电针抑制脊髓内趋化因子CX3CL1减轻炎性痛的机制研究

发布时间：2018-05-13 07:07

  本文选题：慢性痛 + 电针　； 参考：《第四军医大学》2016年硕士论文

【摘要】：慢性痛是一类以迁延不愈的难治性疼痛为主要表现的疾病,给患者带来巨大的生理痛苦及精神折磨;仅在美国,每年由慢性痛而引发的经济损失高达约6000万美元,给家庭和社会带来沉重负担[1]。目前,临床镇痛仍以非甾体类抗炎药和阿片类镇痛药等药物治疗方法为主。由于胃肠道副作用及成瘾性等原因,药物镇痛的应用存在诸多局限[2],寻找有效且副反应小的镇痛方法已成为全社会的广泛诉求。我国传统针刺疗法已被证实对慢性痛具有良好的治疗效应且副作用较小,受到国内外医患的普遍认同。随技术创新衍生出的电针技术由于操作简单、刺激参数稳定,在临床工作中有着较为广泛的应用,但其镇痛机制尚不明确。研究表明,电针能够通过调节神经元内多种生物活性物质的合成与释放发挥镇痛效应[3-6]。近年来,越来越多的证据表明神经胶质细胞参与疼痛的发生发展[7-11]。研究发现,表达于神经元的趋化因子CX3CL1及其表达于小胶质细胞的受体CX3CR1在慢性痛的发生中扮演重要角色[12]。在慢性痛病理条件下,CX3CL1表达受小胶质细胞来源的组织蛋白酶S(Cathepsin S,Cat S)等分子的调节而出现上调,作用于小胶质细胞上的受体CX3CR1,激活p38 MAPK通路并促进多种细胞因子如IL-1β、IL-6和TNF-α的释放并引发疼痛(包括慢性炎性痛与神经病理性痛)[13-16],而电针是否通过调节CX3CL1/CX3CR1表达来影响神经元与胶质细胞的相互作用,进而影响疼痛的发生并发挥镇痛作用,目前尚无研究证据。综上,本实验旨在探究神经元/胶质细胞上CX3CL1/CX3CR1在电针镇痛效应中的作用,为针刺的镇痛效应提供更多科学证据,为进一步巩固针刺在临床应用中的应用提供理论依据。实验一:电针对完全弗氏佐剂所致炎性痛的镇痛作用目的:观察电针对完全弗氏佐剂(Complete freund’s adjuvant,CFA)足底注射所致大鼠炎性痛的镇痛效果。方法:成年雄性SD大鼠40只(体重180-200g),随机分为4组:空白对照(Control)组、炎性痛模型(CFA)组、炎性痛模型电针治疗(EA)组以及炎性痛模型非穴位针刺(Sham)组(n=10)。CFA组、EA组、Sham组动物通过左侧后肢足底皮下注射CFA建立炎性痛模型;EA组动物在造模后第1、3、5、7天分别接受电针刺激双侧足三里穴30min的处理,Sham组在对应时间点接受电针刺激双侧腹股沟区无穴位处的处理。在CFA注射后第1-7天,每24h对各组动物的机械缩足反射阈值(PWMT)与热缩足反应潜伏期(PWTL)进行检测。结果:与Control组相比,CFA、EA和Sham组动物足底注射CFA后,均出现显著的机械痛敏和热痛敏(P0.01),持续时间可达7天以上。与CFA组相比,EA组动物PWMT与PWTL显著提高(P0.05)。同时,Sham组动物PWMT和PWTL较EA组降低,但与CFA组相比无统计学差异。实验二:电针对CX3CL1/CX3CR1及其下游生物分子表达与活化状态的影响目的:分别观察CFA足底注射造模以及电针处理后实验动物脊髓背角CX3CL1、CX3CR1及其下游p38 MAPK和多种细胞因子表达或活化状态的变化。方法:体重180-200 g成年雄性SD大鼠60只,按照实验一所述方法随机分为Control、CFA、EA及Sham组(n=18)。根据前期行为学实验结果,在造模后第1、3天行电针处理后将上述各组动物于深麻醉下处死并灌注,取腰膨大段脊髓组织于-80℃冻存。部分所取标本经匀浆离心等处理后制成蛋白样本,使用Western Blot比较CX3CL1、CX3CR1、p38 MAPK、phospho-p38MAPK的表达差异,同时使用酶联免疫吸附实验(ELISA)试剂盒检测细胞因子IL-1β、IL-6、TNF-α的表达量的变化。另一部分标本制作冰冻切片,使用免疫荧光染色检测CX3CL1的荧光强度的变化作为佐证。