Effects of proteasomal peptide aldehydes inhibitors on expression of inflammation factors induced by LPS in Raw264.7 cell line / 中国病理生理杂志

ABSTRACT

AIM:To investigate the effects of MG132,one of the proteasomal peptide aldehydes inhibitors,on lipopolysaccharide(LPS)-induced nuclear transcription factor-kappa B(NF-?B) activation,the production of nitric oxide(NO) and tumor necrosis factor-alpha(TNF-?),as well as the expression of inducible nitric oxide synthase(iNOS) in murine macrophage line RAW264.7.METHODS:Reporter gene assay was used to examine the activity of NF-?B by pNiFty-SEAP/HEK293 cells,which were transfected with the pNiFty reporter plasmid into human embryo kidney cells(HEK293).Fluorescence substrate DAF-2DA was used to testify NO level in Raw264.7 cell line induced by LPS.Furthermore,the secretion of TNF-? was examined by ELISA.Western blotting was used to reveal the expression of iNOS and I?B-?.RESULTS:MG132 significantly decreased the secretion of TNF-? induced by LPS,with the inhibitory rates of 36.7% and 60.4% to 5 ?mol/L and 10 ?mol/L MG132,respectively.The pro-inflammatory mediator NO production was decreased in a dose-dependent manner with the inhibitory rates increasing from 29.5%(2 ?mol/L) to 55.9%(10 ?mol/L).Pretreatment with MG132 reduced the expression of iNOS,but restored the I?B restrain caused by LPS treatment.Observed by a reporter gene assay,TNF-?-induced NF-?B activity was decreased gradually by addition of increasing concentration of MG132(2.5-10 ?mol/L).CONCLUSION:Our results suggest an anti-inflammation effect of MG132 by the suppression of LPS-induced the production of pro-inflammatory mediators including NO and TNF-?,and the expression of iNOS,which probably mediates the blockage of I?B degradation and NF-?B activation.