天麻素在脊髓背角的镇痛作用及其突触机制研究

发布时间：2018-08-12 09:21

【摘要】：慢性痛是一类慢性疾病,反复发作、迁延难治,严重危害人类身心健康和生活质量,慢性痛相关的医疗花费十分巨大。持续的慢性痛会危及患者的人际关系和社交活动,将逐渐导致患者产生焦虑、抑郁、恐惧等恶性情绪,“痛”不欲生。近年来,慢性痛的机制研究虽然取得长足进展,但是,当前临床上的主流镇痛药非甾体类抗炎药(NSAIDs)和阿片类镇痛药存在许多局限性[1],不能满足患者的需求。因此,研究和揭示慢性痛的本质和发病机制,研制新型镇痛药具有重要的医学意义和社会意义。脊髓背角浅层(SDH)对疼痛等伤害性信息向大脑的传递起重要的整合和敏化作用,其内的神经元及其突起相互联系形成重要的兴奋性环路和抑制性环路[2],我们课题组前期的研究发现脊髓背角I层神经元的突触可塑性改变在慢性痛的发生发展过程中发挥重要作用[3-5]。因此,脊髓背角I层神经元是镇痛药物的潜在作用靶点之一。天麻素(Gastrodin,GAS)是从我国传统中草药“天麻”中分离出的主要活性成分之一,具有抗癫痫、抗惊厥、镇静、镇痛和神经保护等作用[6-9]。近年来,系列临床报道GAS可以显著地缓解三叉神经痛、偏头痛、糖尿病性神经痛、血管性头痛等顽固性慢性痛[9-12]。研究报道GAS对痛觉初级传入神经元——背根节(DRG)的兴奋性发挥显著抑制作用,进一步研究发现其机制可能是通过调节电压依赖性钠、钾通道以及酸敏感性阳离子通道的功能状态来实现的[13,14]。然而GAS对慢性炎性痛有无镇痛作用,发挥镇痛作用的细胞和分子机制是什么,目前的研究报道很少。因此,研究和探讨GAS在慢性痛中的作用及其中枢镇痛作用机制有望为其临床应用提供重要的科学依据和理论指导。第一部分:探讨GAS对炎性痛小鼠自发痛和痛觉过敏的影响目的:观察GAS对炎性痛小鼠的镇痛作用。方法:成年雄性小鼠一侧足底皮下注射(s.c.)蜜蜂毒或完全弗氏佐剂(CFA)建立病理性疼痛模型,自发痛行为在蜜蜂毒注射即刻开始测试,触诱发痛和痛觉过敏分别在CFA注射后不同时间点进行测试。结果:腹腔注射(i.p.)不同浓度的GAS(50,100,200 mg/kg)或生理盐水,与对照组相比GAS呈剂量依赖性地抑制蜜蜂毒诱致的自发痛和CFA诱致的触诱发痛、机械性痛敏和热痛敏(n=5-8 per group,P0.05,one-way ANOVA)。而且GAS发挥的镇痛作用不被纳洛酮所阻断,提示其发挥镇痛作用不依赖于阿片μ受体,并且长期用药无耐受性产生。与对照组相比,鞘内注射GAS(10 m M,i.t.)显著地降低CFA诱致的触诱发痛、机械痛敏和热痛敏(n=6 per group,P0.05,one-way ANOVA)。然而,注射GAS(200 mg/kg,i.p.)对正常小鼠的基础痛阈和运动平衡协调能力均无显著影响(n=5-8 mice per group,P0.05,one-way ANOVA)。第二部分:探讨GAS对炎性痛小鼠脊髓背角c-Fos表达的影响目的:为了进一步获得GAS在脊髓发挥镇痛作用的证据,我们用评估神经元活动的功能标志物进行了第二部分实验。方法:上述小鼠建立蜜蜂毒模型后2 h,灌注取腰膨大处脊髓,后固定,脱水,冰冻切片,免疫组化ABC法染色。结果:蜜蜂毒诱致的病理性痛小鼠L4-L5节段脊髓背角c-Fos表达呈现显著的上调,疼痛的初级传入纤维末梢主要终止的部位脊髓背角浅层(I-II)和深层(IV-V)c-Fos密度明显升高。与对照组相比,腹腔注射GAS(50,100,200 mg/kg,i.p.)呈剂量依赖性地抑制脊髓背角浅层和深层神经元c-Fos的表达(P0.05,one-wayANOVA),并且其对浅层的抑制作用比深层更显著。第三部分:探讨GAS对SDH I层神经元兴奋性突触传递和神经元兴奋性的影响目的:为了探究GAS缓解炎性痛的机制,我们进一步评估了GAS对兴奋性突触传递和神经元兴奋性的影响。方法:取出生后14-18 d小鼠,建立CFA模型,24 h后制作带有背根的脊髓横断面切片,对SDH I层神经元进行全细胞膜片钳记录,观察灌流给予GAS(300μM)对慢性痛小鼠兴奋性突触传递及神经元超兴奋性的影响,记录刺激背根诱发的兴奋性突触后电流(e EPSCs)、微小兴奋性突触后电流(m EPSCs)、C纤维诱发的e EPSCs(C-e EPSCs)的双脉冲比值(PPR)和SDH I层神经元的主动膜特性和被动膜特性。结果:(1)与正常小鼠相比,慢性炎性痛后C-e EPSCs的峰值幅度随刺激强度变化反应曲线(I-O曲线)明显左移和上移,给予GAS慢性炎性痛小鼠脊髓背角66.7%(10/15)I层神经元的C-e EPSCs峰值幅度被显著抑制,统计分析显示GAS的平均抑制率为29.18±3.23%(n=10 neurons/7 mice,P0.05,paired-t test)。(2)与正常小鼠相比,CFA诱致的炎性痛小鼠m EPSCs的放电频率明显增加,其频率被GAS显著抑制,平均抑制率为47.5±7.66%(n=7 neurons/4 mice,P0.05,paired-t test),而GAS对其幅度无显著影响;而且GAS引起慢性炎性痛小鼠C-e EPSCs的PPR发生显著变化。这二者均强烈提示GAS发挥镇痛作用是突触前效应。(3)鉴于GAS对炎性痛小鼠初级传入突触增强起抑制作用,接下来我们探讨GAS对初级传入突触后的脊髓背角I层神经元兴奋性和膜特性的影响。发现与正常小鼠相比,CFA炎性痛小鼠SDH I层神经元在注入电流条件下兴奋性明显升高,具体表现为动作电位(AP)的放电频率增加、半宽减小、最大上升斜率增大、阈值下降、基强度降低。CFA炎性痛后SDH I层神经元的被动膜特性(膜电容Cm,膜电阻Rm,静息膜电位RMP)无显著变化(n=16 neurons/10 inflamed mice vs n=16 neurons/5 control mice,P0.05,one-way ANOVA)。GAS能逆转慢性炎性痛小鼠SDH I层神经元的兴奋性,例如GAS使AP的频率、半宽、最大上升斜率、阈值及基强度恢复正常(n=10 neurons/8 inflamed mice vs n=11 neurons/5 control mice,P0.05,paired-t test)。而且我们发现CFA炎性痛小鼠脊髓背角28%(7/25)I层神经元在未注入电流条件下产生自发放电,GAS显著抑制自发放电的频率,其平均抑制率为33.04±7.67%(n=7 neurons/4mice,paired-t test),GAS对其幅度影响无显著性差异。(4)与对炎性痛小鼠显著抑制作用不同的是GAS对正常小鼠SDH I层神经元的C-e EPSCs的幅度、m EPSCs的频率和幅度、C-e EPSCs的PPR的改变以及主动膜特性和被动膜特性均无显著影响。结论:1,GAS(i.p.)显著抑制炎性痛小鼠的自发痛、触诱发痛和痛觉过敏;GAS(i.t.)在脊髓水平发挥强有力的镇痛作用;GAS(i.p.)不影响正常小鼠的基础痛阈和运动协调能力。提示GAS是选择性地抑制病理痛而对生理痛无显著影响的良好镇痛药。2,GAS(i.p.)呈剂量依赖性地抑制炎性痛小鼠脊髓背角c-Fos的表达上调,提示GAS对伤害性刺激诱致的脊髓背角神经元活动的增强具有显著的抑制作用。3,灌流GAS显著抑制炎性痛后脊髓背角I层神经元兴奋性突触传递的可塑性增强。GAS明显抑制炎性痛小鼠m EPSCs的频率并且诱致C-e EPSCs的PPR发生显著变化,二者均强烈提示GAS抑制突触可塑性增强很可能是突触前机制。4,GAS显著逆转慢性炎性痛小鼠SDH I层神经元的兴奋性增强;GAS显著抑制自发放电的频率。提示GAS可能是通过抑制突触前突触传递的增强进而抑制突触后神经元的兴奋性增强而发挥镇痛作用。5,GAS对正常小鼠SDH I层神经元的C-e EPSCs的幅度、m EPSCs的频率和幅度、C-e EPSCs的PPR的改变以及主动膜特性和被动膜特性均无显著影响,提示GAS选择性地抑制病理痛而不影响正常状态的生理痛。
