Five-lipoxygenase-activating protein-mediated CYLD attenuation is a candidate driver in hepatic malignant lesion.

Hepatocellular carcinoma (HCC) is an inflammation-associated cancer. However, the lipid pro-inflammatory mediators have only been seldom investigated in HCC pathogenesis. Cylindromatosis (CYLD) attenuation is involved in hepatocarcinogenesis. Here, we aimed to evaluate the significance of hepatic lipid pro-inflammatory metabolites of arachidonate-affected CYLD expression via the 5-lipoxygenase (5-LO) pathway. Resection liver tissues from HCC patients or donors were evaluated for the correlation of 5-LO/cysteinyl leukotrienes (CysLTs) signaling to the expression of CYLD. The impact of functional components in 5-LO/CysLTs cascade on survival of HCC patients was subsequently assessed. Both livers from canines, a preponderant animal for cancer research, and genetic-modified human HCC cells treated with hepatocarcinogen aristolochic acid I (AAI) were further used to reveal the possible relevance between 5-LO pathway activation and CYLD suppression. Five-LO-activating protein (FLAP), an essential partner of 5-LO, was significantly overexpressed and was parallel to CYLD depression, CD34 neovascular localization, and high Ki-67 expression in the resection tissues from HCC patients. Importantly, high hepatic FLAP transcription markedly shortened the median survival time of HCC patients after surgical resection. In the livers of AAI-treated canines, FLAP overexpression was parallel to enhanced CysLTs contents and the simultaneous attenuation of CYLD. Moreover, knock-in FLAP significantly diminished the expression of CYLD in AAI-treated human HCC cells. In summary, the hepatic FLAP/CysLTs axis is a crucial suppressor of CYLD in HCC pathogenesis, which highlights a novel mechanism in hepatocarcinogenesis and progression. FLAP therefore can be explored for the early HCC detection and a target of anti-HCC therapy.

Identification of Crucial Genes and Key Functions in Type 2 Diabetic Hearts by Bioinformatic Analysis.

Type 2 diabetes (T2D) patients with SARS-CoV-2 infection hospitalized develop an acute cardiovascular syndrome. It is urgent to elucidate underlying mechanisms associated with the acute cardiac injury in T2D hearts. We performed bioinformatic analysis on the expression profiles of public datasets to identify the pathogenic and prognostic genes in T2D hearts. Cardiac RNA-sequencing datasets from db/db or BKS mice (GSE161931) were updated to NCBI-Gene Expression Omnibus (NCBI-GEO), and used for the transcriptomics analyses with public datasets from NCBI-GEO of autopsy heart specimens with COVID-19 (5/6 with T2D, GSE150316), or dead healthy persons (GSE133054). Differentially expressed genes (DEGs) and overlapping homologous DEGs among the three datasets were identified using DESeq2. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses were conducted for event enrichment through clusterProfile. The protein-protein interaction (PPI) network of DEGs was established and visualized by Cytoscape. The transcriptions and functions of crucial genes were further validated in db/db hearts. In total, 542 up-regulated and 485 down-regulated DEGs in mice, and 811 up-regulated and 1399 down-regulated DEGs in human were identified, respectively. There were 74 overlapping homologous DEGs among all datasets. Mitochondria inner membrane and serine-type endopeptidase activity were further identified as the top-10 GO events for overlapping DEGs. Cardiac CAPNS1 (calpain small subunit 1) was the unique crucial gene shared by both enriched events. Its transcriptional level significantly increased in T2D mice, but surprisingly decreased in T2D patients with SARS-CoV-2 infection. PPI network was constructed with 30 interactions in overlapping DEGs, including CAPNS1. The substrates Junctophilin2 (Jp2), Tnni3, and Mybpc3 in cardiac calpain/CAPNS1 pathway showed less transcriptional change, although Capns1 increased in transcription in db/db mice. Instead, cytoplasmic JP2 significantly reduced and its hydrolyzed product JP2NT exhibited nuclear translocation in myocardium. This study suggests CAPNS1 is a crucial gene in T2D hearts. Its transcriptional upregulation leads to calpain/CAPNS1-associated JP2 hydrolysis and JP2NT nuclear translocation. Therefore, attenuated cardiac CAPNS1 transcription in T2D patients with SARS-CoV-2 infection highlights a novel target in adverse prognostics and comprehensive therapy. CAPNS1 can also be explored for the molecular signaling involving the onset, progression and prognostic in T2D patients with SARS-CoV-2 infection.

