Therapeutic Effect Of Sesquiterpene Lactone Compound IJ-5from Inula Japonica Thunb On Rheumatoid Arthritis And Its Anti-inflammatory Mechanisms

Rheumatoid arthritis （RA） is a chronic inflammatory joint disease characterized by theprogressive destruction of cartilage and underlying bone. Even though the mechanismsresponsible for the developement of RA have not been fully understood, over-activatedimmune cells and transcription-factors have been suggested to contribute to the process ofRA. Natural compounds with anti-inflammatory activity isolated from herbs may providetemplates for potential antiarthritic drug development. Our previous studies have identifiedvarious sesquiterpene lactones from the aerial part of Inula japonica Thunb showing potentanti-inflammatory activity by inhibiting the activation of transcription factor NF-κB.However, the therapeutic potential of such compounds for treatment of RA has not beenevaluated. The molecular mechanism underlying their inhibition on NF-κB signalingpathway has not been eluciated. Here, we choose a sesquiterpene lactone compound IJ-5asa representitive and investigate its potential therapeutic effect in the treatment of RA. Theunderlying mechanisms and the possible molecular target are further explored to lay thefoundation for further development of IJ-5into a new antiarthritic drug.Methods:The therateutic effect of IJ-5for RA was evaluated on collagen-induced arthritis (CIA)mice model. The in vitro anti-inflammatory effect of IJ-5was investigated by assaying itssuppression on TNF-α-induced inflammatory gene expression in human synovialfibroblasts with real-time quantitative PCR and ELISA. The effect of IJ-5on NF-κBpathway activation was tested using a dual-luciferase NF-κB reporter assay. The impact ofIJ-5on NF-κB and other signaling pathways was studied with immunoblot technology. Theinhibitory potential of IJ-5against protein kinases was determined using Millipore’sKinaseProfiler, Invitrogen’s Z’-LYTE, and DiscoveRx’s KINOMEscanTM screeningplatforms. The inhibitory effect of IJ-5on ubiquitination was tested in TNF-α-stimulated293T cells by analyzing polyUb chains on RIP1and NEMO with immunoprecipitation,and further confirmed by in vitro ubiquitination assay. The effect of IJ-5on conjugation ofUb to E2was assayed in in vitro E2ubiquitin-loading systems. The direct interactionbetween IJ-5and UbcH5c was assayed by pull-down experiment with IJ-5-coupled beads.The binding site of IJ-5on UbcH5c was determined using LC/MS/MS analysis and molecular docking.Results:1. To evaluate the therapeutic value of IJ-5for treatment of inflammatory diseases, wetested its in vivo anti-inflammatory effects in CIA model. Daily intraperitoneal injectionof IJ-5(2-10mg/kg per day) beginning on day21postimmunization dose-dependentlydecreased the severity of CIA in comparison with vehicle-treated arthritic mice.Radiological examination and histopathological analyses of joints revealed thatIJ-5-treated mice have significantly reduced synovitis and mononuclear cell infiltration.Analysis of the hind paw joints showed that treatment with IJ-5dose-dependentlyinhibited CIA-induced mRNA expression of the pro-inflammatory factors, includingTNF-α, IL-1β, IL-6, MCP-1, MMP3and MMP13. Accordingly, the serum levels ofseveral cytokines, including TNF-α, IL-1β and IL-6, in the arthritic mice were alsodecreased by IJ-5treatment in a dose-dependent manner. Then, RA synovial fibroblastswere investigated to determine the inhibitory effect of IJ-5on pro-inflammatory factorsexpression in vitro. Real-time quantitative PCR and ELISA tests revealed that IJ-5(2.5-10μM) dose-dependently decreased TNF-α-induced expressions ofNF-κB-targeted inflammatory genes, including IL-6, MCP1, IL-8, GRO-α, MIP-1α,RANTES, MMP3and MMP13in human synovial fibroblasts, as well as IL-6, IL-8,MCP1and MMP3levels in culture supernatants, indicating IJ-5can attenuate TNF-α’spro-inflammatory activity. Collectively, both in vivo and in vitro data demonstrate thatIJ-5reduces the arthritic inflammatory response by down-regulating expression ofpro-inflammatory factors.2. To explore the possible mechanism involved in IJ-5mediated anti-inflammation effect,we investigate the influence of IJ-5on the activation of NF-κB and MAPKs pathways,which is reported to relevant with inflammatory responses. First, we investigated theeffect of IJ-5on the NF-κB activation by examing Luciferase activities inpNF-κB-luc-293T cells. The result showed that IJ-5(2.5-10μM) dose-dependentlysuppressed TNF-α-induced NF-κB reporter expression in293T cells. Furtherimmunoblotting assay revealed that IJ-5(2.5-10μM) did not inhibit TNF-α and IL-1βstimulated phosphorylation of ERK, JNK and p38, but selectively blockedTNF-α-induced IKKβ and IκBα phosphorylation and inhibited NF-κB activation in RAsynovial fibroblasts, indicating that IJ-5exerts anti-inflammatory effect by suppressingTNF-α-induced NF-κB activation and gene expressions. 