PERK-CHOP pathway is mainly involved in ER stress- mediated apoptosis in human breast cancer cells induced by AMP.

<p>(A) The inhibition efficiency of siRNAs against CHOP, ATF6α and PERK, respectively. Cells were transfected with siRNAs targeting CHOP, ATF6α and PERK (100 nM each) for 24-h, and the protein levels of CHOP, ATF6α and PERK was determined by western blot, respectively. (B) The effects of CHOP siRNA on AMP-induced cell apoptosis. Cells were transfected with control (siCT) or 100 nM CHOP siRNA (siCHOP), followed by treatment with 60 µM AMP for 24-h, then cell apoptosis was measured. The data shown represent the mean±S.E.M. of three independent experiments *<i>P</i><0.05. (C) And (D) The effects of PERK or ATF6α siRNA on AMP-induced CHOP expression. Cells were transfected with control (siCT) or 100 nM PERK siRNA (siPERK) or ATF6α siRNA (siATF6α), followed by treatment with 60 µM AMP for 24-h, then the protein levels of CHOP were determined by western blot. All results are representative western blots of three independent experiments with similar results.</p

Image1_Dihydromyricetin-Encapsulated Liposomes Inhibit Exhaustive Exercise-Induced Liver Inflammation by Orchestrating M1/M2 Macrophage Polarization.TIF

Exhaustive exercise (EE) induced hepatic inflammatory injury has been well reported. Dihydromyricetin (DHM) has shown anti-inflammatory bioactivity and hepatoprotective effects but is limited by poor bioavailability. Here, high-bioavailability DHM-encapsulated liposomes were synthesized and explored for their therapeutic potential and regulatory mechanisms in a hepatic inflammatory injury model. The animal model was established by swimming-to-exhaustive exercise in C57BL/6 mice, and the anti-inflammatory effects were detected after administration of DHM or DHM liposome. NIR fluorescence imaging was used to assess the potential of liver targeting. The DHM liposome-induced macrophage polarization was measured by flow cytometry ex vivo. The anti-inflammatory mechanism of DHM was studied in cell line RAW264.7 in vitro. Liposome encapsulation enhanced DHM bioavailability, and DHM liposome could alleviate liver inflammation more effectively. Moreover, DHM liposome targeted hepatic macrophages and polarized macrophages into an anti-inflammatory phenotype. The SIRT3/HIF-1α signaling pathway could be the major mechanism of DHM motivated macrophage polarization. Our study indicates that DHM liposomes can alleviate liver inflammation induced by EE through sustained releasing and hepatic targeting. It is a promising option to achieve the high bioavailability of DHM. Also, this study provides new insights into the regional immune effect of DHM against inflammation.</p

<i>S-</i>(-)equol primarily up-regulated Nrf2 protein expression activating ARE-dependent response.

<p>(A) Whole EA.hy926 cells lysates of cells treated with 250 nM <i>S-</i>(-)equol, 500 nM daidzein, or 50 µM tBHQ for 24 h were subjected to immunoblot analysis with anti-Nrf2, anti-HO-1, anti-NQO1, and anti-β-tubulin antibodies. (B) Total HUVECs lysates of cell treated with 250 nM <i>S-</i>(-)equol for 24 h were immunoblotted with anti-Nrf2 and anti-β-tubulin antibodies. (C) EA.hy926 cells were transfected with plasmids containing an HA-tagged Nrf2 open reading frame and the renilla firefly luciferase gene. Medium was changed after 6 hours of incubation. Where indicated, extracts from HA-Nrf2 EA hy.92 cells were additionally probed with anti-HA antibody, anti-Nrf2, anti-HO-1, anti-NQO1 and anti-β-tubulin antibodies. Values are means of three independent experiments with standard deviations represented by vertical bars. Mean values were significantly different compared to controls (*<i>p</i><0.05). Total RNA was extracted from EA.hy926 cells or HUVECs treated as indicated, and Nrf2 (D), HO-1 (E), and NQO1 (F) mRNA was measured by real-time RT-PCR analysis. The values shown represent the mean ± SD obtained for three independent experiments (*<i>p</i><0.05).</p

AMP inhibits cell growth and induces apoptosis in breast cancer cells.

