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Rheumatoid arthritis (RA) is an autoimmune disease characterized by pronounced inflammation and excessive synovial hyperplasia within affected joint. We previously reported 7, 3'-dimethoxy hesperetin (DMHP) as a highly anti-inflammatory active derivative of hesperidin showed apparent pro-apoptotic effect in vitro on fibroblast-like synoviocytes of rats with adjuvant arthritis (AA), an animal model of RA. Here, we investigated the therapeutic effects of DMHP on inflammation and synovial apoptosis in rats with AA in vivo. Paw swelling, arthritis index, TNF-α and IL-1β serum levels were measured to evaluate the effect of DMHP on inflammation in AA rats. DNA ladder detection and TUNEL assay were used to investigate the pro-apoptotic effect of DMHP on synovial apoptosis in vivo. Bcl-2, Bax mRNA and protein expressions in synovium were determined by real-time Q-PCR and western blot, respectively. We found DMHP inhibited secondary hind paw swelling and arthritis index, and decreased TNF-α and IL-1β serum levels in AA rats. Typical DNA ladder formation was found in DNA extraction of synovium from DMHP treated groups. The number of apoptotic synovial cells was elevated with DMHP treatment in TUNEL assay. DMHP markedly decreased Bcl-2 expression whereas increased Bax expression in synovium of AA rats at both transcription and protein levels. Moreover, DMHP treatment on AA rats significantly decreased the protein ratio of Bcl-2/Bax in synovium. In conclusion, DMHP has an apparent therapeutic effect on inflammation in rats with AA. Mechanisms of this effect are partly related to induction of synovial apoptosis through modulation of Bcl-2 and Bax expression.

In our previous study, we have demonstrated that 7, 3'-dimethoxy hesperetin (DMHP), an active derivative of hesperidin, showed pro-apoptotic effect on synoviocytes in vitro. The present study was to investigate the potential therapeutic effect of DMHP on adjuvant arthritis (AA) in rat and its possible mechanisms. Freund's complete adjuvant was used to induce AA in rats. DMHP were administered intragastrically once a day from days 12 to 21 after AA induction. Secondary paw swelling, arthritis index, and pathological assessments were observed. IL-6 production in serum and IL-6 mRNA expression in synovium was detected by ELISA and real-time RT-PCR respectively. The expression of mRNA (JAK2, STAT3) and protein (JAK2, p-JAK2, STAT3, p-STAT3) in synovium were determined. We found that DMHP significantly inhibited hind paw swelling and arthritis index, and ameliorated pathological changes of ankle joint in AA rats. DMHP suppressed the level of IL-6 in serum and the expression of IL-6 mRNA in synovium of AA rats in a dose-dependent manner. DMHP apparently decreased mRNA expression of JAK2 and STAT3 as well as protein expression of p-JAK2 and p-STAT3 in the synovium of the AA rats. Correlation analysis indicated that p-JAK2 or p-STAT3 protein expression was highly correlated with joint damage severity. In conclusion, DMHP has a powerful therapeutic effect on AA in rats and its mechanisms might be partly related to inhibiting excessive activation of JAK2-STAT3 pathway., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Approaches inducing fibroblast-like synoviocytes (FLS) apoptosis in rheumatoid arthritis (RA) patients have been considered as a promising strategy for treating RA. Here, adjuvant arthritis (AA) in rat was induced by complete Freund's adjuvant and FLS were separated and cultured using a tissue explant cultivation method. The apoptotic effect of 7,3'-dimethoxy hesperetin (DMHP, a highly antirheumatic active derivative of hesperidin) on AA FLS was evaluated with MTT assay, Hoechst staining and flow cytometry analysis. Bcl-2, Bax, caspase 3 gene expressions and caspase 3 activity were assayed to identify whether caspase 3 was involved in the apoptosis induced by DMHP. It was found that DMHP significantly decreased AA FLS proliferation in vitro by MTT assay. The AA FLS treated with DMHP displayed typical apoptotic characteristics including irregularity in shape, nuclear shrinkage and chromatin condensation. Flow cytometry analysis indicated that DMHP could obviously increase the AA FLS apoptosis rate. Compared with the AA-FLS control group, DMHP markedly decreased the mRNA expression of Bcl-2, whereas those of Bax and caspase 3 were increased. Moreover, DMHP significantly increased caspase 3 activity in a dose-dependent manner. In aggregate, the results demonstrate that DMHP effectively induces AA FLS apoptosis through caspase 3 activation and can be considered as a possible antirheumatic agent., (Copyright © 2010 John Wiley & Sons, Ltd.)

