Research On Signaling Pathways Of Interleukin-6 Regulating P-glycoprotein In Peripheral Blood Lymphocytes Of Patients With Rheumatoid Arthritis

Background:Rheumatoid arthritis(RA) is one of autoimmune diseases with high disability. At present, the biggest challenge in the field of RA treatment is the emergence of multidrug resistance(MDR) in the application of disease-modifying anti-rheumatic drugs(DMARDs), which may be the crucial mechanism of increased drug excretion mediated by ATP-binding cassette(ABC) transporter proteins. Therein, the most typical excretion mechanism is a boost of expression of cell membrane-protein——P-glycoprotein(P-gp) coded by multidrug resistance gene 1(MDR1).The preliminary studies on this topic have shown that:1. There is a significant increase of P-gp on peripheral blood lymphocytes of RA patients. P-gp expression is correlated with the disease activity measured by the DAS28, erythrocyte sedimentation rate(ESR), C-reactive protein, which have indicated MDR mediated by P-gp in correlation with the disease activity.2. The expression level of P-gp in different treatment groups was compared,compared with single drug,the expression level of P-gp of combined medication was decreased, which indicated that the expression level of P-gp of MTX/LFE cycle combined CTX were reversed to a certain degree.3. After a stimulus of IL-6, P-gp expression and function are both enhanced with certain time and concentration dependence.The regulation effect of cells by cytokines is mediated by intracellular signaling transduction. Many researches have revealed an increasingly important role of cell signal pathways playing on pathogenesis of autoimmune diseases. And specific inhibitors of key enzymes become one of the most promising targeted therapeutic drugs. This research will explore the specific signal pathways of P-gp by IL-6 in peripheral blood lymphocytes of RA patients from the perspective of the mostly studied MAPK and JAK-STAT pathways, in order to explicit the specific regulation mechanisms of P-gp by IL-6 in peripheral blood lymphocytes of RA patients.Objective:By means of pretreating peripheral blood lymphocytes of RA patients in each group with different cell pathway inhibitors, co-culturing with IL-6 for 72 hours, and then observing the function and expression of the membrane surface protein-P-gp and its mRNA-production levels, we could find out the specific signaling pathways of inflammatory cytokines IL-6 regulating P-gp.Methods:Untreated RA patients(n=20) were chosen, without any DMARDs and biologic therapy. Extract peripheral blood lymphocytes from 20 ml collected blood samples.Establish culture system for peripheral blood lymphocytes. All cells were divided into four groups randomly. Group A was the blank control group. Group C and D were repectively pretreated with JAK2-STAT3 signaling pathway inhibitor AG490(50uM)and ERK1/2 signaling pathway inhibitor PD98059(20uM) for 30 minutes. Add IL-6(2ng / mL) to Group B, C, D. Put them at 37? CO2 culture box in saturated humidity for 72 hours. Then collect the cells.(1) Detect P-gp activity of peripheral blood lymphocytes with Rhoda mine 123 accumulation assay.(2) Detect P-gp mRNA of peripheral blood lymphocytes with RT-PCR.(3) Detect P-gp protein level located on cell membranes of peripheral blood lymphocytes with Western blot. By this way,we could analyze the effect of different pathway inhibitor placed on IL-6 regulating P-gp in peripheral blood lymphocytes of RA patients.Results:1.The P-gp mRNA-production levels in peripheral blood lymphocytes measured by RT-PCR: Compared with the blank control group, P-gp mRNA expression level of IL-6 group increased significantly(P<0.05). Compared with the IL-6 group, P-gp mRNA expression level of AG490 pretreated group and PD98059 pretreated group both decreased. The differences were statistically significant(P<0.05); There were no differences compared in pairs(the blank control group, AG490 pretreated group,PD98059 pretreated group)(P>0.05).2.The P-gp protein levels on membrane surface of peripheral blood lymphocytes detected by Western blot: Compared with the blank control group, P-gp protein level of IL-6 group increased significantly(P<0.05). Compared with the IL-6 group, P-gp protein levels of AG490 pretreated group and PD98059 pretreated group both decreased. The differences were statistically significant(P<0.05); There were no differences compared in pairs(the blank control group, AG490 pretreated group,PD98059 pretreated group)(P>0.05).3.The P-gp function in peripheral blood lymphocytes detected by Rh123 accumulation assay: Compared with the blank control group, Rh123 accumulation of IL-6 group decreased significantly, indicating that P-gp function enhanced(P<0.05).Compared with the IL-6 group, Rh123 accumulation of AG490 pretreated group and PD98059 pretreated group both increased, indicating a weak P-gp function. The differences were statistically significant(P<0.05); there were no differences compared in pairs(the blank control group, AG490 pretreated group, PD98059 pretreated group)(P>0.05).Conclusions:1. IL-6 can enhance the mRNA level, protein level and activity of P-gp in peripheral blood lymphocytes of RA.2. ERK1/2 signaling pathway inhibitor PD98059, JAK-STAT signaling pathway inhibitor AG490 can both down-regulate the P-gp mRNA-production levels, the P-gp protein expression levels, and the P-gp protein function in peripheral blood lymphocytes mediated by IL-6.3. ERKl/2 and JAK- STAT signaling pathway take part in the regulation mediated with IL-6 of P-gp in peripheral blood lymphocytes of RA.