OAT3 Mediates Methotrexate Resistance In The Treatment Of Rheumatoid Arthritis

Rheumatoid arthritis（RA）is an autoimmune disease characterized by erosive arthritis.The main pathological features of RA are the chronic inflammation of synovial tissue and the formation of pannus,the erosion of articular cartilage and bones,which ultimately leads to joint deformities and dysfunction.Methotrexate（MTX）acts as a dihydrofolate reductase inhibitor,due to it has good anti-inflammatory,anti-proliferative effects and low cost,and is often used as the drug of choice for the treatment of RA.However,the prevalence of non-response/resistance to MTX treatment among patients with RA is estimated to be between 30% and 50%.The transport of drugs by transporters is directly related to the efficacy of drugs.Many studies have found that drug transporters are related to drug resistance.At present,there are many studies on the relationship between the ATP binding cassette（ABC）transporter family and MTX resistance.It has been found that the overexpression of P-glycoprotein,multidrug resistance-related protein,and breast cancer resistance protein in tissues or cells can be reduced the curative effect of the drug leads to drug resistance.Compared with the ABC family,there are fewer studies on the relationship between the solute carrier（SLC）transporter family and drug resistance.Organic anion transporter 3（OAT3）,as a member of the SLC transporter family,participates in the absorption of MTX,and the expression of OAT3 is reduced in an inflammatory environment.Therefore,we speculate that OAT3 transporter may be involved in MTX resistance.The previous research of our group found that the expression of OAT3 in the synovium of CIA rats was reduced,and the expression of OAT3 was also decreased after stimulation of PGE2 inflammatory factors in normal rat synovial cells.Furthermore,the absorption capacity of MTX was significantly decreased by knockdown of OAT3 expression in normal rat synovial cells.In order to explore whether OAT3 is involved in the development of MTX treatment of RA resistance,we have verified it from two aspects in vitro and in vivo.Purpose:To investigate whether drug resistance to the therapeutic RA drug MTX is related to OAT3 and to clarify whether OAT3 can be a target to improve the efficacy of MTX in treating RA.Method:1.Immunohistochemistry（IHC）was used to detect the expression of OAT3 in the synovial tissue of patients with non-inflammatory diseases and RA patients;Western blot was used to detect the expression of OAT3 in synovial cells extracted from the synovium of RA patients and Osteoarthritis（OA）patients.2.Normal rat synovial cells were isolated in vitro,and the experiments were divided into normal,NC,and OAT3 overexpression groups,where the NC and OAT3 overexpression groups were transiently transfected with the corresponding plasmids using the transfection reagent.Then giving MTX with self-fluorescence（100ng/ml）,incubate for half an hour,using laser confocal microscopy to detect the absorption of MTX.The above cells were divided into a control group,a TNF-α（20ng/ml）stimulation group,and a TNF-α+MTX treatment group.CCK-8 and Transwell methods were used to detect the proliferation activity and migration ability of each group of cells.3.The normal rat synovial cells were divided into normal group,NC group,and OAT3 silenced group.The NC group and OAT3 silenced group were transiently transfected with corresponding si RNA using si RNA-mate transfection reagent.Then giving MTX with self-fluorescence（100ng/ml）,incubate for half an hour,using laser confocal microscopy to detect the absorption of MTX.The above cells were divided into a control group,a TNF-α（20ng/ml）stimulation group,and a TNF-α+MTX treatment group.CCK-8 and Transwell methods were used to detect the proliferation activity and migration ability of each group of cells.4.A CIA rat model was established,and the animals were randomly divided into normal group,CIA model group and MTX treatment group.The treatment group was given MTX（0.5 mg/kg/3d）for 14 days,and the arthritis inflammation indexes（rat body weight,whole body score,arthritis index,joint swelling degree and joint swelling number）were observed every three days.The natural remission of inflammation in CIA control rats was observed,and a 95% confidence interval was calculated.Observing the scope of remission of inflammatory symptoms in MTX-treated rats.If the remission is within the95% confidence interval of remission in CIA rats or lower than the lowest value of the95% replacement interval,it means that the inflammatory symptoms of the rats have not recovered significantly.If the remission is higher than CIA The highest value of the 95%confidence interval in the rat was relieved,indicating that the inflammatory symptoms of the rat were effectively recovered.The indicators for evaluating the efficacy grouping are the five inflammatory indicators observed in the previous period;If more than 60% of the indicators have significant recovery,MTX