【作者】 张耀；

【导师】 刘旭光；

【作者基本信息】 成都中医药大学 ， 针灸推拿学， 2013， 博士

【摘要】 目的：本研究依据“艾灸治疗RA抗炎效应明确,胆碱能抗炎通路对全身和局部炎症均具有明显的抑制作用”的基本事实,以针灸理论为指导,艾灸家兔“肾俞”、“足三里”治疗实验性RA,围绕滑膜炎症关键病理环节,选择胆碱能抗炎通路作为研究切入点,以“艾灸刺激一胆碱能抗炎通路一抗炎效应”为研究主线,运用神经阻断、免疫组化、实时荧光定量PCR等方法技术,探索胆碱能抗炎通路在艾灸治疗RA抗炎效应中的作用机制,对于深化艾灸治疗RA的原理研究、丰富灸法的科学内涵具有重要的价值和意义。方法：将日本大耳白兔随机分为正常对照组、RA模型组、艾灸治疗组、艾灸治疗迷走神经切断组、艾灸治疗假手术组,用福氏完全佐剂塑造RA疾病模型,运用颈部左侧迷走神经干切断术作为干预手段,艾灸“肾俞”、“足三里”治疗。1.观察RA家兔的整体状况、体重、膝关节周长、滑膜组织炎症病理形态；2.采用Elisa法检测滑膜液中TNF-α、IL-1、IL-6、HMGB1含量；3.采用Elisa法检测血清中a7nAChR和ChAT的含量；分别采用免疫组化法和实时荧光定量PCR法检测滑膜组织中a7nAChR、ChAT的蛋白表达和mRNA表达。4.采用免疫组化法检测滑膜组织中NF-κBp65、NF-κBp50、IκBα、IKKβ的表达。结果：1.艾灸家兔“肾俞”、“足三里”可以明显改善RA家兔全身状况、膝关节肿胀、关节滑膜炎症病理变化,而在切断迷走神经的情况下其作用显著降低。2.滑膜液中细胞因子检测结果显示：模型组与正常对照组相比较,RA模型组关节滑膜液中TNFα、IL-1、IL-6、HMGB1含量显著增高(P<0.01)；与RA模型组比较,艾灸治疗组、艾灸治疗假手术组关节滑膜液中TNFα、IL-1、IL-6、 HMGB1含量显著降低(P<0.01),而艾灸治疗迷走神经切断组无明显降低((P>0.05)；与艾灸治疗组比较,艾灸治疗迷走神经切断组滑膜液中TNFα、IL-1、 IL-6、HMGB1含量显著增高(P<0.01),但艾灸治疗假手术组无明显增高(P>0.05)。3.α7nAChR、ChAT表达结果显示：模型组与正常对照组相比较,模型组血清中α7nAChR、ChAT含量、滑膜组织中α7nAChR、ChAT蛋白表达和mRNA表达均显著降低(P<0.01)；与模型组比较,艾灸组和假手术组血清中α7nAChR、 ChAT含量、滑膜组织中α7nAChR、ChAT蛋白表达和mRNA表达均显著增高(P<0.01),而艾灸治疗迷走神经切断组无明显增高(P>0.05)；与艾灸组比较,艾灸治疗迷走神经切断组血清中α7nAChR、ChAT含量、滑膜组织中α7nAChR、ChAT蛋白表达和mRNA表达均显著降低(P<0.01),但假手术组无明显降低(P>0.05)。4.①NF-κBp65、NF-κBp50、p-IκBα、IKKβ表达结果显示：模型组与正常对照组相比较,模型组滑膜组织中NF-κBp65、NF-κBp50、p-IκBα、IKKβ的表达显著增高(P<0.01)；与模型组比较,艾灸组和假手术组滑膜组织中NF-κBp65、 NF-KBp50、p-IκBα、IKKβ的表达显著降低(P<0.01),而艾灸治疗迷走神经切断组无明显降低(P>0.05)；与艾灸组比较,艾灸治疗迷走神经切断组滑膜组织中NF-KBp65、NF-κBp50、p-IKBα、IKKβ的表达显著增高(P<0.01),但假手术组无明显增高(P>0.05)。②IκBα表达结果显示：模型组与正常对照组相比较,模型组滑膜组织中IκBα的表达显著降低(P<0.01)；与模型组比较,艾灸组和假手术组滑膜组织中IκBα的表达显著增高(P<0.01),而艾灸治疗迷走神经切断组无明显增高(P>0.05)；与艾灸组比较,艾灸治疗迷走神经切断组滑膜组织中IκBα的表达显著降低(P<0.01),但假手术组无明显降低(P>0.05)。结论：1.艾灸家兔“肾俞”、“足三里”可以明显改善实验性RA家兔全身状况、膝关节肿胀、关节滑膜炎症病理变化,表明艾灸具有明确的抗炎消肿、抑制滑膜炎症作用,而在切断迷走神经的情况下其抗炎作用显著降低,提示艾灸治疗RA的抗炎效应可能与兴奋胆碱能抗炎通路密切相关。2.艾灸家兔“肾俞”、“足三里”可以抑制实验性RA家兔滑膜液中细胞因子TNF-α、IL-1、IL-6和HMGB1水平,但在切断迷走神经的情况下其作用显著下降,提示兴奋胆碱能抗炎通路可能是艾灸抑制实验性RA家兔滑膜细胞分泌促炎细胞因子的重要机制之一。3.艾灸家兔“肾俞”、“足三里”可以上调ChAT在RA家兔外周血和滑膜组织中的表达,激活免疫系统的固有胆碱能系统,但在切断迷走神经的情况下其作用显著降低,提示艾灸可能通过兴奋迷走神经释放ACh,并与免疫细胞表面的a7nAChR结合,从而激活免疫细胞的固有胆碱能系统,转化为免疫系统内弥散的、持续的免疫调节。4.艾灸家兔“肾俞”、“足三里”可以抑制RA家兔滑膜组织中NF-κBp65、 NF-κBp50、p-IκBα、IKKβ的蛋白表达,上调IκBα的蛋白表达,但在切断迷走神经的情况下其作用显著降低,因此推测艾灸可能通过抑制IKK/IκB/NF-κB信号转导通路活化,改善RA家兔滑膜NF-κB的高调控状态,抑制滑膜细胞炎性细胞因子和炎性介质的合成和分泌,从而实现其抗炎效应,胆碱能抗炎通路在其中具有重要作用。更多还原

【Abstract】 Objective:The research is based on the fact that "moxibustion has obvious anti-inflammatory effects of treating RA and cholinergic anti-inflammatory pathway can obviously inhibit systemic and local inflammation ". Guided by the theory of acupuncture and moxibustion, experimental RA rabbits were treated by moxibustion at "Shenshu"" Zusanli"points. Focusing on the key pathological character of synovitis, we selected cholinergic anti-inflammatory pathway as the breakthrough point of the research and "moxibustion stimulation-cholinergic anti-inflammatory pathway-anti-inflammatory effecf" as the main line of the research. By the use of nerve block, immunohistochemistry, RT-PCR and other methods and techniques, to explore cholinergic anti-inflammatory pathway’s mechanism of action in treating RA by moxibustion, the research is of important