Inhibition of Cholesteryl Ester Transfer Protein Contributes to the Protection of Ginsenoside Re Against Isoproterenol-Induced Cardiac Hypertrophy.

Background and objectives Ginsenoside Re (Re), a protopanaxatriol-type saponin extracted from ginseng, is known to have potential cardioprotective effects; however, the mechanisms of Re in improving cardiac hypertrophy have not been fully elucidated. This study aimed to investigate the therapeutic effects and underlying mechanism of Re on isoproterenol (ISO)-induced cardiac hypertrophy in vivo and in vitro. Methods Rats were intraperitoneally injected with ISO 30 mg/kg thrice daily for 14 consecutive days to induce cardiac hypertrophy, and these rats were treated with atorvastatin (ATC, 20 mg/kg) or Re (20 mg/kg or 40 mg/kg) once daily for three days in advance until the end of the experiment. Heart weight index, hematoxylin and eosin staining, and hypertrophy-related fetal gene expression were measured to evaluate the effect of Re on cardiac hypertrophy in vivo. Meanwhile, the rat H9c2 cardiomyocyte hypertrophy model was induced by ISO 10 µM for 24 hours. Cell surface area and hypertrophy-related fetal gene expression were determined to assess the effect of Re on ISO-induced cardiomyocyte hypertrophy in vitro. The levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in both serum and cardiomyocytes were detected by enzymatic colorimetric assays. Furthermore, we chose cholesteryl ester transfer protein (CETP) as a target to explore the influence of Re on CETP expression in vivo and in vitro through real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Results Intraperitoneal administration of ISO into rats resulted in increases in cross-sectional cardiomyocyte area, the ratio of heart weight to body weight, the ratio of left ventricular weight to body weight, and the ratio of right ventricular weight to body weight, as well as reactivation of fetal genes; however, treatment with Re or ATC ameliorated most of these hypertrophic responses. Similarly, Re pronouncedly alleviated ISO-induced cardiomyocyte hypertrophy, as evidenced by a decreased cell surface area and downregulation of fetal genes. Moreover, our in vivo and in vitro data revealed that Re reduced TC, TG, and LDL-C levels, and enhanced HDL-C levels. Re improved cardiac hypertrophy mainly associated with the inhibition of mRNA level and protein expression of CETP, to an extent comparable to that of the classical CETP inhibitor, anacetrapib. Conclusions Our research found that CETP inhibition contributes to the protection of Re against ISO-induced cardiac hypertrophy, which provides evidence for the application of Re for cardiovascular disease treatments.

Proximal tubular FHL2, a novel downstream target of hypoxia inducible factor 1, is a protector against ischemic acute kidney injury.

Four-and-a-half LIM domains protein 2 (FHL2) is an adaptor protein that may interact with hypoxia inducible factor 1α (HIF-1α) or ß-catenin, two pivotal protective signaling in acute kidney injury (AKI). However, little is known about the regulation and function of FHL2 during AKI. We found that FHL2 was induced in renal tubular cells in patients with acute tubular necrosis and mice model of ischemia-reperfusion injury (IRI). In cultured renal proximal tubular cells (PTCs), hypoxia induced FHL2 expression and promoted the binding of HIF-1 to FHL2 promoter. Compared with control littermates, mice with PTC-specific deletion of FHL2 gene displayed worse renal function, more severe morphologic lesion, more tubular cell death and less cell proliferation, accompanying by downregulation of AQP1 and Na, K-ATPase after IRI. Consistently, loss of FHL2 in PTCs restricted activation of HIF-1 and ß-catenin signaling simultaneously, leading to attenuation of glycolysis, upregulation of apoptosis-related proteins and downregulation of proliferation-related proteins during IRI. In vitro, knockdown of FHL2 suppressed hypoxia-induced activation of HIF-1α and ß-catenin signaling pathways. Overexpression of FHL2 induced physical interactions between FHL2 and HIF-1α, ß-catenin, GSK-3ß or p300, and the combination of these interactions favored the stabilization and nuclear translocation of HIF-1α and ß-catenin, enhancing their mediated gene transcription. Collectively, these findings identify FHL2 as a direct downstream target gene of HIF-1 signaling and demonstrate that FHL2 could play a critical role in protecting against ischemic AKI by promoting the activation of HIF-1 and ß-catenin signaling through the interactions with its multiple protein partners.

