| Rheumatoid arthritis(RA)is a systemic autoimmune disease,with major pathological features including synovitis and cartilage destruction.The pathogenesis of RA is still unclear,but abundant studies have shown that macrophages are involved in the occurrence and development of RA.A large number of infiltrated macrophages in the synovium of RA patients is considered as an early hallmark of RA and is associated with disease activity.According to the phenotype and function,macrophages are mainly divided into M1(proinflammatory)macrophages and M2(anti-inflammatory)macrophages.Under normal physiological conditions,the phenotype of macrophages is in dynamic balance.However,in RA patients and animal models of arthritis,multiple factors will break this balance,causing the imbalance of the number and proportion of M1/M2 macrophages,leading to the continuous increase of M1 macrophages,thus aggravating the inflammatory response.However,there are no specific drugs for macrophages in clinic.Reportedly,prostaglandin E2(PGE2)can activate the Kruppel like factor4(KLF4)transcription factor through the cAMP-CREB/CRTC pathway to promote the M2 marophage polarization.Our previous research found that the level of PGE2 were elevated in synovial cells of rats with adjuvant-induced arthritis(AA).The constant stimulation of PGE2 resulted in the increased translocation of G protein-coupled receptor kinase 2(GRK2)to membrane,over-desensitization of EP4 and the decreased level of cyclic adenosine monophosphate(cAMP).GRK2 is G protein coupled receptors(GPCRs)negative regulator,can specifically bind to GPCRs and make GPCRs phosphorylation,thus mediating GPCRs desensitization.But,PGE2 mediated-EP receptor desensitization in macrophages,especially the over-desensitization of EP4 receptor has not been reported.There is also no report on whether the imbalance of M1/M2 macrophages in RA patients or in animal models of arthritis is related to over-desensitization of EP receptors in macrophages or not.Paeoniflorin-6-oxygen-benzene sulfonic acid ester(CP-25)developed by our institute,is a new active compound with anti-inflammatory immune regulating effect.CP-25 can improve clinical manifestation of AA/collagen induced arthritis(collagen-inducedarthritis,CIA)animal model,and adjust the balance of macrophage cytokines.Previous studies have found that CP-25 can inhibit the abnormal proliferation of synovial cells of AA rats by inhibiting GRK2 transferring to membrane,recovering the membrane expression of EP4 and increasing the level of cAMP.However,whether CP-25 regulates the imbalance of M1/M2 ratio or not is still unclear.The role of CP-25 on M1/M2 ratio and possible mechanism need to further explore.Objective:To observe the effect of PGE2 and its signal on macrophages polarization in CIA mice and in Raw264.7 cells,and the effect and mechanism of CP-25 on the macrophage polarization,so as to reveal that PGE2 and its signal transduction are involved in the imbalance of M1/M2 ratio in RA,and to provide experimental basis for GRK2 as a target of CP-25 on the regulation of macrophage polarization.Methods:CIA mice model is established by Chicken collagen typeⅡpreparation and complete freund’s adjuvant.Culturing primary peritoneal macrophage(PM)and bone marrow derived macrophage(BMM).The expression of i NOS,Arg1,p-CREB,CREB and EP4 and GRK2 membrane expressions were dectected by western blot.The expression of IRF4,IRF5,Mincle,YM-1,IL-10,TNF-α and EP m RNA were detected by QRT-PCR.The expression of CD86,CD206 and F4/80,the expression of EP4 and GRK2 on the surface of macrophages,and the phagocytosis of macrophages was detected by flow cytometry.The levels of PGE2 and cAMP were detected by ELISA.CP-25(35mg/kg)was given by gavage and Etanercept(4.5mg/kg)was given by intra-abdominal injection.The distribution of EP4 and GRK2 on macrophages was detected by confocal laser microscopy.The interaction