Chronic ß-adrenergic stress contributes to cardiomyopathy in rodents with collagen-induced arthritis.

Patients with rheumatoid arthritis (RA) have a much higher incidence of cardiac dysfunction, which contributes to the high mortality rate of RA despite anti-arthritic drug therapy. In this study, we investigated dynamic changes in cardiac function in classic animal models of RA and examined the potential effectors of RA-induced heart failure (HF). Collagen-induced arthritis (CIA) models were established in rats and mice. The cardiac function of CIA animals was dynamically monitored using echocardiography and haemodynamics. We showed that cardiac diastolic and systolic dysfunction occurred in CIA animals and persisted after joint inflammation and that serum proinflammatory cytokine (IL-1ß, TNF-α) levels were decreased. We did not find evidence of atherosclerosis (AS) in arthritic animals even though cardiomyopathy was significant. We observed that an impaired cardiac ß1AR-excitation contraction coupling signal was accompanied by sustained increases in blood epinephrine levels in CIA rats. Furthermore, serum epinephrine concentrations were positively correlated with the heart failure biomarker NT-proBNP in RA patients (r2 = +0.53, P < 0.0001). In CIA mice, treatment with the nonselective ßAR blocker carvedilol (2.5 mg·kg-1·d-1, for 4 weeks) or the specific GRK2 inhibitor paroxetine (2.5 mg·kg-1·d-1, for 4 weeks) effectively rescued heart function. We conclude that chronic and persistent ß-adrenergic stress in CIA animals is a significant contributor to cardiomyopathy, which may be a potential target for protecting RA patients against HF.

TNF-α impairs EP4 signaling through the association of TRAF2-GRK2 in primary fibroblast-like synoviocytes.

Our previous study showed that chronic treatment with tumor necrosis factor-α (TNF-α) decreased cAMP concentration in fibroblast-like synoviocytes (FLSs) of collagen-induced arthritis (CIA) rats. In this study we investigated how TNF-α impairs cAMP homeostasis, particularly clarifying the potential downstream molecules of TNF-α and prostaglandin receptor 4 (EP4) signaling that would interact with each other. Using a cAMP FRET biosensor PM-ICUE3, we demonstrated that TNF-α (20 ng/mL) blocked ONO-4819-triggered EP4 signaling, but not Butaprost-triggered EP2 signaling in normal rat FLSs. We showed that TNF-α (0.02-20 ng/mL) dose-dependently reduced EP4 membrane distribution in normal rat FLS. TNF-α significantly increased TNF receptor 2 (TNFR2) expression and stimulated proliferation in human FLS (hFLS) via ecruiting TNF receptor-associated factor 2 (TRAF2) to cell membrane. More interestingly, we revealed that TRAF2 interacted with G protein-coupled receptor kinase (GRK2) in the cytoplasm of primary hFLS and helped to bring GRK2 to cell membrane in response of TNF-α stimulation, the complex of TRAF2 and GRK2 then separated on the membrane, and translocated GRK2 induced the desensitization and internalization of EP4, leading to reduced production of intracellular cAMP. Silencing of TRAF2 by siRNA substantially diminished TRAF2-GRK2 interaction, blocked the translocation of GRK2, and resulted in upregulated expression of membrane EP4 and intracellular cAMP. In CIA rats, administration of paroxetine to inhibit GRK2 effectively improved the symptoms and clinic parameters with significantly reduced joint synovium inflammation and bone destruction. These results elucidate a novel form of cross-talk between TNFR (a cytokine receptor) and EP4 (a typical G protein-coupled receptor) signaling pathways. The interaction between TRAF2 and GRK2 may become a potential new drug target for the treatment of inflammatory diseases.

Deficiency of ß-arrestin2 exacerbates inflammatory arthritis by facilitating plasma cell formation.

