A total of 30 adult female patients aged between 25 and 65 years old, undergoing radical hysterectomy were recruited in the present prospective study between August 2013 and January 2014. Patients were excluded if they had a weight <45 kg or >65 kg; a history of allergies to local anesthetics, bradycardia or heart block; severe respiratory, renal or hepatic disease, previous history of opioid medication use or a psychiatric medical history.

The study protocol was approved by the ethics committee of Qilu Hospital of Shandong University (Jinan, China) and performed according to the Declaration of Helsinki. Written informed consent was obtained from all participants prior to enrolment.

All participants were randomized into two groups, according to a computer-generated random number table, in which the patients received either intravenous lidocaine (Shanghai Zhi Pharma Co., Ltd., Shanghai, China) or normal saline (control group). The solutions were prepared in a 20 cc syringe and labeled only with a case number by a nurse in a blinded-manner. The patients assigned to the lidocaine group received an intravenous bolus infusion of 1.5 mg/kg lidocaine 10 min prior to the induction of anesthesia, followed by continuous infusion at 1.5 mg/kg/h using a Graseby 3100 syringe pump (Graseby Medical, Ltd., Watford, UK) until discharge from the operating room. The patients in the control group received the same volume of normal saline. All surgical procedures were performed by the same team of surgeons to avoid individual variability in operative techniques. The characteristics of the patients are listed in Table I.

Following the administration of 0.1 mg/kg midazolam (Jiangsu Nhwa Pharmaceutical Co., Ltd., Xuzhou, China), 1–2 mg/kg propofol (Fresenius Kabi AG, Bad Homburg vor der Höhe, Germany), 2 µg/kg fentanyl (Yichang Humanwell Pharmaceutical Co., Ltd., Yichang, China) and 0.6 mg/kg rocuronium (Taizhou Xianju Pharmaceutical Co., Ltd., Taizhou, China) intravenously, the patients were intu-bated and ventilated to maintain the end-tidal CO₂ volume between 30 and 40 mmHg. Anesthesia was maintained using 1.5–3% sevoflurane (Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) in 1 l/min O₂. Fentanyl and rocuronium were added, according to heart rate or the bispectral index (S/5; GE Healthcare Life Sciences, Helsinki, Finland). Noninvasive arterial blood pressure, electrocardiography and pulse oximetry (S/5) were monitored continuously. During surgery, the patients received intravenous infusion of lactated Ringer's solution (Shandong Hualu Pharmaceutical Co., Ltd., Muping, China) at a rate of 6–12 ml/kg/h.

A 10 ml blood sample was drawn from the median cubital vein of each patient 1 day prior to surgery, following discharge from the operating room and at 48 h post-surgery. Serum was isolated at room temperature by centrifugation (1,000 × g, 5 min) and stored at −20°C prior to assessment. Lymphocytes were isolated using Ficoll-paque density centrifugation (Haoyang Biological Manufacture Co., Ltd., Tianjin, China) at 1,000 x g for 15 min at −20°C. The cell viability was assayed by 0.4% trypan blue exclusion, which was >95% in all cases. The collected cells were resuspended at a density of 1×10⁶ cells/ml in RPMI 1640 medium (Thermo Fisher Scientific, Shanghai, China) supplemented with 10% heat-inactivated fetal calf serum, and cultured at 37°C in a humidified 5% CO₂ atmosphere.

The suspended cells (100 µl; 1×10⁶ cells/ml) were cultivated with 5 µg/ml phytohemagglutinin (PHA) (Sigma-Aldrich, St. Louis, MO, USA) in a 96-well plate at 37°C and 5% CO₂ for 24 h. RPMI 1640 was used as a blank control in each group. Subsequently, 10 µl Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well 4 h prior to the end of stimulation, when the optical density values were measured at 450 nm (Model 722; INESA Instrument, Shanghai, China). Data are expressed as the mean ± standard deviation of four wells.

Detection of early apoptosis in the PBLs was performed, according to the manufacturer's instructions, using an Annexin V FITC kit (Bogoo, Shanghai, China). In brief, the PBLs (2×10⁶) were resuspended in 2 ml ice-cold phosphate-buffered saline and washed twice. The collected PBLs were initially suspended in 400 µl 1X binding buffer, following which 5 µl Annexin V-FITC was added for 15 min in the dark followed by 10 µl PI for 5 min in the dark. A negative control was used for each sample, in which the PBLs were incubatedwith binding buffer alone. For the positive control, the cells were incubated with Annexin V-FITC or PI alone. The cell sample was measured immediately using a flow cytometer.

The FC data were acquired within 24 h of staining using CellFit version 2.0 software and a FACSCalibur cytometer (BD Biosciences, San Jose, CA, USA). An argon ion laser excitation of 488 nm was used. The emitted light was detected by logarithmic amplification through barrier filters, which were specific for the emission range of the different fluorophores: 530/22 nm for FITC (fluorescence channel FL1) and 575/42 nm (FL2) for PI. The results of the lymphocyte typing and lymphocyte early apoptosis detection were obtained by quadrant analyses of FL1, vs. FL2 channel dot plots and presented as the percentage of gated lymphocytes.

The level of high-mobility group protein B1 (HMGB1), interferon (IFN)-γ and interleukin (IL)-4 in the serum were determined using ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA), according to the manufacturer's instructions.

SigmaPlot 12.5 (Systat Software, Inc., San Jose, CA, USA) was used for statistical analysis. The data distribution was evaluated using Levene's test. The normally distributed data are expressed as the mean ± standard deviation and were compared using one-way analysis of variance or an unpaired t-test. Descriptive variables were subjected to χ² analysis or Fisher's exact test, as appropriate. P<0.05 was considered to indicate a statistically significant difference.

All 30 patients recruited in the present study completed the study. As shown in Table I, no significant differences were identified between the groups in terms of the American Society of Anesthesiologists class, age, height, weight, duration of surgery or anesthesia (13).

The number of blood lymphocytes always falls peri-operatively, and decreases in the lymphocyte proliferation rate are considered to be one of its causes (1). Therefore, the present study initially examined the proliferation rate of PBLs using a CCK-8 assay. The results, as shown in Fig. 1, demonstrated that lidocaine improved the proliferation rate of the surgical stressor-induced lymphocytes. Therefore, lidocaine may exert a protective effect on lymphocyte function.

Surgical stressors not only reduce lymphocyte proliferation, but they increase apoptosis of immune cells. To determine whether lidocaine exerted a protective effect on surgery-induced apoptosis of lymphocytes, the PBLs in the present study were stained with Annexin V-FITC/PI and assessed using flow cytometry. As shown in Fig. 2, intraoperative systemic lidocaine attenuated the surgery-induced apoptosis of the PBLs.

The serum level of IFN-γ at 48 h post-surgery in the control group was decreased significantly, compared with the pre-surgical level (3.782±0.282, vs. 4.089±0.339, pg/ml respectively). However, no significant differences were observed in the levels of IFN-γ in the lidocaine group or in the levels of IL-4 in the control and lidocaine group, compared with the pre-surgical value. As shown in Fig. 3, the ratio of IFN-γ to IL-4 was well preserved in the lidocaine group.

HMGB1, as a critical mediator of several inflammatory and non-inflammatory diseases, is important in surgery-associated sepsis and tumor metastasis. To assess whether lidocaine has an inhibitory effect on its expression in patients undergoing surgery, the serum level of HMGB1 was determined in the present study using ELISA. As expected, the serum protein level of HMGB1 in the lidocaine group was reduced, which was significantly difference 48 h-post surgery, compared with the control group (Fig. 4).