Expression Of Β-arrestin2 Research Articles

Insight into the regulatory mechanism of β-arrestin2 and its emerging role in diseases.

β-arrestin2, a member of the arrestin family, mediates the desensitization and internalization of most G protein-coupled receptors (GPCRs) and functions as a scaffold protein in signalling pathways. Previous studies have demonstrated that β-arrestin2 expression is dysregulated in malignant tumours, fibrotic diseases, cardiovascular diseases and metabolic diseases, suggesting its pathological roles. Transcription and post-transcriptional modifications can affect the expression of β-arrestin2. Furthermore, post-translational modifications, such as phosphorylation, ubiquitination, SUMOylation and S-nitrosylation affect the cellular localization of β-arrestin2 and its interaction with downstream signalling molecules, which further regulate the activity of β-arrestin2. This review summarizes the structure and function of β-arrestin2 and reveals the mechanisms involved in the regulation of β-arrestin2 at multiple levels. Additionally, recent studies on the role of β-arrestin2 in some major diseases and its therapeutic prospects have been discussed to provide a reference for the development of drugs targeting β-arrestin2.

β-arrestin2 regulating β2-adrenergic receptor signaling in hepatic stellate cells contributes to hepatocellular carcinoma progression.

Background: β-arrestin2 and β2-adrenergic receptor (β2-AR) have important roles in malignant tumors, the present study aims to investigate the role of activated β2-AR in hepatic stellate cells (HSCs) during hepatocellular carcinoma (HCC) progression and the regulatory effect of β-arrestin2.Methods: Immunofluorescence and Western blot were used to detect the expression of β-arrestin2 and β2-AR in HSCs of liver tissues from human HCC samples and diethylnitrosamine (DEN)-induced HCC model mice. We next used β-arrestin2-/- mice to demonstrate the regulatory role of β-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 β-arrestin2 and β2-AR in HSCs. Effect of β-arrestin2 overexpression on β2-AR downstream signaling pathway was verified by Western blot. The secretion of CCL2 was detected by ELISA.Results: The expression of β2-AR was significantly increased, while β-arrestin2 was decreased in HSCs of HCC tissues. And β-arrestin2 deficiency exacerbates DEN-induced HCC accompanied with increased β2-AR expression. The results of flow cytometry showed that the percentage of activated T cells decreased gradually after DEN injection. β-arrestin2 knockout down-regulated the ratio of activated T cells. In vitro, selective activation of β2-AR in HSCs promoted the proliferation and migration of HCC cells. β-arrestin2 overexpression enhanced co-immunoprecipitation of β-arrestin2 and β2-AR in activated HSCs, and decreased its downstream Akt phosphorylation. Akt inhibitor decreased secretion of CCL2 in activated HSCs.Conclusion: Our study demonstrated that β2-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 β-arrestin2.

ARRB2 promotes colorectal cancer growth through triggering WTAP.

Colorectal cancer (CRC) is one of the most lethal cancers worldwide. The expression of β-arrestin2 (β-Arr2, ARRB2) in CRC has been well investigated; however, its exact mechanism causing the cancer progression remains unclear. In this study, we discovered that the expression level of ARRB2 was significantly upregulated in CRC as compared to the normal tissues by employing the Cancer Genome Atlas (TCGA) data, western blot analysis, and immunohistochemistry. Furthermore, the level of ARRB2 was correlated with the patients' overall survival by Kaplan-Meier analysis. The higher expression of ARRB2 promoted CRC cell growth, enhanced the cell motility, and blocked cell apoptosis, which is crucial for tumor growth. Lastly, the suppression of ARRB2 expression was enough to attenuate the progression of CRC induced by azoxymethane/dextran sodium sulfate. Interestingly, we also found that the knockdown of ARRB2 decreased several cancer pathways mediated by the expression of Wilms tumor 1 associated protein (WTAP), which led to the inhibition of cell proliferation and migration. Altogether, our results demonstrated that ARRB2 promoted the growth and migration of CRC cells by regulating the WTAP expression.