Enhanced Aerobic Glycolysis by S-Nitrosoglutathione via HIF-1α Associated GLUT1/Aldolase A Axis in Human Endothelial Cells.

S-nitrosoglutathione (GSNO)-induced apoptosis is associated with reactive oxygen species and loss of mitochondrial Omi/HtrA2 in human endothelial cells (ECs). But its upstream regulation is still not elucidated. Here, we demonstrate that hypoxia induced factor-1α (HIF-1α)-linked aerobic glycolysis is associated with mitochondrial abnormality by treatment of human EC-derived EA.hy926 cells with GSNO (500 µM) for 6 h. GSNO exposure increased the levels of Aldolase A and glucose transporter-1 (GLUT1) mRNAs and proteins. And selectively enhanced aldolase A activity to form glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, which subsequently increased intracellular levels of methylglyoxal and reactive oxygen species in parallel. Using the biotin switch assay, we found that GSNO increased the S-nitrosylating levels of total protein and HIF-1α. Knockdown of HIF-1α with siRNA attenuated its target aldolase A and GLUT1 expression but not VEGF. In contrast, nitrosylation scanvenger dithiothreitol could decrease all the protein levels. It suggested that aerobic glycolytic flux was more dependent on HIF-1α level, and that HIF-1α S-nitrosylation was crucial for its target expression under the normoxic condition. Moreover, GSNO-induced PI3 K (phosphoinositide 3-kinase)/Akt phosphorylation might contribute to HIF-1α stabilization and nucleus translocation, thereby aiding aldolase A and GLUT1 mRNAs upregulation. Taken together, higher concentration GSNO promotes glycolytic flux enhancement and methylglyoxal formation via HIF-1α S-nitrosylation. These findings reveal the mechanism of enhanced glycolysis-associated mitochondrial dysfunction in ECs by GSNO exposure under normoxic and non-hyperglycemic condition. And offer the early potential targets for vascular pathophysiological evaluation. J. Cell. Biochem. 118: 2443-2453, 2017. © 2017 Wiley Periodicals, Inc.

Premalignant alteration assessment in liver-like tissue derived from embryonic stem cells by aristolochic acid I exposure.

The in vitro predictive evaluation of chemical carcinogenicity based on hepatic premalignance has so far not been established. Here, we report a novel approach to investigate the premalignant events triggered by human carcinogen aristolochic acid I (AAI) in the liver-like tissue derived from mouse embryonic stem cells. By AAI exposure, the liver-like tissue exhibited the paracrine interleukin-6 phenotypic characteristics. Hepatocytes expressed STAT3/p-STAT3, c-Myc and Lin28B in parallel. Some of them displayed the dedifferentiation characteristics, such as full of α-fetoprotein granules, increase in size, and nucleocytoplasmic shuttle of Oct4. When these cells were injected into mice, the xenografts mostly displayed the uniform area of hepatic-like tissue with malignant nuclei. The hepatic malignant markers, α-fetoprotein, cytokeratin 7 and cytokeratin 19, were co-expressed in albumin-positive areas, respectively. In conclusion, we established an approach to predict the hepatic premalignance triggered by carcinogen AAI. This premalignant assay system might aid to evaluate the effects of potential carcinogens in liver, and probably to screen the protecting against hepatocarcinogenic efficacy of pharmaceuticals in vitro.

MicroRNA-674-5p/5-LO axis involved in autoimmune reaction of Concanavalin A-induced acute mouse liver injury.

