Development and validation of a nomogram for predicting atrial fibrillation in patients with acute heart failure admitted to the ICU: a retrospective cohort study.

INTRODUCTION: Acute heart failure is a serious condition. Atrial fibrillation is the most frequent arrhythmia in patients with acute heart failure. The occurrence of atrial fibrillation in heart failure patients worsens their prognosis and leads to a substantial increase in treatment costs. There is no tool that can effectively predict the onset of atrial fibrillation in patients with acute heart failure in the ICU currently. MATERIALS AND METHODS: We retrospectively analyzed the MIMIC-IV database of patients admitted to the intensive care unit (ICU) for acute heart failure and who were initially sinus rhythm. Data on demographics, comorbidities, laboratory findings, vital signs, and treatment were extracted. The cohort was divided into a training set and a validation set. Variables selected by LASSO regression and multivariate logistic regression in the training set were used to develop a model for predicting the occurrence of atrial fibrillation in acute heart failure in the ICU. A nomogram was drawn and an online calculator was developed. The discrimination and calibration of the model was evaluated. The performance of the model was tested using the validation set. RESULTS: This study included 2342 patients with acute heart failure, 646 of whom developed atrial fibrillation during their ICU stay. Using LASSO and multiple logistic regression, we selected six significant variables: age, prothrombin time, heart rate, use of vasoactive drugs within 24 h, Sequential Organ Failure Assessment (SOFA) score, and Acute Physiology Score (APS) III. The C-index of the model was 0.700 (95% CI 0.672-0.727) and 0.682 (95% CI 0.639-0.725) in the training and validation sets, respectively. The calibration curves also performed well in both sets. CONCLUSION: We developed a simple and effective model for predicting atrial fibrillation in patients with acute heart failure in the ICU.

Acetylcholine suppresses LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways.

BACKGROUND AND OBJECTIVE: Endothelial cell activation plays a critical role in leukocyte recruitment during inflammation and infection. We previously found that cholinergic stimulation (via vagus nerve stimulation) attenuates vascular endothelial impairment and reduces the inflammatory profile in ovariectomized rats. However, the specific molecular mechanism is unclear. This study was designed to explore the effects and molecular mechanisms of cholinergic agonists (acetylcholine [ACh]) on lipopolysaccharide (LPS)-induced endothelial cell activation in vitro. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of LPS (10/100/1000 ng/mL) to activate endothelial cells. HUVECs were untreated, treated with ACh (10-5 M) alone, treated with 100 ng/mL LPS alone, or treated with different concentrations of ACh (10-9/10-8/10-7/10-6/10-5 M) before LPS stimulation. HUVECs were also pre-treated with 10-6 M ACh with or without mecamylamine (an nAChR blocker) (10 µΜ) and methyllycaconitine (a specific α7 nAChR blocker) (10 µΜ) and incubated with or without LPS. ELISA, western blotting, cell immunofluorescence, and cell adhesion assays were used to examine inflammatory cytokine production, adhesion molecule expression, monocyte-endothelial cell adhesion and activation of the MAPK/NF-κB pathways. RESULTS: LPS (at 10 ng/mL, 100 ng/mL and 1,000 ng/mL) increased VCAM-1 expression in HUVECs in a dose-dependent manner (with no significant difference between LPS at 100 ng/mL and 1,000 ng/mL). ACh (10-9 M-10-5 M) blocked adhesion molecule expression (VCAM-1, ICAM-1, and E-selectin) and inflammatory cytokine production (TNF-α, IL-6, MCP-1, IL-8) in response to LPS in a dose-dependent manner (with no significant difference between 10-5 and 10-6 M Ach). LPS was also shown to significantly enhance monocyte-endothelial cell adhesion, which was largely abrogated by treatment with ACh (10-6M). VCAM-1 expression was blocked by mecamylamine rather than methyllycaconitine. Lastly, ACh (10-6 M) significantly reduced LPS-induced phosphorylation of NF-κB/p65, IκBα, ERK, JNK and p38 MAPK in HUVECs, which was blocked by mecamylamine. CONCLUSIONS: ACh protects against LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways, which are mediated by nAChR, rather than α7 nAChR. Our results may provide novel insight into the anti-inflammatory effects and mechanisms of ACh.