Ginsenoside metabolite compound-K regulates macrophage function through inhibition of β-arrestin2

Ginsenoside metabolite compound-K (C–K), which is an active metabolite of ginsenoside in vivo, can produce anti-inflammatory affects by activating glucocorticoid receptors (GRs) to inhibit the expression of β-arrestin2. Studies have shown that C–K can inhibit the function of immune cells including macrophage polarization and phagocytosis. However, the mechanism by which C–K regulates macrophage polarization is currently unclear. Toll-like receptors (TLRs) are the pattern recognition receptors on the membrane of immune cells, with TLR4 being especially important in polarization of macrophages. The Gαi-mediated activation of nuclear factor-κB (NF-κB) by TLR4 promotes inflammation and phagocytosis in macrophages by increasing the proportion of type I phenotypic macrophages (M1). Whether C–K inhibits the signal transduction of TLR4-Gαi-NF-κB and how that effects macrophage polarization regulation in murine models of RA is not reported. The coupling of G proteins with receptors is regulated by β-arrestin2, but it has been unclear whether C–K modulates the TLR4 interaction with G proteins by inhibiting the expression of β-arrestin2. To explore these questions, the collagen-induced arthritis (CIA) mouse model was employed, and mice were treated with C–K (112 mg/kg/day). The results depict that C–K treatment inhibits macrophage phagocytosis and reduces the proportion of M1. C–K decreases the overexpressed β-arrestin2, Gαi, TLR4 and NF-κB in macrophages of CIA mice, while increasing the expression of Gαs. Furthermore, C–K promotes TLR4-Gαs coupling and inhibits TLR4-Gαi coupling through β-arrestin2 regulation in macrophages, leading to a decrease in the proportion of M1 to M2 macrophages and improved outcomes in CIA mice.

β-arrestin2 regulates the anti-inflammatory effects of Salmeterol in lipopolysaccharide-stimulated BV2 cells

Microglial activation contributes to chronic inflammation and neuronal loss in progressive neurodegenerative disorders such as Parkinson's disease (PD). Thus, treatments suppressing microglial activation may have therapeutic benefits to prevent neuronal loss in neurodegenerative diseases. Our previous findings show that Salmeterol, a long-acting β2-adrenergic receptor (β2-AR) agonist, is neuroprotective in two distinct animal models of PD, including where lipopolysaccharide (LPS) from E. coli was used to initiate chronic neurodegeneration. Salmeterol was found to be a potent inhibitor of dopaminergic neurodegeneration by regulating the production of pro-inflammatory mediators from activated microglial cells. In the present study, we investigated the molecular basis of the anti-inflammatory effects of Salmeterol on LPS-activated murine microglial BV2 cells. BV2 cells were pretreated with Salmeterol and followed by stimulation with LPS. Salmeterol inhibited LPS-induced release of the pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nitric oxide from BV2 cells. Additionally, Salmeterol suppressed nuclear translocation of nuclear factor kappa-B (NF-κB) p65 by inhibiting the IκB-α degradation and TAK1 (transforming growth factor-beta-activated kinase1) phosphorylation. We have also found that Salmeterol increases the expression of β-arrestin2 and enhances the interaction between β-arrestin2 and TAB1 (TAK1-binding protein), reduced TAK1/TAB1 mediated activation of NFκB and expression of pro-inflammatory genes. Furthermore, silencing of β-arrestin2 abrogates the anti-inflammatory effects of Salmeterol in LPS-stimulated BV2 cells. Our findings suggest that the anti-inflammatory properties of Salmeterol is β-arrestin2 dependent and also offers novel therapeutics targeting inflammatory pathways to prevent microglial cell activation and neuronal loss in neuroinflammatory diseases like PD.

Cardiac chronotropic hypo-responsiveness and atrial fibrosis in rats chronically treated with lithium.