[Abstract]:Chronic pain is a kind of chronic disease, which occurs repeatedly and is difficult to cure. It seriously endangers the physical and mental health and quality of life of human beings. The medical costs associated with chronic pain are enormous. Although great progress has been made in the study of the mechanism of chronic pain, there are many limitations in the current clinical mainstream analgesics, non-steroidal anti-inflammatory drugs (NSAIDs) and opioid analgesics [1], which can not meet the needs of patients. The superficial layer of the spinal dorsal horn (SDH) plays an important role in the integration and sensitization of nociceptive information such as pain to the brain. The neurons and their processes in the superficial layer of the spinal dorsal horn are interrelated to form important excitatory and inhibitory loops [2]. Previous studies of our team found that the synaptic plasticity of the neurons in the lamina I of the spinal dorsal horn was altered. Gastrodin (GAS) is one of the main active components isolated from the traditional Chinese herbal medicine Gastrodin, which has the functions of anti-epilepsy, anti-convulsion, sedation, analgesia and neuroprotection. In recent years, a series of clinical reports have shown that GAS can significantly alleviate intractable chronic pain such as trigeminal neuralgia, migraine, diabetic neuralgia and vascular headache [9-12]. Studies have reported that GAS can significantly inhibit the excitability of primary afferent pain neurons, dorsal root ganglion (DRG). Further studies have found that the mechanism may be mediated by GAS. Overregulation of voltage-dependent sodium, potassium, and acid-sensitive cation channels is achieved [13,14]. However, there are few reports on whether GAS has analgesic effect on chronic inflammatory pain, and what cellular and molecular mechanisms are involved in the analgesic effect. Therefore, the role of GAS in chronic pain and its central analgesic effect are studied and discussed. Part I: To investigate the effects of GAS on spontaneous pain and hyperalgesia in mice with inflammatory pain Objective: To observe the analgesic effect of GAS on mice with inflammatory pain. Pathological pain model, spontaneous pain behavior was tested immediately after injection of honeybee venom. Touch-induced pain and hyperalgesia were tested at different time points after injection of CFA. Results: Different concentrations of GAS (50,100,200 mg/kg) or normal saline were injected intraperitoneally (i.p.). GAS inhibited spontaneous pain induced by honeybee venom in a dose-dependent manner compared with the