Study On Effects Of Iguratimod On Bone Metabolism In Patients With Rheumatoid Arthritis

Rheumatoid arthritis(RA)is a chronic auto-immune inflammation disease characterized by joint destruction and joint function disorder.For now,the detailed mechanism of RA is still unknown,but we have found that many cells,cytokines and signal pathways play great roles in it.One of the most important characters of RA is joint damage,which is caused by the imbalance of bone metabolism.Homeostasis of bone metabolism is regulated by the processes of bone resorption and formation,which are regulated by many cytokines and signaling pathways.Receptor activator of nuclear factor-kappa B ligand(RANKL)/ receptor activator of nuclear factor-kappa B(RANK)/ osteoprotegerin(OPG)system and Wnt pathway are two of them.RANKL is a member of the tumor necrosis factor supperfamily.It can bind to its receptor,RANK,to induce the differentiation and activation of osteoclasts.OPG,as a decoy receptor,can inhibit the interaction between RANKL and RANK to protect against bone resorption.Overexpression of RANKL or underexpression of OPG promotes RANKL-mediated bone loss.RANKL/OPG is higher in patients with RA than that in healthy people and patients with osteoarthritis,and it can be a marker to predict the future damage of joints and it decreases after treatment.Wnt pathway is also important in bone metabolism.Activation of the Wnt pathway increases bone mass through induction of the differentiation and activation of osteoblasts.Dickkopf-1(DKK-1)is an important soluble inhibitor of the Wnt pathway that can block osteoblast function by inhibiting the Wnt pathway.Serum DKK-1 levels are up-regulated in patients with RA and are correlated with bone erosion and inflammation,and they decrease after treatment with biological disease-modifying antirheumatic drugs(b DMARDs).C-telopeptide of type I collagen(CTX-I)and procollagen type I N-terminal propeptide(PINP)are two of the most widely used bone remodeling markers.CTX-I is a very sensitive and specific marker of bone degradation.On the contrary,PINP is a biomarker of bone formation.Iguratimod,a novel DMARD that is an effective treatment for RA,has a positive effect on bone protection.Previous studies showed that iguratimod could promote osteoblast differentiation and inhibit osteoclast differentiation.It has also been proven to suppress local RANKL expression in joint tissues.However,specific mechanisms of iguratimod on bone protection are not clear,especially effects on serum levels of regulators of bone remodeling and bone remodeling markers in patients with RA and on RANKL and OPG expression in interleukin-1?(IL-1?)-induced fibroblast-like synoviocytes(FLS)from patients with RA are still to discover.Objective:To evaluate the effects of iguratimod on serum levels of regulators of bone remodeling(RANKL,OPG and DKK-1)and bone remodeling markers(CTX-I and PINP)in patients with RA and on the regulation of RANKL and OPG expression in IL-1?-induced FLS from patients with RA.And to discover the possible roles of iguratimod on bone metabolism and that may contribute to the treatment for RA in clinical practice.Methods:We collected 67 patients with RA who were treated with iguratimod(25 mg,twice daily)or methotrexate(MTX,10 mg,once weekly)or the combination of iguratimod(25 mg,twice daily)and MTX(10 mg,once weekly).Their clinical characters,laboratory data and radiological data were collected before treatment,6 months after treatment,and 12 months after treatment.All of the patients fulfilled the 2010 American College of Rheumatology(ACR)/ European League Against Rheumatism(EULAR)classification criteria for RA.Blood samples were also collected from patients with RA at the same time and enzyme linked immunosorbent assay(ELISA)and electrochemiluminescence immunoassay were used to value the serum RANKL,OPG,DKK-1,CTX-I and PINP levels.We collected synovial tissues from 3 patients with RA when they underwent total knee replacement surgeries and FLS were isolated from them for further experiments.All patients fulfilled the 1987 ACR criteria for RA.FLS were used for the experiments when they were between the passage 4 and 8.FLS were treated in the culture system with IL-1?