A total of 42 male Sprague-Dawley rats (weight, 200–250 g; age, 8–10 weeks) were provided water and feed ad libitum until 3 days prior to the experiment. The rats were house with a l2 h light/dark cycle at 23±1°C and 55–60% humidity. The rats were provided by the Experimental Animal Center of Nantong University (Nantong, China) and the study procedures were approved by the Animal Care and Use Committee of Nantong University (approval no. 20171015S1051122).

The rats were randomly divided into seven groups (n=6/group). Naive group, no treatment was performed; sham group, the rats received an incision through the skin and muscle; skin/muscle incision and retraction (SMIR) (14) group, the rats underwent 1-h retraction following the skin/muscle incision; SMIR + nicorandil groups, referring to the dose of nicorandil in the relevant literature (15–17), the rats were intraperitoneally injected with either low (1.0 mg/kg), middle (1.5 mg/kg) or high dose nicorandil (2.0 mg/kg) separately 0.5 h prior to the SMIR procedure; SMIR + nicorandil (1.5 mg/kg) + glibenclamide (10.0 mg/kg) group, 0.5 h before SMIR modeling, a mixture of 1.5 mg/kg nicorandil (cat. no. N3539; Sigma-Aldrich; Merck KGaA) and 10.0 mg/kg glibenclamide (cat. no. PHR1287; Sigma-Aldrich; Merck KGaA) was injected intraperitoneally.

The mechanical withdrawal threshold (MWT) was detected prior to, and 1, 3, 7 and 14 days following SMIR surgery. The rats were habituated to the testing environment for at least 30 min before testing. Mechanical allodynia was assessed using the Up-Down paradigm (18) with von Frey filaments (IITC Life Science, Inc.) ranging between 1.4 and 26 g. Shrinking, swinging or paw licking were regarded as positive reactions. Each filament was presented five times within 30 sec to determine the response threshold. If the response was not elicited at least twice, the next ascending von Frey filament was applied until at least two responses were observed.

On day 7 after SMIR, the rats were anesthetized with isoflurane (induction with 3–4%, maintenance with 1–2%) and perfused transcardially with PBS followed by 4% paraformaldehyde in PBS (250 ml; pH 7.0). After perfusion, the L3-L5 spinal cord and DRG tissues from rats in the SMIR group were extracted and post-fixed in the same fixative at 4°C overnight, and then placed in 20% and subsequently in 30% sucrose solution at 4°C overnight. After freezing sequentially, the tissues were continuously sectioned at 6 µm and stored at −20°C. These sections were selected randomly and blocked with 5% serum antibody blocking solution (Beyotime Institute of Biotechnology) for 2 h. The tissue slices were then incubated independently with antibodies against p120 (1:300; Santa Cruz Biotechnology, Inc; cat. no. sc-23873), glial fibrillary acidic protein (GFAP; 1:5,000; EMD Millipore; cat. no. AG230), calcitonin gene-related peptide (CGRP; 1:800; Cell Signaling Technology, Inc.; cat. no. 14959), NeuN (1:5,000; EMD Millipore; cat. no. SAB4300883), ionized calcium-binding adapter molecule 1 (Iba1; 1:800; FUJIFILM Wako Pure Chemical Corporation; cat. no. 019-19741) and isolectin B4 (IB4; 1:1,000; Advanced Targeting Systems, Inc.; cat. no. PR-02) at 4°C overnight, then co-incubated with Cy3- or FITC-conjugated secondary antibodies (1:1,000, Jackson ImmunoResearch Laboratories, Inc.; cat. no. 115-165-205, 115-095-205) in the dark for 2 h at room temperature. A total of five sections were randomly selected from the spinal cord and DRG of each rat. The localizations of p120 in the spinal cord dorsal horn and DRG were examined under a fluorescence microscope (Olympus Corporation) in the dark to capture images.