Lithium is a widely used mood-stabilizing agent; however, it causes a variety of cardiovascular side effects including sinus node dysfunction. In this study we explored the potential adverse effects of lithium on cardiac chronotropic responsiveness, atrial tissue histology and gene expression in rats that were chronically treated with therapeutic doses of lithium. Male Wistar albino rats were given lithium chloride (2.5 g/kg) orally for 2 or 3 months. Following treatment, the atria were isolated and spontaneously beating rate and chronotropic responsiveness to β-adrenergic stimulation was evaluated in an organ bath. Development of cardiac fibrosis was examined by histological methods. The expression of atrial Col1a1 (collagen I, alpha 1) and β-arrestin2 was also assessed using quantitative RT-PCR. Treatment with lithium induced a significant hypo-responsiveness to adrenergic stimulation (P < 0.001) and caused fibrosis in the atrial tissue of treated rats. In addition, the expression of atrial Col1a1 mRNA was significantly increased in atrial tissues of lithium-treated animals, while β-arrestin2 mRNA expression did not show a significant difference compared with control animals. Altogether, these findings indicate that cardiac chronotropic hypo responsiveness and associated cardiac fibrosis are side effects of chronic lithium treatment. Moreover, it seems that lithium treatment does not influence β-arrestin2 mRNA expression.

Alteration of \u03b2-Adrenoceptor Signaling in Left Ventricle of Acute Phase Takotsubo Syndrome: a Human Study

Accumulating evidence indicates alteration of the β-adrenoceptor (AR), such as desensitization and subtype switching of its coupling G protein, plays a role in the protection against catecholamine toxicity in heart failure. However, in human takotsubo syndrome (TTS), which is associated with a surge of circulating catecholamine in the acute phase, there is no histologic evidence of β-AR alteration. The purpose of this study was to investigate the involvement of alteration of β-AR signaling in the mechanism of TTS development. Left ventricular (LV) biopsied samples from 26 patients with TTS, 19 with normal LV function, and 26 with dilated cardiomyopathy (DCM) were studied. G protein-coupled receptor kinase 2 (GRK2) and β-arrestin2, which initiate the alteration of β-AR signaling, were more abundantly expressed in the myocardium in acute-phase TTS than in those of DCM and normal control as indicated by immunohistochemistry. The percentage of cardiomyocytes that showed positive membrane staining for GRK2 and β-arrestin2 was also significantly higher in acute-phase TTS. Sequential biopsies in the recovery-phase for two patients with TTS revealed that membrane expression of GRK2 and β-arrestin2 faded over time. This study provided the first histologic evidence of the involvement of alteration of β-ARs in the development of TTS.

β2-adrenoceptor signaling reduction is involved in the inflammatory response of fibroblast-like synoviocytes from adjuvant-induced arthritic rats.

To investigate the effects of β-AR signaling on fibroblast-like synoviocytes (FLS) from adjuvant-induced arthritis (AA) rats and the partial mechanisms focused on β-AR desensitization mediated by GRK2 and β-arrestin2. Animals were divided into a control group and an AA model group, and FLSs were cultured. Arthritis index, histopathology of joints, epinephrine (Epi) and norepinephrine (NE) were detected in vivo. The effect of the β-AR agonist isoprenaline (ISO) and the β2-AR agonist salbutamol on FLS cell viability were detected by CCK8. Cytokines TNF-α, IL-1β, OPG and RANKL were examined by ELISA. The expression of β2-AR was detected by immunofluorescence and flow cytometry. The cytomembrane expression and desensitization of β2-AR, GRK2, and β-arrestin2 were measured by flow cytometry and western blot. The concentration of NE increased to a peak on day 21, which was consistent with the arthritis index. The levels of Epi and NE in synovial tissues were decreased. ISO inhibited FLS cell viability and TNF-α, IL-1β, and RANKL secretion, and promoted OPG secretion. β2-AR mediated the effects of ISO on FLS cell viability. β2-AR signaling was weaker in AA rats compared to the controls. Elevated GRK2 and β-arrestin2 in cytomembranes promoted β2-AR desensitization and may decrease the anti-inflammatory effect of β2-AR signaling. The activation of β2-AR signaling exerts its anti-inflammatory activities on FLS. β2-AR signaling decreased in the AA model, which might be related to the increased membrane expression of GRK2 and β-arrestin2, and promoted the excessive desensitization of β2-AR. Decreased β2-AR signaling may be relevant to the exacerbation of arthritis inflammation.

