神经钙蛋白在奥沙利铂引起的神经病理性疼痛中的作用

摘要

Objective To investigate the effects of CaN on neuropathic pain induced by oxaliplatin.Methods SPF SD male mice,age of six weeks,weight of about 200-250 g,were randomly divided into two groups (n =5),the control group and CIPN model group.CIPN model group received a single intraperitoneal oxaliplatin administration (6 mg/ kg).PWT was measured by von Frey filament two days before administration and on Day 1,2,3,4,5,7,and 14 after administration.The highest pain points were chose.In formal experimental,the SD rats were randomly divided into 3 groups,control group,CIPN model 7 days group,and CIPN model 14 days group.After observing PWT on Day 7 and 14,the L4-5 DRG and DH were removed.The qPCR and western blot were applied to detect the mRNA and protein expression of CaN in DRG and DH.The expression of astrocytes marker GFAP,microglia marker IB4 and CaN were analyzed by im munofluorescence double labeling method.Results On Day 2,3,4,5,7,and 14 after injection,the PWT of CIPN model rats was significantly decreased than those in Vehicle group at the same point (P ＜ 0.05).In DH,compared with control group,the expression of CaN mRNA was lower in both groups.In DRG,the CaN mRNA was expressed with no significant difference among each groups.Western blot analysis showed that mRNA expression level was consistent with protein expression.In both DH and DRG,GFAP/CaN and IB4/CaN were co-expressed,however,the co-expression was significantly reduced in CIPN model 7 days and CIPN model 14 days groups,with changed shape of cells.Conclusion Oxaliplatin may accumulated mainly in the DH and DRG,induce the damage of surrounding nerve and down-regulation of CaN,reduce the mechanical pain threshold and lead to pain and feeling.%目的 探讨神经钙蛋白(CaN)在奥沙利铂引起的神经病理性疼痛中的作用.方法 取鼠龄为6周的SPF级SD雄性大鼠,体重约200～250 g,前期将大鼠随机分为两组(n=5):生理盐水组和CIPN模型组.CIPN模型组大鼠采用腹腔注射6 mg/kg奥沙利铂诱发其发生外周神经性病变(CIPN),于模型制备前2d及制备后的1、2、3、4、5、7、14 d,采用von frey细丝测定各组大鼠左后足机械痛缩足阈值(PWT),选择疼痛最高点作为实验点.正式实验将大鼠随机分为3组(n=12):对照组(处理前)、CIPN模型7d组、CIPN模型14 d组.于模型制备后7和14 d,痛阈测定结束后,取L4-5段脊髓背角(DH)及背根神经节(DRG),采用qPCR和Western blot方法分别检测DH和DRG中CaN mRNA及其蛋白表达水平,采用免疫荧光双标记法检测DRG中星形胶质细胞标志物胶质纤维酸性蛋白(GFAP)、小胶质细胞标志物植物凝集素(IB4)、CaN的表达.结果 CIPN模型组大鼠术后第2、3、4、5、7、14天的PWT均较生理盐水组的同时间点的PWT显著下降(P＜0.05),且PWT在单次注射6 mg/kg奥沙利铂后的第7和14天到达最低点.在DH中,第7、14天CaN mRNA表达相较于对照组有所下调;在DRG中,第7、14天CaN mRNA的表达水平与对照组相比无明显变化,蛋白表达水平与mRNA表达水平保持一致.在DH和DRG中,对照组中GFAP、CaN及IB4、CaN大量共表达,而在CIPN模型7、14 d组中共表达明显减少,且胞体形态发生变化.结论 奥沙利铂可能主要积累在DRG和DH中,对周围的神经造成损伤,诱导CaN表达下调,降低PWT值,导致痛觉的发生及感受.