Effects Of Total Saponin From Rhizoma Dioscreae Nipponicae On The Expression Of NF-κB P65STAT3and AP-1in Vivo And In Vitro

Rheumatoid Arthritis(RA) is a chronic auto-immune disease of whichmain clinical manifestation is multi-symmetry arthritis. The main pathologicalcharacteristics in RA are the joint synovium inflammatory cell infiltration, thejoint synovium pannus formation, cartilage and bone erosion. The increase ofneonatal vascular is an essential condition for development and maintenanceof the pannus with RA. Then, inhibition of the joint synovium neonatalvascular is regarded as a new target for treatment of RA. Preliminary works ofour group showed that total saponin from Rhizoma Dioscreae Nipponicae(RDN) could decrease the number of joint synovium neonatal vascular incollagen-induced arthritis(CIA) rats, significantly downregulate the expres-sions of VEGF and its receptor in the joint synovium of CIA rats. Furtherconfirmed that inhibition of angiogenesis could be used as a therapeutic targetfor RA and is one of the mechanisms of the total saponin from RDN treatingRA. In vitro, IL-17combining with TNF-α could stimulate the rat synovialcell strain RSC-364to secrete a great quantity of VEGF, which could bepartially blocked by PDTC, the specific blocking agent of nucleartranscription factors-κB(NF-κB) signaling pathway. It suggested that NF-κBsignaling pathway was not the only way to regulate VEGF. In addition, theexperiments confirmed that the total saponin from RDN could inhibit inducingeffect of the inflammatory cytokines on the VEGF secretion of the RSC-364cells.This research is based on preliminary study works of the total saponinfrom RDN inhibit angiogenesis. To observe the effects of the total saponinfrom RDN on the expression of NF-κB p65, signal transducer and activator oftranscription3(STAT3) and activator protein-1(AP-1) in vivo and in vitro, collagen-induced arthritis(CIA) rats and the rat synovial cell strain RSC-364were used. We further clarified the regulatory mechanism of the total saponinfrom RDN on inhibition of angiogenesis in treatment of RA.Part Ⅰ Effects of total saponin from Rhizoma DioscreaeNipponicae on the expression of NF-κB p65，STAT3andAP-1in synovium of CIA ratsObjective:To observe the effects of total saponin from Rhizoma DioscreaeNipponicae (RDN) on NF-κB p65, STAT3and AP-1expressions in synoviumof collagen-induced arthritis (CIA) rats, and investigate the underlyingmechanism about signal transduction pathways of total saponin from RDN onantiangiogenesis in treating rheumatoid arthritis.Methods:Twenty-four rats were selected randomly from one hundred Wistar rats asnormal control group, the remaining seventy-six rats were established CIA ratmodel. The seventy-two successful models of CIA were randomly divided intothree groups(n=24): CIA model group, Tripterygium(TP) treamentgroup(positive control group), total saponin from RDN treament group. Fromthe sixteenth day after the initial immunization, the rats in Tripterygiumtreament group were lavaged with Tripterygium(TP)(12mg·kg﹣1·d﹣1,35d);the rats in total saponin from RDN treament group were lavaged with totalsaponin from RDN(25mg·kg﹣1·d﹣1,35d); The rats of normal control group andCIA model group were lavaged the same volume of solvent (0.5%CMC).The arthritis index(AI) were observed on the day16,23,30,37,44and50after the initial immunization; The NF-κB p65DNA-binding activity insynovium was detected by TransAMTMNF-κB p65Transcription Factor AssayKit. Immunohistochemical staining was used to observe the expression ofSTAT3protein in synovium; In situ hybridization was used to detect theexpressions of c-fos mRNA and c-jun mRNA in synovium; The AP-1 DNA-binding activity in synovium was detected by electrophoretic mobilityshift assay(EMSA).Results:1AI of each group rats’paws in different timeThe inflammatory reaction was approximately appeared on the fourteenthday after the initial immunization. Compared with the normal controlgroup(AI=0), CIA model group rats’ AI significantly increased on alltime(P＜0.01);compared with the CIA model group, the AI weresignificantly decreased after treatment with total saponin from RDN and TP onthe23th,30th,37th,44th and50th day(P＜0.01). There was no significantdifference between the two treatment groups(P＞0.05).2Activity of NF-κB p65in synovium of rats in each groupCompared with the normal control group, the NF-κB p65DNA-bindingactivity in nuclear extract of CIA model group rats’ synovium obviouslyincreased(P＜0.01). The NF-κB p65DNA-binding activity in nuclear extractof Tripterygium treament group and total saponin from RDN treament groupwere remarkably lower than that of CIA model group(P＜0.01). There was nosignificant difference between the two treatment groups(P＞0.05).3Expression of STAT3protein in synovium of rats in each groupSTAT3immunohistochemistry positive products were yellow or brownexquisite granulas and mainly located at cytoplasm, a small quantity located atnucleus. Compared with normal control group, STAT3expression in synoviumof CIA model group rats increased remarkably(P＜0.01).STAT3expressionsin synovium of rats in Tripterygium treament group and total saponin fromRDN treament group were remarkably lower than that in CIA modelgroup(P＜0.01). Moreover, there was no obvious difference between thetwo treatment groups (P＞0.05).4Expression of c-fos mRNA in