Qingtong Wang

Therapeutic effects of TACI-Ig on rats with adjuvant-induced arthritis via attenuating inflammatory responses

OBJECTIVE To investigate the effects of TACI-Ig, a recombinant fusion protein that modulates B- and T-cell activation by binding and neutralizing B-lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), in an established adjuvant-induced arthritis (AA) rat model. METHODS Rats with experimental arthritis were randomly separated into different groups and then treated with TACI-Ig (0.7, 2.1, 6.3 mg/kg), rhTNFR-Fc (2.8 mg/kg), MTX (0.5 mg/kg) or IgG-Fc (6.3 mg/kg), from Day 16 to Day 34 after immunization. Arthritis was evaluated by hind paw swelling, polyarthritis index and histopathological examination. Activities of BLyS, APRIL, IL-1β, IL-2, IL-10, TGF-β1, PGE(2), TNF-α, IFN-γ, immunoglobulin (Ig)G1, IgG2a, IgM and IgA were assessed by ELISA. Cluster of differentiation (CD)20 expression was detected by immunohistochemical analysis. RESULTS TACI-Ig (2.1, 6.3 mg/kg) treatment significantly reduced the severity of established arthritis using the methods of clinical observation and histopathological examination. TACI-Ig treatment inhibited expression of IgM, decreased the expression of BLyS and APRIL and regulated the balance of pro-inflammatory and anti-inflammatory cytokines in serum of AA rats. Immunohistochemical analysis demonstrated that CD20 production was reduced in spleen. CONCLUSIONS Data presented here demonstrate that administration of TACI-Ig significantly attenuates progression of experimental arthritis, with reductions in inflammatory response and bone and joint destruction.

The expression change of β-arrestins in fibroblast-like synoviocytes from rats with collagen-induced arthritis and the effect of total glucosides of paeony

AIM OF THE STUDY To investigate the expression of β-arrestins in fibroblast-like synoviocytes (FLS) from collagen-induced arthritis (CIA) rats and the effect of total glucosides of paeony (TGP). MATERIALS AND METHODS TGP and glucosides of tripterygium wilfordii (GTW) were intragastriclly administrated to collagen-induced arthritis (CIA) rats after immunization. The secondary inflammatory reaction was evaluated by hind paw swelling, polyarthritis index and histopathological changes. Antibodies to type II collagen (CII) were determined by enzyme-linked immunosorbent assay (ELISA). Synoviocyte proliferations were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay. The expression of β-arrestins in synoviocytes from CIA rats was measured by western blot. RESULTS The administration of TGP (25, 50, 100 mg/kg) depressed hind paw swelling and decreased the arthritis scores of CIA rats. TGP improved the pathologic manifestations of CIA. Serum anti-CII antibodies level increased significantly in CIA rats, while TGP had no effect on it. Fibroblast-like synoviocytes (FLS) proliferation was inhibited by TGP (50, 100 mg/kg). On d14, d28 after immunization, β-arrestins expression greatly up-regulated in synoviocytes from CIA rats and then returned to baseline levels on d42 after immunization. TGP (50, 100 mg/kg) significantly reduced the expression of β-arrestins. CONCLUSION An inflammatory process in vivo induces an up-regulation of β-arrestins in synoviocytes from CIA rats while TGP can inhibit this change, which might be one of the important mechanisms for TGP to produce a marked therapeutic effect on RA.

BAFF/BAFF-R involved in antibodies production of rats with collagen-induced arthritis via PI3K-Akt-mTOR signaling and the regulation of paeoniflorin

