Effects And Mechanisms Of Ginsenoside Compound K On Collagen-induced Arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease that preferentiallymediated by T and B lymphocytes, primarily targets the synovial membrane, cartilage,and bone. The abnormal immune regulation is the key in the pathogenesis ofautoimmune diseases. The abnormal proliferation and activation of T lymphocytes,various cytokines and imbalance proportion of Th cells have been recognized as acrucial event in the pathogenesis of RA. Ginsenoside compound K (C-K) exhibitsvarious biological activities, including anti-mutation, inhibition of cancer metastasis,cytotoxicity, and reversal of drug resistance and so on. Meanwhile, itsimmunopharmacological activities such as anti-allergic and anti-carcinogenic arereceiving increasingly attention. In the current study, collagen-induced arthritis (CIA)models were established in DBA/1mice to investigate the therapeutic effects andmechanisms of C-K on CIA mice, especially to reveal its mechanism in inhibiting Tlymphocyte abnormal activation. Objective: The aim of this study was to investigate the anti-arthritis andimmunomodulatory activity of C-K in CIA mice, and in particular, to reveal itsmechanism of inhibiting abnormal T lymphocyte activation.Methods: A preparation of0.1mg of heterologous chiC-Ken type II collagen (CII) wasinjected intradermally into the baC-K and the base of the tail of DBA/1mice to inducedCIA on day0and day21. After the onset of arthritis, animals were divided into8groups randomly, named as normal, CIA, C-K (7,14,28,56,112mg/kg/qd,intragastrically), MTX (2mg/kg, intragastrically, once every3days). CIA mice weretreated from day28to day50. The effects of C-K on CIA mice were evaluated byarthritis scores, joints and spleens histopathology, as well as indices of thymus andspleen. T lymphocytes proliferation was determined by [3H]-TdR incorporation. Levelsof anti-CII antibody, anti-CCP antibody, IgG1, IgG2a, TNF-α, IL-1β, IL-17, RANKLand OPG were detected by ELISA. The subsets of T and B lymphocytes, as well as theexpressions of CD28, T cell receptor (TCR), cytotoxic T lymphocyte-associatedantigen-4(CTLA-4) and programmed death-1(PD-1) in purified splenic T lymphocytewere measured by flow cytometry, western blotting and laser confocal microscop.Results:1Effect and mechanism of C-K on CIA mice.1.1Effects of C-K on the body weight and global assessmentThe weight of the CIA mice increased slowly and was significantly less than that of thenormal DBA/1mice beginning on day3after injection of the emulsion. The C-K (14,28,56mg/kg) mice recovered their weight by day32after the emulsion injection, theC-K (14,28,56, and112mg/kg) mice obviously recovered their weight by day35afterthe emulsion injection. On day50, C-K (112mg/kg) reatment showed significantlyincreased body weight as compared compared with that of CIA group.After immunization, the paw of CIA mice appeared red and swelling, the behaviors of mice were obstructed, and arithritic nodes were observed on ears, nose and tails. Days41after immunization, C-K (112mg/kg) showed significantly decreased scores ofglobal assessment as compared with CIA mice.1.2Effects of C-K on arthritis index and swollen joint count of CIA miceHind paw-swelling began on day24post-immunization. From day45, C-K (112,56mg/kg) had reduced the arthritis index. C-K (7,14mg/kg) did not have a clear effect onthe the arthritis index of CIA miceC-K (112mg/kg) significantly reduced the swollen joint count of the mice from day47to day51, C-K (28,56and112mg/kg) significantly reduced the swollen joint count ofthe mice from day49to day51. C-K (7mg/kg) did not have a clear effect on the swollenjoint count of CIA mice1.3Effects of C-K on the histopathology of the joints and spleens in CIA miceIn CIA mice, synoviocytes proliferated to multiple layers, and the articular cartilageswere destroyed and demonstrated inflammatory infiltration. The hyperplastic synoviumin CIA mice included a large number of fibroblasts and new blood vessels. In micetreated with C-K (56mg/kg), articular cartilage destruction and pannus formation wereslightly inhibited. C-K (112mg/kg) significantly reduced articular cartilage destruction,pannus formation, and synovial hyperplasia, although inflammatory cell infiltration wasobserved to a small extent.In the model mice, the white pulp showed proliferation, GC development, and anincrease in pathology scores. C-K (56,112mg/kg) significantly reduced the increasedpathology scores when