Adult male Sprague-Dawley rats, weighing 180–200 g, were purchased from Changsheng Biotechnology Co. Ltd., Liaoning Province, China. The rats were housed in individual cages with free access to water and food. All rats lived with a 12-hour day-night cycle at a room temperature of 20°C–26°C and a relative humidity of 40%–70%. The method for anesthesia was 10% chloral hydrate administered with an intraperitoneal injection at the dose of 0.004 mL/g body weight. The animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals and approved by the Animal Investigation Committee of General Hospital of Southern Theatre Command of PLA (approval number: IACU19-0320). To build a perioperative stress prolongs incision-induced pain hypersensitivity without changing basal pain perception rat model, there was a need to determine how many days the stress should last. Ten rats were randomized into a stress group and a control group (n = 5). The rats in the stress group were woken up when they were asleep, then wrapped tightly in barbed wire, restricted their activities without water and food for 6 hours. They were kept awake during this period. The control group was not treated. Both groups were given a mechanical pain, cold withdrawal latency, and thermal withdrawal latency test daily for 7 days.

Another 40 rats were randomized into 4 groups (n = 10). The rats in each group were treated as described in Table 1.

To evaluate the recovery time of postoperative pain in each group of rats after stress. The pain behavior of rats to mechanical, thermal, and cold stimuli were measured 1 day before the operation, and on days 1, 3, 5, 7, and 9 after the operation.

The reporter fluorescence of the vector in pmiR-RB-REPORT^TM Double luciferase assay (Promega, Madison, WI) is the renilla luciferase gene (hRluc), and the corrected fluorescence is the firefly luciferase gene (hluc, as an internal reference). The 3’untranslated region (3’UTR) region of oprm1 is cloned at the hrluc gene downstream. The miRNA acts on the target gene through the 3’UTR region. Therefore, the miR-339-5p was co-transformed with the constructed reporter gene vector. The interaction between miRNA and the target gene was confirmed by the down-regulation of the relative fluorescence value of the reporter gene. Confirmation of the interaction between miR-339-5p and oprm1 was proved by the down-regulation of the relative fluorescence value of the reporter gene.

The 3’UTR of oprm1 containing the miR-339-5p binding site was amplified with the forward primer and the reverse primer (Ribobio, Guangzhou, China) from rat genomic DNA samples. The polymerase chain reaction (PCR) products were separated in an agarose gel and extracted, purified, and cloned using the Seamless Cloning Kit (Beyotime, Shanghai, China). The oprm1 3’UTR was linked to the vector pmiR-RB-Report^TM (Ribobio) containing renilla and firefly luciferase using the restriction enzymes XhoI and NotI (information of the reporter gene vector seen in Table 2). The resulting construct was verified by sequencing. 293T cells were seeded at 1 × 10⁴ cells per well in 96-well plates. The cells were transfected using Lipo6000^TM (Beyotime) according to the manufacturer’s suggestions 24 hours after plating. In each well, 50 ng of pmiR-RB-Report^TM-3’UTR vector or Mut-3’UTR vector was co-transfected with 50 nM micrONTM rno-miR-339-5p (Ribobio), while 50 nM micrONTM miRNA mimic Negative Control #24 was used in each experiment. Three replicates of each sample were evaluated, and each experiment was repeated at least three times. The cells were harvested in 70 μL of passive lysis buffer per well 48 hours after transfection. The activity of renilla luciferase in the cell lysate was measured using the Dual-Glo^® Luciferase Assay System (Promega Biotech Co., Ltd, Beijing, China) in a GloMax^® 96 Microplate Luminometer (Promega) and normalized to the activity of firefly luciferase.

The mechanical pain threshold test used Von Frey filaments (North Coast Medical Inc., Morgan Hill, CA), pricking the plantar aspect of the rat’s hind paw with cilia of different strength from weak to strong (1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10, 15, and 26 g). If the rat flinched the paw rapidly or licked the paw, it was recorded as positive. If there was no response, it was recorded as negative. The stimulation intensity of cilia was recorded when a positive reaction appeared.

Thermal withdrawal latency test used a hot and cold plate pain meter (BW-YLS-21A; Shanghai Biowill Co., Ltd., Shanghai, China). The temperature of metal plate was adjusted to 55°C. The rats were placed on the metal surface at 55°C and the rat’s locomotion was restricted by a plexiglass cylinder. The time was started when the rats were placed and stopped when the rats flinched their paw rapidly or licked the paw. This time was defined as thermal withdrawal latency.

The cold withdrawal latency test used a hot and cold plate pain meter (BW-YLS-21A; Shanghai Biowill Co.). The temperature of the metal plate was adjusted to 4°C. The rats were placed on the metal surface at 4°C and the rat’s locomotion was restricted by a Plexiglass cylinder. The time was started when the rats were placed and stopped when the rats flinch their paw rapidly or licked their paw. This time was defined as cold withdrawal latency.