control group. The analgesic effect of GAS was not blocked by naloxone, suggesting that the analgesic effect of GAS did not depend on opioid mu receptor and was not tolerated for a long time. Compared with the control group, intrathecal injection of GAS (10 m, i.t.) was significantly more effective. However, GAS injection (200 mg/kg, i.p.) had no significant effect on basal pain threshold and motor balance coordination i n normal mice (n = 5-8 mice per group, P 0.05, one-way ANOVA). Part II: To investigate the effects of GAS on spinal dorsal horn c-Fos i n mice with inflammatory pain. Objective: To obtain further evidence of the analgesic effect of GAS in the spinal cord, we performed the second part of the experiment with functional markers assessing the activity of neurons. METHODS: Two hours after the establishment of the honeybee venom model, the spinal cord at the lumbar enlargement was perfused, fixed, dehydrated, frozen and stained with immunohistochemical ABC method. The expression of c-Fos in L4-L5 spinal dorsal horn of mice with pathological pain induced by bee venom was significantly up-regulated. The density of c-Fos in superficial (I-II) and deep (IV-V) spinal dorsal horn was significantly increased at the primary afferent terminals of pain. Compared with the control group, GAS (50,100,200 mg/kg, i.p.) was inhibited in a dose-dependent manner. The expression of c-Fos in superficial and deep horn neurons (P 0.05, one-way ANOVA) and its inhibitory effect on superficial neurons were more significant than that on deep neurons. METHODS: CFA model was established in 14-18 days postnatal mice. Spinal cord with dorsal root was sectioned 24 hours later. Whole-cell patch clamp recording of SDH I neurons was performed to observe the effects of GAS (300 mu M) on excitatory synaptic transmission and neuronal hyperexcitability in chronic pain mice. The excitatory postsynaptic currents (e EPSCs), minimal excitatory postsynaptic currents (m EPSCs), the bipulse ratios (PPR) of E EPSCs (C-e EPSCs) induced by C fibers and the characteristics of active and passive membranes of SDH I neurons were studied. Results: (1) Compared with normal mice, the peak amplitude of C-e EPSCs changed with the stimulation intensity. The peak amplitude of C-e EPSCs in the spinal dorsal horn of 66.7% (10/15) I layer neurons in GAS-treated chronic inflammatory pain mice was significantly inhibited. Statistical analysis showed that the average inhibition rate of GAS was 29.18 (+ 3.23%) (n = 10 neurons/7 mice, P 0.05, paired-t test). (2) Compared with normal mice, CFA induced inflammatory pain. The discharging frequency of M EPSCs in mice was significantly increased, and its frequency was significantly inhibited by GAS, with an average inhibition rate of 47.5 (+ 7.66%) (n=7 neurons/4 mice, P 0.05, paired-t test), while GAS had no significant effect on the amplitude of M EPSCs, and the PPR of C-e EPSCs in mice with chronic inflammatory pain was significantly changed by GAS. Pre-effect. (3) In view of the