3. Since TNF-α-induced IKKβ activation is achieved through trans-autophosphorylationor upstream kinase-mediated phosphorylation, the suppression of IKKβ activation byIJ-5might come from its direct inhibition on IKKβ or other upstream kinases. However,IJ-5showed no direct inhibitory effects on all of the protein kinases that may mediateIKKβ phosphorylation, including IKKβ, IKKα, IKKε, TAK1, MAP3K14, MAP3K3,MAP3K8, PI3K, PKBα, PKBβ, PKCθ, PKCζ, PKCε, and TBK1, when assayed at30μM in vitro using Millipore’s KinaseProfiler and Invitrogen’s Z’-LYTE screeningplatform. Furthermore, no interactions between IJ-5and these kinases were detectableat DiscoveRx’s KINOMEscan screening platform. These results exclude the possibilitythat these kinases are direct targets for IJ-5.4. Given the critical role of ubiquitination in TNF-α-induced IKKtrans-autophosphorylation, we reasoned that IJ-5might act by impeding polyUb chainsynthesis. We thus tested the impact of IJ-5on TNF-α-induced ubiquitination of RIP1and NEMO in293T cells. The results showed that IJ-5(2.5-10μM) dose-dependentltsuppressed TNF-α-stimulated ubiquitination of RIP1and NEMO in293T cells,suggesting IJ-5may target one or more components of the ubiquitin system. To narrowdown the potential targets for IJ-5, we further tested its activity against the formation ofpolyUb chains in in vitro ubiquitination systems, in which recombinant c-IAP1orHIOP/RNF3, the core components of LUBAC complex, was used as E3enzyme,recombinant UbcH5c, E2-25K, or UbcH7was used as E2enzyme, and recombinantUBE1as E1enzyme. IJ-5exhibited inhibitory effect against polyUb chain formationcatalyzed by c-IAP1and UbcH5c. IJ-5also inhibited the linear chain formationcatalyzed by HIOP/RNF3in partnership with UbcH5c. Interestingly, when UbcH5cwas substituted with other E2s (UbcH7and E2-25K) in the above in vitroubiquitination system, IJ-5showed no inhibitory effects anymore, indicating UbcH5cmaight be the potential target of IJ-5.5. To further delineate IJ-5mechanism of action, we investigated the effects of IJ-5onloading of Ub to different E2s in in vitro Ub-loading assays. The results demonstratedthat IJ-5dose-dependently inhibited Ub loading to UbcH5c. IJ-5also similarlyinhibited Ub loading to UbcH5a and UbcH5b, but did not affect the Ub loading toUbcH7and E2-25K. These data suggested IJ-5is an E2enzyme inhibitor which isselective for UbcH5family. To test whether IJ-5can target UbcH5c in cells, weperformed pull-down experiments using IJ-5-conjugated sepharose beads. Both endogenous UbcH5in RA synovial fibroblast lysates and recombinant UbcH5c werepulled down by IJ-5-conjugated sepharose beads, but not by IJ-5-free control beads.Together, these data clearly confirm the direct interaction between IJ-5and UbcH5andindicate that UbcH5is a target for IJ-5.6. As a sesquiterpene lactone compound, IJ-5contains a α,β-unsaturated carbonyl moietywhich is known to bind covalently to free cysteine thiol groups of proteins by Michaeladdition. Based on the finding that IJ-5can bind to UbcH5c and inhibit Ub loading to it,we predicted that IJ-5might be able to form a covalent adduct with Cys85of UbcH5cvia Michael addition and thereby inactivating UbcH5c. To test this prediction, weincubated recombinant UbcH5c with or without IJ-5and digested the proteins withtrypsin and analyzed using online LC-Orbitrap CID MS/MS. The incubation with IJ-5led to a defined mass change of one peptide with molecular masses of1145.62derivedfrom the digestion of UbcH5c protein that was directly measured by high-resolutionMS with high confidence. This peptide fragment contains UbcH5c’s active site cysteine:Cys85. The mass shift suggests that UbcH5c was modified by IJ-5at Cys85. CIDfragmentation MS/MS was also used to localize the modification site with single aminoacid resolution. Detection of precursor ions at high resolution and a nearly completeseries of fragmentation ions from CID allowed the accurate sequencing and assignmentof the modification site to Cys85.7. Based on the above results, the terminal carbon atom of α-methylene moiety of IJ-5and the sulfur atom of Cys85were specified as ligand reactive group and the receptorbond. To investigate the binding mode of IJ-5and UbcH5c, we further performed thecovalent docking simulation using Maestro software (Schr dinger Inc, version9.0).The top20docked poses with the lowest estimated binding free energies wereremained for manual inspection. From the proposed binding mode, it can be found thatthe stereo conformation of IJ-5can fit well with the binding site around reactivecysteine residue Cys85, and the hydroxyl and carbonyl of IJ-5can form additionalhydrogen bonds with the amino groups of Gln92and Asn114of UbcH5c, respectivelyConclusion:In summary, the present study revealed for the first time that IJ-5, a SL compoundderived from an anti-inflammatory herb, directly bound to and inactivated UbcH5toinhibit TNF-α-induced IKK and NF-κB activation. We also showed that IJ-5inactivatedUbcH5by selective covalent modification of its catalytic cysteine residue. Moreover, IJ-5 not only inhibited the expressions of inflammatory genes in TNF-α-stimulated endothelialcells and synovial fibroblasts but also exhibited anti-inflammatory effects against RAinduced by collagen, suggesting that pharmacologically inactivating UbcH5may offer aneffective treatment for RA. These new findings emphasize the potential of UbcH5as atherapeutic target for TNF-α-mediated disorders and provide an interesting lead compoundfor the generation of new antiarthritic agents.