【摘要】：慢性痛是一類慢性疾病,反復(fù)發(fā)作、遷延難治,嚴(yán)重危害人類身心健康和生活質(zhì)量,慢性痛相關(guān)的醫(yī)療花費(fèi)十分巨大。持續(xù)的慢性痛會(huì)危及患者的人際關(guān)系和社交活動(dòng),將逐漸導(dǎo)致患者產(chǎn)生焦慮、抑郁、恐懼等惡性情緒,“痛”不欲生。近年來,慢性痛的機(jī)制研究雖然取得長(zhǎng)足進(jìn)展,但是,當(dāng)前臨床上的主流鎮(zhèn)痛藥非甾體類抗炎藥(NSAIDs)和阿片類鎮(zhèn)痛藥存在許多局限性[1],不能滿足患者的需求。因此,研究和揭示慢性痛的本質(zhì)和發(fā)病機(jī)制,研制新型鎮(zhèn)痛藥具有重要的醫(yī)學(xué)意義和社會(huì)意義。脊髓背角淺層(SDH)對(duì)疼痛等傷害性信息向大腦的傳遞起重要的整合和敏化作用,其內(nèi)的神經(jīng)元及其突起相互聯(lián)系形成重要的興奮性環(huán)路和抑制性環(huán)路[2],我們課題組前期的研究發(fā)現(xiàn)脊髓背角I層神經(jīng)元的突觸可塑性改變?cè)诼酝吹陌l(fā)生發(fā)展過程中發(fā)揮重要作用[3-5]。因此,脊髓背角I層神經(jīng)元是鎮(zhèn)痛藥物的潛在作用靶點(diǎn)之一。天麻素(Gastrodin,GAS)是從我國(guó)傳統(tǒng)中草藥“天麻”中分離出的主要活性成分之一,具有抗癲癇、抗驚厥、鎮(zhèn)靜、鎮(zhèn)痛和神經(jīng)保護(hù)等作用[6-9]。近年來,系列臨床報(bào)道GAS可以顯著地緩解三叉神經(jīng)痛、偏頭痛、糖尿病性神經(jīng)痛、血管性頭痛等頑固性慢性痛[9-12]。研究報(bào)道GAS對(duì)痛覺初級(jí)傳入神經(jīng)元——背根節(jié)(DRG)的興奮性發(fā)揮顯著抑制作用,進(jìn)一步研究發(fā)現(xiàn)其機(jī)制可能是通過調(diào)節(jié)電壓依賴性鈉、鉀通道以及酸敏感性陽離子通道的功能狀態(tài)來實(shí)現(xiàn)的[13,14]。然而GAS對(duì)慢性炎性痛有無鎮(zhèn)痛作用,發(fā)揮鎮(zhèn)痛作用的細(xì)胞和分子機(jī)制是什么,目前的研究報(bào)道很少。因此,研究和探討GAS在慢性痛中的作用及其中樞鎮(zhèn)痛作用機(jī)制有望為其臨床應(yīng)用提供重要的科學(xué)依據(jù)和理論指導(dǎo)。第一部分:探討GAS對(duì)炎性痛小鼠自發(fā)痛和痛覺過敏的影響目的:觀察GAS對(duì)炎性痛小鼠的鎮(zhèn)痛作用。方法:成年雄性小鼠一側(cè)足底皮下注射(s.c.)蜜蜂毒或完全弗氏佐劑(CFA)建立病理性疼痛模型,自發(fā)痛行為在蜜蜂毒注射即刻開始測(cè)試,觸誘發(fā)痛和痛覺過敏分別在CFA注射后不同時(shí)間點(diǎn)進(jìn)行測(cè)試。結(jié)果:腹腔注射(i.p.)不同濃度的GAS(50,100,200 mg/kg)或生理鹽水,與對(duì)照組相比GAS呈劑量依賴性地抑制蜜蜂毒誘致的自發(fā)痛和CFA誘致的觸誘發(fā)痛、機(jī)械性痛敏和熱痛敏(n=5-8 per group,P0.05,one-way ANOVA)。而且GAS發(fā)揮的鎮(zhèn)痛作用不被納洛酮所阻斷,提示其發(fā)揮鎮(zhèn)痛作用不依賴于阿片μ受體,并且長(zhǎng)期用藥無耐受性產(chǎn)生。與對(duì)照組相比,鞘內(nèi)注射GAS(10 m M,i.t.)