Ethical Approval and Consent to participate
All procedures were strictly performed in accordance with the regulations of the ethics committee of the International Association for the Study of Pain and the Guide for the Care and Use of Laboratory Animals (The Ministry of Science and Technology of China, 2006). All animal experiments were approved by Nanjing Medical University Animal Care and Use Committee and were approved by the Ethics Committee of Nanjing Medical University (No. IACUC-1908026).
Chemicals and Reagents
Cromakalim (Catalog No. 94470-67-4), Minoxidil (Catalog No.38304-91-5), Diazoxide (Catalog No. 364-98-7), Nicorandil (Catalog No. 65141-46-0), LPS (Catalog No. SMB00610), IL-1β (Catalog No. SRP8033) and IL-6 (Catalog No. SRP3330) was purchased from Sigma-Aldrich (St. Louis, MO, USA). R428 (HY-15150) was purchased from MedChemExpress (Pudong New Area, Shanghai, China). Recombinant Mouse Gas6 Protein (rGas6, Catalog No. 986-GS-025/CF). Kir6.1 small interfering RNA (siRNA) designed and constructed by GenePharma Corporation (Shanghai, China). SOCS3 siRNA (Catalog No. sc-41001), Axl siRNA (Catalog No. sc-29770), Gas6 siRNA (Catalog No. sc-35451) and Control siRNA (Catalog No. sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The lentiviral vectors (LV-SOCS3 or LV-EGFP) were purchased from Obio Technology (Shanghai) Corp.,Ltd. Antibody for Axl (Catalog No. ab215205), IBA-1 (Catalog No. ab178847) SOCS3 (Catalog No. ab16030) was purchased from Abcam (Cambridge, MA, USA). DAPI (Catalog No. D9542) was purchased from Merck Corporation (Darmstadt, Germany). Antibody for c-Fos (Catalog No. 2250), CGRP (Catalog No. 14959), Gas6 (Catalog No. 67202), p-Axl (Catalog No. 44463), STAT3 (Catalog No. 12640) and p-STAT3(Catalog No. 9145) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody for Transferrin (Catalog No. 17435-1-AP) were purchased from proteintech Corporation. Antibody for β-actin (Catalog No. A1978) was purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Gibco, and other cell culture media and supplements were purchased from KenGEN (KenGEN BioTECH, China). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Animals and treatment
Adult male C57BL/6J mice (18-22 g) at 8 weeks of age were provided by the Experimental Animal Center at Nanjing Medical University, Nanjing, China. Animals had free access to food and water and were housed in groups of five to six per cage under pathogen-free conditions with soft bedding under controlled temperature (22 ± 2 °C) and a 12-h light/dark cycle (lights on at 8:00 a.m.). All procedures were conducted in accordance with the guidelines and regulations of the National Institutes of Health (NIH) and were approved by the Ethics Committee of Nanjing Medical University (No. IACUC-1908026).
The plantar incision surgery was performed as previously described (Brennan et al., 1996). All surgeries were done under anesthesia induced by 1 % sodium amobarbital. The plantar aspect of the left hind paw was sterilized with a 10% povidone-iodine solution before and after surgeries and was placed through a hole in a sterile drape. A 1-cm longitudinal incision was made through skin and fascia of the plantar aspect of the foot, starting 0.5 cm from the proximal edge of the heel and extending toward the toes. The plantaris muscle was elevated and longitudinally incised through, leaving muscle origin and insertion intact. After hemostasis with gentle pressure, the skin was opposed with 2 mattress sutures of 5-0 nylon. The animals were allowed to recover. The incision was checked daily, and any sign of wound infection or dehiscence excluded the animal from the study.
Animals were habituated to the testing environment daily for at least 5 days before baseline testing. Mechanical withdrawal threshold was detected by Von Frey Hairs (Woodland Hills, Los Angeles, CA, USA) test. Animals were placed in boxes set on an elevated metal mesh floor and were allowed 30 min for habituation before testing. The plantar surface of each hind paw was stimulated with a series of von Frey hairs with logarithmically incrementing stiffness perpendicularly to the plantar surface. Each mouse was tested for three times and the average of the threshold was measured.
Cell preparation and treatment
BV-2 cells were purchased from the American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s Medium (DMEM, KenGEN Bio TECH, China) supplemented with 10% (v/v) FBS (Gibco), penicillin (100 U/ml), and streptomycin (100 U/ ml). All cells were kept in a humidified chamber with 5% CO2 at 37 °C. For further experiments, BV-2 cells were seeded in 6-well plate at a density of 1 x 105 cells/well. After 24 hours, cells were treated with different tool drugs, and then collected the cells and supernatants to detect the expression SOCS3, Gas6, etc.
