Résumé
Objective@#To investigate the effect of metformin on autophagy in muscle cells exposed to palmitic acid, and to explore its mechanism.@*Methods@#L6 rat myoblasts were incubated with palmitic acid at various concentrations(0.1, 0.2, 0.4, 0.6 mmol/L) and metformin(0.5, 1, 2, 5, 10 mmol/L) for 24 h. CCK8 method was used to detect the survival rate of muscle cells. After muscle cells were treated with palmitic acid and metformin for 24 h, mRNA and protein expressions of microtubule-associated protein11ight chain3(LC3Ⅱ), Beclin 1, p62, and silent mating type information regulation2 homolog-3(SIRT3) were detected by RT-PCR and Western blot, respectively. AMP-activated protein kinase(AMPK) phosphorylation level was detected by Western blot.@*Results@#Palmitic acid dose-dependently decreased the survival rate of muscle cells, which was attenuated by metformin at the concentration of 2 mmol/L. After muscle cells were incubated with 0.4 mmol/L palmitic acid and 2 mmol/L metformin for 24 h, palmitic acid significantly reduced the mRNA and protein expressions of LC3Ⅱ, Beclin1, and SIRT3 as well as phosphorylation level of AMPK(all P<0.05), and increased p62 mRNA and protein expressions(P<0.05). Those effects were all antagonized by metformin(all P<0.05).@*Conclusions@#Metformin treatment may promote the autophagy of muscle cells exposed to palmitic acid through AMPK/STRT3 pathway.
Résumé
Objective:To investigate the effect of metformin on autophagy in muscle cells exposed to palmitic acid, and to explore its mechanism.Methods:L6 rat myoblasts were incubated with palmitic acid at various concentrations(0.1, 0.2, 0.4, 0.6 mmol/L) and metformin(0.5, 1, 2, 5, 10 mmol/L) for 24 h. CCK8 method was used to detect the survival rate of muscle cells. After muscle cells were treated with palmitic acid and metformin for 24 h, mRNA and protein expressions of microtubule-associated protein11ight chain3(LC3Ⅱ), Beclin 1, p62, and silent mating type information regulation2 homolog-3(SIRT3) were detected by RT-PCR and Western blot, respectively. AMP-activated protein kinase(AMPK) phosphorylation level was detected by Western blot.Results:Palmitic acid dose-dependently decreased the survival rate of muscle cells, which was attenuated by metformin at the concentration of 2 mmol/L. After muscle cells were incubated with 0.4 mmol/L palmitic acid and 2 mmol/L metformin for 24 h, palmitic acid significantly reduced the mRNA and protein expressions of LC3Ⅱ, Beclin1, and SIRT3 as well as phosphorylation level of AMPK(all P<0.05), and increased p62 mRNA and protein expressions( P<0.05). Those effects were all antagonized by metformin(all P<0.05). Conclusions:Metformin treatment may promote the autophagy of muscle cells exposed to palmitic acid through AMPK/STRT3 pathway.
Résumé
Objective:To obtain antibodies against amylin from a 'naive' human Fab fragment antibody phage diasplay library and to analyze the specificity of antigen binding activity.Methods:Panning and screening Fab antibody from the antibody library,the positive clones with well reactivity to amylin were selected after five times selection of 'adsorption-elution-enrichment'.Then the plasmid DNA which was extracted from the clones,was digested with Spe Ⅰ and Nhe Ⅰ to delete gⅢ (about 660 bp).The digested 47 000 bp DNA which was purified after separation of bands from agarose gel was ligated with T4-DNA ligase.The constructed expressing phagemids were transformed to the BL21(DE3)pLysS,soluble Fab was expressed in it by the induction of IPTG and its characteristics and specificity were determined by ELISA and Western blot.Results:Soluble Fab antibodies were expressed in E.coli.According with molecular weight of IgG Fab,protein band of about 47 kD was shown by SDS-PAGE.Western blot using the goat anti human IgG-HRP showed their binding activities.ELISA showed their specificity with amylin antigens and they did not react with bovine serum albumin.Conclusion:The high level expression and identification of the soluble human anti- amylin Fab fragment antibodies has been obtained successfully,which lays a solid foundation for further researching about the biological and pathological activities of amylin.