Angiotensin II type-2 receptor signaling facilitates liver injury repair and regeneration via inactivation of Hippo pathway.

The angiotensin II type 2 receptor (AT2R) is a well-established component of the renin-angiotensin system and is known to counteract classical activation of this system and protect against organ damage. Pharmacological activation of the AT2R has significant therapeutic benefits, including vasodilation, natriuresis, anti-inflammatory activity, and improved insulin sensitivity. However, the precise biological functions of the AT2R in maintaining homeostasis in liver tissue remain largely unexplored. In this study, we found that the AT2R facilitates liver repair and regeneration following acute injury by deactivating Hippo signaling and that interleukin-6 transcriptionally upregulates expression of the AT2R in hepatocytes through STAT3 acting as a transcription activator binding to promoter regions of the AT2R. Subsequently, elevated AT2R levels activate downstream signaling via heterotrimeric G protein Gα12/13-coupled signals to induce Yap activity, thereby contributing to repair and regeneration processes in the liver. Conversely, a deficiency in the AT2R attenuates regeneration of the liver while increasing susceptibility to acetaminophen-induced liver injury. Administration of an AT2R agonist significantly enhances the repair and regeneration capacity of injured liver tissue. Our findings suggest that the AT2R acts as an upstream regulator in the Hippo pathway and is a potential target in the treatment of liver damage.

Dextran sulfate sodium-induced gut microbiota dysbiosis aggravates liver injury in mice with S100-induced autoimmune hepatitis.

Recently, the incidence of autoimmune hepatitis (AIH) has gradually increased, and the disease can eventually develop into cirrhosis or even hepatoma if left untreated. AIH patients are often characterized by gut microbiota dysbiosis, but whether gut microbiota dysbiosis contributes to the progression of AIH remains unclear. In this study, we investigate the role of gut microbiota dysbiosis in the occurrence and development of AIH in mice with dextran sulfate sodium salt (DSS) induced colitis. C57BL/6J mice were randomly divided into normal group, S100-induced AIH group, and DSS+S100 group (1 % DSS in the drinking water), and the experimental cycle lasted for four weeks. We demonstrate that DSS administration aggravates hepatic inflammation and disruption of the intestinal barrier, and significantly changes the composition of gut microbiota in S100-induced AIH mice, which are mainly characterized by increased abundance of pathogenic bacteria and decreased abundance of beneficial bacteria. These results suggest that DSS administration aggravates liver injury of S100-induced AIH, which may be due to DSS induced gut microbiota dysbiosis, leading to disruption of the intestinal barrier, and then, the microbiota translocate to the liver, aggravating hepatic inflammation.

ß-arrestin2 deficiency ameliorates S-100-induced autoimmune hepatitis in mice by inhibiting infiltration of monocyte-derived macrophage and attenuating hepatocyte apoptosis.

Autoimmune hepatitis (AIH) is a progressive hepatitis syndrome characterized by high transaminase levels, interface hepatitis, hypergammaglobulinemia, and the presence of autoantibodies. Misdiagnosis or delayed treatment of AIH can lead to cirrhosis or liver failure, which poses a major risk to human health. ß-Arrestin2, a key scaffold protein for intracellular signaling pathways, has been found to be involved in many autoimmune diseases such as Sjogren's syndrome and rheumatoid arthritis. However, whether ß-arrestin2 plays a role in AIH remains unknown. In the present study, S-100-induced AIH was established in both wild-type mice and ß-arrestin2 knockout (Arrb2 KO) mice, and the experiments identified that liver ß-arrestin2 expression was gradually increased, and positively correlated to serum ANA, ALT and AST levels during AIH progression. Furthermore, ß-arrestin2 deficiency ameliorated hepatic pathological damage, decreased serum autoantibody and inflammatory cytokine levels. ß-arrestin2 deficiency also inhibited hepatocyte apoptosis and prevented the infiltration of monocyte-derived macrophages into the damaged liver. In vitro experiments revealed that ß-arrestin2 knockdown suppressed the migration and differentiation of THP-1 cells, whereas ß-arrestin2 overexpression promoted the migration of THP-1 cells, which was regulated by the activation of the ERK and p38 MAPK pathways. In addition, ß-arrestin2 deficiency attenuated TNF-α-induced primary hepatocyte apoptosis by activating the Akt/GSK-3ß pathway. These results suggest that ß-arrestin2 deficiency ameliorates AIH by inhibiting the migration and differentiation of monocytes, decreasing the infiltration of monocyte-derived macrophages into the liver, thereby reducing inflammatory cytokines-induced hepatocytes apoptosis. Therefore, ß-arrestin2 may act as an effective therapeutic target for AIH.

