Research and Development, Lexington Veterans Affairs Health Care System, Lexington, KY, United States; Department of Physiology, University of Kentucky, Lexington, KY, United States.
Department of Natural Sciences, St. Petersburg College, St. Petersburg, FL, United States.
Neurosci Lett. 2019 Apr 23;699:54-58. doi: 10.1016/j.neulet.2019.01.046. Epub 2019 Jan 29.
Repeated intravesical PAR4 (protease activated receptor 4) activation elicits persistent bladder pain lasting 5 days after the last treatment. Persistent bladder pain was fully reversed by a systemic HMGB1 (high mobility group box 1) inhibitor while a MIF (macrophage migration inhibitory factor) antagonist partly reversed it. Since there is growing evidence that spinal MIF and HMGB1 mediate inflammatory and neuropathic pain we examined whether there were spinal changes occurring during persistent bladder pain that may be responsible for maintaining bladder pain. In addition, we tested whether we could modulate persistent bladder pain with spinal MIF or HMGB1 antagonists. Persistent bladder pain was elicited in female C57 mice by repeated (3x) intravesical instillation of PAR4-activating peptide while control animals received scramble peptide treatment. On day 4, spinal cord (L6-S1) changes in c-fos (non-specific marker of spinal activation) was assessed with immunofluorescence while MIF and HMGB1 were assessed with immunofluorescence, western blotting and real-time PCR. On day 7, mice received an intrathecal injection of a neutralizing MIF monoclonal antibody (15 μg in 5 μl PBS) or a HMGB1 inhibitor glycyrrhizin (25 μg in 5 μl of 5% alcohol in PBS) and abdominal mechanical threshold was tested. On day 9, mice were treated with vehicle or control and abdominal mechanical threshold was tested. Immunofluorescence showed that c-fos and MIF in the dorsal horn, dorsal grey commissure and intermediolateral areas significantly increased in PAR4-treated mice while HMGB1 was decreased. In addition, intrathecal treatment with MIF neutralizing mAb or glycyrrhizin significantly alleviated abdominal mechanical hypersensitivity at 1 and 2 h and the analgesic effect diminished at 6 h. Vehicle or control treatment had no effect. Persistent bladder pain is associated with spinal changes in MIF and HMGB1 levels. Furthermore, spinal treatment with MIF monoclonal antibody and HMGB1 inhibitor temporarily reversed bladder pain. Our findings suggest that spinal MIF and HMGB1 participate in persistent bladder pain induced by repeated intravesical PAR4 and may be potential therapeutic targets in chronic bladder pain conditions.
重复的膀胱内 PAR4（蛋白酶激活受体 4）激活会引起持续的膀胱疼痛，这种疼痛在最后一次治疗后持续 5 天。全身高迁移率族蛋白 B1（HMGB1）抑制剂可完全逆转持续性膀胱疼痛，而巨噬细胞移动抑制因子（MIF）拮抗剂部分逆转了这种疼痛。由于越来越多的证据表明脊髓 MIF 和 HMGB1 介导炎症和神经病理性疼痛，我们研究了在持续性膀胱疼痛期间是否存在可能导致膀胱疼痛持续存在的脊髓变化。此外，我们还测试了是否可以通过脊髓 MIF 或 HMGB1 拮抗剂来调节持续性膀胱疼痛。通过重复（3 次）膀胱内 PAR4 激活肽灌注，在雌性 C57 小鼠中引起持续性膀胱疼痛，而对照动物接受 scramble 肽处理。在第 4 天，通过免疫荧光评估脊髓（L6-S1）中 c-fos（脊髓激活的非特异性标志物）的变化，同时通过免疫荧光、western blot 和实时 PCR 评估 MIF 和 HMGB1。在第 7 天，小鼠接受鞘内注射中和 MIF 单克隆抗体（15μg 在 5μl PBS 中）或 HMGB1 抑制剂甘草酸（25μg 在 5%酒精的 5μl PBS 中），并测试腹部机械阈值。在第 9 天，用载体或对照物处理小鼠，并测试腹部机械阈值。免疫荧光显示，在 PAR4 处理的小鼠中，背角、背灰质连合和中间外侧区域的 c-fos 和 MIF 显著增加，而 HMGB1 减少。此外，鞘内给予 MIF 中和单克隆抗体或甘草酸可显著缓解 1 小时和 2 小时时的腹部机械性超敏反应，而 6 小时时的镇痛效果减弱。载体或对照物处理没有效果。持续性膀胱疼痛与脊髓中 MIF 和 HMGB1 水平的变化有关。此外，脊髓给予 MIF 单克隆抗体和 HMGB1 抑制剂可暂时逆转膀胱疼痛。我们的研究结果表明，脊髓 MIF 和 HMGB1 参与了由重复膀胱内 PAR4 引起的持续性膀胱疼痛，并且可能是慢性膀胱疼痛的潜在治疗靶点。