顯著地降低CFA誘致的觸誘發(fā)痛、機(jī)械痛敏和熱痛敏(n=6 per group,P0.05,one-way ANOVA)。然而,注射GAS(200 mg/kg,i.p.)對(duì)正常小鼠的基礎(chǔ)痛閾和運(yùn)動(dòng)平衡協(xié)調(diào)能力均無顯著影響(n=5-8 mice per group,P0.05,one-way ANOVA)。第二部分:探討GAS對(duì)炎性痛小鼠脊髓背角c-Fos表達(dá)的影響目的:為了進(jìn)一步獲得GAS在脊髓發(fā)揮鎮(zhèn)痛作用的證據(jù),我們用評(píng)估神經(jīng)元活動(dòng)的功能標(biāo)志物進(jìn)行了第二部分實(shí)驗(yàn)。方法:上述小鼠建立蜜蜂毒模型后2 h,灌注取腰膨大處脊髓,后固定,脫水,冰凍切片,免疫組化ABC法染色。結(jié)果:蜜蜂毒誘致的病理性痛小鼠L4-L5節(jié)段脊髓背角c-Fos表達(dá)呈現(xiàn)顯著的上調(diào),疼痛的初級(jí)傳入纖維末梢主要終止的部位脊髓背角淺層(I-II)和深層(IV-V)c-Fos密度明顯升高。與對(duì)照組相比,腹腔注射GAS(50,100,200 mg/kg,i.p.)呈劑量依賴性地抑制脊髓背角淺層和深層神經(jīng)元c-Fos的表達(dá)(P0.05,one-wayANOVA),并且其對(duì)淺層的抑制作用比深層更顯著。第三部分:探討GAS對(duì)SDH I層神經(jīng)元興奮性突觸傳遞和神經(jīng)元興奮性的影響目的:為了探究GAS緩解炎性痛的機(jī)制,我們進(jìn)一步評(píng)估了GAS對(duì)興奮性突觸傳遞和神經(jīng)元興奮性的影響。方法:取出生后14-18 d小鼠,建立CFA模型,24 h后制作帶有背根的脊髓橫斷面切片,對(duì)SDH I層神經(jīng)元進(jìn)行全細(xì)胞膜片鉗記錄,觀察灌流給予GAS(300μM)對(duì)慢性痛小鼠興奮性突觸傳遞及神經(jīng)元超興奮性的影響,記錄刺激背根誘發(fā)的興奮性突觸后電流(e EPSCs)、微小興奮性突觸后電流(m EPSCs)、C纖維誘發(fā)的e EPSCs(C-e EPSCs)的雙脈沖比值(PPR)和SDH I層神經(jīng)元的主動(dòng)膜特性和被動(dòng)膜特性。結(jié)果:(1)與正常小鼠相比,慢性炎性痛后C-e EPSCs的峰值幅度隨刺激強(qiáng)度變化反應(yīng)曲線(I-O曲線)明顯左移和上移,給予GAS慢性炎性痛小鼠脊髓背角66.7%(10/15)I層神經(jīng)元的C-e EPSCs峰值幅度被顯著抑制,統(tǒng)計(jì)分析顯示GAS的平均抑制率為29.18±3.23%(n=10 neurons/7 mice,P0.05,paired-t test)。(2)與正常小鼠相比,CFA誘致的炎性痛小鼠m EPSCs的放電頻率明顯增加,其頻率被GAS顯著抑制,平均抑制率為47.5±7.66%(n=7 neurons/4 mice,P0.05,paired-t test),而GAS對(duì)其幅度無顯著影響;而且GAS引起慢性炎性痛小鼠C-e EPSCs的PPR發(fā)生顯著變化。這二者均強(qiáng)烈提示GAS發(fā)揮鎮(zhèn)痛作用是突觸前效應(yīng)。(3)鑒于GAS對(duì)炎性痛小鼠初級(jí)傳入突觸增強(qiáng)起抑制作用,接下來我們探討GAS對(duì)初級(jí)傳入突觸后的脊髓背角I層神經(jīng)元興奮性和膜特性的影響。