Ethics statement.
The present study (registration number: TPHRZ20150412) was approved by the Ethics Committee of the Qiqihar Medical University. All patients agreed to the use of their samples in scientific research and provided written informed consent.
Patients. Between May 2015 and July 2016, patients (n = 122) with gastric cancer requiring radical surgery provided written informed consent prior to the trial. The mean age of clinical patients was 48.5 ± 10.5 years, and the mean body weight was 70.4 ± 10.6 kg. The number of male and female patients (n = 61/61) was almost equal. The operation time for each patient was ~ 3 h. All of the patients were scheduled for radical surgery under propofol general anesthesia (3 mg/kg/h, n = 61) or midazolam anesthesia (3 mg/kg/h, n = 61). For all patients, there was no history of endocrine, immune or circulatory system diseases. Patients with gastric cancer with major surgical complications were excluded from the present study. The standardized protocols for the surgical procedure and postoperative patient care were used to minimize any difference in surgical impact among patients.
Patient survival, tumor metastasis and recurrence. A total of 22 patients with gastric cancer received propofol general anesthesia (3 mg/kg/h) prior to surgery, and were followed-up for 60 months. In addition, another 22 patients with gastric cancer (men/women, 10/12; mean age, 36.5 years; mean body weight, 57.5 kg) received midazolam general anesthesia. Patient survival, and tumor metastasis and recurrence were recorded within the 60-month follow-up period.
ELISA. A sample (10 ml) of central venous blood was collected immediately prior to surgery and 4 days after surgery. Following centrifugation at 3,000 x g for 15 min at 4˚C, the serum was separated to analyze serum levels of IL-1β (cat. no. 201-LB; R&D Systems, Inc., Minneapolis, MN, USA), IL-2 (cat. no. 202-IL; R&D Systems, Inc.), IL-6 (cat. no. D6050; R&D Systems, Inc.), IL-8 (cat. no. D8000C; R&D Systems, Inc.), TNF-α (cat. no. DTA00D; R&D Systems, Inc.), IL-10 (cat. no. D1000B; R&D Systems, Inc.) caveolin (Cav)-1 (cat. no. MAB5736; R&D Systems, Inc.), programmed death-ligand (PDL)-1 (cat. no. DB7H10; R&D Systems, Inc.), methyl-CpG binding protein 2 (MECP2; cat. no. M7443-200UL; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and Y-box-binding protein (YB)-1 (cat. no. LS-B11709-50; LifeSpan BioSciences, Inc., Seattle, WA, USA) by ELISA according to the manufacturers’ protocols. The results were detected using an ELISA microplate reader (Spectra Max 190; Molecular Devices, LLC, Sunnyvale, CA, USA).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Total RNA was extracted from total peripheral blood samples using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and RNA purity and quantity were detected by ultraviolet spectrometry. PCR specific primers (Table I) were synthesized by Invitrogen; Thermo Fisher Scientific, Inc. cDNA was synthesized by RT using RNeasy Mini kit (Qiagen Sciences, Inc., Gaithersburg, MD, USA), according to the manufacturer’s protocol, after which qPCR was conducted using a FastStart Universal SYBR Green Master (cat. no. 4913850001; Roche Diagnostics, Basel, Switzerland) and a LightCycler 480 Real-Time PCR system (Roche Diagnostics) under the following conditions: 95˚C for 30 sec, followed by 28 cycles at 95˚C for 30 sec, 56˚C for 45 sec and 72˚C for 35 sec, and a final extension step at 75˚C for 10 min. Quantification cycle (Cq) values of standard samples were calculated based on the internal reference gene β-actin. PCR results were quantitatively analyzed using the 2−∆∆Cq method, as described previously (17). The Th1/Th2 balance was analyzed using the IL-2+TNF/IL-4+IL-10 ratio.
Blood cell counts. A complete blood count was performed on all blood samples using an automated hemoanalyzer (cat. no. WD-5000; Weier Medical Equipment Co., Ltd., Changchun, China), according to the manufacturer’s protocol. Total and differential leukocyte, lymphocyte, monocyte and neutrophil counts were determined as described previously (18).
Flow cytometry. Blood samples (5 ml) were obtained from individuals and lymphocyte subsets were determined by flow cytometry (Cytomics FC500; Beckman Coulter, Inc., Brea, CA, USA), as previously described (19). CD3-fluorescein isothiocyanate (FITC) (cat. no. ab34275, Abcam, Cambridge, UK)/CD4-phycoerythrin (PE) (cat. no. ab18282, Abcam) (helper T cells, CD4+ T cells) and CD3-FITC (cat. no. ab34275, Abcam)/CD8-PE (cat. no. ab210327, Abcam) (suppressor/cytotoxic T cells, CD8+ T cells) antibodies were used to analyzing percentage of CD4 + T and CD8+ T cells by conducting flow cytometry.
