电针镇痛的累积效应与海马神经元可塑性及胞内MAPK/ERK信号通路活动关系分析
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摘要
研究背景
慢性疼痛包括慢性神经病理性疼痛是临床上常见的病症,目前采用西医药治疗的效果有限。针刺治疗慢性痛具有较好的疗效,但是其作用机制还不太清楚。海马作为边缘系统的一部分,除参与学习记忆、情绪活动等等的功能外,还参与疼痛信息的加工处理和针刺镇痛的过程。我们前期在慢性坐骨神经结扎造成的神经痛(CCI)大鼠模型上的实验结果表明,重复电针产生累积镇痛效应的同时,可显著改善CCI大鼠海马CA3区神经元突触间隙、突触厚膜厚度、突触活性带长度明显减小的变化,上调海马镇痛物质乙酰胆碱及其M受体、γ-氨基丁酸受体等基因的表达变化密切相关。但是,针刺镇痛累积效应的细胞内分子机制尚不清楚。
慢性神经病理性疼痛是神经可塑性的一种表达方式。神经损伤产生的持续性剧烈的伤害性刺激传入导致中枢敏化,该敏化反应是活动-依赖性的功能可塑性的变化；在细胞水平上产生于细胞内各种各样的激酶级联的激活进和许多关键细胞膜受体、离子通道的磷酸化等的变化。MAPK(mitogen-activated protein kinase)/ERK (Extracellular signal-regulated kinases)级联是细胞内一个主要的生化信号通路,参与启动和调节细胞外的各种刺激信息引发的细胞内许多信号通路(蛋白分子转化)加工处理过程,包括神经的可塑性、学习记忆等等,特别是,它还是痛觉过敏中枢敏化过程中细胞内多个信号通路调节的总开关(master switch)。此外,在中枢神经系统中,神经递质和神经营养因子(脑源性神经生长因子brain derived neurophic factor, BDNF等)信号通路之间的相互作用(interplay)可调节中枢神经网络对疼痛过敏条件下环境变化的适应性反应。为此,本研究在CCI大鼠模型上,采用行为学、电生理学、分子生物学等技术,进一步研究针刺累积镇痛效应与海马BDNF/MAPK/ERK/CREB信号通路活动变化的关系。
研究方法
实验一,行为学实验：Wistar雄性大鼠80只,随机分为正常对照组(n=16),慢性压迫性损伤(CCI)组(n=16),CCI+EA2天(D)(n=16),CCI+EAl周(W)(n=16),CCI+EA2W (n=16).采用坐骨神经四道结扎造成CCI神经痛模型,电针双侧“足三里”-“阳陵泉”穴,采用2/15Hz,1-2mA,每天1次,分别为不电针、电针2天、电针1周和电针2周,用热辐射测痛仪测量热痛阈,以手术侧和假手术侧的痛阈值的差值作为痛敏分数来观察动物的痛阈的变化。
实验二,生理学实验：Wistar雄性大鼠43只,随机分为正常对照组(n=15),CCI组(n=13),U0126组(n=15),用脑立体定位仪固定大鼠头部,刺激坐骨神经作为伤害性刺激,刺激脉冲(间隔50ms,波宽0.3ms,电流强度5mA),刺激10s,将微量注射器定位于侧脑室(前囟后1.0mm,旁开1.3-2.0mm,深3.5mm)注射ERK抑制剂U0126,电极尖端定位于海马CA1区(前囟后3.2-3.6mm,旁开2.5-3.0mm,深2.5-3.1mm)记录各组大鼠海马CA1痛相关神经元的自发放电变化,以及电针和U026对自发放电的影响作用。
实验三,分子生物学实验：上述行为学动物,正常对照组(n=8),CCI组(n=8),CCI+EA2D (n=8), CCI+EA1W (n=8), CCI+EA2W (n=8),应用real-time PCR方法检测大鼠海马部位BDNF, trk2PKA和pCREB的mRNA表达的变化；正常对照组(n=8), CCI组(n=8), CCI+EA2D (n=8), CCI+EA1W (n=8), CCI+EA2W (n=8),应用western blot方法检测大鼠海马部位Ras、Raf、MEK、p-ERK1/2、P38、CREB等的蛋白的表达变化。
实验结果
1、累积电针对各组大鼠行为学表现的影响
1.1CCI大鼠行为学表现
术后24小时即可见手术组大鼠,手术侧肢体(左侧后肢)自发性抬起,足趾蜷曲、缩回、足掌极少平伸着地。手术后4d,可以观察到有明显的外翻和避免患肢着地,明显的患肢跛行等的行为异常,但没有出现托行。热辐射轻微照射造成热辐射痛,大鼠即长时间舔足,不停甩足,但没有出现自残现象。
1.2累积电针对CCI大鼠的热辐射痛阈的影响
CCI大鼠在术后4天(3.15±1.05),痛敏分数明显高于正常对照组(-0.063±0.23)(P<0.05)；在术后18天,也就是取材前,CCI对照组痛敏分数(3.69±0.24)仍然明显高于正常对照组(0.011±0.24,P<0.05);CCI+EA2D组痛敏分数(3.01±1.65)明显高于正常对照组,明显低于CCI对照组(P<0.05):CCI+EA1W组痛敏分数(2.34±1.85)明显高于正常对照组(P<0.05),明显低于CCI对照组和CCI+EA2W组(P<0.05)：CCI+EA2W痛敏分数(0.98±0.1)明显低于CCI对照组、CCI+EA2D组(P<0.05),与正常对照比较没有显著差异(P>0.05),说明电针具有明显镇痛效应,且这种镇痛效应具有明显的累积性。
2、电针镇痛对大鼠海马痛相关神经元自发放电的影响
2.1痛刺激坐骨神经及电针对正常动物自发放电的影响
对正常大鼠海马CAl区痛兴奋性神经元(n=17)分析可以看到,在痛刺激后单纯痛刺激组放电频率(134.53%±37)和痛刺激加电针组放电频率(126.1%±20.05)都有明显的增加,且单纯痛刺激组与痛刺激加电针组没有差异(P>0.05)；在痛刺激后2min,单纯痛刺激组的放电频率(168.61%±58.72)明显多于痛刺激加电针组的放电频率(121.76%±21.67,P<0.05)；在痛刺激后4min,单纯痛刺激组放电频率(145.08%±21.67)明显多于痛刺激加电针组的放电频率(108.65%±14.16,P<0.05),痛刺激加电针组已经基本恢复至痛刺激前水平。在痛刺激后6min单纯痛刺激组的放电频率仍然处于增加的水平(125.87%±23.92),明显高于痛刺激加电针组的放电频率(105.72%±22.49,P<0.05)；在痛刺激后8min,10min,单纯痛刺激组的放电频率已接近恢复正常水平(116.86%±25.97,103.22%±6.03),与痛刺激加电针组(102.14%±22.58,97.65%±14.4)相比,没有明显的差异(P>0.05),但电针组的放电频率仍然低于单纯痛刺激组。这说明电针有抑制痛兴奋神经元自发放电增加的作用。
对正常大鼠痛抑制神经元(n=12)分析可以看到,在痛刺激完即刻,单纯痛刺激组放电频率减少(67.88%±14.71),电针加痛刺激组放电频率为痛刺激前的(93.35%±15.83),两组有显著差异(P<0.05)；痛刺激后2min,单纯痛刺激组的放电频率(54.35%±19.37)明显低于电针加痛刺激组(74.36%±16.62)；痛刺激后4min,单纯痛刺激组的放电频率(53.01%±19.37)仍然明显低于电针加痛刺激组(87.37%±9.25,P<0.05)；从痛刺激后6min开始,单纯痛刺激组的放电频率开始增加(67.93%±26.47),慢慢恢复,至10min,已经基本恢复至痛刺激前的放电频率(95.06%±15.92),而痛刺激加电针组痛刺激后6min已经恢复至痛刺激前的放电频率(100.59%±23.28),在痛刺激后8min和l0min的放电频率均与痛刺激前的放电频率一致。而单纯痛刺激组和痛刺激加电针组两组在痛刺激后6min,8min,10min均有明显的差异(P<0.05)。电针镇痛有解除痛抑制神经元放电减少的作用。
2.2痛刺激坐骨神经及电针对CCI动物自发放电的影响
分析在CCI大鼠上记录海马痛兴奋神经元(n=14)可以观察到,在单纯痛刺激后海马神经元放电立刻显著增加(204.24%±95.07),与痛刺激加电针组(203.34%±127.56)无显著差异(P>0.05)；在痛刺激后2min增加率达到了痛刺激前的193.54%±80.45,并且明显高于痛刺激加电针组(133.41%±43.19)比较(P<0.05)；在痛刺激后6min,单纯痛刺激组仍然处于放电频率增多(188.82%±60.45),而与痛刺激加电针组有显著的差异(P<0.05),痛刺激加电针组已经基本恢复到痛刺激前的放电频率(113.6%±64.66),；在痛刺激后8min和10min,单纯痛刺激组的放电频率仍然明显多于痛刺激前,分别为167.27%±55.60和148.82%±71.75,痛刺激加电针组基本在痛刺激前的状态(108.81%±43.07,99.17%±27.21),单纯痛刺激组与痛刺激加电针组有显著差异(P<0.05)。
在CCI大鼠上记录到的痛抑制神经元(n=4)分析可以看到,在单纯痛刺激后海马神经元放电立刻显著减少(56.44%±17.35),与痛刺激加电针组(83.21%±36.84)无显著差异(P>0.05)；在痛刺激后2min仍然保持在痛刺激前的59.14%±22.72,并且与痛刺激加电针组(66.54%±27.32)比较无显著差异(P>0.05)；在痛刺激后4min,单纯痛刺激组(67.17%±12.17)和痛刺激加电针组(73.83%±39.81)仍然处于放电频率减少的状态,并且两组无差异(P>0.05)；在痛刺激后6min,单纯痛刺激组减频的影响仍然存在(59.82%±19.14),而痛刺激加电针组放电频率逐渐恢复(94.22%±41.45)；在痛刺激后8min和l0min,单纯痛刺激组的放电频率逐渐恢复分别为70.65%±20.62和90.75%±18.50,痛刺激加电针组基本在痛刺激前的状态(90.75%±18.50,97.50%±11.28),单纯痛刺激组与痛刺激加电针组无显著差异(P>0.05)。
2.3正常大鼠与CCI大鼠痛刺激和电针对痛相关神经元自发放电变化的比较
