本文選題：慢性痛 + 電針��； 參考：《第四軍醫(yī)大學(xué)》2016年碩士論文

【摘要】：慢性痛是一類以遷延不愈的難治性疼痛為主要表現(xiàn)的疾病,給患者帶來巨大的生理痛苦及精神折磨;僅在美國,每年由慢性痛而引發(fā)的經(jīng)濟損失高達約6000萬美元,給家庭和社會帶來沉重負(fù)擔(dān)[1]。目前,臨床鎮(zhèn)痛仍以非甾體類抗炎藥和阿片類鎮(zhèn)痛藥等藥物治療方法為主。由于胃腸道副作用及成癮性等原因,藥物鎮(zhèn)痛的應(yīng)用存在諸多局限[2],尋找有效且副反應(yīng)小的鎮(zhèn)痛方法已成為全社會的廣泛訴求。我國傳統(tǒng)針刺療法已被證實對慢性痛具有良好的治療效應(yīng)且副作用較小,受到國內(nèi)外醫(yī)患的普遍認(rèn)同。隨技術(shù)創(chuàng)新衍生出的電針技術(shù)由于操作簡單、刺激參數(shù)穩(wěn)定,在臨床工作中有著較為廣泛的應(yīng)用,但其鎮(zhèn)痛機制尚不明確。研究表明,電針能夠通過調(diào)節(jié)神經(jīng)元內(nèi)多種生物活性物質(zhì)的合成與釋放發(fā)揮鎮(zhèn)痛效應(yīng)[3-6]。近年來,越來越多的證據(jù)表明神經(jīng)膠質(zhì)細(xì)胞參與疼痛的發(fā)生發(fā)展[7-11]。研究發(fā)現(xiàn),表達于神經(jīng)元的趨化因子CX3CL1及其表達于小膠質(zhì)細(xì)胞的受體CX3CR1在慢性痛的發(fā)生中扮演重要角色[12]。在慢性痛病理條件下,CX3CL1表達受小膠質(zhì)細(xì)胞來源的組織蛋白酶S(Cathepsin S,Cat S)等分子的調(diào)節(jié)而出現(xiàn)上調(diào),作用于小膠質(zhì)細(xì)胞上的受體CX3CR1,激活p38 MAPK通路并促進多種細(xì)胞因子如IL-1β、IL-6和TNF-α的釋放并引發(fā)疼痛(包括慢性炎性痛與神經(jīng)病理性痛)[13-16],而電針是否通過調(diào)節(jié)CX3CL1/CX3CR1表達來影響神經(jīng)元與膠質(zhì)細(xì)胞的相互作用,進而影響疼痛的發(fā)生并發(fā)揮鎮(zhèn)痛作用,目前尚無研究證據(jù)。綜上,本實驗旨在探究神經(jīng)元/膠質(zhì)細(xì)胞上CX3CL1/CX3CR1在電針鎮(zhèn)痛效應(yīng)中的作用,為針刺的鎮(zhèn)痛效應(yīng)提供更多科學(xué)證據(jù),為進一步鞏固針刺在臨床應(yīng)用中的應(yīng)用提供理論依據(jù)。實驗一:電針對完全弗氏佐劑所致炎性痛的鎮(zhèn)痛作用目的:觀察電針對完全弗氏佐劑(Complete freund’s adjuvant,CFA)足底注射所致大鼠炎性痛的鎮(zhèn)痛效果。方法:成年雄性SD大鼠40只(體重180-200g),隨機分為4組:空白對照(Control)組、炎性痛模型(CFA)組、炎性痛模型電針治療(EA)組以及炎性痛模型非穴位針刺(Sham)組(n=10)。CFA組、EA組、Sham組動物通過左側(cè)后肢足底皮下注射CFA建立炎性痛模型;EA組動物在造模后第1、3、5、7天分別接受電針刺激雙側(cè)足三里穴30min的處理,Sham組在對應(yīng)時間點接受電針刺激雙側(cè)腹股溝區(qū)無穴位處的處理。在CFA注射后第1-7天,每24h對各組動物的機械縮足反射閾值(PWMT)與熱縮足反應(yīng)潛伏期(PWTL)進行檢測。結(jié)果:與Control組相比,CFA、EA和Sham組動物足底注射CFA后,均出現(xiàn)顯著的機械痛敏和熱痛敏(P0.01),持續(xù)時間可達7天以上。與CFA組相比,EA組動物PWMT與PWTL顯著提高(P0.05)。同時,Sham組動物PWMT和PWTL較EA組降低,但與CFA組相比無統(tǒng)計學(xué)差異。實驗二:電針對CX3CL1/CX3CR1及其下游生物分子表達與活化狀態(tài)的影響目的:分別觀察CFA足底注射造模以及電針處理后實驗動物脊髓背角CX3CL1、CX3CR1及其下游p38 MAPK和多種細(xì)胞因子表達或活化狀態(tài)的變化。方法:體重180-200 g成年雄性SD大鼠60只,按照實驗一所述方法隨機分為Control、CFA、EA及Sham組(n=18)。根據(jù)前期行為學(xué)實驗結(jié)果,在造模后第1、3天行電針處理后將上述各組動物于深麻醉下處死并灌注,取腰膨大段脊髓組織于-80℃凍存。部分所取標(biāo)本經(jīng)勻漿離心等處理后制成蛋白樣本,使用Western Blot比較CX3CL1、CX3CR1、p38 MAPK、phospho-p38MAPK的表達差異,同時使用酶聯(lián)免疫吸附實驗(ELISA)試劑盒檢測細(xì)胞因子IL-1β、IL-6、TNF-α的表達量的變化。另一部分標(biāo)本制作冰凍切片,使用免疫熒光染色檢測CX3CL1的熒光強度的變化作為佐證。結(jié)果:1.CFA足底注射造成腰膨大段脊髓組織內(nèi)CX3CL1表達水平顯著增高(P0.05),而電針處理使EA組CX3CL1表達相較CFA組明顯降低(P0.01);同時,脊髓內(nèi)趨化因子受體CX3CR1的表達不因CFA注射或電針處理而發(fā)生變化。2.CX3CL1/CX3CR1下游分子P38 MAPK的表達并不因各組間CX3CL1表達水平存在差異而發(fā)生改變。但在造模后第3天,該分子的活化狀態(tài)改變,具體為CFA組動物脊髓phosph-p38 MAPK表達升高(P0.05);而電針處理能夠抑制CFA所誘導(dǎo)的p38 MAPK磷酸化增加,EA組與Control組phosph-p38 MAPK的表達無統(tǒng)計學(xué)差異。3.由p38 MAPK介導(dǎo)釋放的細(xì)胞因子IL-1β、IL-6和TNF-α在CFA作用下表達均顯著升高(P0.05),而EA組中此3類細(xì)胞因子的表達均顯著低于CFA組。實驗三:CX3CL1參與電針對炎性痛的抗炎鎮(zhèn)痛作用目的:證實電針通過抑制CX3CL1表達發(fā)揮抗炎鎮(zhèn)痛效應(yīng)。方法:選取體重140-160g雄性SD大鼠,行鞘內(nèi)置管術(shù)并確認(rèn)手術(shù)成功后選取36只大鼠入組,隨機分入Ig G組、Ig G+EA組、FKN+EA組。首先進行疼痛行為學(xué)實驗,測量PWMT及PWTL的基線值后,各組動物給予CFA足底注射建立炎性痛模型。在造模后第1、3天,Ig G+EA與FKN+EA組動物分別接受電針刺激雙側(cè)足三里穴的處理(30min)。電針處理后各組動物分別接受羊源性對照Ig G(Ig G組、Ig G+EA組)或CX3CL1(FKN+EA組)的鞘內(nèi)注射,并在注射6小時后進行疼痛行為學(xué)測量(n=6)。