| Rheumatoid arthritis(RA)is a complicated,progressive and chronic inflammatory autoimmune disease with inflammation by inflammatory cells,serious synovial hyperplasia and neovascularization,and progressive destruction of cartilage and bone.Fibroblast-like synoviocytes(FLS)is the main effector cell of RA,it play an important role in the process of joint destruction by producing large amounts of cytokines,matrix metalloproteinases(MMPs)and adhesion molecules.The cytoskeleton is involved in many aspects of vital cellular function,such as maintain cell morphology,cell adhesion and migration and muscle contraction.Filamentous actin(F-actin)is the basic skeleton to maintain cell shape and location.F-actin can undergo the processes of dynamic assembly and spatial organization in response to extracellular signals,such as growth factors and cytokines.These changes participate a range of physiological and pathological activities and increase the permeability of physiological barrier,such as blood-joint barrier(BJB).Studies have shown that F-actin can be expressed by synovial cells and endothelial cells,and its expression is regulated by Rho A,p38 MAPK,and nuclear factor kappa B(NF-?B)signal pathway.Geniposide(GE)is an iridoid glycoside compound,which derived from Gardenia jasminoides Ellis fruit,has anti-inflammatory,analgesic,anti angiogenesis and anti diabetic pharmacological effects.Achyranthes bidentata saponins(ABS)is the main active component of Achyranthes bidentata BI.,which is often use as a drug,with cite the drug down to the lower body of the body,and then play the role of treatment of lower body disease.Our previous studies have confirmed that GE had immunomodulatory effect on adjuvant arthritis(AA)rats.GE could inhibited the proliferation of FLS in AA rats,reduced proinflammatory cytokines(eg.interleukin(IL)-6,IL-17)level and increased the anti-inflammatory cytokines(eg.transcription and growth factor beta(TGF)-?,IL-4)level,decreased the phosphorylation of p38 MAPK.ABS could improve the distributionof GE in joint tissue of AA rats,and it had a targeted and guided effect on GE.In this experiment,we use the model of AA rats and take FLS as the objective,observe the effect of GE,ABS and their combination on the function of FLS,to explore the change of F-actin expression,and it made further studies to check the intervention and target of GE,ABS and their combination on the mechanism of Rho A regulation of p38MAPK/NF-?B/F-actin in FLS on AA rats.This study can provide new experimental basis of GE and ABS,reveal the pathological mechanism of RA,and look for new targets for RA.OBJECTIVE These works were carried out to observe the effect of GE,ABS and their combination on proliferation in FLS of AA rats,to investigate the effect of GE,ABS and their combination on secretion of inflammatory and anti-inflammatory cytokines in FLS of AA rats,to observe the effect of GE,ABS and their combination on expression of Rho A,p38 MAPK,NF-?B,F-actin in FLS.Reveal the mechanisms of GE and ABS in RA,to provide new experimental basis for RA treatment.METHOD Sprague-Dawley(SD)rats were subcutaneously injected with Freund's complete adjuvant(FCA)to prepare AA rats,and to observed systemic score assessment,arthritis index(AI),and paw swelling of AA rats.On d21 after immunization,AA rats were sacrificed,FLSs were cultured from synovinal tissue.There were divided into 10 groups:namely,control group: blank DMEM medium,Lipopolysaccharide(LPS)group: 5?g/m L LPS,GE groups(25,50,100 ?g/m L),ABS group(40 ?g/m L),ABS combined with GE groups(40+25,40+50,40+100 ?g/m L),Rho/ROCK inhibitor group(Y-27632,10 ?M).Proliferation of