正常大鼠与CCI大鼠海马CA1痛兴奋神经元自发放电的比较,单纯痛刺激后即可,痛刺激后2min,两组无差异(P>0.05)；痛刺激后4min,6min,8min,CCI大鼠的痛兴奋神经元的自发放电频率明显高于正常组(P<0.05)；痛刺激后10min,两组无明显差异(P>0.05)。两组大鼠海马CA1痛抑制神经元的自发放电在单纯痛刺激即可,痛刺激后2min,4min,6min,8min,10min都有明显的差异(P<0.05),这可能与CCI组痛抑制神经元数量太少有关。
比较正常大鼠和CCI大鼠痛刺激加电针后,痛兴奋神经元和痛抑制神经元的自发放电频率除了痛刺激即刻,痛刺激后各个时间均无显著的差异(P>0.05)。
2.4给予ERK抑制剂U0126后对大鼠针刺的影响
在大鼠海马部位记录到的痛兴奋神经元(n=9)自发放电的变化,我们可以观察到,在痛刺激后即可,U0126组(128.72%±47.36)与单纯电针组(126.1%±20.05)的放电无差异(P>0.05),在给药后即可即刺激后2min,U0126组的神经元自发放电(190.52%±49.01)明显高于单纯电针组的自发放电(121.76%±11.41,P<0.05),在痛刺激后4min,6min,8min, U0126组的神经元自发放电均明显高于单纯电针组(P<0.05),而在痛刺激后10min,U0126组的神经元自发放电(118.34%±55.03)仍高于单纯电针组(97.65%±15.4),但无明显差异(P>0.05)。在大鼠海马部位记录到的痛抑制神经元(n=6)自发放电的变化,我们可以观察到,在痛刺激后即刻,U0126组(100.6%±47.22)与单纯电针组(93.35%±15.83)的放电无差异(P>0.05),在给药后即刻即痛刺激后2min,U0126组的神经元自发放电(55.6%±29.67)低于单纯电针组的自发放电(74.36%±16.62),但无明显差异(P>0.05)。在痛刺激后4min,U0126组的神经元白发放电(49.67%±19.79)明显低于单纯电针组(87.37%±9.25,P<0.05),在痛刺激后6min,8min, U0126组的神经元自发放电(45.17%±21.05,42.9%±15.92)仍然明显低于单纯电针组(100.59%±23.28,98.06%±15.92,P<0.05)。在痛刺激后10min,U0126组的自发放电(40.43%±23.37)仍然没有恢复至痛刺激前状态,仍然明显低于单纯电针组(103.45%±9.69,P<0.05)。
3、电针镇痛对CCI大鼠海马ERK信号通路的影响
3.1电针对BDNF及其受体表达的影响
海马的BDNF蛋白在CCI模型组(0.12±0.02)表达显著低于正常对照组(0.40±0.06,p<0.05)。与CCI模型组比,海马CCI+EA2D (0.25±0.02), CCI+EAIW(0.24±0.03)及CCI+EA2W组(0.35±0.02)的BDNF表达均显著高于CCI组(P<0.05),其中CCI+EA2D, CCI+EAIW之间BDNF的表达无显著差异(P>0.05),而CCI+EA2W组的BDNF表达明显高于CCI+EA2D和CCI+EA1W组(p<0.05),显示出两周电针有累积效应；与正常对照组(0.84±0.2)比较,CCI组(0.17±0.3)海马trk mRNA的相对表达量显著降低(p<0.05),电针2天(0.13±0.08)、电针1周(0.15±0.06)和电针2周(0.36±0.11)后,海马trkmRNA的相对表达量与CCI对照组比较没有显著差异(P>0.05)。
3.2电针对MEK/ERK表达的影响
3.2.1电针对各组大鼠Ras、Raf和MEK蛋白的影响
CCI组海马组织Ras蛋白(0.74±0.25)和C-Raf蛋白(0.84±0.15)的表达水平均明显的低于正常对照组(1.25±0.38,1.15±0.09,p<0.05)。与CCI模型组比,海马Ras蛋白和c-Raf蛋白的表达在CCI+EA2D (0.89±0.14,0.89±0.18), CCI+EAIW (1±0.13,0.97±0.3)略有升高,但都没有统计学意义(P>0.5)。与CCI组比较,CCI+EA2W组在海马Ras蛋白(1.35±0.31)和c-Raf蛋白(1.12±0.24)的表达明显上调(p<0.05)。CCI+EAIW组与CCI+EA2D组比较,海马组织的Ras蛋白和c-Raf蛋白的表达没有显著差异(P>0.05)。海马组织Ras蛋白的表达在CCI+EA2W组明显高于CCI+EA2D和CCI+EAIW组(P<0.05),而c-Raf蛋白在CCI+EA2W组的表达与CCI+EA2D和CCI+EAIW组没有显著的差异(P>0.05)
而MEK蛋白在海马组织的表达,在正常对照组(1.08±0.07)和CCI模型组(0.99±0.11),都没有明显的差异(P>0.05)；不同的电针时间,电针2天(0.98±0.12)、电针1周(1.02±0.15)和电针2周(1.09±0.09)各组对MEK蛋白也没有明显影响(P>0.05)。
3.2.2电针对各组大鼠ERK1/2及p-ERK1/2
CCI模型组ERKl/2mRNA与ERK1/2蛋白在海马组织的表达与正常对照组比较,均没有显著的差异P>0.05)而电针2天、电针1周与CCI模型组比较,ERK1/2mRNA与ERK1/2蛋白在海马组织的表达均没有显著差异(P>0.05)；CCI+EA2W组与CCI组,CCI+EA2D组,CCI+EA1W组比较,ERKl/2mRNA和ERK2蛋白的表达均没有显著的改变(P>0.05)；仅海马ERK1蛋白在CCI+EA2W组的表达高于CCI组(P<0.05)。与对照组(1.1±0.06)比较,CCI组海马组织磷酸化ERK蛋白(p-ERK)(0.43±0.06)表达水平均显著降低(p<0.05)。与CCI模型组比,海马p-ERK蛋白表达在CCI+EA2D组(0.63±0.03)明显升高(p<0.05)；与CCI+EA2D, CCI+EA1W(0.07±0.05)海马p-ERK蛋白表达没有明显的差异(P>0.05),但CCI+EA1W组明显高于CCI组(p<0.05)；CCI+EA2W海马p-ERK的表达(0.83±0.45)明显高于CCI组和CCI+EA2D组(p<0.05),但与CCI+EA1W组无明显的差异(P>0.05)。这说明针刺在上调p-ERK的表达具有一定的累积性。
3.3累积电针对各组大鼠p38及p-p38表达的影响
与正常对照组比较,海马组织p38MAPKmRNA的表达在CCI组、CCI+EA2D组和CCI+EA1W组都有显著降低(p<0.05)；电针2W组p38MAPKmRNA的表达与CCI,CCI+EA2D组比较都有明显的上调(p<0.05),但仍明显低于正常对照组(p<0.05)。但是在蛋白的表达与mRNA的表达不是非常一致。与正常对照组比较,海马组织P38MAPK蛋白在CCI组略有降低(p>0.05)；电针各组海马组织p38MAPK蛋白的表达量也没有明显影响(P>0.05)。
与正常对照组比较,海马组织的p-p38MAPK蛋白的表达在CCI模型组略有下降,但差异不显著,(p>0.05); p-p38MAPK蛋白的表达在CCI+EA2D和CCI+EA1W组都略有升高(p>0.05), p-p38MAPK蛋白在CCI+EA2W组与CCI组比较有较明显的上调(P<0.05)。
3.4电针镇痛对CCI大鼠海马PKA和CREB的影响
海马PKA mRNA在CCI模型组3.30±1.37的表达明显高于正常对照组(1.62±0.50,P<0.05)；与CCI模型组比较,CCI+EA2D组PKA mRNA的表达(4.65±0.79)明显上调(P<0.05)；CCI+EAIW组(4.30±0.49,P<0.05)PKA mRNA的表达明显高于正常对照组和CCI模型组(P<0.05),略低于CCI+EA2D组,但无统计学意义(P>0.05)；而CCI+EA2W组(3.38±0.73)PKAmRNA的明显低于CCI+EA2D和CCI+EAIW组(P<0.05),但仍明显高于正常对照组(P<0.05)。可以看到电针对PKA mRNA的调节作用呈现先上升后下调的作用。
与正常对照组(23.98±12.94)比较,CCI模型组(59.77±27.35)海马部位的CREB mRNA表达明显上调P<0.05)海马部位的CREB mRNA表达在CCI+EA2D组(44.25±24.64)明显高于正常对照组(P<0.05),与CCI组比较,略低但无统计学意义(P>0.05)；与CCI组比较,CCI+EAIW组(32.46±16.91)和CCI+EA2W组(26.85±15.27),海马部位的CREB mRNA表达量明显降低(P<0.05),而与CCI+EA2D组比较,CCI+EA1W组和CCI+EA2W组海马部位的CREB mRNA表达量略有降低,但无统计学意义(P>0.05)；CCI+EA2W组与CCI+EAIW组,CREB mRNA表达没有显著差异(P>0.05)。以上结果说明电针1周和电针2周明显累积性下调了由于CCI导致的已经升高的CREBmRNA。
与正常对照组比较,海马组织CREB和p-CREB蛋白在CCI组略有降低(p>0.05)；电针2天、1周、2周都有上调,但均无统计学意义(P>0.05)。
结论
本研究从行为学、电生理和分子生物学的角度探讨了电针镇痛与海马神经元可塑性变化及海马神经元细胞内ERK信号通路的关系。在CCI模型下,大鼠海马神经元的电活动发生异常的变化,电针可以缓解热辐射痛敏的状态,且这种缓解作用与电针时间呈正比。电针的镇痛作用可能与海马内神经元的ERK信号通路的活性有关,电针可能通过激活ERK信号通路来发挥镇痛的目的。
Objective