Cardioprotective Potential of Cymbopogon citratus Essential Oil against Isoproterenol-induced Cardiomyocyte Hypertrophy: Possible Involvement of NLRP3 Inflammasome and Oxidative Phosphorylation Complex Subunits.

OBJECTIVE: Cymbopogon citratus (DC.) Stapf is a medicinal and edible herb that is widely used for the treatment of gastric, nervous and hypertensive disorders. In this study, we investigated the cardioprotective effects and mechanisms of the essential oil, the main active ingredient of Cymbopogon citratus, on isoproterenol (ISO)-induced cardiomyocyte hypertrophy. METHODS: The compositions of Cymbopogon citratus essential oil (CCEO) were determined by gas chromatography-mass spectrometry. Cardiomyocytes were pretreated with 16.9 µg/L CCEO for 1 h followed by 10 µmol/L ISO for 24 h. Cardiac hypertrophy-related indicators and NLRP3 inflammasome expression were evaluated. Subsequently, transcriptome sequencing (RNA-seq) and target verification were used to further explore the underlying mechanism. RESULTS: Our results showed that the CCEO mainly included citronellal (45.66%), geraniol (23.32%), and citronellol (10.37%). CCEO inhibited ISO-induced increases in cell surface area and protein content, as well as the upregulation of fetal gene expression. Moreover, CCEO inhibited ISO-induced NLRP3 inflammasome expression, as evidenced by decreased lactate dehydrogenase content and downregulated mRNA levels of NLRP3, ASC, CASP1, GSDMD, and IL-1ß, as well as reduced protein levels of NLRP3, ASC, pro-caspase-1, caspase-1 (p20), GSDMD-FL, GSDMD-N, and pro-IL-1ß. The RNA-seq results showed that CCEO inhibited the increase in the mRNA levels of 26 oxidative phosphorylation complex subunits in ISO-treated cardiomyocytes. Our further experiments confirmed that CCEO suppressed ISO-induced upregulation of mt-Nd1, Sdhd, mt-Cytb, Uqcrq, and mt-Atp6 but had no obvious effects on mt-Col expression. CONCLUSION: CCEO inhibits ISO-induced cardiomyocyte hypertrophy through the suppression of NLRP3 inflammasome expression and the regulation of several oxidative phosphorylation complex subunits.

Increased thoracic fluid content is associated with higher risk for pneumonia in patients undergoing maintenance hemodialysis.

BACKGROUND: Pneumonia is the most common infectious disease in patients undergoing maintenance hemodialysis (MHD). The aim of this study is to determine the possible predictive value of thoracic fluid content (TFC) for pneumonia in this population. METHOD: Clinical data were recorded for 1412 MHD patients who were hospitalized for certain comorbidities or complications. Each patient underwent an impedance cardiography (ICG) examination before next dialysis session after admission. Patients were divided into Having-, Will-have-, and Non-pneumonia groups based on whether they had pneumonia at the time of ICG examination after the admission and within five months after the examination. Hemodynamic parameters and other clinical data were compared and analyzed. RESULTS: Patients who were going to develop pneumonia were older, and had a higher proportion of diabetes, poorer nutritional status, a higher level of inflammatory, poorer cardiac function, and more fluid volume load than those who did not develop pneumonia. Multivariate binary logistic analysis revealed that for each 1/KΩ increase in TFC and 1 increase in neutrophil-to-lymphocyte ratio (NLR), the risk of the development of pneumonia increased by 3.1% (p ˂ 0.01) and 7.2% (p = 0.035), respectively, whereas for each 1 g/L increase in hemoglobin and 1 g/L increase in serum albumin, the risk of the development of pneumonia decreased by 1.3% (p = 0.034) and 5% (p = 0.048), respectively. CONCLUSIONS: TFC, NLR, hemoglobin, and serum albumin were independent risk factors for the development of pneumonia in MHD patients. Given the advantages of ICG, TFC can be used clinically as a helpful predictor of pneumonia in MHD patients.

Momordicine I alleviates isoproterenol-induced cardiomyocyte hypertrophy through suppression of PLA2G6 and DGK-ζ.

