外界环境与不同方药对CIA大鼠证候及T细胞亚型漂移影响的比较研究

【作者】 周刚；

【导师】 王庆国；

【作者基本信息】 北京中医药大学 ， 中医临床基础， 2007， 博士

【摘要】 类风湿性关节炎(Rheumatoid Arthritis，RA)是一种病因不明的慢性、进展性、自身免疫性疾病。其病理特征为单核细胞浸润、滑膜细胞增生、血管翳形成，以及软骨侵蚀和关节破坏。临床表现为关节对称性疼痛、运动障碍、甚至关节强直畸形致残，以及多脏器受累。目前RA的具体发病机制尚不清楚，多数学者认为，RA是由遗传与环境因素共同作用而引起的一种自身免疫性疾病。RA在中医学属痹证范畴。“痹”字最早见于《黄帝内经》，其病因据“风寒湿三气杂至”及“邪之所凑，其气必虚”的理论，为“内外合邪”所致。张仲景秉承《内经》之旨，开痹证辨证论治之先河，后世医家多遵从《内经》及《金匮要略》之论痹，结合各自所处历史环境与个人临床经验，从不同角度深入对痹证病因病机的认识，并立法组方遣药，或配以针灸、导引等外治法，使痹证证治丰富多彩，各具特色。而治痹方药之组成、配伍，以及方药之寒热温凉等属性，只有与痹证之“证候要素”、“病机”高度相符，该方药才能获得最佳疗效。这即是痹证“方证相应”理论的研究内容，也是痹证临床辨治的客观要求。为观察外界环境对痹证证候的影响，验证痹证乃“内外合邪”的病因学理论，并研究治痹方药与痹证证型的相关性及该相关性的生物学内涵，本课题以RA目前最为公认的动物模型，即Ⅱ型胶原诱导的关节炎(CIA)模型作为热痹证大鼠模型，采用“人工气候”试验箱进行外界环境的“风寒湿”或“风湿热”干预，再选用治疗热痹证的代表方剂——宣痹汤以及治疗寒热错杂痹证的代表方剂——桂枝芍药知母汤进行交叉治疗干预，进行了有关痹证病因学理论以及痹证“方证相应”理论的动物实验研究。1．实验用Ⅱ型胶原在Wistar大鼠诱导CIA模型。在二次免疫后，即从第2周开始，M(模型)组大鼠非致炎足(左足)出现炎性改变，其临床体征与人RA体征相似，血清Anti-CII、IgG抗体以及INF-γ、IL-4、TNF-a等细胞因子显著升高，踝关节H-E染色观察发现典型的RA炎症性改变。综合体征及微观生物学指标，可认为CIA造模成功，热痹证模型建立。2．在CIA造模成功的基础上，采用“人工气候”试验箱，对模型大鼠“风寒湿”(C组)或“风湿热”(H组)干预三周，定量分析大鼠舌象，并观察宏观体征。结果显示，C组大鼠可辨为寒痹证；H组大鼠仍可辨为热痹证，但兼夹瘀血因素。与M组比较，C组IgG抗体、INF-γ、IL-4、TNF-a水平均显著下降，Anti-CII抗体亦下降，H组IgG抗体、INF-γ、IL-4水平显著下降，TNF-a水平亦下降，Anti-CII抗体水平未见明显改变；流式细胞术结果显示，C组Th1、Th1／Th2均显著降低，H组仅见Th1／Th2下降，Th1、Th2所占淋巴细胞比率无明显变化；荧光实时定量(Real-time)PCR技术测定显示，C组T-bet mRNA表达下调，IL-4 mRNA表达显著上调，C组和H组INF-γmRNA表达均下调，差异均具有显著性，但T-bet／GATA-3比值两组均无统计学改变；关节H-E染色后观察显示，H组与M组相似，均病变重，C组病变较轻，偶见炎症细胞浸润。综合宏观体征和微观指标分析表明，“风寒湿”和“风湿热”均能改善Th1／Th2漂移，其中“风寒湿”改善更为明显，但二者对T-bet／GATA-3比值均无明显影响。3．采用同样的实验技术，对M组CIA大鼠灌胃给予宣痹汤(X组)及桂枝芍药知母汤(G组)三周后显示，两组大鼠血清IgG抗体、INF-γ、IL-4、TNF-a水平均降低，Th1、Th1／Th2均显著降低，Th2显著上升，T-bet、INF-γmRNA表达显著下调，IL-4 mRNA表达显著上调，T-bet／GATA-3在G组有改变，在X组明显改变。H-E染色显示，G组病变仍较重，X组偶见炎症细胞，病变减轻。此外，使用缺口末端标记法(TUNEL)原位检测关节组织细胞凋亡显示，两组细胞凋亡率均明显下降，但X组更明显。综合宏观体征和微观指标，宣痹汤与桂枝芍药知母汤相比，前者能更好地治疗CIA大鼠，逆转Th1／Th2漂移，下调T-bet／GATA-3比值。4．同样，对“风寒湿”干预三周后的CIA大鼠，灌胃给予宣痹汤(C+X组)及桂枝芍药知母汤(C+G组)干预三周，结果显示，C+X组大鼠血清Anti-CII抗体水平显著上升，Th1，Th1／Th2亦显著上升，C+G和C+X组均见IL-4 mRNA表达明显下调，仅C+X组T-bet／GATA-3比值上调。关节HE染色后观察显示，C+X组炎症细胞大量浸润，与M组病变程度类似，而C+G组单核细胞浸润减少，病变减轻。TUNEL原位检测关节组织细胞凋亡显示，与C相比较，C+G组细胞凋亡率下降，C+X组未见明显改变，但镜下观察显示C+X组炎症细胞、纤维细胞增加，细胞凋亡加重。综合宏观体征和微观指标，宣痹汤与桂枝芍药知母汤均未明显改善经“风寒湿”干预三周的CIA大鼠病情，反而，宣痹汤使临床症状、关节局部病变，细胞凋亡恶化，且上调了T-bet／GATA-3比值，加剧了Th1／Th2漂移。5．此外，对“风湿热”干预三周后的CIA大鼠，灌胃给予宣痹汤(H+X组)及桂枝芍药知母汤(H+G组)三周，结果显示，仅H+G组大鼠血清Anti-CII抗体水平显著下降，H+G组和H+X组Th1、Th1／Th2均显著降低，Th2显著上升，INF-γmRNA表达均下降，另外，仅H+G组GATA-3 mRNA表达上调，T-bet／GATA-3比值下降。关节HE染色后观察显示，H+G组与H组比较，病变减轻，而H+X组病变仍较重。TUNEL法原位检测关节组织细胞凋亡显示，两组细胞凋亡率均未明显改变。综合宏观体征和微观指标，宣痹汤与桂枝芍药知母汤相比，在改善经“风湿热”干预三周后的CIA临床症状和微观指标方面，后者要优于前者。综上，“风寒湿”或“风湿热”外界环境干预可导致热痹证CIA大鼠证候改变，并都能改善CIA热痹证大鼠的病情，逆转Th1／Th2漂移，但前者影响更为明显，后者对CIA大鼠关节局部病变无影响。这证明，外界环境对痹证证候演变有一定程度的影响，验证了痹证乃“内外合邪”的病因学理论。另外，宣痹汤与桂枝芍药知母汤相比，前者与热痹证大鼠的“证候要素”、“病机”更为相符，能更好地治疗CIA热痹证，逆转Th1／Th2漂移，但与寒痹证CIA大鼠的“证候要素”、“病机”相反，使寒痹证大鼠病情恶化，加剧Th1／Th2漂移：桂枝芍药知母汤对寒痹证大鼠病情及Th1／Th2漂移影响不明显，但与热痹证兼夹瘀血因素大鼠的“证候要素”、“病机”更为相符，能更好改善该证型CIA大鼠病情，但二者对Th1／Th2漂移影响相似。这验证了痹证“方证相应”理论中，只有治痹方药与痹证证候之“要素”、“病机”高度相符，该方药才能获得最佳疗效的正确观点。而且，Th1／Th2细胞亚群漂移程度可能是痹证“方证相应”理论中“方”与“证”符合度在细胞水平的物质基础，T-bec／GATA-3 mRNA表达情况，可能是“方”与“证”符合度在分子水平的物质基础。更多还原