ß-arrestin2 (ß-arr2) is, a key protein that mediates desensitization and internalization of G protein-coupled receptors and participates in inflammatory and immune responses. Deficiency of ß-arr2 has been found to exacerbate collagen antibody-induced arthritis (CAIA) through unclear mechanisms. In this study we tried to elucidate the molecular mechanisms underlying ß-arr2 depletion-induced exacerbation of CAIA. CAIA was induced in ß-arr2-/- and wild-type (WT) mice by injection of collagen antibodies and LPS. The mice were sacrificed on d 13 after the injection, spleen, thymus and left ankle joints were collected for analysis. Arthritis index (AI) was evaluated every day or every 2 days. We showed that ß-arr2-/- mice with CAIA had a further increase in the percentage of plasma cells in spleen as compared with WT mice with CAIA, which was in accordance with elevated serum IgG1 and IgG2A expression and aggravating clinical performances, pathologic changes in joints and spleen, joint effusion, and joint blood flow. Both LPS stimulation of isolated B lymphocytes in vitro and TNP-LPS challenge in vivo led to significantly higher plasma cell formation and antibodies production in ß-arr2-/- mice as compared with WT mice. LPS treatment induced membrane distribution of toll-like receptor 4 (TLR4) on B lymphocytes, accordingly promoted the nuclear translocation of NF-κB and the transcription of Blimp1. Immunofluorescence analysis confirmed that more TLR4 colocalized with ß-arr2 in B lymphocytes in response to LPS stimulation. Depletion of ß-arr2 restrained TLR4 on B lymphocyte membrane after LPS treatment and further enhanced downstream NF-κB signaling leading to additional increment in plasma cell formation. In summary, ß-arr2 depletion exacerbates CAIA and further increases plasma cell differentiation and antibody production through inhibiting TLR4 endocytosis and aggravating NF-κB signaling.

ß-arrestin2 deficiency protects against hepatic fibrosis in mice and prevents synthesis of extracellular matrix.

Hepatic fibrosis is a disease of the wound-healing response following chronic liver injury, and activated hepatic stellate cells (HSCs) play a crucial role in the progression of hepatic fibrosis. ß-arrestin2 functions as a multiprotein scaffold to coordinate complex signal transduction networks. Although ß-arrestin2 transduces diverse signals in cells, little is known about its involvement in the regulation of liver fibrosis. Our current study utilized a porcine serum-induced liver fibrosis model and found increased expression of ß-arrestin2 in hepatic tissues with the progression of hepatic fibrosis, which was positively correlated with collagen levels. Furthermore, changes in human fibrotic samples were also observed. We next used ß-arrestin2-/- mice to demonstrate that ß-arrestin2 deficiency ameliorates CCl4-induced liver fibrosis and decreases collagen deposition. The in vitro depletion and overexpression experiments showed that decreased ß-arrestin2 inhibited HSCs collagen production and elevated TßRIII expression, thus downregulating the TGF-ß1 pathway components Smad2, Smad3 and Akt. These findings suggest that ß-arrestin2 deficiency ameliorates liver fibrosis in mice, and ß-arrestin2 may be a potential treatment target in hepatic fibrosis.

IgD-Fc-Ig fusion protein, a new biological agent, inhibits T cell function in CIA rats by inhibiting IgD-IgDR-Lck-NF-κB signaling pathways.

IgD-Fc-Ig fusion protein, a new biological agent, is constructed by linking a segment of human IgD-Fc with a segment of human IgG1-Fc, which specifically blocks the IgD-IgDR pathway and selectively inhibits the abnormal proliferation, activation, and differentiation of T cells. In this study we investigated whether IgD-Fc-Ig exerted therapeutic effects in collagen-induced arthritis (CIA) rats. CIA rats were treated with IgD-Fc-Ig (1, 3, and 9 mg/kg) or injected with biological agents etanercept (3 mg/kg) once every 3 days for 40 days. In the PBMCs and spleen lymphocytes of CIA rats, both T and B cells exhibited abnormal proliferation; the percentages of CD3+ total T cells, CD3+CD4+ Th cells, CD3+CD4+CD25+-activated Th cells, Th1(CD4+IFN-γ+), and Th17(CD4+IL-17+) were significantly increased, whereas the Treg (CD4+CD25+Foxp3+) cell percentage was decreased. IgD-Fc-Ig administration dose-dependently decreased the indicators of arthritis; alleviated the histopathology of spleen and joint; reduced serum inflammatory cytokines levels; decreased the percentages of CD3+ total T cells, CD3+CD4+ Th cells, CD3+CD4+CD25+-activated Th cells, Th1 (CD4+IFN-γ+), and Th17(CD4+IL-17+); increased Treg (CD4+CD25+Foxp3+) cell percentage; and down-regulated the expression of key molecules in IgD-IgDR-Lck-NF-κB signaling (p-Lck, p-ZAP70, p-P38, p-NF-κB65). Treatment of normal T cells with IgD (9 µg/mL) in vitro promoted their proliferation. Co-treatment with IgD-Fc-Ig (0.1-10 µg/mL) dose-dependently decreased IgD-stimulated T cell subsets percentages and down-regulated the IgD-IgDR-Lck-NF-κB signaling. In summary, this study demonstrates that IgD-Fc-Ig alleviates CIA and regulates the functions of T cells through inhibiting IgD-IgDR-Lck-NF-κB signaling.

