| Objective: Rheumatoid arthritis (RA) is a chronic inflammatory diseasethat is characterized by synovitis associated with the progressive destructionof cartilage and bone. But its etiology and pathogenesis are still unclear. Inrecent years, TNF-a inhibitors as a representative of the biological agents isthe important therapeutic progress of RA, provide new targeted therapy choicefor the treatment of rheumatic disease. But biological agents can't fully controlthe progression of disease, clinical remission rate not satisfied, whichexpensive cost and obvious side effect limit the clinical application. The effectof Tripterygium glycoside(TG) as the major medicine plants (including theplant medicine effective component) to RA bone destruction has beenconfirmed. The aim of the study based on rat model of collagen-inducedarthritis to investigate the effect of active components of traditional Chinesemedicines and their compatibility on the expression of CD4+CD25+Foxp3+Regulatory T cells (Treg cells), Interleukin-10(IL-10), Transforminggrowth factor β(TGF-β), Interleukin-17(IL-17) and MMP-3. Explore itspartial mechanism of Osteoimmunology and also observe the influence oninflammatory score of experimental arthritis and the destruction of bone,thereby to supply the evidences of experiment for treating RA with activecomponents of plant amedica.Methods: Choosing Wistar rats weight in150-180g, the number of maleand female were equal. Measuring rats weight by electronic scale andthickness of their rear paws by sliding caliper, they were randomly dividedinto Normal control group and CIA group. CIA groups were injected with0.2ml collagen from chicken sternal cartilage type II in the tail, right hind limb and back together, and boosted at the14th day to make manufacture the modelof CIA．Normal group were injected saline in the same way. On21st day aftermodeling, rats that presented secondary-response were divided randomly intoModel group, TG group, TG+GS group, MTX group, and each group includedeight samples. By intragastric administration, they were respectively given TG,TG+GS and MTX for21days, and Normal group and Model group were givenequivalent volume of saline. On42nd day after modeling, measuring the pawsthickness, weights and AI, rats were drawn blood from orbit, anti-freezing,isolate the peripheral blood mononuclear cells (PBMC), Treg cells weredetected by FCM. Then they were sacrificed and drawn of blood by abdominalaortas under10%chloralic hydras to get serum. By enzyme linkedimmunosorbent assay, the levels of serum transforming growthfactor-β1(TGF-β1), interleukin-10(IL-10), interleukin-17(IL-17) were detected,while the expression of TGF-β1,IL-10and matrixmetalloproteinases-3(MMP3) in tissues were acquired by immuno-histochemical method and progressed semiquantitative analysis. DataStatistics: The results were expressed as±s. All statistical analyses of datawere computed by Statistical Package for Social Sciences (version13.0). Thecomparisons of mean among groups were evaluated by one-way analysis ofvariance (ANOVA). Pearson correlated-test was adopted in correlationanalysis. P<0.05was judged as a statistical difference.Results:1. Before modeling, there were no statistical differences among theweight of each group (P>0.05). But after21days, the weight of Model group,TG group, TG+GS group and MTX group is lower than normal groupremarkably (P<0.05). Compared with Model group, there were no statisticaldifferences among them (P>0.05). After three weeks of treatment, the weightof Model group, TG group, TG+GS group and MTX group is lower thanNormal group remarkably (P<0.01). Compared with Model group, there werestatistical differences among TG+GS group, MTX group and it (P<0.05),TG+GS group is higher than MTX group remarkably (P<0.01). The body weight gain of TG group, MTX group, TG+GS group and Model group islower than Normal group (P<0.05). The body weight gain of TG group,TG+GS group and MTX group is higher than Model group (P<0.05). Therewas no significant difference between TG group and Model group (P>0.05).2. On21stday after modeling, the AI value of Model group was9.88±0.83, TG group9.63±1.06, TG+GS group9.87±1.25, MTX group9.25±1.04. Compared with Model group, there were no statistical differencesamong them (P>0.05). On42ndday after modeling, the AI change value ofModel group was10.00±1.31, TG group4.88±1.13, TG+GS group4.75±1.49,MTX group4.63±1.06. Compared with Model group, there were statisticaldifferences among them (P<0.01). TG+GS group was the biggest, but therewas no statistical differences between MTX and it (P>0.05).3. After three weeks of treatment, the right paw swelling degree (%) ofTG+GS group, and MTX group is lower than Model group remarkably(P<0.01). Compared with MTX group, the inhibition ratio of TG+GS group ishigher, but there was no statistical differences between them (P>0.05), and TGgroup is lower (P<0.05). But the average swelling degree (%) of left paws ofTG+GS group, and MTX group is lower than Model group remarkably(P<0.01). Compared with MTX group, the inhibition ratio of TG+GS group ishigher (P>0.05), and TG group is lower (P<0.01).4.The percentage of CD4+CD25+Regulatory T cells in PBMC of Modelgroup, TG group, TG+GS group and MTX group is lower than normal groupremarkably (P<0.01). Compared with Model group, there were statisticaldifferences among TG group, TG+GS group, MTX group and it(P<0.01).Compared with MTX group, TG+GS group is higher than MTX group, butthere was no statistical