| Objective: Rheumatoid arthritis (RA) is a kind of disease characterized by synovial hyperplasia and destruction of cartilage and bone. At present, it is confirmed that disease modifying antirheumatic drugs (DMARDs) have the control action for bone destruction, but have finite efficacy, obvious side effect and expensive cost. Fibroblast-like synoviocytes (FLS) are direct effectors of tissue injury and matrix remodeling in inflammatory synovitis. The aim of this study in which we culture rat FLS, then treat FLS with recombinant mouse macrophage migration inhibitory factor (rmMIF), and/or Tripterygium glycoside (TG), and/or ginsenoside (GS), then measure cell proliferation, detect osteoprotective cytokine such as receptor activator of nuclear factor kappaB ligand (RANKL), osteoprotegerin (OPG), tumor necrosis factor-α(TNF-α), and interleukin-1(IL-1), is to observe the intervention effect of rmMIF and compatibility of traditional Chinese medicines active components, Tripterygium glycoside (TG) and ginsenoside (GS), to explore the mechanism of destruction of bone induced by MIF and osteoprotective mechanism of compatibility of TG and GS.Methods:1 Rat FLS cloned strain RSC-364 was generally resuscitated, cultured, transferred, was used between passage 3 and 5 in the study.2 FLS were divided into blank group, MIF group, MIF+GS group, MIF+TG group, MIF+GS+TG group, MIF+Methotrexate (MTX) group.3 FLS proliferation was determined by optical density (OD) with MTT cell proliferation and cytotoxicity assay.4 Expression of RANKL and OPG in FLS was detected with immuno-histochemical method and had semiquantitative analysis.5 FLS supernatants were collected and stored at -70℃. Level of RANKL, OPG in FLS supernatants was measured by enzyme linked immunosorbent assay, while TNF-αand IL-1 in supernatants was measured by radio-immunity assay.6 All statistical analyses of data were computed by Statistical Package for Social Sciences (version 13.0). The results were expressed as x±s, the variation for multiple comparisons of data were assessed by one-way analysis of variance (ANOVA). One-Way ANOVA was followed by LSD multiple range tests for comparison between different treatment groups. The differences were considered statistically significant at P<0.05.Results:1 Intervention effect of rmMIF to rat FLS proliferation and expression of osteoprotective cytokine1.1 rmMIF can promote rat FLS proliferation. Compared with blank group, the optical density of MIF group was significantly higher (P=0.004).1.2 It is not showed that rmMIF promote rat FLS to express OPG. There was no significant difference of the labelling index of OPG between blank group and MIF group (P=0.117). The data were analysed with one-way analysis of variance, there was no significant difference of the level of OPG in supernatant in all groups (P=0.400). 1.3 rmMIF can promote rat FLS to express RANKL. In comparison with blank group, the labelling index of RANKL of MIF group (P=0.044) was higher significantly, and the level of RANKL in supernatant was higher, too (P=0.001).1.4 rmMIF can elevate ratios of RANKL/OPG in cultured rat FLS supernatant. There was significant difference between blank group and MIF group, MIF group was higher (P=0.001).1.5 It is not showed that rmMIF promote rat FLS to express TNF-α. The level of TNF-αin supernatant was not significant different between blank group and MIF group (P=0.408). 1.6 It is not showed that rmMIF promote rat FLS to express IL-1β. There was no significant level difference of IL-1βin supernatant in all groups (P=0.549).2 Intervention effect of TG and GS to rmMIF induced rat FLS proliferation and expression of osteoprotective cytokine2.1 TG can inhibit rmMIF induced rat FLS proliferation, but GS can't, and can't promote TG to inhibit rat FLS proliferation. The optical density of MIF+TG group (P=0.000) and MIF+GS+TG group (P=0.000) are lower significantly compared with MIF group, However, there was no significant difference between MIF+TG group and MIF+GS+TG group (P=0.624).2.2 TG can up-regulate expression of OPG in rat FLS, but GS can't, and can't promote the effect of TG. The labelling index of OPG of MIF+TG group (P=0.000, P=0.000) and MIF+GS+TG group (P=0.000, P=0.000) altogether were higher compared with blank group or MIF group significantly, However, there was no significant difference between MIF+TG group and MIF+GS+TG group (P=0.545). There was no significant difference of the level of OPG in supernatant in all groups (P=0.400).2.3 TG can inhibit rmMIF induced expression of RANKL in rat FLS, GS can too. There is synergetic effect between TG and GS. The labelling index of RANKL of MIF+TG group (P=0.003) and MIF+GS+TG group (P=0.000) were lower than MIF group significantly; MIF+GS+TG group (P=0.000, P=0.018) had lower labelling index significantly compared with MIF+GS group or MIF+TG group. Compared with MIF group, the level of RANKL of MIF+GS group (P=0.036), MIF+TG group (P=0.000) and MIF+GS+TG group (P=0.000) were decreased significantly. Compared with MIF+GS group or MIF+TG group, MIF+GS+TG group (P=0.000, P=0.034) was lower significantly on the level of RANKL in supernatant.2.4 TG can degrade elevated ratios of RANKL/OPG in cultured rat FLS supernatant by rmMIF, but GS can't. GS can enhance the effect of TG. In comparison with MIF group, MIF+TG group (P=0.000) and MIF+GS+TG group (P=0.000) were lower significantly. Compared with MIF+GS group or MIF+TG group, MIF+GS+TG group (P=0.000, P=0.023) was lower. 2.5 TG can inhibit rmMIF induced expression of TNF-αin rat FLS. It is not showed that GS inhibit rmMIF induced expression of TNF-αin rat FLS, and enhance the effect of TG. The level of TNF-αof MIF+TG group (P=0.018), MIF+GS+TG group (P=0.039) were decreased compared with MIF group significantly. There was no significant difference between MIF+TG group and MIF+GS+TG groups (P=0.729).2.6 It is not showed that TG and GS inhibit rat FLS to express IL-1β. The data were analysed with one-way analysis of variance, there was no significant difference of the level of IL-1βin all groups (P=0.549).Conclusions:1 The results that rmMIF can promote rat FLS proliferation, expression of RANKL, ratios of RANKL/OPG in supernatant suggest up-regulating FLS proliferation, expression of RANKL in FLS, and ratios of RANKL/OPG maybe is the mechanism that MIF participate the bone destruction in RA.2 TG can inhibit rmMIF induced rat FLS proliferation, but GS can't, and can't promote TG to inhibit rat FLS proliferation. Inhibiting FLS proliferation perhaps is the partial mechanism of TG for RA treatment.3 TG can inhibit rmMIF induced expression of TNF-αand RANKL in rat FLS, can degrade expression of TNF-αand RANKL in FLS, ratios of RANKL/OPG in supernatant. GS can inhibit rmMIF induced expression of RANKL, but can't degrade expression of TNF-α, IL-1β, and ratios of RANKL/OPG.4 TG and GS, active components of traditional Chinese medicines, have synergy to inhibit that rmMIF up-regulate expression of RANKL in rat FLS and ratios of RANKL/OPG in supernatant. This results suggest combination of active components of traditional Chinese medicines is an important pathway to enhance the therapeutic effect for RA.
|
PDF Full Text Request