ARNTL2 is a Prognostic Biomarker and Correlates with Immune Cell Infiltration in Triple-Negative Breast Cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and is associated with poor prognosis. The aberrant expression of circadian genes contributes to the origin and progression of breast cancer. The present study was designed to explore the potential function and prognosis value of circadian genes in TNBC. METHODS: The transcriptome data of circadian genes were downloaded from The Cancer Genomic Atlas (TCGA), GSE25066 and GSE31448 datasets. The differential expressed circadian genes between non-TNBC and TNBC patients were analysed by Wilcoxon test. Univariate and multivariate Cox regression analyses were employed to identify the prognostic circadian genes. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) were performed to study the biological functions of ARNTL2. The composition of 22 immune cells in the tumour samples was estimated with CIBERSORT algorithm. The correlations between ARNTL2 expression and tumour-infiltrating immune cells were evaluated by Spearman correlation coefficient. RESULTS: A total of 8 circadian genes were found to be differentially expressed between non-TNBC and TNBC, but only ARNTL2 has prognostic value. Multivariate Cox analysis identified that ARNTL2 was an independent prognosis factor for overall survival and relapse-free survival in TNBC patients. Functionally, ARNTL2 was mainly involved in immune response processes such as positive regulation of cytokine production, regulation of innate immune response, and cellular responses to molecules of bacterial origin. High expression of ARNTL2 was positively correlated with activated CD4 memory T cells, activated mast cells, and neutrophil infiltration and the expression of markers of neutrophils (ITGAM), dendritic cells (HLA-DRA, HLA-DPA1, ITGAM), Th1 (IL1B, STAT1), Th2 (IL13), Th17 (STAT3) and mast cells (TPSB2, TPSAB1). CONCLUSION: ARNTL2 may be linked with the functional modulation of the tumour immune microenvironment and serve as a potential biomarker for predicting the prognosis of TNBC patients.

[Practical exploration on the Online Four-step Teaching of Encouraging and Sharing during COVID-19 period].

The "Four-step Teaching of Encouraging and Sharing" is a learner-centered teaching method that advocates teamwork and gives full play to the role of the teacher in guiding learning. It is an innovative teaching approach to realize students' self-transcendence by stimulating students' internal motivation for independent learning, applying group task-driven learning, and giving teachers' feedback to students' sharing. It consists of four steps: teachers' guiding, students' self-regulated learning, team learning and practice, experience sharing. We have applied this method to the teaching practice of physiology and experimental physiological science with a significant impact on teaching effects. This teaching method has also been implemented to other courses in other majors. To solve the problems of reduced communication and interaction, low learning enthusiasm and motivation in online teaching course during COVID-19 pandemic, we recruited 21 undergraduates from different schools and majors. Using the "Tencent Meeting" platform, the authors tried to apply the whole process of the "Four-step Teaching of Encouraging and Sharing" to the online teaching of physiology. Group tests and questionnaires were used to evaluate teaching effects. The results showed that the implementation of the "Online Four-step Teaching of Encouraging and Sharing (OFST)" was feasible and effective, and to a certain extent alleviated the problems of loneliness and low learning motivation of students during online learning caused by home quarantine, which was particularly helpful for long-distance inter-school and inter-discipline team learning.

Estrogen-Related Receptor α (ERRα) and G Protein-Coupled Estrogen Receptor (GPER) Synergistically Indicate Poor Prognosis in Patients with Triple-Negative Breast Cancer.