Autoimmune hepatitis is characterized, in part, by the pathways involving cysteinyl-leukotriene metabolites of arachidonic acid, the dynamics of which remain unclear. Here, we explored post-transcriptional regulation in the 5-lipoxygenase (5-LO) pathway of arachidonic acid in a Concanavalin A (Con A) induced mouse model. We found that Con A administration lead to 5-LO overexpression and cysteinyl-leukotriene release in early hepatic injury, which was attenuated by cyclosporin A pretreatment. Subsequent microarray and qRT-PCR analysis further showed that microRNA-674-5p (miR-674-5p) displayed a significant decrease in expression in Con A-damaged liver. Noting that miR-674-5p harbors a potential binding region for 5-LO, we further transfected hepatic cell lines with overexpressing miR-674-5p mimic and discovered a negative regulating effect of miR-674-5p on 5-LO expression in the presence of IL-6 or TNF-α. These findings suggest that miR-674-5p might be a negative regulator in 5-LO mediated autoimmune liver injury, representing a compelling avenue towards future therapeutic interventions.

Peroxiredoxin-2 nitrosylation facilitates cardiomyogenesis of mouse embryonic stem cells via XBP-1s/PI3K pathway.

Protein nitrosylation is a ubiquitous post-translational modification in almost all biological systems. However, its function on stem cell biology is so far incompletely understood. Here, we demonstrated that peroxiredoxin 2 (Prdx-2) nitrosylation was involved in cardiomyocyte differentiation of mouse embryonic stem (ES) cells induced by S-nitrosoglutathione (GSNO). We found that temporary GSNO exposure could promote ES cell-derived cardiomyogenesis. Using a stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics approach, coupled with biotin switch technique, a total of 104 nitrosylated proteins were identified. Specifically, one of the antioxidant enzymes, Prdx-2, was abundantly nitrosylated and temporarily reduced in antioxidant activity, causing transient endogenous hydrogen peroxide (H2O2) accumulation and subsequent X-box binding protein-1s/phosphatidylinositol 3-kinase pathway activation. The present study reveals the mechanism in which GSNO favors cardiomyocyte differentiation. Prdx-2 nitrosylation could be a potent strategy to affect the pluripotent stem cell-derived cardiomyogenesis.

A Flavonoid Compound Promotes Neuronal Differentiation of Embryonic Stem Cells via PPAR-ß Modulating Mitochondrial Energy Metabolism.

Relatively little is known regarding mitochondrial metabolism in neuronal differentiation of embryonic stem (ES) cells. By using a small molecule, present research has investigated the pattern of cellular energy metabolism in neural progenitor cells derived from mouse ES cells. Flavonoid compound 4a faithfully facilitated ES cells to differentiate into neurons morphologically and functionally. The expression and localization of peroxisome proliferator-activated receptors (PPARs) were examined in neural progenitor cells. PPAR-ß expression showed robust upregulation compared to solvent control. Treatment with PPAR-ß agonist L165041 alone or together with compound 4a significantly promoted neuronal differentiation, while antagonist GSK0660 blocked the neurogenesis-promoting effect of compound 4a. Consistently, knockdown of PPAR-ß in ES cells abolished compound 4a-induced neuronal differentiation. Interestingly, we found that mitochondrial fusion protein Mfn2 was also abolished by sh-PPAR-ß, resulting in abnormal mitochondrial Ca2+ ([Ca2+]M) transients as well as impaired mitochondrial bioenergetics. In conclusion, we demonstrated that by modulating mitochondrial energy metabolism through Mfn2 and mitochondrial Ca2+, PPAR-ß took an important role in neuronal differentiation induced by flavonoid compound 4a.

Hepatic Premalignant Alterations Triggered by Human Nephrotoxin Aristolochic Acid I in Canines.