The rats were anesthetized and sacrificed as previously described, and the L3-L5 spinal cord and DRG tissues were homogenized in sodium dodecyl sulfate (SDS) sample buffer containing a mixture of protease and phosphatase inhibitors (Sigma-Aldrich; Merck KGaA), and measured with a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). For separation, 30 µg total protein per gel lane was loaded onto 10% gels (Beyotime Institute of Biotechnology). The separated proteins were then transferred onto nitrocellulose membranes. The membranes were incubated for 2 h at room temperature in tris-buffered saline and Tween-20 blocking solution containing 5% skimmed milk, followed by overnight incubation at 4°C in blocking solution containing primary antibodies against the following proteins: p120 (1:300; Santa Cruz Biotechnology, Inc.; cat. no. sc-23873) and GAPDH (1:5,000; Sigma-Aldrich; Merck KGaA; cat. no. G2267). Membranes were washed three times and incubated with the anti-mouse or anti-rabbit peroxidase-conjugated secondary antibodies (1:2,000; Jackson Immuno Research Laboratories, Inc.; cat. nos. 115-035-003, 111-005-003, respectively) at room temperature for 2 h. After washing, immunolabeling was detected using the Tanon2500 gel imaging system (Tanon Science and Technology Co., Ltd.) and hypersensitive ECL chemiluminescence detection kit (Absin). ImageJ software (version no. 1.8.0, National Institutes of Health) was used to capture and analyze the intensity of the bands.

The data were presented as mean ± standard error of mean of at least three experimental repeats. Data were analyzed using one- or two-way analysis of variance followed by Bonferroni's or Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To study the mechanism underlying CPSP, an SMIR model was established according to a previous study (14) (Fig. 1A). As shown in Fig. 1B, the MWT significantly decreased on postsurgical days 1, 3, 7 and 14 in the sham, SMIR + 1.5 and SMIR + 2.0 mg/kg nicorandil groups, as compared with the SMIR group, in a time-dependent manner. Whereas, no statistical difference was observed in the MWT of the SMIR + 1.0 mg/kg nicorandil group compared with the SMIR group (P>0.05). It was identified that the anti-nociceptive effects of 1.5 and 2.0-mg/kg nicorandil were more pronounced compared with 1.0 mg/kg nicorandil (P<0.05), and no significant difference was observed between 1.5 and 2.0 mg/kg (P>0.05; Fig. 1B). These results suggested that 1.5 and 2.0 mg/kg nicorandil may inhibit SMIR-induced mechanical hyperalgesia, and that 1.5 mg/kg nicorandil may be the most effective dose. Therefore, 1.5 mg/kg nicorandil was used to determine whether the anti-nociceptive effects of a KATP activator could be relieved by the KATP blocker glibenclamide (10.0 mg/kg). As expected, injection of glibenclamide together with nicorandil reversed the anti-mechanical nociceptive effects of nicorandil on postsurgical days 1, 3, 7 and 14. These findings suggested that nicorandil may alleviate CPSP through activating KATP.

To explore the role of p120 in central sensitization of CPSP and its possible mechanism, changes in the expression of p120 in the spinal cord and DRG were examined on postsurgical days 3, 7 and 14 in the SMIR and sham groups. It was identified that SMIR-induced CPSP enhanced the expression of p120 in the spinal cord and DRG of rats compared with in the naive group (Fig. 2A and B). There was also a high expression of p120 on postsurgical days 7 and 14 in the sham group (Figs. S1 and S2). As the 7th day after surgery may be the turning point of the transformation from acute pain to chronic pain, the present study selectively compared the expression of p120 on postsurgical day 7 in the sham group with that in the SMIR group. The results demonstrated that the expression of p120 in the spinal cord of the SMIR group was higher compared with that of the sham group on postsurgical day 7, whereas no significant difference was observed in DRG between the two groups (Fig. 2A and B).

To study the location of p120, spinal cord sections of rats in the SMIR group at postsurgical day 7 were stained with the neuronal marker NeuN, astrocyte marker GFAP and microglia marker Iba1. Results revealed that p120 was distributed in GFAP-positive astrocytes. However, p120-positive staining was not observed in the Iba1- and NeuN-positive neurons (Fig. 3A-C).

At postsurgical day 7, DRG tissue sections were stained with p120, and the medium and large neuronal marker NF200, or the medium and small neuronal markers CGRP and IB4. The results revealed that p120 was mainly distributed in the NF200-positive medium and large neurons, but not in the CGRP- and IB4-positive medium and small neurons (Fig. 4A-C).

To study the mechanism underlying the analgesic effect of KATP agonists, the present study detected the changes in the expression of p120 in the spinal cord and DRG following intraperitoneal injection of 1.5 mg/kg nicorandil 0.5 h before SMIR. The changes in the expression of p120 were observed on postsurgical day 7. The results suggested that SMIR promoted the expression of p120 in the spinal cord and DRG, whereas nicorandil treatment decreased the expression of p120 (Fig. 5A and B).