PURPOSE: The present study aims to demonstrate the correlation between estrogen-related receptor α (ERRα) and G protein-coupled estrogen receptor (GPER) expression and its predictive role in the prognosis of patients with triple-negative breast cancer (TNBC). METHODS: A retrospective review of 199 cases of TNBC was conducted to assess the GPER and ERRα expression, and its clinicopathologic and prognostic implications. Subsequently, the effects of ERRα and GPER on cell viability, migration, and invasion induced by estrogen were also investigated in vitro. RESULTS: Compared to TNBCs with ERRα low expression, ERRα-high patients exhibited higher nuclear grade, more frequent lymph nodal metastasis, a higher rate of local recurrence, and distant metastasis. Survival analyses revealed that ERRα-high patients had decreased overall survival (OS), local recurrence-free survival (LRFS), and distant disease-free survival (DDFS) than ERRα-low patients. The GPER expression level positively correlated with ERRα (R=0.167, P=0.18), and TNBCs with ERRα-low/GPER-low demonstrated the best survival outcomes among groups. In vitro, E2 significantly enhanced cell viability, migration, and invasion in BT-549 and MDA-MB-231 cell lines, which was associated with the increased expression of ERRα. Moreover, the overexpression of ERRα induced by estrogen and G1 (GPER agonist) was reversed by knocking down of GPER and blocking the MAPK signaling with PD98059 in both cell lines. CONCLUSION: Our findings suggest that ERRα and GPER synergistically predict unfavorable prognosis in TNBCs. Mechanically, GPER mediates the upregulation expression of ERRα induced by estrogen and promotes cell viability, migration, and invasion.

The FTO/miR-181b-3p/ARL5B signaling pathway regulates cell migration and invasion in breast cancer.

BACKGROUND: N6-methyladenosine (m6 A) RNA modification has been demonstrated to be a significant regulatory process in the progression of various tumors, including breast cancer. Fat mass and obesity-associated (FTO) enzyme, initially known as the obesity-related protein, is the first identified m6 A demethylase. However, the relationship between FTO and breast cancer remains controversial. In this study, we aimed to elucidate the role and clinical significance of FTO in breast cancer and to explore the underlying mechanism. METHODS: We first investigated the expression of FTO in breast cancer cell lines and tissues by quantitative reverse transcription-PCR (qRT-PCR), Western blotting, and immunohistochemistry. Wound healing assay and Transwell assay were performed to determine the migration and invasion abilities of SKBR3 and MDA-MB453 cells with either knockdown or overexpression of FTO. RNA sequencing (RNA-seq) was conducted to decipher the downstream targets of FTO. qRT-PCR, luciferase reporter assay, and Western blotting were employed to confirm the existence of the FTO/miR-181b-3p/ARL5B axis. The biological function of ADP ribosylation factor like GTPase 5B (ARL5B) in breast cancer cells was evaluated by wound healing assay and Transwell invasion assay. RESULTS: High FTO expression was observed in human epidermal growth factor receptor 2 (HER2)-positive breast cancer, predicting advanced progression (tumor size [P < 0.001], nuclear grade [P = 0.001], peritumoral lymphovascular invasion [P < 0.001), lymph node metastasis [P = 0.002], and TNM stage [P = 0.001]) and poor prognosis. Moreover, FTO promoted cell invasion and migration in vitro. Mechanistically, RNA-seq and further confirmation studies suggested that FTO up-regulated ARL5B by inhibiting miR-181b-3p. We further verified that ARL5B also displayed carcinogenic activity in breast cancer cells. CONCLUSION: Our work demonstrated the carcinogenic activity of FTO in promoting the invasion and migration of breast cancer cells via the FTO/miR-181b-3p/ARL5B signaling pathway.

Preparedness of medical education in China: Lessons from the COVID-19 outbreak.

The COVID-19 outbreak can be seen as a 'big test' for China; a summative assessment of its preparedness on multiple fronts, including medical education. Being intimately involved in the coordinated response, the First Affiliated Hospital of Sun Yat-sen University has been a first-hand witness to the strengths and weaknesses of the current medical education system in China. On the one hand, we believe that the distinguished contributions in disease containment efforts by healthcare professionals indicated that our medical education system has achieved its intended outcomes and is socially accountable. On the other hand, we have also identified three major issues that need to be addressed from an educational standpoint: insufficient emphasis on public health emergency preparedness; unsophisticated mechanisms for interdisciplinary cooperation; and inadequate guidance in medical ethics. Whilst these reflections might be seen in its summative form, we would suggest changing it to that of a formative process, where we learn from our assessment through observation and feedback of the gaps, upon which improvement of our present situation can be made. We hope that these lessons may be helpful to our colleagues in the rest of China and around the world, who are engaged in medical educational reform.