ETHNOPHARMACOLOGICAL RELEVANCE Paeoniflorin (Pae) is extracted from the root of paeonia lactiflora which have attracted attention for anti-rheumatic and immune modulating properties. AIM OF THE STUDY To investigate the role of PI3K/Akt/mTOR signaling mediated by BAFF/BAFF-R in antibodies production and the regulation of Pae on the signaling pathway in rats with collagen-induced arthritis (CIA). MATERIALS AND METHODS CIA rats were randomly separated into different groups and treated with Pae (25, 100mg/kg) from day 18 to day 38 after immunization. The effects of Pae on B lymphocytes of CIA rats were evaluated by the levels of BAFF, anti-CII antibody, IgA, IgG and IgM, and the expressions of BAFF-R, PI3K, p-Akt and mTOR. RESULTS In CIA rats, the levels of anti-CII antibody, IgA, IgG and IgM in serum enhanced, BAFF, BAFF-R, PI3K, p-Akt and mTOR were highly expressed. Pae (100mg/kg) obviously decreased arthritis score, relieved ankle and paw swelling, improved spleen histopathology in CIA rats, decreased the levels of IgA, IgM, IgG and anti-CII antibody, and significantly decreased the expressions of BAFF, BAFF-R, PI3K, p-Akt and mTOR. CONCLUSION PI3K/Akt/mTOR signaling mediated by BAFF/BAFF-R participates in antibodies production by B lymphocytes of CIA rats. Pae had therapeutic effects on rats with CIA. These effects might be relative to regulating PI3K/Akt/mTOR signal mediated by BAFF/BAFF-R, and down regulate the antibodies production further.

The Roles of B Cells and Their Interactions with Fibroblast-Like Synoviocytes in the Pathogenesis of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that may affect many tissues and organs but principally attacks synovial joints. The pathogenesis of RA is complex and encompasses many cell types, including T cells, B cells and fibroblast-like synoviocytes (FLSs); each is believed to play a significant role in the process of RA. Interactions between T cells and B cells and between T cells and FLSs have been studied in depth. However, it has been showed that B cells also play a critical role in rheumatoid synovitis. Therefore, cell-cell contact between B cells and FLSs may be significant in the pathophysiology of RA. This review focuses on recent advances in the research of the interaction between B cells and FLSs. Considering this interaction may provide directions for innovations in RA therapy.

Ginsenoside metabolite compound K attenuates inflammatory responses of adjuvant-induced arthritis rats

Abstract Objective: To investigate the effects of ginsenoside metabolite compound K (CK) on adjuvant-induced arthritis (AA) rats and the partial mechanisms focused on the function of immunocyte (B cell and macrophage) and effectors’ cell (fibroblast-like synoviocyte, FLS). Methods: Animals were divided randomly into nine groups including control, AA, CK (5, 10, 20, 40, 80, and 160 mg/kg, i.g.), and MTX (0.5 mg/kg, i.g.). The effects of CK on AA rats are evaluated by swelling of the paw, histopathology of joint, and inflammatory cytokine production in serum. To further investigate the effects of CK on the function of B cell, peritoneal macrophage, and FLS from AA rats, we examined the proliferation of B cell and FLS by [3H] thymidine incorporation, and the phagocytic function of peritoneal macrophage was measured by neutral red uptake. Cytokines and antibodies in serum and the supernatant from peritoneal macrophage and FLS were measured by ELISA kit. Results: CK suppressed the severity of AA rats by attenuating the paw swelling and histopathology of joint. CK can inhibit the proliferation of B cell and autoantibody levels, and suppressed the phagocytic function of peritoneal macrophage and secreted pro-inflammatory cytokines TNF-α, IFN-γ, and IL-17 and up-regulated the level of protective cytokines IL-10. CK attenuated the proliferation of FLS, and balanced the ratio of RANKL to OPG in AA rats. Conclusion: Our results suggest that CK may attenuate the severity of AA rats, partially by influencing the function of immunocyte (B cell and macrophage) and effectors’ cells (FLS) in AA.

Combined use of etanercept and MTX restores CD4+/CD8+ ratio and Tregs in spleen and thymus in collagen-induced arthritis

ObjectiveTo further explore the mechanism of etanercept (ENT, rhTNFR:Fc) and methotrexate (MTX) in the combined treatment of rheumatoid arthritis (RA), we investigated whether thymic and splenic T-cell subsets and their related cytokines imbalance could be restored by ETN/MTX treatment.MethodsThe effect of ETN/MTX on collagen-induced arthritis (CIA) was evaluated by arthritis scores, joint and spleen histopathology, as well as indices of thymus and spleen. T lymphocytes proliferation was determined by [3H]-TdR incorporation. Levels of TNF-α, LT-α, IL-1β, RANKL, IL-10, IL-17, IFN-γ and IL-6 were detected by enzyme linked immunosorbent assay. The subsets of T lymphocytes including CD4+, CD8+, CD3+CD4+, CD4+CD25+, CD4+CD62L+ and CD4+CD25+Foxp3+ cells were quantified using flow cytometry.ResultsCombined administration of ETN/MTX significantly inhibited the proliferation of T lymphocytes, decreased serum IL-6, TNF-α, IL-1β, RANKL and macrophage supernatant IL-17, LT-α, increased serum IFN-γ and macrophage supernatant IL-10. Moreover, the combined administration could restore CD4+/CD8+ ratio and Treg cells of CIA thymus and spleen.ConclusionTaken together, our findings suggest that ENT/MTX may modify the abnormal T lymphocytes balance from central to peripheral lymphoid organs, which may partially, explained the mechanism of the combined administration.