<p>(A) Dose-dependent effects of Ampelopsin (AMP) on cell viability. MCF-10A, MDA-MB-231 and MCF-7 cells were treated with different concentrations of AMP (20, 40, 60, 80 µM) for 24-h, respectively, and cell viability was measured by CCK-8 assay. (B) Dose-dependent effects of AMP on apoptosis. After treatment with the indicated concentrations of AMP for 24-h, apoptosis was assessed using Annexin V-FITC/PI staining. All data shown are representative of three independent experiments, *<i>P</i><0.05.</p

Efficacy of <i>S-</i>(-)equol in protecting against oxidative stress-induced toxicity through Nrf2.

<p>EA.hy926 cells that were pretreated with 100<i>S-</i>(-)equol or sham treated for 24 h were exposed to H₂O₂ (100–800 µM) (A) or tBHP (20–80 µM) (B) in the presence or absence of 100 nM <i>S-</i>(-)equol for 24 h. HUVECs were pretreated with 100 nM <i>S-</i>(-)equol or sham treated for 24 h were exposed to H₂O₂ (100 µM) (C). Cell survival after oxidative stress was measured using a CCK-8 assay. The data are reported as the mean ± SD, n = 6. (D) Cells untreated or pretreated with 250 nM <i>S-</i>(-)equol for 24 h and then treated with H₂O₂ for another 24 h. Apoptotic cell death was detected using Annexin V-FITC and flow cytometry. The mean ± SD was calculated from three independent experiments. (E) Apoptotic cells were visualized by TUNEL. Immunoblot analysis showing Nrf2 protein in EA.hy926 cells (F, right) or HUVECs (G, right) transfected with a control or Nrf2-siRNA. Nrf2 protein was measured with immunoblot analysis using an anti-Nrf2 antibody to confirm knockdown of Nrf2 expression. (F and G) Untransfected cells and cells transfected with indicated siRNAs were incubated with 100 nM <i>S-</i>(-)equol for 24 h in the presence of 650 µM H₂O₂ or with H₂O₂ alone. The apoptotic index for each sample was determined using a cell death ELISA kit. The data are reported as the mean ± SD, n = 3 (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 compared with control group, ^#<i>p</i><0.05 compared with <i>S-</i>(-)equol treated group).</p

Resveratrol induces autophagy in BCSCs.

<p>(A) Resveratrol treatment (40 µM) for 24 h increased the number of autophagosomes in BCSCs from SUM159 and MCF-7, determined by TEM analysis. (B) Western blotting detection of LC3-II, Beclin1 and Atg 7 in BCSCs treated with different dose of resveratrol for 24 h. (C) GFP-LC3-II puncta formation assay in BCSCs treated with resveratrol for 24 h. Data are presented as mean ± SD (<i>n</i> = 3). <b>^*</b><i>P</i><0.05, <b>^**</b><i>P</i><0.01 compared with the control (0 µM).</p

AMP activates ER stress in breast cancer cells.

<p>(A) Time-dependent effects of Ampelopsin (AMP) on ER stress-associated proteins. MDA-MB-231 and MCF-7 cells were treated with 60 µM AMP for indicated time, and the protein expression levels of GRP78, p-PERK, p-elF2α, cleaved ATF6α, and CHOP were assessed by western blotting. (B) The effect of ER stress inhibitors or activators on AMP-induced ER stress in breast cancer cells. After pre-treatment with 2 mM 4-Phenylbutyric acid (4-PBA) or 150 nM thapsigargin (Thap) for 2-h, respectively, followed by treatment with 60 µM AMP for 24-h, the expression of GRP78 and CHOP were assessed by western blotting. All results are representative western blots of three independent experiments with similar results.</p

Ampelopsin induces ROS and ER stress to form a vicious cycle in breast cancer cells.

