| Objective: Rheumatoid arthritis (RA) is a chronic inflammatory disease that is characterized by synovitis associated with the progressive destruction of cartilage and bone. Its etiopathogenisis and pathogenesy are still unclear. The deformity rate of untreated RA patients is higher, but bone destruction is the key point. At present, disease modifying antirheumatic drugs are confirmed that have the control action for bone destruction, while tumor necrosis-αantagonist is the represent, but which finite efficacy, obvious side effect and expensive cost are their characteristics. On the base of collagen-induced arthritis/adjuvant arthritis (CIA/AA) model and fibroblast like synoviocytes culture, we observed the effects of active components of traditional Chinese medicines and their compatibility on experimental arthritis and the expression of MIF with which include Tripterygium glycoside (TG), triptolide and Ginsenosides (GS). Furthermore, we developed the observation of serum pharmacology aimed at fibroblast like synoviocytes proliferation and MIF of synoviocytes expressed. Even more, we ran the correlation analysis of serum IL-1β, TNF-αand the expression of MIF in CIA rats, and carryed out intervention study about active components of Tripterygium wilfordii. The aim of the study is to explore the correlation of macrophage migration inhibition factor (MIF) and the mechanisms of bone destruction with AA in rats, and the intervention effect of active components compatibility of traditional Chinese medicines and it combine with Methotrexat (MTX), thereby to supply the evidences of experiment for treating RA with active components of plant amedica.Methods: Choosing Wistar rats weight in 180g±20g, the number of male and female were equal. Measuring rats weight by electronic scale and thickness of their rear paws by sliding caliper, they were randomly divided into normal control group and AA group. Eight rats in normal control group were injected 0.1ml saline into their right rear paws under sterilizing regularly, which others were founded AA model by intradermic injection equivalent volume of 0.1ml Freund's complete adjuvant (FCA) containing 1mg of mycobacterium tuberculosis in the same way. On 21st day after modeling, rats that presented secondary-response were divided randomly into Model group, MTX group, TG group, GS group, GS+MTX group, TG+GS group, and each group included eight samples. By intragastric administration, they were respectively given MTX, TG, GS, GS+MTX and GS+TG for 21days, and normal control group and model group were given equivalent volume of saline. On 42nd day after modeling, measuring the paws thickness and weights, rats were sacrificed and drawn of blood by abdominal aortas under 10% chloraldurat to get serum. By enzyme linked immunosorbent assay and radio-immunity, the levels of serum MIF, receptor activator of NF-kappa B ligand (RANKL), osteoprotegerin (OPG), tumor necrosis factor-α(TNF-α) and interleukin-1 were detected, while the expression of MIF, OPG and RANKL in tissues were acquired by immuno-histochemical method and progressed semiquantitative analysis. Data Statistics: The results were expressed as±s. All statistical analyses of data were computed by Statistical Package for Social Sciences (version 13.0). The comparisons of mean among groups were evaluated by one-way analysis of variance (ANOVA). Pearson correlated-test was adopted in correlation analysis. P < 0.05 was judged as a statistical difference.Results:1 The effect of TG+GS on general state in AA ratsBefore modeling, there were no statistical differences among the weight of each group(P>0.05). On 21st day after modeling, the weight-gain value of Model group was 11.63±1.69, MTX group 11.25±2.92, TG group 11.38±3.85, GS group 11.75±2.82, GS+MTX group 13.13±4.32, TG+GS group 13.75±2.87 and normal control group 23.38±4.44, they were lower than normal control group. Compared with model group, there were no statistical differences among them (P > 0.05). On 42nd day after modeling, the weight-gain value of MYX group was 8.87±4.64, TG group 8.87±2.90, GS group 11.75±7.46, GS+MTX group 16.25±3.69, TG+GS group 20.00±7.09 and model group 