The primer sequences used for quantitative qRT-PCR of the differentially expressed genes related to diet type.

<p>The primer sequences used for quantitative qRT-PCR of the differentially expressed genes related to diet type.</p

Western blot analysis of calreticulin (CALR; A) and apolipoprotein A-I (ApoA-I; B) in liver tissue samples from high-grain diet and control groups.

<p>Protein extracts of liver tissue samples were prepared and subjected to immunodetection with the indicated antibodies. Intensities of CALR and ApoA-I bands were normalized to the corresponding β-actin control. Values are presented as means ± SD; n = 5. * <i>P</i><0.05.</p

Gene ontology (GO) analysis of differentially expressed proteins.

<p>GO annotations are presented by category: A) biological process B) cellular components C) molecular function.</p

KEGG pathway analysis of differentially expressed proteins.

<p>Pathway enrichment analysis was performed using the DAVID web application.</p

2-DE patterns of proteins extracted from dairy goat liver.

<p>A. Control group; B. High-grain diet group. Fifty-two differentially expressed proteins showing significant spot intensity changes are marked in A and B. The proteins to which these 52 differentially expressed protein spots correspond are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080698#pone-0080698-t002" target="_blank">Table 2</a>.</p

Identification of differentially expressed liver proteins.

a<p>Numbering corresponds to the 2-DE gel in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080698#pone-0080698-g001" target="_blank">Fig.1</a>.</p>b<p>The total number of identified peptide.</p>c<p>Increased(>) or decreased(<) compared with the control group.</p

Representative 2-DE images of proteins extracted from dairy goat liver.

<p>A) Control group; B) High-grain diet group. Equal amounts of protein (850 µg) were loaded and separated on 17-cm IPG strips (pH 3–10), followed by electrophoresis on 12.5% SDS-PAGE gels for second dimension electrophoresis. The gels were stained with CCB G250. Experiments were performed in triplicate.</p

The schematic diagram of the experimental design for NMBA injection and RPS administration.

<p><b>A</b>. A schematic representation of experimental protocol. The empty arrows indicate subcutaneous injection of saline. The solid arrows indicate subcutaneous injection of NMBA. The shaded area represents oral RPS administration. <b>B</b>. The chemical formula of RPS. The letter “R” indicates that different functional groups can be at that position, resulting in different types of RPS with various molecular structures.</p

Rhizoma Paridis Saponins Suppresses Tumor Growth in a Rat Model of <i>N</i>-Nitrosomethylbenzylamine-Induced Esophageal Cancer by Inhibiting Cyclooxygenases-2 Pathway

<div><p>Rhizoma Paridis Saponins (RPS), a natural compound purified from Rhizoma Paridis, has been found to inhibit cancer growth in vitro and in animal models of cancer. However, its effects on esophageal cancer remain unexplored. The purpose of this study was to investigate the effects of RPS on tumor growth in a rat model of esophageal cancer and the molecular mechanism underlying the effects. A rat model of esophageal cancer was established by subcutaneous injection of <i>N</i>-nitrosomethylbenzylamine (NMBA, 1mg/kg) for 10 weeks. RPS (350 mg/kg or 100mg/kg) was administered by oral gavage once daily for 24 weeks starting at the first NMBA injection. RPS significantly reduced the size and number of tumors in the esophagus of rats exposed to NMBA and inhibited the viability, migration, and invasion of esophageal cancer cells EC9706 and KYSE150 in a dose dependent manner (all <i>P</i> < 0.01). Flow cytometry revealed that RPS induced apoptosis and cell cycle G2/M arrest in the esophageal cancer cells. The expression of cyclooxygenases-2 (COX-2) and Cyclin D1 in rat esophageal tissues and the esophageal cancer cells were also significantly reduced by RPS (all <i>P</i> < 0.01). Consistently, RPS also significantly decreased the release of prostaglandin E2, a downstream molecule of COX-2, in a dose-dependent manner (<i>P</i> < 0.01). Our study suggests that RPS inhibit esophageal cancer development by promoting apoptosis and cell cycle arrest and inhibiting the COX-2 pathway. RPS might be a promising therapeutic agent for esophageal cancer.</p></div

RPS reduced the size and number of tumors on the esophagus of rats exposed to NMBA.

<p><b>A</b>. Photographs and H&E staining of esophageal tissues of rats from healthy control, NMBA, or NMBA+RPS group. Male F344 rats (n = 10 per group) were subcutaneously injected with saline containing DMSO (Healthy control group), NMBA at 1mg/kg (NMBA group), or NMBA plus oral administration of RPS at 350 mg/kg (NMBA + RPS group). Esophagus was dissected, photographed, and examined under a light microscope. Part of esophageal tissues was fixed in 10% formalin solution and stained with H&E. <b>B</b>. RPS significantly reduced the number of tumors on esophagus. Tumors larger than 1mm in diameter were counted, n = 10. <b>C</b>. RPS significantly decreased tumor size. The volume of the lesions was calculated using the standard formula: volume = length × width × height × 0.52, n = 10. <b>D</b>. RPS reduced the number of papilloma and carcinoma. Two pathologists, who were blinded for the treatment allocation, examined the type and number of tumors. The average number of papilloma and carcinoma is presented, n = 10. * represents significant difference between NMBA group vs healthy control group, <i>P</i> < 0.01. # represents significant difference between NMBA +RPS group vs NMBA group, <i>P</i> < 0.01.</p