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中国应用生理学杂志 ›› 2022, Vol. 38 ›› Issue (2): 113-118.doi: 10.12047/j.cjap.6220.2022.023

• 研究论文 • 上一篇    下一篇

鞘内注射干扰素调节因子8小干扰RNA对术后持续性疼痛大鼠痛阈及脊髓小胶质细胞活化的影响*

徐昌顺1,2, 林春2△, 蔡振宇1△   

  1. 1.厦门大学附属第一医院疼痛科, 厦门 361001;
    2.福建医科大学基础医学院 脑老化与神经变性疾病重点实验室 福建医科大学疼痛研究所, 福州 350108
  • 收稿日期:2021-05-14 修回日期:2022-03-19 出版日期:2022-03-28 发布日期:2022-08-29
  • 通讯作者: Tel: 0591-22862439,13358397120; E-mail: 441448233@qq.com, xuchangsn@163.com
  • 基金资助:
    *福建省科技创新联合基金(2018Y9069);福建省财政专项基金(2019B028)

Effects of intrathecal injection of IRF8 SiRNA on pain threshold and activation of spinal cord microglia in rats with postoperative persistent pain

XU Chang-shun1,2, LIN Chun2△, CAI Zhen-yu1△   

  1. 1. Department of Pain Management, the First Affiliated Hospital of Xiamen University, Xiamen 361001;
    2. Pain Institute, Key Laboratory of Brain Aging and Neurodegenerative Diseases, School of Basic Medical Sciences, Fujian Medical University, Fuzhou 350108, China
  • Received:2021-05-14 Revised:2022-03-19 Online:2022-03-28 Published:2022-08-29

摘要: 目的: 探究鞘内注射干扰素调节因子8小干扰RNA(IRF8 SiRNA)对PPsP大鼠痛阈及脊髓小胶质细胞活化的影响。方法: 120只雄性SD大鼠随机分为假手术组(SH,n=12),模型组(SM,n=48),溶媒组(SD,n=12)和IRF8沉默组(SS,n=48),其中,SM组于大鼠后足中部隐静脉内侧按皮肤/肌肉切开牵拉(SMIR)法建立术后持续性疼痛(PPsP)模型,SH组仅切开不牵拉;SD组与SS组建模前一周先于L4/5椎间隙行鞘内置管术, SS组于建模后第5、6日连续鞘内给予IRF8 SiRNA溶液20 μl(溶于DEPC水中,150 pmol),SD组给予等量DEPC水。测量并记录建模前(D0),建模后第1(D1)、3(D3)、7(D7)、12(D12)、22(D22)、33(D33)日等时点各组大鼠术侧后足机械刺激缩足反应阈值 (PWT); 建模后第12日各取6只,Western blot法检测脊髓背角Iba-1蛋白表达情况,并取SH组和SM组各3只,取术野隐神经行电镜观察其超微结构改变;再取SM组和SS组于上述各时点各6只,流式细胞术检测脊髓背角小胶质细胞活化情况。结果: 与D0相比, SM组在D1~D22PWT降低(P<0.05或P<0.01),并在D33恢复至正常水平(P>0.05);与SH组相比,SM组PWT在D1~D22均降低(P<0.05或P<0.01);与SD组相比,SS组PWT在D7~D22增高(P<0.05或P<0.01);与SH组相比,SS组D7~D22降低(P<0.05或P<0.01);隐神经髓鞘平均厚度: SH组为(377.03± 69.60) nm,SM组为(369.50±73.26) nm,两组间相比无统计学意义(P>0.05);与SH组相比,SM组Iba-1明显上调(P<0.01);与SD组相比,SS组Iba-1表达受到抑制(P<0.05),与SH组相比,SS组Iba-1表达也具有统计学差异(P<0.05),而SM组与SD组之间,Iba-1的表达无统计学意义(P>0.05);与D0相比,SM组小胶质细胞活化比率在D3~D22均显著增加(P<0.01),而SS组小胶质细胞活化于D3达到高峰(P<0.01);鞘内给药后,SS组脊髓背角小胶质细胞活化比率明显下降,与SM组相比,在D7~D12显著下降(P<0.01)。结论: SMIR诱导的PPsP大鼠显著且持续的机械痛觉过敏为非明显的外周神经损伤所致,可能是基于脊髓背角小胶质细胞活化所介导,而鞘内给予IRF8小干扰RNA可抑制脊髓背角小胶质细胞的激活,并逆转SMIR诱导的痛觉过敏。

