A total of 40 eight-week-old male Sprague-Dawley rats purchased from Chang Sheng Biotechnology Corp. (Liaoning, China) were used in the present study. Animals were housed in the animal facility at the Shenghai Hospital of China Medical University (Shenyang, China) with standard 12 h light/dark cycles and ad libitum access to food and water. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Shengjing Hospital of China Medical University (Shenyang, China). RSV was purchased from Guanyu Biotechnology Corp. (Shanxi, China). Animals were randomly assigned into five different groups with eight rats in each group: i) Control group: animals were exposed to normal conditions without RSV treatment; ii) CIH group: animals were exposed to intermittent hypoxic conditions without RSV treatment; iii) CIH plus low RSV group: animals were exposed to intermittent hypoxic conditions and administered daily with 3 mg/kg/day of RSV; iv) CIH plus median RSV group: animals were exposed to intermittent hypoxic conditions and administered daily with 30 mg/kg/day of RSV; v) CIH plus high RSV group: animals were exposed to intermittent hypoxic conditions and administered daily with 60 mg/kg/day of RSV. RSV was prepared by adding 3 ml water. Rats were fed via oral gavage with 3 ml RSV daily, for a total of 28 days. Rats in control groups without RSV treatment were administered 3 ml of water as vehicle via an oral gavage. The body weight of animals was recorded every week for all animals. Blood glucose levels and insulin levels were recorded at the end of the experiment for all animals.

Animals were exposed to intermittent hypoxia every day for 8 h, from 9 a.m. to 5 p.m., for 28 days, as described previously (24,25). Briefly, housing cages were placed inside plexiglas chambers where the oxygen level was monitored and regulated by timer-controlled valves connected to room air source and a nitrogen source. The air and nitrogen entered the chamber via separate flow meters. During this period of time, oxygen was reduced from 21% to 10% over 1.5 min, maintained at 10% for 1.5 min, returned to 21% over 1 min and held at 21% for 2 min. This cycle was repeated for 8 h. Control animals were housed in identical chambers for an equivalent amount of time and were exposed to the same timer and valve-controlled changes in air flow as the CIH rats; however, the only source of gas in the control chambers was room air, so they remained at normoxic levels throughout the investigation. Immediately following the final hypoxia session, the cages were returned to the main housing room. The animals were fasted for 12 h prior to day 29 following the treatment and then sacrificed under isoflurane anesthesia. Liver tissues and blood samples were collected.

Animals were sacrificed in a preprandial state under isoflurane anesthesia on day 29. Liver tissues were collected from all animals. All tissues were rapidly frozen in liquid nitrogen and stored at −80°C for future investigation. Paraffin-embedded tissue sections (4 μm thick) were obtained and were stained with hematoxylin and eosin (H&E).

Blood glucose concentration was measured using a Contour glucometer (Bayer, Pittsburgh, PA, USA) and Contour blood glucose test strips (Bayer). Plasma insulin was determined using a Rat Insulin ELISA kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. The value of the homeostatic model assessment (HOMA)-IR index was calculated using the following formula: [Fasting insulin concentration (μU/ml) × fasting glucose concentration (mmol/l)] / 22.5.

Sirt1 protein expression in paraffin-embedded hepatic tissue sections was detected using a standard avidin-biotin immunoperoxidase protocol. Briefly, liver sections were deparaffinized and pretreated with 1% H₂O₂ for 30 min to quench endogenous peroxidase activity, followed by three rinses in phosphate-buffered saline (PBS). The samples were blocked with 10% goat serum (Solarbio, Beijing, China) and subsequently incubated with 1:50 dilution of polyclonal rabbit anti-rat Sirt1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C. This was followed by incubation with 1:500 dilution of polyclonal goat anti-rabbit secondary antibody (Beyotime, Shanghai, China) at room temperature for 1 h. Immune complexes were detected using an avidin-3,3′-diaminobenzidine staining kit (Beijing Zhongshan Biotechnology, Beijing, China). The sections were counterstained with hematoxylin and mounted. Images of the slides were captured using a stereo BX51 light microscope (Olympus, Tokyo, Japan) equipped with a digital camera.

