs was used to investigate the effect of exogenous spermine with different concentrations (50 μmol/L-5 mmol/L) on HUVECs in different times (2 h, 4 h). The morphological changes of HUVECs (by inverted microscope and electron microscope), the cell viability, the level of MDA and activity of SOD were observed. RESULTS: Compared to normal control group, no change of all index detected in the group with spermine (50 μmol/L) was observed (P>0.05). Spermine injured HUVECs in a concentration-dependent manner. After adding spermine for 2 h and 4 h, it was observed that cellular injury in 4 h group was more serious than that in 2 h group. The injury of HUVECs caused by exogenous spermine was characterized by decrease in cellular viability and activity of SOD, ultrastructural injury, increase in MDA level. CONCLUSION: Exogenous spermine induces the injury of HUVECs in concentration and time-dependent manners. Its mechanisms may be related to lipid peroxidation induced by increase in the production of oxygen free radical.  相似文献   

12.
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.  相似文献   

13.
AIM: To investigate the effects of epigallocatechin-3-gallate (EGCG) on 1-methyl-4-phenylpyridinium ion (MPP+)-induced apoptosis in rat pheochromocytoma (PC12) cells and to explore the relationships between its roles of anti-oxidation, intracellular calcium homeostasis and anti-apoptosis. METHODS: Rat PC12 cells were pretreated with vehicle control or EGCG (10, 50, and 100 μmol/L) for 30 min, then cultured with MPP+ (900 μmol/L) for 24 h. The cell viability and apoptosis were monitored by MTT assay and flow cytometry using Annexin V and PI. The activity of intracellular reactive oxygen species (ROS), contents of superoxide dismutase (SOD) and malondialdehyde (MDA), cytoplasmic Ca2+ density and apoptotic morphology of mitochondria were examined by fluorescent plate-based assays, confocal microscope, and transmission electron microscope, respectively. RESULTS: MPP+ impaired the PC12 cells in a concentration-dependent pattern and induced apoptosis of the cells (31% versus control). Compared with the control, the cells pretreated with EGCG showed markedly higher rate of viability and lower apoptosis. Meanwhile, EGCG pretreatment significantly increased the SOD activity and decreased the levels of MDA and ROS. Interestingly, EGCG also decreased the concentration of cytoplasmic Ca2+ and improved the morphology of mitochondria. CONCLUSION: EGCG exhibits inhibitory effects on MPP+-induced apoptosis in rat PC12 cells, which is possibly associated with increasing the cell ability of anti-oxidation and decreasing the concentration of cytoplasmic Ca2+.  相似文献   

14.
AIM:To investigate the characteristics of pathological injury and its relationship with the transformation of energy metabolism of hypertrophic cardiomyocytes after hypoxia-reoxygenation. METHODS:Cultured rat cardiomyocytes were induced to be hypertrophy by angiotensin Ⅱ (Ang Ⅱ) and norepinephrine (NE). Glucose oxidation rate (GOR), glucolysis rate (GLR) and fatty acid oxidation rate (FOR) were determined by liquid scintillation counting, and cell apoptosis was detected by TUNEL. RESULTS:(1) Compared with the normal cardiomyocytes (NC), the GOR and GLR were slightly higher and the FOR was slightly lower in the group of hypertrophic cardiac cells (HC) than that in the group of normal cardiomyocytes cultured under the normal oxygen partial pressure. The apoptosis rate had no difference between the two groups. (2) The apoptosis rate of hypertrophic cardiomyocytes after hypoxia was significantly higher than that of hypertrophic cardiomyocytes in normal culture. It was higher and moreover, some necrosis cardiomyocytes appeared after reoxygenation. (3) GOR and FOR in both group (NC and HC) were slightly lower in a time-dependent manner after hypoxia than that in each group in normal culture condition. GLR had no difference in both group. The GOR was more lower in both NC and HC group when reoxygenation than that at the point of hypoxia for 2 hours, but the GLR and FOR were significantly higher in HC than that in NC when reoxygenation. (4) The GOR was significantly higher and the GLR and FOR were significantly lower in the hypertrophic cardiomyocytes group (HC) with dichloroacetate (DCA, 1 000 μmol/L) or trimetazidine (TMZ, 1 μmol/L) treated respectively than that in the responded hypertrophic cardiomyocytes after stimulation by hypoxia-reoxygenation. In the meanwhile, the apoptosis rate also was markedly lower in the treated hypertrophic cardiomyocytes group. CONCLUSION:The transformation of energetic metabolism pathway plays an important role in the pathogenesis (mainly the apoptosis) of the hypertrophic cardiomyocytes after hypoxia-reoxygenation.  相似文献   

