Effects Of Paeoniflorin-6’-O-benzene Sulfonate On Improving Fibroblast-like Synoviocytes Dysfunction Of Collagen-induced Arthritis Rats By Targeting G Protein Coupled Receptor Kinase 2

Rheumatoid arthritis(RA)is an autoimmune disease characterized by systemic immune dysfunction and synovial inflammation.One of the most typical pathological hallmarks is the abnormal proliferation of fibroblast-like synovial cells(FLS)in the joint.The inflammatory factors,such as prostaglandin E2(PGE2),interleukin 6(IL-6),tumor necrosis factor α(TNF-α)were significantly up-regulated in RA-FLS.Isolating FLS of rats in vitro,PGE2 stimulates FLS for a long time to induce the inflammatory environment,it was found that continuous PGE2 can promote G protein coupled receptor kinase 2(GRK2)translocation to membrane by activating c AMP-PKA signaling pathway,leading to Prostaglandin E receptor 4(EP4)phosphorylation and mediating EP4 receptor desensitization,which play an important role in FLS abnormal proliferation.GRK2 is an important negative regulator in EP4 signaling pathway,and its activity and expression change may play an important role in the abnormal proliferation of FLS.GRK2,as a Ser/Thr protein kinase,is composed of 689 amino acids,and it has three domains including N-terminus,kinase domain(KD),and C-terminus.Among them,the kinase domain has multiple important phosphorylation sites and functional domains,which are involved in the regulation of GRK2 activity.Currently,GRK2 activity regulators include ATP,paroxetine,etc.The mechanism of GRK2 activity regulators is mainly through binding to the GRK2 kinase domain.GRK2 activity regulators are mostly applied to co-crystallization experiments in vitro and heart failure animal model experiments.Our group found that paroxetine,as a GRK2 inhibitor,can improve the synovial inflammation of collagen induced arthritis(CIA)rats.Paeoniflorin-6’-O-benzene sulfonate(CP-25)was synthesized by the esterification of benzene sulfonate of pae in our laboratory.In the CIA or adjuvant arthritis(AA)model,CP-25 can inhibit EP4 desensitization-mediated the abnormal proliferation of FLS by down-regulating PGE2-induced GRK2 translocation signaling pathway.The focus of this study is to clarify whether the administration of CP-25 improves synovial inflammation in joint of CIA rats by regulating the expression and distribution of GRK2? Whether CP-25 regulates GRK2 activity,keeping the balance of EP4 receptor signal to improve the function of FLS? Exploring the binding domains of sites of CP-25 on GRK2?Based on the previous research,CIA rat models were successfully established in this experiment and assigned into CIA model group,CP-25 group,GRK2 inhibitor paroxetine and MTX group according to random number table,and meanwhile there were age-matched normal control groups.HE staining,laser Confocal,flow cytometry,q RT-PCR,Western blot,co-immunoprecipitation(Co-IP),small interfering RNA and other techniques were used to observe the effects of CP-25 on improving synovial cells dysfunction in CIA rats by regulating GRK2 expression and distribution.The direct binding and the binding sites of CP-25 and GRK2 were explored by Microscale thermophoresis(MST),molecular simulation docking,Cellular Thermal Shift Assay(CETSA),etc.Point mutant construction,Co-IP and enzyme activity inhibition experiments were used to observe the effects of CP-25 on the GRK2 enzyme activity and the interaction between GRK2 and EP4 were investigated.In order to identify that mechanism of CP-25 on improving joints synovial hyperplasia of CIA rats through targeting to GRK2.Objective: 1.To explore the relationship between FLS dysfunction and the expression and distribution of GRK2 and EP4,and to clarify that CP-25 improves FLS dysfunction by down-regulating the membrane expression of GRK2,reducing co-expression of GRK2 and EP4,and up-regulating EP4 membrane expression.2.To explore the targeted protein GRK2 for CP-25,and the effects of CP-25 in the regulation of GRK2 enzyme activity and the association of GRK2 and EP4,and to reveal the molecular mechanism of CP-25 in the improvement of FLS dyfunction in CIA rats.Methods: 1.Establish a CIA rat models,assigning into 4 group,CIA group,CP-25 group,GRK2 inhibitor(paroxetine)group and MTX group,and meanwhile there were age-matched normal control groups.The body weight,paw swelling,global assessment,swollen joint count,arthritis index,thymus and spleen index,thymus and spleen lymphocyte proliferation function and other indicators of each group were