Elevated plasma free fatty acid (FFA) levels in obesity may play a pathogenic role in the development of insulin resistance. However, molecular mechanisms linking FFA to insulin resistance remain poorly understood. Oxidative stress acts as a link between FFA and hepatic insulin resistance. NADPH oxidase 3 (NOX3)-derived reactive oxygen species (ROS) may mediate the effect of TNF-␣ on hepatocytes, in particular the drop in cellular glycogen content. In the present study, we define the critical role of NOX3-derived ROS in insulin resistance in db/db mice and HepG2 cells treated with palmitate. The db/db mice displayed increased serum FFA levels, excess generation of ROS, and upregulation of NOX3 expression, accompanied by increased lipid accumulation and impaired glycogen content in the liver. Similar results were obtained from palmitate-treated HepG2 cells. The exposure of palmitate elevated ROS production and NOX3 expression and, in turn, increased gluconeogenesis and reduced glycogen content in HepG2 cells. We found that palmitate induced hepatic insulin resistance through JNK and p38 MAPK pathways, which are rescued by siRNA-mediated NOX3 reduction. In conclusion, our data demonstrate a critical role of NOX3-derived ROS in palmitate-induced insulin resistance in hepatocytes, indicating that NOX3 is the predominant source of palmitate-induced ROS generation and that NOX3-derived ROS may drive palmitate-induced hepatic insulin resistance through JNK and p38 MAPK pathways.

Aim: To investigate the effects of Astragalus polysaccharides (APS) on tumor necrosis factor (TNF)-α-induced inflammatory reactions in human umbilical vein endothelial cells (HUVECs) and to elucidate the underlying mechanisms. Methods: HUVECs were treated with TNF-α for 24 h. The amounts of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined with Western blotting. HUVEC viability and apoptosis were detected using cell viability assay and Hoechst staining, respectively. Reactive oxygen species (ROS) production was measured by DHE staining. Monocyte and HUVEC adhesion assay was used to detect endothelial cell adhesive function. NF-κB activation was detected with immunofluorescence. Results: TNF-α (1-80 ng/mL) caused dose-and time-dependent increases of ICAM-1 and VCAM-1 expression in HUVECs, accompanied by significant augmentation of IκB phosphorylation and NF-κB translocation into the nuclei. Pretreatment with APS (10 and 50 µg/mL) significantly attenuated TNFα-induced upregulation of ICAM-1, VCAM-1, and NF-κB translocation. Moreover, APS significantly reduced apoptosis, ROS generation and adhesion function damage in TNF-α-treated HUVECs. Conclusion: APS suppresses TNFα-induced adhesion molecule expression by blocking NF-κB signaling and inhibiting ROS generation in HUVECs. The results suggest that APS may be used to treat and prevent endothelial cell injury-related diseases.

Doxorubicin (adriamycin), an anthracycline antibiotic, is commonly used to treat many types of solid and hematological malignancies. Unfortunately, clinical usage of doxorubicin is limited due to the associated acute and chronic cardiotoxicity. Previous studies demonstrated that Astragalus polysaccharide (APS), the extracts of Astragalus membranaceus, had strong anti-tumor activities and anti-inflammatory effects. However, whether APS could mitigate chemotherapy-induced cardiotoxicity is unclear thus far. We used a doxorubicin-induced neonatal rat cardiomyocyte injury model and a mouse heart failure model to explore the function of APS. GFP-LC3 adenovirus-mediated autophagic vesicle assays, GFP and RFP tandemly tagged LC3 (tfLC3) assays and Western blot analyses were performed to analyze the cell function and cell signaling changes following APS treatment in cardiomyocytes. First, doxorubicin treatment led to C57BL/6J mouse heart failure and increased cardiomyocyte apoptosis, with a disturbed cell autophagic flux. Second, APS restored autophagy in doxorubicin-treated primary neonatal rat ventricular myocytes and in the doxorubicin-induced heart failure mouse model. Third, APS attenuated doxorubicin-induced heart injury by regulating the AMPK/mTOR pathway. The mTOR inhibitor rapamycin significantly abrogated the protective effect of APS. These results suggest that doxorubicin could induce heart failure by disturbing cardiomyocyte autophagic flux, which may cause excessive cell apoptosis. APS could restore normal autophagic flux, ameliorating doxorubicin-induced cardiotoxicity by regulating the AMPK/mTOR pathway.

Earlier studies have shown that rhein, one of the major bioactive constituents of the rhizome of rhubarb, inhibits the proliferation of various human cancer cells. However, because of its water insolubility, the antitumor efficacy of rhein is limited in vivo. In this study, we studied the antitumor activity of rhein lysinate (the salt of rhein and lysine and easily dissolving in water) and its mechanism. Inhibition of breast cancer cell proliferation was determined by MTT assay and the mechanism of action of rhein lysinate was investigated by western blot analysis. The therapeutic efficacy of rhein lysinate was evaluated by human cancer xenografts in athymic nude mice. Rhein lysinate inhibited the proliferation of breast cancer cells (MCF-7, SK-Br-3, and MDA-MB-231). The IC50 values were 95, 80, and 110 micromol/l, respectively. Rhein lysinate inhibited the phosphorylation of epidermal growth factor receptor, MEK, and ERK with or without EGF stimulation. It also inhibited tumor growth and enhanced the therapeutic effect of Taxol on MCF-7 xenografts in athymic mice. Rhein lysinate inhibited the phosphorylation of epidermal growth factor receptor and MAPK signal pathway. These results suggest that rhein lysinate might be useful as a modulation agent in cancer chemotherapy.