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

Paeoniflorin-6'-O-benzene sulfonate (CP-25) is a new ester derivative of paeoniflorin with improved lipid solubility and oral bioavailability, as well as better anti-inflammatory activity than its parent compound. In this study we explored whether CP-25 exerted therapeutic effects in collagen-induced arthritis (CIA) mice through regulating B-cell activating factor (BAFF)-BAFF receptors-mediated signaling pathways. CIA mice were given CP-25 or injected with biological agents rituximab or etanercept for 40 days. In CIA mice, we found that T cells and B cells exhibited abnormal proliferation; the percentages of CD19+ total B cells, CD19+CD27+-activated B cells, CD19+BAFFR+ and CD19+TACI+ cells were significantly increased in PBMCs and spleen lymphocytes. CP-25 suppressed the indicators of arthritis, alleviated histopathology, accompanied by reduced BAFF and BAFF receptors expressions, inhibited serum immunoglobulin levels, decreased the B-cell subsets percentages, and prevented the expressions of key molecules in NF-κB signaling. Furthermore, we showed that treatment with CP-25 reduced CD19+TRAF2+ cell expressions stimulated by BAFF and decreased TRAF2 overexpression in HEK293 cells in vitro. Thus, CP-25 restored the abnormal T cells proliferation and B-cell percentages to the normal levels, and normalized the elevated levels of IgA, IgG2a and key proteins in NF-κB signaling. In comparison, rituximab and etanercept displayed stronger anti-inflammatory activities than CP-25; they suppressed the elevated inflammatory indexes to below the normal levels in CIA mice. In summary, our results provide evidence that CP-25 alleviates CIA and regulates the functions of B cells through BAFF-TRAF2-NF-κB signaling. CP-25 would be a soft immunomodulatory drug with anti-inflammatory effect.

CP-25, a Novel Anti-inflammatory and Immunomodulatory Drug, Inhibits the Functions of Activated Human B Cells through Regulating BAFF and TNF-alpha Signaling and Comparative Efficacy with Biological Agents.

Paeoniflorin-6'-O-benzene sulfonate (code: CP-25) was the chemistry structural modifications of Paeoniflorin (Pae). CP-25 inhibited B cells proliferation stimulated by B cell activating factor belonging to the TNF family (BAFF) or Tumor necrosis factor alpha (TNF-alpha). CP-25, Rituximab and Etanercept reduced the percentage and numbers of CD19+ B cells, CD19+CD20+ B cells, CD19+CD27+ B cells and CD19+CD20+CD27+ B cells induced by BAFF or TNF-alpha. There was significant difference between CP-25 and Rituximab or CP-25 and Etanercept. CP-25 down-regulated the high expression of BAFFR, BCMA, and TACI stimulated by BAFF or TNF-alpha. The effects of Rituximab and Etanercept on BAFFR or BCMA were stronger than that of CP-25. CP-25, Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept down-regulated the expression of MKK3, P-p38, P-p65, TRAF2, and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P-p65 in B cells stimulated by TNF-alpha. These results suggest that CP-25 regulated moderately activated B cells function by regulating the classical and alternative NF-κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway. This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug.

A novel anti-inflammatory and immunomodulatory drug CP-25 alleviated collagen induced arthritis by down-regulating BAFF-NF-κB signaling pathway / 中国药理学与毒理学杂志

