Oxidized low-density lipoprotein changes the inflammatory status and metabolomics profiles in human and mouse macrophages and microglia.

The conjunctiva of primary open angle glaucoma patients showed high level of oxidized low-density lipoprotein (ox-LDL), which is associated with the inflammatory response. Microglia and macrophages are the immune cells involved in retinal ganglion cell survival regulation; yet, their roles of the ox-LDL-induced inflammation in glaucoma remain elusive. Here we aimed to investigate the lipid uptake, inflammatory cytokine expression, and metabolomics profiles of human and murine-derived microglial and macrophage cell lines treated with ox-LDL. Under the same ox-LDL concentration, macrophages exhibited higher lipid uptake and expression of pro-inflammatory cytokines as compared to microglia. The ox-LDL increased the levels of fatty acid metabolites in macrophages and sphingomyelin metabolites in microglia. In summary, this study revealed the heterogeneity in the inflammatory capacity and metabolic profiles of macrophages and microglia under the stimulation of ox-LDL.

Neuromedin U programs eosinophils to promote mucosal immunity of the small intestine.

Eosinophils are granulocytes that play an essential role in type 2 immunity and regulate multiple homeostatic processes in the small intestine (SI). However, the signals that regulate eosinophil activity in the SI at steady state remain poorly understood. Through transcriptome profiling of eosinophils from various mouse tissues, we found that a subset of SI eosinophils expressed neuromedin U (NMU) receptor 1 (NMUR1). Fate-mapping analyses showed that NMUR1 expression in SI eosinophils was programmed by the local microenvironment and further enhanced by inflammation. Genetic perturbation and eosinophil-organoid coculture experiments revealed that NMU-mediated eosinophil activation promotes goblet cell differentiation. Thus, NMU regulates epithelial cell differentiation and barrier immunity by stimulating NMUR1-expressing eosinophils in the SI, which highlights the importance of neuroimmune-epithelial cross-talk in maintaining tissue homeostasis.

Celastrol inhibits laser-induced choroidal neovascularization by decreasing VEGF induced proliferation and migration.

AIM: To evaluate celastrol's effect on choroidal neovascularization (CNV). METHODS: In this study, neovascular formation in vitro (tube formation and aortic ring culture) and in vivo (laser induced neovascular in mice) was treated with celastrol to evaluate this natural compound's impact on CNV. Western blot was applied to explore the possible mechanism for it. For in vitro assay, triplicate for each group was repeated at least three times. For in vivo assay, each group contains 5 mice. RESULTS: Celastrol supressed tube formation and aortic ring sprout neovascularization. In vitro assay exhibited that celastrol inhibiting vascular endothelial growth factor (VEGF)-induced proliferation and migration of human umbilical vein endothelial cells and human choroidal endothelial cells, and by blocking VEGF signaling. Furthermore, intraperitoneal administration of celastrol significantly reduced the area of laser-induced CNV in an in vivo mouse model. By day 14, the area of CNV had decreased by 49.15% and 80.26% in the 0.1 mg/kg celastrol-treated group (n=5) and in the 0.5 mg/kg celastrol treated group (n=5), respectively, compared to the vehicle-treated group (n=5). CONCLUSION: Celastrol inhibits CNV by inhibiting VEGF-induced proliferation and migration of vascular endothelial cells, indicating that celastrol is a potent, natural therapeutic compound for the prevention of CNV.

Establishment and clinical application of time-resolved immunofluorescence assay of lipoprotein-associated phospholipase A2.

AIM: This study aimed to establish a highly sensitive time-resolved fluorescence immunoassay (TRFIA) for the detection of serum lipoprotein-associated phospholipase A2 (Lp-PLA2) and evaluate the clinical application value of Lp-PLA2 in patients with breast cancer. METHODS: The level of Lp-PLA2 was detected using the double-antibody sandwich method. First, the Lp-PLA2-TRFIA method was established, and the method was evaluated on the basis of linearity, sensitivity, precision, specificity, and recovery rate. Then, the fluorescence counts in serum of healthy subjects and patients with breast cancer were detected by Lp-PLA2-TRFIA, and the levels of Lp-PLA2 were calculated using a standard curve. RESULTS: Lp-PLA2-TRFIA had a wide linear range (43.48-2000 ng/mL). The intra-assay precisions of Lp-PLA2-TRFIA ranged from 2.66% to 4.84% (<10%), and the inter-assay precisions were between 5.39% and 6.95% (<15%). No cross-reaction was observed among Lp-PLA2, Tumor-associated trypsinogen-2, and T-cell immunoglobulin mucin 3. In addition, the recovery rates were between 90% and 100%. The serum Lp-PLA2 levels of patients with breast cancer were significantly higher than those of healthy subjects. CONCLUSIONS: We successfully established a highly sensitive Lp-PLA2-TRFIA method, and found serum Lp-PLA2 may be associated with dyslipidemia in breast cancer and could be used for auxiliary diagnose.