Effect of PLC-ß1/CaM signaling pathway mediated by AT1R on the occurrence and development of hepatocellular carcinoma.

OBJECTIVE: To study the roles of AT1R, PLC-ß1, CaM and other related signal molecules in the formation and development of hepatocellular carcinoma (HCC) and their correlation. METHODS: ELISA and immunohistochemistry were used to analyze the expressions of target proteins in serum and liver tissue of HCC patients, and the correlation between AT1R, PLC-ß1 and CaM and postoperative survival status of patients was followed up and determined. CCK-8 method was used to screen the doses of Ang II and candesartan sensitive to HepG2 and HCCLM3 cells. Transwell experiment was used to observe the effects of different drugs on the migration and invasion activity of HCC cells. Meanwhile, flow cytometry and Western blot were used to detect the expression levels of AT1R, PLC-ß1 and CaM in the cells. Then PLC-ß1 siRNA was selected to transfect HCC cells, so as to further clarify the mechanism of the above signal proteins. HepG2 cells were inoculated under the hepatic capsule of mice to induce the formation of HCC in situ. Ang II and candesartan were used to stimulate HCC mice to observe the difference in liver appearance and measure the liver index. Finally, ELISA and immunofluorescence experiments were selected to analyze the levels of target proteins in mouse serum and liver tissue. RESULTS: The expression levels of target proteins in serum and liver tissue of HCC patients were significantly increased, and the postoperative survival time of patients with high expression of AT1R, PLC-ß1 or CaM was obviously shortened. Ang II and candesartan could significantly promote and inhibit the motility of HCC cells, and had different effects on the levels of AT1R, PLC-ß1 and CaM in cells. However, in hepatocellular carcinoma cells transfected with PLC-ß1 siRNA, the intervention ability of drugs was obviously weakened. Ang II could significantly promote the formation and progression of mouse HCC, while candesartan had the opposite effect. Meanwhile, medications could affect the expressions of target proteins in mouse serum and liver tissue. CONCLUSION: AT1R, PLC-ß1 and CaM may be risk factors affecting the formation and prognosis of HCC, and the PLC-ß1/CaM signaling pathway mediated by AT1R is an important way to regulate the migration and invasion activity of HCC cells.

Inflammasome as an Effective Platform for Fibrosis Therapy.

Fibrosis is the final stage of the development of chronic inflammation. It is characterized by excessive deposition of the extracellular matrix, leading to tissue structure damage and organ dysfunction, which is a serious threat to human health and life. However, the molecular mechanism of fibrosis is still unclear. Inflammasome is a molecular complex of proteins that has been becoming a key innate sensor for host immunity and is involved in pyroptosis, pathogen infection, metabolic syndrome, cellular stress, and tumor metastasis. Inflammasome signaling and downstream cytokine responses mediated by the inflammasome have been found to play an important role in fibrosis. The inflammasome regulates the secretion of IL-1ß and IL-18, which are both critical for the process of fibrosis. Recently, researches on the function of inflammasome have attracted extensive attention, and data derived from these researches have increased our understanding of the effects and regulation of inflammasome during fibrosis. In this review, we emphasize the growing evidence for both indirect and direct effects of inflammasomes in triggering fibrosis as well as potential novel targets for antifibrotic therapies.

