【作者】 汪元；

【导师】 刘健；

【作者基本信息】 湖北中医药大学 ， 中医内科学， 2010， 博士

【摘要】 1.目的以中医理论为指导，总结分析类风湿关节炎(RA)神经-内分泌-免疫(NEI)网络的变化及中医药对其影响；通过动物实验,研究佐剂性关节炎(AA)大鼠神经递质、下丘脑-垂体-肾上腺(HPA)轴功能、血管神经肽、调节性T细胞及Foxp3表达的变化及健脾化湿通络法其影响,基于数据挖掘技术对上述研究结果进行系统分析,全面揭示健脾化湿通络法对AA大鼠NEI网络的调控作用及可能机制,丰富RA“从脾论治,整体调节”的科学内涵,为中医药治疗RA的机理研究提供新思路。2.方法2.1理论研究通过文献研究,总结分析中医理论与NEI网络学说的关系,阐述RANEI网络的变化及中医健脾法对其影响。2.2实验研究将84只Wistar雄性大鼠按随机数字表分为7组：正常对照组、模型对照组、甲氨喋呤(MTX)组、雷公藤多甙片(TPT)组和新风胶囊(XFC)高、中、低剂量组,每组12只,除正常对照组外,向每只动物右后足跖皮内注射弗氏完全佐剂0.1ml致炎,复制AA大鼠模型。致炎后第19天开始给予相应药物治疗30 d。观察各组大鼠的体质量、足跖肿胀度、关节炎指数；酶联免疫吸附法检测各组大鼠血清5-羟色胺(5-HT)、多巴胺(DA)、促肾上腺皮质激素(ACTH)、皮质醇(CORT)、肿瘤坏死因子α(TNF-α)、白介素-10(IL-10),内皮素-1(ET-1)和降钙素基因相关肽(CGRP)含量及脑组织ET-1、CGRP含量；流式细胞术检测大鼠血液CD4+CD25+调节性T细胞表达频率；免疫组化法检测下丘脑糖皮质激素受体(GR)蛋白表达；逆转录-聚合酶链反应法检测下丘脑促肾上腺激素释放激素(CRH)mRNA表达,大脑皮质GR mRNA表达及脾脏细胞Foxp3mRNA表达；免疫印迹法检测大鼠胸腺组织Foxp3蛋白表达水平。3.结果3.1理论研究结果NEI网络学说与中医理论关系紧密,NEI网络的双向调节和动态平衡是中医阴阳平衡理论及整体观念的重要体现,NEI网络在RA发病中发挥重要作用,而中医脾脏是NEI网络的调节中枢,中医健脾法通过调整NEI网络平衡发挥对RA的综合治疗作用。3.2实验研究结果3.2.1健脾化湿通络法对AA大鼠的疗效分析健脾化湿通络中药新风胶囊治疗组AA大鼠的体质量增长显著高于模型对照组、MTX组及TPT组,与正常对照组无显著差异(P>0.05),MTX组及TPT组体质量增长显著低于正常对照组(P<0.01)；新风胶囊治疗组AA大鼠足跖肿胀度和关节炎指数显著低于模型对照组(P<0.01,或P<0.05),与其余各治疗组间无明显差异(P>0.05)。3.2.2健脾化湿通络法能抑制AA大鼠神经递质5-HT分泌研究结果显示：与正常对照组比较,模型组大鼠血清5-HT水平显著升高(R0.05),各治疗组5-HT水平均有所下降,组间比较显示XFC组下调作用更为明显(P<0.05),各组DA水平无显著变化。3.2.3健脾化湿通络法能调整AA大鼠HPA轴功能紊乱研究结果显示：与正常对照组比较,模型组大鼠血清ACTH、CORT水平及大脑皮质GRmRNA表达显著升高(P<0.05,或P<0.01),下丘脑CRHmRNA表达降低(P<0.01)；与模型组比较,新风胶囊组能有效下调AA从大鼠血清ACTH及CORT水平,下调AA大鼠大脑皮质GRmRNA表达,上调下丘脑CRHmRNA表达,作用显著优于MTX和TPT组(P<0.05,或P<0.01)。3.2.4健脾化湿通络法能降低TNF-α水平,促进IL-10分泌研究结果显示：与正常对照组比较,模型对照组大鼠TNF-α显著升高,IL-10值降低(P<0.05或P<0.01)；与模型对照组相比,XFC、MTX、TPT组均能降低TNF-α(P<0.01),升高IL-10(P<0.01);各治疗组相比,XFC组在降低TNF-α,升高IL-10方面优于MTX组和TPT组(P<0.05或P<0.01)。3.2.5健脾化湿通络法能调节AA大鼠血管活性肽ET-1、CGRP水平研究结果显示：与正常对照组比较,模型组大鼠血清及脑组织中ET-1含量明显升高(P<0.01),CGRP含量明显降低(P<0.01)；与模型组比较,各治疗组均能显著降低ET-1水平,升高CGRP水平(P<0.01),XFC中、高剂量组降低ET-1,升高CGRP作用明显优于MTX和TPT组(P<0.05,或P<0.01)。3.2.6健脾化湿通络法能上调CD4+CD25+Treg比例,促进Foxp3表达研究结果显示：与正常对照组比较,模型组大鼠CD4+CD25+Treg比例明显下降(P<0.01),脾脏Foxp3mRNA及胸腺Foxp3蛋白表达明显降低(P<0.01)；与模型组比较,XFC中、高剂量组能明显增加大鼠CD4+CD25+ Treg比例(P<0.01),升高胸腺Foxp3蛋白表达(P<0.01),明显优于MTX组和XFC低剂量组(P<0.05,或P<0.01)。3.2.7NEI网络检测指标的分层聚类分析分层聚类分析结果表明,全部检测指标分为两大类,在AA模型中表现为升高的CORT、TNF-α、5-TH、ACTH、组织ET、AI、ET-1、Treg、GRmRNA、关节肿胀度、DA等11项指标优先聚合,在这些指标中CORT、TNF-α、5-TH三者关系最为紧密；在AA模型中表现为降低的CGRP、胸腺Foxp3、IL-10、脾脏Foxp3、组织CGRP、CRH等6项指标再聚合,在这6项指标中CGRP、IL-10、胸腺及脾脏Foxp3表达关系最为紧密。这表明,在评价NEI网络各指标的变化时,可在两大类指标中选择具有较强相关性的指标即可,其效果与检测全部指标基本是等价的,但更经济快捷,易于发现各指标间的变化规律。4.结论4.1NEI网络学说与中医理论关系紧密NEI网络的双向调节和动态平衡是中医阴阳平衡理论及整体观念的重要体现,NEI网络在RA发病中发挥重要作用,而中医脾脏是调节NEI网络的重要中枢,中医健脾法能从多靶点、多环节、多层次、多途径对RA发挥整体调节作用。