SIRT1 expression was determined by RT-qPCR as described previously (26). Briefly, total RNA was isolated from rat liver using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized by RT using the QuantiTec Reverse Transcription kit (Tiangen Biotech, Beijing, China). Subsequently, qPCR was performed using the following primers: Sirt1, forward 5′-ACCCTCAATTTCTGTTCTGC-3′ and reverse 5′-TTGGACATTACCACGTCTGC-3′; β-actin, forward 5′-GGAGATTACTGCCCTGGCTCCTAGC-3′ and reverse 5′-GGCCGGACTCATCGTACTCCTGCTT-3′. The cycle threshold (Ct) values of the target gene were first normalized to β-actin from the same sample and subsequently the relative differences between the groups were calculated and expressed as relative mRNA levels, setting the control group as 1. Each sample was assessed in triplicate.

Liver tissue was homogenized in radio immunoprecipitation lysis buffer (Beyotime) supplemented with protease inhibitors (PMSF and aprotonin) and protein phosphatase inhibitor (NaF and sodium orthovandate). The protein concentration was determined using a bicinchoninic acid assay protein assay kit (Beyotime). Protein aliquots (40 μg) were separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat milk in PBS and incubated overnight at 4°C with one of the following primary antibodies: Rabbit polyclonal anti-SIRT1 (1:400; Santa Cruz Biotechnology, Inc.); rat polyclonal anti-insulin receptor (InsR; 1:1,000; Wuhan Boster Biological Technology, Ltd., Wuhan, China); rabbit polyclonal anti-glucose transporter 2 (Glut2, 1:400; Santa Cruz Biotechnology, Inc.); rabbit polyclonal anti-phospho-Tyr467-phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K; 1:400; Santa Cruz Biotechnology, Inc.); rabbit polyclonal anti-total-PI3K (1:1,000; Wuhan Boster Biological Technology, Ltd.); rabbit polyclonal anti-phospho-Ser473-AKT (1:400; Santa Cruz Biotechnology, Inc.); rabbit polyclonal anti-total-AKT (1:400; Wuhan Boster Biological Technology, Ltd.). Following incubation with the primary antibody, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies at 37°C for 45 min. Enhanced chemiluminescence reagent (Millipore) was used for detection of protein bands. The densities of the protein bands were determined using a gel imaging system (Beijing Sixty-One Instrument, Beijing Six-One Instrument, Beijing, China). The expression levels of Sirt1, InsR, total PI3K, phosphorylated-PI3K, total AKT, phosphorylated-AKT and Glut2 protein levels were normalized against those of β-actin levels.

Data are presented as the mean ± standard deviation. For a comparison between two groups, Student’s t-test was used. For comparisons among three or more groups, one-way analysis of variance was used. P<0.05 was considered to indicate a statistically significant difference.

Weight gain is associated with chronic hypoxia in animals and humans. To evaluate the effect of RSV on body weight in animals following hypoxia exposure, the weight of each animal was recorded every week. Prior to exposure to hypoxia, all rats had similar body weights. Compared with the control group, the body weight in rats significantly increased following CIH exposure. At day 29, the average body weight of control rats was 297 g and that of CIH-treated rats was 388 g (P=0.000). The body weights in rats treated with RSV decreased; the average weight was 372.125 g for the CIH plus low RSV group, 323.675 g for the CIH plus median RSV group and 370.250 g for the CIH plus high RSV group. Compared with the CIH group, there were significant differences among RSV-treated groups (P<0.01). These results demonstrate that CIH increases the body weight of rats, however, RSV inhibits this weight gain (Fig. 1).

To evaluate the effect of RSV on IR following chronic hypoxia exposure, the blood glucose and insulin levels of animals was measured on day 29 following hypoxia exposure. IR was assessed using the HOMA-IR index. Compared with the control group, the HOMA-IR values of CIH-treated groups were significantly higher (P=0.000). The values were 4.14±0.29 (control group), 11.32±0.47 (CIH group), 9.70±0.24 (CIH plus low RSV group), 7.55±0.29 (CIH plus median RSV group) and 6.16±0.06 (CIH plus high RSV group), respectively. Compared with the CIH without RSV group, the values significantly decreased in other CIH-treated groups following RSV treatment (P=0.000). The reduction in RSV-treated groups was dose dependent. However, the values in CIH-exposed groups remained higher than that observed in the control group regardless of RSV treatment (Fig. 2).