This study aimed to observe the protective effect of momordicine I, a triterpenoid compound extracted from momordica charantia L., on isoproterenol (ISO)-induced hypertrophy in rat H9c2 cardiomyocytes and investigate its potential mechanism. Treatment with 10 µM ISO induced cardiomyocyte hypertrophy as evidenced by increased cell surface area and protein content as well as pronounced upregulation of fetal genes including atrial natriuretic peptide, ß-myosin heavy chain, and α-skeletal actin; however, those responses were markedly attenuated by treatment with 12.5 µg/ml momordicine I. Transcriptome experiment results showed that there were 381 and 447 differentially expressed genes expressed in comparisons of model/control and momordicine I intervention/model, respectively. GO enrichment analysis suggested that the anti-cardiomyocyte hypertrophic effect of momordicine I may be mainly associated with the regulation of metabolic processes. Based on our transcriptome experiment results as well as literature reports, we selected glycerophospholipid metabolizing enzymes group VI phospholipase A2 (PLA2G6) and diacylglycerol kinase ζ (DGK-ζ) as targets to further explore the potential mechanism through which momordicine I inhibited ISO-induced cardiomyocyte hypertrophy. Our results demonstrated that momordicine I inhibited ISO-induced upregulations of mRNA levels and protein expressions of PLA2G6 and DGK-ζ. Collectively, momordicine I alleviated ISO-induced cardiomyocyte hypertrophy, which may be related to its inhibition of the expression of glycerophospholipid metabolizing enzymes PLA2G6 and DGK-ζ.

Resveratrol ameliorates high-phosphate-induced VSMCs to osteoblast-like cells transdifferentiation and arterial medial calcification in CKD through regulating Wnt/ß-catenin signaling.

Vascular smooth muscle cells (VSMCs) to osteoblast-like cells transdifferentiation induced by high-phosphate is a crucial step in the development of arterial medial calcification (AMC) in patients with chronic kidney disease (CKD), and previous studies implicate Wnt/ß-catenin signaling in osteogenic transdifferentiation of VSMCs and AMC. Given that resveratrol's ability to modulate Wnt/ß-catenin signaling in other types of cell, we tested the effect of resveratrol on high-phosphate-induced osteogenic transdifferentiation of VSMCs and AMC in CKD. Resveratrol ameliorated AMC in rats with chronic renal failure and calcium deposition in aortic rings and VSMCs cultured in a high-phosphate environment. Resveratrol also diminished high-phosphate-induced osteogenic transdifferentiation of VSMCs in cultured aortic rings and VSMCs. In vitro, resveratrol attenuated the activation of ß-catenin induced by high-phosphate and inhibited the expression of Runx2, a downstream effector of Wnt/ß-catenin signaling during osteogenic transdifferentiation of VSMCs. Intriguingly, resveratrol inhibited high-phosphate-induced phosphorylation of LRP6 (Ser1490), but didn't inhibit Wnt3a-induced phosphorylation of LRP6 (Ser1490) and Runx2 expression. The expression of several Wnts was induced by high-phosphate, but the expression of Wnt7a, not Wnt2b and Wnt10a could be suppressed by resveratrol. In addition, the expression of both porcupine and wntless, two obligatory proteins for Wnt secretion, was induced by high-phosphate in cultured aortic rings and VSMCs, which could be suppressed by resveratrol. In summary, these findings suggest that resveratrol possesses a vascular protective effect on retarding high-phosphate-induced osteogenic transdifferentiation of VSMCs and AMC in CKD by targeting Wnt/ß-catenin signaling, which may, to a large extent, via impeding Wnt secretion.

Deletion of FHL2 in fibroblasts attenuates fibroblasts activation and kidney fibrosis via restraining TGF-ß1-induced Wnt/ß-catenin signaling.