Aristolochic acid I (AAI) existing in plant drugs from Aristolochia species is an environmental human carcinogen associated with urothelial cancer. Although gene association network analysis demonstrated gene expression profile changes in the liver of human TP53 knock-in mice after acute AAI exposure, to date, whether AAI causes hepatic tumorigenesis is still not confirmed. Here, we show that hepatic premalignant alterations appeared in canines after a 10-day AAI oral administration (3 mg/kg/day). We observed c-Myc oncoprotein and oncofetal RNA-binding protein Lin28B overexpressions accompanied by cancer progenitor-like cell formation in the liver by AAI exposure. Meanwhile, we found that forkhead box O1 (FOXO1) was robustly phosphorylated, thereby shuttling into the cytoplasm of hepatocytes. Furthermore, utilizing microarray and qRT-PCR analysis, we confirmed that microRNA expression significantly dysregulated in the liver treated with AAI. Among them, we particularly focused on the members in let-7 miRNAs and miR-23a clusters, the downstream of c-Myc and IL6 receptor (IL6R) signaling pathway linking the premalignant alteration. Strikingly, when IL6 was added in vitro, IL6R/NF-κB signaling activation contributed to the increase of FOXO1 phosphorylation by the let-7b inhibitor. Therefore, it highlights the new insight into the interplay of the network in hepatic tumorigenesis by AAI exposure, and also suggests that anti-premalignant therapy may be crucial for preventing AAI-induced hepatocarcinogenesis.

Ginsenoside Rg1 exerts a protective effect against Aß25₋35-induced toxicity in primary cultured rat cortical neurons through the NF-κB/NO pathway.

Ginsenoside Rg1 (Rg1) is a multipotent triterpene saponin extracted from ginseng, and has been proven to act as a nootropic agent against various types of neurological damage. The present study was designed to investigate the neuroprotective effect and the underlying mechanisms of Rg1 on apoptosis induced by ß-amyloid peptide 25-35 (Aß25-35) in primary cultured cortical neurons. The primary neurons were preincubated with 20 µM Rg1 for 24 h and exposed to 10 µM Aß25-35 for 72 h. In the present study, we found that Rg1 prevented nuclear factor κ-light-chain­enhancer of activated B cells (NF-κB) nuclear translocation and IκB-α phosphorylation in primary cultured cortical neurons after Aß25-35 exposure by scavenging excess reactive oxygen species (ROS); ROS was measured using DCFDA and examined using a fluorescence microscope. In addition, Rg1 successfully suppressed Aß25­35-inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in a NF-κB-dependent manner; the suppression of NO was clearly illustrated by the NO production assay. Pretreatment of the cells with Rg1 elevated the proportion of Bcl-2/Bax, lessened the release of cytochrome c from mitochondria into cytoplasm and then blocked mitochondrial apoptotic cascades after Aß25-35 insult by lowering NO generation. Taken together, our data demonstrate that Rg1 rescues primary cultured cortical neurons from Aß25-35-induced cell apoptosis through the downregulation of the NF-κB/NO signaling pathway.

Pharmacokinetic and pharmacodynamic analysis of ferulic acid-puerarin-astragaloside in combination with neuroprotective in cerebral ischemia/reperfusion injury in rats.

OBJECTIVE: To investigate the effects of the active ingredients combined therapy on inflammatory factors interleukin 1 beta (IL-1ß) and neuropeptide Y (NPY) based on pharmacodynamics in rats. METHODS: The animal model was built by transient middle cerebral artery occlusion (MCAO). The method for evaluating the concentrations of the FA-Pr-Al components in rat plasma was established by using HPLC and the expression levels of IL-1ß and NPY were determined by ELISA. A new mathematics method of the trend of percentage rate of change (PRC) was used to assess the correlation between pharmacokinetics (PK) and pharmacodynamics (PD). RESULTS: FA-Pr-Al in combination reduced neurological deficits, decreased infarct volume and inhibited the expression levels of IL-1ß and NPY (all P<0.05) compared with the model group. FA, Pr and Al all displayed two compartment open models in rats. Clockwise hysteresis loops were obtained by time-concentration-effect curves. IL-1ß and NPY level changes in the plasma followed an opposite trend to the plasma concentration tendency after Cmax was reached. Astragaloside's PRC value was significantly higher than those of FA and puerarin between 120 to 180 min. CONCLUSIONS: The pharmacokinetics of FA-Pr-Al in combination were closely related its pharmacodynamics in treating ischemia/reperfusion injury, and the components of FA-Pr-Al may have a synergistic pharmacological effect. Astragaloside may play a more pronounced role in regulating IL-1ßand NPY levels compared with puerarin or FA.

Icariin promotes expression of junctophilin 2 and Ca2+ related function during cardiomyocyte differentiation of murine embryonic stem cells.

