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Objective:To investigate the effect of ginkgolide B （GB） on the activation of c-Jun aminoterminal kinase（JNK） signaling pathway and apoptosis in amyotrophic lateral sclerosis cell model. Method:NSC34 cells were infected by slow virus containing expression superoxide dismutase1（SOD1）WT and hSOD1G93A and empty plasmid， and screened with a certain concentration of puromycin， so as to observe the transfection efficiency of slow virus and cell morphology under inverted fluorescence microscope. Western blot method was used to verify whether infected cells were over-expressing SOD1 target proteins. The hSOD1G93A-NSC34 cell lines were established and given GB. Cell cultures were divided into normal group， model group and different concentrations of ginkgolide B groups （25， 50， 75， 100 mg∙L-1）. After 48 h， methyl thiazolyl tetrazolium （MTT） was used to detect cell survival rates， and select the best drug concentration. Subsequent experimental groups were divided into normal group， model group， 75 mg∙L-1 GB group， SP600125 group， and 75 mg∙L-1 GB + SP600125 group. Flow cytometry was used to detect the apoptosis of each group of cells. Western blot was used to detect the expressions of phosphorylation（p）-JNK， c-Jun， p-c-Jun， and cysteine aspartic acid protease -3（Caspase-3） proteins. Result:Compared with normal NSC34 cells， hSOD1G93A-NSC34 cell body became round， the synapses decreased and shortened， but the cell morphology of hSODWT-NSC34 cell and empty plasmid group did not change significantly. Western blot showed that hSOD1G93A-NSC34， hSOD1WT-NSC3 intracellular SOD1 protein levels increased significantly （P<0.01）， and the amyotrophic lateral sclerosis cell model was established. Compared with the normal group， the cell activity in the model group was significantly reduced （P<0.01）. Compared with the model group， the cell activity increased at different concentrations of GB， especially when the drug concentration was 75 mg∙L-1 （P<0.01）. In subsequent experiments， compared with the normal group， the apoptosis， and expressions of p-JNK， p-c-Jun， and cleaved Caspase-3 proteins in the model group increased significantly （P<0.01）. Compared with the model group， the apoptosis and p-JNK， p-c-Jun， released Caspase-3 protein expressions of 75 mg∙L-1 GB group， SP600125 group， 75 mg∙L-1 GB + SP600125 group decreased significantly （P<0.05， P<0.01）. Conclusion:GB has a protective effect on the cell model of atrophy lateral sclerosis， which may be realized by JNK signal pathway.
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Objective:To explore effects and mechanism on glucolipid metabolism of liver of ginsenoside Rb1 in diabetic rats.Methods:Diabetic rat model was induced by streptozotocin (STZ).Hepatic pathological changes were observed by hematoxylin-eosin (HE) staining.Apoptosis was analyzed through Terminal deoxynucleotidyl transferase-mediated dUTP nick labeling (TUNEL) staining.Blood glucose was determined with glucometer.The level of insulin and C peptide was tested by radioimmunoassay.Total cholesterol,low density lipoprotein cholesterol,high-density lipoprotein cholesterol,triglyceride was detected by full automatic biochemical analyzer.The expression of JNK signal pathway related proteins JNK1 and c-Jun was measured by Western blot.The expression of inflammatory factor IL-6,IL-1β and TNF-α was detected by immunohistochemistry.Results:Liver injury was ameliorated by ginsenoside Rb1 in diabetic rats.Compared with control group,the level of blood glucose in STZ-induced diabetic rat group was increased with attenuated level of insulin and C peptide (P<0.05).Compared with STZ-induced diabetic rat group,the level of blood glucose in STZ+ginsenoside Rb1 group was decreased with enhancive level of insulin and C peptide(P<0.05).Compared with control group,the level of total cholesterol,triglyceride and low density lipoprotein cholesterol in STZ-induced diabetic rat group was elevated with attenuated level of high-density lipoprotein cholesterol(P<0.05).Compared with STZ-induced diabetic rat group,the level of total cholesterol,triglyceride and low density lipoprotein cholesterol in STZ+ginsenoside Rb1 group declined with elevated level of high-density lipoprotein cholesterol(P<0.05).The relative expression of JNK1 and c-Jun in STZ-induced diabetic rat group was higher than control group (P<0.05).The relative expression of JNK1 and c-Jun in STZ+ginsenoside Rb1 group was lower than STZ-induced diabetic rat group(P<0.05).Compared with control group,the expression of IL-6,IL-1β and TNF-α in STZ-induced diabetic rat group was enhanced (P<0.05).The expression of IL-6,IL-1β and TNF-α in STZ+ginsenoside Rb1 group was lower than STZ-induced diabetic rat group(P<0.05).Conclusion:Ginsenoside Rb1 ameliorates liver injury,abnormal glucolipid metabolism and inflammatory response of liver through inhibition of JNK signal pathway in diabetic rats.