The aim of this work was to evaluate the effects of glucagon-like peptide-1 (GLP-1) on high-glucose-induced oxidative stress and investigate the possible mechanisms underlying this process. We measured reactive oxygen species (ROS) production, cell apoptosis, the expression of NOX4 and its subunits, and p47phox translocation in human umbilical vein endothelial cells (HUVECs). An experimental type 2 diabetes model was induced using streptozotocin in male Sprague-Dawley rats. Fasting blood glucose (FBG), fasting insulin (FINS), total cholesterol (TC), triglycerides (TGs), and free fatty acid (FFA) were measured. Histomorphological analysis of the aorta was performed using hematoxylin-eosin staining. NOX4 and VCAM-1 expression in the aorta was measured. We found that high-glucose-induced ROS production and apoptosis were inhibited by GLP-1 treatment. High glucose caused upregulation of NOX4, p47phox, and Rac-1 and translocation of p47phox but had no effect on the cells pretreated with GLP-1. Furthermore, in the diabetic group, FBG, FINS, TG, TC, and FFA were increased, and NOX4 and VCAM-1 levels were also elevated. However, GLP-1 attenuated all these changes. GLP-1 ameliorated high-glucose-induced oxidative stress by inhibiting NOX4, p47phox, and Rac-1 expression and translocation of p47phox, suggesting its clinical usefulness in diabetic vascular complications.

Background/Aims: Recent studies have suggested that changes in non-coding mRNA play a key role in the progression of non-alcoholic fatty liver disease (NAFLD). Metformin is now recommended and effective for the treatment of NAFLD. We hope the current analyses of the non-coding mRNA transcriptome will provide a better presentation of the potential roles of mRNAs and long non-coding RNAs (lncRNAs) that underlie NAFLD and metformin intervention. Methods: The present study mainly analysed changes in the coding transcriptome and non-coding RNAs after the application of a five-week metformin intervention. Liver samples from three groups of mice were harvested for transcriptome profiling, which covered mRNA, lncRNA, microRNA (miRNA) and circular RNA (circRNA), using a microarray technique. Results: A systematic alleviation of high-fat diet (HFD)-induced transcriptome alterations by metformin was observed. The metformin treatment largely reversed the correlations with diabetes-related pathways. Our analysis also suggested interaction networks between differentially expressed lncRNAs and known hepatic disease genes and interactions between circRNA and their disease-related miRNA partners. Eight HFD-responsive lncRNAs and three metformin-responsive lncRNAs were noted due to their widespread associations with disease genes. Moreover, seven miRNAs that interacted with multiple differentially expressed circRNAs were highlighted because they were likely to be associated with metabolic or liver diseases. Conclusions: The present study identified novel changes in the coding transcriptome and non-coding RNAs in the livers of NAFLD mice after metformin treatment that might shed light on the underlying mechanism by which metformin impedes the progression of NAFLD.

Over recent years, an increasing number of studies have confirmed that the occurrence and development of vascular pathological changes are closely related to oxidative stress and the inflammatory response of the vascular endothelium. Kaempferol is the most common flavonoid compound found in fruits and vegetables. Our present research identified that kaempferol had the capability to protect the vascular endothelium in a mouse model of vascular injury and explored the specific mechanisms underlying these effects by investigating oxidative stress, the extent of cardiovascular injury, and inflammatory markers such as NF-kB, TNF-a, IL-6, and the Nrf2/HO-1 signaling pathway. Analysis showed that kaempferol reduced oxidative stress and inflammation mediated by H 2 O 2 and paraquat, respectively, both in vitro and in vivo. Furthermore, kaempferol suppressed the levels of TNF-a and IL-6, and the activation of NF-kB, in aortic tissues and human umbilical vein endothelial cells (HUVECs). Finally, we observed that kaempferol corrected the levels of antioxidants and elevated the protein levels of Nrf2 and HO-1 in aortic tissues and HUVECs. Collectively, our findings prove that kaempferol protects blood vessels from damage induced by oxidative stress and inflammation and that the Nrf2/HO-1 signaling pathway plays a key role in mediating these effects.

Background. Sirtuin 1 (SIRT1) is a member of the sirtuin family, which could activate cell survival machinery and has been shown to be protective in regulation of heart function. Here, we determined the mechanism by which SIRT1 regulates Angiotensin II- (AngII-) induced cardiac hypertrophy and injury in vivo and in vitro. Methods. We analyzed SIRT1 expression in the hearts of control and AngII-induced mouse hypertrophy. Female C57BL/6 mice were ovariectomized and pretreated with 17β-estradiol to measure SIRT1 expression. Protein synthesis, cardiomyocyte surface area analysis, qRT-PCR, TUNEL staining, and Western blot were performed on AngII-induced mouse heart hypertrophy samples and cultured neonatal rat ventricular myocytes (NRVMs) to investigate the function of SIRT1. Results. SIRT1 expression was slightly upregulated in AngII-induced mouse heart hypertrophy in vivo and in vitro, accompanied by elevated cardiomyocyte apoptosis. SIRT1 overexpression relieves AngII-induced cardiomyocyte hypertrophy and apoptosis. 17β-Estradiol was able to protect cardiomyocytes from AngII-induced injury with a profound upregulation of SIRT1 and activation of AMPK. Moreover, estrogen receptor inhibitor ICI 182,780 and SIRT1 inhibitor niacinamide could block SIRT1's protective effect. Conclusions. These results indicate that SIRT1 functions as an important regulator of estrogen-mediated cardiomyocyte protection during AngII-induced heart hypertrophy and injury.