Role and mechanism of Lck/Yes-related novel tyrosine kinases in macrophage M1 polarization
10.19405/j.cnki.issn1000–1492.2026.02.004
- VernacularTitle: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
- Author:
Xin YU
1
;
Zhensheng GAO
1
;
Weihua BIAN
1
;
Xiangyong LIU
1
;
Yeying SUN
1
Author Information
1. Department of Pharmacy， Binzhou Medical University， Yantai 264003
- Publication Type:Journal Article
- Keywords:
Lyn kinase;
THP-1;
M1 macrophage;
CRISPR/Cas9;
chronic inflammation;
JAK/STAT signaling pathway
- From:
Acta Universitatis Medicinalis Anhui
2026;61(2):209-216
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo investigate the role and mechanism of Lck/Yes-related novel protein tyrosine kinase （Lyn） on lipopolysaccharide （LPS）-induced M1-type polarization of macrophage. MethodsThe LentiCRISPR-V2 plasmid was digested with the restriction endonuclease BSMBI-V2， and the digested DNA fragments were recovered. The digested plasmid was ligated with Lyn-sgRNA using T4 ligase to generate the Lenti-Lyn-gRNA lentivirus. 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 differentiate 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 differentiation 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）， interleukin-6 （IL-6）， and chemokine （C-X-C motif） ligand 10 （CXCL-10）. Western blot was conducted to assess the protein 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. ResultsThe 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， IL⁃6， CXCL⁃10， protein expression of iNOS and CD80 expression in M1 macrophages （P<0.05）. Western blot assay showed that Lyn knockdown inhibited protein expression of JAK1 and P-STAT1 （P<0.01）. ConclusionAfter 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.