Chronic pain is a long-time stimulation and brings great suffering to the patient. There was lot of attention on the analgesia that to release the pain. Hippocampus which not only has the function of learning and memory, but also play a central role in the regulation of pain is a part of the limbic system. Hippocampus involved in the process of acupuncture analgesia. The acupuncture accumulative analgesia has relationship with Ach, orphanin and the synaptic changes in hippocampus. To further study the effect of the acupuncture analgesia on the hippocampus, we observed the changes in discharge frequency stimulation of hippocampal neurons between the normal rats and the chronic constrictive injury (CCI) rats after trains of the electric impulses applied to the left sciatic nerve and electroacupuncture, and the relationship between the cumulative electroacupuncture and the expression of BDNF, MEK/ERK signal pathway and PKA, CREB in hippocampus.
Methods:
108Wistar rats were randomly divided into normal control(n=31), CCI (n=29), CCI+EA2D(n=16), CCI+EA1W(n=16), CCI+EA2W(n=16). The neuropathic pain model was established by sciatic nerve ligation(CCI). EA(2/15Hz,1-2m A,30min) was applied to "Zusanli"(ST36)-"Yanglingquan"(GB34) for30min, once daily for2days,1week and2weeks, respectively. The thermal pain threshold(PT) of the bilateral paws was detected the next morning after EA. Train of the electric impulses applied to the left sciatic nerve were used as noxious stimuli, discharges of hippocampal neurons were registered by extracellular recording method with metal electrode. The expression of trk2mRNA, PKAmRNA and CREBmRNA in hippocampus were detected by real-time PCR, and hippocampal Raf, MEK, p-ERK1/2, P38, CREB expression were assayed by western blot.
Results:
1The influence of cumulative electroacupuncture on changes of behavioral performance in each groups
1.1The behavioral performance of CCI
The CCI rats spontaneously lifted the surgical limb (left hind limb), curled and retracted the toes24h after operated. In the4days after operated, we observed some behavior abnormalities of eversion of foot, the limb claudication, avoidance a heavy burden on the limb, but not a drag. The heat radiating pain was caused by a slight thermal radiation. The CCI rats had a long time licks and shakes of the limb, but not self-mutilation.
1.2Comparison of the thermal pain threshold(PT) in rats of different groups
In comparison with the normal control group, the PT in CCI rats was increased considerably in4days after operated (P<0.05).In18days after operated, the PT of CCI group was still higher than the normal control group (P<0.05).In the CCI+EA2D group, the PT was higher than the normal control group (P<0.05) and lower than the CCI group (P<0.05). Compare with the CCI and CCI+EA2W group, the RT of CCI+EA1W was down-regulated considerably (P<0.05). The PT of CCI+EA2W group was significant lower than CCI、 CCI+EA2D group (P<0.05). Comparison between CCI+EA2W and CCI groups showed that there was no difference(P>0.05), suggesting a cumulative analgesic effect of repeated EA
2The effect of electroacupuncture analgesia on discharge of hippocampal pain-relation neurons(PEN)
2.1The effect of noxious stimuli and electroacupuncture on neuronal discharge in the normal control group
The discharge frequency of hippocampal PEN (n=17) were increased significantly in the noxious stimuli (134.53%±37) and EA group (126.1%±20.05) after given a noxious stimuli (P<0.05) in normal control rats and there was on difference between the two groups (P>0.05). The discharge frequency of hippocampal PEN in noxious stimuli group were markedly higher than the EA group at2,4,6min after given the noxious stimuli (P<0.05). No significant differences were found between the noxious stimuli group and the EA group at8and10min after given the noxious stimuli(P>0.05).It suggested EA played an inhibited role in regulating the discharge of PEN after given the noxious stimuli.
The discharge frequency of hippocampal PIN (n=12) were decreased significantly in the noxious stimuli (67.88%±14.71) and EA group (93.35%±15.83) after given a noxious stimuli in normal control rats.The frequency of the noxious stimuli group was evidently lower than EA group (P<0.05). In comparison with the noxious stimuli group, the frequency of EA group were increased considerably (P<0.05) at2,4,6,8,10min after given the noxious stimuli.It suggested EA played an excited role in regulating the discharge of PIN after given the noxious stimuli.
