Lck/Yes相关新型酪氨酸激酶在巨噬细胞M1极化中的作用及机制研究OA

Objective To investigate the role and mechanism of Lck/Yes-related novel protein tyrosine kinase(Lyn)on lipopolysaccharide(LPS)-induced M1-type polarization of macrophage.Methods The LentiCRISPR-V2 plasmid was digested with the restriction endonuclease BSMBI-V2,and the digested DNA fragments were recov-ered.The digested plasmid was ligated with Lyn-sgRNA using T4 ligase to generate the Lenti-Lyn-gRNA lentivi-rus.THP-1 cells were infected with the Lenti-Lyn-gRNA lentivirus to obtain a stable cell line with Lyn knockout,and a monoclonal THP-1 cell line with complete Lyn knockout(Lyn⁻/⁻)was established subsequently.Wild-type Lyn(LynWT)and Lyn⁻/⁻ THP-1 cells were induced with 100 ng/mL phorbol myristate acetate(PMA)for 48 h to dif-ferentiate into M0 macrophages,which were further polarized into M1 macrophages by stimulation with 100 ng/mL LPS for 24 h.Quantitative real-time polymerase chain reaction(qPCR)was performed to detect the expression of M0 macrophage markers,including integrin αM(CD11b),macrophage antigen(CD68),and monocyte differen-tiation antigen(CD14).The expression of Lyn in M1 macrophages differentiated from wild-type THP-1 cells(LynWT-M1)was measured by qPCR,and the ratio of phosphorylated Lyn to total Lyn(P-Lyn/Lyn)in LynWT-M1 cells was determined by Western blot.In M1 macrophages differentiated from Lyn-knockout THP-1 cells(Lyn⁻/⁻-M1),qPCR was used to detect the mRNA expression of inducible nitric oxide synthase(iNOS),interleu-kin-6(IL-6),and chemokine(C-X-C motif)ligand 10(CXCL-10).Western blot was conducted to assess the pro-tein expression of iNOS,as well as the protein levels of molecules related to the Janus kinase 1(JAK1)-signal transducer and activator of transcription 1(STAT1)signaling pathway,including JAK1,phosphorylated JAK1(P-JAK1),STAT1,and phosphorylated STAT1(P-STAT1).Additionally,the expression of the M1 macrophage marker cluster of differentiation 80(CD80)was analyzed by flow cytometry.Results The Lyn-/-monoclonal cell line was successfully constructed.The expression of CD11b was significantly elevated in Lyn-/-M0 macrophages,and the differentiation of M1 macrophages was successful.Knockdown of Lyn inhibited mRNA expression of iNOS,I L-6,C X C L-10,protein expression of iNOS and CD80 expression in M1 macrophages(P<0.05).Western blot as-say showed that Lyn knockdown inhibited protein expression of JAK1 and P-STAT1(P<0.01).Conclusion After CRISPR/Cas9-mediated Lyn knockout,the expression levels of JAK1 and P-STAT1,the key molecules in the JAK/STAT signaling pathway of M1 macrophages,are significantly downregulated;concomitantly,the expression of M1 macrophage-specific secretory factors(iNOS,IL-6,CXCL-10 mRNA)and CD80 is also downregulated,which may be achieved via targeted regulation of the JAK1/P-STAT1-mediated JAK/STAT signaling pathway.