根據(jù)行為學(xué)測量結(jié)果,在造模后第3天取各組動物脊髓腰膨大組織檢測p38 MAPK磷酸化水平的變化(n=6)。結(jié)果:1.CFA注射造模后,各組動物均出現(xiàn)機械痛敏和熱痛敏現(xiàn)象。經(jīng)電針處理的Ig G+EA組動物疼痛閾值較未經(jīng)電針處理的Ig G組更高(P0.01)。同時,鞘內(nèi)注射CX3CL1可逆轉(zhuǎn)電針的鎮(zhèn)痛效果:與Ig G+EA組相比,FKN+EA組動物的PWMT與PWTL均顯著下降(P0.05)。2.各組p38 MAPK的表達仍無顯著性差異,但phosph-p38 MAPK的表達差異明顯:Ig G+EA組磷酸化p38 MAPK的表達水平較Ig G組顯著降低(P0.05),而FKN+EA組中,電針抑制磷酸化p38 MAPK表達水平升高的作用被部分逆轉(zhuǎn),其磷酸化p38 MAPK表達量高于Ig G+EA組,但仍較Ig G組略低(P0.05)。結(jié)論:1.電針能夠緩解CFA足底注射造成的機械痛敏和熱痛敏,發(fā)揮鎮(zhèn)痛效應(yīng)。2.CFA炎性痛模型大鼠脊髓組織內(nèi)CX3CL1表達提高,且在不影響表達量的前提下促進p38 MAPK磷酸化水平升高,進而增加細(xì)胞因子表達激活炎性反應(yīng)。而電針可對以上效應(yīng)產(chǎn)生抑制。3.通過行為學(xué)及生物化學(xué)實驗,發(fā)現(xiàn)電針可通過抑制CX3CL1表達抑制CFA介導(dǎo)的炎癥反應(yīng),發(fā)揮抗炎鎮(zhèn)痛效應(yīng)。小結(jié):本實驗采用雄性SD大鼠CFA足底注射炎性痛模型,首先通過疼痛行為學(xué)實驗檢測電針對CFA所致炎性痛是否具有鎮(zhèn)痛效應(yīng),發(fā)現(xiàn)電針雙側(cè)足三里處理可有效改善CFA所誘發(fā)的痛覺敏化。下一步選取行為學(xué)實驗結(jié)果較為穩(wěn)定的時間點處死實驗動物并取腰段脊髓組織,運用Western Blot、ELISA、免疫熒光染色等實驗方法,證實電針處理可以降低CX3CL1的表達,抑制下游細(xì)胞因子的釋放,揭示其可調(diào)節(jié)脊髓內(nèi)炎癥反應(yīng)。最后通過鞘內(nèi)給藥與電針處理相結(jié)合的方式,利用行為學(xué)檢測和分子生物學(xué)實驗方法,證明電針可通過抑制脊髓內(nèi)CX3CL1表達誘導(dǎo)抗炎作用而發(fā)揮鎮(zhèn)痛效應(yīng),表明電針對脊髓CX3CL1表達的調(diào)節(jié)是其發(fā)揮鎮(zhèn)痛效應(yīng)的重要機制。該研究結(jié)果豐富了針刺鎮(zhèn)痛機制的內(nèi)涵,為加深對針刺鎮(zhèn)痛的理解提供了重要支撐。
[Abstract]:Chronic pain is a kind of disease which is mainly manifested by persistent and refractory pain, which brings great physiological and mental suffering to patients. In the United States, the economic loss caused by chronic pain is up to about 60 million dollars a year in the United States, which brings a heavy burden to the family and society [1].. There are many limitations in the application of drug analgesia because of the side effects and addiction of the gastrointestinal tract. The application of the analgesic drug has many limitations in [2]. To find effective and minor side effects has become a widespread demand in the whole society. Traditional acupuncture therapy has been proved to have good therapeutic effect and side effects in slow pain in China. The electroacupuncture technology derived from technological innovation has been widely used in clinical work, but its analgesic mechanism is not clear. The study shows that electroacupuncture can play an analgesic effect by regulating the synthesis and release of various biological active substances in the neuron. Effect [3-6]. in recent years, more and more evidence suggests that neuroglia participates in the development of pain, [7-11]. studies have found that the chemokine CX3CL1 expressed in neurons and the receptor CX3CR1 expressed in microglia play an important role in the pathogenesis of chronic pain, [12]. in the pathological conditions of slow pain, CX3CL1 expression is microglia The regulation of cell derived cathepsin S (Cathepsin S, Cat S) and other molecules is up-regulated, acting on the receptor CX3CR1 on microglia, activating the p38 MAPK pathway and promoting the release of a variety of cytokines