【Abstract】 Rheumatoid Arthritis (RA) is one kind of chronic and progressive autoimmune diseaseswith an unclear etiology. Its pathological characteristics are the infiltration of mononuclearcells, hyperplasia of synoviocytes, formation of new blood vessels, cartilage invasion and jointdestruction. The clinical manifestations include symmetric arthralgia, movement dysfunction,even rigid joint deformation, disability and involvement of multiple organs. At present, thespecific pathogenic mechanism of RA has remained poor-defined. Most scholars think that RAis one kind of autoimmune diseases under the combined effects of genetic and environmentalfactors.RA belongs to Bi-syndrome in TCM. The word of "Bi" was found the earliest in HuangdiNeijin. The etiology of Bi-syndrome was caused by "external evils and internal injuries ", asstated in the book, i.e. "co-attaching of wind, cold, and dampness" and "Where there isinsufficient of qi, where the evils attack". In later times, Zhang Zhongjing followed thestipulations of Neijing and became the first to discuss the syndrome-based treatment ofBi-syndrome. And most physicians in subsequent dynasties adhered to the descriptions ofBi-syndrome based upon Neijing and Jinkui Yaolue. In conjunctions with the historicalenvironment of each individual case and their personal clinical experiences and as viewedfrom difference aspects, an in-depth understanding of etiology and pathogenesis ofBi-syndrome was acquired. And the prescriptions were given according to the stipulations andcomplimented with such external therapeutic methods as acupuncture & moxibustion andphysical-breathing exercises to render the diagnosis and treatment more varied andcharacteristics-filled. And while treating Bi-syndrome, only if the composition, formulationand such features as cold, heat, warm and cool of the TCM ingredients in prescriptions highlycorrespond with the "syndrome factors" and "pathogenesis" of Bi- syndromes could theformula achieve the best therapeutic efficacy. This is also one of the research contents of"correspondence of prescription and syndrome" theory of Bi-syndrome and also one of theobjective requirements of syndrome-based clinical treatment for Bi-syndrome.To observe the effect of external environment on Bi-syndrome so as to justify the theorythat Bi-syndrome caused by "External evils and Internal injuries", and meanwhile to study thecorrelation of Bi-syndrome-treating prescriptions and Bi-syndrome, and understand theunderlying biological mechanism of this kind of correlation, we employed the currently mostaccepted animal model of RA, that is TypeⅡcollagen-induced arthritis (CIA) for rats withheat-Bi-syndrome. The "artificial climate" experiment chamber was employed to perform theexternal environment interventions of "wind-cold & dampness" or "wind-dampness & heat"and then selected the representative prescription of treating heat-Bi-syndrome—Xuan Bi Tang and the representative prescription of treating mixed-cold-heat-Bi-syndrome——GuizhiShaoyao