Additional file 1: of Effects of combined extract of cocoa, coffee, green tea and garcinia on lipid profiles, glycaemic markers and inflammatory responses in hamsters

Histopathology of epididymal fat, kidney, heart and lung tissues. a; epididymal fat tissue. b; kidney tissue. c; heart tissue. d; lung tissue. (H & E stain, magnification: 200×, Scale bar: 40 μm). Vehicle; vehicle control, HCD; high-cholesterol diet control, CCGG-1X; high-cholesterol diet with 311 mg/kg/d of CCGG, CCGG-2X; high-cholesterol diet with 622 mg/kg/d of CCGG, CCGG-5X; high-cholesterol diet with 1555 mg/kg/d of CCGG. (DOCX 3836 kb

DMY inhibits T cells proliferation <i>ex</i><i>vivo</i> and T cells infiltration into mouse liver tissues.

<p>(A) Splenic CD4⁺ T cells were purified from male ICR mouse and stimulated with vehicle alone or anti-CD3/anti-CD28 mAbs plus 0, 25, 50, or 100 μM of DMY for 48 h. [³H]Thymidine incorporation in T cells was determined. (B) The concentration of IL-2 in cultural media of T cells was determined by ELISA. (C) Immunohistochemistry analysis of mouse liver tissues against CD4 mAb. Data are mean ± SEM, <i>n</i> = 3. Means without a common letter differ, <i>P</i> < 0.05.</p

The effect of DMY treatment on NF-κB and MAPK signaling pathway in LPS−stimulated macrophages.

<p>Cells were pre-treated with 50 μM DMY for 1 h, then with LPS treatment for 2, 4, and 8 h. Western blot analysis of (A) Phospho- and total IKK, IκBα, JNK1/2 and ERK1/2 levels. (B) Immunofluorescence staining of nuclear translocation of NF-κB(p65) protein. Cells were stained with DAPI (nuclear marker, blue) and rabbit anti-p65 antibody (red).</p

Working hypothesis of the metabolic mechanisms of LPS/D-GalN– induced hepatic toxicity and protection by DMY.

<p>Symbol circle and square represent the relative metabolite changes in the LPS/D-GalN−challenged group and the DMY treatment group, respectively. The decrease, increase and no difference in levels with statistical significance are presented in green, red and tan, respectively.</p

The effect of DMY treatment on Jak/STAT signaling pathway in LPS−stimulated macrophages.

<p>Cells were pre-treated with 50 μM DMY for 1 h, then with LPS treatment for 2, 4, and 8 h. Western blot analysis of (A) Phospho- and total Jak2 and STAT1 levels, (B) Total STAT3, phospho-STAT3, and SOCS3 levels and the levels of STAT3 and phospho-STAT3 in nuclear and cytosolic fractions. PARP and α-tubulin were used as internal control of nuclear and cytosolic proteins, respectively.</p

Immunohistochemistry of liver tissues from LPS/D-GalN–challenged mice with or without DMY pretreatment.

<p>Immunofluorescence staining and quantification of DMY inhibiting STAT3 and F4/80 (macrophage) infiltration (A) and Ly6G (neutrophil) infiltration (B) in LPS/D-GalN−treated mouse liver. Data are mean ± SEM, <i>n</i> = 4. Means without a common letter differ, <i>P</i> < 0.05.</p

Protective effects of DMY on LPS/D-GalN−induced acute liver dysfunction in mice.

<p>Mice were pretreated with DMY (1 and 10 mg/kg) for three consecutive days, then LPS/D-GalN for 8 h. (A) Serum levels of AST and ALT with or without treatment. Data are mean ± SEM, <i>n</i> = 6. Means without a common letter differ, <i>P</i> < 0.05. (B) Hematoxylin and eosin staining of mouse livers. (C) TUNEL assay of apoptosis in mouse liver with LPS/D-GalN challenge or DMY treatment. Representative image of each treatment group is shown. Brownish cells are TUNEL-positive apoptotic cells. (D) Survival rate of LPS/D-GalN-challenged mice with or without DMY-pretreatment. ^*,#<i>P</i> < 0.05, significant differences within treatment groups and the LPS/D-GalN group (log rank test).</p

Comparative metabolomic analysis of mouse serum.

<p>Score plots (A, C) and corresponding loading plots (B, D) for PCA of UPLC/QTOF MS data from mice treated with vehicle control, LPS/D-GalN, and LPS/D-GalN–challenged mice pretreated with DMY1 and DMY10 (<i>n</i> = 3 in each group). <i>Dashed </i><i>circles</i> group pairs of samples from LPS/D-GalN–challenged group vs. other treatment groups. The ions most responsible for the variance of the score plots are indicated by their distance from the origin. The metabolites are labeled according their retention times in the chromatogram and <i>m</i>/<i>z</i> values. </p