Objective To investigate the effect of silica nanoparticles(SiNPs)induced macrophage pyroptosis on matrix metalloproteinase and fibroblast transdifferentiation to myofibroblasts in primary lung fibroblasts.Methods After constructing the macrophage pyroptosis model induced by SiNPs,the morphological location of SiNPs in macrophages was performed by nano hyperspectral technology,then using optical microscopy to observe macrophage morphology after SiNPs exposure,using Western blot to detect the expression of pyroptosis related protein.Primary lung fibroblasts of mice were extracted by lung-digesting method with trypsin and type Ⅳ collagenase,the characterization and identification of primary lung fibroblasts were performed with optical microscopy and immunofluorescence techniques,respectively.Subsequently,an in vitro co-culture model was constructed to investigate SiNPs-induced macrophage pyroptosis and its interaction with primary lung fibroblasts.The primary lung fibroblasts treated with macrophage culture supernatant was as MF group,while those fibroblast treated with pyroptosis macrophage culture supernatant as PF group,then,qRT-PCR and Western blot techniques were used to detect the expressions of MMP2 and MMP9 at mRNA and protein levels in primary lung fibroblasts,and the expression of myofibroblasts marker α-SMA protein was also detected by immunofluorescence and immunocytochemical staining methods.Results The results of nanometer of hyperspectral imaging,morphological observation and Western blot detection showed that SiNPs successfully induced pyroptosis of J774A.1 macrophages,the optical microscopy observation revealed that compared with trypsin digestion,the purity of primary mice lung fibroblasts extracted by type Ⅳ collagenase method was higher,and the immunofluorescence detection results suggested that the Vimentin of fibroblast obtained by type Ⅳ collagenase method exhibited a typical microfilamentous structure,the cytoskeletal structure was complete as well.The qRT-PCR results showed that the mRNA levels of MMP2 and MMP9 were increased in the PF group compared with that of MF group(P＜0.05),the Western blot results showed that the protein expression levels of MMP2 and MMP9 were also increased in PF group(P＜0.05),the immunofluorescence results showed that the intensity of α-SMA red fluorescence was increased in the PF group compared with the MF group,and the immunocytochemical staining results showed strongly positive in the PF group.Conclusion The results suggested that SiNPs-induced macrophage pyroptosis may promote the up-regulation of MMP2 and MMP9 expression levels in primary lung fibroblasts as well as the fibroblast transdifferentiation to myofibroblasts.
维普
万方数据