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

doi:10.12307/2026.119

Abstract

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.

Key words: IRF9, spinal cord injury, bioinformatics, PC12 cells, inflammatory response, immune regulation, apoptosis, secondary injury, engineered tissue engineering

CLC Number:

Tian Minghao, Liao Yehui, Zhou Wenyang, He Baoqiang, Leng Yebo, Xu Shicai, Zhou Jiajun, Li Yang, Tang Chao, Tang Qiang, Zhong Dejun . Neuroprotective regulation of the IRF9 gene after spinal cord injury: bioinformatics analysis combined with experimental validation[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(11): 2712-2726.

Figures/Tables 14

2.1 脊髓损伤后的差异基因为探讨脊髓损伤后的基因表达变化，此次研究对多数据集进行整合分析。主成分分析结果显示，在去批次处理前，不同数据集的样本在主成分空间中呈现明显分离，表明存在显著的批次效应(图1A左)。经过ComBat方法去批次处理后，样本间的批次效应显著减少，损伤组与对照组在主成分空间中的聚类更加紧密(图1A右)，验证了去批次处理的有效性，为后续的差异基因分析奠定了基础。基于去批次处理后的数据，选择脊髓损伤后3 d以上的样本为实验组，假手术组和空白组样本为对照组，通过差异基因分析共筛选出显著差异表达基因1 041个，其中上调基因699个，下调基因342个(图1B)，该火山图清晰地展示了差异基因的分布：红色点表示上调基因，蓝色点表示下调基因，灰色点为无显著差异的基因。进一步分析绘制热图(图1C)，展示了前100个显著上调和下调基因在损伤组与对照组中的表达趋势，上调基因主要涉及免疫调控和炎症反应激活(例如S100a9、Serpina3n和Lcn2)，其中一些基因与炎症相关过程密切相关(例如Ccl2、Timp1和Cd68)；此外，还发现涉及组织修复和细胞增殖的基因显著上调，例如Mki67和Postn，下调基因主要与神经功能和神经保护相关，例如 Kcnc1和Slc17a6，这些基因涉及突触传递、神经修复及神经元功能的调控(例如 Nefh和Stx1b)。这些结果表明，脊髓损伤可能通过显著激活炎症反应并抑制神经保护机制，导致神经功能障碍的发生。 2.2 GO和KEGG富集分析结果 GO功能富集分析(图2)显示，差异基因显著富集于免疫反应、细胞迁移、神经修复及细胞微环境调控相关的功能条目。在生物过程中，富集条目如“白细胞趋化性”“免疫反应调节”和“炎症反应调节”提示脊髓损伤后免疫系统和炎症反应的显著激活。在细胞组分中，“髓样白细胞迁移”和“髓样白细胞激活”进一步表明免疫细胞在损伤部位的动员和参与，“突触后膜”“远端轴突”和“细胞前缘”暗示突触结构重塑及细胞迁移对修复的关键作用。在分子功能中，“糖胺聚糖结合”和“整合素结合”等条目表明炎症调节、细胞信号传导以及免疫细胞招募的显著激活。 KEGG通路富集分析(图3)表明，差异基因集中于“细胞因子-受体相互作用”“趋化因子信号通路”和“Toll样受体信号通路”等免疫和炎症相关通路，突出免疫细胞招募及炎症调节的重要性。“核因子kB信号通路”和“补体与凝血级联反应”提示局部炎症信号的放大过程。“白细胞经内皮迁移”和“溶酶体”通路进一步揭示免疫细胞迁移与清除的关键意义，同时支持其在神经损伤修复中的潜在功能。 2.3 GSEA富集分析结果 GSEA分析结果显示，脊髓损伤后炎症反应和免疫调控相关基因集显著富集(图4)。在全局分析中，“干扰素γ响应”(NES=2.571，P.adjust=5.41×10-26)和“干扰素α响应”(NES=2.459，P.adjust=6.26 × 10-16)在脊髓损伤组中富集程度最高，提示干扰素信号在炎症反应和免疫调控中的核心地"