Nuclear factor (NF)-κBp65 overexpression. Gastric cancer cells were isolated from gastric cancer tissues obtained from patients that received propofol general anesthesia, as previously described (20). Cells (1 × 105) were cultured in a 6-well plate in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich; Merck KGaA) supplemented with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA), 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C in an atmosphere containing 5% CO2 until they reached 85% confluence. Subsequently, the medium was removed and the cells were washed three times with PBS. Gastric cancer cells (1 × 106) were then infected with plentivirus-NF-κBp65 (pNF-κB; 100 mM; 5’-TTCCCTGAAGTGGAGCTAGGA-3’; Invitrogen; Thermo Fisher Scientific, Inc.) or plentivirus-Vector (control; 100 mM; 5’-GCTTCGGCAGCATATACTCTAAAT-3’; Invitrogen; Thermo Fisher Scientific, Inc.) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer’s protocol. Expression level of NF-κB was analyzed after 48 h transfection using RT-qPCR (supplementary materials Figure S1). NF-κB-overexpressed gastric cells were treated with propofol (2 mg/ml) for 12 h at 37˚C for further analysis.
Western blotting. Gastric cells and NF-κB-overexpressed cells were lysed in radioimmunoprecipitation assay buffer (M-PER reagent for cells; Thermo Fisher Scientific, Inc.) and were homogenized at 4˚C for 10 min. Protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Subsequently, protein extracts (20 µg) were separated by 12.5% SDS-PAGE and were then transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated in blocking buffer (5% milk) for 2 h at 37˚C prior to incubation with primary antibodies at 4˚C overnight. The primary rabbit anti-human antibodies used in the immunoblotting assays were: NF-κBp65 (1:1,200, cat. no. ab16502; Abcam), IL-1β (1:1,000, cat. no. ab2105; Abcam), IL-8 (1:1,200, cat. no. ab7747; Abcam), TNF-α (1:1,000, cat. no. ab6671; Abcam), Janus kinase 1 (JAK1; 1:1,200, cat. no. ab47435; Abcam), signal transducer and activator of transcription 3 (STAT3; 1:500, cat. no. ab68153; Abcam) and β-actin (1:2,000, cat. no. ab8226; Abcam). After incubation, the membranes were washed three times in Tris-buffered saline containing 1.0% Tween (TBST) and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) monoclonal antibody (1:5,000, cat. no. PV-6001; OriGene Technologies, Inc., Beijing, China) for 1 h at 37˚C. After three washes with TBST, the membranes were developed using a chemiluminescence assay system (Roche Diagnostics) and exposed to Kodak films (Kodak, Rochester, NY, USA). Densitometric semi-quantification of the immunoblots was performed using Quantity-One software (version 1.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Immunohistochemistry. Immunohistochemical analysis was performed as described previously (21). Gastric cancer tissues were obtained from patients and were fixed with 10% formalin for 2 h at 25˚C. Subsequently, the tissues were washed with PBS for 10 min, incubated with xylene for 5 min and incubated with an ethanol gradient (80, 90, 95 and 100%) for 3 min at 25˚C. Tissues were then embedded in paraffin at 56˚C and paraffin-embedded gastric cancer tissues sections (4 µm) were prepared for further analysis. The paraffin-embedded sections were treated with hydrogen peroxide (3%) for 10–15 min at 37˚C and were hydrated in a decreasing series of ethanol. Antigen retrieval was performed using an antigen retrieval kit (cat. no. ab93684; Abcam) at 65˚C for 10 min. Subsequently, 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) was used to block nonspecific binding at 37˚C for 2 h and tissue sections were incubated with the following primary antibodies at 4˚C for 12 h: NF-κBp65 (1:1,200, cat. no. ab16502; Abcam), JAK1 (1:1,200, cat. no. ab47435; Abcam) and STAT3 (1:500, cat. no. ab68153; Abcam). The tissue sections were then washed three times and incubated with a HRP-conjugated IgG monoclonal antibody (1:2,000, cat. no. PV-6001; OriGene Technologies, Inc.) for 12 h at 4˚C. Sections were incubated with diaminobenzidine (Sigma-Aldrich; Merck KGaA) at room temperature for 10 sec, in order to detect positive signals, and six random views were observed under a microscope. Images were recorded using an inverted light microscope (Olympus Corporation, Tokyo, Japan). Protein density was analyzed using ImageJ software 4.6 (National Institutes of Health, Bethesda, MD, USA).
Statistical analysis. All data are presented as the means ± standard deviation of triplicate experiments, and were analyzed by SPSS 19.0 statistical software (SPSS, Inc., Chicago, IL, USA). The comparisons of means between two matched groups were conducted using a paired Student’s t-test. Unpaired t-test or ANOVA were used to compare statistical difference between two groups. One-way ANOVA analysis followed by Tukey’s test was used to compare statistical difference among multiple groups. P < 0.05 was considered to indicate a statistically significant difference.