ZXDC基因敲减对脊髓神经元氧化应激损伤的保护作用

Effect of ZXDC knockdown on spinal cord neurons after oxidative stress injury

目的 探讨转录因子ZXDC基因敲减对脊髓神经元(SCNs)氧化应激损伤后的保护作用及机制。方法 应用不同浓度H_(2)O_(2)诱导SCNs氧化应激损伤,CCK8检测细胞活性筛选H_(2)O_(2)诱导最佳浓度;实验细胞分为:Control组、H_(2)O_(2)+AAV-NC组和H_(2)O_(2)+AAV-ZXDC siRNA组,免疫荧光染色鉴定原代SCNs,检测各组ZXDC表达和神经突起平均长度;Western bolt检测各组细胞中ZXDC、CCL2、CCR2表达,qRT-PCR检测细胞中炎症因子TNF-α和IL-1β m RNA表达,应用EdU荧光染色、CCK8检测ZXDC对SCNs增殖和活性的影响。结果 筛选600μmol/L H_(2)O_(2)适用于SCNs氧化应激模型制备。β-Ⅲ-tubulin免疫荧光染色鉴定原代SCNs培养成功;与Control组比较,H_(2)O_(2)+AAV-NC组神经突起平均长度显著降低,ZXDC、CCL2和CCR2表达显著增高,而H_(2)O_(2)+AAV-ZXDC siRNA组较H_(2)O_(2)+AAV-NC组神经突起平均长度显著增高,ZXDC、CCL2和CCR2蛋白表达显著降低(P<0.05);与Control组比较,H_(2)O_(2)诱导组TNF-α和IL-1β m RNA相对表达显著增高,细胞增殖和细胞活力显著降低。与H_(2)O_(2)+AAV-NC组比较,H_(2)O_(2)+AAV-ZXDC si RNA组TNF-α和IL-1β m RNA相对表达显著降低,细胞增殖和细胞活力显著增高(P<0.05)。结论 ZXDC基因敲减通过调控CCL2/CCR2信号通路促进氧化应激损伤SCNs细胞增殖、细胞活力和神经突生长。

Objective To investigate the protective effect and mechanism of transcription factor ZXDC knockdown on spinal cord neurons(SCNs)after oxidative stress injury.Methods Different concentrations of H_(2)O_(2)were implemented to induce oxidative stress injury in SCNs,and the optimal concentration of H_(2)O_(2)induction was screened by CCK8 assay.The cells were divided into:Control group,H_(2)O_(2)+AAV-NC group and H_(2)O_(2)+AAV-ZXDC si RNA group,and the primary SCNs were identified by immunofluorescence staining.The ZXDC expression and neurite length were detected by immunofluorescence staining.The expression of ZXDC,CCL2 and CCR2 in each group were evaluated by using Western Blot,the expression of inflammatory factors TNF-α and IL-1β m RNA in cells was quantified by fluorescence real-time quantitative polymerase chain reaction(qRT-PCR),and EdU fluorescence staining and CCK8 were applied to detect the proliferation and activity of SCNs.Results Screening of 600μmol/L H_(2)O_(2)was suitable for the preparation of SCNs oxidative stress model.β-III-tubulin immunofluorescence staining was successful in identifying primary SCNs in culture.Compared with the Control group,the average neurite length was significantly decreased,and the expression of ZXDC,CCL2 and CCR2 was significantly increased in the H_(2)O_(2)+AAV-NC group.Moreover,the H_(2)O_(2)+AAV-ZXDC siRNA group increased average neurite length and decreased the expression of ZXDC,CCL2 and CCR2 compared with the H_(2)O_(2)+AAV-NC group(P<0.05).Compared with the Control group,the H_(2)O_(2)-induced group significantly increased the expression of TNF-α and IL-1β mRNA and decreased cell proliferation and cell viability,and the H_(2)O_(2)+AAV-ZXDC siRNA group decreased the expression of TNF-α and IL-1β mRNA and increased cell proliferation and viability than those of the H_(2)O_(2)+AAV-NC group(P<0.05).Conclusion ZXDC knockdown promotes cell proliferation,cell viability and neurite growth in oxidative stress-injured SCNs through regulating the CCL2/CCR2 signaling pathway.