inhibitory effect of GAS on primary afferent synaptic enhancement in inflammatory pain mice, we then investigated the effects of GAS on excitability and membrane properties of lamina I neurons in the dorsal horn of the spinal cord after primary afferent synapses. It was found that compared with normal mice, the excitability of SDH I neurons in CFA inflammatory pain mice increased significantly under the condition of injection current. After CFA inflammation pain, the passive membrane properties (membrane capacitance Cm, membrane resistance Rm, resting membrane potential RMP) of SDH I neurons did not change significantly (n = 16 neurons / 10 inflamed mice vs n = 16 neurons / 5 control mice, P 0.05, one-w inflamed mice vs n = 16 neurons / 5 control mice). GAS reversed the excitability of SDH I layer neurons in mice with chronic inflammatory pain. For example, GAS restored AP frequency, half width, maximum slope of rise, threshold and basal strength to normal (n = 10 neurons/8 inflamed mice vs n = 11 neurons/5 control mice, P 0.05, paired-t test). We also found that 28% (7/25) of the spinal dorsal horn of CFA mice with inflammatory pain had lamina I activity. GAS significantly inhibited the frequency of spontaneous discharge without current injection. The average inhibition rate was 33.04 (+ 7.67%) (n=7 neurons/4 mice, paired-t test). GAS had no significant effect on the amplitude of spontaneous discharge. (4) GAS significantly inhibited the C-e EPSCs of SDH I layer neurons in normal mice. Conclusion: 1, GAS (i.p.) significantly inhibits spontaneous pain, palpation-induced pain and hyperalgesia in inflammatory pain mice; GAS (i.t.) plays a strong analgesic role at spinal cord level; GAS (i.p.) does not affect the basis of normal mice. GAS (i.p.) inhibited the up-regulation of c-Fos expression in spinal dorsal horn of inflammatory pain mice in a dose-dependent manner, suggesting that GAS significantly inhibited the enhancement of spinal dorsal horn neuronal activity induced by noxious stimulation. 3. GAS significantly inhibited the plasticity of excitatory synaptic transmission in lamina I neurons of spinal dorsal horn after inflammatory pain. GAS significantly inhibited the frequency of M EPSCs and induced significant changes in PPR of C-e EPSCs in inflammatory pain mice. Both strongly suggested that GAS inhibited synaptic plasticity enhancement may be a presynaptic mechanism. 4. GAS significantly reversed chronic pain. GAS significantly inhibited the frequency of spontaneous discharges, suggesting that GAS may play an analgesic role by inhibiting the increase of presynaptic transmission and then inhibiting the excitability of postsynaptic neurons. The changes of PPR and the characteristics of active and passive membranes of C-e EPSCs had no significant effect, suggesting that GAS selectively inhibited pathological pain without affecting normal physiological pain.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R243.2

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