2.4 关键基因的筛选为进一步探讨脊髓损伤过程中炎症和免疫调控的分子机制，此次研究结合前文差异基因分析结果，聚焦于与炎症和免疫相关基因集。在GO数据库中筛选基因过程如下：以“大鼠”为物种背景，分别使用关键词“inflammatory” 和 “immune” 进行检索，得到与炎症相关的基因共计 841个；免疫相关的基因共计3 115个。将两部分基因合并后，去重得到炎症和免疫相关基因总计3 353个。最后通过与差异基因交集筛选出目标基因(图5A)。这些基因既具有显著差异表达，又在免疫和炎症信号通路中发挥潜在作用，最终筛选出339个候选基因。这些基因作为后续随机森林分析和蛋白质互作网络构建的研究对象。 2.5 蛋白质互作网络分析结果构建的蛋白质互作网络由208个节点和957条边组成，展示了差异基因之间复杂的相互作用关系(图5B)。在网络中，度值最高的5个基因分别是Cd4、Ccl2、Tlr4、Stat1和Itgad，这些基因在免疫调控和炎症反应中起着核心作用，而IRF9处于网络的次核心位置。进一步分析显示，30个Hub 基因(图5C)中同样包括IRF9，这进一步支持IRF9在脊髓损伤后的潜在功能性地位。推测IRF9 通过调控干扰素信号通路及其下游炎症因子的表达，可能在脊髓损伤后的炎症反应中发挥关键作用，调节免疫反应的平衡，进而影响组织修复和损伤进程。 2.6 随机森林分析结果使用随机森林分析蛋白互作网络分析计算出的前30个Hub基因，筛选中重要性排名前10的Hub基因(图6)，分别是：Ccl3、IRF9、Cxcl12、Cxcl1、Tlr4、Ifit3、Cxcl10、Cd4、Cxcr4、Ccl2。其中，Ccl3、Ccl2、Cxcl12、Cxcl10和 Cxcl1 作为关键趋化因子，主要参与免疫细胞募集和炎症放大效应，属于下游执行者；Tlr4是模式识别受体，位于免疫和炎症反应的最上游，通过感知损伤信号触发下游炎症级联；IRF9作为干扰素信号通路的核心转录因子，广泛调控多条炎症和免疫相关通路，是网络中重要的枢纽基因。这些基因的功能分工揭示了脊髓损伤后炎症和免疫调控的分层机制，并为探索潜在的治疗靶点提供了依据。 2.7 体外细胞实验验证结果 2.7.1 确定最适脂多糖质量浓度 Western blot检测结果显示(图7A-C)，经脂多糖处理后，PC12细胞内Bcl-2蛋白表达降低、Cleaved-Caspase3蛋白表达升高，表明细胞凋亡显著增强，其中以500，5 000 ng/mL脂多糖的处理结果更明显。CCK-8细胞检测结果显示，经脂多糖处理后，PC12细胞增殖显著降低，其中以500，5 000 ng/mL脂多糖的处理结果更明显(图7D)。综合上述结果，500 ng/mL被确定为脂多糖诱导PC12细胞炎症反应和凋亡的最适质量浓度。 2.7.2 慢病毒转染率及对PC12细胞的影响流式细胞术、DAPI染色和Western blot检测结果表明，慢病毒成功转染PC12细胞，转染效率满足后续实验需求(图8)。其中，流式细胞术和DAPI染色显示，绿色荧光蛋白阳性细胞占总细胞数量的比例超过70%；Western blot检测结果表明，5组PC12细胞内Cleaved-Caspase3、Bcl-2蛋白表达比较差异无显著性意义(P > 0.05)。划痕实验结果显示，5组PC12细胞迁移能力比较差异无显著性意义(P > 0.05)，见图9A，B。CCK-8检测结果显示，5组PC12细胞增殖比较差异无显著性意义(P > 0.05)，见图9C。结果表明，慢病毒转染后不会对PC12细胞造成损伤或诱导细胞凋亡。 2.7.3 敲低IRF9减轻脂多糖诱导的PC12细胞凋亡和功能损伤 Western blot检测结果显示，与空白对照组相比，损伤对照组Bax和Cleaved-Caspase3蛋白表达升高(P < 0.01或P < 0.05)，Bcl-2蛋白表达降低(P < 0.05)；与损伤对照组比较，IRF9敲低组Bax和Cleaved-Caspase3蛋白表达降低(P < 0.05)，Bcl-2蛋白表达升高(P < 0.05)，IRF9过表达组Bax、Bcl-2和Cleaved-Caspase3蛋白表达与损伤对照组比较差异无显著性意义(P > 0.05)，见图10A-C。表明IRF9在脂多糖诱导的PC12细胞凋亡过程中起促进作用，敲低IRF9能够减轻细胞凋亡，但尚不能证明过表达IRF9能促进炎症刺激后的细胞凋亡。划痕实验结果显示，与空白对照组比较，损伤对照组、IRF9过表达组、IRF9敲低组PC12细胞迁移能力均降低；与损伤对照组比较，IRF9敲低组PC12细胞迁移能力升高，见图10D，E。CCK-8检测结果显示，与空白对照组比较，损伤对照组、IRF9过表达组、IRF9敲低组PC12细胞增殖均降低；与损伤对照组比较，IRF9敲低组PC12细胞增殖升高，见图10F。 2.7.4 IRF9调控炎症损伤后PC12细胞中脑源性神经营养因子和神经营养因子3的表达 Elisa检测结果显示，与空白对照组比较，损伤对照组、IRF9敲低组、IRF9过表达组神经营养因子3水平均降低，损伤对照组、IRF9过表达组脑源性神经营养因子水平降低；与损伤对照组比较，IRF9敲低组脑源性神经营养因子水平升高，见图11。"

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