Tinghuai Wang

Involvement of MAPK/NF-κB Signaling in the Activation of the Cholinergic Anti-Inflammatory Pathway in Experimental Colitis by Chronic Vagus Nerve Stimulation

Background Autonomic nervous system dysfunction is implicated in the etiopathogenesis of inflammatory bowel diseases (IBD). Therapies that increase cardiovagal activity, such as Mind-Body interventions, are currently confirmed to be effective in clinical trials in IBD. However, a poor understanding of pathophysiological mechanisms limits the popularization of therapies in clinical practice. The aim of the present study was to explore the mechanisms of these therapies against 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats using a chronic vagus nerve stimulation model in vivo, as well as the lipopolysaccharide (LPS)-induced inflammatory response in human epithelial colorectal adenocarcinoma cells (Caco-2) by acetylcholine in vitro. Methods and Results Colitis was induced in rats with rectal instillation of TNBS, and the effect of chronic VNS (0.25 mA, 20 Hz, 500 ms) on colonic inflammation was evaluated. Inflammatory responses were assessed by disease activity index (DAI), histological scores, myeloperoxidase (MPO) activity, inducible nitric oxide synthase (iNOS), TNF-α and IL-6 production. The expression of Mitogen-activated protein kinases (MAPK) family members, IκB-α, and nuclear NF-κB p65 were studied by immunoblotting. Heart rate variability (HRV) analysis was also applied to assess the sympathetic-vagal balance. DAI, histological scores, MPO activity, iNOS, TNF-α and IL-6 levels were significantly decreased by chronic VNS. Moreover, both VNS and acetylcholine reduced the phosphorylation of MAPKs and prevented the nuclear translocation of NF-κB p65. Methyllycaconitine (MLA) only reversed the inhibitory effect on p-ERK and intranuclear NF-κB p65 expression by ACh in vitro, no significant change was observed in the expression of p-p38 MAPK or p-JNK by MLA. Conclusion Vagal activity modification contributes to the beneficial effects of the cholinergic anti-inflammatory pathway in IBD-related inflamed colonic mucosa based on the activation of MAPKs and nuclear translocation of NF-κB. Our work may provide key pathophysiological mechanistic evidence for novel therapeutic strategies that increase the cardiovagal activity in IBD patients.

Heart rate variability biofeedback decreases blood pressure in prehypertensive subjects by improving autonomic function and baroreflex.

BACKGROUND Individuals with prehypertension are at risk of hypertension and cardiovascular diseases, and yet efficient interventions are lagging behind. Studies indicate that heart rate variability-biofeedback (HRV-BF) increases HRV and baroreflex sensitivity (BRS) as well as reduces related pathological symptoms, suggesting potentially beneficial effects of HRV-BF on prehypertension, but little is known about these effects. In this study, these effects were investigated and their mechanisms were explored. OBJECTIVES The effect of HRV-BF on prehypertension in young adults and its potential mechanism were explored. DESIGN Forty-three (43) individuals with prehypertension were recruited and classified into three categories: HRV-BF group, slow abdominal breathing group, and control group. All groups were assessed with measurements of noninvasive blood pressure (BP), BRS, respiration, and galvanic skin response (GSR) at pre-intervention, in the entire process of each session, at postintervention, as well as at a 3-month follow-up. INTERVENTIONS Subjects participated in a 10-session HRV-BF protocol or simple slow abdominal breathing protocol conducted over 5 weeks. A 3-month follow-up was also performed on these individuals. RESULTS The incidence of prehypertension was as high as 14.5% in young college students. Individuals with prehypertension were lower in BRS (7.5±5.2 ms/mm Hg) and HRV (log10-transformed of the standard deviation of normal-to-normal beats [SDNN]=1.62±0.13 ms, lgTotal power of spectral density in the range of frequencies between 0 and 0.4Hz (TP)=8.02±0.55 ms2) than those with normal blood pressure (BRS=18.4±7.4 ms/mm Hg, lgSDNN=1.79±0.10 ms, lgTP=8.68±0.85 ms2). HRV-BF reduced blood pressure (from 131.7±8.7/79.3±4.7 mm Hg to 118.9±7.3 mm Hg/71.9±4.9 mm Hg, p<0.01), increased BRS (from 7.0±5.9 ms/mm Hg to 15.8±5.3 ms/mm Hg, p<0.01) and increased HRV (lgSDNN from 1.61±0.11 to 1.75±0.05 ms, and lgTP from 8.07±0.54 to 9.08±0.41 ms2, p<0.01). These effects were more obvious than those of the slow-breathing group, and remained for at least 3 months. HRV-BF also significantly increased vagus-associated HRV indices and decreased GSR (indices of sympathetic tone). CONCLUSIONS These effects suggest that HRV-BF, a novel behavioral neurocardiac intervention, could enhance BRS, improve the cardiac autonomic tone, and facilitate BP adjustment for individuals with prehypertension.

