AOM/DSS induced colitis was scored as the disease activity index (DAI) as described previously . In brief, the DAI was the combined scores of weight Anti-diabetic Compound Library concentration loss (0, none; 1, 0–5%; 2, 5–10%; 3, 10–20%; and 4, >20%), stool consistency change (0, none; 2, loose stool; and 4, diarrhea), and bleeding (0, none; 1, trace; 2, mild hemoccult; 3, obvious hemoccult; and 4, gross bleeding), and then divided by three. The animals were scored for the DAI at the same time of each day, blind to the treatment. The minimal score was 0 and the maximal score was 4. Paraffin-embedded gut tissue samples were serially sectioned, and some sections were stained with hematoxylin and eosin (H&E). The stained sections were subsequently examined
for histopathological changes by a gastrointestinal pathologist. Proteins of the mouse colonic tissue that was collected on Day 14 were extracted with radio-immunoprecipitation assay lysis buffer (Thermo Scientific, Hanover Park, IL, USA) adding 10 μL/mL proteinase inhibitor cocktail and phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA). ELISA was performed with Multi-Analyte ELISArray Kit containing 12 mouse inflammatory cytokines [interleukin (IL)1α, IL1β, IL2, IL4, IL6, IL10, IL12, IL17A, interferon (IFN)-γ, tumor necrosis factor-α (TNF-α),
granulocyte colony-stimulating factor (G-CSF), and granulocyte–macrophage colony-stimulating factor (GM-CSF)] according to the manufacturer’s instructions. Total RNA was isolated from the mouse colonic tissues using the miRNeasy kit (QIAGEN, Valencia, CA, USA) based on the manufacturer’s instructions old and was used as a template Bafilomycin A1 nmr to synthesize cDNA for qRT-PCR. First strand cDNA was synthesized using Thermo Scientific Maxima First Strand cDNA Synthesis Kit. qRT-PCR was performed
on a 7900HT real-time PCR system (Applied Biosystems, Foster City, CA, USA). qRT-PCR with SYBR Green dye (QIAGEN) was used to determine the gene expression. Primers for qRT-PCR are listed in Table 1. β-actin was used as an endogenous control. Each sample was run in triplicate. Data are presented as mean ± standard deviation. Data were analyzed using analysis of variance (ANOVA) for repeated measures and Student t test. The level of statistical significance was set at p < 0.05. The chemical structures of 11 major ginsenosides, in the protopanaxadiol or protopanaxatriol groups, are shown in Fig. 2A. The chromatograph of AG extract is shown in Fig. 2B. As shown in Fig. 2C, the contents of protopanaxatriol type ginsenosides Rg1, Re, Rh1, Rg2, and 20R-Rg2 in AG extract were 0.43%, 11.33%, 0.10%, 0.15%, and 0.13%, respectively, whereas the contents of protopanaxadiol type ginsenosides Rb1, Rc, Rb2, Rb3, Rd, and Rg3 were 38.89%, 2.24%, 0.50%, 0.62%, 2.68%, and 0.28%, respectively. The total ginsenoside content was 57.4%. Starting from Day 4 after DSS treatment, animals in the model group showed apparent diarrhea and rectal bleeding.