synovium of rats in each groupThe positive signals of c-fos mRNA mainly distributed in cytoplasmwhich were yellow or brown exquisite granulas. Compared with normalcontrol group, the CIA model group rats’ synovium tissue of joint can be seen a lot of the cytoplasm stained brown or dark brown, c-fos mRNA expressionincreased significantly(P＜0.01). Compared with the CIA model group, c-fosmRNA expressions in synovium of rats in Tripterygium treament group andtotal saponin from RDN treament group were remarkably decreased (P＜0.01).Moreover, there was no obvious difference between the two treatment groups(P＞0.05).5Expression of c-jun mRNA in synovium of rats in each groupThe positive reaction of c-fos mRNA mainly distributed in cytoplasmwhich were yellow or brown exquisite granulas. Compared with normalcontrol group, the CIA model group rats’ synovium tissue of joint can be seena lot of the cytoplasm stained brown or dark brown, c-jun mRNA expressionincreased significantly(P＜0.01). Compared with the CIA model group, c-junmRNA expressions in synovium of rats in Tripterygium treament group andtotal saponin from RDN treament group were remarkably decreased (P＜0.01).Moreover, there was no obvious difference between the two treatment groups(P＞0.05).6Activity of AP-1in synovium of rats in each groupCompared with the normal control group, the AP-1DNA-binding activityin nuclear extract of CIA model group rats’ synovium obviouslyincreased(P＜0.01). The AP-1DNA-binding activity in nuclear extractof Tripterygium treament group and total saponin from RDN treament groupwere remarkably lower than that of CIA model group(P＜0.01).Conclusion:1Compared with the normal control group, the CIA model group rats’ AIremarkably increased on the16th,23th,30th,37th,44th and50th day. The AIwere significantly decreased after treatment with total saponin from RDN.2This study first confirmed that the total saponin from RDN couldremarkably decrease NF-κB p65, AP-1DNA-binding activity and STAT3expression, obviously downregulate the expressions of c-fos mRNA and c-junmRNA in synovium of CIA rats. It demonstrated that the total saponin fromRDN could influence VEGF secretion through the above-mentioned signal transduction pathway, thereby to restrain angiogenesis of rheumatoid arthritis.PartⅡ Effects of medicated Serum with total saponin from RDNon the expression of NF-κB p65，STAT3and AP-1in ratsynovial cell strain RSC-364induced by IL-17and TNF-αObjective:To observe the effects of medicated Serum with total saponin fromRhizoma Dioscreae Nipponicae (RDN) on NF-κB p65, STAT3and AP-1expressions in rat synovial cell strain RSC-364induced by IL-17and TNF-α.To investigate the underlying mechanism about signal transduction pathwaysof total saponin from RDN on antiangiogenesis in treating rheumatoidarthritis.Methods:Medicated serum of total saponin from RDN and Tripterygium(positivecontrol group) were prepared. Rat synovial cell strain RSC-364werestimulated by IL-17(10μg/L)+TNF-α(10μg/L) in the presence or absence oftotal saponin from RDN medicated serum, Tripterygium medicatedserum.After tween-four hours incubation, the NF-κB p65DNA-bindingactivity in nuclear extract of rat synovial cell strain RSC-364in each groupwas detected by TransAMTMNF-κB p65Transcription Factor Assay Kit, andthe AP-1DNA-binding activity was detected by electrophoretic mobility shiftassay(EMSA). Western blot was used to observe the expression of STAT3protein in each cells group.Results:1Activity of NF-κB p65in rat synovial cell strain RSC-364in eachgroupCompared with the control group, the NF-κB p65DNA-binding activityin nuclear extract of rat synovial cell strain RSC-364induced byIL-17+TNF-α model group obviously increased(P＜0.01). The NF-κB p65DNA-binding activity in nuclear extract of Tripterygium medicated serum group and total saponin from RDN medicated serum group remarkably lowerthan that of IL-17+TNF-α model group(P＜0.01). There was no significantdifference between the two medicated serum groups(P＞0.05).2Expression of STAT3protein in rat synovial cell strain RSC-364ineach groupCompared with control group, STAT3expression in IL-17+TNF-α modelgroup increased remarkably(P＜0.01).STAT3expressions in Tripterygiummedicated serum group and total saponin from RDN medicated serum groupwere remarkably lower than that of IL-17+TNF-α model group(P＜0.01).Moreover, there was no obvious difference between the two medicated serumgroups (P＞0.05).3Activity of AP-1in rat synovial cell strain RSC-364in each groupCompared with the control group, the AP-1DNA-binding activity innuclear extract of IL-17+TNF-α model group obviously increased(P＜0.01).The AP-1DNA-binding activity in nuclear extract of Tripterygium medicatedserum group and total saponin from RDN medicated serum group wereremarkably lower than that of IL-17+TNF-α model group(P＜0.05). Therewas no significant difference between the two medicated serum groups(P＞0.05).Conclusion:The rat synovial cell strain RSC-364induced by IL-17and TNF-α wasused as the research subject in this study. In vitro, first confirmed that totalsaponin from RDN medicated serum could remarkably lower NF-κB p65,AP-1DNA-binding activity and STAT3expression, considering that the totalsaponin from RDN could influence VEGF secrete maybe related to theabove-mentioned signal transduction pathway, thereby to restrain angiogenesisof rheumatoid arthritis.