<p>(A) Effects of NAC on AMP-induced ER stress. After pre-treatment with 5 mM NAC for 2-h, followed by treatment with 60 µM AMP for 24-h, CHOP and GRP78 expression were evaluated by western blotting. (B) The effects of CHOP siRNA on AMP-induced ROS generation. Cells were transfected with control (siCT) or 100 nM CHOP siRNA (siCHOP), followed by treatment with 60 µM AMP for 24-h. Then cellular ROS levels were assessed. The results of A and C represent the mean±S.E.M. of three independent experiments *<i>P</i><0.05.</p

ERβ plays a role in activation of Nrf2 by <i>S-</i>(-)equol in endothelial cells.

<p>(A) EA.hy926 cells were transfected with ARE-dependent firefly luciferase reporter gene (<i>F</i>) and the renilla firefly luciferase gene (<i>R</i>), and treated with 100 nM <i>S-</i>(-)equol in the presence or absence of 10 nM DPN, 10 nM PPT, 100 nM MPP or 100 nM PHTPP for 2 h following further incubation with or without 100 nM <i>S-</i>(-)equol. The potency of induction is expressed as the relative luminescence (R/F) measured using the dual luciferase reporter assay system (mean ± SD, n = 6). *<i>p</i><0.05 versus with control group, ^#<i>p</i><0.05 versus with <i>S-</i>(-)equol treated group. (B) EA.hy926 cells were incubated with 0.1% DMSO (1), 10 nM PPT (3), or 100 nM <i>S-</i>(-)equol (5) for 16 h and then immunostained with an antibody against ERα; and cells also were incubated with 0.1% DMSO (2), 10 nM PPT (4), or 100 nM <i>S-</i>(-)equol (6) for 16 h and then immunostained with an antibody against ERβ, then analyzed by confocal microscopy. (C) Cells were treated with 100 nM <i>S-</i>(-)equol in the presence or absence of 10 nM DPN, 10 nM PPT, 100 nM MPP or 100 nM PHTPP for 2 h following further incubation with or without 100 nM <i>S-</i>(-)equol, and Nrf2 localization was analyzed by confocal microscopy. Values (intensity of nuclear versus cytoplasmic) are means of counting 100 cells with standard deviations represented by vertical bars. *<i>p</i><0.05 versus with control group, ^#<i>p</i><0.05 versus with <i>S-</i>(-)equol treated group.</p

Ampelopsin Improves Insulin Resistance by Activating PPARγ and Subsequently Up-Regulating FGF21-AMPK Signaling Pathway

<div><p>Ampelopsin (APL), a major bioactive constituent of <i>Ampelopsis grossedentata</i>, exerts a number of biological effects. Here, we explored the anti-diabetic activity of APL and elucidate the underlying mechanism of this action. In palmitate-induced insulin resistance of L6 myotubes, APL treatment markedly up- regulated phosphorylated insulin receptor substrate-1 and protein kinase B, along with a corresponding increase of glucose uptake capacity. APL treatment also increased expressions of fibroblast growth factor (FGF21) and phosphorylated adenosine 5’-monophosphate -activated protein kinase (p-AMPK), however inhibiting AMPK by Compound C or <i>AMPK</i> siRNA, or blockage of <i>FGF21 by FGF21</i> siRNA, obviously weakened APL -induced increases of FGF21 and p-AMPK as well as glucose uptake capacity in palmitate -pretreated L6 myotubes. Furthermore, APL could activate PPAR γ resulting in increases of glucose uptake capacity and expressions of FGF21 and p-AMPK in palmitate -pretreated L6 myotubes, whereas all those effects were obviously abolished by addition of GW9662, a specific inhibitor of peroxisome proliferator- activated receptor –γ (PPARγ), and <i>PPARγsiRNA</i>. Using molecular modeling and the luciferase reporter assays, we observed that APL could dock with the catalytic domain of PPARγ and dose-dependently up-regulate PPARγ activity. In summary, APL maybe a potential agonist of PPARγ and promotes insulin sensitization by activating PPARγ and subsequently regulating FGF21- AMPK signaling pathway. These results provide new insights into the protective health effects of APL, especially for the treatment of Type 2 diabetes mellitus.</p></div