基于CCI模型大鼠研究拨法“消炎止痛”效应的内在机制

发布时间：2018-07-09 23:03

本文选题：拨法 + CCI　；参考：《北京中医药大学》2017年硕士论文

【摘要】：[目的]通过行为学和形态学指标,研究拨法改善神经病理性疼痛大鼠痛觉过敏,促进神经修复的可能依据,通过研究促炎性因子IL-6及抗炎保护因子SOCS3在血清及脊髓中的表达情况,探究拨法治疗神经病理性疼痛,起到"消炎止痛"作用的可能起效机制。[方法]以SD雄性大鼠为实验动物,用随机数字表,随机分为正常组、假手术组、模型组和拨法组,选用坐骨神经慢性压迫性损伤(chronic constriction injury,CCI)为模型,采用"按摩推拿手法模拟仪"(中华人民共和国发明专利号:ZL200710187403.1.)模拟拨法操作对大鼠进行干预,穴位选用殷门、承山、阳陵泉,每穴3min,每只总计9min,拨法干预每日1次,每干预10日休息1天,总共干预20天,选取造模7天后和拨法干预20天后两个取材时间点,取材前对大鼠进行光热耐痛阈检测,评价其痛觉过敏的恢复情况。对大鼠术侧坐骨神经进行HE染色,并对结果进行观察分析,寻找拨法促进神经损伤修复的形态学证据;对大鼠血清中IL-6及脊髓背角中IL-6和SOCS3的表达进行检测,定性定量分析指标表达的变化,阐述拨法促进神经病理性疼痛缓解,起到"消炎止痛"作用的可能起效机制。[结果]1行为学结果造模7天后:正常组大鼠活动均正常;假手术组大鼠术侧肢体轻微跛行,无爪挛缩,耐痛阈与正常组大鼠耐痛阈无显著性差异(P0.05);模型组和拨法组大鼠逐渐出现行走时术侧肢体跛行状况,踝部下垂,且脚掌不能平放于地面,足背着地现象,耐痛阈与正常组相比显著下降(P0.05)。拨法干预20天后:正常组大鼠活动均正常;假手术组大鼠轻微跛行现象消失,基本与正常组大鼠行动状态无差别(P0.05);模型组大鼠疼痛症状未见缓解,术侧持续出现爪挛缩,悬空不与地面接触,关节僵硬;拨法组大鼠术侧肢体仍有跋行现象,脚掌仍不能平放于地面,但爪挛缩现象较模型组有好转,光热耐痛阈结果虽与同期正常组仍有差距(P0.05),但与同期模型组相比已有显著性升高(P0.05)。2形态学结果造模7天后,正常组、假手术组大鼠术侧坐骨神经切片HE染色在400倍光镜下可见坐骨神经纤维排列整齐、紧密;模型组和拨法组则可见形态较不规则,神经纤维排列不规则,间质疏松水肿。拨法干预20天后,正常组、假手术组大鼠神经切片染色状况正常,模型组光镜下可见轴索水肿严重,排列紊乱,结构不清。拨法组可见轴索排列整齐度有所恢复,神经纤维排列有序性提高。3酶联免疫吸附试验(ELISA)检测结果造模7天后:经ELISA检测,假手术组大鼠血清中IL-6的含量与正常组相比无显著性差异(p0.05);模型组与同期正常组相比有显著升高(P0.05)。拨法干预20天后:拨法组大鼠血清中IL-6的表达较同期正常组仍有显著性差异(P0.05),但含量明显降低,与造模7天后的同组比较,其含量约下降了 30%,逐渐接近于正常组;拨法组跟同期模型组大鼠血清中IL-6的含量相比明显下降,有显著性差异(P0.05)。4免疫组化染色检测结果(1)IL-6检测结果:造模7天后,假手术组大鼠脊髓背角中IL-6免疫阳性细胞零星分布,与正常组无显著性差异(P0.05),模型组与同期正常组相比显著升高(P0.05);拨法干预20天后,拨法组大鼠脊髓中IL-6的表达与同期正常组相比仍较高(P0.05),但与造模7天后的同组比较,表达量约下降了 30%;拨法组大鼠脊髓背角中IL-6的表达量约为同期模型组的70%,与同期模型组相比有显著性差异(P0.05)。(2)SOCS3检测结果:造模7天后,假手术组大鼠脊髓背角中SOCS3的表达与正常组相比,无显著性差异(P0.05),模型组与同期正常组相比SOCS3的表达显著升高(P0,05)。拨法干预20天后,拨法组大鼠脊髓背角中SOCS3的表达持续升高,约为造模7天后同组表达量的1.4倍,与同期模型组相比有显著性差异(P0.05),约为同期模型组表达量的1.3倍。5蛋白免疫印迹检测结果造模7天后,假手术组大鼠脊髓中IL-6的相对蛋白含量与同期正常组相比无显著性差异(P0.05);模型组IL-6相对蛋白含量与同期正常组相比有差异性升高(P0.05)。拨法干预20天后,拨法组大鼠脊髓中IL-6的相对蛋白含量与正常组相比仍有显著差异(P0.05),但其含量己有明显下降;同期模型组大鼠脊髓中IL-6的相对蛋白含量虽也有下降,但比之于同期拨法组的相对蛋白含量下降程度有较大差异(P0.05)。[结论]1.拨法干预能够提高CCI大鼠光热耐痛阈,缓解CCI大鼠痛觉过敏的症状。2.拨法干预能够促进CCI大鼠受损神经的再生,有利于减轻轴突肿胀,促进轴索及神经纤维的有序性排列。3.拨法干预能够显著降低血清及脊髓中促炎性因子IL-6的表达,抑制外周及中枢炎性反应,起到"消炎止痛"的作用。4.拨法能够显著提高抗炎保护因子SOCS3在脊髓背角的表达,抑制神经炎症反应、中枢敏化和痛觉转导,缓解疼痛。本次实验结果表明,拨法干预能够明显改善痛觉异常,缓解痛觉过敏,促进受损神经细胞的恢复;拨法能够通过下调血清及脊髓中的促炎性因子IL-6的表达,以及上调脊髓中抗炎保护因子SOCS3的表达消除炎症、抑制疼痛传导、改善神经病理性疼痛,从而起到"消炎止痛"的作用。
[Abstract]:[Objective] to study the effect of dialing method to improve the hyperalgesia of neuropathic pain rats and to promote the possibility of nerve repair through the study of behavioral and morphological indexes. Through the study of the expression of proinflammatory factor IL-6 and anti-inflammatory protective factor SOCS3 in the serum and spinal cord, we explore the effect of dialing on neuropathic pain and play the role of "anti-inflammatory and analgesic". The possible mechanism was possible. [Methods] SD male rats were used as experimental animals and randomly divided into normal group, sham operation group, model group and dialing group, chronic constriction injury (CCI) was selected as model, and "massage massage instrument Simulator" (People's Republic of China invention patent number: ZL200710) was used. 187403.1.) the rats were intervened with the simulated operation. The acupoints were selected by Yin gate, Chengshan and Yang Ling spring, each point 3min, each total of 9min, 1 times a day, each intervention was intervened for 1 days for 20 days, and two time points were selected for 7 days and 20 days after the intervention, and the photothermal pain threshold test of the rats before taking the material was evaluated and