| Rheumatoid arthritis(RA) is a chronic, inflammatory and systemic autoimmune disease. Mitogen-activated protein kinase(MAPK) is one of the most vital enzymes in signal network, which plays a significant role in the pathogenesis of RA. Currently, MAPKs are sorted into three main interactive subgroups, including c-Jun N-terminal kinase(JNK), extracellular signal-regulated kinase(ERK), and p38 MAPK.Geniposide(GE), an iridoid glycoside compound derived from Gardenia jasminoides Ellis fruit, is known to have anti-inflammatory activities. Our previous studies have demonstrated: GE exhibited an immunomodulatory effect on AA rats. GE could inhibit the abnormal proliferation of peripheral blood lymphocytes(PBL) and fibroblast like synoviocytes(FLS), repair the lower proliferation activity of mesenteric lymph node lymphocytes(MLNL), reduce the level of pro-inflammatory cytokines and increase the anti-inflammatory cytokines production, down-regulate the expression of p-JNK in PBL and MLNL, p-ERK1/2 in MLNL, and p-p38 MAPK in PBL and FLS, thereby effectively relieve RA.Based on our previous studies, in this experiment, we take immune cells(PBL), peripheral immune cells(MLNL) and effector cells(FLS) as the objective, to explore the intervention and target of GE on three subtypes of MAPKs signaling pathways in PBL, MLNL and FLS, and to work out the correlation analysis and crosstalk between the three pathways. It has an important guiding significance for the pathogenesis of RA and the treatment mechanism of GE.OBJECTIVE According to the improvement of GE on paw swelling, arthritis score and histological alteration of mesenteric lymph nodes and synovial in AA rats, to observe the therapeutic effect of GE on AA rats; to observe the effect of GE on proliferation activity of PBL, MLNL and FLS of AA rats; to observe the effect of GE on secretion of inflammatory and anti-inflammatory cytokines in PBL, MLNL and FLS of AA rats; to observe the effect of GE on expression of ERK1/2, JNK and p38 MAPK in PBL, MLNL and FLS of AA rats, and to analyze the correlation of these pathways, so as to reveal the interaction of these pathways.METHOD AA was induced by a single injection into the left hind metatarsal footpad with 100 μl of Freund’s complete adjuvant(FCA) for each rat. Rats were differentiated to six groups in a random manner: normal group, AA group, GE group(30, 60, 120 mg/kg, i.g., d14-d21) and SIN group(positive control group, 90 mg/kg, i.g., d14-d21). The degree of arthritis was estimated by secondary hind paw swelling and arthritis score. Pathohistology of mesenteric lymph nodes and synovial tissue was observed. The proliferation of PBL, MLNL and FLS was assessed by MTT. The levels of IFN-γ, IL-4, IL-17 and TGF-β were determined by ELISA. Key proteins in MAPK pathway were measured by western blotting.RESULTS 1 Therapeutic effect of GE on AA ratsThe peak of inflammatory response occurred on day 14 after immunization. The right hind paw swelling and arthritis score were evidently raised in AA group compared to normal group(P<0.01); Hyperplastic lymphatic follicle and inflammatory cells infiltration were evidently increased in AA rats; Hyperplastic synovium and inflammatory cells infiltration were also markedly observed in AA rats. GE(60 and 120 mg/kg) apparently suppressed the hind paw swelling and polyarthritis index from day 14 to day 21 after immunization(P<0.01), and improve histological alteration of mesenteric lymph nodes and synovial in AA rats.2 Effects of GE on proliferation, cytokine production and MAPK signalling of PBL in AA ratsThe proliferation activity of PBL in AA rats raised evidently compared to normal rats(P<0.01). GE(60 and 120 mg/kg) could apparently suppress the cell proliferation in AA rats(P<0.01).Compared to normal group, the production of IFN-γ and IL-17 was significantly increased, while that of IL-4 and TGF-β1 was markedly decreased in PBL of AA