between EP4 and GRK2 on macrophages was detected by immune co-precipitation.The expression of EP4 and GRK2 in macrophages was observed by high-content imager.GRK2 gene expression in Raw264.7 was silenced by si RNA and was knocked out by CRISPR/Cas 9.CRISPR/Cas 9 technology was used to construct GRK2 KO mice,and primary PM and BMM of GRK2 KO mice were cultured.Results: 1.The change of the M1/M2 ratio in macrophages of CIA miceIn the PM of CIA mice,the result showed that compared with the normal group,in the model group,the level of i NOS increased,the level of Arg1 decreased,the ratio of CD86/CD206 increased,the macrophage phagocytosis enhanced,m RNA levels of IRF5,Mincle and TNF-α increased and m RNA levels of RF4,YM1 and IL-10 decreased.We also measured the i NOS and Arg1 levels in the BMM of CIA mice,and the results showed that compared with the normal group,the i NOS level increased while the Arg1 level decreased in the model group.2.Effects of PGE2 and its signal on macrophage polarization in CIA miceTo investigate the role of PGE2-EP4-cAMP-CREB in macrophage polarization,we detected the expression of PGE2 and its signal.Compared with the normal group,in the model group,the level of PGE2 in the serum and PM supernatant was increased,the membrane expression of EP4,the level of cAMP and p-CREB were decreased,the membrane expression of GRK2 was increased.cAMP analogue dbcAMP can reduce the ratio of M1/M2 in the PM of normal group and model group,and PGE2(1μM)can reduce the ratio of M1/M2 in the normal group,but has no significant effect on the PM polarization of mice in the model group.These results suggested that PGE2 had different effects on PM polarization in the normal group and the model group,which might be related to the concentration of PGE2,EP receptor sensitivity and cell polarization situation.Which EP receptor work in CIA mice? The results showed that the total expression of EP2 and EP4 increased,and the m RNA level of EP4 increased in the model group.These results suggest that EP2 and EP4 may be involved in the pathogenesis of CIA mice.To further clarify through which receptor PGE2 regulates cAMP-CREB,we stimulated the normal group and the model group PM with EP2 agonist and EP4 agonist under the same conditions.EP4 agonist had similar results with PGE2(1μM),suggesting that PGE2 may regulate cAMP-CREB through EP4 receptor.In the stimulation of PGE2,the membrane expression of EP4 increased,while the membrane expression of GRK2 increased in the PM of normal group.The membrane expression of EP4 showed no significant change,while the membrane expression of GRK2 increased in the PM of model group.It suggested that the over-densitization of EP4 and the increase of GRK2 transmembrane may be related to the of PGE2-induced abnormal PM polarization in CIA mice.Laser confocal microscope observation of PM showed that EP4 and GRK2 were mainly distributed in the cytoplasm and membrane.The Merge diagram suggested that EP4 and GRK2 had certain interaction.CO-IP further showed that GRK2-EP4 had weak effect under normal conditions,and the interaction of GRK2-EP4 enhanced in the model group.3.The effect of PGE2 and its signal on the polarization of Raw264.7 cellsM1 and M2 macrophages were induced by stimulation in vitro.Compared with M0 group,in M1 group,the membrane expression of EP4 decreased,the membrane expression of GRK2 increased,and the level of cAMP and p-CREB decreased.In the M2 group,the membrane expression of EP4 increased,the membrane expression of GRK2 decreased,and the level of cAMP and p-CREB increased.cAMP analogue dbcAMP can reduce the ratio of M1/M2 in M0,M1 and M2 macrophages.In the M0 group stimulated by PGE2,CD86/CD206 decreased,EP4 membrane expression increased,and cAMP level increased in the PGE2(100n M,1μM)group.In the M1 group stimulated by PGE2,CD86/CD206 increased,EP4 membrane expression decreased,and cAMP level decreased in