Neuroprotective regulation of the IRF9 gene after spinal cord injury:bioinformatics analysis combined with experimental validation

Tian Minghao; Liao Yehui; Zhou Wenyang; He Baoqiang; Leng Yebo; Xu Shicai; Zhou Jiajun; Li Yang; Tang Chao; Tang Qiang; Zhong Dejun

doi:10.12307/2026.119

Abstract：BACKGROUND:The precise regulatory mechanisms of inflammation following spinal cord injury remain unclear.In recent years,studies have shown that interferon regulatory factor 9(IRF9)plays a key role in various inflammation-related diseases,but its specific role in spinal cord injury has not been systematically studied. OBJECTIVE:To systematically investigate the role of IRF9 in the inflammatory response after spinal cord injury and its potential molecular mechanisms by integrating bioinformatics analysis and in vitro experiments. METHODS:(1)Gene microarray data from the GEO database were integrated and analyzed using differential gene expression analysis,GO and KEGG functional enrichment analyses,gene set enrichment analysis,and protein-protein interaction network analysis to explore the functions and pathways of differentially expressed genes after spinal cord injury and identify the key genes related to inflammation.(2)The 3rd generation PC12 cells were cultured in four groups:blank control group without any treatment,injury control group with lipopolysaccharide intervention for 24 hours,IRF9 overexpression group transfected with IRF9 overexpression lentivirus for 72 hours followed by lipopolysaccharide intervention for 24 hours,and IRF9 knockdown group transfected with IRF9 knockdown expression lentivirus for 72 hours followed by lipopolysaccharide intervention for 24 hours.After the end of lipopolysaccharide intervention,cell proliferation was detected by cell counting kit-8 assay,brain-derived nerve growth factor and neurotrophic factor 3 levels in cell culture medium were detected by Elisa,intracellular Bcl-2,Bax,Cleaved-Caspase3 protein expression was detected by western blot,and cell migration ability was detected by cell scratch assay. RESULTS AND CONCLUSION:(1)Bioinformatics analysis demonstrated that spinal cord injury significantly activated inflammation-and immune-related pathways,identifying IRF9 as a potential core regulator.High IRF9 expression was found to potentially upregulate Toll-like receptor and nuclear factor-κB signaling pathways while suppressing cyclic adenosine monophosphate and calcium signaling pathways and glutamatergic synaptic function,contributing to the regulation of inflammation and neurological dysfunction.(2)The results of cellular experiments showed that compared with the blank control group,the injury control group had lower cell proliferation,migration ability and Bcl-2 protein expression,lower levels of brain-derived nerve growth factor and neurotrophic factor 3,and higher expression of Bax and Cleaved-Caspase-3 proteins.Compared with the injury control group,the IRF9 knockdown group showed higher cell proliferation,migration ability and Bcl-2 protein expression,higher levels of brain-derived nerve growth factor and neurotrophic factor 3,and lower expression of Bax and Cleaved-Caspase-3 proteins.To conclude,IRF9 may serve as an important regulator of secondary injury and a potential therapeutic target for spinal cord injury.