Junctophilin2 (JP2) is a critical protein associated with cardiogenesis. Icariin (ICA) facilitated the directional differentiation of murine embryonic stem (ES) cells into cardiomyocytes. However, little is known about the effects of ICA on JP2 during cardiac differentiation. Here, we explored whether ICA has effects on the expression and Ca2+ related function of JP2 during cardiomyocyte differentiation of ES cells in vitro. Embryonid bodies (EBs) formed by hanging drop were treated with 10(-7) mol/L ICA from day 5 to promote the cardiac differentiation. Percentage of beating EBs and number of beating area within EBs were monitored. Cardiomyocytes were purified by discontinuous percoll gradient centrifugation from EBs. The expression of JP2, α-actinin and troponin-T within EBs or isolated cardiomyocytes were analyzed by immunocytochemistry, western blot and flow cytometry. The transient Ca2+ release was characterized in cardiomyocytes treated with/without 10 mmol/L caffeine and 8 mmol/L Ca2+. Our results showed that ES cell-derived cardiomyocytes were well characterized with JP2 proteins. ICA promoted cardiomyocyte differentiation as indicated by an increased percentage of beating EBs and number of beating area within EBs. The expression of JP2, α-actinin and troponin-T were up-regulated both in EBs and isolated cardiomyocytes from EBs. Furthermore, ICA-induced JP2 expression was accompanied by a remarkable increase of the amplitude of Ca2+ transients in cardiomyocytes before/after caffeine and Ca2+ stimulating. In conclusion, ICA promotes in cardiac differentiation partly through regulating JP2 and improved the Ca2+ modulatory function of cardiomyocytes.

Nitrosative stress induces peroxiredoxin 1 ubiquitination during ischemic insult via E6AP activation in endothelial cells both in vitro and in vivo.

AIMS: Although there is accumulating evidence that increased formation of reactive nitrogen species in cerebral vasculature contributes to the progression of ischemic damage, but the underlying molecular mechanisms remain elusive. Peroxiredoxin 1 (Prx1) can initiate the antioxidant response by scavenging free radicals. Therefore, we tested the hypothesis that Prx1 regulates the susceptibility to nitrosative stress damage during cerebral ischemia in vitro and in vivo. RESULTS: Proteomic analysis in endothelial cells revealed that Prx1 was upregulated after stress-related oxygen-glucose deprivation (OGD). Although peroxynitrite upregulated Prx1 rapidly, this was followed by its polyubiquitination within 6 h after OGD mediated by the E3 ubiquitin ligase E6-associated protein (E6AP). OGD colocalized E6AP with nitrotyrosine in endothelial cells. To assess translational relevance in vivo, mice were studied after middle cerebral artery occlusion (MCAO). This was accompanied by Prx1 ubiquitination and degradation by the activation of E6AP. Furthermore, brain delivery of a lentiviral vector encoding Prx1 in mice inhibited blood-brain barrier leakage and neuronal damage significantly following MCAO. INNOVATION AND CONCLUSIONS: Nitrosative stress during ischemic insult activates E6AP E3 ubiquitin ligase that ubiquitinates Prx1 and subsequently worsens cerebral damage. Thus, targeting the Prx1 antioxidant defense pathway may represent a novel treatment strategy for neurovascular protection in stroke.

[Characteristics of microsomal phase II metabolic enzymes in mouse embryonic stem cell-derived liver tissue].

OBJECTIVE: To investigate the characteristics of phase II metabolic enzymes in mouse embryonic stem (ES) cell-derived liver tissue. METHODS: Mature hepatocytes were differentiated from embryonic stem cells in cultured mouse embryoid bodies (EB) at d18. Western blot was used to detect the expression of uridine 5'-diphosphate glucronosyl transferase (UGT1a1,UGT1a6) and microsomal glutathione S-transferases 1(mGST1) during the differentiation course.The derived liver tissue was incubated with UDPGA and 7-HFC,the formation of 7-HFC glucuronide was detected by HPLC to examine the total activities of UGT1a1 and UGT1a6. Furthermore, the microsomes were incubated with CDNB and GSH,and the mGST1 activity was measured by spectrometry. RESULTS: An increase tendency of UGT1a1 expression was noticed during the differentiation course. UGT1a6 and mGST1 were not detected in the earlier stage until d18 of differentiation. The metabolic activity of mGST1 in the derived hepatocytes was 7.65 nmol/min/mg on d18. CONCLUSION: The ES cell-derived liver tissue possesses partial metabolic function of phase II enzymes on d18 of differentiation,which might be used as a model for in vitro research on hepatic pathophysiology and phase II drug metabolism.

