The Drift Of The External Environment With Different Herbs On The Cia Rats Syndrome And T Cell Subsets Comparative Study

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.