Abstract：
This study aims to clarify the effects of dihydroartemisinin(DHA) combined with pregabalin(PGB) on neuropathic pain(NP) in mice and explore the neuroinflammatory regulatory mechanism. NP mice model was established using spinal nerve ligation, whereas the sham group exposed the spinal nerve without ligation. The mice were randomly divided into sham group, model group, PGB groups of low, medium, and high doses(PGB-L, PGB-M, and PGB-H, with 22, 45, and 91 mg·kg~(-1)), DHA group(16 mg·kg~(-1)), and DHA combined with PGB groups of low, medium, and high doses(DHA + PGB-L, DHA + PGB-M, and DHA + PGB-H). Administration by gavage 18 days after modeling. Von Frey and cold plate were used to detect mechanical pain threshold and cold pain sensitivity in mice. The tail suspension test and forced swimming test were used to investigate depressive behavior, and the open field test was used to estimate anxiety behavior. The Morris water maze was used to evaluate cognitive function. Liquid suspension chip technology was used to quantitatively analyze immune inflammation-related factors. Immunofluorescence was used to detect the expression of CC chemokine ligand 3(CCL3) and transmembrane protein 119(TMEM119). The results showed that compared with the sham group, the mechanical pain and cold pain sensitivity thresholds of the model group were significantly reduced, and the struggle time was significantly increased in the tail suspension test and forced swimming test. The activity time in the central area was significantly reduced in the open field test. The residence time in the second/fourth quadrant was significantly longer than that in other quadrants, and the latency time of platform climbing significantly increased after platform withdrawal in the Morris water maze experiment. The expression of CCL3 was significantly increased; the number of TMEM119 positive cells and the cell body area were significantly increased. Compared with the model group, the DHA + PGB-M group showed a significant increase in mechanical pain and cold pain sensitivity thresholds, as well as a significant increase in struggle time in the tail suspension test and forced swimming test. The activity time in the central area of the open field test was significantly reduced. The residence time in the second/fourth quadrant was significantly shorter than that in other quadrants, and the latency time of platform climbing after platform withdrawal was significantly reduced. Compared with the PGB-M group, the mechanical pain threshold of D14-17 in the DHA + PGB-M group was significantly increased, and the struggle time during forced swimming was significantly increased. The residence time in the second/fourth quadrant of the Morris water maze was significantly shorter than that in other quadrants. Compared with the model group, the expression of CCL3, the number of TMEM119 positive cells, and the cell body area in the DHA + PGB-M group were significantly decreased. This study indicates that DHA + PGB can enhance the analgesic effect of PGB on NP mice, break through the limitations of PGB tolerance, and make up for the shortcomings of PGB in antidepressant and cognitive improvement. Its mechanism may be related to regulating neuroinflammation by inhibiting the activation of microglial cells and expression of CCL3.
摘要：
本研究旨在阐明双氢青蒿素(DHA)联合普瑞巴林(PGB)对小鼠神经病理性疼痛(NP)的影响,并探讨其神经炎症调控机制。采用脊神经结扎法建立NP小鼠模型,假手术组暴露脊神经，不结扎。将小鼠随机分为假手术组,模型组,PGB组低，中等,和高剂量(PGB-L，PGB-M,和PGB-H,22、45和91mg·kg〜（-1）），DHA组(16mg·kg~(-1)),和DHA结合PGB组的低，中等,和高剂量（DHA+PGB-L，DHA+PGB-M,和DHA+PGB-H)。在建模后18天通过管饲法施用。VonFrey和冷板用于检测小鼠的机械痛阈值和冷痛敏感性。悬尾试验和强迫游泳试验用于研究抑郁行为，并采用空场测验估计焦虑行为。使用Morris水迷宫评估认知功能。采用液体悬浮芯片技术对免疫炎症相关因子进行定量分析。免疫荧光法检测CC趋化因子配体3(CCL3)和跨膜蛋白119(TMEM119)的表达。结果显示,与假手术组相比,模型组机械性疼痛和冷痛敏感阈值明显降低,在尾部悬吊试验和强迫游泳试验中挣扎时间显著增加。在开场试验中，中心区域的活动时间显著减少。第二/第四象限的停留时间明显长于其他象限，在Morris水迷宫实验中，平台退出后平台爬升的潜伏期显着增加。CCL3表达显著增长；TMEM119阳性细胞数和细胞体面积显著增长。与模型组相比，DHA+PGB-M组显着增加机械性疼痛和冷痛的敏感性阈值，以及在尾部悬挂测试和强迫游泳测试中挣扎时间显着增加。开放场试验中心区域的活动时间显著减少。第二/第四象限的停留时间明显短于其他象限，平台退出后平台爬升的延迟时间明显缩短。与PGB-M组相比，DHA+PGB-M组D14-17的机械痛阈值明显升高,强迫游泳期间的挣扎时间显著增加。Morris水迷宫第二/第四象限的停留时间明显短于其他象限。与模型组相比，CCL3的表达，TMEM119阳性细胞数，DHA+PGB-M组细胞体面积显著减小。本研究表明DHA+PGB可以增强PGB对NP小鼠的镇痛作用,突破了PGB容差的限制，弥补了PGB在抗抑郁和认知改善方面的不足。其机制可能与通过抑制小胶质细胞活化和CCL3表达调节神经炎症有关。