treatment was considered effective（MTX-E）,otherwise ineffective treatment（MTX-N）,and efficacy was calculated.Rats were randomly selected from each group to obtain synovial tissue.Western blot and immunohistochemistry（IHC）were used to detect the expression of OAT3 in synovial tissue,and UPLC-MS/MS was used to detect the content of MTX in synovial tissue of rats.5.The MTX-N group was randomly divided into the control,NC,and OAT3 overexpression groups.Among them,the NC and OAT3 overexpression groups were injected with corresponding lentiviruses in the joint cavity,and the enrichment of lentivirus in the joints,peripheral blood,and tissue organs was detected using a small animal live imaging instrument,western blot,and IHC.According to the arthritis evaluation index to determine the improvement of the curative effect.Western blot、IHC and UPLC-MS/MS were used to detect the expression of OAT3 and the content of MTX in rat synovial tissue.6.The joints of each group were retained and the joint pathology and bone erosion were detected using Hematoxylin and eosin（H＆E）and X-ray.Result:1.IHC results showed that the expression of OAT3 in synovial tissue of RA patients was significantly lower than that of patients with non-inflammatory diseases;Western blot results showed that the expression of OAT3 in synovial cells of RA patients was lower than that of OA patients.2.The laser confocal results showed that after overexpression of OAT3 in rat synovial cells,its absorption of MTX was significantly higher than that of the NC group.CCK-8results showed that the proliferation of synovial cells was significantly enhanced after TNF-α stimulation,and MTX could alleviate the excessive proliferation of cells,and the OAT3 overexpression group alleviated more significantly.Transwell results showed that after TNF-α stimulation,the migration ability of synovial cells was significantly improved,and MTX could reduce their migration ability,and the OAT3 overexpression group had a stronger tendency to decrease.3.The laser confocal results showed that after silencing the expression of OAT3 in rat synovial cells,its absorption of MTX was significantly lower than that of the NC group.The results of CCK-8 showed that the effect of MTX in the OAT3 silence group on alleviating the hyperproliferation of synovial cells was significantly lower than that of the NC group.Transwell results showed that the effect of MTX in the OAT3 silence group in reducing the migration ability of synovial cells was significantly lower than that in the NC group.4.The body weight of CIA rats,whole body score,paw volume,paw swelling number and arthritis index,showed that the treatment efficiency of MTX in CIA rats reached48.98%.Western blot and IHC results showed that OAT3 expression in synovial tissue of CIA rats was significantly lower than in normal group and significantly lower in MTX-N group than in MTX-E group;UPLC-M S/MS showed significantly lower MTX content in MTX-N group than in MTX-E group.5.The lentivirus is only concentrated in the joints of rats after testing.After injection of overexpressed lentivirus into the joint cavity of rats,the curative effect is greatly improved,reaching 80%.Western blot and IHC results showed that the OAT3 expression of NC group was significantly lower than the OAT3 overexpression group;UPLCMS/MS results showed that the MTX content in the synovial tissue of the NC group was significantly lower than that of the OAT3 overexpression group.6.H＆E results showed that,compared with normal rats,the ankle cartilage tissue of CIA rats was severely damaged,some parts were inflamed with a large number of inflammatory infiltration,the synovial membrane was abnormally proliferated,and new pannus appeared.The symptoms of MTX-E group were significantly relieved;compared with CIA rats,the joint pathology of MTX-N rats was not significantly improved.After intra-articular injection of overexpression lentivirus,joint inflammation,cell infiltration,neovascularization,and abnormal synovial hyperplasia in the OAT3 overexpression group were significantly improved.X-ray photography showed that compared with the normal group,the secondary paw bone erosion in the CIA model group was very serious,the bone erosion in the MTX-E group was significantly reduced,and the bone erosion in the MTX-N was more serious.The bone space of the secondary paw in the CIA model group has basically disappeared;the bone space of the drug group still exists,but it is relatively narrow.MTX-N is similar to the CIA model group.After additional treatment,the bone erosion in the OAT3 overexpression group was further reduced,the bone space was intact,and there were only minor or suspected lesions.Conclusion:1.OAT3 mediates the uptake of MTX in synovial cells and regulates MTX to inhibit synovial cell proliferation;OAT3 expression is abnormal in RA and experimental arthritis,involved in the production of resistance to the RA therapeutic drug MTX.2.OAT 3 is one of the targets to address MTX therapeutic resistance to RA.