Drugs and chemicals
D-pinitol (purity 95%) was purchased from Sigma-Aldrich Co., St Louis, MO, USA. Rats-specific TNF-α, IL-1β, and IL-6 enzyme-linked immunosorbent assay (ELISA) Kit were obtained from Bethyl Laboratories Inc., Montgomery, TX, USA. The primary antibodies of caspase-3, caspase-9, caspase-12, AFT4 (Activating transcription factor-4), AFT6α (Activating transcription factor-6α), XBP-1 (X-box binding protein 1), ERK-1/2 (Extracellular signal-regulated kinase-1/2), and P38 were purchased from Abcam, Cambridge, MA, USA.
Animals
Adult male Sprague Dawley rats (180-200 g) were purchased from Second Affiliated Hospital of Chongqing Medical University animal house, China, and kept in quarantine for one week in housed at the institute animal house at standard laboratory conditions, i.e., a temperature of 24±1°C, the relative humidity of 45–55% and 12:12 h light/dark cycle. Animals had free access to standard chaw pelleted food and water ad libitum. The experimental protocol was approved by the Institutional Animal Ethics Committee of Second Affiliated Hospital of Chongqing Medical University, China.
Induction of hepatic IRI
Rats were anesthetized with an intraperitoneal (i.p.) injection of pentobarbital (5%), and then they were subjected to surgery as described previously [6, 14]. Briefly, a partial warm hepatic ischemia model (70% of liver mass) was induced by occlusion of the hepatic artery and the portal vein (hepatic pedicle) with a microvascular clamp for 60 min. After removal of the clamps, reperfusion was initiated for next 24 hr.
Experimental groups
Animals were divided randomly into various groups (n=12-16) as follows:
Group 1: Sham group: Rats were subjected to the surgical procedure but without the occlusion of the hepatic pedicle. They received pre-treatment of a vehicle (10 g/kg of 1% aqueous DMSO solution (Dimethyl sulfoxide), p.o.) for 21 days.
Group 2: IRI control group: Rats were subjected to 60 min of partial ischemia (70%) followed by 24 h reperfusion. They received pre-treatment of a vehicle (10 g/kg of 1% aqueous DMSO solution, p.o.) for 21 days.
Group 3: Thymoquinone (30 mg/kg) treated group: Rats were subjected to 60 min of partial ischemia (70%) followed by 24 h reperfusion. They received pre-treatment of Thymoquinone (30 mg/kg, p.o.) for 21 days.
Group 4: D-Pinitol (5 mg/kg) treated group: Rats were subjected to 60 min of partial ischemia (70%) followed by 24 h reperfusion. They received pre-treatment of D-Pinitol (5 mg/kg, p.o.) for 21 days.
Group 5: D-Pinitol (10 mg/kg) treated group: Rats were subjected to 60 min of partial ischemia (70%) followed by 24 h reperfusion. They received pre-treatment of D-Pinitol (10 mg/kg, p.o.) for 21 days.
Group 6: D-Pinitol (20 mg/kg) treated group: Rats were subjected to 60 min of partial ischemia (70%) followed by 24 h reperfusion. They received pre-treatment of D-Pinitol (20 mg/kg, p.o.) for 21 days [18, 19].
Serum biochemistry
On day 21, at the end of the reperfusion period (24 h), blood was withdrawn by a retro-orbital plexus, and serum was obtained by centrifugation at 8350 ×g for 10 min, at 4 °C. The levels of serum Aspartate transaminase (AST) and alanine aminotransferase (ALT) were measured by using reagent assay kits (Accurex Biomedical Pvt. Ltd., Mumbai, India) with an ultraviolet-visible spectrophotometer.
