Macrophage-derived HMGB1 is a key molecule in paclitaxel-induced peripheral neuropathy in mice: involvement of ROS generation and NF-κB activation

[Speaker] Risa Domoto:1
[Co-author] Daichi Yamasoba:1, Hiroki Yamanishi:1, Fumiko Sekiguchi:1, Maho Tsubota:1, Masahiro Nishibori:2, Atsufumi Kawabata:1
1:Lab. Pharmacol. Pathophysiol., Fac. Pharm., Kindai Univ., Japan, 2:Dept Pharmacol, Okayama Univ. Grad. Sch. Med, Japan

Background: High mobility group box 1 (HMGB1), a nuclear protein, is released from various cells including macrophages, which in turn leads to aggravation of inflammation by activating multiple targets including receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4) and also by accelerating CXCL12-induced activation of CXC chemokine receptor 4 (CXCR4). In the present study, we examined the role of macrophage-derived HMGB1 in paclitaxel-induced peripheral neuropathy and analyzed the molecular mechanisms for the PCT-induced HMGB1 release from macrophages.
Methods: Male ddY mice received four repeated i.p. administrations of PCT at 4 mg kg on day 0, 2, 4 and 6. Neuropathic allodynia was evaluated by von Frey test in the right hindpaw. F4/80-positive macrophage accumulation in the isolated sciatic nerve was examined by immunostaning. Mouse macrophage-like RAW264.7 cells were stimulated with PCT for 3-24 h. Intracellular and/or extracellular protein levels were determined by Western blotting. Accumulation of reactive oxygen species (ROS) was measured by a fluorometric assay.
Results: Repeated i.p. administration of PCT caused delayed neuropathic allodynia, which was prevented by recombinant human soluble thrombomodulin, capable of inactivating HMGB1, an anti-HMGB1 neutralizing antibody, ethyl pyruvate (EP), known to inhibit HMGB1 release from macrophages, or liposomal clodronate that depletes macrophages. Macrophage infiltration in the sciatic nerve was confirmed after PCT treatment. In RAW264.7 cells, PCT caused cytoplasmic translocation and extracellular release of nuclear HMGB1. N-Acetyl-L-cysteine (NAC), an antioxidant, SB203580, a p38 MAP kinase inhibitor, or pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, blocked the PCT-induced HMGB1 release. Intracellular ROS accumulation was detected following PCT application. Finally, we found that PCT-induced neuropathic allodynia in mice was inhibited by NAC or PDTC.
Conclusions: Our data thus suggest that PCT causes HMGB1 release from macrophages through ROS production followed by the activation of p38 and NF-κB, leading to peripheral neuropathy.

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