Study Chinese Medicine NDRF Combined With Methotrexate On The Anti-inflammatory And Immunomodulatory Of The Rabbits With Adjuvant Arthritis

Objective:①To improve adjuvant arthritis(AA) model of rabbits and provide a better exper--imental treatment model of AA rabbits;②To observe the effects on the anti-inflammatory and the joint injuries treatment, andto provide the guidelines of the research and the treatment for the anti-inflammatory,separately intervening AA rabbits with Chinese medicine NDRF alone and NDRFcombined with methotrexate (MTX);③To observe the effects on the regulation of CD4+CD25+Treg and Th1/Th2, and toinvestigate the role of immune tolerance in the AA pathogenic mechanism,separate--ly intervening AA rabbits with Chinese medicine NDRF alone and NDRF combinedwith MTX.Methods:①To establishedAAmodel with tumor necrosis factor alpha(TNF-α) emulsified inComplete Freund Adjuvant through plantar subcutaneous injection, the first programadopting the injection method of seven points on the rabbits, and was boosted tendays after, the second program adopting the injection method of nine points on therabbits, and was boosted seven days after and fourteen days after again;②To judge the AAmodel, observing the rabbits’general behaviors, such as their dailyactivities and eating, measuring their joints swelling, body weight, temperature, kneecircumference and arthritis index. Meanwhile, adding the observation on the indicatorof inflammatory cytokines, pathology and X-ray imaging;③To test the cytokines expression of TNF-α, IL-1β, IFN-γ, IL-4, IL-10and TGF-β inthe rabbits,serum, and the expression of MMP-1and MMP-9in the rabbits,synovialfluid by ELISA;④To test the relative expression level of TNF-αmRNA, IL-1β mRNA and MMP-1mRNA in the rabbits’ synovial tissue by RT-PCR;⑤To Detect the change of TNF-α and IL-1β in the rabbits’ synovial tissue by method of immunohistochemistry;⑥To observe histopathological changes through HE staining the sample of the rabbits’synovial tissue;⑦To observe the change in the rabbits’ joints by DR X-ray machine and determinethe extent of damage to the rabbits,joints;⑧To detect the change of CD4+CD25+Treg cells in the rabbits’ peripheral by themethod of flow cytometry.Results:①In the first program, it was the21st day after modeling when the arthritis index ofthe rabbits reached six, while in the second program, it was the18th day aftermodeling when the arthritis index reached six; the perimeter of the right knee joints ofin both programs was greatly increased in the first week, and the difference before andafter injection had statistical significance (p<0.05); the perimeter of the left kneejoints of in the first program was significantly increased on the21stday, while in thesecond program, it was on the16thday when the perimeter of the left knee joints hadthe most obvious increase. The difference before and after injection in both programshad statistical significance(p<0.05).②Adding indicators: on the21stday after modeling, by method of ELISA, theexpression level of TNF-α and IL-1β in the rabbits’ serum was found significantlyhigher than before modeling; the expression level of MMP-1and MMP-9in therabbits’ synovial fluid was significantly higher than before modeling; the expressionof TNF-α and IL-1β in the model group rabbits’ synovial tissue was strong positive bymethod of immunohistochemistry, while the normal group was negative; comparedthe X-ray images of the normal group to that of the model group, some difference wasfound on the bone erosion score, the joint space score and the measure results of thejoint space; From the perspective of pathology, the subcutaneous tissue of the modelgroup rabbits was found obviously inflamed or even ulcerated, the intra-articularspace narrowing, synovial swelling thickening, large quantity of lymphocytes,macrophages, eosinophils and some other chronic inflammatory cells infiltrating.③The test result of the cytokines in rabbits’ serum after the experimental treatment:MMP-1and MMP-9in the synovial fluid of the NDRF group and the NDRFcombined with MTX group was significantly decreased, compared with the modelgroup it had statistical significance (p<0.05); after the treatment, the expression level of IL-10, TGF-β and IL-4were all increased, compared with the model group, theNDRF group got an obvious increase (p<0.05); the IFN-γ level of the rabbits’ serumdecreased, compared with the model group, the NDRF group got an obvious decrease(p<0.05).