2.2The effect of noxious stimuli and electroacupuncture on neuronal discharge in CCI control group
The discharge frequency of hippocampal PEN (n=14) were increased significantly in the noxious stimuli (204.24%±95.07) and EA group (203.34%±127.56) after given a noxious stimuli in CCI rats and there was on difference between the two groups(P>0.05). The frequency of hippocampal PEN was reached193.54%±80.45in noxious stimuli group and markedly higher than EA group (133.41%±43.19, P<0.05).The frequency of hippocampal PEN in EA group (113.6%+64.66) recovered to the normal levels, and observably lower than noxious stimuli group (188.82%±60.45, P<0.05) at6min after given the noxious stimuli.
The discharge frequency of hippocampal PIN (n=4) were decreased significantly in the noxious stimuli66.44%±17.35) and EA group (83.21%±36.84) after given a noxious stimuli (P<0.05) in CCI rats and there was on difference between the two groups(P>0.05). There was no difference between the two groups at2,4min after the noxious stimuli(P>0.05). At6min after the noxious stimuli, the frequency of hippocampal PIN in the noxious stimuli group (59.82%±19.14) was still inhibited and the frequency of EA group (94.22%±41.45) recovered to the normal levels. At8and10min after the noxious stimuli, the frequency of hippocampal PIN reached to70.65%±20.62和90.75%±18.50in noxious stimuli group. The discharge frequency of noxious stimuli group and EA group had no significant changes(P>0.05).
2.3Comparison of the discharges of the normal control rats and CCI rats after noxious stimuli and electroacupuncture
At2min after the noxious stimuli, there was no difference between the normal control group and the CCI group (P>0.05);4min,6min,8min after noxious stimuli, the discharges of PEN in CCI group markedly higher than the normal control group0.05) at10min after noxious stimuli. Except for the moment of given a noxious stimuli,2,4,6,8,10min after noxious stimuli, there were remarkable difference between the two group (P<0.05)
The discharge frequency of normal control group and the CCI group had no had no significant changes (P)0.05), except for the moment of given a noxious stimuli.
2.3The effect of inhibitor of ERK U0126on the changes of discharge induced by electroacupuncture in rats
The discharge frequency of hippocampal PEN of U0126(n=9) group and EA group had no significant changes (P>0.05) after given a noxious stimuli.The frequency of hippocampal PEN in the U0126group (190.52%±49.01) was higher than the EA group (121.76%±11.41, P<0.05) at2min after the noxious stimuli(after injection the U0126).At4,6,8min after the noxious stimuli, the discharge of the U0126group were still higher than the EA group(P<0.05). There were no difference between the U0126group and the EA group at10min after noxious stimuli (P>0.05).
The discharge frequency of hippocampal PIN of U0126(n=6) group and EA group had no significant changes (P>0.05) after given a noxious stimuli At the moment of injection of U0126(2min after noxious stimuli), the U0126group (55.6%±29.67) was lower than the EA group (74.36%±16.62, P>0.05). At4,6,8,10min after noxious stimuli, the U0126group were significantly lower than the EA group(P<0.05)
3The effect of electroacupuncture analgesia on hippocampal MEK/ERK signal pathway in different groups
3.1The effect of EA on hippocampal BDNF and trk2expression Compare with the normal control group, the expression level of hippocampal BDNF in CCI group was decreased considerably (P<0.05).In comparison with the CCI group, the hippocampal BDNF expression in the CCI+EA2D, CCI+EA1W and CCI+EA2W group was upregulated considerably (P<0.05). The expression of hippocampal BDNF in CCI+EA2W was higher than the CCI+EA2D and CCI+EA1W group(P<0.05), suggesting a cumulative effect of repeated EA in up-regulating hippocampal BDNF expression. NO significant difference were found between CCI+EA2D and CCI+EA1W groups in hippocampal BDNF(P>0.05). Compared with normal control group, the expression level of hippocampal trkmRNA in CCI group was decreased significantly (P<0.05). Compare with the CCI group, hippocampal trkmRNA expression in CCI+EA2D, CCI+EA1W and CCI+EA2W group had no significant changes(P>0.05).
3.2The effect of EA on hippocampal MER/ERK signal pathway
3.2.1The effect of EA on hippocampal Ras-. C-Raf and MEK expression Compared with the normal control group, the expression of hippocampal Ras in CCI group was decreased considerably(P<0.05). Compared with the CCI group, hippocampal Ras expression was up-regulated slightly in CCI+EA2D and CCI+EA1W(P>0.05), significantly in CCI+EA2W (P<0.05). In comparison with the CCI+EA2D and CCI+EA1W group, hippocampal Ras expression in CCI+EA2W group was up-regulated considerably(P<0.05).
Compared with the normal control group, the expression level of hippocampal C-Raf was down-regulated markedly (P<0.05). In comparison with the CCI group, the hippocampal C-Raf expression in CCI+EA2D and CCI+EA1W group was increased slightly(P>0.05), and in CCI+EA2W was increased considerably(P<0.05).
Compared with the normal group, the expression level of hippocampal MEK in CCI group had no significant changes(P>0.05). No significant differences were found between CCI, CCI+EA2D, CCI+EA1W and CCI+EA2W group(P>0.05).
3.2.2The effect of EA on hippocampal ERK1/2expression
Compared with the normal control group, the expression level of hippocampal ERK1/2mRNA and ERK1/2in CCI group had no significant changes(P>0.05). No significant differences were found between those groups in hippocampal ERK1/2mRNA and ERK1/2expression (P>0.05) expect the ERK1expression in CCI+EA2W group was higher than in CCI group (P<0.05). In comparison with the normal control group, the hippocampal p-ERK1/2expression in CCI group were down-regulated considerably (P<0.05). Compared with the CCI group, hippocampal p-ERK1/2expression in CCI+EA2D and CCI+EA1W group were increased significantly (P<0.05). The expression level of hippocampal p-ERK1/2in CCI+EA2W group were higher than in CCI group and in CCI+EA2D group(P<0.05). There was no significant difference between CCI+EA1W and CCI+EA2W groups in hippocampal p-ERK1/2expression(P>0.05). It suggested a cumulative effect of repeated EA in up-regulating hippocampal p-ERK1/2expression.
3.3Effect of EA on p38and p-p38expression in the hippocampus
Compared with the normal control group, the p38MAPKmRNA expression of hippocampus in CCI group, CCI+EA2D group and CCI+EA1W group were decreased considerably (P<0.05). The expression level of hippocampal p38MAPKmRNA was markedly higher than those in CCI group and CCI+EA2D group (P<0.05), and markedly lower than that in the normal control group(P<0.05). The expression level of hippocampal p38MAPK had no significant changes between the different groups (P>0.05).
In comparison with the normal control group, the expression of hippocampal p-p38MAPK in CCI group was decreased slightly (P<0.05). Comparison between CCI group and CCI+EA2W group showed that the expression level of hippocampal p-p38MAPK was significantly lower in the former group than in the latter group (P<0.05), suggesting a cumulative effect of repeated EA in up-regulating hippocampal p-p38MAPK expression.
3.4Effect of EA on hippocampal PKA mRNA and CREB mRNA, CREB expression Compared with the normal control group, the expression of hippocampal PKA mRNA in CCI group was increased considerably (P<0.05). Compared with the CCI group, the hippocampal PKA mRNA expression in CCI+EA2D and CCI+EA1W group were significantly up-regulated (P<0.05). The expression of hippocampal PKA mRNA in CCI+EA2W was lower markedly than that of in CCI+EA2D and CCI+EA1W group(P<0.05).
Compared with the normal control group, the expression of hippocampal CREB mRNA in CCI and CCI+EA2D group was increased considerably (P<0.05).In comparison with the CCI group, the hippocampal CREB mRNA expression in CCI+EA1W and CCI+EA2W group were decreased markedly (P<0.05), while comapaed with the CCI+EA2D group, the hippocampal CREB mRNA expression in CCI+EA1W and CCI+EA2W group were decreased lightly (P>0.05).It suggested a cumulative effect of repeated EA for1weeks and2weeks in down-regulating the hippocampal CREB mRNA increase expression caused by CCI. However, there was no significant changes between those different groups in the expression level of hippocampal CREB and p-CREB.
Conclusion
The present study is representing behavioral, electrophysiological and molecular evidence to observe the relationship between electroacupuncture analgesia and hippocampal neuron plasticity changes and hippocampal ERK signaling pathway. The electrical activity of hippocampal pain-relation neurons had abnormal changes in the CCI model. There was a cumulative effect of repeated EA in relieving the state of the thermal radiation hyperalgesia. The effect of EA analgesic had close relationship with the hippocampal ERK signaling pathway.
