Lexington VA Health Care System, Research & Development, Lexington, KY 40502, USA.
Department of Internal Medicine, Yale University, New Haven, CT 06510, USA.
Cells. 2023 May 22;12(10):1440. doi: 10.3390/cells12101440.
Activation of intravesical protease activated receptors-4 (PAR4) results in bladder pain through the release of urothelial macrophage migration inhibitory factor (MIF) and high mobility group box-1 (HMGB1). We aimed to identify HMGB1 downstream signaling events at the bladder that mediate HMGB1-induced bladder pain in MIF-deficient mice to exclude any MIF-related effects. We studied whether oxidative stress and ERK activation are involved by examining bladder tissue in mice treated with intravesical disulfide HMGB1 for 1 h and analyzed with Western blot and immunohistochemistry. HMGB1 intravesical treatment increased urothelium 4HNE and phospho-ERK1/2 staining, suggesting that HMGB1 increased urothelial oxidative stress and ERK activation. Furthermore, we examined the functional roles of these events. We evaluated lower abdominal mechanical thresholds (an index of bladder pain) before and 24 h after intravesical PAR4 or disulfide HMGB1. Intravesical pre-treatments (10 min prior) included: N-acetylcysteine amide (NACA, reactive oxygen species scavenger) and FR180204 (FR, selective ERK1/2 inhibitor). Awake micturition parameters (voided volume; frequency) were assessed at 24 h after treatment. Bladders were collected for histology at the end of the experiment. Pre-treatment with NACA or FR significantly prevented HMGB1-induced bladder pain. No significant effects were noted on micturition volume, frequency, inflammation, or edema. Thus, HMGB1 activates downstream urothelial oxidative stress production and ERK1/2 activation to mediate bladder pain. Further dissection of HMGB1 downstream signaling pathway may lead to novel potential therapeutic strategies to treat bladder pain.
激活膀胱内蛋白酶激活受体 4（PAR4）会通过释放尿路上皮巨噬细胞迁移抑制因子（MIF）和高迁移率族蛋白 B1（HMGB1）导致膀胱疼痛。我们旨在确定 HMGB1 在膀胱中的下游信号事件，以介导 MIF 缺陷型小鼠中 HMGB1 诱导的膀胱疼痛，排除任何与 MIF 相关的影响。我们通过研究用腔内二硫键 HMGB1 处理 1 小时的小鼠的膀胱组织，并用 Western blot 和免疫组织化学分析，来研究氧化应激和 ERK 激活是否参与其中。HMGB1 腔内处理增加了尿路上皮 4HNE 和磷酸化 ERK1/2 染色，表明 HMGB1 增加了尿路上皮的氧化应激和 ERK 激活。此外，我们还研究了这些事件的功能作用。我们在腔内 PAR4 或二硫键 HMGB1 处理前和处理后 24 小时评估了下腹部机械阈值（膀胱疼痛的指标）。腔内预处理（预处理 10 分钟）包括：N-乙酰半胱氨酸酰胺（NACA，活性氧清除剂）和 FR180204（FR，选择性 ERK1/2 抑制剂）。在治疗后 24 小时评估清醒排尿参数（排空量；频率）。实验结束时收集膀胱进行组织学检查。NACA 或 FR 的预处理显著预防了 HMGB1 诱导的膀胱疼痛。对排尿量、频率、炎症或水肿没有显著影响。因此，HMGB1 激活下游尿路上皮氧化应激产物和 ERK1/2 激活，介导膀胱疼痛。对 HMGB1 下游信号通路的进一步剖析可能会导致治疗膀胱疼痛的新的潜在治疗策略。
