Promoting effect of DPP-4 gene silencing on pyroptosis in alveolar macrophages and its mechanism
Objective To observe the effect of dipeptidyl peptidase 4(DPP-4)silencing on lipopolysaccharide(LPS)-induced pyroptosis in alveolar macrophages,and to explore its possible mechanism.Methods MH-S alveolar macrophage cell lines(MH-S cells for short)in the logarithmic growth phase were randomly divided into the Control siRNA group(transfected with Control siRNA),DPP-4 siRNA group(transfected with DPP-4 siRNA),Control siRNA+LPS group(transfected with Control siRNA and then were added with LPS),and DPP-4 siRNA+LPS group(transfected with DPP-4 siRNA and then were added with LPS),respectively.The cell morphology of each group was observed under an in-verted microscope,the proliferative viability of cells was assessed by CCK-8 method,the cytotoxicity of cells was deter-mined by lactate dehydrogenase(LDH)cytotoxicity assay kit(expressed as LDH release),and the cell death proportion was detected by Calcein-AM/PI double staining method(expressed as PI positive cell rate).The relative protein expres-sion levels of NOD-,LRR-and pyrin domain-containing protein 3(NLRP3),apoptosis-associated speck-like protein con-taining a CARD(ASC),cysteinyl aspartate specific proteinase p20(Caspase-1 p20),N-Gasdermin D(GSDMD-N)and inflammatory cytokines interleukin-18(IL-18)and interleukin-1β(IL-1β)in pyroptosis pathway were detected by West-ern blotting(WB).MH-S cells in the logarithmic growth phase were randomly divided into the Control siRNA+LPS group(transfected with Control siRNA and then were added with LPS),DPP-4 siRNA+LPS group(transfected with DPP-4 siR-NA and then were added with LPS),Control siRNA+SB203580+LPS group(transfected with Control siRNA and then were added with p38 inhibitor SB203580 and LPS),and DPP-4 siRNA+SB203580+LPS group(transfected with DPP-4 siRNA and then were added with p38 inhibitor SB203580 and LPS).The activation of p38 and the expression levels of pyroptosis-related proteins were detected by WB.Results There was no pyroptosis in cells of the Control siRNA group.A small number of cells showed pyroptosis in the DPP-4 siRNA group.A large number of cells showed pyroptosis in the Control siRNA+LPS group and the DPP-4 siRNA+LPS group,and the pyroptosis manifestation was more obvious in the DPP-4 siRNA+LPS group.The cell proliferation activity decreased successively in the Control siRNA group,the DPP-4 siRNA group,the Control siRNA+LPS group,and the DPP-4 siRNA+LPS group.The LDH release amount,the PI positive cell rate,and the relative expression levels of NLRP3,ASC,Caspase-1 p20,GSDMD-N,IL-18,and IL-1β proteins in cells all increased successively;significant difference was found between groups(all P<0.05).Compared with the DPP-4 siR-NA+LPS treatment group,the relative expression levels of p-p38/p38,p-NF-κB/NF-κB,NLRP3,Caspase-1 p20,and GSDMD-N proteins decreased in the Control siRNA+LPS treatment group,the Control siRNA+SB203580+LPS treat-ment group,and the DPP-4 siRNA+SB203580+LPS treatment group,and the decrease was more significant in the Con-trol siRNA+SB203580+LPS treatment group and the DPP-4 siRNA+SB203580+LPS treatment group(all P<0.05).Conclusion DPP-4 silencing can promote LPS-induced pyroptosis of MH-S cells,which may be related to the initiation of NLRP3 inflammasome formation and the release of inflammatory cytokines by promoting phosphorylation of p38 and NF-κB,thereby regulating the Caspase-1-mediated classical pyroptosis pathway.
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