高压氧处理诱导小鼠nNOS/NO/γ-GABA信号通路双相镇痛效应的研究  

Research on the effects of hyperbaric oxygen on biphasic analgesic through nNOS/NO/γ-GABA pathways in mice

作　　者：刘书林 姚永杰 刘秋红 戴圣龙 时粉周 朱伟 孙学军 Raymond Quock 

机构地区：[1]海军军医大学海军医学研究所,上海200433 [2]海军军医大学军事预防系海军航空医学教研室 [3]美国华盛顿州立大学心理系

出　　处：《中华航海医学与高气压医学杂志》2017年第6期459-464,484,共7页Chinese Journal of Nautical Medicine and Hyperbaric Medicine

基　　金：美国国家卫生研究院基金（GM-77153）

摘　　要：目的 测定反复高压氧（hyperbaric oxygen,HBO）处理的镇痛效应,探索神经型一氧化氮合酶（neural nitric oxide synthase,nNOS）、一氧化氮（nitric oxide,NO）和 γ-氨基丁酸（γ-aminobutyrate,GABA）的作用机制.方法 建立疼痛动物模型小鼠,将动物随机分为HBO组、高压空气组、常压氧气组和常压空气组,进行反复HBO或空气暴露.反复HBO方案为先向高压氧舱内通纯氧5 min,以0.10 MPa/min的速度增压,增到0.35 MPa后,持续通氧气/空气60 min,然后以0.10 MPa/min的速率减压.每天暴露1次,共进行4次.利用腹部收缩试验进行镇痛效应评估,检测小鼠脑组织和脊髓内NO含量及NOS的蛋白表达.侧脑室给予各型NOS抑制剂,检测其对HBO镇痛效应的影响.荧光标记中脑导水管周围灰质（periaqueductal gray,PAG）内的nNOS＋神经元和谷氨酸脱羟酶（glatamic acid decarboxylase,GAD）阳性（GAD＋）神经元的位置关系.结果 HBO多次处理后,小鼠出现双相镇痛效应,包括：（1）早期镇痛,于HBO处理后1 h后出现,持续时间约8 h;（2）晚期镇痛,于HBO暴露1 d后出现,1周后镇痛效果达到顶峰,持续时间长达近3周.末次HBO暴露结束3 h后给予的非特异性NOS抑制剂N＇-硝基-L-精氨酸甲酯盐酸盐（N＇-Nitro-L-arginine-methyl ester hydrochloride,L-NAME）、nNOS抑制剂S-甲基-L-硫代瓜氨酸（S-methyl-L-thiocitrulline,SMTC）显著抑制早期镇痛效应.L-NAME和SMTC显著抑制晚期镇痛.HBO处理1 h后,小鼠脑组织和脊髓组织中的NO和nNOS的含量显著升高.HBO处理后7 d侧脑室注射CGP35348,高压氧晚期镇痛效应显著下降.免疫荧光结果显示,动物PAG中的nNOS＋神经元和GAD＋神经元出现重合.结论 4次HBO处理诱导双相镇痛效应.早期镇痛效应出现于HBO处理后1 h后,晚期镇痛效应出现于HBO暴露1 d后.早期效应的机制涉及nNOS的激活,而晚期效应的机制涉及nNOS与GABAB受体的相互作用.Objective To study the analgesic effect of repeated hyperbaric oxygen （HBO） exposures and explore the mechanism involving neural nitric oxide synthase （ nNOS）, nitric oxide （ NO） and γ-aminobutyric acid （GABA）.Methods The animal pain model was established and the animals were randomly divided into the HBO group, the hyperbaric air （ HBA） group, the normobaric air （NBA） group and the normobaric oxygen （ NBO） group, and were exposed repeatedly to either HBO or air .The chamber was ventilated with 100% O2 for 5 min, then, the chamber was pressurized to 0.35 MPa at a rate of 0.10 MPa/min.At the pressure of 0.35 MPa, the chamber was again ventilated with oxygen /air for 60 minutes, and then, was decompressed at a rate of 0.10 MPa/min.The animals were exposed in the chamber one session a day for a succession of 4 days.Analgesic effect was evaluated by abdominal contraction test , and nitrate reductase assay was used to determine the expression levels of NO and NOS in the brain tissue and the spinal cord.NOS inhibitors were given by i.c.v injection to measure the effect of NOS on the analgesic effect of HBO.The nNOS ＋ neurons and glatamic acid decarboxylase （ GAD） positive （ GAD ＋） neurons in the periaqueductal gray （ PAG） were labeled by fluorescopy.Results Repeated HBO treatment induced a biphasic analgesic effect, including： （1） early analgesia which was displayed an hour after HBO exposure and lasted for about 8 hours; （2） late analgesia which was displayed one day after HBO exposure , reached peak one week later and lasted for about 3 weeks.Three hours after the termination of last HBO exposure , medication of the non-specific NOS inhibitor N′-Nitro-L-arginine-methyl ester hydrochloride （ L-NAME ） and nNOS inhibitor S-methyl-L-thiocitrulline （SMTC） could obviously inhibit early analgesic effect .L-NAME and SMTC could significantly inhibit late analgesia .One hour after HBO exposure, the levels of NO and nNOS in the brain tissue and spinal cord were co

关 键 词：高压氧 镇痛 nNOS/NO/γ-GABA信号通路 

分 类 号：R459.6[医药卫生—治疗学]