芍药苷对小鼠神经病理性痛的影响

Effect of paeoniflorin on neuropathic pain in mice

目的探讨芍药苷对小鼠神经病理性痛(NP)的影响。方法选择SPF级雄性BALB/c小鼠40只,4~6周龄,体重20~25 g。将小鼠随机分为四组:假手术组(S组)、坐骨神经套管结扎损伤组(C组)、坐骨神经套管结扎损伤+芍药苷50 mg/kg组(P50组)和坐骨神经套管结扎损伤+芍药苷100 mg/kg组(P100组),每组10只。S组仅分离坐骨神经,不进行套管结扎。C组、P50组和P100组行坐骨神经套管结扎术。P50组术后连续14 d腹腔注射芍药苷50 mg/kg,P100组术后连续14 d腹腔注射芍药苷100 mg/kg,S组和C组术后连续14 d腹腔注射相同容量的生理盐水。于术后1、2、5、7、10、14 d测定机械缩足阈值(MWT)和热回缩潜伏期(TWL),于术后13 d行蔗糖偏好实验(SPT)测定糖水消耗百分比,于术后14 d行强迫游泳实验(FST)和悬尾试验(TST)分别测定静止不动时间。于术后14 d取小鼠眼球血,采用ELISA法测定TNF-α、IL-6、IL-1β浓度。采用Western blot法检测海马组织MKP-1蛋白含量、p-ERK/ERK及p-p38/p38比值。采用HE染色观察海马组织CA3区锥体细胞形态,免疫组织化学染色观察海马组织DG区小胶质细胞激活数量。结果与S组比较,C组、P50组和P100组术后2、5、7、10、14 d MWT明显降低、TWL明显缩短,糖水消耗百分比明显降低,FST和TST静止不动时间明显延长,海马组织TNF-α、IL-6和IL-1β浓度、p-ERK/ERK、p-p38/p38比值明显升高,MKP-1蛋白含量明显降低,DG区小胶质细胞激活数量明显增加(P<0.05)。与C组比较,P50组和P100组术后2、5、7、10、14 d MWT明显升高、TWL明显延长,糖水消耗百分比明显升高,FST和TST静止不动时间明显缩短,海马组织TNF-α、IL-6和IL-1β浓度、p-ERK/ERK、p-p38/p38比值明显降低,MKP-1蛋白含量明显升高,DG区小胶质细胞激活数量明显减少(P<0.05)。与P50组比较,P100组术后2、5、7、10、14 d MWT明显升高、TWL明显延长,糖水消耗百分比明显升高,FST和TST静止不动时间明显缩短,海马组织TNF-α、IL-6和IL-1β浓度、p-ERK/ERK、p-p38/p38比值明显降低,MKP-1蛋白含量明显升高,DG区小胶质细胞激活数量明显减少(P<0.05)。结论芍药苷可以缓解小鼠神经病理性痛导致的痛觉过敏和抑郁样行为,降低海马组织炎性因子浓度,升高MKP-1蛋白含量,降低其下游信号分子p-ERK/ERK、p-p38/p38比值,减少小胶质细胞的激活,芍药苷100 mg/kg效果优于50 mg/kg。

Objective To investigate the effect of paeoniflorin(PF)on neuropathic pain in mice.Methods Fourty male pathogen-free BALB/C mice,aged 4-6 weeks,weighing 20-25 g,were selected.They were randomly divided into four groups:sham operation group(group S),sciatic nerve ligation injury group(group C),sciatic nerve ligation injury with PF 50 mg/kg group(group P50),and sciatic nerve ligation injury with PF 100 mg/kg group(group P100),10 mice in each group.Only sciatic nerve were isolated,and no ligation was performed in group S.Groups C,P50 and P100 were underwent sciatic nerve ligation.Group P50 were intraperitoneally injected with paeoniflorin 50 mg/kg for 14 consecutive days after operation,while group P100 were intraperitoneally injected with paeoniflorin 100 mg/kg for 14 consecutive days after operation,groups S and C received the same volume of saline intraperitoneally for 14 consecutive days after operation.Mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)were determined 1,2,5,7,10,and 14 days after operation.Sucrose preference test(SPT)was performed 13 days after operation to measure the ratio of intake of sugar to water,forced swimming test(FST)and tail suspension test(TST)were carried out 14 days after operation to determine the time of mouse motionless.The eyeball blood was taken 14 days after operation and the concentrations of TNF-α,IL-6,and IL-1βwere determined by ELISA.Then the mice were killed,the content of MKP-1 protein,p-ERK/ERK,p-p38/p38 were detected by Western blot.HE staining was used to observe the morphology of pyramidal cells in the CA3 region of the hippocampus,and immunohistochemical staining was used to observe the number of activated microglia in the DG region of the hippocampus.Results Compared with group S,MWT were significantly decreased,TWL were significantly shortened 2,5,7,10,and 14 days after operation,sugar water consumption ratio was significantly decreased,the immobility time of FST and TST were significantly prolonged,the cincentration of TNF-α,IL-6,and IL-1β,the ratio of p-ERK/ERK and p-p38/p38 were significantly increased,the content of MKP-1 protein,the number of activated microglia was significantly decreased in groups C,P50 and P100(P<0.05).Compared with group C,MWT were significantly increased,TWL were significantly prolonged 2,5,7,10,and 14 days after operation,sugar water consumption ratio was significantly increased,the immobility time of FST and TST were significantly shortened,the cincentration of TNF-α,IL-6,and IL-1β,the ratio of p-ERK/ERK and p-p38/p38 were significantly decreased,the content of MKP-1 protein,the number of activated microglia was significantly increased in groups P50 and P100(P<0.05).Compared with group P50,MWT were significantly increased,TWL were significantly prolonged 2,5,7,10,and 14 days after operation,sugar water consumption ratio was significantly increased,the immobility time of FST and TST were significantly shortened,the cincentration of TNF-α,IL-6,and IL-1β,the ratio of p-ERK/ERK and p-p38/p38 were significantly decreased,the content of MKP-1 protein,the number of activated microglia was significantly increased in group P100(P<0.05).Conclusion Paeoniflorin can relieve hyperalgesia and depression-like behavior induced by neuropathic pain,reduce the concentration of inflammatory factors,increase the content of MKP-1 protein,reduce the ratio of downstream signal molecules p-ERK/ERK,p-p38/p38,and the number of activated microglia in mice.Paeoniflorin 100 mg/kg shows better effect than 50 mg/kg.