H2 Protects Against Lipopolysaccharide-Induced Cardiac Dysfunction via Blocking TLR4-Mediated Cytokines Expression.

Background and Purpose: Septic cardiomyopathy, which is one of the features of multi-organ dysfunction in sepsis, is characterized by ventricular dilatation, reduced ventricular contractility, and reduction in ejection fraction and, if severe, can lead to death. To date, there is no specific therapy that exists, and its treatment represents a large unmet clinical need. Herein, we investigated the effects and underlying anti-inflammatory mechanisms of hydrogen gas in the setting of lipopolysaccharide (LPS)-induced cardiomyocytes injury. Experimental Approach: Hydrogen gas was intraperitoneally injected to mice in LPS plus hydrogen group and hydrogen group for 4 days. On fourth, LPS was given by intraperitoneal injection to mice in LPS group and to mice in LPS plus hydrogen group. In addition, H9c2 cardiomyocytes were treated with hydrogen-rich medium for 30 min before LPS. The transthoracic echocardiography was performed at 6 h post-LPS to assess left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction (EF%), fractional shortening (FS%), left ventricular mass average weight (LV mass AW), and LV mass AW (Corrected). The histological and morphological analyses of left ventricular were performed by hematoxylin and eosin (H&E) staining and Masson's trichrome staining. The mRNA levels of ANP and BNP were examined by PCR in vitro. The expression of cytokines were assayed by Enzyme Linked Immunosorbent Assay (ELISA) and PCR. Moreover, Western blotting was performed to examine the expression of TLR4, the activation of ERK1/2, p38, JNK, and the expression of NF-κB in nucleus after 6 h of LPS challenge in vivo and in vitro. Key Results: LPS induced cardiac dysfunction; hydrogen therapy improved cardiac function after LPS challenge. Furthermore, pretreatment with hydrogen resulted in cardioprotection during septic cardiomyopathy via inhibiting the expression of pro-inflammatory cytokines TNFα, IL-1ß, and IL-18; suppressing the phosphorylation of ERK1/2, p38, and JNK; and reducing the nuclear translocation of NF-κB and the expression of TLR4 by LPS. Conclusion and Implications: Hydrogen therapy prevents LPS-induced cardiac dysfunction in part via downregulation of TLR4-mediated pro-inflammatory cytokines expression.

Prognostic role of GPER/Ezrin in triple-negative breast cancer is associated with menopausal status.

The role of G protein-coupled estrogen receptor 1 (GPER) signaling, including promotion of Ezrin phosphorylation (which could be activated by estrogen), has not yet been clearly identified in triple-negative breast cancer (TNBC). This study aimed to evaluate the prognostic value of GPER and Ezrin in TNBC patients. Clinicopathologic features including age, menopausal status, tumor size, nuclear grade, lymph node metastasis, AJCC TNM stage, and ER, PR and HER-2 expression were evaluated from 249 TNBC cases. Immunohistochemical staining of GPER and Ezrin was performed on TNBC pathological sections. Kaplan-Meier analyses, as well as logistic regressive and Cox regression model tests were applied to evaluate the prognostic significance between different subgroups. Compared to the GPER-low group, the GPER-high group exhibited higher TNM staging (P = 0.021), more death (P < 0.001), relapse (P < 0.001) and distant events (P < 0.001). Kaplan-Meier analysis showed that GPER-high patients had a decreased OS (P < 0.001), PFS (P < 0.001), LRFS (P < 0.001) and DDFS (P < 0.001) than GPER-low patients. However, these differences in prognosis were not statistically significant in post-menopausal patients (OS, P = 0.8617; PFS, P = 0.1905; LRFS, P = 0.4378; DDFS, P = 0.2538). There was a significant positive correlation between GPER and Ezrin expression level (R = 0.508, P < 0.001) and the effect of Ezrin on survival prognosis corresponded with GPER. Moreover, a multivariable analysis confirmed that GPER and Ezrin level were both significantly associated with poor DDFS (HR: 0.346, 95% CI 0.182-0.658, P = 0.001; HR: 0.320, 95% CI 0.162-0.631, P = 0.001). Thus, overexpression of GPER and Ezrin may contribute to aggressive behavior and indicate unfavorable prognosis in TNBC; this may correspond to an individual's estrogen levels.