β-Arrestin2 Inhibits Expression of Inflammatory Cytokines in BEAS-2B Lung Epithelial Cells Treated with Cigarette Smoke Condensate via Inhibition of Autophagy

Background/Aims: β-arrestin2 has been shown to have a role in human inflammatory disease. However, the role of β-arrestin2 in cigarette smoke-induced inflammation in the lung remains unknown. The aims of this study were to investigate the effects of β-arrestin2 on cigarette smoke condensate (CSC)-induced expression of inflammatory cytokines in the BEAS-2B human bronchial epithelial cell line in vitro, and the mechanisms involved. Methods: The MTT assay determined cell viability of cultured BEAS-2B cells. Autophagy was assessed by western blot, adenoviral mRFP-GFP-LC3 transfection, and immunofluorescence. The effects of β-arrestin2 shRNA knockdown were studied by western blot and real-time reverse transcription-polymerase chain reaction (RT-PCR). Western blot evaluated the AMPK/mTOR signaling pathway. Levels of inflammatory cytokines, interleukin (IL)-6, IL-8, and MCP-1 were measured in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). Results: CSC suppressed expression of β-arrestin2 in BEAS-2B cells, activated the AMPK/mTOR signaling pathway, increased cell autophagy and the expression of IL-6, IL-8, and MCP-1,pretreatment with the β-arrestin2 biased ligands, propranolol, and ICI118551 reversed these changes. Inhibition of autophagy reduced the expression of inflammatory cytokines following CSC. Conclusion: In the human bronchial epithelial cell line, BEAS-2B, β-arrestin2 reduced the expression of CSC-induced inflammatory cytokines by inhibiting autophagy, most likely via the AMPK/mTOR signaling pathway.

CCR5 expression in inflammatory bowel disease and its correlation with inflammatory cells and β-arrestin2 expression

Objective: To elucidate the correlation of expression of CC chemokine receptor 5 (CCR5) with degrees of inflammatory cells infiltration and expression of β-arrestin2 in biopsic intestinal mucosa of the patients with inflammatory bowel disease (IBD).Methods: Paraffin sections were derived from 53 patients with active IBD, 26 patients with remissive IBD and 30 healthy people. Immunohistochemical envision two-step method was used to test the expression of CCR5 and β-arrestin2 in biopsic intestinal mucosa. HE and toluidine blue staining were used to detect the pathological cytological analysis and classification in lamina propria of colonic mucosa.Results: The positive rate, strong positive rate and immunohistochemical score of CCR5 expression in active IBD were significantly higher than that in normal controls and remissive IBD (p < .05). CCR5 expression had no obvious correlation with clinical severity, lesion distribution and endoscopic classification of active IBD. Neutrophils, eosinophils and lymphocytes in active IBD were significantly higher than that in normal controls and remissive IBD (p < .05), while the lymphocyte grade had a positive correlation with CCR5 expression (p = .042, r = .286). Mastocytes in active IBD, remissive IBD and normal controls had no obvious difference (p > .05). β-arrestin2 expression was significantly lower in active IBD than that in remissive IBD and normal controls, and it had a negative correlation with CCR5 expression (p = .01, r = −.247).Conclusions: CCR5 is highly expressed in active IBD, and it has positive correlation with lymphocyte grade and negative correlation with expression of β-arrestin2.

Potential specific immunological indicators for stroke associated infection are partly modulated by sympathetic pathway activation.

BackgroundEvidence has led to the consideration of immunodepression after stroke as an important contributor to stroke associated infection (SAI). However, so far no specific immunological indicator has been identified for SAI, and the underlying mechanism remains poorly understood.ResultsSAI patients had significantly higher IL-6 and IL-10 levels and lower HLA-DR levels than no-infection patients within 48h after stroke onset. NA significantly increased IL-10 levels, reduced HLA-DR expression, and decreased IL-6 expression by increasing β-arrestin2 expression which reduced the activation of the NF-κB pathway. Propranolol reversed this effect of NA by reducing β-arrestin2 expression.Materials and MethodsA systematic search for eligible clinical studies was applied to pool the differences in peripheral cytokine levels between infection and no-infection stroke patients. The underlying mechanism behind these differences was investigated in vitro by applying norepinephrine (NA) and lipopolysaccharide (LPS) to simulate sympathetic pathway activation and sepsis respectively in THP-1 cells. Propranolol was applied to determine the effect of reversing the activation of the sympathetic pathway. Immunological indicators were also detected to assess the immune activation of THP-1 cells and measurements of the expression of β-arrestin2, NF-κB, IκBα and phosphor-IκBα were performed to assess the activation of the sympathetic pathway.ConclusionIL-6, IL-10 and HLA-DR are good candidate biomarkers for SAI. The activation of the sympathetic pathway could partly account for the specific immunological alterations found in SAI patients including HLA-DR decrease and IL-10 increase, which both could be reversed by propranolol. However, the mechanism underlying IL-6 increase still needs further exploration.