such as IL-1 beta, IL-6 and TNF- alpha and triggering pain (including chronic inflammatory pain and neuropathic pain), and whether electroacupuncture is transferred through the modulation. The expression of CX3CL1/CX3CR1 affects the interaction of neurons and glia, and then affects the occurrence of pain and plays an analgesic role. At present, there is no research evidence. To sum up, the purpose of this study is to explore the role of CX3CL1/CX3CR1 in the analgesic effect of electroacupuncture in neurons / glia and provide more scientific evidence for the analgesic effect of acupuncture. Further consolidate the application of acupuncture in the clinical application of the theoretical basis. Experiment 1: the analgesic effect of Electroacupuncture on complete Freund adjuvant induced inflammatory pain: observe the analgesic effect of electric acupuncture on Complete Freund 's adjuvant (CFA) foot injection in rats. Methods: 40 adult male SD rats (weight 180) -200g), randomly divided into 4 groups: blank control (Control) group, inflammatory pain model (CFA) group, inflammatory pain model electroacupuncture therapy (EA) group and non acupoint acupuncture (Sham) group (n=10).CFA group, EA group, Sham group animal by hypodermic hypodermic injection of CFA in left hind limb to establish inflammatory pain model, EA group received electricity on the 1,3,5,7 day after model building. The treatment of 30min in the bilateral Zusanli point was stimulated by acupuncture. Group Sham was treated with electroacupuncture at the corresponding time point at the non acupoint at the bilateral groin area at the corresponding time point. On day 1-7 after CFA injection, the mechanical contraction reflex threshold (PWMT) and the latent period (PWTL) of the reacting foot reaction (PWTL) were detected by each 24h. Results: compared with the Control group, the animals of CFA, EA and Sham groups were compared. After the injection of CFA, there were significant mechanical and thermal pain sensitivity (P0.01) for more than 7 days. Compared with group CFA, PWMT and PWTL increased significantly in group EA (P0.05). At the same time, PWMT and PWTL of Sham animals were lower than those in EA group, but there was no difference compared with those in CFA group. Experiment two: Electroacupuncture and its downstream biomolecular table Objective: To observe the changes in the expression or activation state of CX3CL1, CX3CR1 and its downstream p38 MAPK and a variety of cytokines in the spinal dorsal horn of experimental animals after CFA plantar injection and electroacupuncture treatment. Methods: 60 adult male SD rats with weight 180-200 g were randomly divided into Control, CFA, EA, according to the experimental method. And group Sham (n=18). According to the results of the early behavioral experiment, the above animals were killed and perfused under the deep anesthesia after the process of electroacupuncture at 1,3 days after the model building. The spinal