Therapeutic Effects Of Anacardic Acid On Rheumatoid Arthritis And Associated Molecular Mechanisms

Part One: Anacardic acid ameliorates collagen-induced arthritis in a mouse modelObject: Anacardic acid,which is abundantly detected in nutshell of Anacardium occidentale,is a general term applied to a family of 6-alkyl salicyclic acids having varying saturation degrees in the 15-carbon alkyl chain.Anacardic acid is attracting increasing attention due to its multiple pharmacological activities including anticancer and antibacterial properties.Anacardic acid acts as an inhibitor of histone acetyltransferase and shows the ability to potentiate apoptosis in tumor cells.In this study,we explored the effects of anacardic acid on collagen-induced arthritis(CIA)in vivo.Methods: Female DBA/1J mice(6 week old)were randomly assigned to four groups(n = 8).For establishment of CIA model,mice were immunized with 100 ?g chicken type II collagen,which was emulsified with an equal volume of complete Freund's adjuvant supplemented with 4 mg/m L heat-killed mycobacterium.A booster immunization was performed on day 21 with 100 ?g chicken type II collagen emulsified with an equal volume of incomplete Freund's adjuvant.To determine therapeutic effects,anacardic acid was administered via intraperitoneal injection at a dose of 1 or 5 mg/kg body weight twice per week for 3 weeks,starting from the day of booster immunization.Arthritis severity was evaluated twice per week in a blinded manner according to the following criteria: 0 = no signs;1 = mild swelling of the wrist or ankle;2 = moderate swelling extending from the ankle to the tarsal bones;3 = moderate swelling extending from the ankle to the metatarsal joints;4 = severe swelling of the wrist and ankle including all digits.On day 42,animals were sacrificed and serum was collected.The hindlimbs were dissected and subjected to histological analysis.The degree of synovitis,pannus formation,bone erosion,and cartilage destruction was scored according to the following criteria: 0,normal;1,mild changes;2,moderate changes,and 3,severe changes.The serum levels of TNF-? and IL-1? were determined using mouse specific ELISA kits.Results: Administration of anacardic acid markedly diminished the severity of arthritis in mice exposed to collagen.The high dose of anacardic acid(5 mg/kg body weight)resulted in a more profound amelioration of disease than a lower dose of 1 mg/kg body weight.As revealed by histopathology,CIA mice displayed severe synovial inflammation,pannus formation,cartilage damage,and bone erosion.Anacardic acid treatment led to a remarkable improvement in histopathological findings and scores.Analysis of inflammatory mediators revealed that the levels of TNF-? and IL-1? were significantly higher in sera from CIA mice than those from control mice.The elevation in the inflammatory cytokines was significantly attenuated in anacardic acid-treated groups.Conclusion: We provide first evidence for the potential antiarthritic activity of anacardic acid,which is associated with suppression of TNF-? and IL-1?.Part Two: Anacardic acid suppresses proliferation and invasion of rheumatoid arthritis fibroblast-like synoviocytesObject: Hyperplasia of fibroblast-like synoviocytes(FLSs)is causally linked to the pathogenesis of RA.RA-FLSs exhibit tumor?like aggressive phenotype including overproliferation and invasion.Therefore,FLSs serve as an important therapeutic target for the treatment of RA.micro RNAs(mi Rs)are a large class of small non-coding regulatory RNAs and repress the expression of multiple genes via cleavage of target m RNAs or inhibition of protein translation.In this study,we explored the effects of anacardic acid on the biological behaviors of RA-FLSs in vitro.The molecular mechanism underlying in the action of anacardic acid was examined.Methods: RA FLSs were incubated for 48 h with various concentrations of anacardic acid(5,30,and 60 ?M),in the presence of recombinant human TNF-?(10 ng/m)or IL-1?(10 ng/m L).In some experiments,MK-2206(1 ?M)was added together with TNF-?(10 ng/m L)or IL-1?(10 ng/m L)to the culture medium and incubated for 48 h before measurement of cell proliferation and invasion.RA FLSs were treated with TNF-?(10 ng/m L)or IL-1?(10 ng/m L)for 48 h.Afterwards,cells were incubated with 20 ?M Ed U for 2 h.The cells were counterstained with Hoechst 33342(5 mg/m L).The Ed U-positive cells were examined under a microscope,and at least 300 cells were counted per well.Cells suspended in serum-free medium containing different concentrations of anacardic acid and TNF-?(10 ng/m L)or IL-1?(10 ng/m L)were seeded into the upper chamber.The culture medium containing 10% FBS was added to the lower chamber.After incubation for 48 h,the invaded cells were determined under a microscope.The Akt protein and m RNA expression levels were determine by Western blot analysis and real-time PCR assay.mi R-633 mimic,anti-mi R-633,and negative control oligonucleotides were transfected.In some experiments,the Akt-expressing construct was co-transfected together with mi R-633 mimic into RA FLSs.HEK293 T cells were co-transfected with Akt 3?-UTR reporters and mi R-633 mimic or control mi RNA.Forty-eight hours later,cells were lysed and luciferase activities were measured.Results: As determined by Ed U incorporation assays,anacardic acid treatment significantly blocked TNF?? and IL?1? induced proliferation in RA-FLSs.Moreover,the anti-proliferative effect of anacardic acid was in a concentrationdependent manner.Transwell invasion assays further demonstrated that TNF?? and IL?1? induced cell invasion was impaired in the presence of anacardic acid.Western blot analysis demonstrated that anacardic acid treatment significantly suppressed the protein expression and phosphorylation of Akt.Real-time PCR analysis revealed that the m RNA expression level of Akt was not altered by anacardic acid treatment.Anacardic acid-treated RA FLSs showed 4.8-fold higher expression levels of mi R-633 than control cells(P < 0.05).Overexpression of mi R-633 significantly reduced the activity of luciferase reporter harboring Akt 3?-UTR.Consistently,mi R-633 overexpression significantly repressed the protein expression of Akt in RA FLSs.Similar to anacardic acid treatment,overexpression of mi R-633 significantly restrained the proliferation and invasion of RA FLSs.Rescue experiments revealed that enforced expression of Akt led to restoration of proliferation and invasion in mi R-633-overexpressing RA FLSs.Pharmacological inhibition of Akt activity also caused a suppression of cell proliferation and invasion in RA FLSs exposed to TNF?? and IL?1?.Depletion of mi R-633 rendered RA FLSs more resistant to anacardic acid-mediated suppression of proliferation and invasion.In addition,downregulation of mi R-633 reversed anacardic acid-mediated inhibition of Akt expression in RA FLSs.Conclusion: Anacardic acid-mediated suppression of RA-FLSs proliferation and invasion is causally linked to induction of mi R-633 and downregulation of Akt expression.These findings suggest that anacardic acid may provide therapeutic benefits in the treatment of RA.