β-Arrestin–biased signaling mediates memory reconsolidation

A long-standing hypothesis posits that a G protein-coupled signaling pathway mediates β-adrenergic nervous system functions, including learning and memory. Here we report that memory retrieval (reactivation) induces the activation of β1-adrenergic β-arrestin signaling in the brain, which stimulates ERK signaling and protein synthesis, leading to postreactivation memory restabilization. β-Arrestin2-deficient mice exhibit impaired memory reconsolidation in object recognition, Morris water maze, and cocaine-conditioned place preference paradigms. Postreactivation blockade of both brain β-adrenergic Gs protein- and β-arrestin-dependent pathways disrupts memory reconsolidation. Unexpectedly, selective blockade of the Gs/cAMP/PKA signaling but not the β-arrestin/ERK signaling by the biased β-adrenergic ligands does not inhibit reconsolidation. Moreover, the expression of β-arrestin2 in the entorhinal cortex of β-arrestin 2-deficient mice rescues β1-adrenergic ERK signaling and reconsolidation in a G protein pathway-independent manner. We demonstrate that β-arrestin-biased signaling regulates memory reconsolidation and reveal the potential for β-arrestin-biased ligands in the treatment of memory-related disorders.

Specific localization of β-Arrestin2 in myenteric plexus of mouse gastrointestinal tract.

β-arrestin2 is a key molecule involved in signaling and internalization of activated G protein-coupled receptors including µ-opioid receptors (MOR). Previously we have shown that decreased expression of β-arrestin2 upon chronic morphine is associated with the development of opioid tolerance in the gastrointestinal tract. However, the localization of β-arrestin2 within the gastrointestinal wall is not known. In this study we found that β-arrestin2 is localized in the soma of a select group of neurons in the myenteric ganglia but not in smooth muscle. The density of β-arestin2 was significantly higher in the ileum than the colon. We identified four variants of β-arrestin2 in the ileum, with ARRB-005 and ARRB-013 being the most abundant. Further, the current study utilized multiple-labeling immunofluorescence to characterize the chemical coding of neurons expressing β-arrestin2 in the murine myenteric plexus and the co-localization of MOR1 and β-arrestin2. β-arrestin2 co-localized with choline acetyltransferase and calretinin. In contrast, β-arrestin2 neither co-localized with substance P, nitric oxide synthase nor calbindin. Genetic deletion of β-arrestin2 did not affect cholinergic neuron activation by nicotine in the isolated ileum (-log M EC50: wild type = 5.8 vs. β-arrestin2 knockout = 5.9). Our findings suggest specificity in the localization of β-arrestin2 in the myenteric plexus within MOR1-expressing neurons and provide a relation for direct intracellular crosstalk between MOR1 receptor activation and β-arrestin2 signaling in the myenteric neurons. β-arrestin2 deletion does not directly alter basal enteric cholinergic neuronal function.

Beta-arrestin2 is involved in the increase of distal colonic contraction in diabetic rats

Colonic dysmotility occurs in diabetes and the patients exhibit diarrhea or constipation. The pathogenetic mechanisms underlying colonic dysmotility in diabetic patients remain poorly understood. The effects of β-arrestin2 on colonic contraction in diabetic rats were investigated for the first time. Male SD rats were treated with a single intraperitoneally injected dose of streptozotocin, and those displaying sustained high blood glucose were selected as diabetes mellitus models. Longitudinal muscle strips of the distal colon were prepared to monitor contraction of the colon in vitro. Expression of β-arrestin2 was investigated by Western blot analysis. Anti-β-arrestin2 antibody had no direct effect on the contraction of distal colonic strips in both normal and diabetic rats. Carbachol-induced contractions of distal colonic strips were higher in diabetic rats than in normal rats. Anti-β-arrestin2 antibody partly blocked carbachol-induced increases of distal colonic strips in diabetic rats. The expression level of β-arrestin2 protein in the colon was higher in diabetic rats than in normal rats. These results suggest that β-arrestin2 is involved in the increase of distal colonic contraction in diabetic rats.