the PGE2(1μM,10μM)group.In the M2 group stimulated by PGE2,CD86/CD206 decreased,EP4 membrane expression increased,and cAMP level increased in the PGE2(10n M,100 n M,1μM,10μM)group.The M0,M1 and M2 macrophages were observed by confocal laser microscopy,and EP4 and GRK2 were mainly distributed in the cytoplasm and membrane.CO-IP further showed that GRK2-EP4 interaction in M1 macrophage enhanced compared with that in M0 group,while there was no significant change in GRK2-EP4 interaction in M2 macrophage.4.Effect of GRK2 on macrophage polarization and over-desensitization of EP4In order to further explore the role of GRK2 in EP4 over-desensitization and the effect of PGE2 on macrophage polarization,the silencing of GRK2 gene expression by si RNA showed that the M1/M2 ratio reduced in GRK2 si RNA-transfected Raw264.7 cells.After knockout of GRK2 gene with CRIPR/Cas9,the ratio of CD86/CD206 in Raw264.7-GRK2-/-cells decreased.Raw264.7 cells were stimulated with PGE2(10μM)at different time points,and the results showed that the CD86/CD206 in Raw264.7 decreased gradually and reached the lowest value at 20 minutes,and the difference was not statistically significant after 1h compared with the control group.EP4 receptor membrane expression gradually increased,reached the highest value at 20 minutes and the difference was not statistically significant after 1h compared with the control group.PGE2(10μM)was also used to stimulate GRK2 si RNA-transfected Raw264.7 cells at different time points.CD86/CD206 in GRK2 si RNA-transfected Raw264.7 cells gradually decreased,reaching the lowest value at 1h,and the difference was not statistically significant after 2h compared with the control group.Under the stimulation of PGE2(10μM),CD86/CD206 in Raw264.7-GRK2-/-cells gradually decreased,reaching the lowest value at 2h,with no significant difference at 12 h.EP4 receptor membrane expression gradually increased,reached the highest value at 2h,and then decreased,and the EP4 membrane expression at 12 h showed no significant difference compared with the control group.5.CP-25 improved CIA through inhibiting GRK2 translocation to membrane to restore normal PGE2-EP4-cAMP-CREB signal transduction to regulate macrophage polarizationCP-25 significantly reduced the multiple arthritis index score of CIA mice,indicating that CP-25 had therapeutic effect on CIA mice.Compared to model group,CP-25 reduced the level of PGE2 of serum and PM supernatant and the expression of i NOS of PM and BMM,and increased the expression of Arg1 of PM and BMM.CP-25 also increased the ratio of CD86/CD206 of PM and weakened macrophage phagocytosis.The m RNA level of IRF5,STAT1,Mincle,and TNF-α decreased,while m RNA level of IRF4,STAT6,YM1,and IL-10 increased in CP-25 group.CP-25 also increased the membrane expression of EP4,reduce the membrane expression of GRK2,and weakened the interaction between EP4 and GRK2.Conclusion: 1.The ratio of M1/M2 in macrophages of CIA mice increased,CP-25(35mg/kg)can reduce the ratio of M1/M2 and restore the balance between M1 and M2.2.The level of PGE2 in the supernatant of serum and PM in CIA mice increased.Under the continuous stimulation of PGE2,the EP4 in the PM over-desensitized,and the level of cAMP and p-CREB expression decreased.The PGE2-EP4-cAMP-CREB signaling pathway mediated the polarization of macrophage to M1 macrophage in CIA mice.3.In the macrophages of CIA mice,GRK2 abnormally elevated and co-expressed with EP4 receptor.GRK2 mediated the over-desensitization of EP4 and participatesd in the abnormal PGE2-EP4-cAMP-CREB signal in CIA macrophages,leading to the polarization of M1 macrophages.4.CP-25 inhibited GRK2 membrane translocation,restored the sensitivity of EP4,increases the level of cAMP and the expression of p-CREB,thus promoting the polarization of M2 macrophages and regulating the M1/M2 ratio balance. |
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