醛肽类蛋白酶体抑制剂对LPS诱导小鼠巨噬细胞炎症介质表达的影响

Effects of proteasomal peptide aldehydes inhibitors on expression of inflammation factors induced by LPS in Raw264.7 cell line

目的:探讨醛肽类蛋白酶体抑制剂MG132对脂多糖(LPS)诱导小鼠巨噬细胞Raw264.7核转录因子-κB(NF-κB)活化、炎性因子一氧化氮(NO)和肿瘤坏死因子α(TNF-α)分泌以及诱导型一氧化氮合酶(iN-OS)表达的影响。方法:利用报告基因检测法分析转染细胞(pNiFty-SEAP/HEK293)中NF-κB的活性;采用DAF-2DA荧光探针法检测Raw264.7细胞内NO的产生;蛋白免疫印迹法探讨细胞中iNOS和IκB-α蛋白表达情况;酶联免疫吸附法测定Raw264.7细胞上清中TNF-α的含量。结果:预先加入MG132能够显著抑制LPS诱导的TNF-α分泌,其抑制率由5μmol/L时的36.7%升高到10μmol/L时的60.4%。反映NO含量的荧光强度值随着MG132给药浓度的增加而降低,其抑制率从2μmol/L时的29.5%达到10μmol/L的55.9%。预刺激后MG132可使胞浆中iNOS蛋白表达减少,IκB-α蛋白却明显增加,NF-κB的活性随着给药浓度的升高而不断降低。结论:MG132能够抑制LPS诱导的TNF-α和NO的产生,减少iNOS表达,具有抗炎作用。其作用机制可能与IκB降解受阻,导致NF-κB活性降低有关。

AIM ： To investigate the effects of MG132, one of the proteasomal peptide aldehydes inhibitors, on lipopolysaccharide （LPS） - induced nuclear transcription factor - kappa B （ NF - kB） activation, the production of nitric oxide （NO） and tumor necrosis factor -alpha （TNF -α）, as well as the expression of inducible nitric oxide synthase （iN- OS） in murine macrophage line RAW264.7. METHODS： Reporter gene assay was used to examine the activity of NF - kB 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 - α was examined by ELISA. Western blotting was used to reveal the expression of iNOS and IKB-α RESULTS： MG132 significantly decreased the secretion of TNF -α induced by LPS, with the inhibitory rates of 36.7% and 60. 4% to 5 μmol/L and 10 μmol/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 μmol/L） to 55.9% （ 10μmol/L）. Pretreatment with MG132 reduced the expression of iNOS, but restored the IKB restrain caused by LPS treatment. Observed by a reporter gene assay, TNF - α - induced NF - kB activity was decreased gradually by addition of increasing concentration of MG132 （2.5 - 10 μmol/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 -α, and the expression of iNOS, which probably mediates the blockage of IKB degradation and NF - kB activation.