双氢青蒿素和普瑞巴林联用对神经病理性疼痛小鼠的干预作用及炎症调控机制

Interventional effect and inflammatory regulation mechanism of dihydroartemisinin combined with pregabalin on neuropathic pain mice

明确双氢青蒿素(dihydroartemisinin,DHA)与普瑞巴林(pregabalin,PGB)联用对小鼠神经病理性疼痛(neuropathic pain,NP)的干预作用,并探索其神经炎症调控的初步机制。以脊神经结扎法建立NP小鼠模型,假手术组暴露脊神经而不结扎。随机分为假手术组,模型组,PGB低、中、高剂量(PGB⁃L、PGB⁃M、PGB⁃H,22、45、91 mg·kg^(-1))组、DHA(16 mg·kg^(-1))组、DHA联合PGB低剂量、中剂量、高剂量(DHA+PGB⁃L、DHA+PGB⁃M、DHA+PGB⁃H)组。造模第1~18天灌胃给药。以Von Frey法检测小鼠机械痛阈值,冷板法检测冷痛敏,悬尾及强迫游泳实验检测抑郁行为,旷场实验检测焦虑行为,Morris水迷宫检测认知功能。液相悬浮芯片技术对免疫炎症相关因子进行定量分析,免疫荧光检测CC趋化因子配体3(CCL3)、跨膜蛋白119(TMEM119)表达。结果显示,与假手术组比较,模型组小鼠机械痛及冷痛敏阈值显著降低;悬尾实验、强迫游泳停止挣扎时间显著增加;旷场实验在中心区域活动时间显著减少;Morris水迷宫在第二/四象限的停留时间显著多于其余象限,撤台后上台潜伏期显著增加,免疫荧光TMEM119阳性细胞数量显著增多,胞体面积显著增大,CCL3表达显著增加。与模型组比较,DHA+PGB⁃M组机械痛、冷痛敏阈值显著增加,悬尾、强迫游泳停止挣扎时间显著增加,旷场实验在中心区域活动时间显著减少,Morris水迷宫在第二/四象限的停留时间显著少于其余象限,撤台后上台潜伏期显著减少。与PGB⁃M组比较,DHA+PGB⁃M组D14⁃17机械痛阈值显著增加,强迫游泳停止挣扎时间显著增加,Morris水迷宫在第二/四象限的停留时间显著少于其余象限。与模型组比较,DHA+PGB⁃M组TMEM119阳性细胞数量明显减少,胞体面积明显减小,CCL3表达明显降低。该研究表明,DHA⁃PGB联用可以增强PGB对NP小鼠的镇痛效果,突破PGB耐受性的局限,弥补PGB在抗抑郁、改善认知方面的不足,相关机制可能与抑制小胶质细胞活化及CCL3表达调控神经炎症有关。

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.