17β-Estradiol enhances breast cancer cell motility and invasion via extra-nuclear activation of actin-binding protein ezrin.

Estrogen promotes breast cancer metastasis. However, the detailed mechanism remains largely unknown. The actin binding protein ezrin is a key component in tumor metastasis and its over-expression is positively correlated to the poor outcome of breast cancer. In this study, we investigate the effects of 17β-estradiol (E2) on the activation of ezrin and its role in estrogen-dependent breast cancer cell movement. In T47-D breast cancer cells, E2 rapidly enhances ezrin phosphorylation at Thr567 in a time- and concentration-dependent manner. The signalling cascade implicated in this action involves estrogen receptor (ER) interaction with the non-receptor tyrosine kinase c-Src, which activates the phosphatidylinositol-3 kinase/Akt pathway and the small GTPase RhoA/Rho-associated kinase (ROCK-2) complex. E2 enhances the horizontal cell migration and invasion of T47-D breast cancer cells in three-dimensional matrices, which is reversed by transfection of cells with specific ezrin siRNAs. In conclusion, E2 promotes breast cancer cell movement and invasion by the activation of ezrin. These results provide novel insights into the effects of estrogen on breast cancer progression and highlight potential targets to treat endocrine-sensitive breast cancers.

Caveolin-3 is involved in the protection of resveratrol against high-fat-diet-induced insulin resistance by promoting GLUT4 translocation to the plasma membrane in skeletal muscle of ovariectomized rats.

Insulin resistance is recognized as a common metabolic factor which predicts the future development of both type 2 diabetes and atherosclerotic disease. Resveratrol (RSV), an agonist of estrogen receptor (ER), is known to affect insulin sensitivity, but the mechanism is unclear. Evidence suggests that caveolin-3 (CAV-3), a member of the caveolin family, is involved in insulin-stimulated glucose uptake. Our recent work indicated that estrogen via ER improves glucose uptake by up-regulation of CAV-3 expression. Here, we investigated the role of CAV-3 in the effect of RSV on insulin resistance in skeletal muscle both in vivo and in vitro. The results demonstrated that RSV ameliorated high-fat-diet (HFD)-induced glucose intolerance and insulin resistance in ovariectomized rats. RSV elevated insulin-stimulated glucose uptake in isolated soleus muscle in vivo and in C2C12 myotubes in vitro by enhancing GLUT4 translocation to the plasma membrane rather than increasing GLUT4 protein expression. Through ERα-mediated transcription, RSV increased CAV-3 protein expression, which contributed to GLUT4 translocation. Moreover, after knockdown of CAV-3 gene, the effects of RSV on glucose uptake and the translocation of GLUT4 to the plasma membrane, as well as the association of CAV-3 and GLUT4 in the membrane, were significantly attenuated. Our findings demonstrated that RSV via ERα elevated CAV-3 expression and then enhanced GLUT4 translocation to the plasma membrane to promote glucose uptake in skeletal muscle, exerting its protective effects against HFD-induced insulin resistance. It suggests that this pathway could represent an effective therapeutic target to fight against insulin resistance syndrome induced by HFD.

Estrogen improved metabolic syndrome through down-regulation of VEGF and HIF-1α to inhibit hypoxia of periaortic and intra-abdominal fat in ovariectomized female rats

Metabolic syndrome (MBS), a cluster of metabolic abnormalities and visceral fat accumulation, increases cardiovascular risks in postmenopausal women. In addition to visceral fat, perivascular adipose tissue has been recently found to play an important role in vascular pathophysiology. Hence, the present study investigates the effects of estrogen on both intra-abdominal fat (visceral fat) and periaortic fat (perivascular fat) accumulation as well as hypoxia in ovariectomized female rats. Female rats were divided into sham operation, ovariectomy and ovariectomy with 17β-estradiol supplementation groups. Twelve weeks later, we found that estrogen improved MBS via reducing body weight gain, the weight of periaortic and intra-abdominal fat, hepatic triglyceride, and total serum cholesterol levels. Estrogen also increased insulin sensitivity through restoring glucose and serum leptin levels. For periaortic fat, western blot showed estrogen inhibited hypoxia by reducing the levels of VEGF and HIF-1α, which is consistent with the results from immunohistochemical staining. The correlation analysis indicated that perivascular fat had a positive correlation with body weight, intra-abdominal fat or serum total cholesterol, but a negative correlation with insulin sensitivity index. For intra-abdominal fat, real-time fluorescent RT-PCR showed estrogen improved fat dysfunction via reducing the levels of relative leptin, MCP-1 but increasing adiponectin mRNA. Estrogen reduced the levels of VEGF and HIF-1α to inhibit hypoxia but restored the levels of PPARγ and Srebp-1c, which are important for lipid capacity function of intra-abdominal fat. These results demonstrated estrogen improved MBS through down-regulating VEGF and HIF-1α to inhibit hypoxia of periaortic and intra-abdominal fat in ovariectomized female rats.

