Study On The Paharmacological Action And The Paharmacological Mechanisms On Cellular Immunity Of The Traditional Chinese Medicine On Treatment Of Rheumatoid Arthritis

The bone and joint disease was an important factor which made patients been pain acutely for long time. Thereby, in order to promote the level of prevention and cure on the bone and joint disease, WHO named the decade from 2000 to 2010 for "bone and joint decade". Rheumatoid Arthritis (RA), the most familiar and disable disease of bone and joint, had effect on millions and millions people from any race all over the world to result in immense lost of individual, family and society. This disease had been main study in bone and joint decade. Doctor and scientists are making great efforts to explore better therapeutics and drugs on RA all over the world.There is nothing to modify illness and prevent joints destruction now. Methotrexate (MTX) could prevent the joints from destroy and had been one of the most commonly used anti-rheumatoid drugs. However MTX couldn't prevent cartilage and bone lost. Traditional Chinese medicine (TCM) had been treated on RA for long history and had good effect. But TCM had been failure to study paharmacological action, superiority and mechanism with scientifically and strictly. The effective substance of TCM compound preparation was not understood to influence on its quality control and development. It is the key to prevent anti-rheumatoid TCM from developing further in clinical now.On the background, the aim of this research was to evaluate the paharmacological action of TCM and clarified its pharmacological mechanism on cellular immunity. Scientific evidence of this research would make TCM on treatment on RA developed further.Firstly, we reviewed the clinical data of RA patients who had been treated with Tongbilin (TBL, the compound preparation of TCM) for two years. It was observed the changes of symptoms, erythrocyte sedimentation rate(ESR), C reacting protein(CRP) of 230 RA patients and X-ray film feature of 35 from 230 RA patients to offer a key to study pharmacological mechanism of TBL. Secondly, Type II collagen-induced-arthritis (CIA)rats were treated by TBL (the compound preparation of TCM), Tripterygium glucosides (TG, the extract preparation of TCM), Sinomenine (SINO, simple substance preparation of TCM) and MTX (the simple substance preparation of chemicals medicine) individually. The pharmacological action of these TCM preparations was study individually in a parallel comparative study with MTX. Furtheremore, it was cellular and molecular degree to investigate the pharmacological mechanism of TCM on cellular immunity on activation, differentiation, proliferation, cell cycle and cell signaling protein of T lymphocytes whish played a important in RA pathological process with immunological methods like flow cytometry, MTT and western-blot.The clinical result showed TBL could modify pain, swell and function of limbs and decrease ERP and CRP of RA (P<0.05 ). There were no changes obviously in X-ray film of 35 RA patients after two years. These clinical cases with large sample and long time hint TBL could modify RA symptoms and prevent joints from destruction and offered a key to study pharmacological action and mechanism of TBL further. However, this clinical research had some deficient without double-blind, random and antithetical design. There was a common problem in TCM research too. As a result, CIA rats were used to study TBL, TG and SINO on pharmacological action and mechanism on cellular immunity.As the classical experimental model of RA, CIA was used to study pathology and pharmacology of RA. We sum up the rule of disease and set up and perfect the radiographic assessment protocol of CIA. In this research different feature between CIA and RA were uncovered firstly. This perfect assessment system was set up with swell, pathology section and radiographic assessment and relations of all factors in assessment system were analyzed. We studied the pharmacological action of these TCM preparations with this assessment system afterward.The result of CIA model showed the inflammation of CIA was onset so fast that four limbs might become the most swollen in one week. And then, the inflammation alleviated and the second climax swell appeared on 21th day. The joint became abnormality finally. There were infiltration of neutrophil, lymphocytes and Plasmacytes, synovial hyperplasia, hyperplasia of fibrous tissue and destruction of cartilage in the pathological section of third proximal toes joints of left fore limbs and right hind limbs. The X-ray film revealed that CIA rat were manifested with symmetrical tissue swelling of joints, diffused osteoporosis, bone erosion, exostosis of bone and narrow or wide joints space. The severe levels of mild and greatest of joints destruction were happened mostly. There were some different between CIA and RA. CIA was characterized by suffering of the joints of DIP, PIP and ankle. MTPs were suffered least and joints destruction which were opposite to RA ofhuman being. Because RA was suffered of the joints of MTP mostly while DIPs were suffered least. 