Compounds isolated from Psoralea corylifolia seeds inhibit protein kinase activity and induce apoptotic cell death in mammalian cells.

OBJECTIVES: Psoralea corylifolia is a plant widely used in traditional Chinese medicine, e.g. for its chemopreventive effect. To identify active substances responsible for this effect, we investigated pharmacological effects of 11 compounds isolated from the seeds of this plant (newly described substances: 7, 2', 4'-trihydroxy-3-arylcoumarin and psoracoumestan). METHODS: The influence of distinct compounds on different signal transduction pathways (cell proliferation, survival, angiogenesis and metastasis) was screened via analysis of the activity of 24 protein kinases, mitogen activated protein kinase phosphorylation via Western blot, cytotoxicity was shown using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and determination of caspase activity. Oxidative stress was detected via 2',7'-dichlorofluorescein fluorescence. KEY FINDINGS: Some compounds showed cytotoxic effects (H4IIE, Hct116, C6 cells) mainly mediated via induction of apoptosis. Distinct compounds caused a strong inhibition of MAPK/ERK kinase (MEK) phosphorylation, weak effects on extracellular-signal regulated kinase (ERK) phosphorylation and no significant effect on p38 and c-Jun amino-terminal kinase. Corylifol C and, to a lesser extent, xanthoangelol are potent protein kinase inhibitors (inhibitory concentration 50% values for epidermal growth factor receptor (EGFR): 1.1 and 4.4 × 10(-6) µg/ml, respectively). Because EGFR, MEK and ERK are kinases involved in cellular proliferation, an inhibition of these enzymes may be useful to cause chemopreventive effects. CONCLUSIONS: Distinct compounds isolated from P. corylifolia showed a high potential to influence cellular pathways, e.g. by inhibition of protein kinases that may be interesting for pharmacological purposes.

Hypoxia preconditioning of mesenchymal stromal cells enhances PC3 cell lymphatic metastasis accompanied by VEGFR-3/CCR7 activation.

Mesenchymal stromal cells (MSCs) in bone marrow may enhance tumor metastases through the secretion of chemokines. MSCs have been reported to home toward the hypoxic tumor microenvironment in vivo. In this study, we investigated prostate cancer PC3 cell behavior under the influence of hypoxia preconditioned MSCs and explored the related mechanism of prostate cancer lymphatic metastases in mice. Transwell assays revealed that VEGF-C receptor, VEGFR-3, as well as chemokine CCL21 receptor, CC chemokine receptor 7 (CCR7), were responsible for the migration of PC3 cells toward hypoxia preconditioned MSCs. Knock-in Ccr7 in PC3 cells also improved cell migration in vitro. Furthermore, when PC3 cells were labeled using the hrGfp-lentiviral vector, and were combined with hypoxia preconditioned MSCs for xenografting, it resulted in an enhancement of lymph node metastases accompanied by up-regulation of VEGFR-3 and CCR7 in primary tumors. Both PI3K/Akt/IκBα and JAK2/STAT3 signaling pathways were activated in xenografts in the presence of hypoxia-preconditioned MSCs. Unexpectedly, the p-VEGFR-2/VEGFR-2 ratio was attenuated accompanied by decreased JAK1 expression, indicating a switching-off of potential vascular signal within xenografts in the presence of hypoxia-preconditioned MSCs. Unlike results from other studies, VEGF-C maintained a stable expression in both conditions, which indicated that hypoxia preconditioning of MSCs did not influence VEGF-C secretion. Our results provide the new insights into the functional molecular events and signalings influencing prostate tumor metastases, suggesting a hopeful diagnosis and treatment in new approaches.

mGluR5 stimulating Homer-PIKE formation initiates icariin induced cardiomyogenesis of mouse embryonic stem cells by activating reactive oxygen species.

