艾司氯胺酮通过调控miR-148a-3p/KLF4通路减轻脊髓损伤大鼠的炎症反应

Esketamine alleviates inflammatory response in rats with spinal cord injury by regulating miR-148a-3p/KLF4 pathway

目的通过构建脊髓损伤大鼠模型,探讨艾司氯胺酮对脊髓损伤和微小RNA-148a-3p(miR-148a-3p)/Kruppel样因子4(KLF4)通路的影响。方法选择SPF级成年雌性SD大鼠48只,7~9周龄,体重215~255 g。将大鼠随机分为四组:假手术组(S组)、脊髓损伤组(SCI组)、艾司氯胺酮50 mg/kg组(E组)和艾司氯胺酮50 mg/kg+miR-148a-3p抑制剂5 mg/kg组(EI组),每组12只。S组予去除椎板处理,SCI组仅建立脊髓损伤大鼠模型,E组和EI组分别于建模后每日腹腔注射艾司氯胺酮50 mg/kg和艾司氯胺酮50 mg/kg+miR-148a-3p抑制剂5 mg/kg,连续注射7 d。于建模后1、4、7、10 d采用斜板实验计算最大倾斜角度、行为学实验计算运动功能BBB评分评估大鼠脊髓神经功能。建模后10 d BBB评分后处死大鼠,采用ELISA法检测脊髓白细胞介素-1β(IL-1β)、丙二醛(MDA)、超氧化物歧化酶(SOD)浓度,qPCR法检测脊髓miR-148a-3p、KLF4 mRNA表达量,Western blot法检测脊髓神经元核抗原(NeuN)、神经胶质纤维酸性蛋白(GFAP)、KLF4蛋白含量,HE染色法观察损伤处脊髓病理情况。结果与S组比较,SCI组、E组和EI组建模后1、4、7、10 d最大倾斜角度明显减小、BBB评分明显降低,脊髓IL-1β和MDA浓度、miR-148a-3p和KLF4 mRNA表达量、GFAP和KLF4蛋白含量明显升高,脊髓SOD浓度、NeuN蛋白含量明显降低(P<0.05)。与SCI组比较,E组和EI组建模后4、7、10 d最大倾斜角度明显增大、BBB评分明显升高,脊髓IL-1β和MDA浓度、GFAP蛋白含量明显降低,脊髓SOD浓度、miR-148a-3p和KLF4 mRNA表达量、NeuN和KLF4蛋白含量明显升高(P<0.05)。与E组比较,EI组建模后4、7、10 d最大倾斜角度明显减小、BBB评分明显降低,脊髓IL-1β和MDA浓度、GFAP蛋白含量明显升高,脊髓SOD浓度、miR-148a-3p和KLF4 mRNA表达量明显降低(P<0.05)。S组脊髓结构完整,细胞间排列紧密;SCI组脊髓结构被破坏,产生多个空洞;E组脊髓结构逐渐恢复,细胞排列较为整齐,空洞数量明显减少;EI组脊髓结构恢复较慢,细胞排列较为松散,仍有少量空洞。结论艾司氯胺酮通过调控miR-148a-3p/KLF4通路,抑制氧化应激反应和炎症反应,升高脊髓miR-148a-3p、KLF4 mRNA表达量和NeuN和KLF4蛋白含量,改善大鼠行为学和脊髓神经功能,保护神经组织和脊髓结构,减缓脊髓损伤。

Objective To explore the influence of esketamine on spinal cord injury and its role in microRNA-148a-3p(miR-148a-3p)/kruppel like factor 4(KLF4)pathway by constructing a rat model of spinal cord injury.Methods Forty-eight SPF grade adult female SD rats,aged 7-9 weeks,weighing 215-255 g,were randomly divided into four groups:sham operation group(group S),spinal cord injury group(group SCI),esketamine 50 mg/kg group(group E)and esketamine 50 mg/kg+miR-148a-3p inhibitor 5 mg/kg group(group EI),12 rats in each group.Rats in group S were removed the vertebral lamina,rats in group SCI only injured spinal cord to setablish spinal cord injury model,rats in group E and group EI were intraperitoneally injected with esmketamine 50 mg/kg and esmketamine 50 mg/kg+miR-148a-3p inhibitor 5 mg/kg for 7 days after modeling.The maximum tilt angle was calculated by the inclined plate experiment and the BBB score was calculated by the behavioral experiment to evaluate the spinal cord nerve function of rats 1,4,7 and 10 days after modeling.The rats were killed after BBB score 10 days after modeling,the concentrations of interleukin-1β(IL-1β),malondialdehyde(MDA)and superoxide dismutase(SOD)in the spinal cord were measured by ELISA,the expressions of mir-148a-3p and KLF4 mRNA in spinal cord were measuredby qPCR,the contents of neuron nuclear antigen(NeuN),glial fibrillary acidic protein(GFAP)and KLF4 protein in spinal cord were detected by Western blot,and the pathological changes of injured spinal cord were observed by HE staining.Results Compared with group S,the maximum tilt angle and BBB score were decreased significantly 1,4,7,and 10 days after modeling,the spinal cord IL-1βand MDA concentrations,miR-148a-3p and KLF4 mRNA expressions,GFAP and KLF4 protein contents were increased significantly,the spinal cord SOD concentration and NeuN protein content were decreased significantly in groups SCI,E and EI(P<0.05).Compared with group SCI,the maximum inclination angle and the BBB score were increased significantly 4,7,and 10 days after modeling,the spinal cord IL-1βand MDA concentrations,and GFAP protein content were decreased significantly,the spinal cord SOD concentration,miR-148a-3p and KLF4 mRNA expressions,NeuN and KLF4 protein contents were increased significantly in groups E and EI(P<0.05).Compared with group E,the maximum inclination angle and BBB score were decreased significantly 4,7,and 10 days after modeling,the spinal cord IL-1βand MDA concentrations,and GFAP protein content were increased significantly,the spinal cord SOD concentration,miR-148a-3p and KLF4 mRNA expressions were decreased significantly in group EI(P<0.05).The structure of spinal cord was complete,and the cells were closely arranged in group S.The structure of the spinal cord in group SCI was destroyed into multiple cavities.The spinal cord structure gradually recovered,the cells were arranged neatly,and the number of cavities was significantly reducedin group E.The structure of the spinal cord recovered slowly,the cells were loosely arranged,and there were still a few cavities in group EI.Conclusion By regulating miR-148a-3p/KLF4 pathway,esmketamine can inhibit oxidative stress and inflammatory response,increase the expression of miR-148a-3p and KLF4 mRNA and the protein contents of NeuN and KLF4 in spinal cord,improve rat behavior and spinal cord nerve function,protect nerve tissue and spinal cord structure and slow down spinal cord injury.