Takeuchi Fuka, Hagiyama Man, Yoneshige Azusa, Wada Akihiro, Inoue Takao, Hosokawa Yoichiroh, Ito Akihiko
Division of Molecular Pathology, Graduate School of Medicine, Kindai University, 377-2 Ohno-higashi, Osaka-sayama, Osaka 589-8511, Japan.
Department of Pathology, Kindai University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-sayama, Osaka 589-8511, Japan.
Life Sci. 2024 Nov 15;357:122997. doi: 10.1016/j.lfs.2024.122997. Epub 2024 Aug 22.
Cell adhesion molecule 1 (CADM1) is a member of the immunoglobulin superfamily and is abundantly expressed on nerve fibers. Recently, the anti-CADM1 ectodomain antibody 3E1 has proven useful as a drug delivery vector for CADM1-expressing cells in vitro. When injected subcutaneously into mice, whether 3E1 accumulates on nerve fibers and serves as an analgesic was examined.
Injected 3E1 was detected by immunohistochemistry and double immunofluorescence. Analgesic effects were verified by a formalin-induced chemical-inflammatory pain test and video-recorded behavior analysis that were performed 6, 12, and 24 h after antibody injection. Primary cultures of mouse dorsal root ganglion (DRG) cells were incubated with 3E1 and expressions of CADM1 and its key downstream molecules were examined by Western blot analyses and live cell imaging. DRG cells were loaded with a Ca fluorescent indicator Fluo-8 and a femtosecond laser pulse was irradiated near the cell body to mechanically stimulate the nerves.
Subcutaneously injected 3E1 was widely localized almost exclusively on peripheral nerve fibers in the dermis. In formalin tests, 3E1-injected mice exhibited less pain-related behavior than control mice. When 3E1 was added to DRG cell cultures, it localized to neurites and resulted in decreased expression of CADM1, increased phosphorylation of Src and Akt, and CADM1-3E1 complex formation. Femtosecond laser-induced stimulation transmission along neurites was clearly visualized by Fluo-8 fluorescence in control cells, whereas it was markedly suppressed in 3E1-treated cells.
3E1 was suggested to be a potential long-acting analgesic based on its high affinity for CADM1.
细胞黏附分子1（CADM1）是免疫球蛋白超家族的成员，在神经纤维上大量表达。最近，抗CADM1胞外结构域抗体3E1已被证明在体外作为表达CADM1细胞的药物递送载体是有用的。当皮下注射到小鼠体内时，检测3E1是否在神经纤维上积累并起到镇痛作用。
通过免疫组织化学和双重免疫荧光检测注射的3E1。在抗体注射后6、12和24小时，通过福尔马林诱导的化学性炎症疼痛试验和视频记录行为分析来验证镇痛效果。将小鼠背根神经节（DRG）细胞的原代培养物与3E1一起孵育，并通过蛋白质印迹分析和活细胞成像检查CADM1及其关键下游分子的表达。用钙荧光指示剂Fluo-8加载DRG细胞，并在细胞体附近照射飞秒激光脉冲以机械刺激神经。
皮下注射的3E1几乎完全广泛地定位在真皮中的外周神经纤维上。在福尔马林试验中，注射3E1的小鼠表现出比对照小鼠更少的疼痛相关行为。当将3E1添加到DRG细胞培养物中时，它定位于神经突，并导致CADM1表达降低、Src和Akt磷酸化增加以及CADM1-3E1复合物形成。在对照细胞中，通过Fluo-8荧光可以清楚地看到飞秒激光诱导的沿神经突的刺激传递，而在3E1处理的细胞中则明显受到抑制。
基于其对CADM1的高亲和力，3E1被认为是一种潜在的长效镇痛药。