evaluated;HE staining was used to observe the pathology of joint and spleen;the proportion of T cell subsets was detected by flow cytometry;ELISA,Luminex detection and q RT-PCR were used for the detection of PGE2,TNF-α,IFN-γ,TGF-β,VEGF,ICAM-1,IL-1β,GM-CSF and IL-6 levels;immunohistochemistry and laser confocal were used for the detection of GRK2 and EP4 expression and distribution in synovial tissue;primary FLS was isolated and identified by flow cytometry,FLS functions were identified by CCK-8,high content cell imaging technology,transwell migration and invasion experiments;cytoplasm and membrane expression of GRK2 and EP4 were detected by flow cytometry and western blot;Co-IP and laser confocal methods were used to detect co-expression of GRK2 and EP4;silencing of GRK2 of CIA-FLS,high content cell imaging technology,transwell migration and invasion assay were used to detect the changes of FLS proliferation function,migration and invasion,laser confocal methods was used to detect GRK2 and EP4 co-expression.2.Studies of CP-25 targeting to GRK2: MST experiment was used to detect the binding ability of CP-25 and GRK2;molecular simulation docking to detect the binding of CP-25 for GRK2 and predict possible binding sites;important amino acids residues of GRK2 were mutated,and GRK2 mutants were successfully constructed.Western blot combined with immunofluorescence was used to detect the expression of GRK2 mutants in HEK 293 T cells.Thermal drift experiment was used to detect the effect of CP-25 on GRK2 protein stability in cells.The effect of CP-25 on GRK2 enzyme activity was detected by ADP-Glo kit.Eukaryotic expression system of GRK2 wild-type and mutant proteins were successfully established,pull down assay was used to detect the binding of GRK2 and EP4 receptor;the effect of CP-25 on the interaction between GRK2 and EP4 was detected by Co-IP method.Results: 1.The therapeutic effect of CP-25 on CIA rats. On the first day of modeling,swelling of the paw joints on the inflamed side was obvious,and secondary lesions appeared on the 17 th day,such as swelling of the contralateral paw and forefoot,redness and inflammation of the tail,redness,and inflammation of ears,nasal congestion,movement disorders,etc.Successfully establishing CIA rats model,CP-25(50 mg/kg/d),paroxetine(15 mg/kg/d),and MTX(0.5 mg/kg/3 d)with continuous administration for 21 days,the results showed that the weight of rats significantly reduced after immunity,after CP-25 treatment,the body weight of rats significantly increased at d23-d32 after immunity;the treatment of CP-25,paroxetine,and MTX significantly relieved paw swelling,global assessment,swollen joint count,and arthritis index of CIA rats;significantly improved the pathology of synovial tissue and spleen tissue of CIA rats;significantly inhibited spleen and thymus index,and spleen and thymus lymphocyte proliferation;significantly down-regulated the ratio of CD3+CD4+ T cells and Th17 cells,and up-regulated the ratio of Treg cells;significantly down-regulated the inflammatory cytokines,such as PGE2,TNF-α,IL-1β,IL-6,IFN-γ,GM-CSF,ICAM-1 and the angiogenesis-related factors VEGF and up-regulated anti-inflammatory factor TGF-β levels.The results suggested that CP-25 has therapeutic effect for CIA rats.2.CP-25 treatment can improve synoviocytes dysfunction by regulating the expression and distribution of GRK2 in synovial cells The primary synoviocytes was isolated and identified by flow cytometry,and the expression of Vimentin in cells was more than 90%,which was identified as FLS.The treatment of CP-25 can significantly down-regulated PGE2,TNF-α,IL-1β,IL-6,IFN-γ,GM-CSF,ICAM-1,TGF-β and VEGF levels in the supernatant of primary FLS.The treatment of CP-25 significantly inhibited the proliferation,migration and invasion of primary FLS.The expression and distribution of GRK2 and EP4 in synovial tissue of CIA,synovial tissue of RA patients,and primary FLS were detected.The results showed that GRK2 was mainly expressed in the cytoplasm and EP4 was mainly expressed in the cell membrane in normal synoviocytes;compared with the normal synovial tissue,GRK2 membrane expression increased,EP4 membrane expression decreased,GRK2 and EP4 co-expression were significantly up-regulated in the synoviocytes of CIA rats and RA patients;CP-25 treatment significantly down-regulated GRK2 membrane expression in synoviocytes of CIA rats,down-regulated the co-expression of GRK2 and EP4,and up-regulated EP4 membrane expression,thereby improving synoviocytes dysfunction.Silencing