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.

Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling

Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt)/glycogen synthase kinase (GSK)-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308) and enhances the phosphorylation of GSK-3α (Ser-21)/β (Ser-9) in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.

Role of β2-adrenoceptor-β-arrestin2-nuclear factor-κB signal transduction pathway and intervention effects of oxymatrine in ulcerative colitis

To investigate the β2-adrenoceptor (β2AR)-β-arrestin2-nuclear factor-κB (NF-κB) signal transduction pathway and the intervention effects of oxymatrine in a rat model of ulcerative colitis. Forty SD rats were randomly divided into four groups, which included the normal control group, the model group, the mesalazine group and the oxymatrine treatment group, with 10 rats per group. Experimental colitis induced with trinitrobenzene sulfonic acid (TNBS) was established in each group except the normal control group. The rats in the oxymatrine treatment group were treated with intramuscular injection of oxymatrine 63 mg/(kg·d) for 15 days and the rats in the mesalazine group were treated with mesalazine solution 0.5 g/(kg·d) by gastric lavage for 15 days. The rats in the normal control group and model group were treated with 3 mL water by gastric lavage for 15 days. Diarrhea and bloody stool were carefully observed. Histological changes in colonic tissue were examined on day 7 in 2 rats per group that were randomly selected. The expression of β2AR, β-arrestin2 and NF-κB p65 in colon tissue and spleen lymphocytes were detected with immunohistochemistry and Western immunoblotting techniques on day 16 after fasting for 24 h. Six rats died of lavage with 2 each in the normal control, the model group and the mesalazine group; and were not included in the analysis. The rats in the model group suffered from looser stool and bloody purulent stool after modeling. But in the oxymatrine and mesalazine groups, looser stool and bloody purulent stool reduced after treatment. And the colonic wall in the model group was thickened and the colon length shortened. The colon mucosa was congested in multiple areas with edema, erosion, superficial or linear ulcer and scar formation, while the intestinal mucosa injury reduced in the mesalazine and oxymatrine groups (P<0.01). In colonic mucosa and in spleen lymphocytes, compared with the normal control group, the expression of NF-κBp65 were significantly increased (P<0.01) in the model group while the expressions of β 2AR and β-arrestin2 were significantly decreased (P<0.01). Compared with the model group, the expression of NF-κ Bp65 was significantly decreased in the mesalazine group (P<0.01) and oxymatrine treatment group (P<0.01) while the expressions of β2AR and β-arrestin2 were significantly increased (P<0.01). There were no statistically significant differences in the expression of β2AR, β-arrestin2 and NF-κBp65 between the mesalazine group and oxymatrine group (P>0.05). The β2AR-β-arrestin2-NF-κB signal transduction pathway participated in the pathologic course of ulcerative colitis. Oxymatrine attenuated ulcerative colitis through regulating the β2AR-β-arrestin2-NF-κB signal transduction pathway.

The Role of β-Arrestin2 in the Mechanism of Morphine Tolerance in the Mouse and Guinea Pig Gastrointestinal Tract

β-Arrestin2 has been reported to play an essential role in analgesic tolerance. Analgesic tolerance without concomitant tolerance to constipation is a limiting side effect of chronic morphine treatment. Because tolerance to morphine develops in the mouse ileum but not the colon, we therefore examined whether the role of β-arrestin2 in the mechanism of morphine tolerance differs in the ileum and colon. In both guinea pig and mouse, chronic in vitro exposure (2 h, 10 μM) to morphine resulted in tolerance development in the isolated ileum but not the colon. The IC(50) values for morphine-induced inhibition of electrical field stimulation contraction of guinea pig longitudinal muscle myenteric plexus shifted rightward in the ileum from 5.7 ± 0.08 (n = 9) to 5.45 ± 0.09 (n = 6) (p < 0.001) after morphine exposure. A significant shift was not observed in the colon. Similar differential tolerance was seen between the mouse ileum and the colon. However, tolerance developed in the colon from β-arrestin2 knockout mice. β-Arrestin2 and extracellular signal-regulated kinase 1/2 expression levels were determined further by Western blot analyses in guinea pig longitudinal muscle myenteric plexus. A time-dependent decrease in the expression of β-arrestin2 and extracellular signal-regulated kinase 1/2 occurred in the ileum but not the colon after 2 h of morphine (10 μM) exposure. Naloxone prevented the decrease in β-arrestin2. In the isolated ileum from guinea pigs chronically treated in vivo with morphine for 7 days, neither additional tolerance to in vitro exposure of morphine nor a decrease in β-arrestin2 occurred. We conclude that a decrease in β-arrestin2 is associated with tolerance development to morphine in the gastrointestinal tract.

