2.1 Animal model and groups
Sprague–Dawley rats (180–220 g) were purchased from Changsha Tianqin Biotechnology Co., Ltd. (Changsha, China). Laboratory animals were provided free access to water and food in a room with a controlled temperature and luminosity (22 ± 2 °C, 50% humidity, and 12-h light/dark cycle). We attempted to reduce the pain of experimental animals and reduce the number of animals during the experiment. All experiments were reviewed and approved by the Animal Management Committee of Nanchang University. Neuropathic pain is produced by inducing chronic constriction injury of the sciatic nerve in rats. Briefly, rats were anaesthetized with urethane combined with xylazine. First, the sciatic nerve in the left thigh was isolated, and then four ligatures were ligated around the sciatic nerve. The distance between each ligature was one millimeter. The ligatures were loosely ligated until slight movement of the ipsilateral hind limb was observed. Finally, the surgical site of the rat was disinfected with iodophor. The operation used for the rats in the sham group was the same as that for the rats in the CCI group, but the sciatic nerve was not ligated. Successful establishment of the model is defined by spontaneous foot elevation, toes curled together, sloping rows and a reduction in mechanical and thermal pain sensitivity of at least 30%.
2.2 Drugs and administration
The Cx43 mimetic (Gap26), which was purchased from GIPBIO, was mixed with saline prior to use. The drug was administered to the animal by intrathecal injection through spinal puncture between the L5 and L6 levels to deliver 10 µl (20 µg) of drug into the cerebrospinal fluid. The drug was injected on Days 7, 9, 11 and 13 after the procedure.
2.3 Behavioral Analysis
Measurement of the mechanical withdrawal threshold (MWT): The rats were placed in a clear plastic box (22×12×22 cm) on top of a stainless-steel wire mesh. The temperature in the room was maintained at 25 °C at all times, and the rats were allowed a few minutes to acclimate. The left hindfoot was randomly stimulated using an electronic analgesia apparatus (BME-404, Tianjin, China) at a pressure of 0–50 g in rats by investigators with no prior knowledge of the groups. The rats were stimulated 10 times at 10 second intervals. The mean value of each group of rats was recorded as their MWT.
Measurement of the thermal withdrawal latency (TWL): The rats were placed in a clear bottomless square acrylic box (22×12×22 cm) on a glass plate. The left hindfoot of the rat was irradiated with a heat beam using a fully automatic plantar analgesia apparatus (BME-410C, Tianjin, China). The heat beam was turned off when the rat withdrew its left hindfoot. The time between heat beam onset and heat beam off (withdrawal of the rat's hind foot) was recorded as the TWL. The left hind foot of rats was randomly alternately stimulated at 5-minute intervals by investigators with no prior knowledge of the groups. Each rat was measured 6 times. The maximum duration of the heat beam exposure was 30 seconds.
2.4 Quantitative real-time reverse transcription-polymerase chain reaction (qPCR)
L4–6 DRGs were isolated from rats on Day 13 after surgery, and the isolated ganglia were immediately washed with 0.01 M PBS, dispensed in RNA stabilization solution (Thermo Fisher Scientific, USA) and stored at -80 °C. Total RNA was extracted from DRGs using the TransZol UP Plus RNA Kit (TransGen Biotech, Beijing, China). One milliliter of TransZol Up was added to 50–1100 mg of sample and homogenized using a nuclease-free glass homogenizer. After an incubation for 5 min, a portion of chloroform was added, shaken vigorously for 30 seconds and incubated for 3 min at room temperature. The mixture was centrifuged at 10,000 g for 15 min at 4 °C. The colorless aqueous phase was transferred to a new EP tube, an equal volume of anhydrous ethanol was added and the sample was mixed by inversion. The resulting solution was added to a centrifuge column and centrifuged at 12,000 g for 30 seconds, discarding the effluent. Five hundred microliters of CB9 were added, the sample was centrifuged twice at 12,000 g for 30 seconds each time and the effluent was discarded. Then, 500 µl of WB9 were added and centrifuged twice at 12,000 g for 30 seconds each, discarding the effluent. The sample was centrifuged at 12,000 g for 2 min at room temperature to completely remove any residual ethanol. The centrifuge column was placed in an RNase-free tube, and 50–200 µl of RNase-free water were added and incubated with the column for 1 min at room temperature. The sample was centrifuged at 12,000 g for 1 min at room temperature to elute the RNA. The RNA was stored at -80 °C. The RNA was then reverse transcribed to complementary DNA (cDNA) templates using EasyScript One-step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) by incubating the sample at 42 °C for 15 minutes. The cDNA templates were further amplified with qPCR using PerfectStart Green qPCR SuperMix (TransGen Biotech, Beijing, China). P2X4 and Cx43 mRNA expression levels were quantified using a Bio–Rad fluorescent quantitative PCR instrument (CFX Connect). The qPCR cycling conditions were set according to the manufacturer's reaction parameters: predenaturation at 94 °C for 30 seconds, 44 cycles of three steps: 94 °C for 5 seconds followed by 55 °C for 15 seconds and 72 °C for 10 seconds for amplification (total Volume 20 µl). ΔCT was calculated as the average threshold cycle (CT) value of Cx43 mRNA or P2X4 mRNA minus the average CT value of GAPDH (∆CT = CT target – CT reference). ∆∆CT = ∆CT test sample − ∆CT calibrator sample. Finally, we determined the relative quantity (RQ) of Cx43 or P2X4 expression according to the acknowledged equation: RQ=2-∆∆CQ. The forward primer sequence of P2X4 was 5’-CCATCTGCATAGTGACGGCT-3’. The reverse primer sequence was 5’-TGTGCACTCTTCCCACTGTC-3’. The forward and reverse primer sequences of GAPDH were 5’-GCATCTTCTTGTGCAGTGCC-3’ and 5’-TACGGCCAAATCCGTTCACA-3’.