4.2AA大鼠存在NEI网络功能紊乱AA大鼠神经系统功能紊乱主要表现为神经递质5-HT水平显著升高；内分泌系统功能紊乱主要表现为血清ACTH、CORT水平及大脑皮质GRmRNA表达显著升高,下丘脑CRHmRNA表达降低；血管活性肽ET-1含量明显升高,CGRP含量明显降低；免疫系统功能紊乱主要表现为致炎细胞因子TNF-α升高,抑炎细胞因子IL-10降低；CD4+CD25+Treg下降,脾脏Foxp3mRNA及胸腺Foxp3蛋白表达明显降低。4.3健脾化湿通络法对AA大鼠NEI网络显示出良好的调控作用健脾化湿通络中药新风胶囊能有效降低AA大鼠足跖肿胀度和关节炎指数,对大鼠体质量无不良影响；新风胶囊能抑制AA大鼠神经递质5-HT分泌,有效下调AA大鼠血清ACTH及CORT水平,下调AA大鼠大脑皮质GRmRNA表达,上调下丘脑CRHmRNA表达,从下丘脑或更高层的中枢结构维持HPA轴环路稳态；降低TNF-α水平,促进IL-10分泌,维持细胞因子网络平衡；降低ET-1水平,升高CGRP水平；上调CD4+ CD25+ Treg比例,促进脾脏Foxp3mRNA及胸腺Foxp3蛋白表达。4.4基于NEI网络调控的健脾化湿通络法治疗AA大鼠的作用机制4.4.1健脾化湿通络法能降低外周神经递质5-HT的释放,减少5-HT的致痛效应,减轻关节症状。4.4.2健脾化湿通络法能有效下调AA大鼠血清ACTH及CORT水平,下调AA大鼠大脑皮质GRmRNA表达,上调下丘脑CRHmRNA表达,从下丘脑或更高层的中枢结构维持HPA轴环路稳态,抑制炎症反应。4.4.3健脾化湿通络法通过调节血管活性肽ET-1与CGRP的平衡,调整关节滑膜新生血管的舒缩功能,改善关节局部血管功能障碍,抑制致炎细胞因子的生成而促进关节局部炎症消退,发挥抗炎作用。4.4.4健脾化湿通络法通过上调CD4+ CD25+ Treg,促进Foxp3表达,促进抑炎细胞因子生成,抑制自身免疫T细胞活化,发挥抗炎抗免疫作用。更多还原

【Abstract】 1.Objective:According to the theory of Tranditional Chinese Medicine (TCM), summing up changes of neuro-endocrine-immune(NEI)network of Rheumatoid Arthritis (RA) and analyzing impacts of Chinese medicine on it.Observing the effect of Strengthening the spleen-Resolving dampness-Promoting collaterals method on neurotransmitters in adjuvant arthritis (AA) rats,and the function of hypothalamus pituitary adrenal (HPA) axis,the changes of vascular neuronpeptide, regulatory T cells and Foxp3 expression, exploring the mechanism of strengthening spleen, resolving dampness and activating collaterals herb on NEI network of AA rats,to enrich the scientific connotation of the treatment of RA based on "Spleen"2.Methods2.1 Theory researchThrough literature review, expounded the relationship between Chinese medicine theory and the NEI network theory,described the changes of RA NEI network and impacts by Chinese medicine. 2.2 Experimental researchEighty-four rats were averagely randomized into normal control(NC) group, model control (MC) group, methotrexate (MTX) treated group, Tripterygium wilfordii polyglycoside Tablet (TPT) treated group, XFC low-dose, middle-dose, high-dose treated groups,12 in each. Except for rats in NC group, the remaining rats in other groups were induced to AA model by intracutaneous injection of 0.1 ml of Freund’s complete adjuvant in the right hindlimb. Serum levels of 5-hydroxytryptamine(5-HT),dopamine(DA),adrenocor ticotro-pic hormone(ACTH),cortisol(CORT),tumor necrosis factor-α(TNF-a),and interleukin-10(IL-10),the levels of endoth-elin-1(ET-1)and calcitionin gene