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.

Assessment of Electrical Resistivity and Oxygen Diffusion Coefficient of Cementitious Materials from Microstructure Features.

A newly proposed modified non-contact electrical resistivity measurement was used to test the resistivity of concrete and cement mortar. The oxygen diffusion coefficients of concrete and mortar were determined by a gas diffusion measurement, and the capillary porosity of concrete and cement mortar was measured by mercury intrusion porosimetry (MIP) measurement. The obtained electrical resistivity and capillary porosity results were verified with other researchers' data, the measured electrical resistivity results can be estimated by a simple equation from the capillary porosity results. The obtained oxygen diffusion coefficient results were quantitatively correlated with capillary porosity and electrical resistivity measurement results. The proposed equations can be practically used to assess the electrical resistivity and oxygen diffusion coefficient.

The GPER1/SPOP axis mediates ubiquitination-dependent degradation of ERα to inhibit the growth of breast cancer induced by oestrogen.

G protein-coupled oestrogen receptor 1 (GPER1), predicted to be a novel oestrogen receptor, has been linked to the development and progression of breast cancer. However, the molecular mechanisms underlying its functions remain elusive. Here, we show that the protein levels of GPER1 are negatively associated with those of ERα and that higher expression of GPER1 correlated with a better clinical outcome in oestrogen receptor-positive (ER+) breast cancer patients. Activation of GPER1 decreases ERα protein levels, which subsequently suppresses ERα-mediated transcription and target gene expression but does not affect its mRNA expression in ER + breast cancer cells. A mechanistic study revealed that GPER1 mediates ubiquitin (Ub)-proteasome-dependent degradation of ERα via upregulation of the Cullin3-based E3 ubiquitin ligase adaptor protein speckle-type POZ protein (SPOP), and depletion of SPOP abrogates GPER1-induced ERα ubiquitination and degradation. Functionally, GPER1 activation inhibits 17ß-oestradiol (E2)-induced ER + breast cancer cell proliferation, migration, and invasion in vitro and tumour growth in vivo. Our findings reveal a novel mechanism by which GPER1 negatively modulates the ERα signalling pathway via promoting its ubiquitin-proteasome-dependent degradation, which may contribute to its inhibition of breast cancer progression.

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.

RNA sequencing analysis of long non-coding RNA expression in ocular posterior poles of guinea pig myopia models.

Purpose: To detect the differential expression of long non-coding RNAs (lncRNAs) in the ocular posterior poles of two guinea pig myopia models and explore the pathogenic role of lncRNAs in myopia. Methods: Form-deprived myopia (FDM) and lens-induced myopia (LIM) models were induced in guinea pig right eyes by wearing a translucent latex balloon head mask and a -10.00 diopter (D) lens, respectively. Ocular biometric parameters were measured biweekly. At 6 weeks after the induction of myopia, the guinea pig eyeballs were processed for hematoxylin and eosin staining to examine the ocular morphology. The ocular posterior poles from the normal control, FDM, and LIM groups were collected to analyze the differential expression of lncRNAs between the groups with high-throughput RNA sequencing (RNA-seq). Further, the lncRNA-mRNA colocation network was delineated to predict the functions of the differentially expressed lncRNAs. Last, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on the colocated mRNAs of the differentially expressed lncRNAs. Additionally, the expression of the most differentially expressed lncRNAs in the myopia-induced eyes and the contralateral eyes was validated with quantitative real-time PCR (qPCR). Results: Compared with the normal controls and the contralateral eyes, the myopia-induced eyes in the FDM and LIM groups exhibited decreased scleral and choroidal thicknesses, reduced refraction, and increased ocular axial length but without changes in the corneal curvature radius at 6 weeks after myopia was induced. RNA-seq showed that 372 and 247 lncRNAs were differentially expressed in the FDM and LIM groups, respectively, in comparison to the normal counterparts. There were 380 differentially expressed lncRNAs in the LIM group compared to the FDM group. The GO and KEGG analyses showed that the colocated mRNAs of the differentially expressed lncRNAs were enriched in cellular components such as the extracellular matrix (ECM) structural constituent; in molecular functions such as kinase activity, metabolism, and growth; as well as in pathways including ECM-receptor interaction, glycosaminoglycan degradation, and mucin type O-Glycan biosynthesis. The expression patterns of the selected lncRNAs were verified with qPCR. Conclusions: High-throughput RNA-seq revealed previously undescribed lncRNA expression profiling in guinea pig FDM and LIM models. These results may shed light on the molecular pathogenesis of myopia and provide clues for interventional targets for this highly prevalent visual disorder.