Biochemical estimation:
Tissue homogenate preparation, estimation of oxidative stress and pro-inflammatory markers
All animals were sacrificed at the end of the study, i.e., on the 22nd day, the liver was immediately isolated. Tissue homogenates were prepared with 0.1 M Tris-HCl buffer (pH 7.4), and supernatant of homogenates was employed to estimate superoxide dismutase (SOD), reduced glutathione (GSH), lipid peroxidation (MDA, i.e., Malondialdehyde content), and nitric oxide (NO content) as described previously [23, 24]. Another portion of aliquot was used for estimation of hepatic pro-inflammatory markers using rats-specific TNF-α, IL-1β, and IL-6 enzyme-linked immunosorbent assay (ELISA) Kit (Bethyl Laboratories Inc., Montgomery, TX, USA).
Mitochondrial enzymes estimation:
Liver mitochondrial isolation was performed according to a previously described method [25]. Mitochondrial complex-I activity was measured spectrophotometrically according to a previously described method [26]. Mitochondrial Complex-II activity Succinate dehydrogenase (SDH) was measured spectrophotometrically according to an already described method [27]. Mitochondrial redox activity (Complex-III) i.e., the MTT reduction rate, was used to assess the activity of mitochondrial respiratory chain in isolated mitochondria. It was determined, according to an already established method [28]. Mitochondrial complex-IV (Cytochrome oxidase assay) activity was assayed in liver mitochondria according to the previously described method [29].
Reverse Transcriptase PCR
The mRNA expressions of GRP78, CHOP, and β-actin were analyzed in liver tissue using quantitative reverse transcription-polymerase chain reaction (qRT–PCR) according to the method described elsewhere [30]. PCR was performed using 1 X forward and reverse primers, and 2.5 U Taq polymerase (MP Biomedicals India Private Limited). Amplification of β-actin served as a control for sample loading and integrity.
Western blot procedure
The protein expressions of caspase-3, caspase-9, caspase-12, AFT4, AFT6α, XBP-1, ERK-1/2, P38, and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were estimated in liver tissue according to the method described elsewhere [31]. Briefly, liver tissue was dissected and sonicated in Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Inc.). The lysates were centrifuged at 10,000 x g for 10 min at 4°C. Protein concentrations were determined using a Bicinchoninic Acid (BCA) assay kit (Beyotime Shanghai, China) on ice for 30 min. An equal amount of extracted protein samples (50 μg) were separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat dry milk at 37°C for 1 h and incubated overnight at 4°C with the respective primary antibodies that recognized caspase-3, caspase-9, caspase-12, AFT4, AFT6α, XBP-1, ERK-1/2, P38 and GAPDH. Anti-rabbit horseradish-linked IgG was used as the secondary antibody, incubated for 37°C for 2 h. Protein bands were visualized using the Chemiluminescent kit. GAPDH served as the loading control.
Preparation of single-cell (SC) suspensions and flow cytometry analysis
Preparation of single-cell (SC) suspensions and determination of apoptotic cell populations were determined as previously described [32]. At the end of treatment, the liver of rats was collected and mixed with 0.4% collagenase and 0.25% Trypsin at 37 °C for 30 min and dissociated, grinded and obtained homogenate was passed through a 70 μm nylon mesh. Single-cell (SC) suspension was washed three times with phosphate-buffered saline (PBS). In order to determine SC apoptosis, the isolated SCs were incubated with rabbit anti-cow S-100 antibody and followed by staining with APC-goat anti-rabbit IgG (both from BD) with FITC-Annexin V and PI (Sigma-Aldrich, St. Louis, MO, USA). The percentages of expression of Fas and Annexin-V on gated S-100 positive SC were analyzed by a FACS Calibur cytometer using CELL Quest software.
Histological and electron microscopic analysis
Histopathological analysis of liver tissue was carried out using hematoxylin and eosin (H&E) stain under a light microscope whereas, the liver ultrastructural studies were performed under a transmission electron microscope according to the method described previously [33].
Statistical analysis
GraphPad Prism (GraphPad, San Diego, CA) was used to perform data analysis. Data are expressed as mean ± standard error mean (SEM) and analyzed by using One-Way ANOVA followed by Tukey’s multiple range post hoc analysis (for parametric tests). A value of p < 0.05 was considered to be statistically significant.