微小RNA-31-5p调控蛋白激酶Cε通路在远程缺血预处理保护兔脊髓缺血再灌注损伤中的作用机制

Mechanism of microRNA-31-5p regulating protein kinase Cε pathway in protection of rabbit spinal cord ischemia-reperfusion injury by remote ischemic preconditioning

目的探讨微小RNA-31-5p(miR-31-5p)在远程缺血预处理(RIPC)保护兔脊髓缺血再灌注损伤(SCIRI)中的作用及其机制。方法将60只日本大耳白兔分为假手术(sham)组、缺血再灌注(I/R)组、RIPC组、RIPC+miR-NC组、RIPC+miR-31-5p mimic组,每组12只。采用夹闭腹主动脉30 min建立SCIRI模型,通过鞘内注射miR-31-5p mimic质粒构建miR-31-5p过表达模型,除sham组和I/R组外,其余各组于闭腹主动脉前1 h实施RIPC。再灌注48 h后,对动物进行后肢运动功能评分和血-脊髓屏障(BSCB)通透性检测;苏木精-伊红(HE)染色观察脊髓组织形态学变化;TUNEL检测脊髓组织神经元凋亡情况;逆转录-实时定量聚合酶链反应(RT-qPCR)检测脊髓组织miR-31-5p和蛋白激酶Cε(PKCε)和脑源性神经营养因子(BDNF)的mRNA表达;Western Blot检测脊髓组织PKCε、BDNF蛋白表达。结果与sham组相比,I/R组后肢运动功能评分降低,伊文思蓝(EB)含量、神经元凋亡率增加,差异有统计学意义(P<0.05),且脊髓神经元数量减少,空泡化明显,胞核固缩;与I/R组相比,RIPC组后肢运动功能评分、PKCε和BDNF的mRNA和蛋白表达增加,EB含量、神经元凋亡率、miR-31-5p表达降低,差异有统计学意义(P<0.05),且正常神经元明显增多,少数细胞空泡变性,核仁明显;使用miR-31-5p mimic上调miR-31-5p的表达可显著减弱RIPC对兔SCIRI的保护作用(P<0.05)。结论 miR-31-5p的表达在RIPC后降低,并可通过激活PKCε蛋白,上调脊髓组织中BDNF的表达,减轻SCIRI。

Objective To investigate the role and mechanism of microRNA-31-5 p(miR-31-5 p) in the protection of rabbit spinal cord ischemia-reperfusion injury(SCIRI) by remote ischemic preconditioning(RIPC). Methods A total of 60 Japanese white rabbits were divided into sham group, ischemia/reperfusion(I/R) group, RIPC group, RIPC+miR-NC group and RIPC+miR-31-5 p mimic group, with 12 rabbits in each group. SCIRI model was established by clamping the abdominal aorta for 30 minutes, and the miR-31-5 p overexpression model was constructed by intrathecal injection of miR-31-5 p mimic plasmid. Except for the sham group and the I/R group, RIPC was performed in other groups one hour before the abdominal aorta closed. After 48 hours of reperfusion, the animals were evaluated for hindlimb motor function score and blood-spinal cord barrier(BSCB) permeability;Hematoxylin-Eosin(HE) staining was used to observe the morphological changes of spinal cord tissue;TUNEL was used to detect neuronal apoptosis in spinal cord tissue;reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR) was used to detect the mRNA expression of miR-31-5 p,protein kinase Cε( PKCε) and brain-derived neurotrophic factor( BDNF) in spinal cord tissue;Western Blot was used to detect the expression of PKCε and BDNF protein in spinal cord tissue.Results Compared with the sham group,the hindlimb motor function score of the I/R group was significantly reduced,and the Evans blue( EB) content and neuron apoptosis rate were significantly increased( P < 0. 05),and the number of spinal cord neurons was reduced,vacuolation was obvious,and nucleus pyknosis was observed;compared with the I/R group,the hindlimb motor function score,PKCε and BDNF mRNA and protein expression of RIPC group were significantly increased,and the EB content,neuronal apoptosis rate and miR-31-5 p expression were significantly decreased( P < 0. 05),the normal neurons increased significantly,a few cells had vacuolar degeneration,and nucleoli were obvious;upregulation of miR-31-5 p expression with miR-31-5 p mimic significantly weakened the protective effect of RIPC on rabbit SCIRI( P < 0. 05). Conclusion The expression of miR-31-5 p decreases after RIPC,and it can increase the expression of BDNF in the spinal cord and relieve SCIRI by activating the PKCε protein.