The rat hepatic cancer cell line Morris hepatoma 3924A was purchased from American Type Culture Collection (Manassas, VA, USA) Morphine, bupivacaine and fentanyl were purchased from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). The cell line was cultured at 37°C in a humidified atmosphere containing 5% CO₂ in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (FBS), 100 U ml⁻¹ penicillin and 100 mg ml⁻¹ streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 30 male American Certification Institute (ACI) rats (age, 12–18 weeks; weight, 200–250 g) were purchased from the Experimental Animal Center of Changchun Biological Institute (Changchun, China), and they were housed at a temperature of 20–22°C, relative humidity of 50–60% and 12/12 h light/dark cycles. They were also provided with free access to food and water. The 7-0 injury-free operative sutures were purchased from Shanghai Surgical Suture Company. ELISA kit and antibodies used in the present study were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Details were as follows: IFN-γ ELISA kit (cat no. BMS621); TGF-α ELISA kit (cat no. EHTGFA); VEGF ELISA kit (cat no. ERVEGFACL); phospho-STAT3 (Tyr705) polyclonal antibody (cat no. 44-380G; dilution, 1:1,000); and phospho-EGFR (Tyr1086) polyclonal antibody (cat no. 36-9700; dilution, 1:500). The anti-rabbit IgG secondary antibody, horseradish peroxidase (cat no. P7899; dilution, 1:100,000) was provided by Sigma-Aldrich (Merck KGaA; Darmstadt, Germany).

All animal experiments were performed in compliance with the Animal Management Rules of the Ministry of Health of the People's Republic of China (http://www.medste.gd.cn/Html/sciedu/sysgl/Class3141/24961720080910094400.html) and approved by the Animal Care and Use Committee of Jilin University (Changchun, China). All animals were pathogen-free and allowed ad libitum access to food and water. To produce the tumor xenograft model, MH3924A cells in the logarithmic phase were injected subcutaneously into the upper axillary fossa of the male ACI rats (12–18 weeks, n=10 per group). After 14 days, subcutaneous tumors were surgically removed. Fresh tumor tissues were chosen and divided into l-mm³ tumor pieces. The male rats were fixed on a frame prior to anesthesia, then the abdominal body hair was removed and the animals were disinfected with iodine. A 5-ml syringe was placed at the back of the fixed rats to raise the abdomen of the rats for surgical convenience. Next, when the size of incision in upper abdomen reached ~0.5 cm, the skin, subcutaneous tissue and peritoneal tissue was consecutively cut and then the rat liver was exposed. The left lobe was cleaned with wet cotton and the liver capsule was punctured with ophthalmic scissors, making a further long cut under the capsule. Next, the as-prepared tumor tissue was inserted into the long cut, which was closed with 7-0 microscopic sutures. Prior to abdominal closure, it should be confirmed that there was no blister tissue out and no bleeding.

When the tumor volume reached ~300 mm³, these tumor-bearing rats were randomly divided into three treatment groups: One group was treated with hepatectomy and general anesthesia combined with epidural block (group G + E), one group was treated with hepatectomy and general anesthesia (group G) and one group, the control group, did not undergo any treatment (group C). There were 10 rats included in each group.

A simple tumor resection was performed under general anesthesia. Specifically, sevoflurane together with oxygen was injected into a self-made anesthesia box. Next, the retraction response of rat toes to stimulus was continuously assessed to determine the degree of anesthesia. When the retraction response of rat toes to stimulus disappeared, tracheal incubation was conducted for the rat by transmission light intubation. Specifically, the 50-W illuminant was placed 5 cm away from the neck of the rat. In this way, the light could pass through the neck skin and trachea wall in sequence. A mini sized spatula with a 145° angle were used to push away the tongue for clear observation of the epiglottis and vocal cord movement under the transmitted light, thus an 18 G cannula was inserted under a bright view. The regular movement of the rat thorax was observed following connection with the ventilator with mechanical ventilation. The parameters of the ventilator were set as follows: Breathing rate, 55/min; tidal volume, 3 ml/100 g; inspiration rate: expiration rate, 1.5:1; sevoflurane concentration, 2–3% (v/v). To maintain the anesthesia effect, morphine (0.2 ml/100 g, 10% solution) was injected intraperitoneally. When the rats woke following surgery, the endotracheal tube was removed.

Perioperative epidural analgesia consisted of a bolus of 6–9 ml of 0.25% bupivacaine followed by a postoperative infusion of bupivacaine (0.1–0.2%) with or without 2 µg/ml of fentanyl at a rate of 6–9 ml/h. Specifically, the limbs and head of the rat were fixed following the success of tracheal incubation under general anesthesia. A self-made sterile PVC epidural catheter with diameter of 0.3 mm was inserted via removing the eighth or ninth thoracic spinous process using a vertical needling and was advanced 1 cm cephalad into the epidural space. Subsequently, 0.25% of bupivacaine was micro-injected via the epidural catheter.

According to observations, the tumor size was suitable for hepatectomy and no distant metastasis was observed at day 14 following inoculation with tumor cells. Hepatectomy was conducted following successful anesthesia with the assistance of undergraduate students majoring in liver surgery (Departments of General Surgery, China-Japan Union Hospital Jilin University, Changchun, China). Specifically, the skin, subcutaneous tissue and peritoneal tissue was consecutively removed from the original upper abdomen incision. Next, the left lobe of the liver was exposed and the megascopic in situ tumor tissue was removed, as was 5 mm normal liver tissue around the edge of tumor. The 7-0 microscopic suture was used to close the abdomen following the confirmation of no bleeding.