15.
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   

16.
WAN Bin  CAO Heng-bin  YU Gen-hua 《园艺学报》2017,33(12):2195-2201
AIM: To investigate the effect of dihydroartemisinin (DHA) adjuvant treatment on enhancing the antitumor effect of 5-fluorouracil (5-FU) against gastric cancer. METHODS: The gastric cancer BGC-823 cells were divided into control group, DHA group, 5-FU group, 5-FU+DHA group and 5-FU+DHA+SIRT1 plasmid group. The viability of BGC-823 cells treated with DHA and 5-FU was measured by MTT assay. The expression of SIRT1 and NADPH oxidase, activation of caspase-9 and caspase-3, and phosphorylation of ASK1 and JNK in the BGC-823 cells treated with DHA and 5-FU were determined by Western blot. The production of ROS and the apoptosis of the BGC-823 cells treated with DHA and 5-FU were analyzed by flow cytometry. RESULTS: Dihydroartemisinin significantly inhibited the expression of SIRT1 and increased NADPH oxidase protein level (P<0.05). DHA increased the sensitivity of BGC-823 cells to 5-FU, thus decreasing the IC50 of 5-FU to the gastric cancer cells. However, transfection with SIRT1 plasmid decreased the cytotoxicity of DHA and 5-FU co-treatment to the BGC-823 cells. DHA promoted the production of ROS and phosphorylation of ASK1 and JNK induced by 5-FU in the BGC-823 cells (P<0.05). However, ROS scavenger N-acetylcysteine (NAC) or JNK specific inhibitor SP600125 inhibited the cell death and activation of caspase-9 and caspase-3 induced by DHA and 5-FU co-treatment (P<0.05). In addition, NAC significantly inhibited the phosphorylation of JNK in the BGC-823 cells co-treated with DHA and 5-FU. However, treatment with SP600125 did not influence the ROS production in the BGC-823 cells, indicating that JNK was the downstream target of ROS pathway. CONCLUSION: Combination of DHA with 5-FU induces caspase-dependent apoptosis in gastric cancer cells through the SIRT1/NADPH oxidase/ROS/JNK signaling pathway.  相似文献   

17.
AIM: To evaluate the effects of angiotensinⅡ (AngⅡ) on autophagy induction in vascular endothelial cells. METHODS: Human vascular endothelial EA.hy926 cells were used in the study. Intracellular reactive oxygen species (ROS) levels were detected by a microplate reader after the cells were treated with AngⅡ (10-7 mol/L) or AngⅡ combined with antioxidant N-acetyl-L-cysteine (NAC,50 μmol/L) for 24 h. The protein levels of LC3-Ⅱ was detected by Western blotting after the cells were stimulated by different concentrations (10-8, 10-7, 10-6 mol/L) of AngⅡ for 24 h or by AngⅡ (10-7mol/L) for different time (0 h, 6 h, 12 h, 24 h, 36 h). The number of autophagosomes was evaluated by fluorescence microscopy after stained with acridine orange. Similarly, the protein level of LC3-Ⅱ and the number of autophagosomes were detected after treated with AngⅡ(10-7mol/L), AngⅡ combined with autophagy inhibitor 3-methyladenine (3-MA) at concentration of 2 mmol/L or AngⅡ combined with NAC at concentration of 50 μmol/L. RESULTS: Intracellular ROS level and LC3-Ⅱprotein level were significantly increased (P<0.05) after the cells were treated with AngⅡ, accompanied by the significant increase in the number of autophagosomes. AngⅡ-induced autophagy (as showed both in LC3-Ⅱprotein level and autophagosomes) was dramatically down-regulated by the treatment with 3-MA or NAC in EA.hy926 cells (P<0.05). CONCLUSION: AngⅡ induces autophagy through elevating ROS levels in EA.hy926 cells.  相似文献   

18.
AIM：To investigate the effect of cholestane-3β, 5α, 6β-triol (Triol) on apoptosis of malignant glioma cells. METHODS：C6 cells and A172 cells were incubated with Triol at different concentrations for different time durations. MTT assay was used to detect the cell viability. Hoechst 3f3342 staining and TUNEL assay were used to analyze the cell apoptosis. The caspase activity was measured. The expression of apoptosis-related proteins, Bcl-2 family members, was determined by Western blotting. RESULTS：Triol decreased the cell viability of C6 and A172 cells in a dose- and time-dependent manner and the IC50 values were (17.8±0.6)μmol/L and (20.6±0.2) μmol/L, respectively. Visible nuclei with apoptotic characteristics, significant increase in TUNEL-positive cells, and the activation of apoptotic execution enzyme caspase-3 indicated that cell apoptosis was induced by Triol in both cell lines. After C6 cells were exposed to Triol for 12 h, 24 h and 48 h, the activity of caspase-8 in extrinsic apoptotic pathway and caspase-9 in intrinsic apoptotic pathway was increased time-dependently. Meanwhile, the levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL, was down-regulated, while pro-apoptotic protein Bak was up-regulated in a time-dependent manner. CONCLUSION：Triol induces apoptosis of malignant glioma cells by activating intrinsic and extrinsic apoptotic pathways, and Bcl-2 family members are involved in Triol-induced apoptosis.  相似文献   

19.
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   

20.
AIM：To determine the effect of endogenous hydrogen sulfide (H2S) on the production of reactive oxygen species (ROS) in medullary neurons induced by angiotensin II (Ang II). METHODS：Primary cultured rat medullary neurons were used in the study. Identification of medullary neurons and the co-expression of cystathionine β-synthetase (CBS) were detected by double-labeling immunofluorescence. Medullary neurons were treated with Ang II in the presence or absence of sodium butyrate (NaBu, a CBS agonist; 100 μmol/L, 250 μmol/L and 500 μmol/L). ROS production was measured by dihydroethidium staining. The activity of total superoxide dismutase (SOD) was detected by ELISA. The mRNA expression of CBS was determined by real-time PCR. RESULTS：The medullary neurons in the cultured cells were over 90%. Ang II (1 μmol/L) significantly increased ROS level in the medullary neurons. Ang II inhibited the activity of total SOD in the medullary neurons. CBS was expressed in the medullary neurons. Ang II decreased the mRNA expression of CBS. NaBu (250 μmol/L and 500 μmol/L) inhibited ROS production induced by Ang II with a dose-dependent manner, while NaBu alone had no influence on the ROS level in the medullary neurons. CONCLUSION：Ang II increases the level of ROS in medullary neurons partly by inhibiting the activity of total SOD and the mRNA expression of CBS. Endogenous H2S inhibits the ROS level increased by Ang II in the medullary neurons.  相似文献   

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