This was a prospective study conducted between January 2019 and August 2020. Patients who had received elective abdominal operations with a physical status of class 1 or 2 based on the American Society of Anesthesiologists physical status classification system were enrolled in this study at the First Affiliated Hospital of Jinan University. The exclusion criteria were as follows: Coagulation ailments; present or past histories of drug misuse; alcoholism; and childbirth and pregnancy issues.

All subjects were randomly divided into two groups. The patients in the control group (n=36) received EA therapy at 1 cm below the acupoints and the patients in the treatment group (n=36) received EA at the exact acupoints. The acupoints were located as the Taichong (LR3) site at the distal end of the first metatarsal space on the dorsum of the foot and the Hegu (LI4) site at the radial side of the middle of the second metacarpal bone of the hand. To avoid over-stimulation, first the Hegu site and then the Taichong site were stimulated instead of stimulating them at the same time. The time lapse between the stimulation of the Hegu site and Taichong site was 15 min per session. The acupoints of both groups were ~6.4-12.8 mm deep and the acupuncture apparatus was an Acuhealth Guru 900 (Eastern Currents, Ltd.). For the acupuncture treatment, the acupuncture needles were inserted either at 1 cm below the acupuncture point or at the exact acupuncture point. Following needling, fentanyl (0.001 mg/kg) or midazolam (0.04 mg/kg) was administered orally to reduce pain. After acupuncture, an anesthesiologist intubated the individuals receiving the treatment and a monitoring device was inserted immediately to measure the indices for 5 min. The demographics and features of all subjects were collected and compared between the two groups. In addition, the systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate (HR) of patients in the two groups were measured before and after needling and the collected data was presented in Table I. Furthermore, peripheral blood monocytes (PBMCs) and peripheral blood samples were collected from all patients for later analyses. The Ethics Committee of the First Affiliated Hospital of Jinan University approved the protocol of the present study (approval no. JNDX007546228FSY). Written informed consent was obtained from all participants before the initiation of the present study.

Complete RNA was isolated from the human and cell samples (1×10⁵ cells) with TRIzol^® (Thermo Fisher Scientific, Inc.) according to the manufacturer's guidelines. RNA concentration and purity were tested by UV spectrophotometer. RNA (300 µl) extracted from each sample was precipitated with ethanol, washed, resuspended and converted to cDNA using reverse transcription following manufacturer's instructions. cDNA was then subjected to RT-qPCR using the SYBR-green method (MilliporeSigma) and a 7900HT real-time PCR cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). An annealing temperature of ~62°C for 15 min was used. Subsequently, the relative expression of miR-155 (Forward: 5′-TGCTAATCGTGATAGGGG-3′; Reverse: 5′-GAACATGTCTGCGTATCTC-3′), miR-335 (Forward: 5′-AAGAGCAATAACGAAAAATG-3′; Reverse: 5′-GAACATGTCTGCGTATCTC-3′), miR-383 (Forward: 5′-TCAGAAGGTGATTGTGGC-3′; Reverse: 5′-GAACATGTCTGCGTATCTC-3′) and eNOS mRNA (Forward: 5′-GAAGGCGACAATCCTGTATGGC-3′; Reverse: 5′-TGTTCGAGGGACACCACGTCAT-3′) was determined through the 2^−ΔΔCq method (23). The thermocycling protocol for RT-PCR was 10 min at 95°C, followed by 40 cycles of 30 sec at 95°C, 30 sec at 60°C and 30 sec at 72°C. U6 (Forward: 5′-CTCGCTTCGGCAGCACA-3′; Reverse: 5′-AACGCTTCACGAATTTGCGT-3′) and β-actin mRNA (Forward: 5′-CACCATTGGCAATGAGCGGTTC-3′; Reverse: 5′-AGGTCTTTGCGGATGTCCACGT-3′) were used as the internal controls for miRNAs and mRNA respectively. Each experiment was repeated 3 times.

