Effect of Cathepsin-L on Microglia Polarization After Spinal Cord Injury

Li Mingwu; Qin Shuchao; Zhou Ruyu; Duan Jun; Li Jingjing

Abstract：Objective This study aims to identify genes influencing spinal cord function by regulating microglial cell polarization,based on phenotypic changes in mouse microglia before and after spinal cord injury(SCI).Methods Utilizing the GSE172167 dataset from the gene expression omnibus(GEO),we analyzed single-cell sequencing data of mouse spinal cord tissue pre-and post-SCI.Principal component analysis(PCA)and t-distributed stochastic neighbor embedding(t-SNE)reduced dimensionality,identifying 10 cell subgroups.Subsequently,differential genes highly expressed in microglia post-SCI were isolated.A prospective study from February to April 2023 employed BV2 cells and C57BL/6 mice to validate the function and mechanism of cathepsin-L(CTSL).BV2 cells were categorized into seven groups:control,lipopolysaccharide(LPS),interleukin(IL)-4,negative control(NC)-shRNA,knockdown(Sh)-CTSL,NC-overexpression(OE),and OE-CTSL.The animal model included five groups:sham surgery,SCI,transfection with sh-CTSL adenovirus,transfection with OE-CTSL adenovirus,and a control group.Real-time quantitative polymerase chain reaction(qPCR)and western blotting(WB)assessed CTSL mRNA and protein levels,along with qPCR for tumor necrosis factor-alpha(TNF-α)and IL-1β.Flow cytometry determined M1-type(CD86+/CD80+)and M2-type(CD163+/CD206+)microglia proportions.Immunofluorescence staining quantified iNOS-positive and Arg-1-positive cells in mouse spinal cord tissue.Additionally,the Basso-Beattie-Bresnahan(BBB)scale evaluated motor neuron function in all five mouse groups at 0,1,2,3,and 4 weeks post-injury.Results In vitro,the control group exhibited lower proportions of M1 microglia,IL-1β,TNF-α,and M2 microglia compared to the LPS group.The LPS group showed higher M1 microglia,IL-1β,and TNF-α levels than the IL-4 group,with a lower M2 microglia proportion.In the sh-CTSL group,M1 microglia,IL-1β,and TNF-α levels were lower than in the LPS and NC-shRNA groups,while M2 microglia were higher.Conversely,in the OE-CTSL group,M1 microglia,IL-1 β,and TNF-α levels were higher than in the IL-4 and NC-OE groups,with a lower M2 microglia proportion.In vivo,the CTSL knockdown group had higher BBB scores at 3 and 4 weeks post-surgery compared to the SCI and blank groups,while the CTSL overexpression group had lower scores.In spinal cord tissues,the sham group exhibited lower M1 microglia,iNOS-positive cells,IL-1β,TNF-α,M2 microglia,and Arg-1 positive cell counts compared to the SCI and blank groups.The CTSL overexpression group showed higher M1 microglia,iNOS-positive cells,IL-1β,and TNF-α,with lower M2 microglia and Arg-1 positive cell counts than the SCI and blank groups.In contrast,the CTSL knockdown group had lower M1 microglia,iNOS-positive cells,IL-1β,and TNF-α,with higher M2 microglia and Arg-1 positive cell counts than these groups.All differences were statistically significant(P＜0.05).Conclusion CTSL knockdown promotes the transformation of M1 microglia to M2 in mouse spinal cord,alleviates the inflammatory response,and enhances mouse spinal cord injury repair.