OBJECTIVE To investigated the regulatory effect of paeoniflorin-6'-O-benzene sulfonate (CP-25) on B cell activating factor (BAFF)/BAFF receptor-nuclear factor of kappa B (NF-κB) signaling in B cell of collagen induced-arthritis (CIA) mice. METHODS Mice CIA was induced by injection of typeⅡcollagen (CⅡ). The arthritis index (AI) and swollen joint count (SJC) were assessed, and histopathology of spleen and joints were observed. The percentage of B cells subsets, BAFF receptor expressions were analyzed by flow cytometry. BAFF and immunoglobulin (Ig) levels were measured by protein antibody array. The expressions of TRAF2, MKK3, MKK6, p-P38, and p-NF-κB65 in NF-κB signaling mediated by BAFF were analyzed by western blot. RESULTS CP-25 decreased AI and SJC, restored abnormal weights, reduced thymus index and spleen index, inhibited T/B cells proliferation, alleviated the histopathology of spleen and joints in CIA mice. CP-25 also reduced high levels of serum BAFF and immunoglobulin, decreased CD19+B cells, CD19+CD27+B cells, and CD19-CD27+CD138+ plasma cells, inhibited BAFFR and TACI expressions, decreased the expressions of TRAF2, MKK3, MKK6, p-P38, and p-NF-κB65. Compared with biological agents etanercept and rituximab, CP-25 restored high T cells proliferation and percentages of B subsets to normal level, and recovered the high levels of IgA, IgD, IgG1, IgG2a and high expressions molecules in NF- κB signaling to normal levels. The action intensity of rituximab and etanercept was more strong than CP- 25. The inhibitor effects of rituximab and etanercept on AI and SJC, thymus index, proliferation of T cells and B cells subsets were strong, and down-regulated the indexes to under normal levels. CONCLUSION CP-25 might be a promising anti- inflammatory immune and regulation drug, which alleviated CIA and regulated the functions of B cells through BAFF/BAFF receptor-NF-κB signaling.

Ginsenoside metabolite compound K alleviate collegen-induced arthritis through impairing dendritic cells function / 中国药理学与毒理学杂志

OBJECTIVE Ginsenoside metabolite compound K (CK) is a degradation product of ginsenoside in the intestine by bacteria. The anti-inflammatory and immunomodulatory activities of CK have been reported. This study investigated whether CK exerted its immunoregulatory effect through modulation of dendritic cells (DCs) function. METHODS In vivo, severity of collegen-induced arthritis (CIA), T cells and DCs subsets, phenotype of DC were assayed by flow cytometry, CCL19 and CCL21 level in lymph nodes assayed by ELISA. In vitro, bone marrow-derived DCs from normal mice were matured with lipopolysaccharide and treated with CK for 48 h. In vivo, bone marrow-derived DCs were generated from CIA mice before and 2 weeks into CK treatment. DCs were analyzed for migration, phenotype and T- cell stimulatory capacity. RESULTS CK alleviated the severity of CIA, decreased pDCs and mo-DCs, increased na?ve T cells in CIA mice lymph nodes, and suppressed CCL21 expression in lymph nodes. CK suppressed DCs migration induced by CCL21 and T cells-stimulatory capability of DC, down-regulated LPS-induced expression of CD80, CD86, MHCII and CCR7 on DCs. CONCLUSION This study elucidated the novel immunomodulatory property of CK via impairing function of DCs in priming T cells activation. These results provide an interesting novel insight into the potential mechanism by which CK contribute to the restoration of immunoregulation in autoimmune conditions.

Down-regulation of ß-arrestin2 promotes tumour invasion and indicates poor prognosis of hepatocellular carcinoma.

ß-arrestins, including ß-arrestin1 and ß-arrestin2, are multifunctional adaptor proteins. ß-arrestins have recently been found to play new roles in regulating intracellular signalling networks associated with malignant cell functions. Altered ß-arrestin expression has been reported in many cancers, but its role in hepatocellular carcinoma (HCC) is not clear. We therefore examined the roles of ß-arrestins in HCC using an animal model of progressive HCC, HCC patient samples and HCC cell lines with stepwise metastatic potential. We demonstrated that ß-arrestin2 level, but not ß-arrestin1 level, decreased in conjunction with liver tumourigenesis in a mouse diethylnitrosamine-induced liver tumour model. Furthermore, ß-arrestin2 expression was reduced in HCC tissues compared with noncancerous tissues in HCC patients. ß-arrestin2 down-regulation in HCC was significantly associated with poor patient prognoses and aggressive pathologic features. In addition, our in vitro study showed that ß-arrestin2 overexpression significantly reduced cell migration and invasion in cultured HCC cells. Furthermore, ß-arrestin2 overexpression up-regulated E-cadherin expression and inhibited vimentin expression and Akt activation. These results suggest that ß-arrestin2 down-regulation increases HCC cell migration and invasion ability. Low ß-arrestin2 expression may be indicative of a poor prognosis or early cancer recurrence in patients who have undergone surgery for HCC.

