Animals and Treatments. Adult male Wistar rats weighing 190 to 200 g were obtained from the Vivarium "Claude Bernard" belonging to the Autonomous University of Puebla. Rats were housed in a temperature-controlled room, with 12-h light-dark cycles and free access to food (standard chow) and drinking water. All procedures described in this study were under the Guide for the Care and Use of Laboratory Animals of the Mexican Council for Animal Care NOM-062-ZOO-1999. In the development of this study, it was ensured that rats do not suffer pain and discomfort. Fifty rats were divided into control and surgery groups (n = 25), and each group was subdivided into five groups (n = 5) to be administered with different signaling pathway inhibitors or vehicle solutions in the control groups (see inhibitors section).
Abdominal Surgery and Samples Obtention. The animals belonging to the surgery group were anesthetized with xylazine/ketamine (20/137 mg/Kg) and surgically operated to a 3-cm abdominal incision, as has been described previously [6]. Briefly, anterolateral aponeurosis and muscle were retracted to expose the liver for 1 min, and then rats were sewed with silk (4-O) in two layers (peritoneal and muscular). Control animals were anesthetized but did not subject to surgery. Nine hours after surgery, rats were re-anesthetized and sacrificed by exsanguination. Blood was drawn by cardiac puncture, and serum was prepared by centrifugation. Livers were excised, thoroughly perfused with ice phosphate-buffered saline (PBS) and stored at -70°C until further analysis.
Inhibitors Administration. Groups of 5 animals were administered with different inhibitors of each signaling pathway. Matched control groups (n = 5) were administered with an equivalent amount of vehicle used for dissolving the inhibitors. The inhibitors were administered in the following way: two hours before the experimental surgery, was administered AG490 (5 µg/kg bwt in 10% DMSO), an inhibitor of Jak2. Ninety minutes before surgery, Wortmannin (100 µg/kg bwt in 15% DMSO), an inhibitor of PI3K, was administered. One hour after surgery was administered PDTC (100 mg/kg bwt in saline solution), an inhibitor of NF-kB. Six hours before surgery was administered S3I-201 (5 mg/kg bwt in DMSO 10%), an inhibitor of STAT3. Finally, one hour before and again 4 hours after surgery SB239063 (10 mg/kg bwt in acidified 0.5% Tragacanth) was administered in combination with SP600125 (30 mg/kg bwt in olive oil), which was administered ninety min before surgery, each one of these inhibitors of MAPp38 and JNK signaling pathways, respectively.
Inhibitors were administered via intraperitoneal except for S3I-201 and SP600125, those which were administered intravenously and orally, respectively.
Zip14 RT-qPCR. Total RNA was isolated from hepatic tissue using TRI-reagent (Ambion) according to the manufacturer's instruction and then treated with DNase I, Amp Grade (Invitrogen). RNA was quantified by spectrophotometry (ND-1000 [V3.12], NanoDrop, Houston, TX, USA), and the integrity was confirmed using a 1.5% agarose gel. Total RNA (400 ng) was reverse transcribed using 200 U with MMLV reverse transcriptase, 0.2 µg of random primer, 10 mM dNTPs, and 20 U RiboLock RNase inhibitor (Thermo Scientific) in a total volume of 15 µL. TaqMan Gene Expression Assays Master mix (Applied Biosystems) was used for qPCR. Beta-actin (Actb) was used as the housekeeping gene. For quantification, the comparative CT method (ΔΔCT) was used, in which the resulting mRNA levels of the samples were normalized against the housekeeping gene mRNA levels as an endogenous reference. PCR reactions were performed in triplicate, and at least three independent experiments were developed.
Western Blot Analysis. Portions of the liver were homogenized in RIPA buffer with phosphatase and protease inhibitors (Pierce). Homogenates were centrifuged at 16,000 X g for 20 min at 20º C. Total soluble protein was determined by the Lowry method, and 40 µg protein per well was electrophoresed through 4–20% sodium dodecyl sulfate (SDS) polyacrylamide gels (Bio-Rad, México). After electrophoresis, proteins were electro-transferred to PVDF membranes. Immunodetection was performed using the following primary antibodies: ZiP14 (ab106568; Abcam), STAT3(sc482; Santa Cruz Biotechnology), p-STAT3 (sc8059; Santa Cruz Biotechnology), NF-kB p65 (sc7151; Santa Cruz Biotechnology), p-JNK (sc6254; Santa Cruz Biotechnology), p-p38 (sc-7973; Santa Cruz Biotechnology), p-Akt (sc101629; Santa Cruz Biotechnology) and β-actin (sc13065; Santa Cruz Biotechnology), and then incubated with a secondary antibody conjugated with horseradish peroxidase. The images of the western blots were analyzed using ImageJ (NIH image) analysis software.
Immunohistochemical Analysis. Hepatic tissue was fixed with 4% formaldehyde in PBS. All samples were dehydrated in a graded ethanol series and embedded in paraffin. Tissue sections of 5 µm were cut and mounted on slides coated with 3-aminopropyltriethoxysilane. Slides were heated at 60°C for 30 minutes, cooled at room temperature, deparaffinized, and rehydrated. Antigen retrieval was performed with Diva Decloaker (Biocare Medical) according to the manufacturer's instructions. Then slides were immersed in a 0.3% H2O2 solution for 10 minutes to inhibit endogenous peroxidase activity and washed three times with PBS (pH 7.4). After the washes, slides were treated with 2% bovine albumin (Sigma) solution at room temperature for one h in a humidified chamber. Affinity-purified rabbit anti-Zip14 antibody (Abcam) was diluted (1:170) in 1% bovine albumin (Sigma) solution. Specimens were incubated at 4°C overnight with the diluted primary antibody in a humidified chamber. The primary antibody was detected after incubating with an anti-rabbit conjugated with horseradish peroxidase secondary antibody (HRP, 1:200, Santa Cruz Biotechnology) for 2-h in a humidified chamber. The tissue in the slides was counterstained with hematoxylin for nuclei localization. Zip14 protein was identified by depositing a brown product developed using the Betazoid DAB Buffer (Biocare Medical).
Zinquin and Dithizone staining. A solution of Zinquin ethyl ester in DMSO (5 mM), freshly prepared, was diluted with PBS to a final concentration of 25 µM and applied to slides mounted with hepatic tissue, as described previously. After using this solution, slides were washed three times with PBS to remove the Zinquin excess, dried, and an anti-fade mounting medium (Vectashield) was added. The fluorescence of Zinquin was visualized by microscopy at excitation 351–358 nm and emission at 460 nm wavelength.
The dithizone staining was performed by dissolving 100 mg of dithizone (Sigma) in acetone and adding it to an equal volume of Zn-free water. Slides with hepatic tissue were exposed to a dithizone solution for 15 min, followed by a Zn-free water rinse, and later were covered with a cover slip and examined by light microscopy.
Statistical Analysis
Data are expressed as means +/- SEM, and statistical differences between control and experimental groups were determined using the student’s unpaired t-test.