| BackgroudRheumatoid arthritis(RA)is a autoimmune disease which is characterized by chronic joint swelling,synovial inflammation and cartilage destruction.The synovial membrane is infiltrated by CD4+T cells and there is functional disorder under the background of particular genetic and inflammatory environment,which play a key role in the synovial inflammation of RA.With the insightful understanding of the two subsets(regulatory T cells,Treg and T-helper 17 cells Th17)in CD4+T cells,the imbalance of Treg/Th17 might be the one of leading causes in the RA development.At present,the NSAIDS,DMARDS,glucocorticoids and biological agent are used to treat RA.These medications have certain therapeutic effects.However,patients could not take them for a long term due to the side effects and high cost.The Qiang Huo Er Huang decoction has good clinical therapeutic effect and lower side effect,and has been widely used to treat RA in our department,however the mechanism remains to be explored.ObjectiveTo observe the effect of different Qiang Huo Er Huang decoction dosages on adjuvant arthritis(AA)rats and explore the mechanism in the light of Treg/Th17.MethodsRats were subcutaneously injected by the adjuvant emulsion which was composed of inactivated mycobacterium tuberculosis and mineral oil.The model evaluation system is as follows: a)0,normal;b)1,with one toe joint swelling;c)2,with over two toe joints or whole claw swelling;d)3,the whole paw swelling more serious;e)4,the whole paw swelling serious and inelastic.The AA model is established while the arthritis score is greater than 1.All rats were divided into six groups,including control group,AA group,AA + methotrexate(MTX)group(weekly),AA + Qiang Huo Er Huang decoction groups(low dose: 4.5g/kg/day,moderate dose: 9g/kg/day and high dose: 18g/kg/day,respectively).On the first day of joints swelling,rats received treatments by daily gavage.Control rats were provided with tap water alone.The changes of arthritis score,body weight,paw thickness and ankle circumference were observed during the following 4 consecutive weeks.The proliferation of splenic lymphocytes after stimulated by Phorbol myristate acetate(PMA)was measured ex vivo by CCK-8.The hematoxylin-eosin(HE)staining was used to evaluate joint histological changes.The flow cytometry method was applied to detect Treg/Th17 frequency in spleen.The serum Interleukin(IL)-6,IL-17,TNF-? and TGF-? were measured by Enzyme-linked immunosorbent assay(ELISA).Results1.The AA model of rats was established after injected adjuvant while the arthritis score was greater than 1,and the successful rate of AA rats was 75%.The characteristic of model joint showed that synovial proliferation,articular cartilage damage,and a large amount of newborn pannus formation by HE staining.2.The study results showed that the arthritis score significantly decreased in the MTX group(P<0.01)and high dose decoction group(P<0.01)at the 24 th,28th day,respectively.3.Compared with the AA rats,the MTX group and high dose decoction group gained body weight more rapidly at the 24 th,28th day(P<0.01),respectively.4.Compared with the AA rats,the paws swelling of the MTX group and high dose decoction group were alleviated,the paws thickness were reduced as well as at the 24 th,28th day(P<0.05).5.Compared with the AA rats,the ankle circumference of high dose decoction group after treated 28 days were decreased((2.84 ± 0.29)cm,P=0.006).6.The MTX group and high dose decoction group significantly inhibited the proliferation of splenic lymphocytes(P<0.01).7.The ratio of Treg/Th17 was imbalanced in the AA group: a decrease of Treg frequency(P<0.05)and a significant increase of Th17(P<0.01).After four weeks of treatment,the Treg ratio of high dose decoction group((11.19 ± 2.01)%,P=0.000)was significantly elevated,while the Th17 ratio((0.98 ± 0.35)%,P=0.041)was significantly decreased.Compared with AA group,there were no differences in the moderate and low dose decoction group(P>0.05).8.The serum contents of IL-6((134.21 ± 16.58)pg/mL,P=0.000),IL-17((36.26 ± 5.62)pg/mL,P =0.001)and TNF-?((540.01 ± 36.94)pg/mL,P =0.013)in AA group were obviously higher than normal group,however the level of TGF-?((41.81 ± 6.35)pg/mL,P =0.020)was much lower.After the intervention of MTX and high dose Qiang Huo Er Huang decoction,the serum contents of IL-6,IL-17 A and TNF-? significantly were decreased,while TGF-? apparently increased.Conclusion:The high dose of Qiang Huo Er Huang decoction has a therapeutic effect for treating adjuvant arthritis,and down-regulate the serum contents of IL-6,IL-17 and TNF-?,up-regulate the TGF-?.The underlying mechanism is possibly associated with correcting the imbalance of Treg/Th17 subsets,CD4+ T splenic lymphocytes. |
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