Oestrogen Inhibits VEGF Expression And Angiogenesis In Triple-Negative Breast Cancer By Activating GPER-1.

Triple-negative breast cancer (TNBC) is the most malignant type of breast cancer with ample vascularisation and high vascular endothelial growth factor (VEGF) expression. The sex steroid hormone oestrogen is involved in several cellular activities associated with TNBC regulation. However, the role of oestrogen in VEGF expression and angiogenesis in TNBC remains unclear. In this study, we found that treatment with 17ß-oestradiol (E2) inhibited VEGF mRNA and protein expression in the TNBC cell lines MDA-MB-468 and MDA-MB-436. To further elaborate on the phenomenon of E2-regulated angiogenesis, we showed that conditioned medium from the TNBC cell line MDA-MB-468 treated with E2 inhibits the tube formation ability of human umbilical vein endothelial cells (HUVECs). Additionally, the G-protein-coupled oestrogen receptor-1 (GPER-1)-specific agonist G-1 has a function similar to that of E2. While G-15, the selective antagonist of GPER-1, notably reversed the inhibitory effects of E2 and G-1 on VEGF expression and tube formation, suggesting that GPER-1 is involved in the E2-induced angiogenesis suppression in TNBC cells. Moreover, E2 inhibited in vivo tumour growth and angiogenesis and reduced the expression levels of VEGF, NF-κB/p65, STAT3, and the endothelial marker CD34 in MDA-MB-468 xenograft tumours. Our findings provide important evidence that E2 can inhibit VEGF expression and angiogenesis in TNBC by activating GPER-1, offering additional insight into tumour angiogenesis and targets for drug intervention in TNBC.

Endothelial function and dysfunction: Impact of metformin.

Cardiovascular and metabolic diseases remain the leading cause of morbidity and mortality worldwide. Endothelial dysfunction is a key player in the initiation and progression of cardiovascular and metabolic diseases. Current evidence suggests that the anti-diabetic drug metformin improves insulin resistance and protects against endothelial dysfunction in the vasculature. Hereby, we provide a timely review on the protective effects and molecular mechanisms of metformin in preventing endothelial dysfunction and cardiovascular and metabolic diseases.

Hydrogen Therapy in Cardiovascular and Metabolic Diseases: from Bench to Bedside.

Hydrogen (H2) is colorless, odorless, and the lightest of gas molecules. Studies in the past ten years have indicated that H2 is extremely important in regulating the homeostasis of the cardiovascular system and metabolic activity. Delivery of H2 by various strategies improves cardiometabolic diseases, including atherosclerosis, vascular injury, ischemic or hypertrophic ventricular remodeling, intermittent hypoxia- or heart transplantation-induced heart injury, obesity and diabetes in animal models or in clinical trials. The purpose of this review is to summarize the physical and chemical properties of H2, and then, the functions of H2 with an emphasis on the therapeutic potential and molecular mechanisms involved in the diseases above. We hope this review will provide the future outlook of H2-based therapies for cardiometabolic disease.

Hydrogen inhibits isoproterenol­induced autophagy in cardiomyocytes in vitro and in vivo.