17β-estradiol down-regulates lipopolysaccharide-induced MCP-1 production and cell migration in vascular smooth muscle cells

Atherosclerosis is an inflammatory disease where lipopolysaccharide (LPS) triggers the release of inflammatory cytokines that accelerate its initiation and progression. Estrogen has been proven to be vasoprotective against atherosclerosis; however, the anti-inflammatory function of estrogen in the vascular system remains obscure. In this study, we investigated the effect of estrogen on LPS-induced monocyte chemoattractant protein-1 (MCP-1; listed as CCL2 in the MGI database) production in vascular smooth muscle cells (VSMCs). LPS significantly enhances MCP-1 production and this is dependent on nuclear factor kappa B (NFkappaB) signaling, since the use of NFkappaB inhibitor pyrrolidine dithiocarbamate or the silencing of NFkappaB subunit p65 expression with specific siRNA largely impairs LPS-enhanced MCP-1 production. On the contrary, 17beta-estradiol (E(2)) inhibits LPS-induced MCP-1 production in a time- and dose-dependent manner, which is related to the suppression of p65 translocation to nucleus. Furthermore, p38 MAPK is rapidly activated in response to LPS, while E(2) markedly inhibits p38 MAPK activation. Transfection with p38 MAPK siRNA or the use of p38 MAPK inhibitor SB203580 markedly attenuates LPS-stimulated p65 translocation to nucleus and MCP-1 production, suggesting that E(2) suppresses NFkappaB signaling by the inactivation of p38 MAPK signaling. LPS promotes VSMCs migration and this is abrogated by MCP-1 antibody, implying that MCP-1 may play a major role as an autocrine factor in atherosclerosis. In addition, E(2) inhibits LPS-promoted cell migration by downregulation of MCP-1 production. Overall, our results demonstrate that E(2) exerts anti-inflammatory property antagonistic to LPS in VSMCs by reducing MCP-1 production, and this effect is related to the inhibition of p38 MAPK/NFkappaB cascade.

Non-genomic effects of 17β-estradiol in activation of the ERK1/ERK2 pathway induces cell proliferation through upregulation of cyclin D1 expression in bovine artery endothelial cells

Objective. Growing evidence indicates that estrogens non-genomic effects play important roles in cellular functions and backs up the hypothesis of the existence of a membrane estrogen receptor (mER) in a number of cell types, but little is known about the complementary effects between traditional genomic and novel non-genomic effects of estrogen. The aim of the present study was to explore the non-genomic activation of ERK1/2 mitogen-activated protein kinase (MAPK) by 17β-estradiol (E2) through mER and its role in cell proliferation. Methods. On cultured bovine artery endothelial cells (BAECs) we used the [3H]thymidine incorporation assay to evaluate the influence of E2 on cell proliferation and fluorescence microscopy to show the presence of mER on the cell membrane. Scatchard analysis was performed to identify and characterize the mER on a purified membrane fraction of BAECs. Results. E2 upregulated cyclin D1 protein expression and enhanced cell proliferation. Inhibition of the MAPK cascade with PD98059 or of G protein with pertussis toxin (PTX) completely abolished the above effects, while the estrogen receptor antagonist tamoxifen attenuated E2-dependent upregulation of cyclin D1 and cell proliferation. Accordingly, E2 rapidly led to ERK1/ERK2 activation, which was prevented by tamoxifen or PTX and was entirely reproduced by membrane-impermeable estradiol–bovine serum albumin conjugate (E2coBSA). Immunofluorescent staining with E2coBSA–fluorescein isothiocyanate resulted in a punctuate staining pattern of the plasma membrane and Scatchard analysis of the E2-binding protein in a purified membrane fraction of BAECs showed that E2 binds to the membrane fraction with a dissociation constant of 0.2394 nmol/l. Conclusion. Our findings showed that E2 induces cell proliferation through upregulation of cyclin D1 via non-genomic activation of the ERK1/ERK2 pathway mediated by mER and G protein.

Progesterone enhances vascular endothelial cell migration via activation of focal adhesion kinase.

