Ethics information
All procedures were carried out in accordance with the European Communities Council Directive (86/609/ CEE) on animal experiments and with approval from the ethical committee on animal welfare of our institution (Comité Etico de Experimentación Animal del Centro de Investigación Biomédica de La Rioja, CEEA-CIBIR). In addition, all the study was carried out in compliance with the ARRIVE guidelines (https://arriveguidelines.org). Consent to participate not applicable.
The Materials and Methods section has been published elsewhere [20]. Briefly, a total of 80 male homozygous IL-10-deficient mice (B6.129P2-IL10tm1Cgn/J) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). When the animals were approximately 6 weeks old, they were randomly assigned (n = 20) to one of 4 groups and fed for 24 weeks: i) the IL-10KO group (IL-10KO) received a standard rodent diet and tap water; ii) the preventive MVC group received the same diet as the IL-10KO group and were administered MVC (Pfizer, New York, N York) in their drinking water (300 mg/L) [4,19,20]; iii) the preventive RAPA group received the same diet as the IL-10KO group and were administered RAPA in their drinking water (1.5 mg/kg/day) [20]; and iv) the preventive MVC plus RAPA group (MVC-RAPA) received the same diet as the IL-10KO group and were administered MVC plus RAPA in their drinking water at the same concentration as in the MVC and RAPA groups.
The mice were observed daily, and all observations were recorded. All animals were sacrificed at Week 24. Blood samples were collected under anesthesia after a 4-hour fasting period. Internal organs were examined macroscopically and weighed.
Gene expression quantification
Total RNA was extracted and purified from liver samples using an RNA RNeasy Mini Kit (Qiagen, Valencia, CA) and treated with DNase I (Qiagen) according to the manufacturer’s instructions [20]. cDNA was synthesized by reverse transcription of 1 mg of total RNA using the SuperScript III First-Strand Synthesis kit (Invitrogen, Carlsbad) in a total volume of 20 µl according to the manufacturer’s instructions, followed by amplification using SYBR Green (Takara Bio Inc, Shiga, Japan) The PCR primer sequences are listed in Supplemental Table 1. The amplification and detection of specific products were performed using an ABI PRISM 7300 system (Applied Biosystems, Foster City, CA, USA). All reactions were run in duplicate for each sample. The expression of respective genes was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control [20].
Western blot analysis
Liver samples were lysed using a homogenizer in RIPA lysis buffer (Sigma Aldrich, St. Louis, MO). The cell lysate was centrifuged at 10,000 g for 10 minutes at 4 °C [20]. The concentration of total protein in each sample was determined by the Bradford method. 5´adenosine monophosphate-activated protein kinase (AMPK) [35], phosphorylated AMPK (pAMPK) [36], protein kinase-B (Akt) [37], phosphorylated Akt (pAkt) [38], nuclear factor-kB (NFKB) [39], phosphorylated NFKB (pNFKB) [40], mammaliam target of rapamycin (mTOR) [41] and phosphorylated mTOR (pmTOR) [42] were evaluated by Western blotting (WB). GAPDH was used as an internal control [43] (Supplemental Table 2).
Proteins were analyzed by colorimetry using a secondary antibody bound to peroxidase (anti-rabbit IgG, Cell Signaling, Danvers, MA) and incubated with the corresponding substrate [20]. After high-resolution scanning, the concentration of each protein was evaluated by densitometry using Image J software. The density values for each of the test samples were normalized to the values of GAPDH, which was used as a loading control [20].
Histopathological Studies
Hematoxylin-eosin (HE) Staining
Following fixation, tissues were dehydrated and embedded in paraffin. Tissue sections (3 mm thick) were rehydrated and stained with HE according to standard protocols.
Immunohistochemical Analysis of Ki67
Deparaffinized sections were hydrated in a graded series of alcohol solutions. The sections were incubated in antigen retrieval buffer (the sections were boiled at 98 °C for 20 minutes in 10 mmol/L sodium citrate buffer) and treated with 3% H2O2 to block endogenous peroxidase [44]. A monoclonal antibody (anti-Ki67, Santa Cruz Biotechnology, Inc.) was applied to the slides and incubated in a humidified chamber overnight in a refrigerator at 4 °C [44]. Biotinylated secondary antibodies were then applied, followed by incubation with streptavidin peroxidase. The sections were washed with phosphate-buffered saline (PBS) three times after each step. The sections were stained with diaminobenzidine (DAB) chromogen solution and counterstained with hematoxylin [44].
Statistical analysis
The data are presented in the figures as the mean ± SEM (standard error of the mean). Body weight data were analyzed with analysis of variance followed by the Dunnett post hoc test. For all other data, the Kruskal–Wallis test was used followed by the Mann–Whitney U test. Correlations between variables were determined using the Spearman rank-sum test. All data were analyzed with GraphPad Prism 6 software and were considered statistically significant when p<0.05.