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Objective To investigate the effects of disulfiram (DS) combined with Cu on the human Burkitt lymphoma cell xenografts in nude mice.Methods Burkitt lymphoma xenograft was established by subcutaneous injection of Raji cell into nude mice after 2 Gy whole body X-irradiation (1×107 Raji cells were resuspended in 200 μl saline).18 bearing tumor mice were randomly divided into control group,DS group and DS/Cu group.During the experiment,the effects of DS/Cu on the nude mice with tumors were examined,including the tumor volumes,weights and the growth curves of xenograft tumor.Histopathological examination of tumor tissue was observed with optical microscape.The protein expression levels of p-JNK and c-jun were also detected by Western blot.Results Subsequent tumor size and weight in DS or DS/Cu-treated animals were (67.71±2.15) mm3,(33.35±7.74) mm3 and (43.35±4.22) mg,(18.05±2.88) mg.One-way ANOVA analysis indicated that the tumor size and weight in DS or DS/Cu-treated animals were reduced significantly relative to tumors in vehicle-treated animals (F =27.579,P =0.000;F =16.369,P =0.000).Furthermore,multiple comparisons revealed that the DS or DS/Cu-treated animals had significantly reduced tumor size and weight compared with control animals (all P ＜ 0.05).There were significant differences in tumor size and weight between DS or DS/Cu-treated animals (both P ＜ 0.05).Tumor inhibition rates in DS or DS/Cu group were 63.48 ％ and 80.24 ％,respectively.An increase of apoptosis changes in the xenograft tumor cells in DS or DS/Cu treated mice were more significant.Westem blot showed that the p-JNK and c-jun protein expressions in the tumors were improved after the DS or DS/Cu treatment,more obvious in DS/Cu treatment.Conclusion DS/Cu can inhibit the growth of xenografts,and one possible mechanism may involve the regulation of JNK signal pathway.
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Objective: To investigate the regulatory effect of C-Jun N-terminal kinase (JNK) signal transduction pathway on multi-resistance in the human hepatic cancer Bel-7402/5-fluorouracil (FU) cells, and provide a possible novel target for study on the mechanism of multidrug-resistance in human hepatic cancer cells. Methods: The protein expression levels of JNK and phospho-JNK (p-JNK) in parental human hepatic cancer Bel-7402 cells and the drug-resistant Bel-7402/FU cells were detected by Western blotting. After the inhibition of JNK pathway induced by specific inhibitior SP600125, the expressions of multi-drug resistance 1 (MDR1) mRNA and P-glycoprotein (P-gp) in Bel-7402/FU cells were detected by RT-PCR and immunocytochemistry, respectively. The accumulation and efflux of rhodanmine 123 (Rh123) in the cells were examined by flow cytometry, and the sensitivity to 5-FU of Bel-7402/FU cells was detected by MTT method. Results: The expression of JNK protein was not significantly different between the Bel-7402/FU cells and the parental Bel-7402 cells, but the expression of p-JNK protein in the Bel-7402/FU cells was significantly increased. After inhibition of JNK pathway, the expressions of MDR1 mRNA and P-gp protein were obviously decreased, with a markedly increased accumulation and decreased efflux of Rh123, leading to enhhanced sensitivity to 5-FU of Bel-7402/FU cells. Conclusion: JNK signal transduction pathway is involved in the expressions of MDR1 gene and P-gp in drug-resistant human hepatic cancer Bel-7402/FU cells, and it can regulate the sensitivity to 5-FU of Bel-7402/FU cells.
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AIM: To investigate the effect of high glucose toxicity on JNK pathway and cell function of INS-1 cells.METHODS: Cultured INS-1 cells with or without IGF-1 exposure, were treated with glucose at 3 concentrations (5.6 mmol/L, 11.2 mmol/L and 33.3mmol/L), respectively. MTT was used to measure the cell viability. Apoptosis was determined by immuno-fluorescence and flow-cytometry analysis. The serine 270 phosphorylation of IRS and phosphorylation of JNK in INS-1 cells were detected in the presence or absence of SP600125 treatment.RESULTS: The cell viability decreased and apoptosis increased with elevated glucose concentrations. The percentage of apoptosis cells was 11.3% in 5.6 G group, 12.7% in 11.2 G group and 28.2% in 33.3 G group. There was remarkable increase in apoptosis in 33.3 G group with a 2.49-fold increase to the cells in the basal 5.6 mmol/L glucose. High glucose activated the serine 270 phosphorylation of IRS correlates with JNK phosphorylation in INS-1 cells. Using Western blotting analysis, the levels of JNK phosphorylation were 3.33 fold increased and serine 270 phosphorylation of IRS was 1.17 fold increased in 33.3 G group compared to 11.2 G group (P<0.01). IGF-1 treatment inhibited phosphorylation of JNK and IRS. SP600125 treatment completely blocked JNK phosphorylation in 11.2 G group and reduced JNK phosphorylation by 90% in 33.3 G group. In addition, SP600125 treatment partly reduced serine 270 phosphorylation of IRS by 88.3% in 11.2 G group and 80% in 33.3 G group, the viability of INS-1 cells increased and the apoptosis decreased.CONCLUSION: The toxicity of chronic high glucose, which inhibits the cells viability and induces the cell apoptosis, might be related to suppress IRS signal by activating the JNK pathway. Blocking the JNK pathway might relieve the effect of glucose toxicity to the β cell function by improving the IRS signal pathway.
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AIM:To investigate the effect of high glucose toxicity on JNK pathway and cell function of INS-1 cells.METHODS: Cultured INS-1 cells with or without IGF-1 exposure,were treated with glucose at 3 concentrations (5.6 mmol/L,11.2 mmol/L and 33.3mmol/L),respectively. MTT was used to measure the cell viability. Apoptosis was determined by immuno-fluorescence and flow-cytometry analysis. The serine 270 phosphorylation of IRS and phosphorylation of JNK in INS-1 cells were detected in the presence or absence of SP600125 treatment.RESULTS: The cell viability decreased and apoptosis increased with elevated glucose concentrations. The percentage of apoptosis cells was 11.3% in 5.6 G group,12.7% in 11.2 G group and 28.2% in 33.3 G group. There was remarkable increase in apoptosis in 33.3 G group with a 2.49-fold increase to the cells in the basal 5.6 mmol/L glucose. High glucose activated the serine 270 phosphorylation of IRS correlates with JNK phosphorylation in INS-1 cells. Using Western blotting analysis,the levels of JNK phosphorylation were 3.33 fold increased and serine 270 phosphorylation of IRS was 1.17 fold increased in 33.3 G group compared to 11.2 G group (P