To evaluate the morphological alterations in the rat liver following CIH exposure and the effect of RSV on these changes, liver tissue sections were stained with H&E. The hepatocytes in the control rat tissue were tightly arranged together with clearly visible nuclei. The hepatocytes in the CIH-treated rats were swollen accompanied with nuclear pyknosis, degeneration and only partial cytoplasmic vacuoles were present. The changes in the group treated with a low dose of RSV were similar to that observed in the CIH without RSV group, for example, swollen hepatocytes, fuzzy cell boundaries, nuclear pyknosis and the presence of partial cytoplasmic vacuole were observed. The cell boundaries in the median RSV treated rats were less fuzzy than those observed in the control group. The changes in the group treated with a high dose of RSV appeared normal. These observations suggested that RSV may protect the rat against CIH-induced liver damage (Fig. 3).

RSV is hypothesized to be the most potent activator of Sirt1. To evaluate the effect of RSV on Sirt1 levels in liver following CIH exposure, mRNA and protein levels of Sirt1 in liver tissue were analyzed. Compared with the control group, Sirt1 protein levels in CIH-treated groups significantly reduced following CIH exposure (P=0.000). However, the levels in the RSV-treated CIH groups were elevated compared with that in the CIH group without RSV treatment (P=0.000). These changes were dose dependent (Fig. 4A). The mRNA changes of SIRT1 were similar to that observed in the protein levels. SIRT1 mRNA levels in the hepatic tissue were significantly reduced following CIH (P=0.000), however, RSV treatment significantly increased the mRNA levels (P=0.000; Fig. 4B). The IHC staining revealed a significant reduction in the SIRT1-positive cells in hepatic tissue sections from rats subjected to CIH compared with that detected in the control group, however, RSV treatment led to an increase in Sirt1-positive cells (Fig. 4C). These results suggested that RSV significantly upregulated the levels of Sirt1 in rats following CIH exposure.

InsR and Glut2 are important in regulating glucose metabolism. The levels are positively correlated with IR and diabetes. To evaluate the effect of RSV on these levels in the liver, the levels of the two proteins were analyzed using western blotting. The gel densities were quantitated and expressed as fold-changes following normalizing against the levels of β-actin. The relative protein InsR level was significantly higher in the CIH group compared with that in the control group (P=0.000; Fig. 5A). Compared with the CIH without RSV group, RSV treatment significantly reduced InsR levels (P=0.042, 3 mg/kg/day RSV; P=0.000, 30 mg/kg/day RSV; P=0.000, 60 mg/kg/day RSV). The level of InsR in the group treated with a low dose of RSV was higher than that in the normal control, while, the level of InsR in the groups treated with a median and high dose of RSV were similar to that in the normal control (Fig. 5A). The alterations in Glut2 levels were similar to those of InsR. Glut2 levels significantly increased in the CIH group compared with that in the control group (P=0.000). Compared with the CIH without RSV group, RSV treatments significantly reduced Glut2 levels at 30 and 60 mg/kg/day of RSV, but not 3 mg/kg/day of RSV (P=0.422, 3 mg/kg/day RSV; P=0.004, 30 mg/kg/day RSV; P=0.000, 60 mg/kg/day RSV; Fig. 5B).

To further investigate the underlying mechanisms by which RSV reduces IR in chronic hypoxic animals, the phosphorylation of PI3K and AKT was analyzed. The data demonstrated that CIH exposure significantly inhibited the phosphorylation of PI3K and AKT (P=0.000). RSV treatment significantly upregulated the phosphorylation of PI3K and AKT (P=0.000). RSV treatment had no effect on total PI3K and total AKT protein levels following CIH exposure (P>0.05) with the exception of 3 mg/kg/day RSV (P<0.05). Compared with the control group, the total levels of PI3K and AKT in the CIH group increased (P=0.000; Fig. 6).