miR-19a对慢性压迫性损伤大鼠神经病理性疼痛的影响及其机制

Effect of miR-19a on neuropathic pain in chronic constriction injury rats and its mechanism

目的观察miR-19a对慢性压迫性损伤(chronic constriction injury,CCI)大鼠神经病理性疼痛的作用,并探讨其潜在机制。方法 75只雄性SD大鼠按随机数字表法分为3组:假手术组,行假手术;CCI组,坐骨神经结扎法构建CCI大鼠模型;CCI+inhibitor组,CCI大鼠鞘内注射miR-19a inhibitor。于术前当天(0 d)和术后3、7、14、21 d观察机械缩足反射阈值(mechanical withdrawal threshold,MWT)和热刺激反射潜伏期(thermal withdrawal latency,TWL)的变化。采用RT-qPCR检测腰椎脊髓中促炎因子IL-6、TNF-α、IL-1βmRNA及JAK2和STAT3表达的变化,Western blot检测SOCS3蛋白水平的表达,荧光素酶报告基因技术验证miR-19a的靶基因。结果与假手术组比较,CCI组大鼠脊髓的miR-19a表达显著上升(P<0.05),MWT显著降低,TWL显著缩短(P<0.05)。与CCI组比较,CCI+inhibitor组MWT显著升高,TWL显著延长(P<0.05)。RT-qPCR检测结果表明,CCI可显著促进促炎因子IL-6、TNF-α、IL-1βm RNA的表达;而与CCI组比较,CCI+inhibitor组IL-6、TNF-α、IL-1βm RNA表达水平均显著降低(P<0.05)。另一方面,与假手术组比较,CCI组大鼠术后脊髓中JAK2和STAT3 m RNA水平明显增加;与CCI组比较,CCI+inhibitor组术后脊髓中JAK2和STAT3的表达显著降低(P<0.01)。荧光素酶报告基因检测结果表明JAK2/STAT3上游的负调控因子SOCS3是miR-19a的靶基因。Western blot检测结果表明CCI组大鼠脊髓中SOCS3水平较假手术组升高,且SOCS3水平在CCI术后第7天达到最高,而后呈下降趋势;与CCI组比较,CCI+inhibitor组SOCS3表达明显上升(P<0.05)。结论 miR-19a inhibitor能缓解CCI大鼠机械性触诱发痛和热痛觉过敏,该作用与SOCS3介导的JAK2/STAT3通路有关。

Objective To determine the effect of miR-19a on neuropathic pain in rats and investigate the potential mechanisms. Methods A rat model of chronic constriction injury （CCI） was established by sciatic nerve ligation. A total of 75 male SD rats were randomly divided into 3 groups, that is, sham group, CCI group, CCI＋inhibitor group （intrathecal injection of miR-19a inhibitor）. The mechanical withdrawal threshold （MWT） and thermal withdrawal latency （TWL） of each group were observed on the surgery day （regarded as 0 d） and 3, 7, 14 and 21 d post-operatively. Simultaneously, the mRNA expression levels of IL-6, TNF-α, IL-1β, JAK2 and STAT3 in the lumbar spinal cord were detected with RT-qPCR. Then, luciferase report gene assay was used to verify the target genes of miR-19a. Results Compared to the sham group, miR-19a was highly expressed in the spinal cord tissue of CCI group （P〈0.05）, while MWT and TWL were significantly decreased （P〈0.05）. However, MWT and TWL were markedly increased in the CCI＋inhibitor group when compared with the CCI group （P〈0.05）. What is more, RT-qPCR analysis showed that compared with the sham group, the pro-inflammatory factors, such as IL-6, TNF-α and IL-1β were highly increased in the CCI group, while they were decreased in the CCI＋inhibitor group. Compared with the sham group, the mRNA expression levels of JAK2 and STAT3 were significantly enhanced in the spinal cord tissues of CCI rats, while inhibition of miR19a reversed these effects （P〈0.05）. Further luciferase assay identified that SOCS3, an upstream suppressor of JAK2/STAT3, was a target gene of miR-19a. Western blot assay showed the level of SOCS3 was highly expressed in the CCI group when compared with sham group, and it reached the highest at day 7 post-operatively. Compared with CCI group, the SOCS3 expression was markedly increased in CCI＋inhibitor group （P〈0.05）. Conclusion miR-19a inhibitor alleviates the mechanical withdrawal pain and thermal hyperalgesia, and this function is associated with SOCS3mediated JAK2/STAT3 pathway.