Therapeutical Effect Of 7,3'-dimethoxy Hesperetin On Adjuvant Arthritis In Rats And Its Mechanisms

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint swelling, synovial membrane inflammation, and cartilage destruction, which has high morbidity and is one of the major diseases caused loss of labor crowd. Although the etiology and pathogenesis of RA have not been completely cleared, numerous studies have confirmed that immune function disorder, synoviocyte extensive proliferation and articular cartilage or bone destroy are key etiological factors in RA development. Although there are a few anti-rheumatic drugs showing effectiveness on treating RA, their side effects and toxicity call for new and more effective natural drugs.In previous studies, hesperidin, a flavanone glycoside comprising the flavanone hesperetin and the disaccharide rutinose, had been reported to have anti-rheumatic effects on adjuvant arthritis (AA) or collagen-induced arthritis rat. However, its poor water soluble ability and low bioavailability limited the application in medicine. With the deep research on chemical modification of hesperidin, many hesperidin derivatives exerted better water soluble ability and bioavailability than hesperidin itself. In our previous studies, we aimed the structural feature of hesperidin and synthesized a series of hesperidin derivatives. On the basis of our previous work, the aim of the current study was first to screen the anti-inflammatory activity of hesperidin derivatives in vivo and vitro. Then, the anti-rheumatic effect of hesperidin derivative (7, 3'-dimethoxy hesperetin, DMHP) with potent anti-inflammatory activity was observed in AA rat model. We explored the possible mechanisms of DMHP on AA rat from the aspects of immune function and synoviocyte proliferation or apoptosis. Therefore, this study provided experimental data for the development of new therapeutic interventions and targets of RA. The main contents of the current study are divided into three sections as follows:Part one: Anti-inflammatory activity screening of hesperidin derivativesObjective: To screen the anti-inflammatory activity of hesperidin derivatives in vivo and vitro. Methods: The content of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) from peritoneal macrophage (PMΦ) of AA rats were measured by radioimmunity and HPLC method respectively. We used the screening model (dimethylbenzene-induced mouse ear edema) to evaluate the anti-inflammatory activity of hesperidin derivates in vivo. Then we observed the effects of bioactive derivatives on carrageenan induced rat hind paws swelling and the secondary inflammation of AA rat. Results: Derivative 2 (5, 7, 3'-triacetyl hesperidin, TAHP) and 5 (DMHP) of hesperidin significantly decreased the productions of PGE2 and LTB4 from PMΦof AA rats in a dose-dependent manner. TAHP and DMHP also remarkably inhibited the ear edema induced by dimethylbenzene in mice, as the same with naproxen. Furthermore, TAHP and DMHP had therapeutical effect on hind paw swelling induced by carrageenan and secondary inflammation of AA rat. Remarkably, equimolar DMHP exhibited better effective antirheumatic activity than its parent compound hesperidin in the above two models. Conclusion: TAHP and DMHP have significant anti-inflammatory activity in vivo or in vitro. DMHP shows better effect than hesperidin itself and needs further research and development.Part two: Anti-inflammatory and immunomodulatory actions of 7, 3'-dimethoxy hesperetin on AA rat and its mechanismsObjective: To clarify the therapeutic effects of DMHP on AA rat according to the changes of secondary paw swelling, polyarthritis, body weight and histological morphological in AA rat. Meanwhile, targeting at immunocyte, such as splenic lymphocyte, peritoneal macrophage (PMΦ) and synoviocyte, we explored the roles of DMHP in modulating the abnormal immune functions of AA rats and its possible mechanisms. Methods: Rats were immunized by intradermal injection of Freund's complete adjuvant (FCA). DMHP (22, 44, 88 mg/kg) was intragastric administrated after AA induction. Hind paw volume was determined with a digiti pedis volume meter. Body weight, thymus and spleen weight were measured. Histopathologic examinations were observed under a light microscope on ankle joints by HE staining. VEGF Expression of the synovial tissue was estimated with immunohistochemistry assay. The ConA-induced T lymphocyte proliferation and IL-2 production by splenocytes were measured by MTT assay and radioimmunity method respectively. IL-1β, IL-6, IL-10 and TNF-αproductions of PMΦand their mRNA expressions were determined with ELISA and RT-PCR respectively. Synoviocytes were separated by the method of collagenase and trypsin digestion. PGE2, IL-1βand TNF-αlevels produced by synoviocytes were also measured by corresponding methods. Furthermore, the changes of ConA-induced