Involvement of PKC-δ and p38 signaling pathways in crotonaldehyde-stimulated HO-1 expression.

<p>Cells were pretreated with PD 98059 (MEK1 inhibitor), LY 294002 (Akt/PI3K inhibitor), SP 600125 (c-Jun N-terminal Kinases (JNKs) inhibitor), SB 203580 (p38 inhibitor) or Rottlerin (PKC-δ inhibitor) for 1 h, followed by incubation with 25 µM crotonaldehyde for 16 h. Whole cell lysates were prepared and subjected to western blot analysis with antibodies against anti-HO-1 and GAPDH, as indicated (A and C). HepG2 cells were pretreated with inhibitors at the indicated concentrations for 1 h; followed by incubation with 25 µM crotonaldehyde for 2 h. Total RNA was prepared and subjected to RT-PCR for HO-1 and GAPDH (B and D). Cell lysates were immunoblotted with antibodies for the phosphorylated form of p38 MAPK and PKC-δ (E and F). Transient transfection of cells with PKC-δ or p38 siRNA inhibited up-regulation of crotonaldehyde-stimulated HO-1 protein expression (G and H). Representative Western blots of three independent experiments are shown. +, crotonaldehyde alone treated group.</p

Effect on cell cycle distribution after treatment with ZnPP or HO-1 siRNA in crotonaldehyde-stimulated HepG2 cells.

<p>Cell cycle analysis was performed by PI staining. HepG2 cells were treated with 25 µM crotonaldehyde and absence or presence of 1 µM ZnPP or HO-1 siRNA for 16 h. After treatment, the cells were stained with propidium iodide. Fluorescence activated cell sorting (FACS) analysis using PI staining was performed for DNA content measurement. Apoptosis was measured as the percentage of total cell population with the sub-G1 DNA content and was in the region labeled M1. Results are expressed as a dot plot and represent three independent experiments.</p

Crotonaldehyde-stimulated induction of HO-1 expression is mediated by Nrf2-EpRE/ARE.

<p>Cells were treated with crotonaldehyde at the indicated concentrations for 4 h. Nuclear extracts were prepared, and protein samples (40 µg) were subjected to Western blotting using an anti-Nrf2 antibody or an anti-Lamin B (a nuclear protein marker) antibody (A). Transient transfection of HepG2 cells with Nrf2 specific siRNA inhibited expression of the HO-1 protein. Nrf2 siRNA abrogated induction of crotonaldehyde-stimulated HO-1 protein expression (B). +, crotonaldehyde alone treated group. Cells transfected with an EpRE/ARE-luciferase construct were treated with various concentrations of crotonaldehyde for 4 h, and the lysates were mixed with a luciferase substrate. A luminometer was used to measure luciferase activity (C). Data represent the mean ± SD of 4 independent experiments. *<i>p</i><0.001 vs. control.</p

Effect of crotonaldehyde (CRA) on HO-1 expression.

<p>Cells were incubated with the indicated concentration of crotonaldehyde for 16 h. Protein in cell lysates was analyzed by Western blot using HO-1 specific antibody (A). RT-PCR was conducted to measure the levels of HO-1 mRNA transcript (C). Cells were harvested at various time intervals (B and D). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels were measured to ensure equal amounts of protein and mRNA loaded. Cell viability was estimated by the MTT method (E). Data represent the mean ± SD of results in three independent experiments.</p

Effect of crotonaldehyde-induced HO-1 inhibition on cell death.

<p>Cells were incubated in the absence or presence of ZnPP or HO-1 siRNA for 16 h before the indicated tests were performed. Crotonaldehyde-stimulated HepG2 cells were pretreated for 1 h with 1 µM ZnPP or HO-1 siRNA. Protective effect of HO-1 induction on cell death as determined by <i>in situ</i> terminal nick end-labeling (TUNEL). Treatment with H₂O₂ (0.5 mM) served as a positive control. Representative images illustrating fluorescent TUNEL (green) staining of cells cultured for 16 h before the indicated tests were performed (A). The graph indicates that inhibition of HO-1 expression in crotonaldehyde-stimulated HepG2 cells show a significant increase in the number of TUNEL-positive cells compared with those of normal and crotonaldehyde-treated cells (B). TUNEL-positive cells were quantified in five random fields in each culture well, and converted in percentages by referring to the total number of cells. Data represent the mean ± SD of three independent experiments. *<i>p</i><0.005 vs. CRA 25 µM treated cells.</p

Euscaphic acid relieves fatigue by enhancing anti-oxidative and anti-inflammatory effects

Oxidative stress and inflammation are involved in chronic fatigue. Euscaphic acid (EA) is an active compound of Eriobotrya japonica (Loquat) and has anti-oxidative effect. The goal of present study is to prove whether EA could relieve fatigue through enhancing anti-oxidant and anti-inflammatory effects in in vitro/in vivo models. EA notably improved activity of superoxide dismutase (SOD) and catalase (CAT), while EA reduced levels of malondiadehyde (MDA) and inflammatory cytokines without cytotoxicity in H2O2-stimulated in myoblast cell line, C2C12 cells. EA significantly reduced levels of fatigue-causing factors such as lactate dehydrogenase (LDH) and creatin kinase (CK), while EA significantly incresed levels of anti-fatigue-related factor, glycogen compared to the H2O2-stimulated C2C12 cells. In treadmill stress test (TST), EA significantly enhanced activities of SOD and CAT as well as exhaustive time and decreased levels of MDA and inflammatory cytokines. After TST, levels of free fatty acid, citrate synthase, and muscle glycogen were notably enhanced by oral administration of EA, but EA decreased levels of lactate, LDH, cortisol, aspartate aminotransferase, alanine transaminase, CK, glucose, and blood urea nitrogen compared to the control group. Furthermore, in forced swimming test, EA significantly increased levels of anti-fatigue-related factors and decreased excessive accumulations of fatigue-causing factors. Therefore, the results indicate that potent anti-fatigue effect of EA can be achieved via the improvement of anti-oxidative and anti-inflammatory properties, and this study will provide scientific data for EA to be developed as a novel and efficient component in anti-fatigue health functional food.</p

Effects of anti-hTM4SF5 monoclonal antibody on mouse HCC cells.

