南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (12): 1421-1426.doi: 10.12122/j.issn.1673-4254.2019.12.05

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曲古菌素A抑制神经病理性痛大鼠脊髓HDAC4上调并逆转差异表达的miRNAs

欧阳碧函，唐朝辉，侯新冉，陈 旦，郭曲练，翁莹琪   

  • 出版日期:2019-12-27 发布日期:2019-12-20
  • 基金资助:

Trichostatin A suppresses up-regulation of histone deacetylase 4 and reverses differential expressions of miRNAs in the spinal cord of rats with chronic constrictive injury

  

  • Online:2019-12-27 Published:2019-12-20

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摘要： 目的 探讨曲古菌素A（TSA）缓解神经病理性痛的可能作用机制。方法 成年雄性SD大鼠，采用坐骨神经慢性缩窄性损伤（CCI）方法建立大鼠神经病理性疼痛模型，随机分为3组：sham+DMSO组（S组）、CCI+DMSO组（C组）、CCI+TSA组（T组）。于术后第7至第9天，每日1次向鞘内注射5% DMSO溶液或TSA 10 µg（TSA溶于10 µL 5% DMSO）。术后第10 天行为学测量后取各组大鼠腰段脊髓，检测HDAC4 蛋白及 mRNA 表达水平，采用Sanger miRBase V19.0芯片筛选各组差异表达的微小RNA，选取miR-190b-5p和miR-142-3p，采用实时荧光定量PCR进行验证。结果 与S组比较，CCI术后3~10 d机械痛阈均明显降低（P<0.05），而鞘内注射TSA痛阈回升（P<0.05）。HDAC4表达于大鼠腰段脊髓灰质，主要位于胞质，CCI术后HDAC4蛋白表达增高(P<0.05），而 TSA可逆转其增高趋势（P<0.05）。与S组比较，C组大鼠腰段脊髓差异表达（fold change≥2或≤0.5，P< 0.05）的miRNAs共有83种；鞘注TSA可逆转CCI术后差异表达的miRNAs共58种（差异表达倍数fold change≥2或≤0.5），其中CCI术后表达下调，鞘注TSA后表达上调的miRNAs有41 种。通过real-time PCR，我们验证了miR-190b-5p和miR-142-3p的变化。结论 鞘内注射TSA能抑制神经病理性痛大鼠脊髓HDAC4上调及改变部分miRNAs的表达，这种改变可能参与TSA的镇痛作用。

Abstract: Objective To explore the analgesic mechanism of intrathecal trichostatin A (TSA) injection in a rat model of neuropathic pain induced by chronic constrictive injury (CCI). Methods Male SD rats were randomized into sham operation+ DMSO group (group S) , CCI +DMSO group (group C), CCI + 10 μg TSA group (group T), and in the latter two groups, rat models of neuropathic pain were established induced by CCI. The rats were given intrathecal injections of 10 μL 5% DMSO or 10 μg TSA (in 5% DMSO) once a day on days 7 to 9 after CCI or sham operation. The rats were euthanized after behavioral tests on day 10, and the lumbar segment of the spinal cord was sampled to determine the expression of histone deacetylase 4 (HDAC4) protein and mRNA and detect the differentially expressed miRNAs using a miRNA chip. MiR-190b-5p and miR-142-3p were selected for validation of the results using RT-qPCR. Results Compared with those in group S, the rats in group C showed significantly decreased paw withdrawal mechanical threshold (PWMT) from day 3 to day 10 after CCI (P<0.05); intrathecal injection of TSA significantly reversed the reduction of PWMT following CCI (P<0.05). Positive HDAC4 expression was detected mainly in the cytoplasm of the neurons in the gray matter of the spinal cord, and was obviously up-regulated after CCI (Ρ<0.05). Intrathecal injection of TSA significantly suppressed CCI-induced up-regulation of HDAC4 at 10 days after the operation (P<0.05). Compared with the miRNA profile in group S, miRNA profiling identified 83 differentially expressed miRNAs in group C (fold change ≥2 or ≤0.5, P<0.05); TSA treatment reversed the expressions of 58 of the differentially expressed miRNAs following CCI, including 41 miRNAs that were decreased after CCI but up-regulated following TSA treatment. The results of real-time PCR validated the changes in the expressions of miR-190b-5p and miR-142-3p. Conclusion TSA suppresses CCI-induced up-regulation of HDAC4 and reverses differential expressions of miRNAs in the spinal cord of rats, which may contribute to the analgesic effect of TSA on neuropathic pain.