T lymphocyte proliferation and IL-1β, IL-6 productions of PMΦwere observed after DMHP treated in vitro. Results: Intragastrical administration of DMHP significantly attenuated the primary or secondary paw swelling and reduced polyarthritis index of AA rats. DMHP also could relieve the decrease of body weight and down-regulate the organ indexes of AA rats. DMHP clearly ameliorated the pathological changes of AA rats from the following aspects: synovial hyperplasia, inflammatory cells infiltration, vascular proliferation and bone or cartilage destruction. DMHP also decreased the expression of VEGF in synovial membrane. In addition, DMHP remarkably recovered T lymphocyte proliferation and IL-2 production which was suppressed in AA rats. DMHP down-regulated the productions of IL-1, IL-6 and TNF-αfrom PMΦin AA rats at protein and transcription levels, while IL-10 expression was increased after DNHP treatment. DMHP could inhibit the over-activated synoviocytes activity of AA rats by reducing the productions of PGE2, IL-1βand TNF-α. In addition, DMHP could recovere T lymphocyte proliferation and decrease the levels of IL-1βand IL-6 in vitro. Conclusion: The above results demonstrate that DMHP has a therapeutic effect on AA. DMHP recovers the balance of cytokine network by decreasing the production of inflammatory mediators and increasing anti-inflammatory cytokine level. The mechanisms of the anti-rheumatic effect of DMHP are, at least in part, related to adjusting abnormal immune function in AA rats.Part three: The pro-apoptotic effect of 7, 3'-dimethoxy hesperetin on synoviocytes in rats with adjuvant arthritis and its possible mechanismsObjective: To observe the pro-apoptotic effect of DMHP on synoviocytes in vivo and vitro. Meanwhile, we explored the possible mechanisms of inducing-apoptosis effect of DMHP from the following several aspects including Bcl-2 gene family, Caspase3 and cytosolic ionized free calcium ([Ca2+]i). Methods: DMHP (22, 44, 88 mg/kg) was intragastric administrated after AA induction. The apoptosis indexes of synoviocyte were measured by TUNEL method. The expressions of Bcl-2 and Bax in synovium were assessed and quantitated by immunohistochemistry method. The primary synoviocyte proliferation was measured by MTT assay. Fibroblast-like synoviocytes (FLS) was separated and cultured using tissue explant cultivation method. After different concentrations of DMHP treatment on AA-FLS in vitro, FLS proliferation was measured by MTT assay. In addition, we determined the pro-apoptotic effect of DMHP in vitro by the methods of Hoechst 33342 staining, DNA ladder and transmission electron microscope. The FLS apoptosis rates in different groups were assayed with Annexin V-FITC/PI double staining analysis by flow cytometry. The mRNA and protein expressions of Bcl-2, Bax and Caspase3 were assayed by RT-PCR and Western blot respectively. Caspase3 activity was also assayed by ultraviolet spectrophotometry. Meanwhile, intracellular [Ca2+]i concentration was detected with Fluo-3 AM fluorescence assay by living cell workstation. Results: Intragastrical administration of DMHP significantly inhibited the primary synoviocyte proliferation and induced synoviocyte apoptosis in a dose dependent manner. Besides, DMHP could inhibit the expression of Bcl-2 and increase the expression of Bax, as well as decrease the ratio of Bcl-2 to Bax in synovium compare to AA group. DMHP could remarkably inhibit AA-FLS proliferation in vitro. A DNA ladder formation and typical nuclear morphological changes were clearly observed in DMHP-treated AA-FLS groups. Analysis of flow cytometry indicated that DMHP obviously increased AA-FLS apoptosis rate in a dose-dependent manner. Treatment with DMHP in vitro apparently increased the mRNA levels of Bax and Caspase3 while markedly decreased the expression of Bcl-2 mRNA in contrast to AA-FLS control group. The Bcl-2, Bax and Caspase3 p20 proteins in different groups exhibited the similar changes as corresponding mRNA expression. In addition, DMHP treatment caused a significant dose-dependent increase in the Caspase3 activity. DMHP (50μM) did not show significant effect on [Ca2+]i of AA-FLS in non-calcium extracellular solution. However, in containing-calcium extracellular solution, after the treatment of DMHP (50μM) for 20 min, the intracellular [Ca2+]i of AA-FLS was significantly increased in contrast to the beginning (0 min) (p<0.01). Conclusion: DMHP inhibits synoviocytes proliferation and exert definitely inducing-apoptotic effect on synoviocytes from AA rats in vivo and vitro, which might be a key factor of DMHP in controlling hyperplastic synovitis of AA. The mechanisms of pro-apototic effect of DMHP on synoviocytes might be related to modulating expressions of Bcl-2/Bax, up-regulating Caspase3 expression or activation and promoting the extracellular calcium influx causing calcium overload.