A total of 36 male six-week-old Sprague-Dawley rats, weighing 200–250 g, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and allowed to acclimatize for one week prior to experiments. The rats were randomly assigned into one of three groups: A control group (n=12, exposed to mock anesthesia), an isoflurane group (n=12, exposed to 2% isoflurane for 90 min) and an isoflurane + IGF-1 group (n=12, exposed to 2% isoflurane for 90 min and then administered IGF-1 intervention). The rats were maintained under a standard 12/12-h light/dark cycle in temperature-controlled cages (22±5°C) with a relative humidity of 60±10% and received standard food pellets and distilled water ad libitum. All animals were treated in accordance with the standards of the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Bethesda, MD, USA) and all protocols were approved by the Ethics Committee on Animals at Sun Yat-Sen University (Zhuhai, China).

The rats in the isoflurane groups were placed in an induction chamber and received 2% isoflurane (Forene; Abbott, Queenborough, Kent, UK)/98% medical grade air (vol/vol) from an anesthesia machine (Fabius CE, Dräger Medical, Lübeck, Germany) for 90 min. Rats in the control group received air/oxygen at identical flow rates. The concentration of isoflurane was monitored continuously with a Datex Capnomac gas-analyzer (Datex-Ohmeda, Helsinki, Finland) throughout anesthesia. The rectal temperature was maintained at 37.0±0.5°C by a heating lamp and pulse oximeter oxygen saturation (SpO₂) was routinely monitored. The rats in the isoflurane + IGF-1 group received anesthetic exposure, then intravenous injections of IGF-1 (20 µg/kg; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) at different amounts of time (0 and 72 h and 7 days). Thereafter, all animals were sacrificed by decapitation after administration of IGF-1 at 0 and 72 h and 7 days and liver tissues were collected for further analyses.

The extent of liver necrosis was determined in 10% neutral buffered formaldehyde-fixed (4°C for 6 h) paraffin-embedded liver sections stained with hematoxylin and eosin, according to a previously described method (21). Briefly, sections were harvested at different times following the application of IGF-1 (0 h, 72 h and 7 days). Sections were deparaffinzed, hydrated, stained in alum hematoxylin, differentiated with acid alcohol, washed with tap water, stained with eosin and dehydrated. Sections were then observed under a light microscope (BX51; Olympus Corp., Tokyo, Japan) by a pathologist who was blinded to the groups. Ten high-power fields were randomly collected. The area of necrosis was quantified using ImageJ software version 1.45 (National Institutes of Health). The necrosis rate was calculated as the area of necrosis divided by the total area.

A TUNEL assay was performed to determine the apoptosis of hepatic cells in rat livers using a commercially available TUNEL kit (APO-DIRECT™ kit; BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. Briefly, sections were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with phosphate-buffered saline (PBS) twice, maintained with 0.1% Triton X-100 in PBS and then incubated with H₂O₂ for 10 min in methanol at room temperature. Sections were then washed with PBS, followed by incubation in 50 µl biotin labeling solution at 37°C for 1 h in a humidified chamber. Following washing in PBS, sections were incubated with labeling termination solution for 5 min at room temperature and washed again. Streptavidin-horseradish peroxidase working solution (50 µl; Novolink Polymer; Leica Microsystems, Ltd., Milton Keynes, UK) was added to the sections at room temperature for 30 min. Subsequently, sections were incubated in diaminobenzidine working solution (Novolink Polymer; Leica Microsystems, Inc.) at room temperature, washed twice with PBS and photographed using a fluorescence microscope (BX50/BX-FLA/DP70; Olympus Corp.).

Levels of IGF-1 and IGF-1R mRNA were measured using standard RT-qPCR techniques. RT-qPCR was performed at least three times. Total RNA was derived from liver tissues using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA according to the manufacturer's instructions. First-strand cDNA was synthesized with the EasyScript^® First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Inc., Beijing, China). Amplification reactions were determined using the GeneAmp PCR System 2400 thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed using a QuantiTect SYBR Green RT-PCR kit (Qiagen SA, Courtaboeuf, France). Primer sequences were as follows: IGF-1, forward 5′-GTTCCGATGTTTTGCAGGTT-3′ and reverse 5′-TCTGAGGAGGCTGGAGATGT-3′; IGF-1R, forward 5′-ACTATGCCGGTGTCTGTGTG-3′ and reverse 5′-TGCAAGTTCTGGTTGTCGAG-3′; and GAPDH, forward 5′-TCCTGCACCACCAACTGCTTAG-3′ and reverse 5′-AGTGGCAGTGATGGCATGGACT-3′. PCR conditions were as follows: Initial denaturation was performed at 95°C for 15 sec, followed by 30 cycles of denaturation for 30 sec at 95°C, annealing at 61°C for 5 sec, extension at 72°C for 15 sec, and subsequent final extension was performed at 72°C for 10 min. PCR products were run on an 1.2% agarose gel. The GAPDH gene was used as a reference gene. Fold changes relative to GAPDH were calculated using the 2^−ΔΔCq method (22).

