Laboratory of Pharmacology and Pathophysiology, Faculty of Pharmacy, Kindai University (formerly known as Kinki University), 3-4-1 Kowakae, Higashi-osaka, 577-8502, Japan.
Department of Pharmacology, Okayama University Graduate School of Medicine, Okayama, 700-8558, Japan.
J Neuroinflammation. 2019 Oct 30;16(1):199. doi: 10.1186/s12974-019-1581-6.
Macrophage-derived high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) protein, plays a key role in the development of chemotherapy-induced peripheral neuropathy (CIPN) caused by paclitaxel in rodents. Endothelial thrombomodulin (TM) promotes thrombin-induced degradation of HMGB1, and TMα, a recombinant human soluble TM, abolishes peripheral HMGB1-induced allodynia in mice. We thus examined whether HMGB1, particularly derived from macrophages, contributes to oxaliplatin-induced neuropathy in mice and analyzed the anti-neuropathic activity of the TM/thrombin system.
CIPN models were created by the administration of oxaliplatin in mice and rats, and the nociceptive threshold was assessed by von Frey test or paw pressure test. Macrophage-like RAW264.7 cells were stimulated with oxaliplatin in vitro. Proteins were detected and/or quantified by Western blotting, immunostaining, or enzyme-linked immunosorbent assay.
Intraperitoneal administration of an anti-HMGB1-neutralizing antibody (AB) at 1 mg/kg prevented the oxaliplatin-induced allodynia in mice and rats. Antagonists of Toll-like receptor (TLR) 4, receptor for advanced glycation end products (RAGE) and CXCR4 among the HMGB1-targeted pro-nociceptive receptors, also mimicked the anti-neuropathic activity of AB in mice. Macrophage accumulation in the sciatic nerve was observed in mice treated with paclitaxel, but not oxaliplatin, and neither macrophage depletion nor inhibitors of macrophage activation affected oxaliplatin-induced allodynia. Oxaliplatin was 10- to 100-fold less potent than paclitaxel in releasing HMGB1 from macrophage-like RAW264.7 cells. Like AB, TMα at 10 mg/kg prevented the oxaliplatin-induced allodynia in mice as well as rats, an effect abolished by argatroban at 10 mg/kg, a thrombin inhibitor. The anti-neuropathic activity of TMα in oxaliplatin-treated mice was suppressed by oral anticoagulants such as warfarin at 1 mg/kg, dabigatran at 75 mg/kg, and rivaroxaban at 10 mg/kg, but not antiplatelet agents such as aspirin at 50 mg/kg and clopidogrel at 10 mg/kg. Repeated administration of the anticoagulants gradually developed neuropathic allodynia and elevated plasma HMGB1 levels in mice treated with a subeffective dose of oxaliplatin.
Our data thus suggests a causative role of HMGB1 derived from non-macrophage cells in oxaliplatin-induced peripheral neuropathy and a thrombin-dependent anti-neuropathic activity of exogenous TMα and, most probably, endogenous TM.
高迁移率族蛋白 B1（HMGB1）是一种来源于巨噬细胞的损伤相关分子模式（DAMP）蛋白，在紫杉醇诱导的啮齿动物化疗诱导性周围神经病（CIPN）的发展中起关键作用。内皮血栓调节蛋白（TM）可促进凝血酶诱导的 HMGB1 降解，重组人可溶性 TMα可消除小鼠外周 HMGB1 诱导的痛觉过敏。因此，我们研究了 HMGB1，特别是来源于巨噬细胞的 HMGB1 是否参与了奥沙利铂诱导的小鼠周围神经病，并分析了 TM/凝血酶系统的抗神经病活性。
通过奥沙利铂在小鼠和大鼠中的给药来建立 CIPN 模型，并通过冯弗雷试验或足底压力试验评估痛觉阈值。用奥沙利铂体外刺激巨噬细胞样 RAW264.7 细胞。通过 Western blot、免疫染色或酶联免疫吸附试验检测和/或定量蛋白质。
腹腔内给予 1mg/kg 的抗 HMGB1 中和抗体（AB）可预防奥沙利铂诱导的小鼠和大鼠痛觉过敏。HMGB1 靶向的促伤害性受体（TLR）4、晚期糖基化终产物受体（RAGE）和 CXCR4 的拮抗剂在小鼠中也模拟了 AB 的抗神经病活性。在接受紫杉醇治疗的小鼠中观察到坐骨神经中巨噬细胞的积累，但在接受奥沙利铂治疗的小鼠中则没有，并且巨噬细胞耗竭或巨噬细胞激活抑制剂均不影响奥沙利铂诱导的痛觉过敏。奥沙利铂从巨噬细胞样 RAW264.7 细胞中释放 HMGB1 的效力比紫杉醇低 10 至 100 倍。与 AB 一样，10mg/kg 的 TMα 也可预防奥沙利铂诱导的小鼠痛觉过敏，而 10mg/kg 的凝血酶抑制剂 argatroban 可消除其作用。TMα 在奥沙利铂处理的小鼠中的抗神经病活性被口服抗凝剂如华法林（1mg/kg）、达比加群（75mg/kg）和利伐沙班（10mg/kg）抑制，但不被抗血小板药物如阿司匹林（50mg/kg）和氯吡格雷（10mg/kg）抑制。重复给予抗凝剂会逐渐引起接受亚有效剂量奥沙利铂治疗的小鼠产生神经病性痛觉过敏，并导致血浆 HMGB1 水平升高。
我们的数据因此表明，非巨噬细胞来源的 HMGB1 在奥沙利铂诱导的周围神经病中起因果作用，外源性 TMα 和可能的内源性 TM 具有依赖于凝血酶的抗神经病活性。
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