Four-and-a-half LIM domains protein 2 (FHL2) has been proposed involving in ß-catenin activity. We previously reported that FHL2 mediates TGF-ß1-induced tubular epithelial-to-mesenchymal transition through activating Wnt/ß-catenin signaling. However, the potential role and mechanism for FHL2 in TGF-ß1-induced fibroblast activation and kidney fibrosis remains unknown. Here, we initially observed higher levels of FHL2 expression in fibrotic kidneys from both patients and mice, especially in α-smooth muscle actin (α-SMA)-positive cells in the interstitium. In cultured interstitial fibroblasts, FHL2 expression was induced by TGF-ß1. Knockdown of FHL2 remarkably suppressed TGF-ß1-induced α-SMA, type I collagen, and fibronectin expression, while overexpression of FHL2 was sufficient to activate fibroblasts. In mice, fibroblast-specific deletion of FHL2 diminished renal induction of α-SMA, type I collagen, and fibronectin and interstitial extracellular matrix deposition at 2 weeks after ureteral obstruction. We next investigated Wnt/ß-catenin activity and found that ß-catenin was activated in most FHL2-positive cells in renal interstitium from mice with obstructive nephropathy. In vitro, TGF-ß1 induced a physical interaction between FHL2 and ß-catenin, especially in the nucleus. Downregulation of FHL2 inhibited TGF-ß1-induced active ß-catenin upregulation, ß-catenin nuclear translocation, and ß-catenin-mediated transcription, whereas overexpression of FHL2 was able to activate Wnt/ß-catenin signaling. FHL2 overexpression-induced ß-catenin-mediated gene transcription could be hindered by ICG-001, but FHL2 overexpression-induced upregulation of active ß-catenin could not be. Collectively, this study reveals that the signal regulatory effect of FHL2 on ß-catenin plays an important role in TGF-ß1-induced fibroblast activation and kidney fibrosis.

The regulatory role of HIF-1 in tubular epithelial cells in response to kidney injury.

The high sensitivity to changes in oxygen tension makes kidney vulnerable to hypoxia. Both acute kidney injury and chronic kidney disease are almost always accompanied by hypoxia. Tubular epithelial cells (TECs), the dominant intrinsic cells in kidney tissue, are believed to be not only a victim in the pathological process of various kidney diseases, but also a major contributor to kidney damage. Hypoxia inducible factor-1 (HIF-1) is the main regulator of adaptive response of cells to hypoxia. Under various clinical and experimental kidney disease conditions, HIF-1 plays a pivotal role in modulating multiple cellular processes in TECs, including apoptosis, autophagy, inflammation, metabolic pattern alteration, and cell cycle arrest. A comprehensive understanding of the mechanisms by which HIF-1 regulates these cellular processes in TECs may help identify potential therapeutic targets to improve the outcome of acute kidney injury and delay the progression of chronic kidney disease.

FHL2 promotes tubular epithelial-to-mesenchymal transition through modulating ß-catenin signalling.

ß-Catenin signalling plays an important role in regulating tubular epithelial-to-mesenchymal transition (EMT), an indispensable programme for driving renal fibrosis. As an adapter protein, four and a half LIM domain protein 2 (FHL2) acts as a coregulator of ß-catenin in several other cell types. To determine whether FHL2 affects ß-catenin signalling and thus is involved in tubular EMT, we examined its expression and function in the process of TGF-ß1-induced EMT. FHL2 mRNA and protein were induced by TGF-ß1 in rat tubular epithelial cells (NRK-52E), an effect that intracellular Smad signalling was required. Ectopic expression of FHL2 inhibited E-cadherin and enhanced α-smooth muscle actin (α-SMA) and fibronectin expression, whereas knockdown of FHL2 partially restored E-cadherin and reduced α-SMA and fibronectin induction stimulated by TGF-ß1. Overexpression of FHL2 increased ß-catenin dephosphorylation (Ser37/Thr41), nuclear translocation and ß-catenin-mediated transcription and up-regulated expression of ß-catenin target, EMT-related genes, such as Snail, Twist, vimentin, plasminogen activator inhibitor-1 and matrix metalloproteinase-7. Conversely, knockdown of FHL2 increased ß-catenin phosphorylation (Ser33/37/Thr41), decreased its nuclear translocation and inhibited ß-catenin-mediated transcription and target genes expression. TGF-ß1 induced a FHL2/ß-catenin interaction in NRK-52E cells, especially in the nuclei. In a mouse model of obstructive nephropathy, FHL2 mRNA and protein were induced in a time-dependent fashion, and the extent and pattern of renal ß-catenin activation were positively correlated with FHL2 induction. Collectively, this study suggests that FHL2, via modulating ß-catenin signalling, may implicate in regulation of TGF-ß1-mediated tubular EMT and could be a potential therapeutic target for fibrotic kidney disease.