GRK2 can regulate the function of CIA-FLS cells.The results show that the long-term stimulation of PGE2(1 μM)promotes the up-regulation of GRK2 translocation,increases the co-expression of GRK2 and EP4,leading to the decreasing of the sensitivity of EP4 to PGE2,thereby promoting CIA-FLS proliferation,migration and invasion;silencing GRK2 can reduce expression and activity,decrease the co-expression of GRK2 and EP4,and restore the sensitivity of EP4 to PGE2,thereby improving PGE2-induced FLS dysfunction,suggesting GRK2 may mediate PGE2-induced CIA-FLS dysfunction.The above results suggest that the up-regulating of GRK2 membrane expression promoted the binding of GRK2 and EP4,and down-regulated the membrane expression of EP4,resulting in FLS dysfunction;CP-25 can improves FLS dysfunction by down-regulating the membrane expression of GRK2,reducing the co-expression of GRK2 and EP4,and up-regulating the membrane expression of EP4.3.CP-25 directly targets GRK2 proteinThe compounds CP-25,pae,paeoniflorin,paroxetine binding to GRK2 protein yielded Kd values of 1.75 ± 0.38 μM,2.11 ± 0.47 μM.,10.9 ± 2.06 μM and 4.950 ± 1.26 μM.The results showed that the combining capacity in the order of CP-25> Pae> Paroxetine> Paeoniflorin.The spatial structure of CP-25 is similar to GRK2 inhibitor ATP and paroxetine,both of which have a ‘V-shape’ structure,it has been reported that the benzodioxole ring of paroxetine can form hydrogen bond with Asp272 and Met274 in GRK2 hinge regions,thereby speculating that the cyclohexane and two oxygen heterocyclic rings of CP-25 may bind to some amino acids in kinase domain of GRK2.The docking simulation of compound CP-25 with GRK2 complex,the interaction energy(C-DOCKER)is-60,and the binding site is kinases domain of GRK2,and CP-25 can form hydrogen bond with Gly201,Lys220,Lys230,Ala321,and Asp335 of GRK2.Seven point mutation plasmids GRK2-G201 A,K220R,K230 R,A321G,D335 A,D272A,M274 A and five multi-point mutation plasmids GRK2-G201A-A321 G,G201A-A321G-D335 A,G201A-A321G-D335A-K220 R,G201A-A321G-D335A-K220R-K230 R and D272-M274 were successfully constructed using the point mutation kit,the mutant plasmid was successfully expressed in HEK 293 T cells;Cellular Thermal Shift Assay showed that CP-25 and GRK2 inhibitors can up-regulate Tm50 of endogenous GRK2 and GRK2-wt protein,increasing the thermal stability of GRK2 protein.They didn’t affect Tm50 of GRK2-G201A-A321G-D335A-K220R-K230 R protein.The results showed that CP-25 can directly bind to the kinases domain of GRK2 and form the hydrogen bonds with Gly201,Lys220,Lys230,Ala321 and Asp335 of GRK2.4.CP-25 affects the enzyme activity of GRK2 and the interaction between GRK2 of EP4 receptorCP-25 can inhibit GRK2 enzymatic activity,with IC50 of 236.4 n M and 390.0 n M,respectively.Eukaryotic expression system of GRK2(His-tagged)was successfully constructed,GRK2-wt and GRK2-G201A-A321G-D335A-K220R-K230 R proteins were successfully expressed and purified using HEK 293 T cells and 6FF agarose beads,and GRK2 translocation to EP4 receptor was detected,suggesting that both GRK2-wt and GRK2 mutant can bind to EP4.GRK2-wt and GRK2 mutants were successfully expressed in HEK 293 T cells,the association of GRK2-wt,G201 A,K220R,K230 R,A321G,D335 A,D272A,M274 A,D272-M274 with EP4 was up-regulated with the stimulation of PGE2 and CAY10598,CP-25 significantly down-regulated agonist-induced the association,but didn’t affect the association of GRK2-A321 G with EP4;GRK2-G201A-A321 G,G201A-A321G-D335 A,G201A-A321G-D335A-K220 R,G201A-A321G-D335A-K220R-K230 R can bind to EP4 with the stimulation of agonists.CP-25 didn’t affect agonists-induced the association of GRK2 mutant and EP4.The results showed that CP-25 can inhibit GRK2 enzymatic activity,down-regulate the association of GRK2 and EP4 may be through controlling Ala321 of GRK2.Conclusions: 1.The up-regulation of GRK2 translocation in FLS can promote the binding of GRK2 and EP4,and down-regulate the membrane expression of EP4,thereby promoting FLS dysfunction;the treatment of CP-25 can down-regulate GRK2 translocation,reduce the co-expression of GRK2 and EP4,and up-regulate the membrane expression of EP4,thereby improving FLS dysfunction.2.It was found that CP-25 directly targets GRK2,inhibits GRK2 activity,and down-regulates co-expression of GRK2 and EP4 receptors by controlling the key amino acid residue of Ala321.The molecular mechanism of CP-25 regulation of GRK2 may be involved in improving EP4 receptor desensitization mediated FLS dysfunction.