关键词: 干扰素调节因子8, 小胶质细胞, 术后持续性疼痛, 脊髓, 痛觉过敏, 大鼠小干扰RNA(SiRNA)

Abstract: Objective: To investigate the effects of intrathecal injection of IRF8 SiRNA on the pain threshold and activation of spinal cord microglia in rats with postoperative persistent pain. Methods: One hundred and twenty male Sprague-Dawley rats were randomly divided into sham group (SH, n=12), SMIR group (SM, n=48), SMIR + DEPC group (SD, n=12) and SMIR + irf8 SiRNA group (SS, n=48). In the SM group, the persistent postsurgical pain(PPsP) model was established according to the skin/muscle incision and retraction (SMIR), and the SH group was only incised without retracted. The SD group and SS group received intrathecal catheterization one week before SMIR, the SS group was injected with 20 μl of IRF8 SiRNA solution (dissolved in DEPC-treated water, 150 pmol) intrathecally on the 5th and 6th day after SMIR, and the SD group was injected with the same amount of DEPC-treated water. The paw withdrawal threshold (PWT) of each group was measured and recorded before SMIR and on the 1st, 3rd, 7th, 12th, 22nd and 33rd days after SMIR. Western blot was used to detect the expression of Iba-1 in the dorsal horn of spinal cord on the 12th days after SMIR, and the saphenous nerves in the SH group and SM group were collected to observe their ultrastructural changes under electron microscope. The flow cytometry was used to detect the activation of microglia in spinal cord dorsal horn before SMIR and on the 1st, 3rd, 7th, 12th, 22nd and 33rd days after SMIR in the SM group and SS group. Results: Compared with D0, the PWT of SM group was decreased on the 1th to 22nd day after SMIR (P<0.05 or P<0.01), and returned to normal level on the 33rd day after SMIR (P> 0.05). Compared with the SH group, the PWT of the SM group was decreased on the 1th to 22nd day after SMIR (P<0.05 or P< 0.01). However, compared with the SD group, the PWT of the SS group was increased on the 7th to 22nd day after SMIR (P<0.05 or P<0.01). Compared with SH group, the PWT of SS group was decreased on the 7th to 22nd day after SMIR (P<0.05 or P<0.01). The average thickness of saphenous nerve myelin was (377.0 3±69.60) nm in the SH group and (369.50±73.26) nm in the SM group, and there was no significant difference between the two groups (P>0.05). Compared with the SH group, the expression level of Iba-1 was increased significantly (P<0.01) in the SM group. Compared with the SD group, the expression of Iba-1 was inhibited (P<0.05) in the SS group, and compared with the SH group, the expression of Iba-1 was also statistically different (P<0.05) in the SS group, while the expression of Iba-1 was not statistically significant between the SM group and the SD group (P>0.05). Compared with D0, the activation ratio of microglia was increased significantly on the 3rd to 22nd day after SMIR (P<0.01) in the SM group , while the activation of microglia reached a peak on 3rd day after SMIR (P<0.01) in the SS group. After intrathecal administration, the activation rate of microglia in the spinal dorsal horn of the SS group was decreased significantly, and compared with the SM group, it was decreased significantly on the 7th to 12th day after SMIR (P<0.01). Conclusion: The significant and persistent mechanical hyperalgesia in PPsP induced by SMIR was caused non-obvious peripheral nerve injury, which may be mediated by the activation of microglia in the dorsal horn of the spinal cord. IRF8 SiRNA administrated by intrathecal injection could inhibit the activation of microglia and reverse SMIR-induced hyperalgesia.

Key words: IRF8, microglia, persistent postsurgical pain, spinal, hyperalgesia, rat SiRNA

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