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Wang H.,Anhui Medical University | Zhu H.-Q.,Anhui Medical University | Zhu H.-Q.,Key Laboratory of Gene Research of Anhui Province | Wang F.,Anhui Medical University | And 5 more authors.
Molecular and Cellular Biochemistry | Year: 2013

The development of atherosclerosis (AS) is a multifactorial process in which elevated plasma cholesterol levels play a central role. As a new class of players involved in AS, the regulation and function of microRNAs (miR) in response to AS remain poorly understood. This study analyzed the effects of miR-1 (antagomir and mimic) on endothelial permeability and myosin light chain kinase (MLCK) expression and activity in the artery wall of apoE knock-out mice after feeding them a high-cholesterol diet. Further, we tested to determine whether that effects are involved in ERK phosphorylation. Here, we show that a high-cholesterol diet induces a significant decrease of miR-1 expression. Histopathologic examination demonstrated that miR-1 antagomir enhances endothelial permeability induced by high cholesterol and miR-1 mimic attenuated endothelial barrier dysfunction. Consistent with endothelial permeability, Western blotting, qPCR, and γ-32P-ATP phosphate incorporation showed that MLCK expression and activity were further increased inmiR- 1 antagomir-treated mice and decreased in miR-1 mimictreated mice compared with those of mice receiving control miR. Further mechanistic studies showed that high-cholesterol- induced extracellular signal regulated kinase (ERK) activation was enhanced by miR-1 antagomir and attenuated by miR-1mimic. Collectively, those results indicate that miR- 1 contributes to endothelial barrier function via mechanisms involving not only MLCK expression and activity but also ERK phosphorylation. © The Author(s) 2013. Source


Zhu H.-Q.,Anhui Medical University | Zhu H.-Q.,Key Laboratory of Gene Research of Anhui Province | Wang F.,Anhui Medical University | Dong L.-Y.,Anhui Medical University | And 4 more authors.
Life Sciences | Year: 2014

Aims This study was aimed to determine whether microRNA1 (miR1) plays a role in the activation of myosin light chain kinase (MLCK) mediated by oxLDL in human umbilical vein endothelial cells (HUVECs). Main methods HUVECs were treated with oxLDL along with a control miR or miR1 mimic. MiR1 expression was assayed by miRNA plate assay kit and mirVana™ miRNA isolation kit. The MLCK protein, transcript, and kinase activity were measured by Western blot, real-time-polymerase chain reaction and γ-32P-ATP phosphate incorporation, respectively. In addition, phosphorylation of MLC, ERK and p38 was analyzed by Western blot. Key findings The results showed that upon treatment with oxLDL, miR1 expression was decreased, whereas MLCK expression was increased, in a time- and dose-dependent manner. Consistent with this, miR1 mimic prevented MLCK expression and activation and attenuated the phosphorylation of MLC and ERK/p38 in oxLDL-treated HUVECs. Furthermore, we showed that miR1 was able to bind a site located at the 3′un-translational region of MLCK mRNA and inhibited its expression. Significance Taken together, this study demonstrated that the effect of miR1 on hyperlipidemia is mediated through down-regulation of MLCK and the ERK/p38 MAPK pathway. © 2014 Elsevier Inc. Source


Jiang Q.,Anhui Medical University | Yuan Y.,Anhui Medical University | Yuan Y.,Jiujiang University | Zhou J.,Anhui Medical University | And 6 more authors.
Molecular and Cellular Biochemistry | Year: 2015

To investigate whether endoplasmic reticulum (ER) stress participates in the induction of apoptosis in HepG2 cells exposed to high glucose and explore its probable mechanism. A series of experiments were performed following HepG2 cells treated with different concentrations of glucose for 48 h. The apoptosis was detected by means of Hoechst staining and flow cytometry. Caspase-3 activity assay was performed by measuring the pNA (p-nitroaniline) to indirectly reveal the catalytic activity of caspase-3. The expression levels of apoptosis-, ER stress-associated proteins and MAPKs were analyzed by western blot. To further characterize the molecular mechanisms, the effects of antioxidant alpha-lipoic acid (ALA) and specific inhibitors for JNK and p38 (SP600125 and SB203580, respectively) were examined by Hoechst staining, immunofluorescence, and western blot. After HepG2 cells were incubated with high glucose for 48 h, both Hoechst staining and flow cytometry analyses unveiled the apoptosis of HepG2 cells. Caspase-3 activity assay revealed that the activity of caspase-3 was enhanced. Western blot showed an enhancement of pro-caspase-9 degradation, a reduction of Bcl-2/Bax ratio, a decrease in GRP78 expression, and increases in CHOP and p47/phox levels. In addition, western blot analysis presented that phosphorylation of p38 and JNK was triggered and that the expression of ASK1 was elevated. In the case of the contributions of oxidative stress and the MAPK signaling pathways, all ALA, SP600125 and SB203580 were able to largely rescue high glucose-induced apoptosis. High glucose induced the apoptosis in HepG2 cells through the activation of ASK1-p38/JNK pathway mediated by ER stress and oxidative stress. © 2014, Springer Science+Business Media New York. Source