發(fā)現(xiàn)與正常小鼠相比,CFA炎性痛小鼠SDH I層神經(jīng)元在注入電流條件下興奮性明顯升高,具體表現(xiàn)為動(dòng)作電位(AP)的放電頻率增加、半寬減小、最大上升斜率增大、閾值下降、基強(qiáng)度降低。CFA炎性痛后SDH I層神經(jīng)元的被動(dòng)膜特性(膜電容Cm,膜電阻Rm,靜息膜電位RMP)無顯著變化(n=16 neurons/10 inflamed mice vs n=16 neurons/5 control mice,P0.05,one-way ANOVA)。GAS能逆轉(zhuǎn)慢性炎性痛小鼠SDH I層神經(jīng)元的興奮性,例如GAS使AP的頻率、半寬、最大上升斜率、閾值及基強(qiáng)度恢復(fù)正常(n=10 neurons/8 inflamed mice vs n=11 neurons/5 control mice,P0.05,paired-t test)。而且我們發(fā)現(xiàn)CFA炎性痛小鼠脊髓背角28%(7/25)I層神經(jīng)元在未注入電流條件下產(chǎn)生自發(fā)放電,GAS顯著抑制自發(fā)放電的頻率,其平均抑制率為33.04±7.67%(n=7 neurons/4mice,paired-t test),GAS對(duì)其幅度影響無顯著性差異。(4)與對(duì)炎性痛小鼠顯著抑制作用不同的是GAS對(duì)正常小鼠SDH I層神經(jīng)元的C-e EPSCs的幅度、m EPSCs的頻率和幅度、C-e EPSCs的PPR的改變以及主動(dòng)膜特性和被動(dòng)膜特性均無顯著影響。結(jié)論:1,GAS(i.p.)顯著抑制炎性痛小鼠的自發(fā)痛、觸誘發(fā)痛和痛覺過敏;GAS(i.t.)在脊髓水平發(fā)揮強(qiáng)有力的鎮(zhèn)痛作用;GAS(i.p.)不影響正常小鼠的基礎(chǔ)痛閾和運(yùn)動(dòng)協(xié)調(diào)能力。提示GAS是選擇性地抑制病理痛而對(duì)生理痛無顯著影響的良好鎮(zhèn)痛藥。2,GAS(i.p.)呈劑量依賴性地抑制炎性痛小鼠脊髓背角c-Fos的表達(dá)上調(diào),提示GAS對(duì)傷害性刺激誘致的脊髓背角神經(jīng)元活動(dòng)的增強(qiáng)具有顯著的抑制作用。3,灌流GAS顯著抑制炎性痛后脊髓背角I層神經(jīng)元興奮性突觸傳遞的可塑性增強(qiáng)。GAS明顯抑制炎性痛小鼠m EPSCs的頻率并且誘致C-e EPSCs的PPR發(fā)生顯著變化,二者均強(qiáng)烈提示GAS抑制突觸可塑性增強(qiáng)很可能是突觸前機(jī)制。4,GAS顯著逆轉(zhuǎn)慢性炎性痛小鼠SDH I層神經(jīng)元的興奮性增強(qiáng);GAS顯著抑制自發(fā)放電的頻率。提示GAS可能是通過抑制突觸前突觸傳遞的增強(qiáng)進(jìn)而抑制突觸后神經(jīng)元的興奮性增強(qiáng)而發(fā)揮鎮(zhèn)痛作用。5,GAS對(duì)正常小鼠SDH I層神經(jīng)元的C-e EPSCs的幅度、m EPSCs的頻率和幅度、C-e EPSCs的PPR的改變以及主動(dòng)膜特性和被動(dòng)膜特性均無顯著影響,提示GAS選擇性地抑制病理痛而不影響正常狀態(tài)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R243.2

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