Modulation of tumor cell migration, invasion and cell-matrix adhesion by human monopolar spindle-one-binder 2.

Human monopolar spindle-one-binder 2 (hMOB2) is a member of the hMOB family of proteins, and it has been reported to regulate the nuclear-Dbf2-related kinase (NDR) activation. However, the function of hMOB2 expression in tumor cell adhesion and motility has not been addressed. Herein, the lentiviral-mediated overexpression and the knockdown of hMOB2 in HepG2 and SMMC-7721 cells was established. It was demonstrated that overexpression of hMOB2 significantly reduced the cell motility and enhanced the cell-matrix adhesion, while the hMOB2 knockdown decreased not only the cell motility, but also the cell-matrix adhesion. Immunofluorescence results showed that both hMOB2 overexpression and knockdown altered assembly of the focal adhesions and the actin cytoskeleton rearrangement. Furthermore, the focal adhesion kinase (FAK)-Src-paxillin signal pathway activated by hMOB2 was confirmed to be involved in controlling the cell motility and the cell-matrix adhesion. These results demonstrated that the altered cell-matrix adhesion and cell motility induced by hMOB2 expression was caused by the assembly of focal adhesions as well as the actin cytoskeleton rearrangement through the activation of the FAK-Src-paxillin signal pathway, unveiling a novel mechanism of cell motility and cell-matrix adhesion regulation induced by hMOB2 expression.

An adenosine A3 receptor agonist inhibits DSS-induced colitis in mice through modulation of the NF-κB signaling pathway.

The role of the adenosine A3 receptor (A3AR) in experimental colitis is controversial. The A3AR agonist N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) has been shown to have a clinical benefit, although studies in A3AR-deficient mice suggest a pro-inflammatory role. However, there are no studies on the effect of 2-Cl-IB-MECA and the molecular mechanism of action of A3AR in murine colitis models in vivo. Is it the same as that observed in vitro? The interaction between 2-CL-IB-MECA and A3AR in a murine colitis model and the signaling pathways associated with this interaction remain unclear. Here we demonstrate a role for the NF-κB signaling pathway and its effect on modifying the activity of proinflammatory factors in A3AR-mediated biological processes. Our results demonstrated that A3AR activation possessed marked effects on experimental colitis through the NF-κB signaling pathway.

Activation of adenosine A3 receptor alleviates TNF-α-induced inflammation through inhibition of the NF-κB signaling pathway in human colonic epithelial cells.

To investigate the expression of adenosine A3 receptor (A3AR) in human colonic epithelial cells and the effects of A3AR activation on tumor necrosis factor alpha (TNF-α-) induced inflammation in order to determine its mechanism of action in human colonic epithelial cells, human colonic epithelial cells (HT-29 cells) were treated with different concentrations of 2-Cl-IB-MECA prior to TNF-α stimulation, followed by analysis of NF-κB signaling pathway activation and downstream IL-8 and IL-1ß production. A3AR mRNA and protein were expressed in HT-29 cells and not altered by changes in TNF-α or 2-Cl-IB-MECA. Pretreatment with 2-Cl-IB-MECA prior to stimulation with TNF-α attenuated NF-κB p65 nuclear translocation as p65 protein decreased in the nucleus of cells and increased in the cytoplasm, inhibited the degradation of IκB-α, and reduced phosphorylated-IκB-α level significantly, compared to TNF-α-only-treated groups. Furthermore, 2-Cl-IB-MECA significantly decreased TNF-α-stimulated IL-8 and IL-1ß mRNA expression and secretion, compared to the TNF-α-only treated group. These results confirm that A3AR is expressed in human colonic epithelial cells and demonstrate that its activation has an anti-inflammatory effect, through the inhibition of NF-κB signaling pathway, which leads to inhibition of downstream IL-8 and IL-1ß expression. Therefore, A3AR activation may be a potential treatment for gut inflammatory diseases such as inflammatory bowel disease.

