Effect And Mechanism Of TNF-? On P-glycoprotein In Rheumatoid Arthritis

Background:Rheumatoid arthritis (RA) is a chronic systemic inflammatory autoimmune disease with high rate of disability.The main manifestation is progressive and erosive arthritis. Progression of RA results in severe disability and loss of function with severe pain. The main treatments currently of RA include non-steroidal anti-inflammatory drug (NSAIDs), disease modifying antirheumatic drug (DMARDs) and biological agents. Although combination of two or three DMARDs was administered to the patients with RA in the earlier stage of the disease, some patients displayed Poor efficacy to the treatment. Moreover, as the extension of the treatment, drug efficacy gradually reduced, show low response or even no response and drug resistance occurred. Emergence of multiple drug resistance (MDR) plays an important part in therapeutic efficacy of RA. As one of the cause of treatment failure, MDR was take account in the domain of carcinoma and infective disease. The research of MDR in RA was less.Molecular mechanisms underlying drug resistance include the action of transmembrane transporters, which mediate the cellular extrusion of a large variety of therapeutic drugs. P-glycoprotein (P-gp) is the most important and classic transmembrane transporter up to now. Overexpressi -on of P-gp results in reduction of intracellular concentrations of substrates involving xenobiotics and various drugs such as vinca alkaloids, anthracyclines, verapamil, some DMARDs (eg, hydroxyl chloroquine, D-penicillamine, colchicines) and corticosteroids. It was reported that the expression and activity of P-gp increased in refractory RA and play a part in MDR of RA. Tumor necrosis factor-alpha (TNF-?) is proinflammatory factor which play a core role in the pathogenesis of RA. Many studies confirmed that TNF-?regulates P-gp in different cell lines and involved in MDR. It is unknown that whether TNF-?regulate P-gp and involved in MDR in RA. The main target of DMARDs is the lymphocyte, so we choose peripheral blood mononuclear cells (PBMC) as research object. In this study, we investigated the level of the expression and activity of P-gp, TNF-?and analyzed their correlation. On this basis, we also investigated the effect of TNF-?on the expression and activity of P-gp of PBMC in vitro and explored the possible mechanisms. Our study is helpful to elucidate the character of MDR in RA and provides a basis of the molecular mechanisms of MDR in RA, which can offer a new idea and strategy for the treatment of RA especially refractory RA. Objective:To investigate the expression and function of P-gp, TNF-?mRNA of PBMC and the level of TNF-?in serum in normal and RA patients (refractory, effective to treatment or untreated). To analyses correlations among multidrug resistance, TNF-?and expression and function of P-gp.Methods:20 normal control and 60 RA patients (20 untreated,20 ffective to treatment,20 refractory for each group) were selected as object of this study. The clinical activity of RA was assessed by the Disease Activity Score (DAS)28 in the selected RA patients. PBMC from the selected RA patients and normal control was analyzed by flow cytometry for P-gp expression, rhodamine123 accumulation test for P-gp function and RT-PC R for TNF-?mRNA. The serum was analyzed by ELISA for TNF-?.Results:1. The expression and function of P-gp on PBMC was lower in normal control group compared with that in RA groups. Among the groups of RA, the expression and function of P-gp in RA refractory group was higher than that in RA untreated group and RA effective to treatment group(P<0.01). The expression and function of P-gp in RA untreated group was higher than that in RA effective to treatment group(P<0.05)2. The level of PBMC TNF-?mRNA and serum TNF-?in RA groups were significantly higher than that of normal control group(P< 0.01). Among the groups of RA, the level of PBMC TNF-?mRNA and serum TNF-?in RA refractory group was highest, and that in RA effective to treatment group was lowest.3. The score of DAS28 in RA untreated group(5.70±1.46) was higher than that of RA effective to treatment group(3.78±0.79,P< 0.01). The score of DAS28 in RA refractory group(7.02±0.93) was higher than that of RA effective to treatment group and RA untreated group(P< 0.01).4. The expression of P-gp was positively correlated with the level of PBMC TNF-?mRNA and serum TNF-?(r=0.29, P<0.01; r=0.758, P <0.01); Intracellular fluorescence intensity of rhodamine123 was negatively correlated with the level of PBMC TNF-?mRNA and serum TNF-?