| Organic anion transporters(OATS)are composed of more than 10 transmembrane transporters and are an important member of the solute carrier superfamily.Their representative transporters are OAT1,OAT2,and OAT3.Organic anion transporter 1(OAT1)plays an important role in the process of endogenous and exogenous organic ion reabsorption and excretion,and mediates many drugs(such as antibiotics,non-steroidal anti-inflammatory drugs,diuretics and Antivirals,etc.)metabolites and transmembrane transport.Studies have found that in diabetic rat models,non-alcoholic fatty liver models,and hyperuricemia models,the expression of OAT1 in kidney tissues is significantly reduced,and the expression of cytoplasmic expression in renal tubular epithelial cells is significantly reduced.In addition,OAT1 expression was also found to be low in tissues of patients with clinical diabetic nephropathy and patients with acute kidney injury,and the level of OAT1 in the urine of patients was significantly higher than that of the normal group.Prostaglandin E2(PGE2),an inflammatory mediator,can significantly reduce the expression of OAT1 in rat kidney cells and inhibit its transport activity.It is known that PGE2 is involved in signal pathways mediated by PKA and PKC,and whether it can affect the expression of OAT1,cause endocytosis of OAT1,and how it affects it is unknownRheumatoid arthritis(RA)is a chronic,inflammatory synovitis-based systemic disease,and its etiology has not been clarified.RA is an arthritis characterized by polyarticular,symmetry,and invasiveness of the small joints of the hands and feet,which can cause joint deformities and loss of function in severe cases.Fibroblast-like synoviocytes(FLS),as one of the main effector cells for the treatment of arthritis,does OAT1 be expressed in rheumatoid arthritis?How are its expression changes regulated?These are unclear.Paeoniflorin-6 ’-O-benzene sulfonate(code:CP-25)is a new active monomer.The previous research group found that CP-25 can significantly inhibit collagen-induced arthritis(CIA)inflammatory response in rats and has a good therapeutic effect on arthritis.CP-25 can inhibit the abnormal proliferation and inflammatory response of inflammatory FLS to varying degrees,and can inhibit the abnormal desensitization of EP receptors in rheumatoid arthritis synovial cells and moderately restore the membrane expression of EP4 in synovial cells.In addition,CP-25 can restore EP4 receptor-mediated signaling pathways in synovial cells,increase intracellular cAMP levels and downstream PKA activity.Then whether CP-25 can regulate the expression of OAT1.Studying the regulatory mechanism of OAT1 and the effect of CP-25 on its expression will help to understand the transport of OAT1 to drugs and provide clues for the rational clinical use of drugs in the future.Therefore,this study mainly investigated the effect of CP-25 on the expression of OAT1 in synovial tissues of CIA rats,and also investigated whether PGE2 regulates the cell membrane transport of OAT1 in FLS and the role of CP-25 through the EP4/cAMP/PKA signaling pathway.Objective:To investigate the effect of CP-25 on the expression of OAT1 in synovial tissues of CIA rats,and to investigate whether PGE2 regulates the expression of OAT1 in synovial cells through the EP4/cAMP/PKA signaling pathway and the role of CP-25Methods:1)The synovial tissues of clinical OA and RA patients were collected,and the expression of OAT1 in the synovial tissues of clinical patients was detected by Western blot.2)Rat CIA models were established and randomly divided into normal group,CIA model group,CP-25 group(50mg/kg/day),MTX group(0.5mg/kg/3 day),and OAT1 was detected in each group by immunohistochemistry Distribution and expression in rat synovial tissue;ELISA was used to detect the level of PGE2 in the serum of each group of rats3)PGE2 stimulated FLS in normal rats.After 0min,5min,10min,20min,30min,and 60min stimulation,respectively,Western blot was used to detect the effect of OAT1 cell membrane expression in normal rat FLS after PGE2 stimulation4)PGE2 stimulated FLS in normal rats.Western Blotting was used to detect the effect of different concentrations of CP-25(0.1 μM,1.0 μM,10 μM)on the expression of OAT1 cytoplasm in FLS of normal rats stimulated by PGE2.The laser confocal method was used to detect the difference.Effects of the concentration of CP-25(0.1 μM,1.0μM,10 μM)on the expression of OAT1 in the FLS of normal rats stimulated by PGE25)PGE2 stimulated FLS in normal rats and was divided into normal group,PGE2(2μM)group,PGE2+CP-25(1.0μM)group,PGE2+CP-25+EP4 antagonist group EP4 antagonists were detected by laser confocal detection On PGE2-induced OAT1 cell membrane expression and CP-25 effect in normal rat FLS6)The effect of siRNA EP4 on the expression of OAT1 in FLS of normal rats was divided into negative control group and siRNA EP4 group.Western blot was used to detect the expression of OAT1 in each group7)PGE2 stimulated FLS in normal rats and was divided into normal group,PGE2(2μM)group,PGE2+CP-25(1.0μM)group,PGE2+CP-25+PKA inhibitor(5μM)group,each group was given different treatments.Western blot was used to detect