The mechanisms of progesterone on endothelial cell motility are poorly investigated. Previously we showed that progesterone stimulated endothelial cell migration via the activation of actin‐binding protein moesin, leading to actin cytoskeleton remodelling and the formation of cell membrane structures required for cell movement. In this study, we investigated the effects of progesterone on the formation of focal adhesion complexes, which provide anchoring sites for cell movement. In cultured human umbilical endothelial cells, progesterone enhanced focal adhesion kinase (FAK) phosphorylation at Tyr397 in a dose‐ and time‐dependent manner. Several signalling inhibitors interfered with progesterone‐induced FAK activation, including progesterone receptor (PR) antagonist ORG 31710, specific c‐Src kinase inhibitor PP2, phosphatidylinosital‐3 kinase (PI3K) inhibitor wortmannin as well as ρ‐associated kinase (ROCK‐2) inhibitor Y27632. It suggested that PR, c‐Src, PI3K and ROCK‐2 are implicated in this action. In line with this, we found that progesterone rapidly promoted c‐Src/PI3K/Akt activity, which activated the small GTPase RhoA/ρ‐associated kinase (ROCK‐2) complex, resulting in FAK phosphorylation. In the presence of progesterone, endothelial cells displayed enhanced horizontal migration, which was reversed by small interfering RNAs abrogating FAK expression. In conclusion, progesterone promotes endothelial cell movement via the rapid regulation of FAK. These findings provide new information on the biological actions of progesterone on human endothelial cells that are relevant for vascular function.

EEG biofeedback improves attentional bias in high trait anxiety individuals

BackgroundEmotion-related attentional bias is implicated in the aetiology and maintenance of anxiety disorders. Electroencephalogram (EEG) biofeedback can obviously improve the anxiety disorders and reduce stress level, and can also enhance attention performance in healthy subjects. The present study examined the effects and mechanisms of EEG biofeedback training on the attentional bias of high trait anxiety (HTA) individuals toward negative stimuli.ResultsEvent-related potentials were recorded while HTA (n=24) and nonanxious (n=21) individuals performed the color-word emotional Stroop task. During the emotional Stroop task, HTA participants showed longer reaction times and P300 latencies induced by negative words, compared to nonanxious participants.The EEG biofeedback significantly decreased the trait anxiety inventory score and reaction time in naming the color of negative words in the HTA group. P300 latencies evoked by negative stimuli in the EEG biofeedback group were significantly reduced after the alpha training, while no significant changes were observed in the sham biofeedback group after the intervention.ConclusionThe prolonged P300 latency is associated with attentional bias to negative stimuli in the HTA group. EEG biofeedback training demonstrated a significant improvement of negative emotional attentional bias in HTA individuals, which may be due to the normalization of P300 latency.

Hydrogen (H2) Inhibits Isoproterenol-Induced Cardiac Hypertrophy via Antioxidative Pathways

Background and Purpose: Hydrogen (H2) has been shown to have a strong antioxidant effect on preventing oxidative stress-related diseases. The goal of the present study is to determine the pharmacodynamics of H2 in a model of isoproterenol (ISO)-induced cardiac hypertrophy. Methods: Mice (C57BL/6J; 8–10 weeks of age) were randomly assigned to four groups: Control group (n = 10), ISO group (n = 12), ISO plus H2 group (n = 12), and H2 group (n = 12). Mice received H2 (1 ml/100g/day, intraperitoneal injection) for 7 days before ISO (0.5 mg/100g/day, subcutaneous injection) infusion, and then received ISO with or without H2 for another 7 days. Then, cardiac function was evaluated by echocardiography. Cardiac hypertrophy was reflected by heart weight/body weight, gross morphology of hearts, and heart sections stained with hematoxylin and eosin, and relative atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) mRNA levels. Cardiac reactive oxygen species (ROS), 3-nitrotyrosine and p67 (phox) levels were analyzed by dihydroethidium staining, immunohistochemistry and Western blotting, respectively. For in vitro study, H9c2 cardiomyocytes were pretreated with H2-rich medium for 30 min, and then treated with ISO (10 μM) for the indicated time. The medium and ISO were re-changed every 24 h. Cardiomyocyte surface areas, relative ANP and BNP mRNA levels, the expression of 3-nitrotyrosine, and the dissipation of mitochondrial membrane potential (MMP) were examined. Moreover, the expression of extracellular signal-regulated kinase1/2 (ERK1/2), p-ERK1/2, p38, p-p38, c-Jun NH2-terminal kinase (JNK), and p-JNK were measured by Western blotting both in vivo and in vitro. Results: Intraperitoneal injection of H2 prevented cardiac hypertrophy and improved cardiac function in ISO-infused mice. H2-rich medium blocked ISO-mediated cardiomyocytes hypertrophy in vitro. H2 blocked the excessive expression of NADPH oxidase and the accumulation of ROS, attenuated the decrease of MMP, and inhibited ROS-sensitive ERK1/2, p38, and JNK signaling pathways. Conclusion: H2 inhibits ISO-induced cardiac/cardiomyocytes hypertrophy both in vivo and in vitro, and improves the impaired left ventricular function. H2 exerts its protective effects partially through blocking ROS-sensitive ERK1/2, p38, and JNK signaling pathways.