Regulation Of CP-25 On P-glycoprotein In Synovial Cells Of Rats With Adjuvant Arthritis

Rheumatoid arthritis?RA?is a chronic systemic autoimmune disease of unknown etiology.Its pathological features are multi-joint erosion of the small joints of the hands and feet,and inflammatory lesions of the synovial tissue of the symmetry.At present,there are many drugs for rheumatoid arthritis.Among them,methotrexate?MTX?as a disease-modifying antirheumatic drugs?DMARDs?has been recognized as the first-line treatment drug in the world.However,the drug sensitivity of some patients decreased after taking MTX for about 6 months,namely MTX resistance.MTX resistance may be related to a variety of factors,among which MTX efflux mediated by drug transporters has been widely studied in tumor resistance,but it is rarely reported in RA resistance.P-glycoprotein?P-gp?is one of the important members of the ATP-binding cassette?ABC?superfamily.P-gp is an efflux transporter,which can pump drugs out of the cell depending on ATP hydrolysis energy,so that the intracellular drug concentration remains at a low level.P-gp is widely distributed in the body,and the expression of P-gp and its coding gene MDR1 are present in liver,kidney,blood-brain barrier,small intestine,placental tissue,synovial tissue and so on.It plays an important role in the resistance of malignant tumors to MTX.Benzene-sulfonyl paeoniflorin?code:CP-25?is a novel active monomer derived from Paeoniflorin?Pae?structure synthesized by our research group.The previous research group found that CP-25 can significantly inhibit the inflammatory response of adjuvant arthritis rats?AA?,and has a good therapeutic effect on arthritis.CP-25 can significantly regulate the expression and function of P-gp in rat PBMC and Caco-2 cells.Therefore,this study focused on the regulation of CP-25on P-gp in AA rats,and to investigate whether CP-25 can improve MTX resistance mediated by high P-gp expression.Objective:To clarify the regulation of CP-25 on P-gp in synovial cells of AA rats,reveal that CP-25 can improve the expression of P-gp and reduce the mechanism of MTX resistance.Methods:1)Rats AA model was established and randomly divided into normal group,model group,CP-25 group?50mg/kg/day?,MTX group?0.5mg/kg/3 day?,CP-25?50mg/kg/day?combined with MTX?0.5mg/kg/3 day?group and dexamethasone group?1mg/kg/day?.The distribution and expression of P-gp in rat tissues were detected by immunohistochemistry and Western blot.2)Stimulation of normal rat synoviocytes by TNF-?,the effect of CP-25 at different time and concentration on the expression of P-gp in normal rat synovial cells stimulated by TNF-?was detected by western blot.3)MTT assay was used to investigate the resistance of P-gp overexpressed hepatocellular carcinoma drug-resistant cell lines?Bel7402/5-Fu?to MTX and the resistance of CP-25 reversed drug-resistant cell lines to MTX.At the same time,the effect of CP-25 on the expression of P-gp in Bel7402/5-Fu cells was detected by Western blot.4)The Bel7402 and Bel7402/5-FU cells collected in the logarithmic growth phase were cultured with different concentrations of CP-25 and verapamil,and then cultured in RPMI1640 complete medium containing rhodamine 123.Flow cytometry was used to detect the accumulation of rhodamine 123.Bel7402 and Bel7402/5-FU cells were first cultured in RPMI1640 complete medium containing rhodamine 123,and then cultured with different concentrations of CP-25 and verapamil.Flow cytometry was used to detect the pumping of Rhodamine 123.Results:1)CP-25 can significantly alleviate the overall indicators of arthritis index,paw swelling and paw swelling in AA rats,and the CP-25+MTX combination treatment is better than MTX alone.CP-25 can significantly alleviate the pathological changes of joint and spleen tissues.The effect of CP-25+MTX combination group is better than that of MTX alone.By immunohistochemistry,we found that P-gp was expressed in the synovium,spleen and small intestine of rats,and the expression of P-gp in AA rats was significantly higher than that in the normal group.In the tissues of the MTX group,the expression of P-gp was also up-regulated compared with that of the normal group,and after administration of CP-25,CP-25 could significantly down-regulate the expression of P-gp.After CP-25 and MTX were combined,The expression of P-gp was significantly down-regulated in each tissue compared to the MTX group.Therefore,CP-25 can significantly reduce the overall arthritis index of AA rats,CP-25 can down-regulate the abnormal expression of P-gp in AA rat tissues,and the effect of CP-25+MTX combination therapy is more obvious than MTX alone.2)After stimulation with TNF-?,the expression of P-gp was up-regulated in normal rat synoviocytes,and the expression of P-gp in normal rat synovial cells was the most significant after stimulation with 20 ng/mL TNF-?for 12h.Different concentrations of CP-25 act on TNF-?-stimulated normal rat synovial cells at different times.After concentration of CP-25 for 6h,12h,24h and 36h,the concentration of CP-25 could decrease the expression of P-gp in normal rat synoviocytes stimulated by TNF-?.However,the effects of CP-25 at different concentrations did not significantly reduce P-gp expression after 2h.3)After 48 hours of treatment with Bel7402 and Bel7402/5-FU cells by MTX,the IC₅₀of MTX to Bel7402 cells was 3.507?g/mL,while the IC₅₀0 of Bel7402/5-FU cells was13.054?g/mL.MTX has a significant difference in IC₅₀0 for both Bel7402 and Bel7402/5-FU cells,and the drug resistance fold is about 4-fold.Different concentrations of CP-25 had no obvious direct cytotoxic effect on Bel7402/5-FU cells,and did not affect cell growth and proliferation.Only high concentration of 10^-5mol/L showed inhibition.Reversal of CP-25 was combined with different concentrations of MTX,which significantly reduced the IC₅₀?5.656?g/mL?of MTX-inhibited Bel7402/5-FU cells,and decreased the level of cell resistance.It is indicated that CP-25can partially reverse the drug resistance of MTX,and its reversal multiple is 2.31 times,suggesting that CP-25 can reverse the resistance of MTX and increase the therapeutic effect.The results of Western Blotting showed that the expression of P-gp was significantly increased in Bel7402/5-FU resistant cells compared with Bel7402 sensitive cells.CP-25 could down-regulate the expression of P-gp in Bel7402/5-FU cells.Flow cytometry was used to detect the effects of Bel7402 and Bel7402/5-FU cells on the accumulation and pumping of Rhodamine 123.CP-25 can significantly increase the cell accumulation of Rhodamine 123 in Bel7402/5-FU cells and reduce the extracellular efflux of Rhodamine 123.It indicated that CP-25 can inhibit the efflux transport function of P-gp.Conclusions:1)CP-25 can down-regulate the abnormal expression of P-gp in the synovium,spleen and small intestine of AA rats,and CP-25 can inhibit the high expression of P-gp induced by MTX;2)TNF-?can up-regulate the expression of P-gp in normal rat synoviocytes,and CP-25can reduce the expression of P-gp in normal rat synoviocytes after TNF-?stimulation.3)CP-25 can reverse the resistance of Bel7402/5-Fu cells to MTX,and CP-25 can significantly down-regulate the expression of P-gp in Bel7402/5-Fu cells and inhibit its efflux function.CP-25 may reverse the resistance of MTX by regulating P-gp.