ß-arrestin2 regulating ß2-adrenergic receptor signaling in hepatic stellate cells contributes to hepatocellular carcinoma progression.

Background: ß-arrestin2 and ß2-adrenergic receptor (ß2-AR) have important roles in malignant tumors, the present study aims to investigate the role of activated ß2-AR in hepatic stellate cells (HSCs) during hepatocellular carcinoma (HCC) progression and the regulatory effect of ß-arrestin2. Methods: Immunofluorescence and Western blot were used to detect the expression of ß-arrestin2 and ß2-AR in HSCs of liver tissues from human HCC samples and diethylnitrosamine (DEN)-induced HCC model mice. We next used ß-arrestin2-/- mice to demonstrate the regulatory role of ß-arrestin2 in DEN mice. The subsets of T cells were quantified by flow cytometry. MTT and wound healing assay were applied to detect the proliferation and migration of cells. Co-immunoprecipitation assay was used to detect the link of ß-arrestin2 and ß2-AR in HSCs. Effect of ß-arrestin2 overexpression on ß2-AR downstream signaling pathway was verified by Western blot. The secretion of CCL2 was detected by ELISA. Results: The expression of ß2-AR was significantly increased, while ß-arrestin2 was decreased in HSCs of HCC tissues. And ß-arrestin2 deficiency exacerbates DEN-induced HCC accompanied with increased ß2-AR expression. The results of flow cytometry showed that the percentage of activated T cells decreased gradually after DEN injection. ß-arrestin2 knockout down-regulated the ratio of activated T cells. In vitro, selective activation of ß2-AR in HSCs promoted the proliferation and migration of HCC cells. ß-arrestin2 overexpression enhanced co-immunoprecipitation of ß-arrestin2 and ß2-AR in activated HSCs, and decreased its downstream Akt phosphorylation. Akt inhibitor decreased secretion of CCL2 in activated HSCs. Conclusion: Our study demonstrated that ß2-AR activation in HSCs induces the proliferation and migration of HCC cells may be through Akt signaling, and this effect appears to be regulated by ß-arrestin2.

GRK2 Suppresses Hepatocellular Carcinoma Metastasis and Invasion Through Down-Regulation of Prostaglandin E Receptor 2.

BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive form of human liver cancer and the fifth most common malignancy worldwide. Novel effective treatment strategies for HCC are urgently in clinical because of its poor response to conventional therapies. G protein-coupled receptor kinases (GRKs), including GRK2 and GRK3, are known that involves in various essential cellular processes and regulates numerous signaling pathways. However, the role of GRK2/3 in invasion and metastasis of HCC still remains unclear. MATERIALS AND METHODS: Immunohistochemistry, Western blot, laser confocal microscopy and qRT-PCR were used to detect the expression of GRK2/3 and EP2 in liver tissues of HCC patients and DEN-induced HCC mice. Wound healing and transwell assay were applied to measure the migration and invasion of HCC cells after transfected with GRK2 siRNA. The downstream pathway of Akt and ERK was verified by Western blot. RESULTS: The expression of GRK2 was significantly decreased, while GRK3 was not significantly changed in HCC tissues compared with noncancerous tissues of HCC patients. Moreover, GRK2 expression was reduced during liver tumorigenesis in diethylnitrosamine-induced liver tumor model. In addition, our in vitro study showed that GRK2 expression was gradually decreased with increasing HCC cell line metastatic potential, and GRK2 knockdown significantly promoted the migration and invasion of HCC cells. Furthermore, low GRK2 expression was associated with increased expression of EP2 receptor translocation to HCC cell membrane, and the activation of Akt pathway. CONCLUSION: These data suggest that GRK2 inhibits HCC metastasis and invasion may be through regulating EP2 receptor translocation, and this effect appears to be mediated by Akt pathway.

