摘要
目的:研究醛糖还原酶(AR)在小鼠脊髓损伤(SCI)的作用及分子机制。方法:利用野生型(WT)小鼠和AR敲除(AR KO)小鼠,制备小鼠脊髓钳夹伤模型,通过BMS评分对比观察损伤后两种小鼠在不同时间点(3、7、14和28 d)的运动功能恢复情况;采用免疫荧光染色观察小胶质细胞活化状态和损伤面积恢复程度;利用试剂盒和Western Blot检测损伤后小鼠脊髓组织中诱导型一氧化氮合酶(iNOS)、磷酸化核因子-κB(NF-κB)家族p65、4-羟基壬烯醛(4-HNE)和活性氧(ROS)的表达。通过体外培养来源于小鼠小胶质细胞系BV2,给予脂多糖(LPS)及4-HNE联合刺激,利用免疫荧光染色观察BV2细胞极化状态,Western Blot检测iNOS和p-p65蛋白表达水平。结果:与WT小鼠相比,AR基因敲除促进小鼠脊髓损伤后的运动功能恢复,损伤区面积显著减小,并且抑制小鼠脊髓损伤区小胶质细胞活化,NF-κB信号通路相关炎性分子iNOS表达水平明显下调。WT小鼠和AR KO小鼠在脊髓损伤后ROS、4-HNE的含量逐渐上升,在损伤后7 d表达较高,并且具有相似的表达变化模式。LPS联合4-HNE刺激BV2细胞系,可抑制M1型相关分子白细胞分化抗原16/32(CD16/32)、iNOS和p-p65的表达,促进M2型相关分子精氨酸酶1(Arg-1)的表达。结论:AR敲除促进小鼠脊髓损伤后4-HNE的累积,减少NF-κB信号通路的激活,抑制小胶质细胞的活化,从而促进运动功能的恢复。
Objective:To investigate the role of aldose reductase(AR)and the relevant molecular mechanisms in a spinal cord injury(SCI)mouse model.Methods:Wild-type(WT)mice and AR gene knockout(AR KO)mice were used to establish a mouse model of compression SCI.The restoration of mouse locomotor functions at different time points(3,7,14,and 28 d)after the induction of injury was assessed and compared using the Basso Mouse Scale Score(BMS);immunofluorescence(IF)staining was used to determine the status of microglial cell activation and the recovery of the lesion area;test kits and Western Blot were used to detect the expression levels of inducible nitric oxide synthase(iNOS),nuclear factor-κB(NF-κB)family phosphorylated p65(p-p65),4-hydroxynonenal(4-HNE),and reactive oxygen species(ROS)in the post-injury mouse spinal cord tissues.The in vitro experiments were performed on the mouse BV2 microglial cell line stimulated with lipopolysaccharide(LPS)and 4-HNE.The polarization states of BV2 cells were determined by IF staining,while the expression levels of iNOS and p-p65 were evaluated by Western Blot.Results:Compared with WT mice,knockout of the AR gene promoted the restoration of locomotor functions,significantly reduced the lesion areas,and inhibited microglia activation after SCI.AR knockout also significantly downregulated the expression level of iNOS,an inflammatory molecule associated with the NF-κB signaling pathway,in the spinal cord lesions of mice.Both WT mice and AR KO mice exhibited increasing ROS and 4-HNE levels gradually,which were highest at 7 d post-SCI,with similar changes in the patterns of expression.Stimulation of BV2 cells using LPS combined with 4-HNE inhibited the expression of molecules related to the M1-type,including cluster of differentiation 16/32(CD16/32),iNOS,and p-p65,and promoted the expression of arginase 1(Arg-1),a molecule related to the M2-type.Conclusion:Knockout of AR increases the accumulation of 4-HNE after SCI in mice,thereby decreases activation of NF-κB signaling pathway and inhibits
作者
姚艳
王玉珏
李渊博
高小川
刘芳芳
张静
YAO Yan;WANG Yujue;LI Yuanbo;GAO Xiaochuan;LIU Fangfang;ZHANG Jing(Medical Research and Experiment Center,Medical College,Yan'an University,Yan'an 716000;the twenty-fourth team of the Sixth Battalion,School of Basic Medicine,Air Force Medical University,Xi'an 710032,China;Department of Neurobiology,School of Basic Medicine,Air Force Medical University,Xi'an 710032,China)
出处
《神经解剖学杂志》
CAS
CSCD
2023年第3期303-308,共6页
Chinese Journal of Neuroanatomy
基金
国家自然科学基金(81860444)
陕西省自然科学基础研究计划(2023-JC-YB-819)
陕西省卫生健康科研基金项目(2021D053)。
关键词
脊髓损伤
小胶质细胞
醛糖还原酶
4-羟基壬烯醛
小鼠
spinal cord injury(SCI)
aldose reductase(AR)
microglia
4-hydroxynonenal(4-HNE)
mouse
