Potential Application of Boldine in Alleviating SCI-induced Neuropathic Pain

脊髓损伤(SCI)影响着291,000多名美国人，其中约47,000人是退伍军人。提供高质量 照顾患有脊髓损伤的退伍军人是退伍军人事务部的优先事项。对于脊髓损伤患者来说，疼痛是 最难治疗的并发症，尤其是神经病理性疼痛(NP)，众所周知 是慢性的，对目前可用的许多治疗方法都是无效的。到目前为止，还没有完全有效的 脊髓损伤后神经病理性疼痛的治疗。最近的研究表明，神经损伤后，Toll样受体(TLRs) 都与神经病理性疼痛的产生有关。我们在原代星形胶质细胞中的可行性研究表明 冰片碱是一种天然化合物，可以抑制TLR-炎症体信号转导。进行的试验研究 在中度挫伤的小鼠脊髓损伤模型中，冰片碱改善了脊髓损伤后的运动功能 减少脑组织中TLR家族主要成分和炎症体复合体的表达 与疼痛处理相关的区域。基于这些初步发现和Toll样受体的重要作用 和炎症体复合体在神经病理性疼痛中的作用，我们建议使用感觉进行临床前研究 功能结局测量和分子机制研究，以测试口服给药是否 博尔丁可减轻脊髓损伤小鼠模型的疼痛反应。结果将支持临床前和临床前的随访 该天然化合物在减轻神经病理性疼痛中的潜在应用的临床翻译研究 患有SCI的退伍军人。 目的1.检测冰片对原代培养的TLR和炎性小体通路的调节作用。 理论基础：TLRs负责刺激星形胶质细胞和小胶质细胞，并将诱导 这些细胞的促炎介质和细胞因子，因此导致产生和维持 神经性疼痛。我们在小鼠原代星形胶质细胞培养中的初步发现表明，冰片碱治疗 显著降低TLR-炎症小体通路中一些主要成分的表达，包括 TLR2、核因子κB、半胱氨酸天冬氨酸蛋白酶-1、IL-1β。假设：博尔丁在体外可抑制TLR-炎症体途径。 方法：采用原代培养的小鼠星形胶质细胞、小胶质细胞、背角细胞和背根节细胞，用不同浓度的内毒素作用于培养的小鼠背根神经节。 是否含有25或50微克/毫升的冰片。细胞毒性将用乳酸脱氢酶测定。MR-NA和 TLR-炎症体的主要成分的蛋白质水平将通过qPCR或蛋白质印迹来确定， 分别进行了分析。用ELISA法测定培养液中IL-1β水平，测定核转录因子κB的核定位 采用免疫荧光法，并测定Caspase-1活性。 目的2.测定冰片对中度高血压小鼠神经病理性疼痛的影响 严重挫伤，脊髓损伤，并剖析可能的机制。基本原理：小鼠脊髓损伤有 是一个广泛使用的模型来评估不同的实验治疗策略。我们在一项 中度挫伤小鼠脊髓损伤模型提示冰片碱可改善小鼠的运动功能 脊髓损伤后TLR家族和炎症体的几个主要组成部分的mRNA水平降低 大脑中的复合体。假设：博尔丁通过正常化脊髓损伤诱导的神经病理性疼痛 TLR-炎症小体信号。方法：C57BL/6小鼠接受椎板切除或中度挫伤。 在T9-10受伤。动物将被随机分成4组：1)椎板切除术，2)椎板切除术+冰片，3) SCI，SCI加波尔丁。博尔丁每天灌胃两次，总剂量为50毫克/公斤/天。 动物将在手术前接受运动功能评估，然后每周使用Basso小鼠进行评估 标度法(BMS)。手术前将进行冯-弗雷长丝试验和甩尾试验。 受伤后每周学习一次，直到28dpi。然后动物将被安乐死，脑、脊髓、背根 将收集神经节(DRG)和血清进行机制研究，包括定量聚合酶链式反应(QPCR)、免疫印迹、免疫染色、 Luminex多重分析评估TLR-炎症体信号通路的潜在调节。

Spinal cord injury (SCI) affects over 291,000 Americans of whom ~47,000 are veterans. Providing high quality care to veterans with SCI is a priority within the Department of Veterans Affairs. For SCI patients, pain is one of the most difficult complications to treat, and this is especially true for neuropathic pain (NP) which is known to be chronic and refractory to many currently available treatments. Until now, there has been no fully effective therapy for neuropathic pain in SCI. Recent studies have shown that after nerve injury, Toll-like receptors (TLRs) are involved in the generation of neuropathic pain. Our feasibility studies in primary astrocytes suggested that boldine, a naturally occurring compound, could inhibit the TLR-inflammasome signaling. Pilot studies conducted in a mouse model of moderate contusion SCI revealed that boldine improved locomotor function following SCI and reduced the expression of major components of the TLR family and inflammasome complexes in brain regions linked to pain processing. Based on these preliminary findings and the important role of toll-like receptors and inflammasome complexes in neuropathic pain, we propose to conduct preclinical studies using sensory functional outcome measurements and molecular mechanistic studies to test whether oral administration of boldine will alleviate pain responses in a mouse