Depletion of ß-arrestin 2 protects against CCl4-induced liver injury in mice.

Acute liver injury can be caused by oxidative stress within a short period and is a common pathway to many liver diseases. The liver is vulnerable to reactive oxygen species (ROS) and free radical-mediated disorders. ß-arrestin2 was initially discovered to be a negative regulator of G protein-coupled receptor signaling. Recently, ß-arrestin2 has been found to act as a multifunctional adaptor protein and play new roles in regulating intracellular signaling networks. However, the role of ß-arrestin2 in the pathogenesis of acute liver injury is unclear. In this study, we hypothesize that ß-arrestin2 regulates acute liver injury via modulation of oxidative stress. ß-arrestin2 knockout mice were used to investigate the impacts of ß-arrestin2 on carbon tetrachloride (CCl4)-induced acute liver injury and oxidative stress. Results here suggested that ß-arrestin2 deficiency decreased serum activities of aminotransferase and alleviated liver injury induced by CCl4 injection as compared with wildtype mice. ß-arrestin2 knockout mice exhibited stronger tolerance in oxidative stress compared with wild-type mice, which was demonstrated by decreased ROS level and increased superoxide dismutase (SOD) and glutathione (GSH) in the liver. Furthermore, ß-arrestin2 deficiency significantly inhibited NOX4 (a major source of ROS) expression and the activation of the extracellular regulated kinase (ERK) and, c-Jun NH2-terminal kinase (JNK) pathways. These results suggest that ß-arrestin2 deficiency protects against CCl4-induced acute liver injury through attenuating oxidative damage and decreased ERK and JNK phosphorylation.

G protein-coupled receptor kinase 2 regulating ß2-adrenergic receptor signaling in M2-polarized macrophages contributes to hepatocellular carcinoma progression.

Background: ß2-adrenoceptors (ß2-ARs) are expressed on the surface of immune cells, including tumor-associated macrophages (TAMs). Previous studies have demonstrated that the expression of ß2-ARs in hepatocellular carcinoma (HCC) is significantly increased in vitro. However, the role of ß2-AR in M2-polarized macrophages remains unclear. G protein-coupled receptor kinase 2 (GRK2) can regulate G protein-coupled receptor (GPCR). Previous studies showed that down-regulation of GRK2 in HCC contributes the HCC progression, but it still remains unclear whether the regulation of ß2-AR in M2-polarized macrophages by GRK2 can promote HCC. Purpose: The present study was designed to investigate the role of activated ß2-AR in M2-polarized macrophages in the HCC progression and GRK2 regulatory effect, as well as the underlying mechanisms involved. Results: The results demonstrated that the M2-polarized macrophages were increased with HCC progression. In vitro, the activation of ß2-AR by terbutaline in M2-polarized macrophages elevated the proliferative, migratory and invasive attributes of HCC cells. Furthermore, GRK2 down-regulation in ß2-AR activated M2-polarized macrophages activated the downstream cyclic adenosine monophosphate (cAMP)/protein kinase A/cAMP-response element binding protein and cAMP/interleukin-6/signal transducer and the activator of transcription 3 signaling pathways, contributing to the secretion of tumor-associated cytokines, and thus resulting in the promotion of malignant biological behavior in HCC cells. Conclusion: These findings suggest that the regulation of ß2-AR occurs through the silencing of GRK2 in M2-polarized macrophages, which is conducive to HCC development, through its engagement in the activation of downstream signaling.

The role of G protein-coupled receptor kinases in the pathology of malignant tumors.