④After the experimental treatment, TNF-α mRNA、IL-1βmRNA and MMP-1mRNA(2-(Ct)) was analyzed by method of relative quantitative statistical analysis.Compared with the model group, the TNF-αmRNA of the NDRF group and theNDRF combined with MTX group significantly decreased (p<0.05), the IL-1βmRNAof the NDRF group and the NDRF combined with MTX group significantly decreased(p<0.05), the MMP-1mRNA of the NDRF group and the NDRF combined with MTXgroup also significantly decreased(p<0.05).⑤X-ray imaging observation of the rabbits’knee after the experimental treatment: thebone erosion score and the joint space score of the modeling group were greatlyhigher than before, and its joint space width narrowed. After the treatment, alltreatment groups recovered at different levels. Both the bone erosion score and thejoint space measurements were improved. There were significant differences(p<0.05)between the NDRF group and the model group, as well as NDRF combined withMTX group and the model group; There was significant differences(p<0.05) betweenNDRF combined with MTX group and the model group in the joint space score.⑥The immunohistochemical results of the rabbits after the experimental treatment:compared with the model group, the expression level and expression intensity ofTNF-α and IL-1β of the NDRF combined with MTX group was much lower, and thedifference between the two groups had statistical significance(p<0.05); comparedwith the model group, the expression intensity of TNF-α and IL-1β of the NDRFgroup was a litter low, and the difference between the two groups had statisticalsignificance(p<0.05);but no difference(p>0.05) in the statistical analysis of theexpression level.⑦The test results of T cell subsets by flow cytometry: Compared with the normalgroup, the percentage of CD4+CD25+Treg cells in the peripheral blood of the modelgroup was much lower(p<0.05). After the treatment, the percentage of Chinesemedicine NDRF increased and the difference between the two groups had statisticalsignificance(p<0.05). Conclusion:①Results of this experimental model: using CFA combined with TNF-α as inducer,the first program adopting the injection method of seven points twice in ten days, itsmodel rate was78%, and it was the21st day after modeling when the arthritis indexof the rabbits reached six; the second program adopting the injection method of ninepoints three times in fourteen days, its model rate was83%, and it was the18th dayafter modeling when the arthritis index reached six. The test results of inflammatorycytokine levels, pathological changes and knee X-ray imaging were all abnormal, itindicated that the AA model was successful.②The experimental treatment results of this study: Chinese medicine NDRFcombined with MTX can reduce MMP-1and MMP-9level of AA rabbits’ synovialfluid; Chinese medicine NDRF combined with MTX can reduce the level of TNF-αmRNA, IL-1βmRNA and MMP-1mRNA in the AA rabbits’ synovial fluid; bylowering the level of TNF-α, IL-1β, MMP-1and MMP-9, Chinese medicine NDRFcombined with MTX inhibits proinflammatory effect and cures bone and jointdamage.③The X-ray imaging observation of the experimental treatment in this study: aftertreating by Chinese medicine NDRF combined with MTX, the X-ray result showedthat the rabbits’ knee joints had clear boundaries and smooth articular surface, thejoint space narrowing problem had been improved to a certain extent and thepathological severity had been eased. As for the bone erosion score, bone and jointspace score and joint space measurement, the result showed that the treatment groupwas better than the model group(p<0.05). The X-ray imaging result indicates thattreating RA by combining traditional Chinese and western medicine has very goodprospects and it is worth further study.④The test result of this study showed that after treating by Chinese medicine, thelevel of CD4+CD25+Treg cells increased, the level of Th1factor IFN-γ dropped, thelevel of Th2factors IL-4and IL-10increased, it indicates that Chinese medicine hascertain effects on immunomodulation and autoimmune tolerance recovery.