Role Of Src Homology 2 Domain-containing Protein Tyrosine Phosphatase-2 On G Protein-coupled Receptor Kinases 2 In Synoviocytes Of Rats With Adjuvant Arthritis And The Effects Of Paeoniflorin

OBJECTIVEAccording to the changes that secondary paw swelling, polyarthritis index,and histological morphological of adjuvant arthritis(AA)rats,this study tends to clarify the therapeutic effects of paeoniflorin(Pae) on AA rats.Meanwhile,our study aimed to confirm the role of GRK₂ and SHP-2 playing in fibroblase-like synoviocytes(FLS) of AA rats and the effects of Pae.And further to analyze mechanism of Pae on AA rats,we investigate the role of SHP-2 on GRK₂ and its signalling pathways.METHODSFreund's complete adjuvant (FCA)was used to induce AA in rats.Therapeutic treatment of interagastric administration Pae(12.5,25,50,100 mg·kg ^-1·d^-1, d14-d24 )were given after immunization. Hind paw volumes of rats were measured by volume meter.Arthritis index,spleen index were recorded. IL-1 level produced by thymus T cells was measured by ELISA and the proliferation of T and B lymphocytes were measured by [3H]-TdR incorporation. Pathological changes in synovium of joint were observed by light microscope. The proliferation of fibroblase-likesynoviocytes was assayed using the MTT assay.cAMP were measured by radioimmunoassay.The expression of GRK₂,SHP-2 were detected by westernblot analysis.RESULTS1. therapeutic effects of Pae on AA ratsPae(12.5,25,50,100 mg·kg ^-1·d^-1, d14-d24) significantly suppressed significantly the paw swelling as well as down-regulated the index of polyarthritis,spleen index,IL-1 produced by thymus T cells and proliferation of T and B lymphocytes in AA rats. The articular histopathological changes were also improved.This suggested that Pae had therapeutic effects on AA rats.2. Effects of SHP-2 inhibitor NSC-87877 on expression of GRK₂ and SHP-2,the level of cAMP,proliferation in synoviocytes from AA rats.SHP-2 inhibitor NSC-87877 significantly inhibited proliferation of FLS,increased the level of cAMP in synoviocytes and decreased the expression of GRK₂,SHP-2 in FLS.The result of western blot analysis demonstrated that SHP-2 inhibitor NSC-87877(10^-5mol·L^-1) in vitro significantly decreased the expression of GRK₂ in membrane and increased GRK₂ in cytoplasm.3. Effects of Pae on expression of GRK₂ and SHP-2,the level of cAMP,proliferation of FLS from AA rats.Pae significantly inhibited proliferation of FLS,increased the level of cAMP in FLS and decreased the expression of GRK₂,SHP-2.The result of western blot analysis demonstrated that SHP-2 inhibitor NSC-87877(10^-5mol·L^-1) in vitro significantly decreased the expression of GRK₂ in membrane and increased GRK₂ in cytoplasm. CONCLUSIONS1. Pae could alleviate the inflammation of joint at different degree and decrease the spleen index ,the secretion of IL-1,proliferation of T,B lymphocyte and articular histopathological changes in rats with AA.2. SHP-2 inhibitor could decrease the expression of GRK₂ and SHP-2 in FLS from AA rats; SHP-2 inhibitor inhibited proliferation of FLS by incpreasing the level of cAMP and improving the expresstion of GRK₂ in cytoplasm meanwhile decreased the expression of GRK₂ in membrane.Which indicated that SHP-2 inhibited proliferation of FLS by inhibiting transmembrane of GRK₂ and then increasing the level of cAMP in FLS.3.Pae could decreased the expression of SHP-2 in FLS,which may be one of the important mechanisms for the Pae on AA rats.