rats(P<0.01). GE(60 and 120 mg/kg) in a dose-dependent manner obviously declined the level of IL-17 and IFN-γ, and elevated the level of IL-4 and TGF-β in PBL of AA rats(P<0.01).The protein levels of non-phosphorylated MAPK isoforms had no change significantly between groups, but the relative expression of p-ERK1/2, p-JNK and p-p38 was significantly heightened in PBL of AA rats when compared with normal group(P<0.01). GE(60 and 120 mg/kg) in a dose-dependent manner significantly diminished that of p-JNK, p-ERK1/2 and p-p38(P<0.01).3 Effects of GE on proliferation, cytokine secretion and MAPK signal pathway of MLNL, and MAPK signalling of mesenteric lymph nodes in AA ratsThe proliferation of MLNL in AA rats diminished markedly compared to normal rats(P<0.01). GE(60 and 120 mg/kg) in a dose-dependent manner could apparently heighten the proliferation of MLNL in AA rats(P<0.01).Compared to normal group, the production of IFN-γ and IL-17 was significantly increased, while that of IL-4 and TGF-β was markedly decreased in MLNL of AA rats(P<0.01). GE(60 and 120 mg/kg) in a dose-dependent manner obviously declined the level of IL-17 and IFN-γ, and elevated the level of IL-4 and TGF-β in MLNL of AA rats(P<0.01). GE at 30 mg/kg diminished IFN-γ and elevated IL-4 in MLNL of AA rats but had no obvious effect on IL-17 and TGF-β.The total protein levels of ERK1/2, JNK and p38 in MLNL and mesenteric lymph nodes had no change significantly between groups, but the relative expression of p-ERK1/2, p-JNK and p-p38 was significantly heightened in MLNL and mesenteric lymph nodes of AA rats when compared with normal group(P<0.01). GE(60 and 120 mg/kg) in a dose-dependent manner significantly diminished that of p-JNK, p-ERK1/2 and p-p38(P<0.01).FLS, and MAPK signal pathway of synovium in AA rats 4 Effects of GE on proliferation, cytokine secretion and MAPK signal pathway ofThe proliferation of MLNL in AA rats diminished markedly compared to normal rats The proliferation of FLS in AA rats raised markedly compared to normal rats(P<0.01). GE(60 and 120 mg/kg) could apparently inhibited the proliferation of FLS in AA rats(P<0.01).Compared to normal group, the production of IFN-γ and IL-17 was significantly increased, while that of IL-4 and TGF-β was markedly decreased in FLS of AA rats(P<0.01). GE(60 and 120 mg/kg) in a dose-dependent manner obviously declined the level of IL-17 and IFN-γ, and elevated the level of IL-4 and TGF-β in FLS of AA rats(P<0.01). GE at 30 mg/kg only markedly elevated IL-4 in FLS of AA rats(P<0.05) but had no obvious effect on IFN-γ, IL-17 and TGF-β.The total protein levels of ERK1/2, JNK and p38 in FLS and synovium did not vary significantly between groups, but the relative expression of p-ERK1/2, p-JNK and p-p38 was significantly heightened in FLS and synovium of AA rats when compared with normal group(P<0.01). GE(60 and 120 mg/kg) in a dose-dependent manner significantly diminished that of p-JNK, p-ERK1/2 and p-p38(P<0.01).CONCLUSIONS1 GE(60 and 120 mg/kg) obviously cut secondary paw swelling and arthritis score of AA rats; improved histological changes of synovial and mesenteric lymph nodes in AA rats; inhibited the over proliferation of PBL and FLS in AA rats and repaired the lower proliferation activity of MLNL. The outcome hinted that GE could relieve inflammatory reaction of AA due to cellular immunity.2 On the one hand, GE(60 and 120 mg/kg) down-regulated the production of IFN-γ and IL-17, up-regulated the level of IL-4 and TGF-β in PBL, MLNL and FLS of AA rats. On the other hand, GE(60 and 120 mg/kg) decreased the phosphorylation of ERK1/2, JNK and p38 MAPK in PBL, MLNL and FLS of AA rats. The mechanism of GE for exerting anti-inflammatory and immunoregulatory effects maybe involve causing Th1 cells and Th17 cells immune tolerance and enhancing Th2 cells and Treg cells-mediated activities. |
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