Wuyi Sun

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.

Involvement of β-arrestins in cancer progression

Abstractβ-arrestins, including β-arrestin1 and β-arrestin2, are ubiquitous cytosolic proteins which localize in the cytoplasm and plasma membrane, initially be regarded as an potential character in G protein-coupled receptors (GPCR) desensitization, sequestration, and internalization. Besides, recent many studies increasingly revealed that β-arrestins served widely as versatile adapter proteins for scaffolding many intracellular signaling networks to modulate the strength and duration of signaling by diverse types of receptors and downstream kinases. As we known, the biologic and clinical behaviors of many tumors are largely determined by multiple molecular signal pathways. More recently, accumulating evidences established that β-arrestins got widely involved in many cancer developmental signaling events which responsible for tumor viability and metastasis, suggesting an impressive role of β-arrestins in tumor progression. Because of the regulation and biological output of β-arrestins is so complex, the role of β-arrestins in cancer development still remains enigmatic. However, the further understanding with the clinical prognosis and oncogenic potential of β-arrestins might facilitate the identification of diagnosis biomarkers and development of drug targets in cancer. In this article, we reviewed a comprehensive summary of the β-arrestins-mediated functions in human cancers.

TGF-β signaling pathway as a pharmacological target in liver diseases

Transforming growth factor β (TGF-β) belongs to a class of pleiotropic cytokines that are involved in the processes of embryonic development, wound healing, cell proliferation, and differentiation. Moreover, TGF-β is also regarded as a central regulator in the pathogenesis and development of various liver diseases because it contributes to almost all of the stages of disease progression. A range of liver cells are considered to secrete TGF-β ligands and express related receptors and, consequently, play a crucial role in the progression of liver disease via different signal pathways. In this manuscript, we review the role of the TGF-β signaling pathway in liver disease and the potential of targeting the TGF-β signaling in the pharmacological treatment of liver diseases.

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.

Angiotensin II type 2 receptor correlates with therapeutic effects of losartan in rats with adjuvant‐induced arthritis

The angiotensin II type 1 receptor (AT1R) blocker losartan ameliorates rheumatoid arthritis (RA) in an experimental model. In RA, AT2R mainly opposes AT1R, but the mechanism by which this occurs still remains obscure. In the present study, we investigated the role of AT2R in the treatment of rats with adjuvant‐induced arthritis (AIA) by losartan. Adjuvant‐induced arthritis rats were treated with losartan (5, 10 and 15 mg/kg) and methotrexate (MTX; 0.5 mg/kg) in vivo from day 14 to day 28. Arthritis was evaluated by the arthritis index and histological examination. Angiotensin II, tumour necrosis factor‐α, and VEGF levels were examined by ELISA. The expression of AT1R and AT2R was detected by western blot and immunohistochemistry analysis. After stimulation with interleukin‐1β in vitro, the effects of the AT2R agonist CGP42112 (10−8–10−5 M) on the chemotaxis of monocytes induced by 10% foetal calf serum (FCS) were analysed by using Transwell assay. Subsequently, the therapeutic effects of CGP42112 (5, 10 and 20 μg/kg) were evaluated in vivo by intra‐articular injection in AIA rats. After treatment with losartan, the down‐regulation of AT1R expression and up‐regulation of AT2R expression in the spleen and synovium of AIA rats correlated positively with reduction in the polyarthritis index. Treatment with CGP42112 inhibited the chemotaxis of AIA monocytes in vitro, possibly because of the up‐regulation of AT2R expression. Intra‐articular injection with CGP42112 (10 and 20 μg/kg) ameliorated the arthritis index and histological signs of arthritis. In summary, the present study strongly suggests that the up‐regulation of AT2R might be an additional mechanism by which losartan exerts its therapeutic effects in AIA rats.

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.

A standardized extract from Paeonia lactiflora and Astragalus membranaceus induces apoptosis and inhibits the proliferation, migration and invasion of human hepatoma cell lines

Paeonia lactiflora and Astragalus membranaceus are two traditional Chinese medicines, which are commonly used in Chinese herb prescription to treat liver diseases. The protective effects of the extract prepared from the roots of Paeonia lactiflora and Astragalus membranaceus (PAE) on liver fibrosis have been demonstrated in previous studies. However, its effect on hepatocellular carcinoma (HCC) has not been investigated to date. In this study, the effects of PAE on the apoptosis, proliferation, migration and invasion of the human hepatoma cell lines HepG2 and SMMC-7721 were investigated. Our data demonstrated that treatment with PAE (50-200 mg/l) caused an inhibitory effect on the proliferation of the hepatoma cell lines HepG2 and SMMC-7721. Furthermore, PAE induced apoptosis of HepG2 cells and SMMC-7721 cells, which was demonstrated by PI staining. In addition, immunocytochemistry and western blotting showed that PAE significantly decreased the expression of Bcl-2, while the expression of Bax and cleaved caspase-3 in HepG2 cells and SMMC-7721 cells was significantly increased after treatment with PAE. These results clearly demonstrated that PAE induced hepatoma cell apoptosis through increasing the Bax-to-Bcl-2 ratio and upregulating the activation of caspase-3. In addition, the results of wound healing assay and Matrigel invasion assay showed that PAE displayed inhibitory activity on the migration and invasion of HCC cells. Taken together, the present data provides evidence that PAE is a potent antineoplastic drug candidate for the treatment of HCC.

