Regulation Of Expression Of P-glycoprotein In Eripheral Blood Lymphocytes With Rheumatoid Arthritis Exposed To The Pro-inflammatory Cytokine Interleukin-6

BackgroundRheumatoid arthritis (RA) is characterized by chronic inflammation of the synovial tissue and destruction of the joint. It is consensus in the international scope that patients need to be given diseases-modifying anti-rheumatic drugs (DMARDs) in the early stage, such as methotrexate (MTX), leflunomide (LEF), sulfasalazine(SSZ) and hydroxychloroquine (HCQ). DMARDs begin to play a role after several weeks or months, but in the process of clinical treatment. Some RA patients with no or low reaction to drugs, this phenomenon is called multidrug resistance (MDR) phenomenon Multidrug resistance is the important reason for the failure of cancer chemotherapy, which also may be the key factor for the failure of rheumatoid arthritis treatment. The mechanism of MDR is related to ATP-binding cassette(ABC) transporters family members. P-glycoprotein (P-gp) was first discovered in1976, it belongs to the ABC transporters super family members.The regulation mechanism of ABC transporters expression is very complicated. Many’factors (drugs, ultraviolet, X-ray radiation, cytokines, cancer gene, andanticancer) can influence the expression levels of ABC transporters, but the role of cytokines is is very important. Many studies have shown that the progression of rheumatoid arthritis and abnormal immune system is closely related to the release of inflammatory cytokines, which is leading to persistent synovial inflammation and destruction of articular cartilage or bone. Interleukin-6(IL-6) is a pleiotropic inflammatory cytokines, which is also one of most abundant cytokines in the serum and synovial fluid of RA patients. IL-6is the source of pathogenic factors of RA, it is involved in the process of B and T cell activation, and participate in the entire process of the disease. Some studies have shown that IL-6can reduce the expression of P-glycoprotein and function in tumor.At present, there is no regulation of expression of ABC transporters in rheumatoid arthritis exposed to the pro-inflammatory cytokine such as IL-6. In this topic, we carried on the research of expression and function of P-glycoprotein in rheumatoid arthritis exposed to the interleukin-6, and it may provide new targets for MDR reversal of RA patients.Objective1. To explore the differences of early rheumatoid arthritis and normal peripheral blood lymphocytes of the expression of P-glycoprotein.2. To study the affection of IL-6to the expression level、the function and the secretion of P-glycoprotein in the peripheral blood lymphocytes with rheumatoid arthritis.To investigate the role and relevance of P-gp and IL-6in RA multidrug resistance.MethodsRA patients(n=20) and normal controls(n=15) were respectively selected as object of this study. The clinical activity of RA was assessed by the Disease Activity Score(DAS)28in the selected RA patients. PBMC from the selected RA patients and normal control peripheral blood lymphocyte culture system was established. After the stimulation of IL-6in different concentrations (2ng,20ng), respectively in0h,48h and72hours to collect peripheral blood lymphocytes was analyzed by flow cytometry for P-gp expression, rhodamine-123accumulation test for P-gp function. The serum was analyzed by ELISA for P-gp.Results1. Compared to the Healthy controls,the relative fluorescence intensity(RFI) of P-glycoprotein significantly increased in patients with rheumatoid arthritis (P <0.05. and the expression levels of P-glycoprotein was correlated with with activity of disease.(r=0.877,P=0.02; r=0.85,P=0.031; r=0.8755,P=0.023)2. With the time of IL-6stimulus, the expression of P-gp levels had time dependence, peripheral blood lymphocyte were exposed to IL-6at the concentration of0,20ng/ml, after0h、48h、72h, the expression of P-gp was increased, but there was no statistically significant difference (P>0.05); At the concentration of2ng/ml, compared to the Oh and48h, the expression of p-gp levels at72h showed a significant increase (P<0.05);3. With the increase of concentration of IL-6, the expression of p-gp levels had certain concentration dependence. Peripheral blood lymphocyte were exposed to IL-6at the concentration of0,2,20ng/ml. Compared to the0、20ng/ml, the expression of p-gp levels at2ng/ml showed a significant increase (P<0.05);4. After the stimulation of IL-6.the function of P-gp had certain time dependence, peripheral blood lymphocyte were exposed to IL-6at the concentration of2ng/ml, after0h、48h、72h, rhodamine123fluorescence intensity was decreasing, but there was no statistically significant difference (P>0.05); At the concentration of20ng/ml, the difference had statistical significance (P<0.05);5. After the stimulation of IL-6, at48h, peripheral blood lymphocyte were exposed to IL-6at the concentration of0,2,20ng/ml, rhodamine123fluorescence intensity was decreasing, but there was no statistically significant difference (P>0.05); at72h, the difference had statistical significance (P<0.05);6. Four patients of P-gp secretion was detected in patients with rheumatoid arthritis. Compared with patients who were not detected P-gp, which disease activity was high (DAS28>3.2). It can affect large joints (elbow, shoulder, knee joint). The blood flow signals integral of energy doppler ultrasound was significantly higher than other patients.Conclusions1.Compared with healthy controls, patients with rheumatoid arthritis of P-glycoprotein expression level in peripheral blood lymphocyte increased significantly, and the expression levels of P-glycoprotein was correlated with with activity of disease (ESR,CRP,DAS28). It suggests that P-glycoprotein mediated MDR, also may be a index of disease activity. 2. The expression level of P-gp and IL-6have certain time dependence in patients with rheumatoid arthritis.3. After the IL-6stimulus, the function of P-gp Patients with rheumatoid arthritis has ovious time dependence and concentration dependent.4. P-gp is not only expressioned in the lymphocyte membrane of patients with rheumatoid arthritis (RA), but aso have the secretion of P-gp in blood serum. It may be mediated MDR, associated with the activity of disease.