BAFF upregulates CD28/B7 and CD40/CD154 expression and promotes mouse T and B cell interaction in vitro via BAFF receptor.

AIM: B cell-activating factor belonging to the TNF family (BAFF) is a member of TNF family and required for peripheral B cell survival and homeostasis. BAFF has been shown to promote the proliferation of T and B cells. In this study we examined whether and how BAFF mediated the interaction between mouse T and B cells in vitro. METHODS: BAFF-stimulated B or T cells were co-cultured with T or B cells. The interactions between T and B cells were analyzed by measuring the expression of co-stimulatory molecules (CD28/CD80 or CD40/CD154), the proliferation and secretion of T and B cells and other factors. Two siRNAs against the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and BAFF receptor (BAFF-R) were used to identify the receptors responsible for the actions of BAFF. RESULTS: BAFF-stimulated B cells significantly promoted the proliferation and activity of co-cultured T cells, and increased the percentages of CD4(+)CD28(+) and CD4(+)CD154(+) T cells. Similarly, BAFF-stimulated T cells significantly promoted the proliferation and activity of co-cultured B cells, and increased CD19(+)CD80(+) and CD19(+)CD40(+)B cell subpopulations. BAFF-R siRNA-silenced B cells showed significantly lower expression of CD40 and CD80 than the control B cells. When the BAFF-R siRNA-silenced B cells were stimulated with BAFF, then co-cultured with T cells, the expression of CD28 and CD154 on T cells was not increased. TACI siRNA-silenced B cells exhibited higher expression of CD40 and CD80 than the control B cells. When the TACI siRNA-silenced B cells were stimulated with BAFF, then co-cultured with T cells, the expression of CD28 and CD154 on T cells was significantly increased. CONCLUSION: BAFF upregulates CD28/B7 and CD40/CD154 expression, and promotes the interactions between T and B cells in a BAFF-R-dependent manner.

Prednisone treatment inhibits the differentiation of B lymphocytes into plasma cells in MRL/MpSlac-lpr mice.

AIM: A number of evidence shows that the differentiation of B lymphocytes into plasma cells plays an important role in lupus pathogenesis. In this study we investigated how prednisone, a classical therapeutic drug for autoimmune diseases, regulated plasma cell differentiation in MRL/MpSlac-lpr mice. METHODS: MRL/lpr mice were treated with prednisone (2.5 or 5 mg·kg(-1)·d(-1), ig) for 13 weeks, and the proteinuria levels and survival times were monitored. After the mice were euthanized, blood sample, spleen and thymus were collected. The serum levels of anti-dsDNA antibody, anti-nuclear antibody, IL-21, and IL-10 were detected using ELISA kits. Subsets of splenic B and T lymphocytes were quantified with flow cytometry. Transcription factor Blimp-1 and Bcl-6 expression was determined using qPCR and Western blot. RESULTS: Prednisone treatment dose-dependently attenuated the lupus symptoms in MRL/lpr mice with decreased proteinuria levels, prolonged survival times, decreased serum anti-nuclear antibody levels, and reduced spleen and thymus indices. Prednisone treatment also significantly decreased the elevated percentages of plasma cells and plasma cell precursors, decreased the percentages of activated T cells, and increased the frequency of CD4(+)CD62L(+) cells, demonstrated that decreased anti-nuclear antibodies and improvements in lupus symptoms were associated with decreased plasma cells. Furthermore, prednisone treatment decreased serum IL-21 and IL-10 levels and reduced the expression of splenic Blimp-1 and Bcl-6 (two key regulatory factors for plasma cell differentiation) in MRL/lpr mice. CONCLUSION: Prednisone treatment restricts B lymphocyte differentiation into plasma cells in MRL/lpr mice, which may be correlated with the inhibition of IL-21 production and the restoration of the balance between Blimp-1 and Bcl-6.

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.

Paeoniflorin inhibits inflammatory responses in mice with allergic contact dermatitis by regulating the balance between inflammatory and anti-inflammatory cytokines.

