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

  • 摘要:
      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。

     

    Abstract:
      Objective  To investigate the role and mechanism of microRNA-31-5p (miR-31-5p) 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-31-5p mimic group, with 12 rabbits in each group. SCIRI model was established by clamping the abdominal aorta for 30 minutes, and the miR-31-5p overexpression model was constructed by intrathecal injection of miR-31-5p 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-31-5p, 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-31-5p 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-31-5p expression with miR-31-5p mimic significantly weakened the protective effect of RIPC on rabbit SCIRI(P < 0.05).
      Conclusion  The expression of miR-31-5p decreases after RIPC, and it can increase the expression of BDNF in the spinal cord and relieve SCIRI by activating the PKCε protein.

     

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