基于铂蓄积机制探讨黄芪桂枝五物汤对奥沙利铂致周围神经病变的防治作用

发布时间：2018-11-17 15:29

【摘要】：目的通过观察O-IPN模型大鼠一般情况、机械性刺激敏感程度、背根神经节细胞形态学改变,背根神经节铂含量及铂转运蛋白OCT2和ATP7A的表达情况,探讨黄芪桂枝五物汤对奥沙利铂神经毒性的干预作用及机制。方法选用42只雌性Wistar大鼠随机分为4组,空白组、模型组、西咪替丁(CIM)组和黄芪桂枝五物汤(中药)组。适应性喂养7天后,除空白组(n=6)予以腹腔注射5%葡萄糖注射液2ml/kg外,其余各组(n=12)按照2ml/kg (5mg/kg)的剂量予以腹腔注射奥沙利铂,每周2次,共4周。同时,空白组、模型组予以0.9%NaCl溶液灌胃1ml/次,每日1次。CIM组予以3ml/kg (30mg/kg)西咪替丁腹腔注射,每日1次,OXA给药当天,西咪替丁于给药前1小时腹腔注射。中药组予以黄芪桂枝五物汤汤剂2.80g/ml,10ml/kg灌胃,每日1次,OXA给药当天,中药于给药前1小时灌胃。持续性给药至第28天。每周进行2次体重称量,2次机械性疼痛阈值检测,每日观察大鼠给药后反应、饮食、排泄物等。第29天解剖各组大鼠,取L4-5双侧背根神经节。组织切片经尼氏染色后在油镜下观察背根神经节细胞及核仁形态,运用ICP-MS法对背根神经节内的铂含量进行定量检测,进行PCR实验对背根神经节中铂转运蛋白OCT2和ATP7A的mRNA表达情况进行检测。结果通过大鼠行为学观察、机械性刺激实验以及细胞形态学检测证实O-IPN模型制备成功。结果表明,使用OXA能够显著增加大鼠对机械性刺激的敏感度(P0.01),黄芪桂枝五物汤或西咪替丁可在一定程度上缓解大鼠对机械性刺激过度敏感表现,但这一效果短期内并不显著,大约在给药第三周后效果逐渐明显。细胞形态学观察结果显示,使用奥沙利铂后背根神经元出现细胞体积缩小,核仁缩小或分裂,尼氏体淡染、紊乱等形态学改变,使用黄芪桂枝五物汤和西咪替丁均能够改善奥沙利铂导致的大鼠背根神经元细胞及核仁体积缩小、尼氏体结构改变。经背根神经节内铂含量检测发现,使用OXA后背根神经节内可检测出较高含量的铂,而在黄芪桂枝五物汤或西咪替丁干预下,背根神经节内铂含量均显著降低(P0.05)。PCR实验结果表明：在OXA作用下,大鼠背根神经节Oct2的表达略升高,Atp7a的表达稍降低,而在黄芪桂枝五物汤干预下则出现了相反的结果,即Oct2的表达略降低,Atp7a的表达明显升高(P0.05),有趣的是,西咪替丁干预后的结果却是Oct2表达偏高,Atp7a的表达则基本不变。结论黄芪桂枝五物汤可以减轻奥沙利铂慢性神经毒性,但对于急性神经毒性效果不显著,其机制可能是通过少量下调背根神经节细胞铂转入蛋白OCT2mRNA的表达,适当减少铂流入背根神经元,同时主要上调铂转出蛋白ATP7A mRNA的表达而促进铂的流出,起到对抗铂蓄积于背根神经节的效果,从而缓解O-IPN慢性神经毒性表现。
[Abstract]:Objective to observe the general situation, the sensitivity of mechanical stimulation, the morphological changes of dorsal root ganglion cells, the content of platinum in dorsal root ganglion and the expression of platinum transporter OCT2 and ATP7A in O-IPN model rats. To investigate the effect and mechanism of Huangqi Guizhi Wuwu decoction on oxaliplatin neurotoxicity. Methods 42 female Wistar rats were randomly divided into 4 groups: blank group, model group, cimetidine (CIM) group and Huangqi Guizhi Wuwu decoction group. After 7 days of adaptive feeding, oxaliplatin was injected intraperitoneally in the control group (nong6) by intraperitoneal injection of 5% glucose injection (2ml/kg) according to the dose of 2ml/kg (5mg/kg), twice a week for 4 weeks. At the same time, in the blank group, the model group was given 1ml/ with 0.9%NaCl solution once a day, and the CIM group was given 3ml/kg (30mg/kg) cimetidine intraperitoneally, once a day, on the same day as OXA. Cimetidine was injected intraperitoneally 1 hour before administration. The traditional Chinese medicine group was treated with astragalus guizhiwu decoction 2.80 g / ml 10 ml / kg, once a day, one hour before the administration of OXA. The drug was given continuously until the 28 th day. The body weight and mechanical pain threshold were measured twice a week, and the reaction, diet and excretion of rats were observed daily. On the 29 th day, the rats in each group were dissected and L-4-5 bilateral dorsal root ganglion (DRG) was taken. The morphology of dorsal root ganglion cells and nucleoli were observed under oil microscope, and the content of platinum in dorsal root ganglion was quantitatively detected by ICP-MS method. The expression of platinum transporter OCT2 and ATP7A in dorsal root ganglion (DRG) was detected by PCR assay. Results the O-IPN model was successfully prepared by behavioral observation, mechanical stimulation and cell morphology. The results showed that OXA could significantly increase the sensitivity of rats to mechanical stimuli (P0.01). Huangqi Guizhi Wuwu decoction or cimetidine could alleviate the oversensitivity of rats to mechanical stimulation to some extent. However, this effect is not significant in the short term, about the third week after administration of the effect gradually obvious. The morphological changes of dorsal root neurons after oxaliplatin were observed, such as cell volume shrinking, nucleolus shrinking or splitting, Nissl body light staining, disorder and so on. Both Huangqi Guizhi Wuwu decoction and cimetidine could improve the size of dorsal root neurons and nucleoli and change the structure of Nissl body in rats induced by oxaliplatin. A higher level of platinum was detected in dorsal root ganglion (DRG) with OXA, and was treated with astragalus guizhiwuwu decoction or cimetidine. The content of platinum in dorsal root ganglion was significantly decreased (P0.05). PCR experiment showed that the expression of Oct2 in dorsal root ganglion was slightly increased, and the expression of Atp7a was slightly decreased under the action of OXA. However, under the intervention of Huangqi Guizhi Wuwu decoction, the contrary results were found, that is, the expression of Oct2 decreased slightly and the expression of Atp7a increased significantly (P0.05). Interestingly, the result of cimetidine intervention was that the expression of Oct2 was higher than that of control group. The expression of Atp7a was basically unchanged. Conclusion Huangqi Guizhi Wuwu decoction can attenuate the chronic neurotoxicity of oxaliplatin, but it has no significant effect on acute neurotoxicity. The mechanism of Huangqi Guizhi Wuwu decoction may be to down-regulate the expression of platinum transferred protein OCT2mRNA in dorsal root ganglion cells. At the same time, it mainly upregulated the expression of platinum transferrin ATP7A mRNA and promoted the outflow of platinum, which inhibited the accumulation of platinum in dorsal root ganglion and alleviated the chronic neurotoxicity of O-IPN.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R273

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