model of SCI. Outcomes will support follow-up preclinical and clinical translational studies for potential application of this natural compound in reducing neuropathic pain in veterans with SCI. Aim 1. Test the effect of boldine on regulating TLR and inflammasome pathways in primary cultures. Rationale: TLRs are responsible for the stimulation of astrocytes and microglia and will induce expression in these cells of proinflammatory mediators and cytokines, therefore leading to the generation and maintenance of neuropathic pain. Our preliminary findings in mouse primary astrocyte cultures suggested that boldine treatment significantly reduced the expression of some major components in the TLR-inflammasome pathways, including TLR2, NFκB, Caspase-1 and IL-1β. Hypothesis: Boldine will inhibit TLR-inflammasome pathway in vitro. Methods: Mouse primary astrocyte, microglia, dorsal horn and DRG cultures will be treated with LPS in the presence or absence of 25 or 50 µg/ml boldine. Cytotoxicity will be determined by LDH assay. The mRNA and protein levels for major components of the TLR-inflammasome will be determined by qPCR or western blot, respectively. IL-1β level in the medium will be measured by ELISA, NFκB nuclear localization will be determined by immunofluorescence, and Caspase-1 activity will also be measured. Aim 2. Determine the effects of boldine on reducing neuropathic pain in a mouse model of moderate severity contusion SCI and dissect possible mechanisms. Rationale: Spinal cord injuries in the mice have been a widely used model to evaluate different experimental treatment strategies. Our preliminary findings in a mouse model of moderate contusion SCI suggested that administration of boldine improved locomotor function following SCI and reduced the mRNA levels of several major components of the TLR family and inflammasome complexes in the brain. Hypothesis: Boldine will reduce SCI-induced neuropathic pain through normalizing the TLR-inflammasome signaling. Methods: C57BL/6 mice will undergo either a laminectomy or moderate contusion injury at T9-10. Animals will be randomized among 4 groups: 1) Laminectomy, 2) Laminectomy plus boldine, 3) SCI, 4) SCI plus boldine. Boldine will be administered twice daily by gavage to reach a total dose of 50 mg/kg/day. Animals will be evaluated for locomotor function before surgery then weekly thereafter using the Basso Mouse Scale (BMS) method. Von Frey filament test and tail-flick test will be performed before surgery for baseline studies and weekly after injury till 28 dpi. Animals will then be euthanized and brain, spinal cord, dorsal root ganglia (DRG) and serum will be collected for mechanistic studies including qPCR, western blot, immunostaining, Luminex multiplex assay to evaluate the potential regulation of TLR-inflammasome signaling pathways.