基于iTRAQ技术探究过表达Ptpn2对SiO2介导的小鼠肺泡巨噬细胞炎症反应的调控作用OA

Objective To investigate the regulatory effect of overexpressed protein tyrosine phosphatase non-receptor type 2(Ptpn2)on the inflammatory response of mouse alveolar macrophages(MH-S)induced by SiO₂.Methods Cells with overexpressed Ptpn2 were constructed and induced by SiO₂.The experimental groups were divided into four groups:the negative control group with an empty vector(NC),the overexpressed Ptpn2 group(P),the negative control group with an empty vector+SiO₂ induction(NS),and the overexpressed Ptpn2+SiO₂induction group(PS).Isobaric tags for relative and absolute quantification(iTRAQ)combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS)were used to screen differential proteins,followed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)database analyses.Immunofluores-cence staining was used to detect the expressions of Tumor necrosis factor(TNF)α,Gasdermin D(GSDMD),and Transforming growth factor(TGF)-β1.Western blot was used to detect the protein expression levels of PTPN2,Toll-like receptor 4(TLR4),tumor necrosis factor-α(TNF-α),nucleotide-binding oligomerization domain-like re-ceptor protein 3(NLRP3),and proteins related to the TGF-β1 signaling pathway in the cells of each group.Re-sults iTRAQ results identified 144 differential proteins among the four groups.GO analysis showed that in bio-logical processes(BP),these differential proteins were mainly enriched in IκB kinase/nuclear factor-κB(NF-κB)signaling,cell activation and signal transduction involved in immune responses,and regulation of receptor signal-ing pathways by signal transducer and activator of transcription(STAT),etc.KEGG analysis revealed that the dif-ferential proteins were mainly enriched in Toll-like receptor signaling pathway,NF-κB signaling pathway,NOD-like receptor signaling pathway,TGF-β signaling pathway,and TNF signaling pathway.The results of immunofluo-rescence staining showed that compared with the NC group,the expressions of TNF α,GSDMD,and TGF-β1 in the cells of the NS group increased(P<0.05);compared to the NS group,the expression of the aforementioned proteins in the PS group decreased in cellular proteins(P<0.05).The results of Western blot showed that com-pared with the NC group,the protein expression levels of PTPN2,p-NF-κB,MyD88,TLR4,NLRP3,GSDMD,Cas-pase-1,IL-1β,TGF-βR1,TGF-βR,p-Smad2/3 in the NS group were significantly upregulated(P<0.05);com-pared with the NS group,the expression levels of the aforementioned proteins in the PS group were significantly downregulated(P<0.05).Conclusion Overexpression of Ptpn2 can inhibit the protein expressions of TLR4-TNF-α signaling,NLRP3 signaling,and TGF-β1 signaling closely related to inflammatory response in SiO₂-medi-ated MH-S macrophages.