6.12±2.53. Compared with model group, there were no statistical differences among MTX group, TG group and it (P>0.05), but there were statistical differences among GS group, GS+MTX group, TG+GS group and it (P<0.05), TG+GS group was the biggest.2 The effect of TG+GS on paws arthritic index(AI) in AA ratsOn 21st day after modeling, the AI value of Model group was 10.00±1.69, MTX group 9.63±1.41, TG group 8.75±1.28, GS group 10.13±1.73, GS+MTX group 9.00±1.31 and TG+GS group 9.13±1.46. Compared with model group, there were no statistical differences among them (P>0.05). On 42nd day after modeling, the AI change value of MTX group was 5.13±1.46, TG group 4.38±1.51, GS group 4.13±1.36, GS+MTX group 4.25±1.75 and TG+GS group 4.37±1.85. Compared with model group, there were statistical differences among them (P<0.05). MTX group was the biggest, but there was no statistical difference between TG+GS group and it (P>0.05).3 The effect of TG+GS on paws swelling-inhibition ratio in AA ratsOn 21st day after modeling, the both paws swelling-degree of model group were respectively (80.84±10.99, 58.45±14.88), MTX group (81.25±11.00, 59.84±15.84), TG group (79.00±9.77, 59.95±15.66), GS group (82.24±12.72, 57.78±16.18), GS+MTX group (81.27±11.43, 57.85±16.02) and TG+GS group (83.82±13.81, 59.66±15.76). Compared with Model group, there were no statistical differences among them(P>0.05). On 42nd day after modeling, the both paws average swelling-inhibition ratio of MTX group was 29.2%, TG group12.5%, GS group 4.9%, GS+MTX group 33.4% and TG+GS group 29.6%.4 The effect of TG+GS on serum TNF-α, IL-1β, MIF, OPG, RANKL in AA rats4.1 Comparison of serum TNF-α(ng/ml): The level of serum TNF-αin Model group was 1.37±0.16, MTX group 1.15±0.15, TG group 1.17±0.17, GS group 1.19±0.17, GS+MTX group 0.98±0.14, TG+GS group 1.02±0.15 and normal control group 0.87±0.11. Compared with normal control group, there were statistical differences among Model group, MTX group, TG group, GS group and it (P<0.01), but there were no statistical differences among GS+MTX group, TG+GS group and it (P>0.05).4.2 Comparison of serum IL-1β(ng/ml): The level of serum IL-1βin Model group was 0.18±0.03, MTX group 0.16±0.02, TG group 0.15±0.02, GS group 0.16±0.03, GS+MTX group 0.13±0.02, TG+GS group 0.13±0.03 and normal control group 0.59±0.02. Compared with normal control group, there were statistical differences among them(P<0.01). Compared with Model group, there were statistical differences among the MTX group, TG group, GS+MTX group, TG+GS group and it(P<0.01). GS+MTX group and TG+GS group were the lowest.4.3 Comparison of serum MIF(pg/ml): The level of serum MIF in Model group was 453.99±53.53, MTX group 417.73±38.90, TG group 431.49±41.08, GS group 423.89±45.01, GS+MTX group 408.28±33.82, TG+GS group 411.59±41.58 and normal control group 349.54±16.94. Compared with normal control group, there were statistical differences among them(P < 0.01). Compared with Model group, there were statistical differences among MTX group, GS+MTX group, TG+GS group and it (P<0.05), but there were no statistical differences among TG group,GS group and it (P>0.05). GS+MTX group was the lowest, but the was no statistical difference between TG+GS group and it (P>0.05).4.4 Comparison of serum OPG(ng/ml): The level of serum OPG in Model group was 86.40±12.27, MTX group 74.80±6.80, TG group 79.14±8.90, GS group 80.73±9.44, GS+MTX group 75.75±6.65, TG+GS group 76.01±7.67 and normal control group 69.41±3.36. Compared with normal control group,there were differences among Model group, TG group, GS group and it (P<0.05), but there were no statistical differences among MTX group, GS+MTX group, TG+GS group and it (P>0.05). 