A previous study from our group has demonstrated that hydrogen administration can attenuate cardiovascular hypertrophy in vivo by targeting reactive oxygen species­dependent mitogen­activated protein kinase signaling. The aim of the present study is to determine the effect of hydrogen on cardiomyocyte autophagy during ß­adrenoceptor activation in vivo and in vitro. We prepared hydrogen­rich medium, and the concentration of hydrogen was measured by using the MB­Pt reagent method. For the in vitro study, H9c2 cardiomyocytes were stimulated with isoproterenol (ISO; 10 µM) for 5, 15 and 30 min, and then the protein expression levels of the autophagy marker microtubule­associated protein 1 light chain 3ß II (LC3B II) were examined by western blotting. The effect of hydrogen­rich medium was then tested by pretreating the H9c2 cardiomyocytes with hydrogen­rich medium for 30 min, then stimulating with ISO, and examining the protein expression levels of the autophagy marker LC3B II. For the in vivo study, mice received hydrogen (1 ml/100 g/day, by intraperitoneal injection) for 7 days prior to ISO administration (0.5 mg/100 g/day, by subcutaneous injection), and subsequently received hydrogen with or without ISO for another 7 days. Hypertrophic responses were examined by heart weight (HW) and heart weight/body weight (HW/BW) measurements. The protein expression of autophagy markers Beclin1, autophagy­related protein 7 (Atg7) and LC3B II were examined. The results demonstrated that excessive autophagy occurred following 5 min of ISO stimulation in vitro. This enhanced autophagy was blocked by pretreatment with hydrogen­rich medium. Furthermore, hydrogen improved the deteriorated hypertrophic responses and inhibited the enhanced autophagic activity mediated by ISO administration in vivo, as indicated by decreasing HW and HW/BW, and suppressing the protein expression levels of Beclin1, Atg7 and LC3B II. Therefore, the results of the present study demonstrated that hydrogen inhibited ISO­induced excessive autophagy in cardiomyocyte hypertrophy models in vitro and in vivo.

Elucidating the role of the FoxO3a transcription factor in the IGF-1-induced migration and invasion of uveal melanoma cancer cells.

Uveal melanoma (UM) is the most common primary intraocular malignant tumor of adults. It has high mortality rate due to liver metastasis. However, the epidemiology and pathogenesis of liver metastasis in UM are not elucidated and there is no effective therapy available for preventing the development of this disease. IGF-1 is a growth factor involved in cell proliferation, malignant transformation and inhibition of apoptosis. In previous report, IGF-1 receptor was found to be highly expressed in UM and this was related to tumor prognosis. FoxO3a is a Forkhead box O (FOXO) transcription factor and a downstream target of the IGF-1R/PI3K/Akt pathway involved in a number of physiological and pathological processes including cancer. However, the role of FoxO3a in UM is unknown. In the present study, we investigated fundamental mechanisms in the growth, migration and invasion of UM and the involvement of FoxO3a. IGF-1 increased the cell viability, invasion, migration and S-G2/M cell cycle phase accumulation of UM cells. Western blot analysis showed that IGF-1 led to activation of Akt and concomitant phosphorylation of FoxO3a. FoxO3a phosphorylation was associated with its translocation into the cytoplasm from the nucleus and its functional inhibition led to the inhibition of expression of Bim and p27, but an increase in the expression of Cyclin D1. The effects of IGF-1 on UM cells were reversed by LY294002 (a PI3K inhibitor) or Akt siRNA, and the overexpression of FoxO3a also attenuated basal invasion and migration of UM. Taken all together, these results suggest that inhibition of FoxO3a by IGF-1 via the PI3K/Akt pathway has an important role in IGF-1 induced proliferation and invasion of UM cells. These findings also support FoxO3a and IGF signaling may represent a valid target for investigating the development of new strategies for the treatment and prevention of the pathology of UM.

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.

[Advances of the Role of Ezrin in Migration and Invasion of Breast Cancer Cells].