G protein-coupled receptor kinases (GRKs) constitute seven subtypes of serine/threonine protein kinases that specifically recognize and phosphorylate agonist-activated G protein-coupled receptors (GPCRs), thereby terminating the GPCRs-mediated signal transduction pathway. Recent research shows that GRKs also interact with non-GPCRs and participate in signal transduction in non-phosphorylated manner. Besides, GRKs activity can be regulated by multiple factors. Changes in GRKs expression have featured prominently in various tumor pathologies, and they are associated with angiogenesis, proliferation, migration, and invasion of malignant tumors. As a result, GRKs have been intensively studied as potential therapeutic targets. Herein, we review evolving understanding of the function of GRKs, the regulation of GRKs activity and the role of GRKs in human malignant tumor pathophysiology.

Emerging Roles of G Protein-Coupled Receptors in Hepatocellular Carcinoma.

Among a great variety of cell surface receptors, the largest superfamily is G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors. GPCRs can modulate diverse signal-transduction pathways through G protein-dependent or independent pathways which involve β-arrestins, G protein receptor kinases (GRKs), ion channels, or Src kinases under physiological and pathological conditions. Recent studies have revealed the crucial role of GPCRs in the tumorigenesis and the development of cancer metastasis. We will sum up the functions of GPCRs—particularly those coupled to chemokines, prostaglandin, lysophosphatidic acid, endothelin, catecholamine, and angiotensin—in the proliferation, invasion, metastasis, and angiogenesis of hepatoma cells and the development of hepatocellular carcinoma (HCC) in this review. We also highlight the potential avenues of GPCR-based therapeutics for HCC.

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.

Targeted blockade of JAK/STAT3 signaling inhibits proliferation, migration and collagen production as well as inducing the apoptosis of hepatic stellate cells.

Protein tyrosine kinases belonging to the Janus kinase (JAK) family are associated with many cytokine receptors, which, on ligand binding, regulate important cellular functions such as proliferation, apoptosis and differentiation. The protective effects of JAK inhibitors on fibrotic diseases such as myelofibrosis and bone marrow fibrosis have been demonstrated in previous studies. The JAK inhibitor SHR0302 is a synthetic molecule that potently inhibits all members of the JAK family, particularly JAK1. However, its effect on hepatic fibrosis has not been investigated to date, to the best of our knowledge. In the present study, the effects of SHR0302 on the activation, proliferation, migration and apoptosis of hepatic stellate cells (HSCs) as well as HSC collagen production were investigated. Our data demonstrated that treatment with SHR0302 (10-9-10-5 mol/l) exerted an inhibitory effect on the activation, proliferation and migration of HSCs. In addition, the expression of collagen I and collagen III were significantly decreased following treatment with SHR0302. Furthermore, SHR0302 induced the apoptosis of HSCs, which was demonstrated by Annexin V/PI staining. SHR0302 significantly increased the activation of caspase-3 and Bax in HSCs whereas it decreased the expression of Bcl-2. SHR0302 also inhibited the activation of Akt signaling pathway. The pharmacological inhibition of the JAK1/signal transducer and activator of transcription (STAT)3 pathway led to the disruption of functions essential for HSC growth. Taken together, these findings provide evidence that SHR0302 may have the potential to alleviate hepatic fibrosis by targeting HSC functions.

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.

Decreased expression of the type III TGF-ß receptor enhances metastasis and invasion in hepatocellullar carcinoma progression.

The transforming growth factor ß (TGF-ß) superfamily of cytokines is multifunctional and involved in the regulation of cell growth and differentiation. TGF-ß can induce an epithelial-mesenchymal transition (EMT) of both epithelial and endothelial cells. This has consequences for cancer progression in regards to both migration and invasion abilities. The type III TGF-ß receptor (TßRIII) is a ubiquitously expressed TGF-ß co-receptor which regulates TGF-ß signaling and the progression of various types of cancer. Previous studies have shown that TßRIII exhibits abnormal expression and plays an essential role in regulating cancer invasion and metastasis, while little is known in regards to its role in hepatocellular carcinoma (HCC) progression. In the present study, we designed the present research to study the role of TßRIII in the invasion and metastasis of HCC and the possible mechanisms involved. The results demonstrated decreased expression of TßRIII in HCC patient tissues and human HCC cell lines. TGF-ß1 stimulation led to the increased migratory ability and reduced expression of TßRIII in HCC cells. In addition, knockdown of TßRIII by small interfering RNA (siRNA) promoted the migration and invasion of HCC cells and induced activation of the Smad2 and Akt pathways. All the results suggest that TßRIII is a novel suppressor of HCC progression.