Zhimu Tang to perform crossover treatment interventions. The studies ofexperimental animals upon the etiology of Bi-syndrome and "correspondence of prescriptionand syndrome" for Bi-syndrome were performed as follows.1. The CIA model was experimentally induced by TypeⅡcollagen in Wistar rats. Afterthe second immunization in rats, i.e. starting from the second week, the non-inflammatory feet(left) began to show inflammatory changes in the Group M (model) rats and the clinicalmanifestations were similar to those of RA patients. Furthermore, the serum Anti-CII, lgGantibody and such cytokines as INF-γ,, IL-4 and TNF-a increased markedly in Group M. AfterH-E staining of the rat ankles, the histological observation revealed that there were suchtypical inflammatory changes. Combining the symptomatic and microscopic biologicalparameters, the CIA model could be considered successful and the model of heat-Bi-syndromeestablished.2. Based upon a successful CIA modeling, the "artificial climate" experiment chamberwas employed. After performing a 3-week intervention of external environment upon themodel rat for "wind-cold & dampness" (Group C) or "Wind-dampness & heat" (Group H),the tongue appearance of rats was analyzed quantitatively and the macroscopic parameters ofrats were simultaneously observed. The results showed that the rats in Group C could bedifferentiated as the cold-Bi-syndrome; The rats in Group H could still be differentiated as theheat-Bi-syndrome, but complicated with the feature of blood stasis. Furthermore, as comparedwith Group M, the levels of IgG antibody, 1NF-γ, IL-4 and TNF-a all decreased markedly inGroup C, Anti-CII antibody also decreased; The levels of Group H IgG antibody, INF-γandIL-4 all decreased markedly, TNF-a level also decreased and the Anti-CII antibody level hadno marked change. The results of FACS analysis demonstrated that both Th1 and Th1/Th2decreased markedly in Group C; In Group H, only Th1/Th2 decreased with statisticsignificance and there was no marked change in ratio of Th1, Th2 lymphocytes. Themeasurement results of real-time quantitative fluorescent PCR technology demonstrated thatT-bet mRNA was down-regulated in Group C, IL-4 markedly up-regulated(P＜0.01); In GroupC and Group H, the INF-γmRNA was both down-regulated and the difference both hadsignificance. But in both groups, the T-bet/GATA-3 ratio had neither the changes with statisticsignificance. After H-E staining of ankles, the microscopic observation showed that the lesionswere both serious in Group H and Group M. The lesions of Group C were relatively milder.And the infiltration of inflammatory cells was seen occasionally. The combination of analyticresults of macroscopic and microscopic parameters indicated that both "wind-cold &dampness" and "wind-dampness & heat" could improve the Thl/Th2 shift. And theimprovement of "wind-cold & dampness" was more marked. But these two had no markedeffect upon the T-bet/GATA-3 ratio. 