Ezrin, also known as cytovillin or vilin 2, is one of the members of ERM (Ezrin/Radixin/Moesin) protein family. Ezrin, which is a tyrosine kinase substrate, functions to bridge membrane proteins and the actin cytoskeleton. Recent studies have demonstrated that Ezrin regulates the proliferation, apoptosis, adhesion, invasion, metastasis and angiogenesis of breast cancer cells. These processes are not only associated with changes in expression level and subcellular localization of Ezrin itself, but also influenced by alteration in microenvironment of primary breast cancer cells. The regulation of Ezrin in mammary carcinoma cells involves interactions among signaling pathways mediated by adhesion molecules (CD44, ICAM, E-cadherin) and the tyrosine kinase growth factors, Epidermal Growth Factor (EGF), and Platelet-derived Growth Factor (PDGF) and their receptors. The determination of the functions and mechanism(s) of action of Ezrin in the migration and invasion of breast cancer cells will provide new information on the basic mechanisms of metastasis of breast cancer cells and has the potential to identify a novel drug target for the prevention and treatment of breast cancer. This article addresses the role of Ezrin in the migration and invasion of breast cancer cells.

Oestrogen exerts anti-inflammation via p38 MAPK/NF-κB cascade in adipocytes.

BACKGROUND: Oestrogen has anti-inflammatory property in obesity. However, the mechanism is still not defined. OBJECTIVE: To investigate the effect of oestrogen on LPS-induced monocyte chemoattractant protein-1 (MCP-1) production in adipocytes. METHODS: Lipopolysaccharides (LPS) was used to imitate inflammatory responses and monocyte chemotactic protein-1 (MCP-1) was selected as an inflammatory marker to observe. 17ß-Estradiol (E2), SB203580 (SB), pyrrolidine dithiocarbamate (PDTC), pertussis toxin (PTX), wortmannin (WM), p65 siRNA and p38 MAPK siRNA were pre-treated respectively or together in LPS-induced MCP-1. Then p38 MAPK and NF-κB cascade were silenced successively to observe the change of each other. Lastly, oestrogen receptor (ER) α agonist, ERß agonist and ER antagonist were utilised. RESULTS: LPS-induced MCP-1 largely impaired by pre-treatment with E2, SB, PDTC or silencing NF-κB subunit. E2 inhibited LPS-induced MCP-1 in a time- and dose-dependent manner, which was related to the suppression of p65 translocation to nucleus. Furthermore, LPS rapidly activated p38 MAPK, while E2 markedly inhibited this activation. It markedly attenuated LPS-stimulated p65 translocation to nucleus and MCP-1 production by transfecting with p38 MAPK siRNA or using p38 MAPK inhibitor. The oestrogen's inhibitory effect was mimicked by the ERα agonist, but not by the ERß agonist. The inhibition of E2 on p38 MAPK phosphorylation was prevented by ER antagonist. CONCLUSIONS: E2 inhibits LPS-stimulated MCP-1 in adipocytes. This effect is related to the inhibition of p38 MAPK/NF-κB cascade, and ERα appears to be the dominant ER subtype in these events.

Estrogen stimulated migration and invasion of estrogen receptor-negative breast cancer cells involves an ezrin-dependent crosstalk between G protein-coupled receptor 30 and estrogen receptor beta signaling.

Estrogen mediates important cellular activities in estrogen receptor negative (ER-) breast cancer cells via membrane associated G protein-coupled receptor 30 (GPR30). However, the biological role and mechanism of estrogen action on cell motility and invasion in this aggressive kind of tumors remains poorly understood. We showed here that treatment with 17ß-estradiol (E2) in ER-negative cancer cells resulted in ezrin-dependent cytoskeleton rearrangement and elicited a stimulatory effect on cell migration and invasion. Mechanistically, E2 induced ezrin activation was mediated by distinct mechanisms in different cell contexts. In SK-BR-3 cells with a high GPR30/ERß ratio, silencing of GPR30 was able to abolish E2 induced ERK1/2, AKT phosphorylation and ezrin activation, whereas in MDA-MB-231 cells with low GPR30/ERß ratio, E2 stimulated ezrin activation was mediated by the ERß/PI3K/AKT signaling pathway. Importantly, we showed that activation of GPR30 signaling significantly prevents ERß activation induced ezrin phosphorylation, cell migration and invasion, indicating an antagonist effect between GPR30 and ERß signaling in MDA-MB-231 cells. These findings highlight the important interplay between different estrogen receptors in estrogen induced cell motility and invasiveness in ER-negative breast cancer cells.