GRK2 overexpression inhibits IGF1-induced proliferation and migration of human hepatocellular carcinoma cells by downregulating EGR1

G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase that is involved in a variety of important signaling pathways and alternation of GRK2 protein level or activity causes diseases such as heart failure, rheumatoid arthritis, and obesity. However, the role and mechanism of GRK2 in hepatocellular carcinoma (HCC) progression is not fully investigated. In this study we found that GRK2 plays an inhibitory role in IGF1-induced HCC cell proliferation and migration. Overexpression of GRK2 causes a decrease in early growth response-1 (EGR1) expression, while knockdown of GRK2 leads to marked increase in EGR1 expression in the treatment of IGF1. Through co-immunoprecipitation and western blot assay, we confirmed that GRK2 can interact with insulin-like growth factor 1 receptor (IGF-1R) and inhibits IGF1-induced activation of IGF1R signaling pathway. Silencing EGR1 attenuates GRK2 overexpression-caused inhibition of cell proliferation, tumor colony number and migration activity, while overexpressing of EGR1 restores the anti-proliferative and migratory effect by GRK2 overexpression in HCCLM3 cells. Collectively, these results suggest that GRK2 may inhibit IGF1-induced HCC cell growth and migration through downregulation of EGR1 and indicate that enforced GRK2 may offer a potential therapeutic approach against HCC.

Depletion of β‐arrestin2 in hepatic stellate cells reduces cell proliferation via ERK pathway

β‐Arrestins are multifunctional adaptor proteins. Recently, some new roles of β‐arrestins in regulating intracellular signaling networks have been discovered, which regulate cell growth, proliferation, and apoptosis. Though, the role of β‐arrestins expression in the pathology of hepatic fibrosis remains unclear. In this study, the possible relationship between the expression of β‐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 β‐arrestin1, β‐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 β‐arrestin2 in the hepatic tissues, but not β‐arrestin1. Further, as hepatic fibrosis worsens, β‐arrestin2‐expressing activated HSCs accounts for an increasingly larger percentage of all activated HSCs. And the expression of β‐arrestin2 had a significant positive correlation with the expression of α‐SMA, an activated HSCs marker. In vitro studies, the dynamic expression of β‐arrestin1 and β‐arrestin2 in platelet derived growth factor‐BB (PDGF‐BB) stimulated HSCs was assessed by Western blotting. The expression of β‐arrestin2 was remarkably increased in PDGF‐BB stimulated HSCs. Furthermore, the small interfering RNA (siRNA) technique was used to explore the effect of β‐arrestins on the proliferation of HSCs and the activation of ERK1/2. Transfection of siRNA targeting β‐arrestin2 mRNA (siβ‐arrestin2) into HSCs led to a 68% and 70% reduction of β‐arrestin2 mRNA and protein expression, respectively. siβ‐arrestin2 abolished the effect of PDGF‐BB on the proliferation of HSCs. In addition, siβ‐arrestin2 exerted the inhibition of the activation of ERK1/2 in HSCs. The present study provided strong evidence for the participation of the β‐arrestin2 in the pathogenesis of hepatic fibrosis. The β‐arrestin2 depletion diminishes HSCs ERK1/2 signaling and proliferation stimulated by PDGF‐BB. Selective targeting of β‐arrestin2 inhibitors to HSCs might present as a novel strategy for the treatment of hepatic fibrosis. J. Cell. Biochem. 114: 1153–1162, 2013.

Paroxetine alleviates T lymphocyte activation and infiltration to joints of collagen-induced arthritis

T cell infiltration to synovial tissue is an early pathogenic mechanism of rheumatoid arthritis (RA). In the present work, we reveal that G protein coupled receptor kinase 2 (GRK2) is abundantly expressed in T cells of collagen-induced arthritis (CIA). A GRK2 inhibitor, paroxetine protects the joints from inflammation and destruction, primarily through inhibition of both CD4+ helper T (Th) cell and CD8+ cytotoxic T (Tc) cell migration to synovial tissue. Meanwhile, paroxetine restores the balance of Th/Tc, effector Th (Theff)/ naïve Th (Thnaive) and effector Tc (Tceff)/ naïve Tc (Tcnaive) to equilibrium by elevating the frequency of Thnaive, Tcnaive and regulatory Th cells; reducing the increased Theff, activated Th and Tceff, having a similar effect as methotrexate (MTX). In addition, both serum and synovial IL-1β, TNF-α and CX3CL1 expression was effectively inhibited in treated rats. In vitro assay confirmed that paroxetine inhibits CX3CL1-induced T cell migration through blocking the activity of GRK2. Among three MAPK families, paroxetine was found to be able to decrease the phosphorylation of ERK. This study elucidates that paroxetine attenuates the symptoms of CIA rats due to its inhibitory effect on T cell activation and infiltration to synovial tissue via suppression of ERK pathway.