(r=-0.843, P<0.01; r=-0.863, P<0.01). The expression of P-gp was positively correlated with the scores of DAS28 (r=0.588, P< 0.01). Intracellular fluorescence intensity of rhodamine123 was negatively correlated with the scores of DAS28(r=-0.702, P<0.01).5. No correlation was found among the expression and function of P-gp, duration of disease and time of therapy. Conclusions:1. The expression and function of P-gp on PBMC was highest in RA refractory group and lowest in RA effective to treatment group. The increase of expression and function of P-gp was associated with refractoriness of RA. P-gp played a role in MDR of RA.2. The expression and function of P-gp on PBMC were correlated with disease activity of RA. No correlation was found among the expressi-on and function of P-gp, duration of disease and time of therapy.3. The expression and function of P-gp were positively correlated with the level of PBMC TNF-?mRNA and serum TNF-?.4. We speculated that primary drug resistance, secondary drug resistance, the change of internal environment in disease activities involve in regulation of P-gp and development of MDR in RA. The change of internal environment in disease activities plays an important part in in regulation of P-gp. Objective:To investigate the influence of TNF-?on expression and function of P-gp on PBMC. To study the effect of TNF-?on the development of MDR in RA.Methods:20 normal control and 60 RA patients (20 untreated,20 effective to treatment,20 refractory for each group) were selected as object of this study. PBMC was isolated and treated with TNF-a (0.5ng/ml terminal concentration) in 24-well-plate at 37?,5% CO2, and 95% relative humidity. PBMC was collected 0,2,6,12, or 24 h after TNF treatment, the 0 time being the untreated control. PBMC analyzed by flow cytometry for P-gp expression, rhodamine123 accumulation test for P-gp function.Results:Results from the experiment showed that 2h after TNF-?treatment, the function of P-gp enhanced and the expression of P-gp did not change significantly.12h after TNF-?treatment, the expression and function of P-gp reached it its maximum and then maintain a high level. No significant difference of the expression and function of P-gp were found between 12h and 24h after TNF-?treatment. At the time of 6h and 12h after TNF-?treatment, onset time and peak time of expression of P-gp were different in different groups. In normal control group and RA untreated group, onset time was 2h and peak time was 12h. In RA effective to treatment group, onset time and peak time was 12h. In refractory group onset time and peak time was 6h. We compared the expression and function of P-gp 12h after TNF-?treatment in each group. The expression and function of P-gp in RA groups were significantly higher than that of normal control group. Among the groups of RA, the expression and function of P-gp in RA refractory group was highest, and that in RA effective to treatment group was lowest.Conclusions:1.TNF-?can up regulate the expression and function of P-gp on PBMC in RA, and play a part in development of refractory RA and MDR in RA.2. The onset time and peak time of induction of P-gp expression by TNF-?was short in in RA refractory group and was long in RA effective to treatment group. RA refractory group was more sensitive to TNF-?compared with RA effective to treatment group.3. The expression and function of P-gp reached it its maximum and then maintain a high level 12h after TNF-?treatment.4. The mechanisms of the expression and function of P-gp elevated in RA and RRA were complicated and the effect of TNF-?on P-gp was one of the mechanisms. Objective:To study the contribution of two signal transduction pathways of TNF-?-MAPKs(ERK1/2, JNK and p38) and NF-?B in regulation of the expression and function of P-gp on PBMC by TNF-?in RA.Methods:20 RA untreated patients were selected as object of this study. PBMC was isolated one PBMC sample was divided into 6 groups:group A, group B, group C, group D, group E and group F. Group A is blank control. Group C, D, E, F were pretreated with NF-?B, JNK, ERK1/2 and p38 inhibitor (PDTC, SP600125, U0126 and SB202190) for 30min. Then Group B, C, D, E, F were treated with TNF-?(0.5ng/ml terminal concentration) in 24-well-plate at 37?,5% CO2, and 95% relative humidity for 12h. PBMC was collected and analyzed by flow cytometry for P-gp expression, rhodamine123 accumulation test for P-gp function.Results:The expression and function of P-gp in group B were significantly higher than that in group A. Compared with group B, the expression and function of P-gp decreased in group C and D. No significant differences of the expression and function of P-gp were found among group B, group E and group F.Conclusions:1. Inhibition of NF-?B and JNK can suppress the increased expression and function of P-gp induced by TNF-?signal transduction pathways of NF-?B and JNK play a role in regulation of the expression and function of P-gp on PBMC by TNF-?in RA.2. Inhibition of ERK1/2 and p38-MAPK had no influence on the increased expression and function of P-gp induced by TNF-?. Signal transduction pathways of ERK1/2 and p38 probably did not play a role in regulation of the expression and function of P-gp on PBMC by TNF-?in RA directly.3. TNF-?regulated the expression and function of P-gp on PBMC through activation of NF-?B and JNK signal transduction pathways.