CP-25 attenuates the inflammatory response of fibroblast-like synoviocytes co-cultured with BAFF-activated CD4(+) T cells.

Total glucosides of paeony (TGP) is the first anti-inflammatory immune regulatory drug approved for the treatment of rheumatoid arthritis in China. A novel compound, paeoniflorin-6'-O-benzene sulfonate (code CP-25), comes from the structural modification of paeoniflorin (Pae), which is the effective active ingredient of TGP. The aim of the present study is to investigate the effect of CP-25 on adjuvant arthritis (AA) fibroblast-like synoviocytes (FLS) co-cultured with BAFF-activated CD4(+) T cells and the expression of BAFF-R in CD4(+) T cells. The mRNA expression of BAFF and its receptors was assessed by qPCR. The expression of BAFF receptors in CD4(+) T cells was analyzed by flow cytometry. The effect of CP-25 on AA rats was evaluated by their joint histopathology. The cell culture growth of thymocytes and FLS was detected by cell counting kit (CCK-8). The concentrations of IL-1β, TNF-α, and IL-6 were measured by Enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of BAFF and BAFF-R were enhanced in the mesenteric lymph nodes of AA rats, TACI expression was reduced, and BCMA had no change. The expression of BAFF-R in CD4(+) T cells was also enhanced. CP-25 alleviated the joint histopathology and decreased the expression of BAFF-R in CD4(+) T cells from AA rats in vivo. In vitro, CP-25 inhibited the abnormal cell culture growth of BAFF-stimulated thymocytes and FLS. In the co-culture system, IL-1β, IL-6 and TNF-α production was enhanced by FLS co-cultured with BAFF-activated CD4(+) T cells. Moreover, BAFF-stimulated CD4(+) T cells promoted the cell culture growth of FLS. The addition of CP-25 decreased the expression of BAFF-R in CD4(+) T cells and inhibited the cell culture growth and cytokine secretion ability of FLS co-cultured with BAFF-activated CD4(+) T cells. The present study indicates that CP-25 may repress the cell culture growth and cytokine secretion ability of FLS, and its inhibitory effects might be associated with its ability to inhibit the expression of BAFF-R in CD4(+) T cells in a co-culture. These observations might provide a scientific basis for the development of new drugs for the treatment of autoimmune diseases by CP-25.

Jia X ，Wei F ，Sun X ，Chang Y ，Xu S ，Yang X ，Wang C ，Wei W ... -  《-》

Paeoniflorin inhibits function of synoviocytes pretreated by rIL-1α and regulates EP4 receptor expression.

To investigate the effect of the Paeoniflorin (Pae), a main active component of total glucosides of paeony (TGP) extracted from the root of Paeonia lactiflora, on regulation of synoviocytes cultured from rats collagen-induced arthritis (CIA) in vitro. CIA was induced in male Sprague-Dawley rats immunized with chicken type II collagen (CCII) in Freund's complete adjuvant. The levels of interleukin-1 (IL-1), tumor necrosis factor α (TNF-α), prostaglandin E(2) (PGE(2)) and cyclic adenosine monophosphate (cAMP) were measured by radioimmunoassay. The proliferation responses was determined by the 3-(4,5-2dimethylthiazal-2yl) 2,5-diphenyltetrazoliumbromide (MTT) assay. Expression of E-prostanoid (EP(4)) receptor was detected by Western blotting technique. Treatment of Pae (2.5, 12.5, 62.5 μg/ml) significantly decreased the production of IL-1 and TNF-α. Recombinant interleukin-1 (rIL-1α) (10 ng/ml) apparently stimulated synoviocyte, thymocyte and splenocyte proliferation, and Pae (12.5, 62.5 μg/ml) inhibited abnormal proliferation responses stimulated by rIL-1α. Moreover, rIL-1α time- and concentration-dependently increased production of PGE(2). The production of PGE(2) produced by synoviocytes from CIA rats significantly inhibited by administration of Pae (12.5, 62.5 μg/ml). rIL-1α (10 ng/ml) decreased cAMP of synoviocytes cells treated for 24h. Similarly rIL-1α (0.1, 1, 10 ng/ml) induced a concentration-dependent decrease in the production of cAMP at 24h. Pae (12.5, 62.5 μg/ml) increased the production of cAMP in synoviocytes. The immunoblot, Pae (12.5, 62.5 μg/ml) apparently increased the expression of EP(4) receptor in synoviocytes stimulated by rIL-1α (10 ng/ml). The present study indicates that Pae might exert its anti-inflammatory effects through suppressing synoviocytes function and regulating immune cells responses in CIA rats, which might be associated with its ability to up-regulate the E-prostanoid (EP(4)) receptor protein expression and modulate intracellular cAMP level.