THP-1 cells and human umbilical vein endothelial cells (HUVECs) from ATCC were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) containing 100 µg/ml streptomycin, 100 U/ml penicillin (Thermo Fisher Scientific, Inc.) and 10% inactivated fetal bovine serum (Harlan Sera-Lab, Ltd.). Cells were cultured in 24-well plates at a density of 5×10⁵ cells/well for the establishment of two different sets of experiments. In one set, cells were divided into the following three groups: NC group in which cells were transfected with 50 nM negative controls (NC; 5′-UUCUCCGAACGUGUCACGUTT-3′ for miR-155 precursors and 5′-CAGUACUUUUGUGUAGUACAA-3′ for anti-miR-155), miR-155 precursors group in which cells were transfected with miR-155 precursors (5′-CTGTTAATGCTAATCGTGATAGGGGTTTTTGCCTCCAACTGACTCCTACATATTAGCATTAACAG-3′) and anti-miR-155 group in which cells were transfected with anti-miR-155 (5′-ACCCCUAUCACGAUUAGCAUUAA-3′). In the other set, cells were divided into the following three groups: NC group in which cells were transfected with NC, miR-383 precursors group in which cells were transfected with miR-383 precursors and anti-miR-383 group in which cells were transfected with anti-miR-383. All cells were transfected with the corresponding miRNA precursors or antagomirs with TransIT-LT1 (Mirus Bio, LLC) 4°C overnight according to the manufacturer's instructions. The cells were collected via trypsinization and assayed for the expression of the corresponding genes of interest 24 h post-transfection.

The sequence analysis results by bioinformatic tool TargetScan (Relase 8.0, http://www.targetscan.org/vert_80/) indicated that eNOS may be a potential target gene shared by miR-155, miR-335 and miR-383. To further explore the inhibitory role of miR-155, miR-335 and miR-383 in the expression of eNOS, i.e., the regulatory relationship of miR-155/eNOS, miR-335/eNOS and miR-383/eNOS, luciferase vectors containing wild-type and mutant eNOS 3′ UTRs were established. In brief, the eNOS 3′ UTRs containing the binding sites for miR-155, miR-335 and miR-383 were amplified by PCR and cloned into pmirGLO dual-luciferase miRNA target expression vectors (cat. no. E1330; Promega Corporation) downstream of the reporter gene to generate wild-type eNOS vectors which contained according binding sites for miR-155, miR-335 and miR-383. In addition, site-directed mutagenesis was performed using a Stratagene Quick Change II mutagenesis assay kit (Stratagene; Agilent Sumitomo Dainippon Pharma Co., Ltd.) following the manufacturer's guidelines to generate mutant eNOS 3′ UTR sequences which contained according binding sites for miR-155, miR-335 and miR-383. Finally, both the mutant and wild-type eNOS vectors were transfected into the THP-1 cells and HUVECs with along with the mimics and antagomirs of miR-155, miR-335 and miR-383 using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's guidelines. At 24 h after transfection, a Bright Glo luciferase assay kit (Promega Corporation) was used to analyze the luciferase activity of the transfected cells which were normalized to Renilla luciferase activity following the manufacturer's guidelines.

Western blots were used in conjunction with an Odyssey Infrared Imaging system (LI-COR Biosciences) using conventional western blotting procedures. In brief, collected PBMCs and cultured cells were subjected to RIPA lysis treatment to collect the proteins, the concentrations of which were determined using a BCA assay kit (Bio-Rad Laboratories, Inc.) following the manufacture's guidelines. Protein samples (50 µg) were then resolved by 12% SDS-PAGE and blotted onto NC membranes (Thermo Fisher Scientific, Inc.). Subsequently, the membranes were blocked with 5% non-fat milk for 60 min at room temperature, followed by overnight incubation at 4°C with primary anti-eNOS antibodies (1:1,000; cat. no. ab76198; Abcam). GAPDH (1:1,000; cat. no. ab8245; Abcam) was used as the loading control. Furthermore, the membranes were incubated with anti-rabbit IgG secondary antibodies conjugated to horseradish peroxidase (HRP) (1:1,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 2 h at room temperature and then developed using an ECL reagent (Thermo Fisher Scientific, Inc.). The relative protein expression of eNOS was calculated using Quantity One software (v4.6.7; Bio-Rad Laboratories, Inc.).

The supernatants of peripheral blood samples collected from different patient groups were retrieved by centrifugation (250 × g) at 4°C for 5 min, and the content of NO in each sample was assayed by a sandwich ELISA kit (cat. no. ab233628; Abcam) following the manufacturer's protocol.