OBJECTIVE: Paeoniflorin (Pae) was previously reported to inhibit inflammation in the skin of mice with allergic contact dermatitis (ACD); however, the mechanism remains unclear. The primary purpose of this study was to investigate the effect of Pae on the regulation of cytokine production in a murine model of ACD. METHODS: ACD was induced in the mice by repeated application of dinitrochlorobenzene (DNCB) to their skin. Cutaneous inflammation was evaluated by measuring ear swelling and by histological examination. The cytokine levels were measured by enzyme-linked immunosorbent assays. RESULTS: The results showed that topical application of DNCB caused obvious swelling and inflammatory cell infiltration. Treatment with Pae (70 or 140 mg/kg/d) significantly inhibited the cutaneous inflammation and decreased thymocyte proliferation in the mice with ACD. Additional data indicated that Pae increased interleukin-4 (IL-4) and IL-10 production but reduced IL-2 and IL-17 levels in the serum as well as in thymocyte and splenocyte culture supernatants. As expected, IL-2 and IL-17 levels in the serum displayed a significant positive correlation with the severity of skin inflammation. In contrast, IL-4 and IL-10 levels were negatively correlated with the inflammation. CONCLUSIONS: The anti-inflammatory action of Pae in the murine model of ACD may be related to its regulation of an imbalanced cytokine production.

BF02, a recombinant TNFR2 fusion protein, alleviates adjuvant arthritis by regulating T lymphocytes in rats.

AIM: To investigate the therapeutic effects of BF02 on adjuvant arthritis (AA) in rats and the regulatory effects of BF02 on T lymphocyte function. METHODS: SD rats received a single intradermal injection of Freund's complete adjuvant emulsion into the right hind metatarsal footpad. After the onset of AA, the rats were injected BF02 (1, 3, or 9 mg/kg, sc) every 3 d for a total of 15 d. Intragastric administration of methotrexate (MTX, 0.5 mg/kg, every 3 d for a total of 15 d) was taken as the positive control drug. Arthritis index, swollen joint count, ankle joint histopathology, spleen histopathology and the paw radiography were used for evaluating the drug effects on AA rats. T lymphocyte function was assessed by measuring T lymphocyte cytokine levels, IL17 and TNF-α mRNA expression levels, and percentage of T lymphocyte subsets. RESULTS: In the AA rats, remarkable secondary inflammatory responses exhibited, accompanied by significantly higher levels of IL-1, IL-6, TNF-α, IL-17, LTα, RANKL, and MMP-13. The expression of IL17 and TNF-α mRNAs was also substantially higher than in normal rats. The percentages of CD3(+)CD4(+) and CD4(+)CD25(+) T lymphocytes were increased, whereas the percentages of CD4(+)CD62L(+) and CD4(+)CD25(+)FoxP3(+) T lymphocytes were decreased. Treatment of the AA rats with BF02 (9 mg/kg) or MTX significantly decreased the arthritis index, swollen joint count and arthritis global assessment. Moreover, both BF02 (9 mg/kg) and MTX significantly inhibited T lymphocyte proliferation, and blocked the above mentioned aberrance in T lymphocyte cytokine levels, IL17 and TNF-α mRNA expression, and percentages of T lymphocyte subsets. CONCLUSION: BF02 exerts therapeutic effects on AA rats via the regulation of T lymphocytes.

Depletion of ß-arrestin2 in hepatic stellate cells reduces cell proliferation via ERK pathway.