Chang Y ，Zhang L ，Wang C ，Jia XY ，Wei W ... -  《-》

APRIL promotes proliferation, secretion and invasion of fibroblast-like synoviocyte from rats with adjuvant induced arthritis.

Fibroblast-like synoviocyte (FLS) is the ultimate effectual cells in the pathogenesis of rheumatoid arthritis (RA). The current study was undertaken to investigate whether a proliferation-inducing ligand (APRIL) mediates the function of FLS in an animal model of RA. Rat adjuvant-induced arthritis (AA) was induced by intradermal injection of 0.1 ml complete Freund's adjuvant. Synovium APRIL expression was detected by immunohistochemical analysis. The level of APRIL and matrix metalloproteinase (MMP)-9 were assayed by enzyme-linked immunosorbent assay. The expression of APRIL and its receptors (TACI, BCMA and BAFF-R) were assessed by immunofluorescence staining, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR. The effects of APRIL on the function of FLS were investigated by MTT, Quantibody Rat Inflammation Array 1 and transwell assays, respectively. A higher concentration of APRIL was detected in AA synovium homogenate compared with normal group. AA FLS expressed APRIL, TACI, BAFF-R and BCMA at the mRNA levels, whereas only APRIL and BCMA were confirmed to be expressed on membrane by flow cytometry. APRIL stimulated AA FLS proliferation, migration and invasion and the secretion of proinflammatory factors. In addition, FLS cocultured with APRIL-treated B cells or T cells had a significantly greater proliferation than FLS cultured alone. Neutralization of APRIL by the TACI-Ig fusion protein attenuated these stimulating effects of APRIL on FLS. Our data indicate that APRIL may act as an important mediator for facilitating the function of FLS. Blockade of APRIL thus may be a valuable adjunct in the treatment of RA.

Chang Y ，Jia X ，Sun X ，Xu S ，Wu Y ，Zhang L ，Wei W ... -  《-》

Total glucosides of paeony inhibit the proliferation of fibroblast-like synoviocytes through the regulation of G proteins in rats with collagen-induced arthritis.

The aim of this study was to investigate the expression of G proteins in fibroblast-like synoviocytes (FLSs) from rats with collagen-induced arthritis (CIA) and to determine the effect of total glucosides of paeony (TGP). CIA rats were induced with chicken type II collagen (CCII) in Freund's complete adjuvant. The rats with experimental arthritis were randomly separated into five groups and then treated with TGP (25, 50, and 100mg/kg) from days 14 to 35 after immunization. The secondary inflammatory reactions were evaluated through the polyarthritis index and histopathological changes. The level of cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. The FLS proliferation response was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The toxin-catalyzed ADP-ribosylation of G proteins was performed through autoradiography. The results show that TGP (25, 50, and 100mg/kg) significantly decreased the arthritis scores of CIA rats and improved the histopathological changes. TGP inhibited the proliferation of FLSs and increased the level of cAMP. Moreover, the FLS proliferation and the level of Gαi expression were significantly increased, but the level of Gαs expression was decreased after stimulation with IL-1β (10ng/ml) in vitro. TGP (12.5 and 62.5μg/ml) significantly inhibited the FLS proliferation and regulated the balance between Gαi and Gαs. These results demonstrate that TGP may exert its anti-inflammatory effects through the suppression of FLS proliferation, which may be associated with its ability to regulate the balance of G proteins. Thus, TGP may have potential as a therapeutic agent for the treatment of rheumatoid arthritis.

Jia XY ，Chang Y ，Sun XJ ，Wu HX ，Wang C ，Xu HM ，Zhang L ，Zhang LL ，Zheng YQ ，Song LH ，Wei W ... -  《-》

Paeoniflorin-6'-O-benzene sulfonate alleviates collagen-induced arthritis in mice by downregulating BAFF-TRAF2-NF-κB signaling: comparison with biological agents.

Shu JL ，Zhang XZ ，Han L ，Zhang F ，Wu YJ ，Tang XY ，Wang C ，Tai Y ，Wang QT ，Chen JY ，Chang Y ，Wu HX ，Zhang LL ，Wei W ... -  《-》