Icariin (ICA) has been reported to facilitate cardiac differentiation of mouse embryonic stem (ES) cells; however, the mechanism by which ICA induced cardiomyogenesis has not been fully elucidated yet. Here, an underlying signaling network including metabotropic glutamate receptor 5 (mGluR5), Homer, phosphatidylinositol 3-Kinase Enhancer (PIKE), phosphatidylinositol 3-Kinase (PI3K), reactive oxygen species (ROS) and nuclear factor-kappaB (NF-κB) was investigated in ICA induced cardiomyogenesis. Our results showed that the co-expression of mGluR5 together with α-actinin or Troponin T in embryoid bodies (EBs) treated with ICA was elevated to 10.86% and 9.62%, compared with the case in the control (4.04% and 3.45%, respectively). Exposure of EBs to ICA for 2 h remarkably increased the dimeric form of mGluR5, which was inhibited by small interfering RNA targeting mGluR5 (si-mGluR5). Moreover, the extracellular glutamate concentration in ICA treatment medium was elevated to 28.9±3.5 µM. Furthermore, the activation of mGluR5 by ICA triggered the formation of Homer-PIKE complex and activated PI3K, stimulating ROS generation and NF-κB nuclear translocation. Knockdown of mGluR5 or inhibition of PI3K by LY294002 blocked ICA induced cardiomyogenesis via repressing mGluR5 pathway, reducing ROS and NF-κB activation. These results revealed that the inducible mechanisms of ICA were related to activate mGluR5 pathway.

Activating glucocorticoid receptor-ERK signaling pathway contributes to ginsenoside Rg1 protection against ß-amyloid peptide-induced human endothelial cells apoptosis.

The deposition of ß-amyloid (Aß) in neurons and vascular cells of the brain has been characterized in Alzheimer's disease. Ginsenoside Rg1 (Rg1) is an active components in Panax ginseng, a famous traditional Chinese medicines recorded in Compendium of Materia Medica. Present study attempted to evaluate the potential mechanisms of Aß-mediated insult and the protective effects of Rg1 on human endothelial cells. Rg1 attenuated the Aß25-35-associated mitochondrial apoptotic events, accompanied by inhibiting HIF-1α expression followed by intracellular reactive nitrogen species generation, and protein nitrotyrosination. These protective effects were abolished by glucocorticoid receptor (GR) antagonist RU486 or p-ERK inhibitor U0126 rather than estrogen receptor α antagonist ICI 82,780. Taken together, our results suggested that Rg1 protected against Aß25-35-induced apoptosis at least in part by two complementary GR-dependent ERK phosphorylation pathways: (1) down-regulating HIF-1α initiated protein nitrotyrosination, and (2) inhibiting mitochondrial apoptotic cascades. These data provided a novel insight to the mechanisms of Rg1protective effects on Aß25-35-induced endothelial cells apoptosis, suggesting that GR-ERK signaling pathway might play an important role in it.

Ischemic injury promotes Keap1 nitration and disturbance of antioxidative responses in endothelial cells: a potential vasoprotective effect of melatonin.

