Effects and mechanisms of higenamine on polarization and pyroptosis of M1 macrophages
Objective To investigate the effects and mechanisms of higenamine(HG)on polarization and pyroptosis of M1 macrophages(induced by RAW264.7 cells).Methods The proliferation inhibition rate of RAW264.7 cells was determined by the CCK-8 method to screen the optimal experimental concentration of HG.The cells were randomly divided into blank group,interleukin(IL)-4+interleukin(IL)-13(IL-4+IL-13)group,lipopolysaccharide(LPS)group,LPS+adenosine triphosphate(ATP)(LPS+ATP)group,IL-4+IL-13+HG group,LPS+HG group,and LPS+ATP+HG group.Equal amounts of PBS(IL-4 20 ng/mL,IL-13 20 ng/mL),1 μg/mL LPS,1 μg/mL LPS+5 mmol/L ATP interventions were given to the blank,IL-4+IL-13,LPS,and LPS+ATP groups,respectively;50 μmol/L HG treatment was given to each dosing group based on the above interventions.The expression of mRNA related to macrophage polarization and pyroptosis was detected by Real-time PCR,and that of macrophage polarization and pyroptosis-related proteins was detected by Western blotting.Macrophage surface markers were detected by flow cytometry.Whole gene transcriptome sequencing analysis,Gene Ontology(GO)enrichment analysis,and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis of M1 macrophages were performed.Cell morphology was observed under a microscope.Enzyme-linked immunosorbent assay(ELISA)was performed to detect transforming growth factor(TGF)-β signaling pathway-related proteins TGF-β1 and platelet-responsive protein(THBS)-1.Results Compared with the blank group,the relative expression of interleukin(IL)-1β,inducible nitric oxide synthase(iNOS),tumor necrosis factor(TNF)-α mRNA,and iNOS and TNF-α proteins was higher in the LPS group(all P<0.01).Compared with the LPS group,the relative expression of interleukin(IL)-1β,iNOS,TNF-α mRNA,and iNOS and TNF-α proteins was lower in the LPS+HG group(all P<0.01).Compared with the blank group,the relative expression of CD206 and arginase(Arg)-1 proteins in the LPS group was lower(both P<0.05).Compared with the LPS group,the relative expression of the above indexes in the LPS+HG group was higher(both P<0.01).Compared with the blank group,the relative expression of CD86 protein and CD86/CD206 in the LPS group was higher(both P<0.01).Compared with the LPS group,the above indexes in the LPS+HG group were lower(P<0.01)and higher(P<0.01),respectively.Compared with the blank group,the relative expression of Arg-1,interleukin(IL)-10 mRNA,CD206 and Arg-1 proteins in the IL-4+IL-13 group was higher(P<0.05,P<0.01).Compared with the IL-4+IL-13 group,the relative expression of the above indexes in the IL-4+IL-13+HG group was higher(P<0.05,P<0.01).HG led to the down-regulation of 337 genes and up-regulation of 148 genes expression in M1 macrophages.Among the ten core genes,the THBS-1 gene with the greatest correlation was selected for KEGG enrichment analysis,and the results showed that the THBS-1/TGF-β1 signaling pathway had an important role in the regulation of M1 macrophages by HG.Compared with the blank group,the relative expression of THBS-1 and TGF-β1 mRNA in the LPS group was higher and lower,respectively(both P<0.01).Compared with the LPS group,the relative expression of the above indexes in the LPS+HG group was lower and higher,respectively(both P<0.01).After treating LPS+ATP-induced macrophage pyroptosis with HG,microscopic observation revealed that HG significantly improved cell swelling and rupture of pyrogenic macrophages.Compared with the blank group,NOD-like receptor protein(NLRP)3,cysteine aspartate protease(Casp)-1 and IL-1β mRNA and NLRP3,Gasdermins-D(GSDMD)Full and N-terminal bodies(GSDMD-F and GSDMD-N),and Casp-1 protein relative expression was higher(P<0.05,P<0.01).Compared with the LPS+ATP group,the relative expression of the above indexes was lower in the LPS+ATP+HG group(P<0.05,P<0.01).Compared with the blank group,the relative expression and protein level of TGF-β1 and THBS-1 mRNA in the LPS+ATP group were lower and higher,respectively(both P<0.01).The relative expression and protein level of the above indexes in the LPS+ATP+HG group were higher and lower,respectively,compared with those in the LPS+ATP group(P<0.05,P<0.01).Conclusion HG inhibits M1 macrophage polarization and pyroptosis and promotes M1 to M2 conversion,and the HG inhibition of M1 macrophage polarization and pyroptosis is associated with the THBS-1/TGF-β1 signaling pathway.
万方数据
维普