Pro-apoptotic effect of epigallo-catechin-3-gallate on B lymphocytes through regulating BAFF/PI3K/Akt/mTOR signaling in rats with collagen-induced arthritis.

To investigate the role of PI3K/Akt/mTOR signaling mediated by B cell-activating factor belonging to the TNF family (BAFF) involved in anti-apoptosis of B lymphocytes in rats with collagen-induced arthritis (CIA) and the regulation of epigallo-catechin-3-gallate (EGCG). Sprague-Dawley rats were immunized to induce CIA. CIA rats were randomly separated into different groups and treated with EGCG (40, 80 mg/kg), Paeoniflorin (100mg/kg) from day 18 to day 38 after immunization. The effects of EGCG on B lymphocytes were evaluated by the levels of BAFF, anti-CII antibody, IgA, IgG and IgM, and the expressions of BAFF receptor, P110δ, p-Akt, mTORC1, Bcl-xL and Bim. B lymphocyte proliferations were analyzed by MTT assay. Apoptosis of B lymphocyte were assayed by flow cytometry. Results showed that, in CIA rats, the levels of BAFF, anti-CII antibody, IgA, IgG and IgM enhanced. BAFF receptor, P110δ, p-AKT, mTORC1 and Bcl-xL were expressed highly, while Bim expression decreased. EGCG (40, 80 mg/kg) and Paeoniflorin decreased the levels of BAFF, anti-CII antibody, IgA, IgG, IgM and the expressions of BAFF receptor, P110δ, p-AKT, mTORC1, Bcl-xL in CIA rats, and increased Bim expression. Further studies showed that EGCG could reduce the expression of P110δ and mTORC1 in vitro. EGCG inhibited B lymphocyte proliferation and induced B lymphocyte apoptosis. In conclusion, BAFF/BAFF receptor might regulate B cell anti-apoptosis through PI3K/Akt/mTOR pathway. EGCG had therapeutic effects on CIA rats, which might be relative to the inhibition effects of EGCG on BAFF and PI3K/Akt/mTOR signaling, and then the apoptosis of B lymphocytes was promoted further.

Involvement of the prostaglandin E receptor EP2 in paeoniflorin-induced human hepatoma cell apoptosis.

Prostaglandin E2 (PGE2) has been shown to play an important role in tumor development and progression. PGE2 mediates its biological activity by binding any one of four prostanoid receptors (EP1 through EP4). The present study was designed to determine the role of the EP2 receptor during the proliferation and apoptosis of human HepG2 and SMMC-7721 hepatoma cell lines and the effect of paeoniflorin, a monoterpene glycoside. The proliferation of HepG2 and SMMC-7721 cells was determined by methyl thiazolyl tetrazolium after exposure to the selective EP2 receptor agonists butaprost and paeoniflorin. Apoptosis of HepG2 and SMMC-7721 cells was also quantified by flow cytometry with annexin V–fluorescein isothiocyanate and propidium iodide staining. The expression levels of Bcl-2 and Bax were quantified by western blotting and immunohistochemistry. The expression of the EP2 receptor and cysteine–aspartic acid protease (caspase)-3 was determined by western blotting. Butaprost significantly increased proliferation in HepG2 and SMMC-7721 cells. Paeoniflorin significantly inhibited the proliferation of HepG2 and SMMC-7721 cells stimulated by butaprost at multiple time points (24, 48, and 72 h). Paeoniflorin induced apoptosis in HepG2 and SMMC-7721 cells, which was quantified by annexin-V and propidium iodide staining. Our results indicate that the expression of the EP2 receptor and Bcl-2 was significantly increased, whereas that of Bax and cleaved caspase-3 was decreased in HepG2 and SMMC-7721 cells after stimulation by butaprost. Paeoniflorin significantly decreased the expression of the EP2 receptor and Bcl-2 and increased Bax and caspase-3 activation in HepG2 and SMMC-7721 cells on addition of butaprost. Our results show that the PGE2 receptor subtype EP2 may play a vital role in the survival of both HepG2 and SMMC-7721 cells. Paeoniflorin, which may be a promising agent in the treatment of liver cancer, induced apoptosis in hepatocellular carcinoma cells by downregulating EP2 expression and also increased the Bax-to-Bcl-2 ratio, thus upregulating the activation of caspase-3.