Venous blood samples were collected from orbits of rats immediately prior to surgery and at 2, 7 and 30 days following surgery. The blood samples were left to clot at room temperature and then stored overnight in a refrigerator to contract the clot. The clot was removed and the remaining material was centrifuged at 4,000 × g for 5 min and the resulting serum was stored at −80°C until analysis. Plasma levels of IFN-γ, TGF-α and VEGF were measured by an ELISA. Plasma levels of each cytokine were measured in duplicate from one aliquot of each sample and the mean value from these two measurements was used as the final concentration. Quantification was achieved using an automated microplate reader. Concentrations of all cytokines were reported in pg/ml. The rats were sacrificed 30 days following surgery. The lungs were removed and collected for the following experiments. The metastatic nodes in the lungs of mice were visualized by fixing them in Bouin's solution (saturated picric acid: Formalin: Acetic acid, 15:5:1) at room temperature for 24 h and photographed using a high-definition digital camera (IXUS 110, Canon Inc., Tokyo, Japan) to evaluate the anti-metastatic effects of general anesthetic and general anesthetic with epidural block.

For each group, 0.1 g of the lung tissue was collected for protein extraction and lysed using a Dounce homogenizer (20 strokes) in 400 µl of buffer containing 50 mM tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), pH 7.6, 42 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol (DTT), 0.5% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate hydrate, 1 mM phenylmethylsulfonyl-fluoride and 1 µg/ml leupeptin (Sigma-Aldrich; Merck KGaA), and then centrifuged at 15,000 × g for 20 min at 4°C. The protein concentrations of the lysates were determined by BCA protein assay. Equal amounts (50 µg) of protein were separated on a 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline/0.1% Tween-20 for 2 h, subsequently blotted with respective primary antibodies overnight at 4°C as described previously in the materials section, and then blotted with horseradish peroxidase-conjugated secondary antibody at 37°C for 1 h. The protein bands were visualized using an enhanced chemiluminescent agent (Fuzhou Maixin Biotech Co., Ltd., Fuzhou, China). Protein levels were quantified by density analysis using ImageJ software version 1.62 (National Institutes of Health, Bethesda, MD, USA), and expressed as the interest protein/internal control.

Data from ≥3 groups were compared using one-way analysis of variance followed by Dunnett's post-hoc test using the SPSS software package (version 13.0; SPSS Inc., Chicago, IL, USA). The experiments were performed in triplicate and the data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

The evaluation on the in vivo anti-metastatic activity of general anesthesia and epidural anesthesia was performed. In the in vivo experiment, the rats were sacrificed 30 days after surgery and the lungs were dissected. The average numbers of metastatic nodules on the lung surface in different groups were counted. Representative photos of the lungs with the metastatic nudes are presented in Fig. 1. As depicted in Table I, the number of metastasis nodules in the G + E group was 18±6, which was significantly (P<0.05) less than that in group C (40±6) or group G (38±5). This result indicated that GEA could effectively inhibit lung metastasis compared with GA following hepatectomy. The conditions of abdominal lymph node metastasis, the rate of malignant ascites and number of abdominal wall planting nodules also validated this conclusion. As presented in Table I, the number of animals for which there was a presence of abdominal lymph node metastasis was significantly lower for group G + E (1/10) than for group C (7/10) or group G (6/10). For group C and group G, the rates of malignant ascites were higher than that of group G + E, which demonstrated the low metastatic rate following surgery with epidural anesthesia. Abdominal wall-implanted nodules represented another important evaluation index for metastatic condition. According to Table I, the number of rats with abdominal wall-implanted nodules in group G + E was 2/10, which was significantly lower than that for group C (8/10) or group G (5/10). Hepatectomy with different anesthetic techniques revealed different rates of tumor metastasis. Notably, GEA could effectively inhibit tumor metastasis compared to GA following hepatectomy.

As shown in Fig. 2A, there was no significant difference in IFN-γ levels between groups C, G and G + E on day 0. On day 2, the concentration of IFN-γ in group G + E (3,411±232 pg/ml) was significantly higher than that in group C (1,497±146 pg/ml) and G (2,578±165 pg/ml). On day 7 and day 30, there was no significant difference between group C and G, but these concentrations were significantly higher than those in group G + E. For group G + E, the concentration of IFN-γ at day 7 and day 30 was slightly lower than that on day 2. Similarly, there was no significant difference in regard to concentrations of TGF-α and VEGF between group C, G and G + E on day 0 (Fig. 2B and C). On the following days, concentrations of TGF-α and VEGF were significantly lower in group G + E than in groups C and G (P<0.05). Notably, the concentration of TGF-α increased slowly over time following surgery. However, the concentration of VEGF was at its minimum on day 7. Groups C and G exhibited increases in the concentration of TGF-α and VEGF, whereas the concentration in group G + E rats remained lower than those in groups C and G.

To investigate the mechanism of the anesthesia-induced anti-metastatic effect further, levels of p-STAT3 and p-EGFR lung metastatic tissues were determined by western blot analysis (Fig. 3). The levels of p-STAT3 and p-EGFR in group G were slightly lower than those in group C. It was shown that there were significantly (P<0.05) lower levels of p-STAT3 and p-EGFR in group G + E than in groups C and G. Evidence indicates that aberrant STAT3 signaling promotes the initiation and progression of human cancer by either inhibiting apoptosis or inducing cell proliferation, angiogenesis, invasion and metastasis (32–34). Additionally, expression of EGFR is altered in a variety of human cancer types, including carcinoma of the lung, breast, head and neck, ovary and bladder, as well as in glioma (35–37). STAT3 and EGFR serve notable roles in tumor development and metastasis (34,38). The results of the present study indicated that GEA significantly inhibited tumor metastasis by reducing levels of p-STAT3 and p-EGFR.