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.

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.

Expression and function of ß-arrestin 2 stimulated by IL-1ß in human fibroblast-like synoviocytes and the effect of paeoniflorin.

The aim of this study was to explore the expression and function of ß-arrestin 2 in human fibroblast-like synoviocytes (FLS) stimulated by IL-1ß and the effect of paeoniflorin (Pae). We isolated and cultured human FLS, which were stimulated by IL-1ß. The FLS proliferations were detected by [3H] thymidine incorporation. The level of cAMP stimulated by IL-1ß on different times was investigated by radioimmunoassay, and the activity of PKA was measured by luminescent kinase assay. The expression of ß-arrestin 2 was measured by western blot. We found that the human FLS proliferation increased apparently in 24 h, and the activities of PKA and cAMP accumulation increased significantly in 6 h after stimulated by IL-1ß, while cAMP accumulation and the activities of PKA decreased especially in 24 h when the expression of ß-arrestin 2 increased significantly. Decreased cAMP accumulation and the increased expression of ß-arrestin 2 may reveal a positive correlation with the FLS proliferation. Pae (10(-5), 10(-6), 10-7 mol•L(-1)) in vitro could suppress the FLS proliferation and the high expression of ß-arrestin 2. The expression of ß-arrestin 2 may have a positive correlation with the human FLS proliferation, while the activities of PKA and cAMP accumulation have a negative correlation with the proliferation. The increased ß-arrestin 2 may down-regulate cAMP-PKA signaling pathway and promote FLS proliferation. Pae may suppress the expression of ß-arrestin 2 and up-regulate cAMP-PKA signaling, which may be one of the mechanisms for the effects of Pae on inhibiting human FLS proliferation.

A standardized extract from Paeonia lactiflora and Astragalus membranaceus attenuates liver fibrosis induced by porcine serum in rats.

Paeonia lactiflora and Astragalus membranaceus are two popular traditional Chinese medicines, commonly used in Chinese herb prescription to treat liver disease. The extract prepared from the roots of Paeonia lactiflora and Astragalus membranaceus (PAE) demonstrated better hepatoprotective activity than the herbs used individually as shown in our previous studies. This study was carried out to investigate the effects of PAE on liver fibrosis induced by porcine serum (PS) in rats and to explore its possible mechanisms. Liver fibrosis was induced in male Wistar rats by injection with PS intraperitoneally. The rats were randomly divided into a normal control group, a liver fibrosis model group and a PAE (40, 80, 160 mg•kg-1) treated group. After a 16-week treatment, PAE-treated rats showed significantly reduced liver damage and symptoms of liver fibrosis upon pathological examination. Administration of PAE significantly decreased serum HA, PC III levels, and content of hydroxyproline in the liver tissue of fibrotic rats. It also restored the decrease in SOD and GSH-Px activities and inhibited the formation of lipid peroxidative products during PS treatment. In vitro, PAE also significantly decreased [3H]-thymidine incorporation in hepatic stellate cells (HSCs) stimulated with platelet-derived growth factor-B subunit homodimer (PDGF-BB). Moreover, PAE significantly decreased the expression of PDGF receptor beta (PDGFR-ß) and p-ERK1/2, p-p38, p-JNK. The results showed that PAE displays antifibrotic effects in rats induced by PS, the mechanism by which might be associated with its ability to scavenge free radicals, decreasing the expression of PDGFR-ß, inhibition of HSC proliferation and MAPK activation. These findings indicate that PAE is a potential agent for the prevention of liver fibrosis.