compared with those observed in CIA mice.1.4Effects of C-K on indices of thymus and spleen in CIA miceResults showed that thymus index and spleen index of model mice were increased compared with normal mice. C-K (56,112mg/kg) group moderated the spleen indicesof CIA mice significantly. C-K did not have a clear effect on the indices of thymus inCIA mice.1.5Effects of C-K on T and B lymphocyte proliferation in CIA miceThe results showed that the T and B lymphocyte proliferation induced by the differentstimulators in CIA mice was increased compared with that in normal mice. C-K clearlyreduced ConA-induced T lymphocyte proliferation. C-K (28,56,112mg/kg) mildlyreduced LPS-induced B lymphocyte proliferation in CIA mice. These data suggestedthat C-K primarily affects the function of T cells, so, the characteristics and mechanismof C-K’s effect need to be elucidated.1.6Therapeutic effects of C-K by regulating cytokines balance in serum and jointsof CIA miceIn CIA mice, the concentrations of serum IgG1, IgG2a, anti-CII antibody, anti-CCPantibody were significantly elevated. Treatment with C-K (28,56and112mg/kg)significantly reduced the concentration of IgG1and IgG2a. C-K (56,112mg/kg)significantly reduced the concentration of anti-CII antibody. C-K did not have a cleareffect on the expression of anti-CCP antibody in CIA mice.The concentrations of IL-1β, TNF-α, IL-17were significantly elevated whereas theconcentrations of OPG were significantly decreased in joints of CIA mice. C-K (56,112mg/kg) reduced the concentration of IL-1β, TNF-α. C-K (14,28,56, and112mg/kg)reduced the expression of IL-17. The concentration of OPG in C-K-treated (28,56and112mg/kg) mice was recovered. C-K did not have a clear effect on the expression ofRANKL in CIA mice.1.7Therapeutic effects of C-K on CIA mice by regulating B and T lymphocytes.We chose three groups treatment with C-K (28,56and112mg/kg) to elucidate the characteristics and mechanism of C-K’s effect.The percentages of CD19+IgM+cells (analogous to immature B cells), CD19+IgD+cells(analogous to mature B cells) and CD19+CD27+cells (analogous to memory B cells) inthe spleens of CIA mice were significantly higher compared with those in normal mice,whereas the percentages of CD19+CD5+CD1dhi cells (analogous to regulatory B cells,Bregs) were significantly lower.Treatment with C-K (28,56and112mg/kg) did not hada clear effect on modulated the activation and differentiation of B lymphocytes in CIAmice,The results showed that the percentages of CD3+CD4+cells (analogous to helper T cells,Th cells), CD4+CD154+cells (Th cells expressing co-stimulatory molecules CD154),CD4+CD69+cells (analogous to activated T cells) and CD4+CD62L-CD44hicells(analogous to effector memory T cells, TEM) in the spleens of CIA mice weresignificantly higher compared with those in normal mice, whereas the percentages ofCD4+CD62L+CD44lowcells (analogous to naive cells) and CD4+CD25+Foxp3+cells(analogous to regulatory T cells, Tregs) were significantly lower. C-K treatmentdecreased the proportions of CD4+CD154+, CD4+CD69+and CD4+CD62L-CD44hicellsand increased the percentages of CD4+CD62L+CD44lowand CD4+CD25+Foxp3+cells.The proportion of CD3+CD4+Th cells was not affected by C-K or MTX administration.The data we obtained indicated that C-K substantially modulated the activation anddifferentiation of T lymphocytes in CIA mice, which might be a novel mechanismunderlying the effects of C-K in the treatment of RA.1.8Effects of C-K on the expression of CD28, TCR, CTLA-4and PD-1in the Tlymphocytes of CIA miceIn this study, the expression of CD28and the TCR in the T lymphocytes of CIA miceincreased, whereas the levels of CTLA-4and PD-1in T lymphocytes were significantlydecreased. Compared with the CIA mice, C-K greatly decreased the expression of CD28and the TCR, whereas it significantly increased the expression of CTLA-4andPD-1.Conclusions:1. Data presented here demonstrate that administrations of ginsenoside C-Ksignificantly recover the depressed weight of CIA mice, reduced the arthritis scoressignificantly, and improved the pathological changes were observed in spleen andjoints. C-K could attenuated the progression of experimental arthritis.2. The therapeutic effects might be related to the ability of C-K to regulate the functionof T lymphocytes and balance inflammatory cytokines.3. The therapeutic effects might be related to the ability of C-K to inhibit the activationand differentiation of T cells via by modulating co-stimulatory and co-inhibitorysignals.