Celastrol Protects RPE Cells from Oxidative Stress-Induced Cell Death via Activation of Nrf2 Signaling Pathway.

PURPOSE: Oxidative stress to retinal pigment epithelial (RPE) cells and inflammation are closely related to the pathogenesis of age-related macular degeneration (AMD). Celastrol is a natural compound isolated from the root of Tripterygium wilfordii. Celastrol has been shown to have potent anti-inflammatory and anti-tumor effects in multiple disease models. The objective of this study was to test the anti-oxidative effects of celastrol in RPE cells and to investigate the underlying mechanisms. METHODS: ARPE-19 cells were treated with hydrogen peroxide (H2O2) and menadione alone or in combination with celastrol. Cell viability and apoptosis were examined by CCK-8 and TUNEL assay, respectively. The expression of Nrf2 and its target genes, such as GCLM and HO-1 was determined by Western blotting. The knockdown of Nrf2 was done by transfecting ARPE-19 cells with lentivirus encoding shRNA against Nrf2. The knockdown efficiency was determined by real-time quantitative PCR and Western blotting. RESULTS: Treatment of ARPE-19 cells with celastrol significantly attenuated the toxic effects of both H2O2 and menadione. Treatment with celastrol enhanced the expression of transcription factor Nrf2 and its targets, GCLM and HO-1. Knockdown of Nrf2 expression by shRNA partially abolished the protective effects of celastrol. Chemical inhibition of glutathione synthesis by L-buthionine-S,R-sulfoximine (BSO) completely abolished the protective effects of celastrol against H2O2 and menadione-induced damage. However, chemical inhibition of HO-1 activity by ZnPPIX did not reduce the protective effects of celastrol. CONCLUSION: This study provides evidence that treatment of RPE cells with celastrol shows potent protective effects against oxidative insults via activation of Nrf2 signaling pathway and upregulation of GCLM expression. This finding suggests that celastrol might be used as a potential therapeutic agent for oxidative stress-related eyes diseases, such as AMD.

Celastrol Ameliorates Inflammation in Human Retinal Pigment Epithelial Cells by Suppressing NF-κB Signaling.

PURPOSE: Celastrol is a triterpenoid quinine methide that exerts important biological effects on a variety of disease models. In this study, we aim to assess the ability of celastrol to inhibit lipopolysaccharide (LPS)-induced inflammation in retinal pigment epithelial (RPE) cells. METHODS: Primary cultures of human RPE (HRPE) cells and ARPE-19 cell lines were treated with celastrol alone or in combination with LPS. The cytotoxic effect of celastrol on RPE cells was determined by the CCK-8 assay. Protein and mRNA levels of inflammatory cytokines, including IL-6, IL-8, and MCP-1, were detected by flow cytometry or by real-time fluorescent quantitative PCR, respectively. The levels of phosphorylated intermediates in the NF-κB signaling pathway (such as IκBα/ß and p65) and MAPK signaling pathway (p38MAPK, SAPK/JNK, and p42/p44MAPK) were detected by western blotting. RESULTS: Celastrol significantly inhibited LPS-induced expression of protein and mRNA expression levels encoding the proinflammatory cytokines, IL-6, IL-8, and MCP-1, in both HRPE and ARPE-19 cells. Cell viability and apoptosis assays revealed that celastrol had no apparent cytotoxic effect and it inhibited apoptosis of RPE cells at concentrations of less than 1 µM. Mechanistically, RPE cells that were pretreated with celastrol exhibited a substantial decrease in phosphorylation of the NF-κB pathway regulators, IKKα/ß and IκBα, and subsequently inactivated P65, suggesting that celastrol ameliorates LPS-induced inflammation by suppressing the NF-κB signaling pathway. CONCLUSION: Our results provide evidence that celastrol is a potent anti-inflammatory agent in RPE cells and it may have potential applications in prevention and treatment of age-related macular degeneration.

Fabrication and Mechanical Properties of Tungsten Inert Gas Welding Ring Welded Joint of 7A05-T6/5A06-O Dissimilar Aluminum Alloy.