Down-regulation of miR-126 is associated with colorectal cancer cells proliferation, migration and invasion by targeting IRS-1 via the AKT and ERK1/2 signaling pathways.

BACKGROUND: Colorectal carcinoma (CRC) is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs, miRs) play important roles in carcinogenesis. MiR-126 has been shown to be down-regulated in CRC. In this study, we identified the potential effects of miR-126 on some important biological properties of CRC cells and clarified the regulation of insulin receptor substrate 1 (IRS-1) and its possible signaling pathway by miR-126. METHODS: The effect of miR-126 on IRS-1, AKT, and ERK1/2 expression was assessed in the CRC cell lines HT-29 and HCT-116 with a miR-126 mimic or inhibitor to increase or decrease miR-126 expression. Furthermore, the roles of miR-126 in regulation of the biological properties of CRC cells were analyzed with miR-126 mimic or inhibitor-transfected cells. The 3'-untranslated region (3'-UTR) of IRS-1 regulated by miR-126 was analyzed by using a dual-luciferase reporter assay. RESULTS: We found that IRS-1 is the functional downstream target of miR-126 by directly targeting the 3'-UTR of IRS-1. Endogenous miR-126 and exogenous miR-126 mimic inhibited IRS-1 expression. Furthermore, gain-of-function or loss-of-function studies showed that over-expression of miR-126 down-regulated IRS-1, suppressed AKT and ERK1/2 activation, CRC cells proliferation, migration, invasion, and caused cell cycle arrest, but had no effect on cell apoptosis. Knockdown of miR-126 promoted these processes in HCT-116 cells and promoted AKT and ERK1/2 activation by up-regulating the expression of the IRS-1 protein. CONCLUSIONS: MiR-126 may play roles in regulation of the biological behavior of CRC cells, at least in part, by targeting IRS-1 via AKT and ERK1/2 signaling pathways.

The relationship between and clinical significance of MicroRNA-32 and phosphatase and tensin homologue expression in colorectal cancer.

MicroRNAs (miRNAs, miRs) are suspected to play important roles in carcinogenesis. MiR-32 has altered expression in colorectal cancer (CRC); however, the clinical significance of miR-32 expression in the process of carcinogenesis is poorly understood. In this study, we determined the levels of, the correlation between, and the clinical significance of the expression of miR-32 and phosphatase and tensin homologue (PTEN), a tumor suppressor targeted by miR-32, in CRC. The levels of miR-32 and PTEN gene expression in 35 colorectal carcinoma samples, 35 corresponding cancer-adjacent tissue samples, 27 colorectal adenoma samples, and 16 normal tissue samples were quantified using real-time quantitative reverse transcriptase-polymerase chain reaction. PTEN protein expression was determined using western blot and immunohistochemistry (IHC). The relationship between the miR-32 and PTEN protein expression and clinicopathological factors was analyzed. Significant upregulation of miR-32 expression and reduction of PTEN were identified in CRC tissues. High miR-32 levels were significantly associated with lymph node and distant metastasis, and Kaplan-Meier analysis indicated that patients with high miR-32 expression had a poor overall survival. Low PTEN protein expression was also significantly correlated with distant metastasis. An inverse relationship between miR-32 and PTEN protein expression was identified. In addition, IHC analysis revealed weak or indiscernible PTEN staining in tumor tissue. MiR-32 overexpression was correlated with specific CRC clinicopathological features and may be a marker of poor prognosis in CRC patients. MiR-32 and PTEN expression were inversely correlated, and miR-32 may be associated with the development of CRC.

EONS: an online synaptic modeling platform.

Chemical synapses, although representing the smallest unit of communication between two neurons in the nervous system constitute a complex ensemble of mechanisms. Understanding these mechanisms and the way synaptic transmission occurs is critical for our comprehension of CNS functions in general and learning and memory in particular. Here we describe a modeling platform called EONS (Elementary Object of Neural System) accessible online, which allows neuroscientists throughout the world to study qualitatively, but also quantitatively the relative contributions of diverse mechanisms underlying synaptic efficacy: the relevance of each and every elements that comprise a synapse, the interactions between these components and their subcellular distribution, as well as the influence of synaptic geometry (presynaptic terminal, cleft and postsynaptic density).