靶向μ阿片受体的单克隆抗体通过增强吗啡诱导的受体内吞作用减弱吗啡耐受

IF 6.1 1区 医学 Q1 PHARMACOLOGY & PHARMACY
Jia-Jia Zhang , Chang-Geng Song , Miao Wang , Gai-Qin Zhang , Bin Wang , Xi Chen , Peng Lin , Yu-Meng Zhu , Zhi-Chuan Sun , Ya-Zhou Wang , Jian-Li Jiang , Ling Li , Xiang-Min Yang , Zhi-Nan Chen
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引用次数: 0

摘要

吗啡是一种常用的镇痛药,可激活阿片受体(MOR),具有明显的耐受性副作用。虽然吗啡诱导MOR内吞作用的无效是导致吗啡耐受的根本原因,但目前尚无有效的治疗吗啡耐受的方法。在本研究中,我们旨在开发一种精确靶向MOR的单克隆抗体(mAb),并确定其对吗啡耐受的治疗效果及其潜在的分子机制。我们采用脱氧核糖核酸免疫与细胞免疫相结合的策略,利用杂交瘤技术成功制备了一种靶向MOR的单抗,命名为3A5C7,并鉴定出该单抗是一种对MOR具有高特异性和亲和力的免疫球蛋白G单抗,对具有空间构象的抗原具有结合能力。通过免疫荧光染色、流式细胞术、Western blotting、共免疫沉淀和小干扰核糖核酸(siRNA)敲低证实,3A5C7增强吗啡诱导的MOR内吞作用通过G蛋白偶联受体激酶2 (GRK2)/β-抑制蛋白2依赖机制作用于HEK293T和SH-SY5Y两株细胞系。该单抗还允许MOR从细胞质再循环到质膜,并减弱吗啡诱导的MOR磷酸化。采用维甲酸诱导SH-SY5Y细胞分化,建立体外吗啡耐受模型。Western blot、酶联免疫吸附试验和基于sirna的敲除显示,3A5C7 mAb通过GRK2/β-arrestin2途径降低腺苷酸环化酶(吗啡耐受性的体外生物标志物)的过度活化。此外,体内热板实验显示,慢性鞘内给药3A5C7可显著减轻小鼠吗啡耐受,戒断跳跃实验显示,慢性和急性鞘内给药3A5C7均可减轻吗啡依赖。最后,鞘内电穿孔沉默短发夹RNA说明3A5C7的体内抗耐受和抗依赖作用是通过GRK2/β-arrestin2途径增强吗啡诱导的MOR内吞作用介导的。总之,我们的研究提供了一种靶向MOR的治疗性单抗3A5C7,通过增强吗啡诱导的MOR内吞作用来治疗吗啡耐受。mAb 3A5C7在治疗临床吗啡耐受方面显示出有希望的转化价值。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis

Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis

Morphine is a frequently used analgesic that activates the mu-opioid receptor (MOR), which has prominent side effects of tolerance. Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance, currently, there is no effective therapy to treat morphine tolerance. In the current study, we aimed to develop a monoclonal antibody (mAb) precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms. We successfully prepared a mAb targeting MOR, named 3A5C7, by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization, and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation. Treatment of two cell lines, HEK293T and SH-SY5Y, with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2 (GRK2)/β-arrestin2-dependent mechanism, as demonstrated by immunofluorescence staining, flow cytometry, Western blotting, coimmunoprecipitation, and small interfering ribonucleic acid (siRNA)-based knockdown. This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR. We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid. Western blot, enzyme-linked immunosorbent assays, and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase, the in vitro biomarker of morphine tolerance, via the GRK2/β-arrestin2 pathway. Furthermore, in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice, and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence. Finally, intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/β-arrestin2 pathway. Collectively, our study provided a therapeutic mAb, 3A5C7, targeting MOR to treat morphine tolerance, mediated by enhancing morphine-induced MOR endocytosis. The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.

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来源期刊
Journal of Pharmaceutical Analysis
Journal of Pharmaceutical Analysis Chemistry-Electrochemistry
CiteScore
16.20
自引率
2.30%
发文量
674
审稿时长
22 weeks
期刊介绍: The Journal of Pharmaceutical Analysis (JPA), established in 2011, serves as the official publication of Xi'an Jiaotong University. JPA is a monthly, peer-reviewed, open-access journal dedicated to disseminating noteworthy original research articles, review papers, short communications, news, research highlights, and editorials in the realm of Pharmacy Analysis. Encompassing a wide spectrum of topics, including Pharmaceutical Analysis, Analytical Techniques and Methods, Pharmacology, Metabolism, Drug Delivery, Cellular Imaging & Analysis, Natural Products, and Biosensing, JPA provides a comprehensive platform for scholarly discourse and innovation in the field.
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