| Background:Rheumatoid arthritis(RA)is a kind of chronic systemic autoimmune disease characterized with synovitis and pannus formation,resulting in joints' mutilation.Disease-modifying anti-rheumatic drugs(DMARDs)which are represented by Methotrexate(MTX)are classic drugs of RA,but there is phenomenon of multiple drug resistance in clinical practice.Drug efflux mediated by ATP-binding cassette(ABC)transmembrane transporters,especially P-glycoprotein(P-gp)coded by MDR1 gene may be the critical mechanism of MDR.Recent researches have found that the pivotal factor of RA pathogenesis-Interleukin-6(IL-6)and downstream signal transduction pathways may play an important role in MDR.However,in fibroblast-like synocytes(FLS),how IL-6 affects P-gp and what is the relation between MTX-resistance and P-gp are still unclear.From these points,this research established RA-FLS culture system in vitro,studied the regulation of IL-6 on P-gp,compared and analyzed the difference and the specific mechanism of MTX monotherapy and MTX combined with small dose of CTX inducing resistant protein P-gp.Objective:By observing levels of P-glycoprotein,JAK2 and STAT3,we could analyze the difference of three kinds of concentrations of IL-6 or MTX combined with small dose of CTX regulating P-gp and relevant signaling pathways,finally find out the effect and pathways paticipated in the process of IL-6 inducing P-gp as well as the specific mechanism of multidrug resisitance reversal by MTX combined with CTX mediated by P-gp in synoviocytes.Methods:Synovial tissues of untreated RA patients(n=3)were extracted by means of fine needle aspiration biopsy,FLS culture system in vitro was established and passaged to the fifth generation or so to do the experiments.Part one: effect and mechanism of IL-6inducing P-gp in synocytes of RA patients(1)FLS cells were divided into A,B,C,D four groups randomly with different treatments: Group A was the cell control group.Group B,C,D were respectively co-cultured with low(4ng/ml),middle(20ng/ml),high(100ng/ml)concentrations of IL-6.The effect of IL-6 in different concentrations on P-gp was observed.(2)FLS cells were divided into A,B,C,D four groups randomly with different treatments:Group A was the cell control group.Group B was co-cultured with IL-6(100ng/ml).Group C,D were respectively pretreated with JAK2-STAT3 signaling pathway inhibitor AG490(50u M)and ERK1/2 signaling pathway inhibitor PD98059(20u M)for 30 minutes,then added with IL-6(2ng/m L).The signal transduction pathways which were involved in IL-6 regulating P-gp were observed.Part two: effect and mechanism of MTX cambined with CTX inducing P-gp in synocytes of RA patients(3)FLS cells were divided into A,B,C,D,E five groups randomly with different treatments: Group A was the cell control group.Group B,C were respectively co-cultured with MTX(0.01?g/ml),MTX+CTX(1?g/ml).Group D,E were pretreated with JAK2-STAT3 signaling pathway inhibitor AG490(50u M)for 30 minutes,then added with MTX,MTX+CTX respectively.The effect of MTX±CTX on P-gp and the signal transduction pathway which was involved in MTX±CTX regulating P-gp were observed.Put all the FLS cells at 37? CO2 culture box in saturated humidity for 72 hours.Then collect the cells:(1)Detect FLS cellular proliferation inhibition rates with MTT assays;(2)Detect P-gp's expression in FLS with flow cytometry;(3)Detect m RNA production of P-gp,JAK2 and STAT3 with RT-PCR;(4)Detect protein contents of p-STAT3 in FLS with cell-based ELISA.The effect of different concentrations of IL-6,MTX±CTX and differernt kinds of pathway inhibitors on P-gp in FLS were analyzed.Results:1.Compared with the cell control group,P-gp expression and P-gp m RNA-production were higher in groups with IL-6,the differences were statistically significant(P<0.05),while the other groups showed differences between groups(P<0.05);Comparision between groups showed that cellular inhibitory rate of the group with high concentration of IL-6was significiantly higher than other groups,the differences were statistically significant(P<0.05),while the other groups showed no difference(P>0.05).2.Compared with the cell control group,IL-6 group showed higher level of P-gp expression and P-gp,JAK,STAT3 m RNA-production,the differences were statistically significant(P<0.05);Compared with IL-6 group,P-gp expression and P-gp m RNA-production were lower in the AG490+IL-6 group and PD98059+IL-6 group,the differences were statistically significant(P<0.05);There was significantly difference in AG490+IL-6 group and PD98059+IL-6 group,AG490+IL-6 group tended to be lower(P<0.05);Compared with IL-6 group,JAK2,STAT3 m RNA-production,p-STAT3 protein content were lower in AG490+IL-6 group,the differences were statistically significant(P<0.05);Comparision between groups showed no differences in cellular inhibitory rates(P>0.05);3.Compared with the cell control group,the other medicine groups showed higher level of P-gp expression and P-gp m RNA-production,the differences were statistically significant(P<0.05);Compared with MTX group,MTX+CTX group showed lower level of P-gp expression and P-gp m RNA-production,the differences were statistically significant(P<0.05);After adding pathway inhibitors,P-gp expression,P-gp,JAK,STAT3 m RNA-production,and p-STAT3 protein content were lower than MTX±CTX group,the differences were statistically significant(P<0.05);Comparision between groups showed no differences in cellular inhibitory rates(P>0.05).Conclusions:1.IL-6 can enhance P-gp expression of synocytes in RA patients in a concentration dependent manner to some extent;2.JAK2-STAT3 signaling pathway and ERK1/2 signaling pathway paticipated in the process of IL-6 inducing P-gp and JAK2/STAT3 pathway shows a more obvious inhibitory effect;3.MTX or MTX combined with CTX can enhance P-gp expression of synocytes in RA patients,JAK2-STAT3 pathway plays a vital role in the process.4.Compared with MTX,MTX combined with CTX weakens the activation of JAK2-STAT3 pathway and P-gp,so P-gp expression can be reversed to some extent;... |
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