3. The same experimental technique was adopted to perform a 3-week intervention oftreatment upon the CIA rats in Group M by gavaging Xuan Bi Tang (Group X) and GuizhiShaoyao Zhimu Tang (Group G) respectively. The results showed that both the serum IgGantibody and the levels of INF-γ, IL-4, TNF-a decreased in both rat groups. Furthermore, bothTh1 and Th1/Th2 decreased markedly, Th2 increased and there was a marked down-regulationof T-bet, INF-γmRNA in both groups. There was a marked up-regulation of IL-4 mRNA andT-bet/GATA-3 changed in Group G where there was marked change in Group X. Asdemonstrated by H-E staining, the lesion in Group G remained relatively serious. Theinflammatory cells were seen occasionally in Group X and the lesion lessened. Furthermorethe TUNEL method was employed to detect the apoptosis of articular tissue cells. The resultsshowed that the apoptotic rates of both cell group cells decreased markedly but it was moreobvious in Group X. Combining the macroscopic and microscopic parameters and comparingXuan Bi Tang and Guizhi Shaoyao Zhimu Tang, the former could better treat the CIA rats,reversed the Th1/Th2 shift and down-regulated the T-bet/GATA-3 ratio.4. Similarly, a 3-week intervention of "wind-cold & dampness" was performed upon theCIA rats and further treated for another 3 weeks by gavaging Xuan Bi Tang ( Group C+X) andGuizhi Shaoyao Zhimu Tang (Group C+G). The results showed that the serum Anti-CIIantibody level increased markedly in Group C+X rats, Th1, Th1/Th2 also increased markedly.A marked down-regulation of IL-4 mRNA was both seen in Group C+G and Group C+X.Only the T-bet/GATA-3 ratio was up-regulated in Group C+X. The observation of H-Estaining of ankles revealed that there was a large amount of infiltration of inflammatory cellsin Group C+X. Similar to the lesion degree in Group M, the infiltration of mononuclear cellsdecreased in Group C+G and the lesion lessened. As demonstrated by the in situ TUNELmethod used to detect the apoptosis of tissues and cells in joints, compared with Group C, thecell apoptotic rate decreased in Group C+G while there was no marked change in Group C+X.But the microscopic observation revealed that there were increases of inflammatory cells andfibroblasts and the cellular apoptosis became more severe in Group C+X. Combining themacroscopic and microscopic parameters, neither Xuan Bi Tang nor Guizhi Shaoyao ZhimuTang could markedly improve the disease conditions of CIA rats receiving a 3-weekintervention of "wind-cold & dampness". On the contrary, Xuan Bi Tang made the clinicalsymptoms, local articular pathology and cellular apoptosis worsen, accentuated the Th1/Th2shift and up-regulated the T-bet/GATA-3 ratio.5. Furthermore, after the CIA rats received a 3-week intervention of "wind-dampness &heat", another 3-week treatment intervention of Xuan Bi Tang (Group H+X) and GuizhiShaoyao Zhimu Tang (Group H+G) was given by gavaging. The results showed that only theserum Anti-CII antibody level decreased markedly in the rats of Group H+G. In Groups H+Gand H+X, both