Clinical epidemiology has indicated that the endothelial injury is a potential contributor to the pathogenesis of ischemic neurovascular damage. In this report, we assessed S-nitrosylation and nitration of Keap1 to identify downstream nitric oxide redox signaling targets into endothelial cells during ischemia. Here, oxygen-glucose deprivation (OGD) exposure initiates the nuclear import of Keap1 in endothelial cells, which interacted with nuclear-localized Nrf2, as demonstrated through co-immunoprecipitation and immunocytochemical assay. Paralleling the ischemia-induced nuclear import of Keap1, increased nitrotyrosine immunoreactivity in endothelial cells was also observed. Consistently, the addition of peroxynitrite provoked nuclear import of Keap1 and a concomitant Nrf2 nuclear import in the endothelial cells. Importantly, pharmacological inhibition of nitrosative stress by melatonin partially inhibited the OGD-induced constitutive nuclear import of Keap1 and subsequently disturbance of Nrf2/Keap1 signaling. Moreover, the effect of melatonin on nitration and S-nitrosylation of keap1 was examined in endothelial cells with 6 hr OGD exposure. Here, we demonstrated that OGD induced tyrosine nitration of Keap1, which was blocked by melatonin treatment, while there were no significant changes in S-nitrosylation of Keap1. The specific amino acid residues of Keap1 involved in tyrosine nitration were identified as Y473 by mass spectrometry. Moreover, the protective role of melatonin against damage to endothelial tight junction integrity was addressed by ZO-1 expression, paralleled with the restored heme oxygenase-1 levels during OGD. Together, our results emphasize that upon nitrosative stress, the protective effect of melatonin on endothelial cells is likely mediated at least in part by inhibition of ischemia-evoked protein nitration of Keap1, hence contributing to relieve the disturbance of Nrf2/Keap1 antioxidative signaling.

Ginsenoside Rg1 facilitates neural differentiation of mouse embryonic stem cells via GR-dependent signaling pathway.

Ginsenoside Rg1, a steroidal saponin of high abundance in ginseng, possesses the neuroprotective effects. In this study, we tried to explore the effect of Rg1 on promoting differentiation of mouse embryonic stem (ES) cells towards the neuronal lineage and its potential role involved in glucocorticoid receptor (GR) activation. Rg1 treatment induced a remarkable increase in the population of neuron-like cells in a time-dependent manner. More than 80% of Rg1-treated embryoid bodies (EBs) differentiated into neuron-like cells on d 8+10. Furthermore, the gradually increased protein expression of neurofilament (NEFM) and ß-tubulin III (a neuronal specific protein) was determined. GR expression gradually increased during the differentiation course. RU486, an antagonist of GR, could efficiently block the neurogenesis-promoting activity of Rg1. On the other side, Rg1 stimulated the phosphorylation of ERK1/2 and Akt at different time points through GR activation-dependent mechanisms. Treatment of both U0126 (an inhibitor of MEK) and LY294002 (an inhibitor of PI3 K), hampered the neuronal differentiation induced by Rg1. Meantime, U0126 further decreased Rg1-induced p-Akt expression. In conclusion, Rg1 possesses the effects on inducing differentiation of mouse ES cells into neurons in vitro via the GR-MEK-ERK1/2-PI3 K-Akt signaling pathway.

The γ-secretase blocker DAPT reduces the permeability of the blood-brain barrier by decreasing the ubiquitination and degradation of occludin during permanent brain ischemia.

BACKGROUND: Tight junction protein degradation is a principal characteristic of the blood-brain barrier (BBB) damage that occurs during brain ischemia. AIMS: We investigated the mechanisms of occludin degradation that underlie permanent middle cerebral artery occlusion (pMCAO) in rats. METHODS AND RESULTS: Western blot and Co-immunoprecipitation data indicated ubiquitination and degradation of occludin in brain after pMCAO, which was consistent with ZO-1 degradation in penumbra regions as observed at 24 h after pMCAO. We further investigated candidate protease(s) responsible for the degradation of occludin during pMCAO. The intraventricular administration of γ-secretase blocker DAPT significantly inhibited the pMCAO-induced neurovascular damage, whereas ALLM and Batimastat, which are inhibitors of calpain and metalloproteinase proteases, respectively, were less effective. Notably, we found that DAPT significantly inhibited BBB disruption in comparison with vehicle treatment, as assessed by Evans blue excretion. Interestingly, the confocal immunostaining revealed that activation of the E3 ubiquitin ligase Itch is associated with degradation of occludin in brain microvessels following ischemia. Furthermore, our data demonstrate that the inhibition of γ-secretase signaling and the itch-mediated ubiquitination of occludin likely underlie the vasoprotective effect of DAPT after pMCAO. CONCLUSION: The γ-secretase blocker DAPT reduces the permeability of the BBB by decreasing the ubiquitination and degradation of occludin during permanent brain ischemia, suggesting that γ-secretase may represent a novel therapeutic target for preventing neurovascular damage.