ß-Arrestins are multifunctional adaptor proteins. Recently, some new roles of ß-arrestins in regulating intracellular signaling networks have been discovered, which regulate cell growth, proliferation, and apoptosis. Though, the role of ß-arrestins expression in the pathology of hepatic fibrosis remains unclear. In this study, the possible relationship between the expression of ß-arrestins with the experimental hepatic fibrosis and the proliferation of hepatic stellate cells (HSCs) were investigated. Porcine serum induced liver fibrosis was established in this study. At five time points, the dynamic expression of ß-arrestin1, ß-arrestin2, and α-smooth muscle actin (α-SMA) in rat liver tissues, was measured by immunohistochemical staining, double immunofluorescent staining, and Western blotting. This study showed that aggravation of hepatic fibrosis with gradually increasing expression of ß-arrestin2 in the hepatic tissues, but not ß-arrestin1. Further, as hepatic fibrosis worsens, ß-arrestin2-expressing activated HSCs accounts for an increasingly larger percentage of all activated HSCs. And the expression of ß-arrestin2 had a significant positive correlation with the expression of α-SMA, an activated HSCs marker. In vitro studies, the dynamic expression of ß-arrestin1 and ß-arrestin2 in platelet derived growth factor-BB (PDGF-BB) stimulated HSCs was assessed by Western blotting. The expression of ß-arrestin2 was remarkably increased in PDGF-BB stimulated HSCs. Furthermore, the small interfering RNA (siRNA) technique was used to explore the effect of ß-arrestins on the proliferation of HSCs and the activation of ERK1/2. Transfection of siRNA targeting ß-arrestin2 mRNA (siß-arrestin2) into HSCs led to a 68% and 70% reduction of ß-arrestin2 mRNA and protein expression, respectively. siß-arrestin2 abolished the effect of PDGF-BB on the proliferation of HSCs. In addition, siß-arrestin2 exerted the inhibition of the activation of ERK1/2 in HSCs. The present study provided strong evidence for the participation of the ß-arrestin2 in the pathogenesis of hepatic fibrosis. The ß-arrestin2 depletion diminishes HSCs ERK1/2 signaling and proliferation stimulated by PDGF-BB. Selective targeting of ß-arrestin2 inhibitors to HSCs might present as a novel strategy for the treatment of hepatic fibrosis.

Etanercept attenuates collagen-induced arthritis by modulating the association between BAFFR expression and the production of splenic memory B cells.

Anti-tumour necrosis factor-α (TNF-α) drugs are approved for the treatment of rheumatoid arthritis (RA). Many studies have investigated the effect of these drugs on the T cell response; however, some clues have indicated that it may also target B cells. This study was carried out to explore the potential effects and mechanisms of etanercept, a soluble TNF-α receptor, on the function of B cells and their development into memory B cells in type II collagen (CII)-induced arthritis (CIA). Beginning on day 24 after CII immunisation, the mice were evaluated every 2-3 days to determine two clinical parameters: their arthritis global assessment and swollen joint count (SJC). The serum concentrations of IgG1, IgG2a and anti-CII antibodies and the splenic pathology and proliferation of B cells were measured. The percentage of total memory B cells in the spleen was analysed with flow cytometry. BAFFR was detected by immunohistochemistry. In CIA mice, etanercept markedly suppressed the arthritis global assessment and the SJC, reduced the production of anti-CII, IgG1 and IgG2a antibodies, and prevented spleen histopathology to varying degrees; however, it had no obvious effect on splenic B cell proliferation. Etanercept also decreased the percentage of total CD27(+) memory B cells in the spleen. Treatment with etanercept was associated with a further increase in BAFFR expression, a significant reduction in CD27 expression, and a negative correlation between the levels of BAFFR and the percentage of memory B cells. Our findings showed that increased BAFFR expression has a regulatory effect on the activation of B cells and the generation of memory B cells, which may be one of the mechanisms of the therapeutic effects of etanercept.

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.

Paeoniflorin inhibits proliferation of fibroblast-like synoviocytes through suppressing G-protein-coupled receptor kinase 2.

Paeoniflorin (Pae) is a monoterpene glucoside and the main component of the total glucosides of paeony (TGP) extracted from the roots of Paeonia lactiflora. Its anti-inflammatory effect is associated with regulating G-protein-coupled receptors (GPCRs) signaling. The aim of this study was to explore the expression change of G-protein-coupled receptor kinase 2 (GRK2) in fibroblast-like synoviocytes (FLS) and the effect of Pae. Pae was obtained and purified from the roots of Paeonia lactiflora. We investigated the expression of GRK2 in synovium during the inflammatory process and assessed the effects of a specific GRK2 inhibitor and Pae on proliferation, cAMP level, and protein kinase A (PKA) activity of FLS in vitro. Additionally, the effect of Pae on GRK2 expression in FLS was detected in vitro. Expression of GRK2 in synovium from CIA rats increased during the inflammatory process. The specific GRK2 inhibitor suppressed proliferation and increased the cAMP level as well as PKA activity of FLS, and Pae had the same effects. Furthermore, Pae decreased GRK2 expression in FLS in vitro. Our results indicate that a chronic inflammatory process in CIA induces upregulation of GRK2 expression in FLS, and Pae can reverse this change, which might be one of the important mechanisms for Pae regulating GPCRs signaling and suppressing the proliferation of FLS in CIA.