After anesthesia with 10% chloralhydrate, a sterile glass capillary was inserted into the sinus vein plexus of the rat. About 500 μL of blood was collected and centrifuged at 4°C/4,000 rpm for 10 minutes, and the serum stored at –80°C. Corticosterone levels were tested using enzyme-linked immunosorbent assay (USCN Co., Ltd., Wuhan, China).

The rats were euthanized after anesthesia, and the whole brain was separated and put on ice immediately. The amygdala was isolated under a microscope and stored at –80°C.

The amygdalas of the rats were fixed with 4% paraformaldehyde for 24 hours. After dehydration, the amygdalas were sliced into 4 μm thick sections. Slices were put into phosphate buffered saline (PBS) for 3 minutes, repeated 3 times. Each slice was added with 200 uL of 5% bovine serum albumin (BSA) and blocked for 30 minutes. Incubated with primary antibodies diluted in blocking solution at 4°C overnight (Anti-Mu Opioid Receptor Antibody; Cohesion Biosciences, London, UK, catalog number CPA3285, 1:500). Then, sections were washed four times with PBS (0.01 M) and incubated with secondary antibodies (fluorescein isothiocyanate conjugated sheep anti-rabbit antibody; Abcam Inc., Cambridge, UK, catalog number ab7086, 1:400), incubated in a 37°C oven for 30 minutes, and washed again four times with phosphate buffered solution with tween-20. Diluted 4’,6-diamidino-2-phenylindole was added and incubated at room temperature for 10 minutes. Images were taken with a laser-scanning confocal microscope (Fluoview FV1000; Olympus, Tokyo, Japan) and processed with Image J (National Institutes of Health, Bethesda, MD) and photoshop CS3 (Abode, San Jose, CA) software.

The proteins were extracted using tissue protein extraction reagent (Webiao Biotech, Shanghai, China). Protein concentrations were determined with a Pierce BCA Protein Assay Kit (Beyotime). The proteins were transferred to a polyvinylidene difluoride membrane. The membranes were blocked with a 5% milk solution prepared in PBS for 60 minutes. Then incubated at 4°C overnight with 1:1,000 diluted primary antibody (Anti-Mu Opioid Receptor Antibody: Cohesion Biosciences, catalog number CPA3285; Anti-phosphorylated extracellular regulatory protein kinase [p-ERK1/2] Antibody: Cohesion Biosciences, catalog number CPA4924; Anti-glial fibrillary acidic protein [GFAP] Antibody: Cohesion Biosciences, catalog number CPA4418). The membranes were washed three times for 5 minutes each with Tween 20 (1:1,000 dilution in PBS), then incubated for 45 minutes with the appropriate peroxidase-conjugated secondary antibody (1:5,000 dilution). The membranes were washed three times with Tween 20-PBS and were developed using an Odyssey two-color infrared laser imaging system (LI-COR, Lincoln, NE). The signal generated by GADPH was used as an internal control.

The paraffin-embedded sections of rat amygdala tissue were immersed in 3% hydrogen peroxide solution for 10 minutes to block the endogenous peroxidase after it was de-paraffinized and rehydrated using xylene and ethanol. Sections were boiled for 30 minutes in 10 mM citrate buffer solution (pH 6.0) for antigen retrieval. Slides were incubated for 45 minutes with 5% BSA and incubated overnight at 4°C with Anti-p-ERK1/2 antibody (Cohesion Biosciences, catalog number CPA4924, 1:500), Anti-GFAP antibody (Cohesion Biosciences, catalog number CPA4418, 1:200) and Anti-Mu Opioid Receptor antibody (Cohesion Biosciences, catalog number CPA3285, 1:500). The specimens were incubated for 45 minutes at 37°C with the appropriate peroxidase-conjugated secondary antibody and visualized using the DAB Detection Kit (BOSTER, Shanghai, China) following the manufacturer’s instructions. All sections with immunohistochemical staining were observed and the pictures of 6 randomly selected fields (but which were homogeneous in staining and cell numbers) (×200) were photographed by an Olympus microscope (CK31; Olympus) under high power view. The integrated optical density in each image was measured with the same setting for all the slides, and the results were analyzed with Image-ProPlus 6.0 software (Media Cybernetics, Rockville, MD).