Tissue was harvested, washed with PBS and homogenized in 10 mM Tris buffer (pH 7.5). Total tissues homogenates were sonicated then centrifuged at 1,000 × g for 20 min at 4°C to extract protein. Protein concentration was assessed using a bicinchoninic acid protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein samples (20 µg per lane) were resolved with 10% SDS-PAGE. The separated proteins were transferred onto a polyvinylidene fluoride ultrafiltration membrane and the membrane was washed twice with Tris-buffered saline with Tween (TBST). The membranes were immersed in 5% fresh nonfat dry milk in TBST for 2 h at room temperature, washed with TBST and incubated with following polyclonal primary antibodies overnight at 4°C: Anti-IGF-1 antibody (sc-14221; 1:1,000), anti-IGF-1R antibody (sc-80985; 1:1,000), anti-caspase-3 antibody (sc-271759; 1:1,000), anti-B-cell lymphoma-extralarge (Bcl-xL) antibody (sc-136132; 1:1,000) or anti-GAPDH (sc-293335; 1:1,000). All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Following washing with TBST, the membrane was incubated with horseradish peroxidase-labeled goat anti-mouse secondary antibody (ZDR-5307; 1;5,000) (Beijing Zhongshan Golden Bridge Biotechnology, Co., Ltd., Beijing, China) for 2 h at room temperature. Finally, bands were visualized using an enhanced chemiluminescence western blotting substrate (Pierce; Thermo Fisher Scientific, Inc.), exposed to an X-ray film (Midwest Scientific, St. Louis, MO, USA), followed by densitometric analysis.

Data are represented as the mean ± standard deviation. The differences were compared using Student's t-test (for two groups) or one-way analysis of variance (for three or more groups). All statistical analyses were performed using SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To evaluate the role of IGF-1 in the liver injury induced by isoflurane, levels of IGF-1 and IGF-1R mRNA and protein were measured following isoflurane exposure for 90 min. The results indicated that compared with the control group, levels of IGF-1 and IGF-1R mRNA (Fig. 1) and protein (Fig. 2) were significantly decreased following isoflurane exposure (all P<0.05), indicating that isoflurane significantly inhibited the expression of IGF-1 and IGF-1R.

To assess the protective function of IGF-1 on the liver, IGF-1 was applied following exposure to isoflurane. At 0, 72 h and 7 days, the liver tissues were harvested and the necrosis rate was determined. At 7 days, the necrosis rate was significantly increased in the isoflurane group compared with the control group (P<0.05); however, compared with the isoflurane group, the necrosis rate was significantly decreased following the application of IGF-1 (P<0.05; Fig. 3). The results suggest that IGF-1 application may reduce tissue necrosis to some extent.

Subsequently, the effect of IGF-1 application on liver apoptosis was evaluated. As presented in Fig. 4, the number of apoptotic cells detected at day 7 were significantly higher following isoflurane exposure than those in the control group (P<0.05). However, compared with the isoflurane group, the number of apoptotic cells was reduced in the group receiving IGF-1 (P<0.05), demonstrating that IGF-1 application may effectively decrease the rate of apoptosis in the liver.

The mechanism of apoptosis was further investigated. The expression levels of the apoptosis-related proteins caspase-3 and Bcl-xL were determined. It was demonstrated that isoflurane significantly increased levels of caspase-3 compared with the control group (P<0.05; Fig. 5A), but decreased levels of Bcl-xL (P<0.05; Fig. 5B). However, application of IGF-1 significantly reduced levels of caspase-3 compared with the isoflurane group (P<0.05), but elevated levels of Bcl-xL (P<0.05; Fig. 5A and B). These results indicate that isoflurane induces apoptosis by elevating the expression of pro-apoptotic proteins (caspase-3), and reducing the expression of anti-apoptotic proteins (Bcl-xL). By contrast, IGF-1 treatment appeared to inhibit apoptosis by decreasing the expression of the pro-apoptotic protein caspase-3, and increasing the expression of the anti-apoptotic protein Bcl-xL (Fig. 5).