All statistical analyses were conducted with SPSS 16.0 statistical computer software (SPSS, Inc.). Non-parametric parameters were compared using the χ² test. Wilcoxon sum rank test or Student's t test were used to analyze differences between two groups, while one-way analysis of variance (ANOVA) in conjunction with Tukey's post hoc test were used to analyze differences among multiple groups. Pearson correlations were used to determine the values of correlation coefficients. All statistical values are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

The participants of this study were divided into two groups. One group of patients (n=36) received EA therapy 1 cm below the acupoints and the other group (n=36) received EA at the exact acupoints. The average age for the control group was 47.2±6.8 years old, while the average age for the treatment group was 51.8±5.6 years old. There were 25 male participants in the control group and 28 male participants in the treatment group. There were no obvious differences in these indicators between the two groups.

The diagrams which indicated the locations of the acupuncture points, Taichong site and Hegu site, are shown in Fig. 1A. The SBP, DBP, MAP and HR of patients in the above two groups were measured before and after needling and 3 min of intubation. It was found that the SBP, DBP, MAP and HR were changed following needling in both groups, whereas no obvious difference was observed for the SBP, DBP, MAP and HR before needling. The SBP (Fig. 1B), DBP (Fig. 1C) and MAP (Fig. 1D) in patients receiving EA therapy at 1 cm below the acupoints were significantly elevated compared with those in patients receiving EA at the exact acupoints following the time-point of needling. However, the HR (Fig. 1E) in patients receiving EA therapy at 1 cm below the acupoints was markedly reduced compared with that in patients receiving EA at the exact acupoints following the time-point of induction.

The expression of miR-155, miR-335, miR-383 and NO was examined in the peripheral blood collected from patients receiving EA therapy at 1 cm below the acupoints or on the exact acupoints. The expression of miR-155 (Fig. 2A), miR-335 (Fig. 2B) and miR-383 (Fig. 2C) was significantly inhibited in peripheral blood samples from patients receiving EA therapy at the acupoints compared with that in patients receiving EA therapy at 1 cm below the acupoints. In addition, ELISA was performed to evaluate the production of NO in the peripheral blood of patients receiving EA therapy at 1 cm below the acupoints or on the exact acupoints. The production of NO (Fig. 2D) in the peripheral blood of patients receiving EA therapy at the acupoints was increased compared with those receiving EA therapy at 1 cm below the acupoints. Furthermore, PBMCs were isolated from patients receiving EA therapy at 1 cm below the acupoints or on the exact acupoints and the expression of miR-155, miR-335, miR-383 and eNOS analyzed using qPCR. The expression of miR-155 (Fig. 3A), miR-335 (Fig. 3B) and miR-383 (Fig. 3C) was significantly reduced in the PBMCs from patients receiving EA therapy at the exact acupoints compared with those receiving EA therapy at 1 cm below the acupoints, while the expression of eNOS mRNA (Fig. 3D) was significantly enhanced in the PBMCs of patients receiving EA therapy at the exact acupoints.

Sequence analysis indicated that eNOS was a potential binding target of miR-155 (Fig. 4A), miR-335 (Fig. 4D) and miR-383 (Fig. 4G). Luciferase vectors containing wild-type and mutant eNOS 3′ UTR were generated and cotransfected into THP-1 cells and HUVECs with miR-155, miR-335 and miR-383 mimics and antagomirs. The luciferase activity of wild-type eNOS was efficiently inhibited by miR-155 (Fig. 4B and C), miR-335 (Fig. 4E and F) and miR-383 (Fig. 4H and I) mimics in THP-1 cells and HUVECs, whereas miR-155, miR-335 and miR-383 antagomirs significantly enhanced the luciferase activity of wild-type eNOS vectors. No apparent changes were observed for the luciferase activity of mutant eNOS.

To further explore the inhibitory role of miR-155, miR-335 and miR-383 in the expression of eNOS, miR-155, miR-335 and miR-383 precursors and antagomirs were transfected into THP-1 cells and HUVECs. The successful transfection of miR-155, miR-335 and miR-383 precursors and antagomirs were confirmed by observing the changes of miR-155 (Supplementary Fig. S1A and B), miR-335 (Supplementary Fig. S1C and D) and miR-383 (Supplementary Fig. S1E and F). Subsequently, the expression of eNOS mRNA and protein was analyzed using qPCR and western blot analysis, respectively. The expression of eNOS mRNA and protein was significantly suppressed by miR-155 (Figs. 5A and B; 6A and B), miR-335 (Figs. 5C and D; 6C and D) and miR-383 (Figs. 5E and F; 6E and F) precursors in THP-1 cells and HUVECs, while the expression of eNOS mRNA and protein was activated by miR-155 (Figs. 5A and B; 6A and B), miR-335 and miR-383 antagomirs in THP-1 cells and HUVECs.