结果:1.CFA足底注射造成腰膨大段脊髓组织内CX3CL1表达水平显著增高(P0.05),而电针处理使EA组CX3CL1表达相较CFA组明显降低(P0.01);同时,脊髓内趋化因子受体CX3CR1的表达不因CFA注射或电针处理而发生变化。2.CX3CL1/CX3CR1下游分子P38 MAPK的表达并不因各组间CX3CL1表达水平存在差异而发生改变。但在造模后第3天,该分子的活化状态改变,具体为CFA组动物脊髓phosph-p38 MAPK表达升高(P0.05);而电针处理能够抑制CFA所诱导的p38 MAPK磷酸化增加,EA组与Control组phosph-p38 MAPK的表达无统计学差异。3.由p38 MAPK介导释放的细胞因子IL-1β、IL-6和TNF-α在CFA作用下表达均显著升高(P0.05),而EA组中此3类细胞因子的表达均显著低于CFA组。实验三:CX3CL1参与电针对炎性痛的抗炎镇痛作用目的:证实电针通过抑制CX3CL1表达发挥抗炎镇痛效应。方法:选取体重140-160g雄性SD大鼠,行鞘内置管术并确认手术成功后选取36只大鼠入组,随机分入Ig G组、Ig G+EA组、FKN+EA组。首先进行疼痛行为学实验,测量PWMT及PWTL的基线值后,各组动物给予CFA足底注射建立炎性痛模型。在造模后第1、3天,Ig G+EA与FKN+EA组动物分别接受电针刺激双侧足三里穴的处理(30min)。电针处理后各组动物分别接受羊源性对照Ig G(Ig G组、Ig G+EA组)或CX3CL1(FKN+EA组)的鞘内注射,并在注射6小时后进行疼痛行为学测量(n=6)。根据行为学测量结果,在造模后第3天取各组动物脊髓腰膨大组织检测p38 MAPK磷酸化水平的变化(n=6)。结果:1.CFA注射造模后,各组动物均出现机械痛敏和热痛敏现象。经电针处理的Ig G+EA组动物疼痛阈值较未经电针处理的Ig G组更高(P0.01)。同时,鞘内注射CX3CL1可逆转电针的镇痛效果:与Ig G+EA组相比,FKN+EA组动物的PWMT与PWTL均显著下降(P0.05)。2.各组p38 MAPK的表达仍无显著性差异,但phosph-p38 MAPK的表达差异明显:Ig G+EA组磷酸化p38 MAPK的表达水平较Ig G组显著降低(P0.05),而FKN+EA组中,电针抑制磷酸化p38 MAPK表达水平升高的作用被部分逆转,其磷酸化p38 MAPK表达量高于Ig G+EA组,但仍较Ig G组略低(P0.05)。结论:1.电针能够缓解CFA足底注射造成的机械痛敏和热痛敏,发挥镇痛效应。2.CFA炎性痛模型大鼠脊髓组织内CX3CL1表达提高,且在不影响表达量的前提下促进p38 MAPK磷酸化水平升高,进而增加细胞因子表达激活炎性反应。而电针可对以上效应产生抑制。3.通过行为学及生物化学实验,发现电针可通过抑制CX3CL1表达抑制CFA介导的炎症反应,发挥抗炎镇痛效应。小结:本实验采用雄性SD大鼠CFA足底注射炎性痛模型,首先通过疼痛行为学实验检测电针对CFA所致炎性痛是否具有镇痛效应,发现电针双侧足三里处理可有效改善CFA所诱发的痛觉敏化。下一步选取行为学实验结果较为稳定的时间点处死实验动物并取腰段脊髓组织,运用Western Blot、ELISA、免疫荧光染色等实验方法,证实电针处理可以降低CX3CL1的表达,抑制下游细胞因子的释放,揭示其可调节脊髓内炎症反应。最后通过鞘内给药与电针处理相结合的方式,利用行为学检测和分子生物学实验方法,证明电针可通过抑制脊髓内CX3CL1表达诱导抗炎作用而发挥镇痛效应,表明电针对脊髓CX3CL1表达的调节是其发挥镇痛效应的重要机制。该研究结果丰富了针刺镇痛机制的内涵,为加深对针刺镇痛的理解提供了重要支撑。
[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.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R246.2
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本文编号：1882168