2 The lesion and destruction of radius and ulna of fore limb were lower in CIA, while that were destructed severely in RA. We used ninety-six joints in scoring system of erosion and one hundred joints in scoring system of JSN to comprehensively and objectively evaluate the joints destruction of CIA. Moreover, to simplify assessment protocol, we analyzed correlation coefficients, ratio and the proportion of joints destruction (greatest, mild and least) of both the new scheme and the original scheme. The best evaluation scheme was decided. The protocol included seventy-four bones in the scoring system of JSN and The protocol included eighty-eight joints was thought as perfect. The result showed with analysis of correlation coefficients and linear regression: The swell, the infiltration of neutrophil and synovial hyperplasia were on linear regression(P<0.05). The destruction of cartilage, synovial hyperplasia and hyperplasia of fibrous tissue were on linear regression(P<0.05). Both the joints destruction and synovial hyperplasia were on linear regression^O. 05)There were the results of the experiment of the effective time. TG (45.5mg-kg"1-d1) and SJJSfO (nOmgkg'd1) could inhibit joints swell of CIA rats treated for 17 days. TBL (3-Og-kg"1^) and MTX (V^mg-kg^w"1) inhibited joints swell on 19th day. That hint the effective time of TG (45.5mg-kg"1-d1) and SINO (^Omg-kg'-d1) were shorter than TBL (S.Ogkg'd1) and MTX (V^mgkg^w1) .Score of pathological section showed TBL (S.Og-kg'd1) could inhibit the infiltration of neutrophil, lymphocytes and Plasmacytes, synovial hyperplasia, hyperplasia of fibrous tissue and destruction of cartilage(P<0.01). SINCKllOmg-kg^-d"1) inhibited infiltration of plasmacytes of joint, synovial hyperplasia and destruction of cartilage(P<0.05); TG(45.5mg-kg"1-d"1) and MTX (V^mg-kg'-w1) inhibited synovial hyperplasia and destruction of cartilage of CIA rats. The result of effective strength showed that TBL, MTX, TG and SINO could inhibit swell and destruction of joints (P <0.05). However, the effect of all research drugs had no significant (P <0.05). MTX (7.6mg-kg"1-w"1) depressed the amount of red blood cells(RBC) and haematochrome compare with the other groups.We researched the main active compound of TBL in vivo and intro after made sure of TBL effect. It is the strongest effect of the overall alkali of TBL included strychnine and vauqueline to inhibit CD69 expressed on T lymphocytes which were stimulated by Concanavalina A(ConA) and Phorboll2,13-dibutyrate(PDB) in the extract from TBL. The overall alkali of TBL could inhibit the inflammation, synovial hyperplasia and destruction of cartilage and bone (PO.05).In this study of pharmacological mechanism, CD69 expressed on T lymphocytes was 3%and HLA-DR expressed on T lymphocytes was 2.7% in peripheral blood mononuclear cells (PBMC) from RA. It was found that the percentage of T cells bearing CD69 was significantly up-regulated (77%)in the mononuclear cells of the Synovial fluid (SFMC) patients, while that of T cells bearing HLA-DR was 44% in SFMC. TBL could significantly down-regulate CD69 and CD71 expressed on T cells stimulated by PDB and CD25, CD69 and CD71 expressed on T cells stimulated by ConA. SINO down-regulated CD69 expressed on T cells stimulated by ConA. It was found the expression of CD3+IFN-y+ cells was markedly elevated when the activated T cells in joint of RA patients was re-stimulated by PDB and Ionomycin with significant predominance of the Thl type subset. Overall alkaloid of TBL in the concentrations of 200mg/L, lOOmg/L, 50mg/L and 25mg/L significantly inhibited the expression of intracellular cytokine IFN-y by activated T cells(/*<0.01), with marked dose-response dependency, but there was no clear effect on IL-4(P>0.05). SINO significantly inhibited the expression of intracellular cytokine IFN-y by activated T cells of mouse too. Overall alkaloid of TBL in the concentrations of 200mg/L, lOOmg/L and 50mg/L significantly inhibited