related peptide(CGRP)in serum and brain tissue in all groups were assayed using enzyme linked immunosorbent assay(ELISA).The frequency of CD4+ CD25+ Treg were measured by flow cytometry;The expressions of glucocorticoid receptor(GR) in hypothalamic were measured by immunohistochemistry;The expressions of corticotrophin relea-seeing hormone(CRH) mRNA in hypothalamic,GR mRNA in the cerebral cortex and Foxp3 mRNA in spleen cell were measured by reverse transcription-polymerase chain reaction(RT-PCR);The levels of Foxp3 protein expression in rat thymus tissue were detected by Western blot.3.1 The results of theory researchNEI network theory and TCM theory have very close 1 inks,two-way adjustment and dynamic equilibrium of NEI network were an important manifestation of Yin-Yang theory and the overall concept of TCM, NEI network play an important role in the pathogenesis of RA, while the Spleen in Chinese medicine is an important regulation center of NEI network. The treatment of RA based on "Spleen" can regulate the function of NEI network effectively.3.2 The results of experimental research3.2.1 The treatment based-on "Spleen" can inhibit the neuron-transmitter 5-HT secretion of AA ratsCompared with those in NC group, serum level of 5-HT in AA rats was significantly increased (P<0.05).XFC could signify-cantly decrease serum level of 5-HT,its effect being signify-cantly better than that of MTX and TPT (P<0.05 or P<0.01).No significant difference in DA levels was found among seven groups (P>0.05).3.6.2 The treatment based on "Spleen" can adjust the dys-function of HPA axis in AA ratsCompared with normal group, serum levels of ACTH, CORT and the expression of GRmRNA of cerebral cortex in AA rats were significantly increased (P<0.05,or P<0.01),while the expre-ssion of CRHmRNA in hypothalamic was decreased (P<0.01). Compared with control group,XFC could significantly reduced the levels of ACTH and CORT in serum, and the expression of GRmRNA in cerebral cortex,advanced the expression of CRHmRNA in hypothalamus,better than MTX and TPT group (P<0.05 or P<0.01).3.6.3 The treatment based on "Spleen" can regulate the levels of vascular peptide ET-1,CGRP in AA ratsCompared with the control,ET-1 level increased markedly (P<0.01),and CGRP level decreased markedly (P<0.01);XFC middle and high doses groups can reduce the release of ET-1 and increase the level of CGRP in serum significantly (P<0.01);XFC high and middle dose groups can reduce the release of ET-1 and increase the level of CGRP