Trizol was used to extract total RNA from the rat amygdala according to the instructions of the kit (Tiangen Biochemical Technology Co., Ltd, Beijing, China). The RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA) was used to synthesis complementary DNA. qRT-PCR was performed using the LightCycler^®480 II system (Roche, Basel, Switzerland). The FastStart Universal SYBR Green Master kit (Roche) was used to detect MOR mRNA with the following PCR amplification cycles: initial denaturation, 95°C for 5 minutes followed by 44 cycles with denaturation at 95°C for 15 seconds, annealing at 65°C for 30 seconds, and extension at 72°C for 20 seconds. B-actin was used as internal references for both MOR mRNA and miR-339-5p. The relative level of expression was calculated using the comparative (2Ct) method. All samples were evaluated in triplicate. The primer sequences were as follows:

The data are presented as the mean ± SEM. GraphPad Prism v8.0 (GraphPad Software, San Diego, CA) was used for statistical analyses. The normality of the data was assessed using the Shapiro–Wilk and Kolmogorov–Smirnov tests, with P < 0.050 accepted as a non-normal distribution. Equality of variance was assessed using an F test. An unpaired Student’s t-test was used for normally distributed datasets. Comparisons of a non-normally distributed group with any other group were made using the Mann–Whitney U-test. Comparisons of more than two groups, all of which were normally distributed, were made using one-way analysis of variance with Tukey’s post-hoc tests for pairwise comparisons. The data from behavior test were statistically analyzed with two-way analysis of variance. P < 0.05 was considered significant.

To determine how many days the stress should last, the rats were stressed in the stress group for 7 days, and the control group were given no stress. At the end of the experiment, the authors found that there was no significant difference in the mechanical pain, thermal, and cold threshold between two groups (P > 0.050, Fig. 1A-C) during the first three days. From the 4th day, the mechanical pain and cold withdrawal latency significantly declined in the stress group than that in the control group (P < 0.050, Fig. 1A, B). From the 5th day, the thermal withdrawal latency was significantly declined in group stress than that in group control (P < 0.050, Fig. 1C). The results showed that stress for 3 days did not affect the basic pain threshold of the rats. Therefore, 3 days was chosen as the duration of stress.

Then the recovery duration of postoperative pain was evaluated in each group. The incision was healed on the 3rd day after operation in the incision (IN) and stress + incision (S + IN) groups. It was found that the recovery time for postoperative incision pain was significantly delayed in group S + IN compared with the other groups. At the 9th day after the operation, compared with other groups, group S + IN still had significant differences in pain behavior compared with the other groups (P < 0.050, Fig. 1D–F), although the incision had been completely healed. There were significantly pain behavioral differences between group S + IN and group IN from the 5th day after the operation. There were no significant pain behavioral differences among group IN, group S, and the sham group from the 7th day after the operation (P < 0.050, Fig. 1D–F). These results show that the short-term perioperative stress prolongs incision-induced pain hypersensitivity without changing basal pain perception rat model was built successfully.

The results of the enzyme-linked immunosorbent assay show that the concentration of serum corticosterone in group IN + S was significantly higher than that in other groups (P < 0.050, Fig. 2A, B). The concentration of serum corticosterone in group S and group IN were significantly higher than in the sham group (P < 0.050, Fig. 2B).

To determine whether miR-339-5p could act on oprm1 as a target, pmiR-RB-REPORT^TM Double luciferase reporter vector was used. A reporter gene vector was constructed (Fig. 3A). Two objective sequences (Fig. 3B) in oprm1 were cloned into the vector and transfected into 239T cells.

After transfected with rno-mir-339-5p, the reported fluorescence expression of r-oprm1-MUT was significantly higher than that of r-oprm1-WT (Fig. 3C), while transfected with negative control, the reported fluorescence expression of r-oprm1-MUT did not change significantly compared with that of r-oprm1-WT. The results showed that there was an obvious interaction between rno-mir-339-5p and the site on r-oprm1 3’UTR.

To determine if perioperative stress could increase spontaneous pain, the expression of p-ERK1/2 and GFAP in the amygdala of rats were tested. These two proteins are common markers for evaluating spontaneous pain. Based on the result of western blotting, the expression of both proteins was increased significantly more in the amygdala of the rats in group IN + S than other groups (Fig. 2A, C). Furthermore, immunohistochemistry staining was performed in the amygdala tissue of the rats, and the results showed the expression of p-ERK1/2 and GFAP in the amygdala tissue of the rats was more significantly increased in group IN + S than other groups (Fig. 4).

The results of western blotting, immunohistochemistry staining, and immunofluoresence in the amygdalas of the rats showed that the expression of MOR was decreased significantly in the amygdalas of rats in group S + IN compared with other groups (P < 0.050, Fig. 4 and Fig. 5A, B), the expression of MOR decreased more significantly in group S and group IN than in the sham group, and there was no significant differences between group S and group IN (P < 0.050, Fig. 4 and Fig. 5A, B).

Furthermore, we performed qRT-PCR to investigate the expression of oprm1 and miR339-5p. The results show that oprm1 mRNA expression decreased and miR-339-5p expression decreased significantly in group S + IN compared with the other groups (P < 0.050, Fig. 5C). The oprm1 mRNA expression decreased and miR-339-5p expression decreased significantly in group S and group IN in comparison to that in the sham group (P < 0.050, Fig. 5C). However, these differences did not exist between group S and group IN (P < 0.050, Fig. 5C).