proliferation of Jurkat cells. These Jurkat cells in Gl cycle treated with overall alkaloid of (TBLlOOmg/L), SINO(0.5mM) and TG(20mg/L) were 70%, 59.7% and 52% individually while that treated with PBS was 37%. Jurkat cells in S cycle treated with MTX(5mg/L) were 54.6% while that treated with PBS was 41.7%. Overall alkaloid of TBL prevented ERK1/2 from phosphorylation and had no effect on calcium ion.The result hint there was cellular immune and humoral immune reaction in pathological process of CIA. The infiltration of neutrophil and synovial hyperplasia had effect on joint swell. Synovial hyperplasia and hyperplasia of fibrous tissue had effect on destruction of cartilage. It was the key for destruction of joints that there was synovial hyperplasia in CIA joints. TG and SINO had effect on inflammation of CIA more quickly than TBL and MTX. TBL could inhibit the inflammation, synovial hyperplasia and destruction of CIA which showed compound prescription of TCM with much composition have many targets of effect. SINO inhibited infiltration of plasmacytes of joint, synovial hyperplasia and destruction of cartilage-, TG and MTX inhibited synovial hyperplasia and destruction of cartilage of CIA rats. MTX (7.6mg-kg"1w1) had some side effect on RBC and haematochrome. It confirmed the overall alkali of TBL was the main active compound and effective substance of TBL that improved the study of quality control and pharmacological mechanism.T lymphocytes in SFMC of RA joints were activate. We suggest that TBL inhibited early, middle and late activation of T cells by inhibiting the activation of PTKs(includedp56fc*, p59fyn and ZAP-70, et al) or the activation PKC directly. SINO could inhibit the activation of PTKs to inhibit activation of T cells. It seems to be that overall alkaloid of TBL can suppress the production of Thl type cytokine and Thl cells differentiation to regulate the Thl/Th2 ratio, thus reducing the immune damages induced by the excessive activation of T cells and relieving the early inflammatory reactions of synovium and the proliferation of synoviocytes. From standpoint of Traditional Chinese medicine, TBL could regulate Yin and Yang imbalance and control immunity of RA. It was effective mechanism of SINO to inhibit the cell signaling molecule except for PKC. It hint TCM studied could prevent T cells from enter G2 cycle by reduce synthesis of energy and material in T cells. MTX inhibited proliferation of Jurkat cells and blocked Jurkat cells in G2 cycle by inhibiting DNA synthesis. We suggested overall alkaloid of TBL might interrupt cell signal pathway of RPTK-ras-raf-MEKi/2-ERKi/2, or not cellular calcium ion, thus to reduce production of the type cytokine of Thl or not Th2.There were the peculiarity and new idea in this study. We proposed the new idea of study the pathological mechanism for the compound preparation of TCM on anti-rheumatoid with the whole theory in TCM and modernistic empirical method. It was confirmed the compound preparation of TCM had effect on multiple channels, pharmacological action and targets though investigating the effect for TCM on CIA model, tissue of joints, cells and molecule. This research explored the pharmacological action of the compound preparation, the extract preparation and monomer preparation of TCM in a parallel comparative study with MTX firstly. And then, these preparation of TCM had difference pharmacological action and mechanism on cytoimmunity individually. This research developed some new methods. Different radiographic manifestations between RA and CIA were known in this study firstly. We set up an assessment system of joints destruction with swell, pathological section and X-ray film to improve the accuracy, objectivity and entirety of evaluation to drugs effect. Meanwhile, we set up a system to study the pharmacological mechanism on cellular immunity for TCM include activation, differentiation, proliferation, cell cycle and signal molecule of T lymphocytes. This research applied the CIA rat model and sensitive and quickly experiment on T cell activation to be sure that overall alkaloid of TBL was the main effective substance and substance foundation of TBL with chemical and pharmacological method.This study provided scientific experimental data for the preparations of TCM to be used in clinical. We had established a scientific experimental platform to evaluate the effect for TCM on RA and study on its pharmacological mechanism on cellular immunity. This research would improve TCM to be applied for treatment to autoimmunity diseases like...