慢性疼痛包括慢性神经病理性疼痛是临床上常见的病症,目前采用西医药治疗的效果有限。针刺治疗慢性痛具有较好的疗效,但是其作用机制还不太清楚。海马作为边缘系统的一部分,除参与学习记忆、情绪活动等等的功能外,还参与疼痛信息的加工处理和针刺镇痛的过程。我们前期在慢性坐骨神经结扎造成的神经痛(CCI)大鼠模型上的实验结果表明,重复电针产生累积镇痛效应的同时,可显著改善CCI大鼠海马CA3区神经元突触间隙、突触厚膜厚度、突触活性带长度明显减小的变化,上调海马镇痛物质乙酰胆碱及其M受体、γ-氨基丁酸受体等基因的表达变化密切相关。但是,针刺镇痛累积效应的细胞内分子机制尚不清楚。
慢性神经病理性疼痛是神经可塑性的一种表达方式。神经损伤产生的持续性剧烈的伤害性刺激传入导致中枢敏化,该敏化反应是活动-依赖性的功能可塑性的变化；在细胞水平上产生于细胞内各种各样的激酶级联的激活进和许多关键细胞膜受体、离子通道的磷酸化等的变化。MAPK(mitogen-activated protein kinase)/ERK (Extracellular signal-regulated kinases)级联是细胞内一个主要的生化信号通路,参与启动和调节细胞外的各种刺激信息引发的细胞内许多信号通路(蛋白分子转化)加工处理过程,包括神经的可塑性、学习记忆等等,特别是,它还是痛觉过敏中枢敏化过程中细胞内多个信号通路调节的总开关(master switch)。此外,在中枢神经系统中,神经递质和神经营养因子(脑源性神经生长因子brain derived neurophic factor, BDNF等)信号通路之间的相互作用(interplay)可调节中枢神经网络对疼痛过敏条件下环境变化的适应性反应。为此,本研究在CCI大鼠模型上,采用行为学、电生理学、分子生物学等技术,进一步研究针刺累积镇痛效应与海马BDNF/MAPK/ERK/CREB信号通路活动变化的关系。
研究方法
实验一,行为学实验：Wistar雄性大鼠80只,随机分为正常对照组(n=16),慢性压迫性损伤(CCI)组(n=16),CCI+EA2天(D)(n=16),CCI+EAl周(W)(n=16),CCI+EA2W (n=16).采用坐骨神经四道结扎造成CCI神经痛模型,电针双侧“足三里”-“阳陵泉”穴,采用2/15Hz,1-2mA,每天1次,分别为不电针、电针2天、电针1周和电针2周,用热辐射测痛仪测量热痛阈,以手术侧和假手术侧的痛阈值的差值作为痛敏分数来观察动物的痛阈的变化。
实验二,生理学实验：Wistar雄性大鼠43只,随机分为正常对照组(n=15),CCI组(n=13),U0126组(n=15),用脑立体定位仪固定大鼠头部,刺激坐骨神经作为伤害性刺激,刺激脉冲(间隔50ms,波宽0.3ms,电流强度5mA),刺激10s,将微量注射器定位于侧脑室(前囟后1.0mm,旁开1.3-2.0mm,深3.5mm)注射ERK抑制剂U0126,电极尖端定位于海马CA1区(前囟后3.2-3.6mm,旁开2.5-3.0mm,深2.5-3.1mm)记录各组大鼠海马CA1痛相关神经元的自发放电变化,以及电针和U026对自发放电的影响作用。
实验三,分子生物学实验：上述行为学动物,正常对照组(n=8),CCI组(n=8),CCI+EA2D (n=8), CCI+EA1W (n=8), CCI+EA2W (n=8),应用real-time PCR方法检测大鼠海马部位BDNF, trk2PKA和pCREB的mRNA表达的变化；正常对照组(n=8), CCI组(n=8), CCI+EA2D (n=8), CCI+EA1W (n=8), CCI+EA2W (n=8),应用western blot方法检测大鼠海马部位Ras、Raf、MEK、p-ERK1/2、P38、CREB等的蛋白的表达变化。
实验结果
1、累积电针对各组大鼠行为学表现的影响
1.1CCI大鼠行为学表现
术后24小时即可见手术组大鼠,手术侧肢体(左侧后肢)自发性抬起,足趾蜷曲、缩回、足掌极少平伸着地。手术后4d,可以观察到有明显的外翻和避免患肢着地,明显的患肢跛行等的行为异常,但没有出现托行。热辐射轻微照射造成热辐射痛,大鼠即长时间舔足,不停甩足,但没有出现自残现象。
1.2累积电针对CCI大鼠的热辐射痛阈的影响
CCI大鼠在术后4天(3.15±1.05),痛敏分数明显高于正常对照组(-0.063±0.23)(P<0.05)；在术后18天,也就是取材前,CCI对照组痛敏分数(3.69±0.24)仍然明显高于正常对照组(0.011±0.24,P<0.05);CCI+EA2D组痛敏分数(3.01±1.65)明显高于正常对照组,明显低于CCI对照组(P<0.05):CCI+EA1W组痛敏分数(2.34±1.85)明显高于正常对照组(P<0.05),明显低于CCI对照组和CCI+EA2W组(P<0.05)：CCI+EA2W痛敏分数(0.98±0.1)明显低于CCI对照组、CCI+EA2D组(P<0.05),与正常对照比较没有显著差异(P>0.05),说明电针具有明显镇痛效应,且这种镇痛效应具有明显的累积性。
2、电针镇痛对大鼠海马痛相关神经元自发放电的影响
2.1痛刺激坐骨神经及电针对正常动物自发放电的影响
对正常大鼠海马CAl区痛兴奋性神经元(n=17)分析可以看到,在痛刺激后单纯痛刺激组放电频率(134.53%±37)和痛刺激加电针组放电频率(126.1%±20.05)都有明显的增加,且单纯痛刺激组与痛刺激加电针组没有差异(P>0.05)；在痛刺激后2min,单纯痛刺激组的放电频率(168.61%±58.72)明显多于痛刺激加电针组的放电频率(121.76%±21.67,P<0.05)；在痛刺激后4min,单纯痛刺激组放电频率(145.08%±21.67)明显多于痛刺激加电针组的放电频率(108.65%±14.16,P<0.05),痛刺激加电针组已经基本恢复至痛刺激前水平。在痛刺激后6min单纯痛刺激组的放电频率仍然处于增加的水平(125.87%±23.92),明显高于痛刺激加电针组的放电频率(105.72%±22.49,P<0.05)；在痛刺激后8min,10min,单纯痛刺激组的放电频率已接近恢复正常水平(116.86%±25.97,103.22%±6.03),与痛刺激加电针组(102.14%±22.58,97.65%±14.4)相比,没有明显的差异(P>0.05),但电针组的放电频率仍然低于单纯痛刺激组。这说明电针有抑制痛兴奋神经元自发放电增加的作用。
对正常大鼠痛抑制神经元(n=12)分析可以看到,在痛刺激完即刻,单纯痛刺激组放电频率减少(67.88%±14.71),电针加痛刺激组放电频率为痛刺激前的(93.35%±15.83),两组有显著差异(P<0.05)；痛刺激后2min,单纯痛刺激组的放电频率(54.35%±19.37)明显低于电针加痛刺激组(74.36%±16.62)；痛刺激后4min,单纯痛刺激组的放电频率(53.01%±19.37)仍然明显低于电针加痛刺激组(87.37%±9.25,P<0.05)；从痛刺激后6min开始,单纯痛刺激组的放电频率开始增加(67.93%±26.47),慢慢恢复,至10min,已经基本恢复至痛刺激前的放电频率(95.06%±15.92),而痛刺激加电针组痛刺激后6min已经恢复至痛刺激前的放电频率(100.59%±23.28),在痛刺激后8min和l0min的放电频率均与痛刺激前的放电频率一致。而单纯痛刺激组和痛刺激加电针组两组在痛刺激后6min,8min,10min均有明显的差异(P<0.05)。电针镇痛有解除痛抑制神经元放电减少的作用。
2.2痛刺激坐骨神经及电针对CCI动物自发放电的影响
分析在CCI大鼠上记录海马痛兴奋神经元(n=14)可以观察到,在单纯痛刺激后海马神经元放电立刻显著增加(204.24%±95.07),与痛刺激加电针组(203.34%±127.56)无显著差异(P>0.05)；在痛刺激后2min增加率达到了痛刺激前的193.54%±80.45,并且明显高于痛刺激加电针组(133.41%±43.19)比较(P<0.05)；在痛刺激后6min,单纯痛刺激组仍然处于放电频率增多(188.82%±60.45),而与痛刺激加电针组有显著的差异(P<0.05),痛刺激加电针组已经基本恢复到痛刺激前的放电频率(113.6%±64.66),；在痛刺激后8min和10min,单纯痛刺激组的放电频率仍然明显多于痛刺激前,分别为167.27%±55.60和148.82%±71.75,痛刺激加电针组基本在痛刺激前的状态(108.81%±43.07,99.17%±27.21),单纯痛刺激组与痛刺激加电针组有显著差异(P<0.05)。
在CCI大鼠上记录到的痛抑制神经元(n=4)分析可以看到,在单纯痛刺激后海马神经元放电立刻显著减少(56.44%±17.35),与痛刺激加电针组(83.21%±36.84)无显著差异(P>0.05)；在痛刺激后2min仍然保持在痛刺激前的59.14%±22.72,并且与痛刺激加电针组(66.54%±27.32)比较无显著差异(P>0.05)；在痛刺激后4min,单纯痛刺激组(67.17%±12.17)和痛刺激加电针组(73.83%±39.81)仍然处于放电频率减少的状态,并且两组无差异(P>0.05)；在痛刺激后6min,单纯痛刺激组减频的影响仍然存在(59.82%±19.14),而痛刺激加电针组放电频率逐渐恢复(94.22%±41.45)；在痛刺激后8min和l0min,单纯痛刺激组的放电频率逐渐恢复分别为70.65%±20.62和90.75%±18.50,痛刺激加电针组基本在痛刺激前的状态(90.75%±18.50,97.50%±11.28),单纯痛刺激组与痛刺激加电针组无显著差异(P>0.05)。
2.3正常大鼠与CCI大鼠痛刺激和电针对痛相关神经元自发放电变化的比较