differences between them (P>0.05). The percentage ofCD4+CD25+Foxp3+Regulatory T cells in PBMC of Model group, TG groupand MTX group is lower than normal group remarkably(P<0.05), TG+GSgroup is lower than normal group, but there was no statistical differencesbetween them(P>0.05). Compared with Model group, there were statisticaldifferences among TG group, TG+GS group, MTX group and it(P<0.01). Compared with MTX group, TG+GS group is higher than MTX group, butthere was no statistical differences between them (P>0.05).5.Comparison of serum TGF-β1(pg/ml):The level of serum TGF-β1inModel group was412.64±87.26,TG group518.68±152.93,TG+GS group596.60±148.59, MTX group587.45±107.06and normal group609.86±100.93.Compared with normal group, there were statistical differences among Modelgroup and it (P<0.01), but there was no statistical differences among TGgroup, TG+GS group, MTX group and it(P>0.05). Compared with Modelgroup, there were statistical differences among TG+GS group, MTX groupand it (P<0.01), TG+GS group is higher than MTX group, but there was nostatistical differences between them (P>0.05).Comparison of serum IL-10(pg/ml):The level of serum IL-10in Modelgroup was37.88±12.14,TG group40.60±11.45,TG+GS group51.28±11.58,MTX group49.35±14.59and normal group54.92±8.81. Compared withnormal group, there were statistical differences among Model group, TGgroup and it (P<0.05), but there was no statistical differences among TG+GSgroup, MTX group and it(P>0.05). Compared with Model group, there werestatistical differences among TG+GS group and it (P<0.05).Comparison of serum IL-17(pg/ml):The level of serum IL-17in Modelgroup was52.48±17.07, TG group42.67±11.26, TG+GS group37.77±10.60,MTX group438.52±15.49and normal group31.24±9.20. Compared withnormal group, there were statistical differences among Model group and it(P<0.01), but there was no statistical differences among TG group, TG+GSgroup, MTX group and it(P>0.05). Compared with Model group, there werestatistical differences among TG+GS group, MTX group and it (P<0.05).TG+GS group is lower than MTX group, but there was no statisticaldifferences between them (P>0.05).There were positive correlations among The percentage of Treg cells inPBMC and the level of serum TGF-β1and IL-10(r=0.488,0.425,respectively,P<0.01). There were negative correlations among The percentage of Treg cellsin PBMC and the level of serum IL-17and pathological score(r=-0.423, -0.360, respectively, P<0.05)6.There were no pathological changes of ankles in normal group. InModel group, there were synovial cells proliferating, a great quantity ofinflammatory cells infiltrating in synovium, synovium liners graduallythickening, synovial cells proliferating in the join region of synovium andcartilage, cartilages damaged, bone destruction in subchondral and trabecularbone, and even the integrity structures of cartilage and trabecular bone werenot observed. The pathological changes of ankles were all alleviated in eachtreatment group. The pathological score in Model group was2.00±0.76, TGgroup1.25±0.71,TG+GS group1.00±0.76and MTX group1.13±0.83.Compared with Model group, there were statistical differencesamong TG+GS group, MTX group and it (P<0.05), but there was nostatistical differences among the other groups and it (P>0.05).7.The expression of synovium TGF-β1in Model group was0.30±0.03, TG group0.22±0.04, TG+GS group0.19±0.03, MTX group0.20±0.04and normal group0.18±0.02. Compared with normal group, there werestatistical differences among Model group, TG group and it (P<0.05), butthere was no statistical differences among TG+GS group, MTX group and it(P>0.05). TG+GS group was least, but there was no statistical differencebetween MTX group and it(P>0.05). The expression of synovium IL-10inModel group was0.11±0.01, TG group0.13±0.04, TG+GS group0.20±0.03, MTX group0.17±0.05and normal group0.23±0.04. Compared withnormal group, there were statistical differences among Model group, TGgroup, MTX group and it (P<0.01), but there was no statistical differencesamong TG+GS group and it(P>0.05). There were no statistical differencesbetween MTX group and TG+GS group(P>0.05). The expression of MMP-3in cartilage tissue of ankles in Model group was0.77±0.06, TG group0.60±0.12, TG+GS group0.50±0.12, MTX group0.46±0.09and normal group0.28±0.07. Compared with normal group, there were statistical differencesamong Model group, TG group, TG+GS group, MTX group and it (P<0.01).MTX group was least, but there was no statistical difference between TG+GS group and it(P>0.05).Conclusion:(1) There are positive correlations among the level ofCD4+CD25+Foxp3+Tregs and serum TGF-β1,IL-10, negative correlationsamong the level of serum IL-17, pathological score. By regulating up the levelof serum TGF β-1, IL-10enhance the immuno-modulating effects ofCD4+CD25+Foxp3+Tregs, CD4+CD25+Foxp3+Tregs may protect bonedestruction of RA.(2) By elevating the level of CD4+CD25+Foxp3+Tregs,serum TGF-β1,IL-10,degrading the level of serum IL-17, and diminishingthe expression of TGF-β1,elevating the expression of IL-10in synoviumand MMP-3in cartilage tissues, Tripterygium glycoside combine withGinsenosides can protect bone tissue in CIA rats. In addition, may also byadjusting the balance between Treg/Th17to play its immunosuppressionfunction.(3) Tripterygium glycoside combine with Ginsenosides haveanti-inflammatory and protective effect of the bone, and supply a new idea andmethod for treating RA. |
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