神经性疼痛(NPP)的治疗被认为具有挑战性,而寻找替代药物正在努力。NPP病理与嘌呤能2X7(P2X7)受体和瞬时受体电位香草酸受体1(TRPV1)的表达有关。Bufalin是一种传统的中药,来自蟾蜍毒液,具有明显的抗肿瘤作用,镇痛药,和抗炎特性。然而,溶解性差,快速新陈代谢,以及对其疼痛缓解机制的认识空白限制了bufalin的临床应用。因此,本研究的目的是阐明蟾蜍灵通过慢性收缩性损伤(CCI)缓解NPP的机制。为了解决对快速新陈代谢的关注,通过膜乳化法制备bufalin-PLGA微球(MS),以达到延长的疼痛缓解作用。蛋白质印迹,实时PCR,免疫荧光,和分子对接技术被用来证明bufalin对NPP的治疗作用。结果表明,在CCI大鼠中同时施用bufalin和bufalin-PLGAMS后,热退缩潜伏期(TWL)和机械退缩阈值(MWT)增强。通过bufalin-PLGAMS实现了长达3天的缓解疼痛效果,并降低了剂量频率。在用bufalin-PLGAMS治疗的CCI大鼠中,TRPV1和P2X7蛋白和mRNA的表达水平均位于背根神经节(DRG),减少了。此外,布法林-PLGAMS有效降低IL-1β水平,CCI组的IL-18、IL-6和TNF-α。分子对接的结果表明,通过直接与P2X7受体结合,蟾蜍灵减轻NPP的可能机制。通过膜乳化制备的bufalin-PLGAMS的给药显示了对缓解NPP的持续作用的有希望的应用。
The treatment of neuropathic pain (NPP) is considered challenging, while the search for alternative medication is striving. NPP pathology is related with the expression of both the purinergic 2X7 (P2X7) receptor and the transient receptor potential vanilloid 1 receptor (TRPV1). Bufalin is a traditional Chinese medication derived from toad venom with pronounced antitumor, analgesic, and anti-inflammatory properties. However, poor solubility, rapid metabolism, and the knowledge gap on its pain alleviation mechanism have limited the clinical application of bufalin. Hence, the purpose of this study is to illustrate the NPP alleviation mechanism of bufalin via chronic constriction injury (CCI). To address the concern on fast metabolism, bufalin-PLGA microspheres (MS) were prepared via membrane emulsification to achieve prolonged pain-relieving effects. Western blot, real-time PCR, immunofluorescence, and molecular docking were employed to demonstrate the therapeutic action of bufalin on NPP. The results showed enhanced thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) after the administration of both bufalin and bufalin-PLGA MS in the CCI rats. Prolonged pain-relieving effects for up to 3 days with reduced dose frequency was achieved via bufalin-PLGA MS. In the CCI rats treated with bufalin-PLGA MS, the expression levels of protein and mRNA in TRPV1 and P2X7, both localized in the dorsal root ganglion (DRG), were reduced. Moreover, bufalin-PLGA MS effectively reduced the levels of IL-1β, IL-18, IL-6, and TNF-α in the CCI group. The results from molecular docking suggested a possible mechanism of NPP alleviation of bufalin through binding to P2X7 receptors directly. The administration of bufalin-PLGA MS prepared by membrane emulsification demonstrated promising applications for sustained effect on the alleviation of NPP.