正常大鼠与CCI大鼠海马CA1痛兴奋神经元自发放电的比较,单纯痛刺激后即可,痛刺激后2min,两组无差异(P>0.05)；痛刺激后4min,6min,8min,CCI大鼠的痛兴奋神经元的自发放电频率明显高于正常组(P<0.05)；痛刺激后10min,两组无明显差异(P>0.05)。两组大鼠海马CA1痛抑制神经元的自发放电在单纯痛刺激即可,痛刺激后2min,4min,6min,8min,10min都有明显的差异(P<0.05),这可能与CCI组痛抑制神经元数量太少有关。
比较正常大鼠和CCI大鼠痛刺激加电针后,痛兴奋神经元和痛抑制神经元的自发放电频率除了痛刺激即刻,痛刺激后各个时间均无显著的差异(P>0.05)。
2.4给予ERK抑制剂U0126后对大鼠针刺的影响
在大鼠海马部位记录到的痛兴奋神经元(n=9)自发放电的变化,我们可以观察到,在痛刺激后即可,U0126组(128.72%±47.36)与单纯电针组(126.1%±20.05)的放电无差异(P>0.05),在给药后即可即刺激后2min,U0126组的神经元自发放电(190.52%±49.01)明显高于单纯电针组的自发放电(121.76%±11.41,P<0.05),在痛刺激后4min,6min,8min, U0126组的神经元自发放电均明显高于单纯电针组(P<0.05),而在痛刺激后10min,U0126组的神经元自发放电(118.34%±55.03)仍高于单纯电针组(97.65%±15.4),但无明显差异(P>0.05)。在大鼠海马部位记录到的痛抑制神经元(n=6)自发放电的变化,我们可以观察到,在痛刺激后即刻,U0126组(100.6%±47.22)与单纯电针组(93.35%±15.83)的放电无差异(P>0.05),在给药后即刻即痛刺激后2min,U0126组的神经元自发放电(55.6%±29.67)低于单纯电针组的自发放电(74.36%±16.62),但无明显差异(P>0.05)。在痛刺激后4min,U0126组的神经元白发放电(49.67%±19.79)明显低于单纯电针组(87.37%±9.25,P<0.05),在痛刺激后6min,8min, U0126组的神经元自发放电(45.17%±21.05,42.9%±15.92)仍然明显低于单纯电针组(100.59%±23.28,98.06%±15.92,P<0.05)。在痛刺激后10min,U0126组的自发放电(40.43%±23.37)仍然没有恢复至痛刺激前状态,仍然明显低于单纯电针组(103.45%±9.69,P<0.05)。
3、电针镇痛对CCI大鼠海马ERK信号通路的影响
3.1电针对BDNF及其受体表达的影响
海马的BDNF蛋白在CCI模型组(0.12±0.02)表达显著低于正常对照组(0.40±0.06,p<0.05)。与CCI模型组比,海马CCI+EA2D (0.25±0.02), CCI+EAIW(0.24±0.03)及CCI+EA2W组(0.35±0.02)的BDNF表达均显著高于CCI组(P<0.05),其中CCI+EA2D, CCI+EAIW之间BDNF的表达无显著差异(P>0.05),而CCI+EA2W组的BDNF表达明显高于CCI+EA2D和CCI+EA1W组(p<0.05),显示出两周电针有累积效应；与正常对照组(0.84±0.2)比较,CCI组(0.17±0.3)海马trk mRNA的相对表达量显著降低(p<0.05),电针2天(0.13±0.08)、电针1周(0.15±0.06)和电针2周(0.36±0.11)后,海马trkmRNA的相对表达量与CCI对照组比较没有显著差异(P>0.05)。
3.2电针对MEK/ERK表达的影响
3.2.1电针对各组大鼠Ras、Raf和MEK蛋白的影响
CCI组海马组织Ras蛋白(0.74±0.25)和C-Raf蛋白(0.84±0.15)的表达水平均明显的低于正常对照组(1.25±0.38,1.15±0.09,p<0.05)。与CCI模型组比,海马Ras蛋白和c-Raf蛋白的表达在CCI+EA2D (0.89±0.14,0.89±0.18), CCI+EAIW (1±0.13,0.97±0.3)略有升高,但都没有统计学意义(P>0.5)。与CCI组比较,CCI+EA2W组在海马Ras蛋白(1.35±0.31)和c-Raf蛋白(1.12±0.24)的表达明显上调(p<0.05)。CCI+EAIW组与CCI+EA2D组比较,海马组织的Ras蛋白和c-Raf蛋白的表达没有显著差异(P>0.05)。海马组织Ras蛋白的表达在CCI+EA2W组明显高于CCI+EA2D和CCI+EAIW组(P<0.05),而c-Raf蛋白在CCI+EA2W组的表达与CCI+EA2D和CCI+EAIW组没有显著的差异(P>0.05)
而MEK蛋白在海马组织的表达,在正常对照组(1.08±0.07)和CCI模型组(0.99±0.11),都没有明显的差异(P>0.05)；不同的电针时间,电针2天(0.98±0.12)、电针1周(1.02±0.15)和电针2周(1.09±0.09)各组对MEK蛋白也没有明显影响(P>0.05)。
3.2.2电针对各组大鼠ERK1/2及p-ERK1/2
CCI模型组ERKl/2mRNA与ERK1/2蛋白在海马组织的表达与正常对照组比较,均没有显著的差异P>0.05)而电针2天、电针1周与CCI模型组比较,ERK1/2mRNA与ERK1/2蛋白在海马组织的表达均没有显著差异(P>0.05)；CCI+EA2W组与CCI组,CCI+EA2D组,CCI+EA1W组比较,ERKl/2mRNA和ERK2蛋白的表达均没有显著的改变(P>0.05)；仅海马ERK1蛋白在CCI+EA2W组的表达高于CCI组(P<0.05)。与对照组(1.1±0.06)比较,CCI组海马组织磷酸化ERK蛋白(p-ERK)(0.43±0.06)表达水平均显著降低(p<0.05)。与CCI模型组比,海马p-ERK蛋白表达在CCI+EA2D组(0.63±0.03)明显升高(p<0.05)；与CCI+EA2D, CCI+EA1W(0.07±0.05)海马p-ERK蛋白表达没有明显的差异(P>0.05),但CCI+EA1W组明显高于CCI组(p<0.05)；CCI+EA2W海马p-ERK的表达(0.83±0.45)明显高于CCI组和CCI+EA2D组(p<0.05),但与CCI+EA1W组无明显的差异(P>0.05)。这说明针刺在上调p-ERK的表达具有一定的累积性。
3.3累积电针对各组大鼠p38及p-p38表达的影响
与正常对照组比较,海马组织p38MAPKmRNA的表达在CCI组、CCI+EA2D组和CCI+EA1W组都有显著降低(p<0.05)；电针2W组p38MAPKmRNA的表达与CCI,CCI+EA2D组比较都有明显的上调(p<0.05),但仍明显低于正常对照组(p<0.05)。但是在蛋白的表达与mRNA的表达不是非常一致。与正常对照组比较,海马组织P38MAPK蛋白在CCI组略有降低(p>0.05)；电针各组海马组织p38MAPK蛋白的表达量也没有明显影响(P>0.05)。
与正常对照组比较,海马组织的p-p38MAPK蛋白的表达在CCI模型组略有下降,但差异不显著,(p>0.05); p-p38MAPK蛋白的表达在CCI+EA2D和CCI+EA1W组都略有升高(p>0.05), p-p38MAPK蛋白在CCI+EA2W组与CCI组比较有较明显的上调(P<0.05)。
3.4电针镇痛对CCI大鼠海马PKA和CREB的影响
海马PKA mRNA在CCI模型组3.30±1.37的表达明显高于正常对照组(1.62±0.50,P<0.05)；与CCI模型组比较,CCI+EA2D组PKA mRNA的表达(4.65±0.79)明显上调(P<0.05)；CCI+EAIW组(4.30±0.49,P<0.05)PKA mRNA的表达明显高于正常对照组和CCI模型组(P<0.05),略低于CCI+EA2D组,但无统计学意义(P>0.05)；而CCI+EA2W组(3.38±0.73)PKAmRNA的明显低于CCI+EA2D和CCI+EAIW组(P<0.05),但仍明显高于正常对照组(P<0.05)。可以看到电针对PKA mRNA的调节作用呈现先上升后下调的作用。
与正常对照组(23.98±12.94)比较,CCI模型组(59.77±27.35)海马部位的CREB mRNA表达明显上调P<0.05)海马部位的CREB mRNA表达在CCI+EA2D组(44.25±24.64)明显高于正常对照组(P<0.05),与CCI组比较,略低但无统计学意义(P>0.05)；与CCI组比较,CCI+EAIW组(32.46±16.91)和CCI+EA2W组(26.85±15.27),海马部位的CREB mRNA表达量明显降低(P<0.05),而与CCI+EA2D组比较,CCI+EA1W组和CCI+EA2W组海马部位的CREB mRNA表达量略有降低,但无统计学意义(P>0.05)；CCI+EA2W组与CCI+EAIW组,CREB mRNA表达没有显著差异(P>0.05)。以上结果说明电针1周和电针2周明显累积性下调了由于CCI导致的已经升高的CREBmRNA。
与正常对照组比较,海马组织CREB和p-CREB蛋白在CCI组略有降低(p>0.05)；电针2天、1周、2周都有上调,但均无统计学意义(P>0.05)。
结论
本研究从行为学、电生理和分子生物学的角度探讨了电针镇痛与海马神经元可塑性变化及海马神经元细胞内ERK信号通路的关系。在CCI模型下,大鼠海马神经元的电活动发生异常的变化,电针可以缓解热辐射痛敏的状态,且这种缓解作用与电针时间呈正比。电针的镇痛作用可能与海马内神经元的ERK信号通路的活性有关,电针可能通过激活ERK信号通路来发挥镇痛的目的。
Objective