The present study was designed to investigate the effect of hesperidin on rat adjuvant arthritis (AA) and to elucidate the involvement of synoviocytes in this process. Freund's complete adjuvant was used to induce AA in rats. Secondary paw swelling and the index of immune organs of AA rats were measured. Macrophage-like synoviocytes were separated by the method of collagenase and trypsin digestion. Synoviocyte proliferation was assayed by MTT assay. TNF-alpha, IL-1beta and IL-10 production of synoviocytes were examined with ELISA. The levels of TNF-alpha, IL-1beta, IL-10 mRNA were determined using RT-PCR. It was found that intragastric hesperidin administration (80, 160 mg/kg) significantly inhibited secondary paw swelling and restored the index of immune organs of AA rats. Furthermore, synoviocyte proliferation in AA rats was apparently suppressed after hesperidin treatment, which was accompanied by decreased transcription as well as production of TNF-alpha and IL-1beta from synoviocytes. Hesperidin also markedly increased IL-10 at both protein and transcription levels in AA rats. In aggregate, the above results demonstrate that hesperidin has a therapeutic effect on AA and the mechanisms are partly related to inhibiting synoviocyte activity and modulating inflammatory cytokine production of synoviocytes which play a crucial role in the pathogenesis of AA.

Objective: To investigate the effect on the extract of total flavonoids of Chrysanthemum indicum (TFC) on adjuvant arthritis synovial cells., Method: SD rats were divided randomly into six groups including normal, model, TFC (84, 168, 336 mg x kg(-1)) and control drug Tripterygium glycosides (30 mg x kg(-1)) groups. Adjuvant arthritis rat model was induced by a single intradermal injection of 0.1 mL of the complete Freund's adjuvant into the right hind feet pads of the SD rats. The proliferation of synoviocyte was measured by MT; The apoptosis rates of synovial cells were evaluated using TUNEL and FCM analysis., Result: TFC resulted in a dose-dependent way in inhibiting the proliferation of synovial and inducing the apoptosis of synovium and synoviocytes in vivo., Conclusion: TFC can inhibit proliferation and induce apoptosis in synovial cells, and exert therapeutical effect on rheumatoid arthritis.

Chrysanthemum is a traditional Chinese medicine used in China to treat inflammatory diseases. The total flavonoids Chrysanthemum indicum (TFC) were extracted from the dried bud of Chrysanthemum indicum. Our previous study had demonstrated that TFC was a new class of effective anti-inflammation, analgesia and immunoloregulation agents. In this study, we established an adjuvant arthritis (AA) model by injection of Freund's Complete Adjuvant (FCA) to investigate the effect of TFC on the apoptosis of synoviocytes in AA Rats. Synoviocytes isolated from knee joint of rats were treated with different doses of TFC in vitro. Synoviocytes proliferation was measured by MTT assay, and DNA fragmentations were evaluated on agarose gel electrophoresis. The levels of caspase-3 cleaved fragments were analyzed by Western blot. The annexin V stain assay was used to explore the inhibition of caspase-3 on the amelioration of synoviocytes apoptosis. The results showed that TFC inhibited the proliferation of synoviocytes. Electrophoresis showed higher ladders of DNA bands in the TFC group. Cleaved fragments of caspase-3 were increased significantly. Furthermore, the apoptotic synoviocytes were markedly decreased by the caspase-3 specific inhibitor. These results suggest that TFC could induce synoviocytes apoptosis and suppress proliferation of synoviocytes in adjuvant-induced arthritis rats.