The "Drug-guide" Effect Of Achyranthes Bidentata Saponins On Geniposide In Regulation Of Ras-Erk1/2 Signal Induced By Integrin ?1 And F-actin In Fibroblast-like Synoviocytes Of Adjuvant Arthritis Rat

1 Objective To observe the “drug-guide” effect of achyranthes bidentata saponins(ABS)on geniposide(GE)in treatment of adjuvant arthritis(AA)induced by Freund's completed adjuvant(FCA)and to explore its possible mechanism.Through intragastric administration of GE and ABS alone or combined to AA rats,observing the improvement of paw swelling,arthritis score,systemic performance score,movement score and histological alteration of synovial in AA rats;observing the effects of GE and ABS on secretion of inflammatory cytokines interleukin(IL)-1?,IL-17 and anti-inflammatory cytokines IL-4,transforming growth factor-?1(TGF-?1)in fibroblast like synoviocytes(FLS)of AA rats;observeing the effect of GE and ABS on immunocytochemistry studies of F-actin in FLS of AA rats;observeing the effect of GE and ABS on expression of Itg ?1,Ras,Phospho-Erk1/2(P-Erk1/2),Erk1/2 and F-actin.2 Methods Each AA rat was induced by a single intradermal injection of 0.1 m L Freund's complete adjuvant(FCA)into the right hind paw.Rats were randomly divided into the following 10 groups: normal group,AA group,ABS groups(30,60,120 mg/kg,i.g.d17~d 24),GE group(60 mg/kg,i.g.d 17~d 24),GE+ABS groups(60+30,60+60,60+120 mg/kg,i.g.d 17~d 24)and sinomenine hydrochloride(SIN)group(as positive control group,90 mg/kg,i.g.,d 17~d 24).The secondary hind paw swelling was estimated by volume meter and the arthritis score,systemic performance score and movement score were evaluated by two independent observers who were blinded to the experimental design.Histopathological changes of synovial tissue with hematoxylin-eosin staining(HE)staining were observed by light microscope.The cellular morphologic changes of F-actin in FLS were observed,and then,images were taken with a confocal laser scanning microscope.The proliferation of FLS was assessed by cell counting kit-8(CCK-8).The levels of IL-1?,IL-4,IL-17 and TGF-?1 of FLS were determined by enzyme linked immunosorbent assay(ELISA).The expression of Itg ?1,Ras,P-Erk1/2,Erk1/2 and F-actin in FLS were measured by western blotting.3 Results3.1 Therapeutic effect of each administration group on AA rats On day 10 after immunization,the limb joints of AA rats became redness,oedema,ankyloses and joint deformity,futhermore,marked synovium hyperplasia and together with the infiltration of inflammation cells of AA synovium tissue.The ABS groups(30,60,120 mg/kg)have no significant effect on AA rats.While,GE(60 mg/kg),GE+ABS(60+30,60+60,60+120 mg/kg)and SIN(90 mg/kg)could significantly relieve paw swelling,lower arthritis index,systemic performance score and increase the movement score of AA rats(P<0.01),improve the status of inflammation and pathological of synovial tissue.Interesting,the effects of GE+ABS(60+60,60+120 mg/kg)groups were obviously better than the GE(60 mg/kg)group(P<0.01).3.2 Effects of each administration group on FLS proliferation of AA rats The proliferation of FLS in AA group was increased significantly compared with normal group(P<0.01).GE(60 mg/kg),GE+ABS(60+30,60+60,60+120 mg/kg)and SIN(90 mg/kg)could apparently decrease the FLS proliferation in AA rats(P<0.01).Moreover,the effects of medium and high combination groups(GE+ABS: 60+60,60+120)were superior to that in GE group(P<0.01).3.3 Effects of each administration group on distribution and morphology of F-actin in FLS of AA rats F-actin of normal FLS was found predominantly in cortical structures around cell periphery,with only a few thin stress fibers located within cell body.In FLS of AA rats,a clear change of the F-actin cytoskeleton was observed from at the cell periphery to stress fibers network that spanned the cell body and the stress fibers became dramatically thick and robust.In ABS(30,60,120 mg/kg)treated groups,the symptoms were not significantly changed;In GE(60 mg/kg),combination groups(GE+ABS:60+30,60+60,60+120 mg/kg)and SIN(90 mg/kg)treated group,the stress fibers were recovered to normal condition.3.4 Effects of each administration group on IL-1?,IL-4,IL-17 and TGF-?1expression in FLS of AA rats In FLS of AA rats,the production of IL-1? and IL-17 were increased,while IL-4 and TGF-?1 were decreased when compared with normal FLS(P<0.01).GE(60 mg/kg),GE+ABS(60+30,60+60,60+120 mg/kg)and SIN(90 mg/kg)could significantly decrease the production of IL-1?,IL-17,and increase the production of IL-4,TGF-?1when compared to AA group(P<0.01).Furthermore,the effects of medium and high combination groups(GE+ABS: 60+60,60+120 mg/kg)were superior to that in GE(60mg/kg)group(P<0.05,P<0.01).3.5 Effects of each administration group on regulation of Ras-Erk1/2 signal pathway induced by Itg ?1 and the expression of F-actin in FLS of AA rats Our results showed that the total protein level of Erk1/2 had no change significantly between groups,but the expression of Itg ?1,Ras,P-Erk1/2 and F-actin in FLS of AA rats were much higher than those in normal FLS(P<0.01),but these changes were reversed by GE(60 mg/kg),GE+ABS(60+30,60+60,60+120 mg/kg)and SIN(90mg/kg)(P<0.01).In addition,the effect of combination medium and high groups(GE+ABS: 60+60,60+120 mg/kg)were superior to that in GE(60 mg/kg)group(P<0.05,P<0.01).4 Conclusions4.1 ABS had no significant therapeutical effect when it be used alone to AA rats,while the combination medium and high groups(GE+ABS: 60+60,60+120 mg/kg)could relieve the paw swelling,lower arthritis index,systemic performance score and movement score,moreover,improve the status of inflammation and pathological of synovial tissue and these effects were superior to that in GE(60 mg/kg)group.The results demonstrated that the ABS might through increasing the concentration of GE in synovial tissue to promote the therapeutical effect of GE to AA rats.4.2 The possible mechanisms of GE to cure AA rats have been proved.On the one hand,GE might through decreasing the expession of F-actin combine to extracellular matrix(ECM)which induced by Itg ?1 to inhibit the activated Ras-Erk1/2 signal pathway,furthermore,that may related to the down-regulation of pro-inflammatory cytokines IL-1?,IL-17 and up-regulation of the anti-inflammatory cytokines IL-4,TGF-?1 in FLS.On the other hand,through reducing the expression of F-actin to decrease the blood-joint barrier(BJB)permeability of AA rats,that would block the harmful substances into the joint cavity,and then alleviate the symptom of AA.