value for deepening the study of the principles of moxibustion treatment of RA and enriching scientific connotation of moxibustion.Methods:Japanese big-eared white rabbits were randomly divided into normal control group, RA model group, moxibustion treatment group, normal treatment of vagotomy group, moxibustion treatment of sham operation group. RA animal models were produced by Freunds1Complete Adjuvant (FCA) and left truncal vagotomy of the neck was as a means of intervention. RA rabbits were treated by moxibustion at "Shenshu"" Zusanli"points.1. RA rabbits’overall condition, body weight, knee joint perimeter, pathological morphology of synovial membrane tissue inflammation were observed.2. TNF-a, IL-1, IL-6, HMGB1in synovial fluid were detected by enzyme-linked immunosorbent assay (ELISA).3.Content of α7nAChR, ChAT in blood serum were detected by enzyme-linked immunosorbent assay (ELISA). Expression of a7nAChR, ChAT in synovial membrane tissue were detected by immunohistochemistry method and mRNA expression of a7nAChR, ChAT in synovial membrane tissue were detected by RT-PCR method.4. Expression of NF-KBp65, NF-κBp50, IκBα, p-IκBa, IKKβ in synovial membrane tissue were detected by immunohistochemistry method.Results:1. Moxibustion at "Shenshu" and "Zusanli" points of rabbits could obviously improve RA rabbits" overall condition, body weight, knee joint perimeter, pathological morphology of synovial membrane tissue inflammation, but its effect was decreased significantly after cutting off the vagus nerve.2. The expression results of cytokine in synovial fluid showed:Compared with the normal control group, RA model group’s expression of TNF a, IL-1, IL-6, HMGB1in synovial fluid were significantly increased (P<0.01); Compared with model group, moxibustion group and sham operation group were decreased significantly (P<0.01) and vagotomy group was not decreased significantly (P>0.05); Compared with moxibustion group, vagotomy group were significantly increased (P<0.01), but the sham operation group was not significantly increased (P>0.05).3. The expression results of a7nAChR and ChAT showed:Compared with the normal control group, RA model group’s expression of α7nAChR, ChAT in blood serum and proteinum expression of α7nAChR, ChAT in synovial membrane tissue mRNA expression of a7nAChR, ChAT in synovial membrane tissue were decreased significantly (P<0.01); Compared with model group, moxibustion group and sham operation group’s expression of a7nAChR, ChAT in blood serum and proteinum expression of a7nAChR, ChAT in synovial membrane tissue mRNA expression of a7nAChR, ChAT in synovial membrane tissue were significantly increased (P<0.01) and vagotomy group was not increased significantly (P>0.05); Compared with moxibustion group, vagotomy group were significantly decreased (P<0.01), but the sham operation group was not significantly decreased (P>0.05).4.