Chronic pain is a long-time stimulation and brings great suffering to the patient. There was lot of attention on the analgesia that to release the pain. Hippocampus which not only has the function of learning and memory, but also play a central role in the regulation of pain is a part of the limbic system. Hippocampus involved in the process of acupuncture analgesia. The acupuncture accumulative analgesia has relationship with Ach, orphanin and the synaptic changes in hippocampus. To further study the effect of the acupuncture analgesia on the hippocampus, we observed the changes in discharge frequency stimulation of hippocampal neurons between the normal rats and the chronic constrictive injury (CCI) rats after trains of the electric impulses applied to the left sciatic nerve and electroacupuncture, and the relationship between the cumulative electroacupuncture and the expression of BDNF, MEK/ERK signal pathway and PKA, CREB in hippocampus.
Methods:
108Wistar rats were randomly divided into normal control(n=31), CCI (n=29), CCI+EA2D(n=16), CCI+EA1W(n=16), CCI+EA2W(n=16). The neuropathic pain model was established by sciatic nerve ligation(CCI). EA(2/15Hz,1-2m A,30min) was applied to "Zusanli"(ST36)-"Yanglingquan"(GB34) for30min, once daily for2days,1week and2weeks, respectively. The thermal pain threshold(PT) of the bilateral paws was detected the next morning after EA. Train of the electric impulses applied to the left sciatic nerve were used as noxious stimuli, discharges of hippocampal neurons were registered by extracellular recording method with metal electrode. The expression of trk2mRNA, PKAmRNA and CREBmRNA in hippocampus were detected by real-time PCR, and hippocampal Raf, MEK, p-ERK1/2, P38, CREB expression were assayed by western blot.
Results:
1The influence of cumulative electroacupuncture on changes of behavioral performance in each groups
1.1The behavioral performance of CCI
The CCI rats spontaneously lifted the surgical limb (left hind limb), curled and retracted the toes24h after operated. In the4days after operated, we observed some behavior abnormalities of eversion of foot, the limb claudication, avoidance a heavy burden on the limb, but not a drag. The heat radiating pain was caused by a slight thermal radiation. The CCI rats had a long time licks and shakes of the limb, but not self-mutilation.
1.2Comparison of the thermal pain threshold(PT) in rats of different groups
In comparison with the normal control group, the PT in CCI rats was increased considerably in4days after operated (P<0.05).In18days after operated, the PT of CCI group was still higher than the normal control group (P<0.05).In the CCI+EA2D group, the PT was higher than the normal control group (P<0.05) and lower than the CCI group (P<0.05). Compare with the CCI and CCI+EA2W group, the RT of CCI+EA1W was down-regulated considerably (P<0.05). The PT of CCI+EA2W group was significant lower than CCI、 CCI+EA2D group (P<0.05). Comparison between CCI+EA2W and CCI groups showed that there was no difference(P>0.05), suggesting a cumulative analgesic effect of repeated EA
2The effect of electroacupuncture analgesia on discharge of hippocampal pain-relation neurons(PEN)
2.1The effect of noxious stimuli and electroacupuncture on neuronal discharge in the normal control group
The discharge frequency of hippocampal PEN (n=17) were increased significantly in the noxious stimuli (134.53%±37) and EA group (126.1%±20.05) after given a noxious stimuli (P<0.05) in normal control rats and there was on difference between the two groups (P>0.05). The discharge frequency of hippocampal PEN in noxious stimuli group were markedly higher than the EA group at2,4,6min after given the noxious stimuli (P<0.05). No significant differences were found between the noxious stimuli group and the EA group at8and10min after given the noxious stimuli(P>0.05).It suggested EA played an inhibited role in regulating the discharge of PEN after given the noxious stimuli.
The discharge frequency of hippocampal PIN (n=12) were decreased significantly in the noxious stimuli (67.88%±14.71) and EA group (93.35%±15.83) after given a noxious stimuli in normal control rats.The frequency of the noxious stimuli group was evidently lower than EA group (P<0.05). In comparison with the noxious stimuli group, the frequency of EA group were increased considerably (P<0.05) at2,4,6,8,10min after given the noxious stimuli.It suggested EA played an excited role in regulating the discharge of PIN after given the noxious stimuli.
