Therapeutical Effect Of Penta-acetyl Geniposideon Adjuvant Arthritis Rats And Its Pro-apoptotic Effect On Fibroblast-like Synoviocytes

Objectives:The pathogenesis of rheumatoid arthritis（RA）is still unknown,overproliferation and inadequate apoptosis of fibroblast-like synovial cells（FLS）are important causes of RA,inhibiting the proliferation of FLS and inducing its apoptosis is an effective strategy for the treatment of RA.Geniposide,the main active ingredient of Gardenia jasminoides,has many pharmacological activities,such as anti-inflammatory,anti-oxidation and anti-tumor effects.Because geniposide has high polarity,low bioavailability,it is hard to separate and prepare monomers from Gardenia effectively,which greatly limits its application.Penta-acetyl geniposide（（Ac）₅GP）is an active compound derived from geniposide as nucleus.Its activity is higher than that of nucleus geniposide.It has been reported that（Ac）₅GP can induce apoptosis of tumor cells.In this study,adjuvant arthritis（AIA）FLS isolated from AIA rats and cultured in vitro were used to study the possible anti-rheumatic effect of（Ac）₅GP and its mechanism,providing new ideas and drug targets for the prevention and treatment of RA.Methods:Rat AIA was induced by complete Freund’s adjuvant.（Ac）₅GP（30,60,120mg/kg）was given to AIA rats by intragastric administration.Paw swelling,polyarthritis index,serum pro-inflammatory cytokines levels,histological assessments of ankle joint,and proteoglycan expression were respectively measured to evaluate the therapeutic effect of（Ac）₅GP on rat AIA.Immunohistochemistry for Ki67 and TUNEL assay were performed to reveal the anti-proliferative and pro-apoptotic effects of（Ac）₅GP on AIA synoviocytes in vivo.AIA FLS was isolated and cultured in vitro,（Ac）₅GP（50,100,200μM）was administered in vitro.Assays of MTT,hoechst staining,DNA electrophoresis and flow cytometry were performed to assess the apoptotic potential of（Ac）₅GP on AIA FLS.Western blot assay was applied to measure the expression levels of apoptosis-related proteins（Bax,Bcl-2 and caspase 3）and key molecules（IκBα,p-IκBαand NF-κB p65）in NF-κB signal pathway.The effect of（Ac）₅GP on NF-κB p65 nuclear translocation in AIA FLS was detected by immunofluorescence method.Results:（Ac）₅GP administration in vivo can inhibit secondary paw swelling,reduce polyarthritis index,decrease TNF-αand IL-1βserum levels,attenuate pathological damage of ankle joint,and promote proteoglycans expression.（Ac）₅GP administration in vivo also could reduce Ki67 positive expression rate and raise the synovial apoptosis index in synovial tissues.（Ac）₅GP（120 mg/kg）significantly decreased the protein level of Bcl-2,increased the protein level of Bax,and decreased the ratio of Bcl-2 to Bax.In addition,（Ac）₅GP（120 mg/kg）inhibited the degradation and phosphorylation of IκBαand reduced the protein level of NF-κB p65 in nuclear extracts.In vitro,（Ac）₅GP could dose-dependently inhibit the proliferation of AIA FLS.Hoechst staining indicated that AIA FLS treated with（Ac）₅GP showed typical apoptotic morphological characteristics.DNA ladder assay confirmed the apoptotic effect of（Ac）₅GP on synovial cell apoptosis.Flow cytometry showed that（Ac）₅GP could significantly increase the apoptotic rate of AIA FLS.The pro-apoptotic effect of（Ac）₅GP on synovial apoptosis was confirmed by DNA ladder detection.Flow cytometric apoptosis assay indicated that（Ac）₅GP significantly increased the apoptosis rate of AIA FLS.Similar to the results in vivo,（Ac）₅GP administration in vitro could decrease the expression of Bcl-2 protein and increase the expression of Bax and caspase-3 protein in AIA FLS.（Ac）₅GP administration in vitro inhibited the degradation and phosphorylation of IκBαin AIA FLS and reduced the protein level of NF-κB p65 in nuclear extract.Immunofluorescence analysis showed that（Ac）₅GP could inhibit the activation of NF-κB by inhibiting the nuclear translocation of NF-κB p65.Conclusion:（Ac）₅GP（active derivative of geniposide）has obvious therapeutic effect on AIA rats.Its mechanism may be related to inhibiting the proliferation of FLS,inducing apoptosis and inhibiting the NF-kappa B pathway.（Ac）₅GP administration in vitro can significantly promote AIA FLS apoptosis,which may be related to the regulation of apoptosis-related protein expression.（Ac）₅GP can inhibit the nuclear entry of NF-κB p65 and the activation of NF-κB pathway in vitro.