FLS was determined by CCK-8 kit.The leves of IL-1?,IL-17,IL-4 and TGF-?1 were detected by ELISA kits.The key proteins Rho A,p38 MAPK,p-p38 MAPK,NF-?B p65,NF-?B p-p65 and F-actin in were determined by western blot.RESULTS1 Effects of GE,ABS and their combination on proliferation of FLS The study results of CCK-8 suggested that the optimal concentration of GE were25,50,100 ?g/m L and ABS was 40 ?g/m L for FLS proliferation.The proliferation activity of FLS in LPS group enhanced significantly compared to control group(P<0.01).Compared with LPS group,ABS(40 ?g/m L)had no significant effect on the proliferation of FLS;GE(25,50,100 ?g/m L)and ABS combined with GE(40+25,40+50,40+100 ?g/m L)showed different degrees of inhibitory effect on proliferative of FLS(P<0.05,P<0.01);Y-27632(10 ?M)also showed significantly inhibition on proliferation of FLS(P<0.01).Compared with GE groups(25,50,100 ?g/m L),only ABS combined with medium,and high dose of GE(40+50,40+100 ?g/m L)had significantly effect on proliferation of FLS(P<0.05).2 Effects of GE,ABS and their combination on cytokines secretion of FLS Compared with control group,the level of IL-1? and IL-17 was obviously increased,while IL-4 and TGF-?1 was declined in FLS of LPS group(P<0.01).Compared with LPS group,GE(25,50,100 ?g/m L),ABS combined with GE(40+25,40+50,40+100 ?g/m L)and Y-27632(10 ?M)significantly decreased the level of IL-1?and IL-17,and enhanced the level of IL-4 and TGF-?1 in FLS of AA rats(P<0.05,P<0.01),but ABS group(40 g/m L)had no significant effect on IL-1?,IL-17,IL-4 and TGF-?1.And compared with GE groups,only ABS combined with medium,and high dose of GE(40+50,40+100 ?g/m L)had obvious effect on IL-1? and IL-4(P<0.05).3 Effects of GE,ABS and their combination on expression of Rho A,p38 MAPK,NF-?B and F-actin of FLS The relative expression of Rho A,p-p38 MAPK,NF-?B p-p65 and F-actin was significantly over-expressed in FLS of LPS group(P<0.01).Compared with LPS group,GE(50,100 ?g/m L)significantly diminished the expression of NF-?B p-p65(P<0.05,P<0.01),GE(25,50,100 ?g/m L)obviously inhibited the expression of Rho A,p-p38 MAPK and F-actin(P<0.05,P<0.01);ABS combined with GE(40+25,40+50,40+100 ?g/m L)and Y-27632(10 ?M)significantly decreased the expression of Rho A,p-p38 MAPK,NF-?B p-p65 and F-actin(P<0.05,P<0.01);ABS(40 ?g/m L)had no obvious effect on Rho A,p-p38 MAPK,NF-?B p-p65 and F-actin.The protein levels of p-p38 MAPK and F-actin in FLS had a significantly between GE(50 ?g/m L)and ABS combined with GE(40+50 ?g/m L)(P<0.05),while the levels of Rho A,p-p38 MAPK,NF-?B p-p65 and F-actin in FLS had vary significantly between GE(100 ?g/m L)and ABS combined with GE(40+100 ?g/m L)(P<0.05).CONCLUSIONS1.In LPS group,FLS proliferation obviously;the production of IL-1? and IL-17 in FLS were significantly increased,the level of IL-4 and TGF-?1 in FLS dramatically declined;the expression of Rho A,p-p38 MAPK,NF-?B p-p65 and F-actin in FLS were also significantly enhanced.2.The inhibitory mecahanism of GE maybe caused by inhibiting the proliferation of FLS,decreasing the secretion of IL-1? and IL-17,increasing the level of IL-4 and TGF-?1,regulating the balance of proinflammatory cytokines and anti-inflammatory cytokines and suppressing the regulation of p38MAPK/NF-?B/F-actin by Rho A in FLS of AA rats.3.ABS combined with low dose of GE(40+25?g/m L)had no significant effect on the anti-inflammatory and immunoregulatory effect of GE,while ABS combined with middle and high dose of GE(40+50,40+100 ?g/m L)could enhanced the anti-inflammatory and immunoregulatory effect of GE in FLS of AA rats. |
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