Quantitative PCR was performed on BV-2 cell samples and on spinal cord samples obtained from mice. Total RNA was isolated by a standard method with TRIZOL reagent (Invitrogen Life Technologies). Isolated RNA was reverse-transcribed into cDNA using PrimeScript™ RT Reagent Kit (TaKaRa) following standard protocols. Real-time quantitative PCR (qPCR) was performed with synthetic primers and SYBR Green (TaKaRa) with a QuantStudio 5 Real-Time PCR Detection System (Thermo Fisher Scientific). The relative expression level of SOCS3 and SOCS1 was calculated and quantified with the 2−ΔΔCt method after normalization with the reference β-actin. All primers used are listed as follows:
SOCS1: Forward CTGCGGCTTCTATTGGGGAC
SOCS3: Forward GCGGGCACCTTTCTTATCC
β-actin: Forward GGCTGTATTCCCCTCCATCG
Samples (cells or spinal cord) were collected and washed with PBS before being lysed in radio immunoprecipitation assay (RIPA) lysis buffer. The protein concentrations were determined by BCA Protein Assay (Thermo Fisher, Waltham, MA, USA) and 40-80 μg of proteins were loaded and separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA, USA). The membranes were blocked with 5% bovine serum albumin for 2 h at room temperature, probed with antibodies overnight at 4 °C with the primary antibodies and then incubated with HRP-coupled secondary antibodies. The primary antibodies used included Axl (1:1000), p-Axl (1:1000), Gas6 (1:1000), SOCS3 (1:1000), p-STAT3 (1:1000), STAT3 (1:1000), and Transferrin (1:1000). For loading control, the blots were probed with antibody for β-actin (1:1000). The filters were then developed by enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA) with secondary antibodies (Chemicon, Billerica, MA). Data were acquired with the Molecular Imager (Gel DocTM XR, 170-8170) and analyzed with Quantity One-4.6.5 (Bio-Rad Laboratories, Berkeley, CA, USA).
After deep anesthesia, the animals were perfused transcardially with normal saline followed by 4% paraformaldehyde in 0.1 M PB, pH 7.4, each for 20 min. Then L4 and or L5 lumbar segment was dissected out and post-fixed in 4% paraformaldehyde. The embedded blocks were sectioned as 25 μm thick. Sections from each group (five animals in each group) were incubated with rabbit antibodies for c-Fos (1:200), CGRP (1:200) and SOCS3 (1:200), goat antibodies for IBA-1 (1:200). Then the free-floating sections were washed with PBS, and incubated with the secondary antibody (1:300, Jackson Laboratories, USA) for 2 h at room temperature. After washing out three times with PBS, the samples were studied under a confocal microscope (Olympus FV1000 confocal system, Olympus, Japan) for morphologic details of the immunofluorescence staining. Examination was blindly carried out.
NF-κB Activation Assay
BV-2 cells were plated in class bottom cell culture dishes and pretreated with cromakalim (100 μM) 6 h, and then co-cultured with LPS (1 μg/ml) or IL-6 (10 ng/ ml) for 3 h. BV-2 Cells were fixed with 4% paraformaldehyde for 30 min, and then fixed with ice-cold methanol and were permeabilized with 0.25 % Triton X-100/PBST. After blocking with 1 % bovine serum albumin (BSA) in PBST for 1 h, the coverslips with BV-2 cells were incubated for 2 h at room temperature with the p65/RelA antibody diluted in 1 % BSA (1:50). Then the coverslips were exposed to the fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (1:100, at room temperature for 1 h) and then were rinsed three times with PBS. Finally, the coverslips were stained with 1 μg/ml DAPI (4′,6-diamidino-2-phenylindole, a fluorescent DNA dye to mark nucleus) for 1 min. Confocal microscopy analyze was carried out using Olympus FV1000 confocal system
3.3 nmol siRNA was dissolved in 330 μl RNase-free water. Control siRNA was used as a negative control. For the transfection of siRNA, cells were cultured in six-well plates with antibiotic-free medium the day before transfection. The transfection was conducted when cells reached 60~80% confluence using Lipofectamine 2000 (Invitrogen, USA) and serum-free medium according to the manufacturer’s instructions. After 6 h, the transfection medium was replaced with the culture medium containing 10% FBS and then incubated at 37 °C in 5% CO2. For the animals’ experiments, i.t. administration of siRNA into mice for 48 h just before plantar incision surgery. The mechanical thresholds were measured to determine whether interference with SOCS3, Gas6 and Axl genes could abolish the analgesia of cromakalim.
SOCS3 overexpression Assay
The lentiviral vectors (LV-SOCS3 or LV-EGFP) were purchased from Obio Technology (Shanghai) Corp.,Ltd. The titer of lentiviral vectors (LV-SOCS3) was 3.00E + 10 PFU/ml. LV-EGFP is as a negative control. 10 μl of lentiviral vectors (LV-SOCS3 or LV-EGFP) was intrathecally injected into mice three days before plantar incision surgery.
GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA) was used to conduct all the statistical analyses. Alteration of expression of the proteins detected and the behavioral responses were tested with one-way ANOVA, and the differences in latency over time among groups were tested with two-way ANOVA. Bonferroni post hoc tests were conducted for all ANOVA models. Results were represented as mean ± SEM of three independent experiments. P < 0.05 was deemed to be statistically significant.