in brain tissue significantly,better than the control group and XFC low-dose group(P<0.05 or P<0.01),showed a definite dose-effect relationship.3.6.4 The treatment based on "Spleen" can upregulate CD4+CD25+Treg, promote’the expression of Foxp3Compared with normal group,the proportion of CD4+CD25+Treg was significantly decreased (P<0.01),the expression of Foxp3 mRNA in spleen and thymus was significantly lower (P<0.01);Compared with control group,XFC high and middle dose groups can significantly increased CD4+CD25+Treg ratio (P<0.01),increased expression of Foxp3 (P<0.01),significantly better than the MTX group and the XFC low dose group (P<0.05,or P<0.01).3.6.5 Hierarchical cluster analysis of relevant indicators of NEI networkAll indicators is divided into two categories,CORT, TNF-α,5-TH, ACTH, ET in tissue, AI,ET-1, Treg, GRmRNA, joint swelling, and dopamine were elevated in AA rats,they are aggregated priority.In these indicators,the relationships of CORT, TNF-αand 5-TH are most closely; CGRP in tissue and serum, F0Xp3 in thymus and spleen,IL-10 and CRH showed reduced in AA rats,in which 6 indicators CGRP, IL-10 and F0Xp3 are most closely.4.conclusionNEI network theory have very close links with YinYang balance theory and the overall concept of TCM, NEI network play an important role in the pathogenesis of RA.The treatment of strengthening spleen, resolving dampness and activating collaterals can regulated the dysfunction of NEI network. Its effective mechanism relate to the following factors: 4.1 The treatment of strengthening spleen, resolving dampness and activating collaterals can inhibit the secretion of neurotransmitter 5-HT and reduce pain caused by 5-HT,relieve joint symptoms.4.2 The treatment of strengthening spleen, resolving dampness and activating collaterals can maintain the steady-state of HPA axis from the hypothalamus or higher central structures.4.3 The treatment of strengthening spleen, resolving dampness and activating collaterals can effectively adjust the ET-1 and CGRP balance, adjust the synovial angiogenesis systolic and diastolic function, improve joint local vascular dysfunction, play an anti-inflammatory role.4.4 The treatment of strengthening spleen, resolving dampness and activating collaterals by improving CD4+CD25+Treg ratio,enhancing the spleen and thymus Foxp3mRNA and protein expression, inhibit secretion of proinflammatory cytokines,inhibit autoimmune T-cell activation, to achieve the effect of anti-inflammatory and anti-immune.更多还原