Potential Application of Boldine in Alleviating SCI-induced Neuropathic Pain

脊髓损伤（SCI）影响了超过291，000名美国人，其中约47，000人是退伍军人。提供高质量 照顾脊髓损伤的退伍军人是退伍军人事务部的优先事项。对于SCI患者来说，疼痛是 这是最难治疗的并发症，尤其是神经性疼痛（NP）， 是慢性的，并且对许多目前可用的治疗是难治的。到目前为止，还没有完全有效的 治疗SCI中的神经病理性疼痛。最近的研究表明，神经损伤后，Toll样受体（TLR） 与神经性疼痛的产生有关。我们对原代星形胶质细胞的可行性研究表明， 天然存在的化合物boldine可以抑制TLR-炎性体信号传导。进行的试点研究 在中度挫伤的小鼠模型中，SCI显示Boldine改善了SCI后的运动功能 并降低脑中TLR家族和炎性小体复合物的主要组分的表达 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 和炎性复合物在神经病理性疼痛，我们建议进行临床前研究， 功能结果测量和分子机制研究，以测试口服给药 Boldine将减轻SCI小鼠模型中的疼痛反应。结果将支持临床前随访和 这种天然化合物在减轻神经性疼痛中的潜在应用的临床转化研究， 退伍军人SCI 目标1.在原代培养物中测试boldine对调节TLR和炎性体途径的作用。 基本原理：TLR负责刺激星形胶质细胞和小胶质细胞，并将诱导星形胶质细胞和小胶质细胞中的表达。 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， 显著降低TLR-炎性体途径中一些主要成分的表达，包括 TLR2、NFκB、Caspase-1和IL-1β。假设：Boldine在体外可抑制TLR-炎性体通路。 方法：将小鼠原代星形胶质细胞、小胶质细胞、背角和DRG培养物用LPS处理， 存在或不存在25或50 μg/ml boldine。将通过LDH试验测定细胞毒性。mRNA和 TLR-炎性体的主要组分的蛋白质水平将通过qPCR或western印迹测定， 分别ELISA法检测培养液中IL-1β水平，NFκB核定位 通过免疫荧光法，还将测量胱天蛋白酶-1活性。 目标2.在小鼠模型中测定boldine对减轻神经性疼痛的作用， 严重挫伤SCI和解剖可能的机制。基本原理：小鼠脊髓损伤 已被广泛使用的模型，以评估不同的实验治疗策略。我们的初步发现， 中度挫伤脊髓损伤小鼠模型提示给予boldine改善了运动功能 并降低TLR家族和炎性小体的几种主要成分的mRNA水平 大脑中的复合体。假设：Boldine将通过使SCI引起的神经病理性疼痛正常化来减少SCI引起的神经病理性疼痛。 TLR-炎性体信号传导。方法：C57 BL/6小鼠将接受椎板切除术或中度挫伤 在T9-10受伤。将动物随机分为4组：1）椎板切除术，2）椎板切除术+boldine，3） SCI，4）SCI + boldine。Boldine将通过灌胃每日两次给药，以达到50 mg/kg/天的总剂量。 在手术前评价动物的运动功能，之后每周使用Basso小鼠 量表（BMS）方法。术前将进行Von Frey细丝试验和甩尾试验作为基线 研究和每周受伤后，直到28 dpi。然后对动物实施安乐死，并对脑、脊髓、背根 将收集神经节（DRG）和血清用于机制研究，包括qPCR、蛋白质印迹、免疫染色， Luminex多重测定以评估TLR-炎性体信号传导途径的潜在调节。

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.