Observation on alveolar macrophage polarization and activation of JNK/AP-1 signaling pathway in mice transfected with DPP-4 siRNA or(and)added with SP600125
Objective To observe the polarization of alveolar macrophages and the activation of JNK/AP-1 signaling pathway in mice transfected with dipeptidyl peptidase-4(DPP-4)siRNA or(and)added with c-Jun N-terminal kinase(JNK)inhibitor(SP600125).Methods Mouse alveolar macrophages(MH-S)were cultured in vitro and randomly grouped into:control group(transfected with empty siRNA),siDPP-4 group(transfected with DPP-4 siRNA),lipopoly-saccharide(LPS)group(transfected with empty siRNA+ LPS),siDPP-4+LPS group(transfected with DPP-4 siRNA+ LPS),LPS+SP600125 group(transfected with empty siRNA+ LPS combined with SP600125),and siDPP-4+LPS+ SP600125 group(transfected with DPP-4 siRNA+ LPS combined with SP600125),respectively.The expression levels of M1 and M2 polarization markers CD86 and CD206 proteins and the phosphorylation of JNK and activator protein-1(AP-1)transcription proteins(c-Jun and c-Fos)in macrophages were detected by Western blotting,the mRNA levels of M1 mark-ers[CD86,tumor necrosis factor(TNF)-α,iNOS,IL-1β]and M2 markers[CD206,arginine kinase-1(ARG-1),IL-4,IL-10]in macrophages were detected by RT-qPCR,and M1 proinflammatory factor NO and intracellular reactive oxygen species(ROS)production in macrophage supernatant were detected by nitric oxide(NO)assay kit and immunofluores-cence,respectively.Results Compared with the control group,the production of M1 proinflammatory factors NO and ROS,and the expression levels of M1 polarization markers(CD86 protein and CD86,TNF-α,iNOS,IL-1β mRNA)in-creased,and the expression levels of M2 polarization markers(CD206 protein and CD206,ARG-1,IL-4,IL-10 mRNA)decreased in the LPS group(all P<0.05).Compared with the LPS group,the production of M1 proinflammatory factors NO and ROS,and the expression levels of M1 polarization markers(CD86 protein and CD86,TNF-α,iNOS,IL-1β mRNA)increased,and the expression levels of M2 polarization markers(CD206 protein and CD206,ARG-1,IL-4,IL-10 mRNA)decreased in the siDPP-4+LPS group(all P<0.05).Compared with the LPS group,the relative expression lev-els of p-JNK/JNK,p-c-Jun/c-Jun and p-c-Fos/c-Fos protein phosphorylation increased in the siDPP-4 +LPS group(all P<0.05).Compared with the siDPP-4+LPS group,the relative expression levels of p-JNK/JNK,p-c-Jun/c-Jun,p-c-Fos/c-Fos protein phosphorylation decreased in the siDPP-4+LPS+SP600125 group(all P<0.05).Conclusions Transfection of DPP-4 siRNA can promote the M1 polarization of mouse alveolar macrophages induced by LPS,inhibit the M2 polariza-tion of alveolar macrophages,and increase the phosphorylation expression of JNK,c-Jun and c-Fos proteins.Addition of JNK inhibitor may reduce the increase in phosphorylation expression of JNK,c-Jun and c-Fos proteins caused by transfec-tion of DPP-4 siRNA.The mechanism of DPP-4 siRNA transfection promoting M1 polarization in mouse alveolar macro-phages induced by LPS may be related to the activation of JNK/AP-1 signaling pathway.
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