<p><b>A.</b> The expression levels of mTM4SF5 mRNA in the indicated mouse HCC cell lines and normal mouse hepatocytes were analyzed by RT-PCR. <b>B.</b> Detection of mTM4SF5 in BNL-HCC cells and H2.35 cells by anti-hTM4SF5R2-3 peptide monoclonal antibody according to a FACS analysis. Normal mouse IgG was used as a control. <b>C.</b> Reactivity of anti-hTM4SF5R2-3 monoclonal antibody with the mTM4SF5R2-3 peptide. The hTM4SF5R2-3 peptide was immobilized on a plate, and competitive ELISA was performed using increasing amounts of soluble mTM4SF5R2-3 peptide. <b>D.</b> Effect of anti-hTM4SF5R2-3 peptide monoclonal antibody on the growth of BNL-HCC cells and H2.35 cells. Cell growth was measured by an MTT assay. <b>E.</b> Effect of anti-hTM4SF5R2-3 peptide monoclonal antibody on the proliferation of BNL-HCC cells and H2.35 cells. The DNA synthesis activity was monitored by BrdU incorporation assay. Each bar is expressed as the Mean ± SD of three experiments. **<i>P</i><0.01.</p

Prophylactic efficacy of a vaccine containing hTM4SF5R2-3 peptide and Lipoplex(O) complex in HCC implanted mouse.

<p>BALB/c mice were immunized with a complex of hTM4SF5R2-3 peptide and Lipoplex(O). The immunized mice were implanted with the BNL-HCC cells (n = 12 per group; n = 10 per PBS-treated control). <b>A.</b> Induction of a strong serologic response to BNL-HCC cell implantation by vaccination with epitope and Lipoplex(O). The sera were collected, and the amounts of mTM4SF5R2-3 peptide-specific total IgG were assayed using an ELISA kit. Each bar is expressed as the Mean ± SD of 10 or 12 mice. <b>B–D.</b> Tumor formation in mice implanted with BNL-HCC cells was inhibited by vaccination with the hTM4SF5R2-3 peptide and Lipoplex(O) complex 60 days after the implantation of BNL-HCC cells. Tumor volumes were calculated as (length×width²)/2 (<b>B</b>). Macroscopic appearance of HCC tumor tissues (<b>C</b>). Tumor growth was measured by tumor weight (<b>D</b>). <b>E.</b> Body weights were measured at the indicated time intervals. <b>F.</b> Histology of normal liver and tumor tissue derived from BNL-HCC cell-implanted mice was observed by staining with hematoxylin and eosin (H&E, left panel). An immunohistochemical analysis (IHC) was performed with anti-hTM4SF5R2-3 monoclonal antibody (right panel). TM4SF5 positive area was expressed as brown color. Scale bars, 500 µm. **<i>P</i><0.01.</p

Production of IgG by immunization with a complex of hTM4SF5R2-3 peptide and Lipoplex(O).

<p><b>A.</b> Optimal dose of peptide in the complex of hTM4SF5R2-3 peptide and Lipoplex(O). BALB/c mice (n = 3/group) were immunized with increasing amounts of hTM4SF5R2-3 peptide and Lipoplex(O) complex. <b>B.</b> Effect of the injection protocol. We immunized one group of mice with hTM4SF5R2-3 peptide after injection with Lipoplex(O) (Lipoplex(O)/TM4SF5R2-3) and another group with MB-ODN 4531(O) after injection with hTM4SF5R2-3 peptide and the DOPE∶CHEMS complex (DOPE∶CHEMS+TM4SF5/4531(O)) three times with a 10 day interval (n = 3/group). The sera were collected, and titers of the peptide-specific total IgG were assayed with an ELISA kit. These experiments were performed 3 times with similar results. **<i>P</i><0.01 (<i>vs</i> PBS control).</p

Contribution of Th1 differentiation on IgG production.

<p>STAT4 but not STAT6 is required for IgG production by a complex of peptide and Lipoplex(O). <b>A,B.</b> BALB/c mice, BALB/c STAT4−/− mice (<b>A</b>), and BALB/c STAT6−/− mice (<b>B</b>) (n = 3/group) were injected i.p. three times with a 10 day interval with the hTM4SF5R2-3 peptide and Lipoplex(O) complex. The sera were collected, and the amounts of the peptide-specific total IgG, IgG1, and IgG2a were assayed with an ELISA kit. These experiments were performed 3 times with similar results. <b>C.</b> BALB/c mice, BALB/c STAT4−/− mice, and BALB/c STAT6−/− mice (n = 5/group) were injected i.p with hTM4SF5R2-3 peptide and Lipoplex(O) complex, and sera from the mice were harvested at 24 h after injection. The levels of IL-12 in the serum were measured with an ELISA assay. Each bar is expressed as the Mean ± SD of three mice. *<i>P</i><0.05, **<i>P</i><0.01.</p