evaluated. The recovery of hyperalgesia. HE staining of the sciatic nerve in rats was carried out and the results were observed and analyzed to find morphological evidence for the repair of nerve injury by dialing method. The expression of IL-6 and SOCS3 in IL-6 and spinal dorsal horn in the rat serum was detected, the changes of the expression of qualitative and quantitative analysis index were observed, and the dialing method promoted neuropathy. The mechanism of rational pain relief played a possible effect mechanism of "anti-inflammatory and analgesic" effect. [results]1 behavior results of 7 days: normal group rats were normal, the sham operation group had slight limp, no claw contracture, and no significant difference in pain threshold between the normal group and the normal group (P0.05); the model group and the dialing group gradually appeared to walk. At the time of lame, the ankle was drooping, and the ankle was not flat on the ground, the foot was back to the ground, the pain threshold was significantly lower than the normal group (P0.05). 20 days after the intervention, the normal group rats were normal; the sham group's slight limp disappeared, and the basic group was not different from the normal group (P0.05); model group was large. The pain symptoms of the rats were not relieved, the side of the operation continued to appear claw contracture, the suspension was not exposed to the ground, and the joint was stiff. The lateral limbs of the rats in the dialing group still had a postscript, but the paws were still unable to be placed on the ground, but the contracture of the paws was better than the model group. The result of the light and heat tolerance threshold still had a gap with the normal group (P0.05), but it was with the same period model group. 7 days later than the existing significant increase (P0.05).2 morphological results, the normal group and the sciatic nerve section of the sham operation group were stained with the sciatic nerve under the 400 times light microscope, and the sciatic nerve fibers were arranged neatly and tightly under the microscope. The model group and the dialing group showed irregular shape, irregular arrangement of nerve fibers, interstitial edema and edema. The dialing method intervened for 20 days. After the normal group, the nerve section of the rats in the sham operation group was normal. The model group showed that the axon edema was serious, the arrangement was disorderly and the structure was not clear. The arrangement of axonal arrangement was recovered and the arrangement of nerve fibers was improved by the.3 enzyme linked immunosorbent assay (ELISA) for 7 days: ELISA test and sham operation There was no significant difference in the content of IL-6 in the serum of the rats compared with the normal group (P0.05); the model group was significantly higher than the normal group (P0.05). 20 days after the intervention, the expression of IL-6 in the serum of the dialing group was significantly different from that of the normal group (P0.05), but the content of the serum was significantly lower than that in the same group after the 7 days of the model. The decrease of about 30%, gradually approaching to the normal group; the dialing group was significantly lower than the IL-6 content in the serum of the model rats. There was a significant difference (P0.05).4 immunohistochemical staining results (1) IL-6 detection results: 7 days after the model, the IL-6 immunoreactive cells in the spinal dorsal horn of the sham operation