Inhibitory Effects of Hydrogen on Proliferation and Migration of Vascular Smooth Muscle Cells via Down-Regulation of Mitogen/Activated Protein Kinase and Ezrin-Radixin-Moesin Signaling Pathways.

Molecular hydrogen (H2) has recently attracted considerable attention for the prevention of oxidative stress-related vascular diseases. The purpose of this study is to evaluate the effects of hydrogen on proliferation and migration of vascular smooth muscle cells (VSMCs) stimulated by angiotensin II (Ang II) in vitro, and on vascular hypertrophy induced by abdominal aortic coarctation (AAC) in vivo. Hydrogen-rich medium (0.6~0.9 ppm) was added 30 min before 10⁻7 M Ang II administration, then the proliferation and migration index were determined 24 h after Ang II stimulation. Hydrogen gas (99.999%) was given by intraperitoneal injection at the dose of 1 ml/100 g/day consecutively for one week before AAC and lasted for 6 weeks in vivo. Hydrogen inhibited proliferation and migration of VSMCs with Ang II stimulation in vitro, and improved the vascular hypertrophy induced by AAC in vivo. Treatment with hydrogen reduced Ang II- or AAC-induced oxidative stress, which was reflected by diminishing the induction of reactive oxygen species (ROS) in Ang II-stimulated VSMCs, inhibiting the levels of 3-nitrotyrosine (3-NT) in vascular and serum malondialdehyde (MDA). Hydrogen treatment also blocked Ang II-induced phosphorylation of the extracellular signal-regulated kinase1/2 (ERK1/2), p38 MAPK, c-Jun NH2-terminal kinase (JNK) and the ezrin/radixin/moesin (ERM) in vitro. Taken together, our studies indicate that hydrogen prevents AAC-induced vascular hypertrophy in vivo, and inhibits Ang II-induced proliferation and migration of VSMCs in vitro possibly by targeting ROS-dependent ERK1/2, p38 MAPK, JNK and ERM signaling. It provides the molecular basis of hydrogen on inhibiting the abnormal proliferation and migration of VSMCs and improving vascular remodeling diseases.

Chronic vagus nerve stimulation attenuates vascular endothelial impairments and reduces the inflammatory profile via inhibition of the NF-κB signaling pathway in ovariectomized rats.

Vagus nerve stimulation (VNS), a method for activating cholinergic anti-inflammatory pathways, could suppress endothelial activation and minimize tissue injury during inflammation. The aim of this study was to investigate the effects of chronic VNS on endothelial impairments and the inflammatory profile in ovariectomized (OVX) rats. Sprague-Dawley rats (7-8 months old) were randomly assigned to the following four groups: sham-OVX, OVX, OVX+sham-VNS, and OVX+VNS. Throughout the experimental period, the OVX+VNS group received VNS for 3h (20.0 Hz, 1.0 mA, and 10.00 ms pulse width) at the same time every other day. After 12 weeks of VNS, blood samples and thoracic aortas were collected for further analyses. Light microscopy and electron microscopy analyses showed that chronic VNS prevented endothelial swelling, desquamation and even necrosis in the OVX rats. In addition, it obviously improved endothelial function in the OVX rats by restoring the endothelial nitric oxide synthase (e-NOS) and serum endothelin-1 level. Increased expression of cell adhesion molecules (VCAM-1, ICAM-1 and E-selectin) in the thoracic aortas and increases in the levels of circulating cytokines (TNF-α, IL-6, MCP-1, and CINC/KC) were also observed in the OVX rats. Chronic VNS significantly restored these detrimental changes partly by increasing the ACh concentrations in vascular walls and blocking NF-κB pathway activity. The results of this in vivo study have shown that the administration of chronic VNS during, in the early stage of estrogen deficiency, protects OVX rats from endothelial impairments and the inflammatory profile. These findings indicate that activation of the vagus nerve could be a promising supplemental therapy for reducing the risks of suffering from further CVDs in postmenopausal women.