The Correlation Study Of MDR1/P-gp And Multidrug Resistance 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.MDR was take account in the domain of carcinoma disease and carried out a deeper study on it, however the study about 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. Overexpression of P-gp results in reduction of intracellular concentrations of substrates involving some DMARDs. It was reported that the expression and activity of P-gp increased in refractory RA and play a part in MDR of RA. P-gp encoded by Multidrug resistance gene-1(MDR-1). We hypothesized that the polymorphism or change of expression lever of MDR1may influence the expression and activity of P-gp, so in this study, we explored the possible association between MDRland drug resistance in RA. Chapter1The correlation study of MDR1gene polymorphisms and multidrug resistance in rheumatoid arthritisObjective:To evaluate whether the two single nucleotide polymorphisms (G2677T/A and C1236T) in the MDR1gene were associated with multidrug resistance in rheumatoid arthritis (RA) in a Chinese population,Methods:A total of204patients with rheumatoid arthritis who had been treated with more than two disease-modifying antirheumatic drugs (DMARDs) for at least half a year. Of this patent group112were drug responsive and92were drug resistant according to the remission criteria of the American college of rheumatology (ACR20). Genotypes of G2677T/A and C1236T were detected in104age-and sex-matched normal people and204RA patients using the polymerase chain reaction followed by restriction digestion (PCR-RFLP).Results:1. No significant differences were observed in allelic and genotype frequencies of MDR1polymorphisms between RA patients and controls.2. Compared to drug-responsive subjects, the drug-resistant subjects carried less2677TT genotype (P=0.014)3. No significant differences were observed in allelic and genotype frequencies of C1236T between drug responsive subjects and drug resistant subjects.Conclusion: 1. MDR1gene polymorphism may not be related with the RA susceptibility.2. The MDR1G2677T/A gene polymorphism may influence the efficacy of RA therapy with DMARDs, and the2677TT genotype may protect RA patients from drug resistance.3. The MDR1C1236T gene polymorphism may not be related with multidrug resistance in RA. Chapter2The correlation study of highly expressing MDR1and multidrug resistance in rheumatoid arthritisObjects:To establish a RA drug resistance Synovial cell model highly expressing MDR1(RA/MDR1cells) in vitro, in order to study further on the molecular mechanism.Methods:1. Synovial cells were isolated by collagenase digestion and proved by immunocytochemistry, then infected by recombination adenovirus Ad-EGFP-MDR1.2. The expression level of MDR1mRNA and P-gp of RA/MDR1cells were detected by real-time PCR and western blot respectively.3. The drug efflux activity of RA/MDR1cells was detected by rhodamine123and its resistance to MTX was detected by MTT.Result:1.72hours after synoviocyte infected by Ad-EGFP-MDR1, we found the cells became bigger and grow slower, and particles in cells increased, the infection rate is more than90%.2. The results of real-time PCR and western blot showed that the expression of MDR1and P-gp in RA/MDR1cells significantly were increased (p<0.01; p<0.05)3. The drug efflux activity was also significantly increased (p<0.01). The inhibition of different concentrations of MTX to RA/MDR1 cells were decreased (p<0.05or p<0.01), and the resistance to MTX was increased37.36times.Conclusion:The RA drug resistance Synovial cell model highly expressing MDR1was established, and the increasing of its drug efflux activity and the resistance to MTX may be mediated by P-gp.