the effect of PKA inhibitor on the cytoplasmic expression of OAT1 in normal rat FLS induced by PGE2 and the effect of CP-25;laser confocal detection was used to detect the PKA inhibitor in normal rat FLS induced by PGE2 Cell membrane expression of OAT 1 and the role of CP-258)PGE2 stimulated FLS in normal rats and was divided into normal group,PGE2(2μM)group,PGE2+CP-25(1.0μM)group,and each group was given different treatments.Cellular membrane red fluorescent probe(Dil)dye was used to localize the expression and distribution of OAT1 in synovial cells.Results:1)The expression level of OAT1 in synovial tissue of RA patients was significantly lower than that of OAT1 in synovial tissue of OA patients.2)CP-25 can significantly reduce the overall index,arthritis index and foot volume of CIA rats;CP-25 can significantly improve the pathological changes of ankle and spleen tissues in arthritic rats.The distribution and expression of OAT1 in the synovial tissue of each group of rats were detected by immunohistochemical methods.We found that the expression of OAT1 was in the synovial tissue of the rats,and the expression of OAT1 in the synovial tissue of the CIA model group was normal Compared with the control group,the expression of OAT1 in the synovial tissue of the CP-25 administration group and the MTX administration group was significantly lower than that of the CIA model group.In summary,CP-25 can significantly reduce the clinical manifestations of arthritis in the CIA model group,and can up-regulate the abnormal expression of OAT1 in the synovial tissue of CIA rats.3)ELISA was used to detect the PGE2 level in the serum of rats in each group Compared with the normal group,the PGE2 concentration in the serum of the CIA model group increased,while the PGE2 concentration decreased after CP-25 and MTX administration.4)PGE2(2 μM)stimulated FLS in normal rats.After 0min,5min,10min,20min,30min,and 60min stimulation,respectively,the cell membrane expression of OAT1 in FLS of normal rats was first down-regulated and then up-regulated.Among them,2μM PGE2 was stimulated for 10min.The expression of OAT1 in the FLS of normal rats was most significantly down-regulated.5)PGE2(2 μM)stimulated FLS in normal rats,and different concentrations of CP-25(0.1 μM,1.0 μM,10 μM)affected the expression of OAT1 cytoplasmic membrane in normal rat FLS stimulated by PGE2.Compared with the normal group,the cytoplasmic expression of OAT1 in the PGE2 stimulated group was significantly increased,and compared with the PGE2 stimulated group,the cytosolic expression of OAT1 decreased with the increase of CP-25 after administration of different concentrations of CP-25;Compared with the control group,the expression of OAT1 cell membrane in the PGE2(2 μM)stimulation group was significantly reduced,and compared with the PGE2(2μM)stimulation group,the expression of OAT1 cell membrane increased with the increase of CP-25 concentration.6)PGE2(2μM)stimulated FLS in normal rats and was divided into normal group,PGE2(2μM)group,PGE2+CP-25(1.0μM)group,and PGE2+CP-25+EP4 antagonist group.Compared with the normal group,the expression of OAT1 cell membrane in the PGE2(2 μM)stimulation group was significantly reduced.Compared with the PGE2(2 μM)stimulation group,the expression of OAT1 cell membrane increased after CP-25 administration.Compared with the CP-25 administration group,PGE2 The cell membrane expression of OAT1 was decreased in the PGE2+CP-25+EP4 antagonist(5 μM)group.7)The experiment was divided into negative control and siRNA EP4 groups.The expression of OAT1 in the interference group was lower than that in the negative control group.8)PGE2(2μM)stimulated FLS in normal rats and was divided into normal group,PGE2(2μM)group,PGE2+CP-25(1.0μM)group,PGE2+CP-25+PKA inhibitor(5μM)group.Compared with the normal group,the OAT1 cytoplasmic expression in the PGE2(2 μM)stimulation group increased significantly.Compared with the PGE2(2μM)stimulation group,the OAT1 cytoplasmic expression decreased after CP-25 administration.Compared with the CP-25 administration group,PGE2 Increased cytoplasmic expression of OAT1 in the PGE2+CP-25+PKA inhibitor(5μM)group;Compared with the normal group,the expression of OAT1 cell membrane in the PGE2(2 μM)stimulation group was significantly reduced.Compared with the PGE2(2μM)stimulation group,the expression of OAT1 cell membrane increased after CP-25 administration.Compared with the CP-25 administration group,PGE2 The cell membrane expression of OAT1 was decreased in the PGE2+CP-25+PKA inhibitor(5μM)group.9)Compared with the normal group,the expression and distribution of OAT1 cell membranes in the PGE2(2 μM)stimulation group were reduced.Compared with the PGE2(2 μM)stimulation group,the expression and distribution of OAT 1 cell membranes increased after CP-25 administration.And OAT1 is mainly distributed on the cell membrane.Conclusions:1)CP-25 can significantly upregulate the expression of OAT1 in synovial tissue of CIA rats.2)CP-25 can regulate the membrane and cytoplasmic expression of OAT1 in FLS by activating PGE2-mediated EP4/cAMP/PKA signaling pathway. |
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