Thl and Th1/Th2 decreased markedly, Th2 increased markedly and the expression of INF-γmRNA both decreased. Furthermore, there was up-regulated GATA-3mRNA and the decreased ratio of T-bet/GATA-3 in Group H+G. The observation after HEstaining showed that the lesion lessened when Groups H+G and H were compared; And thelesion remained relatively severe in Group H+X. The TUNEL method was employed to detectthe apoptosis of articular tissue ceils. The results showed that there was no marked change incellular apoptotic rate of these groups. Combining the macroscopic and microscopicparameters and comparing Xuan Bi Tang and Guizhi Shaoyao Zhimu Tang, the latter could beatthe former in terms of improving the clinical symptoms and microscopic parameters in CIArats receiving a 3-week intervention of "Wind-dampness & heat".Summing up the above, the external environment interventions of "wind-cold &dampness" or "Wind-dampness & heat" could cause the syndrome changes in CIA rats withheat-Bi-syndrome and improve the disease conditions and reverse the Th1/Th2 shift, but theinfluence of the former was more pronounced and the latter had no effect upon the localarticular lesions in CIA rats, which demonstrated that the external environments had certaineffects on Bi-Syndrome, and justify the theory that Bi-syndrome is caused by "external evilsand internal injuries". And more, when Xuan Bi Tang and Guizhi Shaoyao Zhimu Tang werecompared, the former conformed more to the "syndrome factors" and "pathogenesis"of ratswith heat-Bi-syndrome and could better treat the CIA rats with heat-Bi-syndrome and reversethe Th1/Th2 shift, but it run against the "syndrome factors" and "pathogenesis" of CIA ratswith cold-Bi-syndrome, worsened their disease conditions and accentuated the Th1/Th2 shift;Where, the effects of Guizhi Shaoyao Zhimu Tang upon the disease conditions of rats withcold-Bi-syndrome and the Th1/Th2 shift were not marked, but it conformed more to the"syndrome factors" and "pathogenesis" of rats with heat-Bi-syndrome but complicated withfeatures of blood stasis, and could better improve the disease conditions of CIA rats. But theinfluences of the two upon the Th1/Th2 shift were similar, all of abovementioned justify thetheory of "Correspondence of prescription and syndrome" of Bi syndrome, i.e. only if thecomposition, formulation and such features as cold, heat, warm and cool of the TCMingredients in prescriptions highly correspond with the "syndrome factors" and"pathogenesis" of Bi-syndromes could the formula achieve the best therapeutic efficacy, andthe shift degree of Th1/Th2 subpopulation is probably the substance basis of the correspondingdegree of "prescription" and "syndrome" as stated within the theory of "correspondence ofprescription and syndrome" for Bi-syndrome at the cellular level; The expression status ofT-bet/GATA-3 mRNA is probably the substance basis of the corresponding degree of"prescription" and "syndrome" as stated within the theory at the molecular level.更多还原