group were scattered, and there was no significant difference between the normal group and the normal group. The difference (P0.05), the model group was significantly higher than the normal group in the same period (P0.05). The expression of IL-6 in the spinal cord of the dialing group was still higher than that of the normal group (P0.05) after 20 days of dialing intervention, but compared with the same group for the 7 days after the model, the expression amount decreased by 30%, and the expression of IL-6 in the spinal dorsal horn of the dialing group was about 70 in the same period model group. Compared with the model group in the same period (P0.05). (2) SOCS3 detection results: the expression of SOCS3 in the dorsal horn of the spinal cord in the sham operation group had no significant difference compared with the normal group (P0.05), and the expression of SOCS3 in the model group was significantly higher than that of the normal group (P0,05). The expression of the SOCS3 in the spinal dorsal horn of the rats in the dialing group was SO after 20 days. The expression of CS3 continued to rise, which was about 1.4 times that of the same group in the same group after 7 days of modeling (P0.05). There was no significant difference in the protein content of IL-6 in the spinal cord of the sham operation group compared with the normal group at the same time (1.3 times of the expression of.5 protein in the model group). The relative protein content of IL-6 in the model group was significantly higher than that in the normal group (P0.05). The relative protein content of the IL-6 in the spinal cord of the dialing group was significantly different from that of the normal group (P0.05) after 20 days of dialing intervention (P0.05), but the content of the protein in the spinal cord of the group was significantly decreased, although the relative protein content of the IL-6 in the spinal cord of the model group of the same stage model group also had the relative protein content. There is a significant difference in the decrease of the relative protein content in the same period group (P0.05). [conclusion]1. dial intervention can improve the light and heat tolerance threshold of CCI rats, and alleviate the symptoms of CCI rats' hyperalgesia by.2. dialing intervention can promote the regeneration of damaged nerve in CCI rats, reduce axonal swelling and promote axon and nerve fiber. The orderly arrangement of.3. dialing can significantly reduce the expression of proinflammatory factor IL-6 in the serum and spinal cord, inhibit the peripheral and central inflammatory reactions, and play the role of "anti-inflammatory and analgesic",.4. dialing can significantly improve the expression of anti-inflammatory protective factor SOCS3 in the dorsal horn of the spinal cord, inhibit the inflammatory reaction of the deity, central sensitization and pain transduction, and relieve pain. The results of this experiment showed that the dialing method can obviously improve the abnormal pain, relieve the hyperalgesia and promote the recovery of damaged nerve cells. The dialing can reduce the expression of IL-6 in serum and spinal cord, and up the expression of anti inflammatory protective factor SOCS3 in the spinal cord to eliminate inflammation, inhibit the conduction of pain and improve neuropathy. Rational pain, so as to play the role of "anti-inflammatory and analgesic".
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R244.1;R-332

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