4.5 Comparison of serum RANKL(pg/ml): The level of serum RANKL in Model group was 33.80±2.48, MTX group 27.13±2.74, TG group 27.90±2.75, GS group 28.11±2.89, GS+MTX group 23.88±2.20, TG+GS group 24.55±2.10 and normal control group 21.25±1.57. Compared with normal control group, there were statistical differences among Model group, MTX group, TG group, GS group, TG+GS group and it (P<0.05), but there was no statistical difference between GS+MTX group and it (P>0.05).4.6 Comparison the proportionality of serum RANKL/OPG: The proportionality of serum RANKL/OPG in Model group was 0.40±0.06, MTX group0.34±0.04, TG group 0.36±0.05, GS group 0.35±0.07, GS+MTX group 0.32±0.06, TG+GS group 0.32±0.02 and normal control group 0.28±0.06. Compared with normal control group, there were statistical differences among (P<0.05), Model group, MTX group, TG group, GS group and it, but there were no statistical differences among GS+MTX group, TG+GS group and it(P>0.05).4.7 The correlation of TNF-α, IL-1β, RANKL, pathological score and MIF: There were positive correlations among the levels of serum TNF-α, IL-1β, RANKL, pathological score and MIF (r=0.938, 0.748, 0.763, 0.908, respectively, P<0.05).5 The effect of TG+GS on pathological change of ankles in AA ratsThere were no pathological changes of ankles in normal control group. In model group, there were synovial cells proliferating, a great quantity of inflammatory cells infiltrating in synovium, synovium liners gradually thickening, synovial cells proliferating in the join region of synovium and cartilage, cartilages damaged, bone destruction in subchondral and trabecular bone, and even the integrity structures of cartilage and trabecular bone were not observed. The pathological changes of ankles were all alleviated in each treatment group. The pathological score in Model group was 2.00±1.07, MTX group 1.88±1.07, TG group 1.63±1.19, GS group 1.88±0.99, GS+MTX group 0.88±0.83 and TG+GS group 1.38±1.19. Compared with Model group, there was statistical difference between GS+MTX group and it (P<0.05), but there were no statistical differences among the other groups and it (P>0.05).6 The effect of TG+GS on OPG,RANKL in bone tissue of ankles and synovium MIF in AA ratsThe expression of OPG in bone tissue of ankles in Model group was 0.19±0.07, MTX group 0.11±0.07, TG group 0.15±0.07, GS group 0.16±0.05, GS+MTX group 0.12±0.06, TG+GS group 0.13±0.06 and normal control group 0.07±0.03. Compared with normal control group, there were statistical differences among TG group, GS group, TG+GS group and it (P<0.05), but there were no statistical differences among MTX group, GS+MTX group and it(P>0.05). MTX group was least, but there was no statistical difference betwwen TG+GS group and it (P>0.05). The expression of RANKL in bone tissue of ankles in Model group was 0.28±0.08, TG group 0.19±0.10, GS group 0.21±0.07, MTX group 0.13±0.08, GS+MTX group 0.14±0.08, TG+GS group 0.15±0.07 and normal control group 0.08±0.03. Compared with normal control group, there were statistical differences among Model groupTG group,GS group and it (P<0.05), but there were no statistical defferences among MTX group, GS+MTX group, TG+GS group and it (P>0.05). The expression of synovium MIF in Model group was 0.30±0.06, MTX group 0.21±0.07, TG group 0.25±0.10, GS group 0.22±0.08, GS+MTX group 0.19±0.08, TG+GS group 0.21±0.08 and normal control group 0.07±0.03. Compared with normal control group, there were statistical differences among them(P<0.01). Compared with Model group, there were statistical differences among MTX group, GS+MTX group, TG+GS group and it (P<0.05), but there were no statistical differences among GS group , TG group and it(P>0.05). There were no statistical differences among MTX group, GS+MTX group and TG+GS group (P>0.05).Conclusions:1 There are positive correlations among the level of serum MIF and TNF-α, IL-1β, RANKL, pathological score. By regulating up the level of serum TNF-αand IL-1βand aggravating pathological changes of bone tissues, MTF can participate the mechanism of bone destruction in RA. 2 By degrading the level of serum TNF-α, IL-1β, MIF, RANKL, OPG and the proportionality of serum RANKL/OPG, and diminishing the expressions of MIF in synovium and OPG, RANKL in bone tissues, Tripterygium glycoside combine with Ginsenosides can protect bone tissue in AA rats.3 Tripterygium glycoside combine with Ginsenosides can antagonize the effect of MIF and decrease the proportionality of serum RANKL/OPG. Further, these confirm their regulation in anti-inflammatory and protective effecct of bone, and supply a new idea and method for treating RA.
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