2.5 Western blot
The isolated L4–6 DRGs were stored in a freezer at -80 °C. Tissue samples were homogenized by mechanical disruption in RIPA lysis buffer containing protease inhibitors and protein phosphatase inhibitors using a glass homogenizer. After the tissue was completely lysed, the liquid was transferred to a new EP tube and centrifuged at 4 °C and 14,000 g for ten minutes, and the supernatant was collected as the protein extract. Protein loading buffer was added to the protein extract in proportion, boiled in boiling water for 5 min, allowed to cool and stored in aliquots at -20 °C. Protein concentrations were measured using the BCA protein assay (Applygen, Beijing, China). Each lane was spiked with thirty micrograms of protein and electrophoretically separated on SDS–PAGE gels (8%-15%). After electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Afterwards, the PVDF membrane was incubated for 2 hours at room temperature with a blocking buffer containing 5% skim milk powder or 5% bovine serum albumin. Next, the blots were incubated with an antibody against P2X4 (1:1000, rabbit, Alomone), Cx43 (1:1000, goat, SICGEN), CXCR2 (1:200, rabbit, abcam), P38 (1:1000, rabbit, Cell Signaling), phosphor-P38 (1:500, rabbit, Cell Signaling), ERK1/2 (1:1000, rabbit, Cell Signaling), phospho-ERK1/2 (1:1000, rabbit, Cell Signaling), JNK (1:1000, rabbit, Cell Signaling), phospho-JNK (1:500, rabbit, Cell Signaling), P65 (1:500, rabbit, Cell Signaling), or phospho-P65 (1:500, rabbit, Cell Signaling) , IL-1β(1:200, rabbit, Affinity) and TNF-α(1:400, rabbit, Boster) at 4 °C overnight. The blots were probed with an antibody against GAPDH (1:1000, Mouse, BOSTER) to ensure consistent protein loading. The next day, these blots were further incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000, BOSTER) and then exposed to ChemiDocTMXRS+ (Bio–Rad). Finally, the intensity of the protein bands was determined using NIH ImageJ software.
2.6 Immunohistochemistry and immunocytochemistry
The isolated L4–6 DRGs were immediately treated with 0.01 M PBS on Day 13 after surgery, and then the DRGs were fixed with 4% paraformaldehyde (PFA) for 2 hours. After fixation, the tissues were washed 3 times with 0.01 M PBS and then dehydrated overnight at 4 °C in 10%, 20% and 30% sucrose solutions. Finally, the tissue was infiltrated with an OCT mixture (optimum cutting temperature compound) and sectioned with a cryostat for final immunohistochemical treatment. For multiplex immunofluorescence staining, we chose primary antibodies raised in different species and secondary antibodies against the same species to avoid interference with immunofluorescence. Tissue sections were first washed 3 times with 0.01 M PBS, permeabilized with 0.3% Triton-X for 15 minutes, and then washed 3 times with 0.01 M PBS. Next, the sections were incubated with 10% donkey serum at 37 °C for 1 hour. Afterwards, the sections were incubated overnight at 4 °C with the following primary antibody mixture: P2X4(1:100, rabbit, Alomone), Cx43(1:100, goat, Sigma), and GFAP (1:100, mouse, Biolegend). The next day, the sections were washed 3 times with 0.01 Sections were incubated for 1 hour at room temperature with the following mixture of secondary antibodies: TRITC-conjugated donkey anti-rabbit (1:200, donkey, Jackson ImmunoResearch), FITC-conjugated donkey anti-goat (1:200, donkey, Jackson ImmunoResearch), and allophycocyanin-conjugated donkey anti-mouse (1:200, donkey, Jackson ImmunoResearch). The images were finally captured using an Olympus laser scanning confocal microscope (FV3000).
2.7 Enzyme-linked immunosorbent assay (ELISA)
ELISA kits for ATP and CXCL1 were purchased from CAMILO. On Day 13 after surgery, we obtained blood samples from the carotid artery of rats and centrifuged them to obtain serum. Rat serum was stored at -80 °C prior to use. ELISAs were performed using a microplate reader (PerkinElmer, USA) according to the ELISA reagent manufacturer's instructions. A standard curve was included with each experiment.
2.8 Molecular Docking
The Cx43.pdb file were downloaded from http://www.rcsb.org/pdb/home/home.do, and the gap26. sdf file was downloaded fromhttps://pubchem.ncbi.nlm.nih.gov/.Autodock Tools soft was used for molecular docking.
2.9 Statistical method
GraphPad Prism v8.4.0 software was used for graphing and data analysis. The experimental results were expressed as mean ± standard error (mean ± SEM). Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) combined with least significant difference (LSD). P values less than 0.05 were considered statistically significant.