cord tissue of the lumbar enlargement segment was frozen at -80 C. Some specimens were processed by homogenate centrifugation to make a protein sample, and Western Blot was used to compare CX3CL1, CX3CR1, p38 MAPK, Pho. The expression of spho-p38MAPK was different, and the expression of cytokine IL-1 beta, IL-6, and TNF- alpha was detected by enzyme linked immunosorbent assay (ELISA) kit. Another part of the specimens made frozen section, and the fluorescence intensity of CX3CL1 was detected by immunofluorescence staining as evidence. Results: 1.CFA plantar injection caused the lumbar spinal cord group. The expression level of CX3CL1 in the weave was significantly higher (P0.05), while the electroacupuncture treatment made the expression of CX3CL1 in EA group significantly lower than that in the CFA group (P0.01). At the same time, the expression of chemokine receptor CX3CR1 in the spinal cord was not changed by CFA injection or electroacupuncture treatment, and the expression of P38 MAPK in the lower.2.CX3CL1/CX3CR1 downstream molecules was not due to the difference in the level of CX3CL1 expression among the groups. But in the third day after the model, the activation state of the molecule was changed, the expression of phosph-p38 MAPK in the spinal cord of the CFA group increased (P0.05), and the electroacupuncture treatment could inhibit the increase of the phosphorylation of p38 MAPK induced by CFA, and there was no statistical difference between the EA group and the Control group phosph-p38 MAPK. The expression of IL-1 beta, IL-6 and TNF- alpha increased significantly under the action of CFA (P0.05), and the expression of these 3 kinds of cytokines in group EA was significantly lower than that in group CFA. Experiment three: CX3CL1 participated in the anti-inflammatory and analgesic effects of Electroacupuncture on inflammatory pain. 36 rats were selected and randomly divided into group Ig G, Ig G+EA group and FKN+EA group. First, the pain behavior experiment was carried out, and the baseline values of PWMT and PWTL were measured. The animals were given the inflammatory pain model by the CFA foot injection. At the third day after the model, the Ig G+EA and the FKN+EA groups received electroacupuncture stimulation respectively. The treatment of bilateral Zusanli point (30min). After electroacupuncture treatment, each group of animals received intrathecal injection of sheep derived Ig G (Ig G group, Ig G+EA group) or CX3CL1 (FKN+EA group), and performed a painful behavioral measurement after 6 hours of injection (n=6). According to the results of behavioral measurements, the spinal lumbar enlargement tissues of each group were detected p38 MA at third days after the model. The changes in the phosphorylation level of PK (n=6). Results: after 1.CFA injection molding, all the animals in each group had mechanical pain sensitivity and thermal pain sensitivity. The pain threshold of group Ig G+EA treated by Electroacupuncture