Zhu H.-Q.,Anhui Medical University | Zhu H.-Q.,Key Laboratory of Gene Research of Anhui Province | Li Q.,Anhui Medical University | Li Q.,Key Laboratory of Gene Research of Anhui Province | And 6 more authors.
International Journal of Cardiology | Year: 2014

Methods and results This study examined whether microRNA-29b (miR-29b) regulates high-fat diet induced endothelial permeability and apoptosis by targeting MT1, a known melatonin membrane receptor. In apoE knock-out mice, a high-fat diet increased miR-29b expression and induced apoptosis as determined by up-regulation of caspase-3 activity. However, a standard diet did not alter apoptosis. miR-29b antagomir decreased endothelial permeability and apoptosis in high-fat diet-stimulated mice. In contrast, a miR-29b mimic enhanced endothelial permeability and apoptosis. The induction of miR-29b correlated with a reduction in Bcl-2 and MT1 in high-fat diet-stimulated mice. miR-29b have an effect on the marker of inflammation (NF-κB) and cell adhesion molecule (ICAM-1). We further showed that miR-29b targeted and inhibited MT1 expression through a target site located in the 3′un-translational region of MT1 mRNA. This study demonstrates a role of miR-29b in atherosclerosis and identifies MT1 as a direct target of miR-29b.Conclusions The effect of miR-29b on endothelial permeability and apoptosis is mediated through the down-regulation of MT1. Thus, miR-29b may be a new therapeutic target for atherosclerosis.Background High-fat diet has been reported to be associated with cardiovascular diseases which is implicated in atherosclerosis. However, the underlying mechanisms remain unknown. MicroRNAs (miRNAs) are non-coding small RNAs that control gene expression at the post-transcriptional level. Dysregulated miRNAs have been shown to be involved in atherosclerosis. © 2014 Elsevier Ireland Ltd. All rights reserved. Source


Yang X.,Anhui Medical University | Zou D.,Anhui Medical University | Tang S.,Anhui Medical University | Fan T.,Anhui Medical University | And 11 more authors.
Molecular and Cellular Biochemistry | Year: 2016

The increased intestinal permeability and functional impairment play an important role in type 2 diabetes (T2D), and melatonin may possess enteroprotection properties. Therefore, we used streptozotocin-induced diabetic rat model to investigate the regulation of intestinal permeability by melatonin. Rats were randomly divided into three groups, including control, diabetes mellitus (DM), and DM rats treated with melatonin. Melatonin was administered (10 mg/kg/day) by gavage for 24 weeks. The DM rats significantly increased the serum fasting blood glucose and lipid levels, which were alleviated by melatonin treatment. Importantly, the intestinal epithelial permeability was significantly increased in DM rats but was ameliorated following treatment with melatonin. These findings also indicated the expression of myosin light chain kinase (MLCK) and phosphorylation of MLC targeting subunit (MYPT) induced myosin light chain (MLC) phosphorylation level was markedly elevated in hyperglycemic and hyperlipidemic status. They were partly associated with down-regulated membrane type 1 and 2 (MT1 and MT2) expression, and up-regulated Rho-associated protein kinase (ROCK) expression and increased extracellular signal-regulated kinase (ERK) phosphorylation. However, the changes in target protein expression were reversed by melatonin. In conclusion, our results show melatonin beneficial effects on impaired intestinal epithelial permeability in T2D by suppressing ERK/MLCK- and ROCK/MCLP-dependent MLC phosphorylation. © 2016 Springer Science+Business Media New York Source

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