Total glucosides of paeony inhibit the inflammatory responses of mice with allergic contact dermatitis by restoring the balanced secretion of pro-/anti-inflammatory cytokines

The present study aimed to investigate the regulation exerted by the total glucosides of paeony (TGP) on the production of interleukin-2 (IL-2), IL-4, IL-10 and IL-17 in the serum and lymphocytes of mice with allergic contact dermatitis (ACD). ACD in mice was induced by the repeated application of 2,4-dinitrochlorobenzene (DNCB) to their skins. The mice were orally administered TGP (35, 70, and 140mg/kg/d) and prednisone (Pre, 5mg/kg/d) from day 1 to day 7 after immunization. The inflammatory responses were evaluated by ear swelling and histological examination. Thymocyte proliferation was assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide assay. The cytokine production in the serum and lymphocytes supernatant was measured by enzyme-linked immunosorbent assay. The results indicated that the topical application of DNCB to the skin provoked obvious inflammatory responses. The oral administration of TGP (70 and 140mg/kg/d) and Pre (5mg/kg/d) significantly inhibited skin inflammation, decreased the thymus and spleen indices, and inhibited thymocyte proliferation in mice treated with DNCB. Further study indicated that TGP increased IL-4 and IL-10 production but decreased the production of IL-2 and IL-17 in the serum and lymphocyte supernatant. The correlation analysis suggested significantly positive correlations between IL-2 and IL-17 production and the severity of skin inflammation, whereas negative correlations were obtained for IL-4 and IL-10 production and skin inflammation. In summary, these results suggest that the therapeutic effects of TGP on ACD may result from its regulation of the imbalanced secretion of IL-2/IL-4 and IL-10/IL-17.

Ginsenoside compound K suppresses the abnormal activation of T lymphocytes in mice with collagen-induced arthritis

Aim:To investigate the anti-arthritis and immunomodulatory activities of ginsenoside compound K (C-K) in mice with collagen-induced arthritis (CIA).Methods:DBA/1 mice with CIA were treated with C-K (28, 56 or 112 mg·kg−1 ·d−1, ig) or the positive control methotrexate (2 mg/kg, ig, every 3 d) for 34 d. Splenic T and B lymphocytes were positively isolated using anti-CD3-coated magnetic beads or a pan B cell isolation kit. T lymphocyte subsets, and CD28, T cell receptor (TCR), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1) expression in purified splenic T lymphocytes were analyzed using flow cytometry, Western blotting and laser confocal microscopy.Results:C-K treatment significantly ameliorated the pathologic manifestations of CIA mice, remarkably inhibited T lymphocyte proliferation, and marginally inhibited the proliferation of B lymphocytes. C-K treatment significantly suppressed TNF-α and anti-CII antibody levels, and increased IFN-γ level in the joints of CIA mice, but did not alter IL-4 production. Treatment of CIA mice with C-K significantly decreased the percentages of activated T cells, co-stimulatory molecule-expressing T cells and effector memory T cells, and increased the frequencies of naive T cells and regulatory T cells. Furthermore, C-K treatment significantly decreased the expression of CD28 and TCR, whereas it increased the expression of CTLA-4 and PD-1 on T lymphocytes of CIA mice. Methotrexate treatment exerted comparable effects in all these experiments.Conclusion:C-K suppresses the progression of CIA through regulating TCR, CD28, CTLA-4 and PD-1 expression, thus inhibiting the abnormal activation and differentiation of T lymphocytes.