(10 ng/mL)in the presence of different concentrations of iguratimod(5,10 or 20 ?g/m L),or MTX(100 nM),or iguratimod(20 ?g/m L)combined with MTX(100 nM)for 48 hours.The culture supernatants and cells were collected.ELISA was used to test the OPG levels in the culture supernatants.Quantitative real-time polymerase chain reaction(qPCR)and western blot analysis were used to test the RANKL and OPG messenger ribonucleic acid(mRNA)and protein expression in FLS.Results:Disease activity score 28(DAS28)decreased significantly 6 months and 12 months post-treatment in all three groups of patients with RA(all p<0.001).Even though there was no difference on DAS28 levels among 3 groups before treatment,the combination therapy group showed lower DAS28 than the other two groups 6 months post-treatment.There was no significant difference 12 months post-treatment.There was no significant difference in the levels of all serum regulators of bone remodeling and bone remodeling markers at baseline among the three groups.RANKL levels were decreased in all three groups 6 months and 12 months after treatment(all p<0.01)and the combination therapy group showed a greater suppress on RANKL than the other two groups 6 months post-treatment(both p<0.05),but there was no significant difference 12 months post-treatment.The change in the ratio of RANKL/OPG was similar to that in RANKL,but despite that all three group had a significant decrease in the RANKL/OPG ratio 12 months after treatment compared with baseline(all p<0.001),only the combination therapy group showed a significant decrease 6 months post-treatment(p<0.001).The difference of the other two groups was not significant.OPG and DKK-1 levels did not change significantly after treatment in all three groups.PINP levels increased after treatment but only significantly 12 months post-treatment in all three groups(group iguratimod p<0.01,group MTX p<0.05 and the combination therapy group p<0.001,respectively).For CTX-I,only the combination therapy group showed a significant decrease 6 months and 12 months after treatment(p <0.05 and p<0.001,respectively),whereas the other two groups did not experience a significant change.Associations between changes in DAS28 and changes in regulators of bone remodeling and bone remodeling markers were also examined.?DAS28-erythrocyte sedimentation rate(ESR)and ?DAS28-C-reactive protein(CRP)correlated with ?CTX-I positively(r=0.557,p<0.01 and r=0.652,p<0.01,rsepectively)and ?DAS28-ESR correlated with ?PINP negatively(r=-0.443,p<0.05)in group iguratimod after 6 months of treatment.In the combination therapy group,?DAS28-ESR and ?DAS28-CRP correlated negatively with ?PINP(r=-0.433,p<0.05 and r=-0.554,p<0.01,respectively)at 12 months after treatment.No significant differences of radiological score changes from baseline to 6 and 12 months after treatment among groups were noticed.OPG levels in the cell culture supernatants increased after stimulated by IL-1?(p<0.05)but showed no responses to the stimulation of iguratimod and MTX.A increase was found in RANKL mRNA,OPG mRNA and RANKL m RNA/OPG mRNA ratio(p<0.01,p<0.01 and p<0.05,respectively)after stimulated by IL-1? in FLS from patients with RA.For OPG mRNA,iguratimod and MTX had no significant effects but for RANKL mRNA,they both can suppress its expression(both p<0.01)and the combination therapy showed stronger effects than any drug did(both p<0.05).The change in the ratio of RANKL mRNA/OPG mRNA was similar to that in RANKL mRNA,but there was a difference,which is for RANKL mRNA/OPG mRNA,the combination therapy showed significant difference compared with group MTX(p<0.05)but not group iguratimod.There was an increase in intracellular OPG protein expression following stimulation with IL-1? but no more changes corresponding to iguratimod and MTX in FLS from patients with RA.Both iguratimod(10 and 20 ?g/m L)and MTX inhibited the increase in RANKL induced by IL-1?(all p<0.05)and the effects of iguratimod were concentration-dependent.The 20 ?g/mL concentration showed significantly greater suppression than 5 and 10 ?g/m L did(p<0.01 and p<0.05,respectively).In addition,the combination treatment was more efficient in suppressing RANKL expression than either drug was alone(group MTX p<0.01 and group iguratimod p<0.05,respectively).The ratio of RANKL/OPG protein showed similar results to those obtained with RANKL.Conclusions:1.Iguratimod could suppress the disease activity of RA and keep it at a low or moderate degree.2.Iguratimod shows great effects on stimulating bone formation,and when combined with MTX,it shows effects on preventing bone resorption.Iguratimod could decrease serum RANKL levels,which is a regulator of bone remodeling,in patients with RA 6 and 12 months after treatment and cause the decrease of RANKL/OPG ratio 12 months after treatment.It could also suppress RANKL expression in FLS in a concentration-dependent way.Its effects can be achieved through regulating the RANKL/RANK/OPG system and suppressing inflammation.3.Iguratimod could not affect the serum DKK-1 levels,which is a regulator of bone remodeling.Its effects are not achieved through regulating the DKK-1 mediated Wnt pathway.4.The effects of iguratimod on disease activity,on joint protection,and on regulators of bone remodeling and bone remodeling markers are comparable with those of MTX.The combination of both iguratimod and MTX shows faster and stronger effects than single drug does.