was higher than that of the Ig G group without electroacupuncture treatment (P0.01). Meanwhile, intrathecal CX3CL1 could reverse the analgesic effect of the electroacupuncture: compared with Ig G+EA group, the FKN+EA group animals There was no significant difference in the expression of p38 MAPK between MT and PWTL (P0.05), but the expression difference of phosph-p38 MAPK was obvious: the expression level of p38 MAPK was significantly lower in Ig G+EA group than in the Ig G+EA group. The expression level was higher than that of the Ig G+EA group, but it was still slightly lower than that of the Ig G group (P0.05). Conclusion: 1. electroacupuncture can relieve the mechanical pain sensitivity and thermal pain sensitivity caused by the CFA foot injection. The expression of CX3CL1 in the spinal cord of the rat model of the analgesic effect.2.CFA is improved, and the increase of the phosphorylation level of p38 MAPK is promoted without the influence of the expression amount, and then the increase of the level of the phosphorylation of p38 MAPK is increased, and then the fines are increased. Cytokine expression activates the inflammatory response. And electroacupuncture can inhibit the effects of.3. through behavioral and biochemical experiments. It is found that electroacupuncture can inhibit the inflammatory response mediated by CFA by inhibiting the expression of CX3CL1 and exerts anti-inflammatory and analgesic effects. Summary: this experiment used the CFA plantar inflammatory pain model in male SD rats, first through the pain behavior. It was found that electroacupuncture had analgesic effect on inflammatory pain caused by CFA. It was found that electroacupuncture bilateral Zusanli treatment could effectively improve the pain sensitization induced by CFA. The next step was to select the experimental animals and take the spinal cord tissue from the stable time point of the behavioral experiment, and to use Western Blot, ELISA, immunofluorescence staining and so on. Methods it is proved that electroacupuncture can reduce the expression of CX3CL1, inhibit the release of the downstream cytokines and reveal the regulation of the inflammatory reaction in the spinal cord. Finally, by combining the intrathecal administration with the electroacupuncture treatment, it is proved that the electroacupuncture can induce anti inflammation by inhibiting the expression of CX3CL1 in the spinal cord by the methods of behavioral and molecular biological experiments. The effect of analgesic effect shows that the regulation of Electroacupuncture on the expression of CX3CL1 in the spinal cord is an important mechanism for its analgesic effect. The results of this study enrich the connotation of the mechanism of acupuncture analgesia and provide important support for deepening the understanding of acupuncture analgesia.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R246.2
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本文編號：1882168