2.2The effect of noxious stimuli and electroacupuncture on neuronal discharge in CCI control group
The discharge frequency of hippocampal PEN (n=14) were increased significantly in the noxious stimuli (204.24%±95.07) and EA group (203.34%±127.56) after given a noxious stimuli in CCI rats and there was on difference between the two groups(P>0.05). The frequency of hippocampal PEN was reached193.54%±80.45in noxious stimuli group and markedly higher than EA group (133.41%±43.19, P<0.05).The frequency of hippocampal PEN in EA group (113.6%+64.66) recovered to the normal levels, and observably lower than noxious stimuli group (188.82%±60.45, P<0.05) at6min after given the noxious stimuli.
The discharge frequency of hippocampal PIN (n=4) were decreased significantly in the noxious stimuli66.44%±17.35) and EA group (83.21%±36.84) after given a noxious stimuli (P<0.05) in CCI rats and there was on difference between the two groups(P>0.05). There was no difference between the two groups at2,4min after the noxious stimuli(P>0.05). At6min after the noxious stimuli, the frequency of hippocampal PIN in the noxious stimuli group (59.82%±19.14) was still inhibited and the frequency of EA group (94.22%±41.45) recovered to the normal levels. At8and10min after the noxious stimuli, the frequency of hippocampal PIN reached to70.65%±20.62和90.75%±18.50in noxious stimuli group. The discharge frequency of noxious stimuli group and EA group had no significant changes(P>0.05).
2.3Comparison of the discharges of the normal control rats and CCI rats after noxious stimuli and electroacupuncture
At2min after the noxious stimuli, there was no difference between the normal control group and the CCI group (P>0.05);4min,6min,8min after noxious stimuli, the discharges of PEN in CCI group markedly higher than the normal control group
The discharge frequency of normal control group and the CCI group had no had no significant changes (P)0.05), except for the moment of given a noxious stimuli.
2.3The effect of inhibitor of ERK U0126on the changes of discharge induced by electroacupuncture in rats
The discharge frequency of hippocampal PEN of U0126(n=9) group and EA group had no significant changes (P>0.05) after given a noxious stimuli.The frequency of hippocampal PEN in the U0126group (190.52%±49.01) was higher than the EA group (121.76%±11.41, P<0.05) at2min after the noxious stimuli(after injection the U0126).At4,6,8min after the noxious stimuli, the discharge of the U0126group were still higher than the EA group(P<0.05). There were no difference between the U0126group and the EA group at10min after noxious stimuli (P>0.05).
The discharge frequency of hippocampal PIN of U0126(n=6) group and EA group had no significant changes (P>0.05) after given a noxious stimuli At the moment of injection of U0126(2min after noxious stimuli), the U0126group (55.6%±29.67) was lower than the EA group (74.36%±16.62, P>0.05). At4,6,8,10min after noxious stimuli, the U0126group were significantly lower than the EA group(P<0.05)
3The effect of electroacupuncture analgesia on hippocampal MEK/ERK signal pathway in different groups
3.1The effect of EA on hippocampal BDNF and trk2expression Compare with the normal control group, the expression level of hippocampal BDNF in CCI group was decreased considerably (P<0.05).In comparison with the CCI group, the hippocampal BDNF expression in the CCI+EA2D, CCI+EA1W and CCI+EA2W group was upregulated considerably (P<0.05). The expression of hippocampal BDNF in CCI+EA2W was higher than the CCI+EA2D and CCI+EA1W group(P<0.05), suggesting a cumulative effect of repeated EA in up-regulating hippocampal BDNF expression. NO significant difference were found between CCI+EA2D and CCI+EA1W groups in hippocampal BDNF(P>0.05). Compared with normal control group, the expression level of hippocampal trkmRNA in CCI group was decreased significantly (P<0.05). Compare with the CCI group, hippocampal trkmRNA expression in CCI+EA2D, CCI+EA1W and CCI+EA2W group had no significant changes(P>0.05).
3.2The effect of EA on hippocampal MER/ERK signal pathway
3.2.1The effect of EA on hippocampal Ras-. C-Raf and MEK expression Compared with the normal control group, the expression of hippocampal Ras in CCI group was decreased considerably(P<0.05). Compared with the CCI group, hippocampal Ras expression was up-regulated slightly in CCI+EA2D and CCI+EA1W(P>0.05), significantly in CCI+EA2W (P<0.05). In comparison with the CCI+EA2D and CCI+EA1W group, hippocampal Ras expression in CCI+EA2W group was up-regulated considerably(P<0.05).
Compared with the normal control group, the expression level of hippocampal C-Raf was down-regulated markedly (P<0.05). In comparison with the CCI group, the hippocampal C-Raf expression in CCI+EA2D and CCI+EA1W group was increased slightly(P>0.05), and in CCI+EA2W was increased considerably(P<0.05).
Compared with the normal group, the expression level of hippocampal MEK in CCI group had no significant changes(P>0.05). No significant differences were found between CCI, CCI+EA2D, CCI+EA1W and CCI+EA2W group(P>0.05).
3.2.2The effect of EA on hippocampal ERK1/2expression
Compared with the normal control group, the expression level of hippocampal ERK1/2mRNA and ERK1/2in CCI group had no significant changes(P>0.05). No significant differences were found between those groups in hippocampal ERK1/2mRNA and ERK1/2expression (P>0.05) expect the ERK1expression in CCI+EA2W group was higher than in CCI group (P<0.05). In comparison with the normal control group, the hippocampal p-ERK1/2expression in CCI group were down-regulated considerably (P<0.05). Compared with the CCI group, hippocampal p-ERK1/2expression in CCI+EA2D and CCI+EA1W group were increased significantly (P<0.05). The expression level of hippocampal p-ERK1/2in CCI+EA2W group were higher than in CCI group and in CCI+EA2D group(P<0.05). There was no significant difference between CCI+EA1W and CCI+EA2W groups in hippocampal p-ERK1/2expression(P>0.05). It suggested a cumulative effect of repeated EA in up-regulating hippocampal p-ERK1/2expression.
3.3Effect of EA on p38and p-p38expression in the hippocampus
Compared with the normal control group, the p38MAPKmRNA expression of hippocampus in CCI group, CCI+EA2D group and CCI+EA1W group were decreased considerably (P<0.05). The expression level of hippocampal p38MAPKmRNA was markedly higher than those in CCI group and CCI+EA2D group (P<0.05), and markedly lower than that in the normal control group(P<0.05). The expression level of hippocampal p38MAPK had no significant changes between the different groups (P>0.05).
In comparison with the normal control group, the expression of hippocampal p-p38MAPK in CCI group was decreased slightly (P<0.05). Comparison between CCI group and CCI+EA2W group showed that the expression level of hippocampal p-p38MAPK was significantly lower in the former group than in the latter group (P<0.05), suggesting a cumulative effect of repeated EA in up-regulating hippocampal p-p38MAPK expression.
3.4Effect of EA on hippocampal PKA mRNA and CREB mRNA, CREB expression Compared with the normal control group, the expression of hippocampal PKA mRNA in CCI group was increased considerably (P<0.05). Compared with the CCI group, the hippocampal PKA mRNA expression in CCI+EA2D and CCI+EA1W group were significantly up-regulated (P<0.05). The expression of hippocampal PKA mRNA in CCI+EA2W was lower markedly than that of in CCI+EA2D and CCI+EA1W group(P<0.05).
Compared with the normal control group, the expression of hippocampal CREB mRNA in CCI and CCI+EA2D group was increased considerably (P<0.05).In comparison with the CCI group, the hippocampal CREB mRNA expression in CCI+EA1W and CCI+EA2W group were decreased markedly (P<0.05), while comapaed with the CCI+EA2D group, the hippocampal CREB mRNA expression in CCI+EA1W and CCI+EA2W group were decreased lightly (P>0.05).It suggested a cumulative effect of repeated EA for1weeks and2weeks in down-regulating the hippocampal CREB mRNA increase expression caused by CCI. However, there was no significant changes between those different groups in the expression level of hippocampal CREB and p-CREB.
Conclusion
The present study is representing behavioral, electrophysiological and molecular evidence to observe the relationship between electroacupuncture analgesia and hippocampal neuron plasticity changes and hippocampal ERK signaling pathway. The electrical activity of hippocampal pain-relation neurons had abnormal changes in the CCI model. There was a cumulative effect of repeated EA in relieving the state of the thermal radiation hyperalgesia. The effect of EA analgesic had close relationship with the hippocampal ERK signaling pathway.
引文
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