In this paper, Tungsten Inert Gas Welding (TIG) and single-sided welding and double-sided forming have been used to weld the 7A05-T6/5A06-O dissimilar aluminum alloy circular welded joint of a ring-stiffened closed cylindrical sandwich shell. Microstructural characterization and mechanical properties of welded joints were investigated by use of scanning electron microscopy (SEM), backscatter electron diffraction (EBSD) and transmission electron microscopy (TEM), respectively. Hardness distribution and tensile properties of the welded joints were examined. The results showed the failure of the welded joints produced in the fusion zone (FZ). The tensile strength and yield strength of the welded joints were, respectively, 78.87% and 97.24% of the 5A06-O base metal (BM), and the elongation reached 84.29% of 7A05-T6 base metal. Welding high heat input led to the coarse grain size in the fusion zone, and the long-term similar quenching effect can lead to the full dissolution of the strengthening zone of the fusion zone resulting in the reductions of strength and hardness. Around 7A05 heat-affected zone (HAZ), there is an obvious hardening zone and softening zone, the solid solution precipitates into the Rayleigh brilliant η′ (MgZn2) phase, which results in natural aging strengthening, thus obtaining high hardness. η′ (MgZn2) enhanced phase dissolved fully and the dislocation density decreased rapidly in the HAZ region resulted in a softening zone with a lower hardness at about (10⁻18) mm from the center of the weld center.

Expression of cold-inducible RNA-binding protein (CIRP) in renal cell carcinoma and the effect of CIRP downregulation cell proliferation and chemosensitivity to gemcitabine.

The aim of the present study was to investigate the expression of cold-inducible RNA-binding protein (CIRP) in renal cell carcinoma (RCC) and to determine the effects of downregulation of CIRP on cell proliferation and chemosensitivity to gemcitabine. The expression of CIRP was detected by western blot analysis, quantitative polymerase chain reaction and immunohistochemistry (IHC) in 17 RCC and peri-cancerous tissue samples. Subsequently, the RCC 786-0 cell line was selected in order to investigate the function of CIRP using RNA interference (RNAi) technology, which was able to inhibit the expression of CIRP in vitro. Furthermore, the chemosensitivity to gemcitabine of each group [CIRP small interfering RNA (siCIRP), negative control small interfering RNA (siNC) and blank control] was compared. There were marked differences between the RCC and peri-cancerous tissues. IHC demonstrated that the CIRP expression in 13/17 (76.50%) tumor samples was markedly positive compared with that in the peri-cancerous tissues and the most common pathological type was clear cell RCC (92.30%). This observation was further confirmed through western blot analysis of protein expression levels. CIRP downregulation by RNAi in the RCC 786-0 cell line significantly decreased RCC proliferation. Additionally, when RNAi was coupled with gemcitabine treatment, there was a significant increase in apoptosis in the siCIRP group. CIRP was overexpressed in RCC tissues and in the 786-0 cell line. Downregulation of CIRP by siRNA inhibited the proliferation of the 786-0 cell line and enhanced the chemosensitivity of the cells to gemcitabine. Therefore, CIRP downregulation may provide a novel pathway for the treatment of metastatic RCC.

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.

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.

17ß-estradiol induces vasorelaxation by stimulating endothelial hydrogen sulfide release.

Estrogen exerts vascular protective effects, but the underlying mechanisms remain to be understood fully. In recent years, hydrogen sulfide (H(2)S) has increasingly been recognized as an important signaling molecule in the cardiovascular system. Vascular H(2)S is produced from L-cysteine, catalyzed by cystathionine γ-lyase (CSE). In our study, apolipoprotein E (ApoE)-deficient mice were ovariectomized and implanted with placebo (OVX mice) or 17ß-estradiol (E(2)) pellets (OVX + E(2) mice). Compared with OVX mice, OVX + E(2) mice showed increased plasma H(2)S levels (P = 0.012) and decreased aortic lesion area (P = 0.028). These effects were largely reversed when supplementing with the irreversible CSE inhibitor DL-propargylglycine (PPG) in the OVX + E(2) + PPG mice. Meanwhile, the nitric oxide and prostacyclin-resistant responses to cumulative application of acetylcholine (ACh) were studied among all the three groups of femoral arteries. Compared with the arteries in the OVX group, the vasodilator sensitivity of arteries to ACh was increased in the OVX + E(2) group and attenuated in the OVX + E(2) + PPG group. E(2) and estrogen receptor (ER) α agonist 4',4″,4'″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol rapidly increased H(2)S release in human endothelial cells, but not partially selective ERß agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile. These effects were inhibited by ER antagonist ICI 182780 or by protein kinase G (PKG) inhibitor KT5823. Furthermore, endothelial PKG activity was increased by E(2) (P = 0.003) and E(2)-induced vasodilation was inhibited by KT5823 (P = 0.009). In conclusion, the endothelial CSE/H(2)S pathway is activated by E(2) through PKG, which leads to vasodilation. These actions may be relevant to estrogen's anti-atherogenic effect.