β-arrestin2 stimulates interleukin-17 production and expression of CD4+ T lymphocytes in a murine asthma model.

Allergic asthma is a complex and chronic inflammatory airway disease. Interleukin-17 is a pro-inflammatory cytokine which plays critical role in the pathogenesis of allergic asthma. It has been reported that β-arrestin2 regulated the development of allergic asthma at a proximal step in the inflammatory cascade. In this study, the influence of β-arrestin2 on Interleukin-17 production and expression of CD4+ T lymphocytes in a murine asthma model was investigated. Splenic CD4+ T lymphocytes from wild-type mice and those from a murine asthma model were purified. CD4+ T lymphocytes from a murine asthma model were transfected with siRNAs targeting the β-arrestin2 or were pretreated with the ERK1/2 inhibitor, PD98059. After stimulation, the protein expression of β-arrestin2、phosphorylated-ERK1/2 and IL-17 were detection by Western blot; the mRNA expression of IL-17 were detected by real-time PCR; the accumulation of IL-17 in supernatants were detected by ELISA. We found that β-arrestin2、phosphorylated-ERK1/2 and IL-17 expression in CD4+ T lymphocytes from a murine asthma model were increased compared with those from wild-type mice (p < 0.01). Treatment of CD4+ T lymphocytes with siRNAs targeting the β-arrestin2 down-regulated phosphorylated- ERK 1/2 and IL-17 expression (p < 0.01). PD98059 decreased IL-17 production and expression in CD4+ T lymphocytes in a murine asthma model (p < 0.05). We conclude that β-arrestin2 stimulated IL-17 production and expression of CD4+ T lymphocytes in a murine asthma model. The effect was partly mediated by ERK 1/2 activation. Targeting β-arrestin2 biological activity could be a valid therapeutic approach for the treatment of allergic asthma.

β-arrestin2 stimulates interleukin-17 production and expression of CD4+ T lymphocytes in a murine asthma model.

Allergic asthma is a complex and chronic inflammatory airway disease. Interleukin-17 is a pro-inflammatory cytokine which plays critical role in the pathogenesis of allergic asthma. It has been reported that β-arrestin2 regulated the development of allergic asthma at a proximal step in the inflammatory cascade. In this study, the influence of β-arrestin2 on Interleukin-17 production and expression of CD4+ T lymphocytes in a murine asthma model was investigated. Splenic CD4+ T lymphocytes from wild-type mice and those from a murine asthma model were purified. CD4+ T lymphocytes from a murine asthma model were transfected with siRNAs targeting the β-arrestin2 or were pretreated with the ERK1/2 inhibitor, PD98059. After stimulation, the protein expression of β-arrestin2、phosphorylated-ERK1/2 and IL-17 were detection by Western blot; the mRNA expression of IL-17 were detected by real-time PCR; the accumulation of IL-17 in supernatants were detected by ELISA. We found that β-arrestin2、phosphorylated-ERK1/2 and IL-17 expression in CD4+ T lymphocytes from a murine asthma model were increased compared with those from wild-type mice (p < 0.01). Treatment of CD4+ T lymphocytes with siRNAs targeting the β-arrestin2 down-regulated phosphorylated- ERK 1/2 and IL-17 expression (p < 0.01). PD98059 decreased IL-17 production and expression in CD4+ T lymphocytes in a murine asthma model (p < 0.05). We conclude that β-arrestin2 stimulated IL-17 production and expression of CD4+ T lymphocytes in a murine asthma model. The effect was partly mediated by ERK 1/2 activation. Targeting β-arrestin2 biological activity could be a valid therapeutic approach for the treatment of allergic asthma.