①The expression results of NF-κBp65, NF-κBp50, p-IκBα, IKKα showed: Compared with the normal control group, RA model group’s expression of NF-κBp65, NF-κBp50, p-IκBα, IKKP in synovial membrane tissue were significantly increased (P<0.01); Compared with model group, moxibustion group and sham operation group’s expression of NF-κBp65, NF-KBp50, p-IκBα, IKKP in synovial membrane tissue were decreased significantly (P<0.01) and vagotomy group was not decreased significantly (P>0.05); Compared with moxibustion group, vagotomy group were significantly increased (P<0.01), but the sham operation group was not significantly increased (P>0.05).②The expression results of IκBα showed:Compared with the normal control group, RA model group’s expression of IκBαin synovial membrane tissue were significantly decreased (P<0.01); Compared with model group, moxibustion group and sham operation group’s expression of IκBα in synovial membrane tissue were increased significantly (P<0.01) and vagotomy group was not increased significantly (P>0.05); Compared with moxibustion group, vagotomy group were significantly decreased (P<0.01), but the sham operation group was not significantly decreased (P>0.05).Conclusions:1. Moxibustion at "Shenshu" and "Zusanli" points of rabbits can obviously improve RA rabbits’overall condition, knee joint perimeter,pathological morphology of synovial membrane tissue inflammation,which shows that moxibustion has a definite effect of anti-inflammatory, detumescence and inhibition of synovial inflammation. But its effect is decreased significantly after cutting off the vagus nerve. It prompts that anti-inflammatory effect of moxibustion for treatment of RA may be closely related to stimulating cholinergic anti-inflammatory pathway.2. Moxibustion at "Shenshu" and "Zusanli" points of rabbits can inhibit expression of TNF a, IL-1, IL-6, HMGB1in synovial fluid of RA rabbits, but its effect is decreased significantly after cutting off the vagus nerve. It prompts that stimulating cholinergic anti-inflammatory pathway may be one of the important mechanisms that moxibustion inhibit expression of proinflammatory cytokines in synovial fluid of experimental RA rabbits.3.Moxibustion at "Shenshu" and "Zusanli" points of rabbits can increase the expression of a7nAChR、ChAT in blood serum and synovial membrane tissue of RA rabbits, and activate the inherent cholinergic system in immune system, but its effect is decreased significantly after cutting off the vagus nerve. It prompts that moxibustion may promote the vagus nerve to release ACh and make ACh combine with a7nAChR of immune cells, thereby activating the inherent cholinergic system in immune cells and converting to diffuse, sustained immune regulation within the immune system.4. Moxibustion at "Shenshu" and "Zusanli" points of rabbits can inhibit the expression of NF-κBp65、NF-icBp50、p-IκBα、IKKP in synovial membrane tissue of RA rabbits,and increase the expression of IicBa, but its effect is decreased significantly after cutting off the vagus nerve. Therefore we speculate that by inhibiting the activation of IKK/IκB/NF-κB signal transduction pathway moxibustion may restrain high regulation of NF-κB in RA rabbits’synovium and inhibit the expression of Inflammatory cytokines in synovial cells, and then achieve its anti-inflammatory effect, in which cholinergic anti-inflammatory pathway plays an important role.更多还原