Male C57BL/6J mice (6-8 weeks, 23-25 g) were used (Tongji Medical College, Wuhan, China). All mice were placed in a temperature-controlled environment, followed a 12-hour light-dark cycle, 50% humidity, and were randomly fed a standard diet and water. The experimental procedures were performed in accordance with the approval of the ethics committee of the Animal Care and Use Committee of Huazhong University of Science and Technology.
Drugs and anesthesia
All operations were conducted under 50 mg/kg sodium pentobarbital anesthesia (i.p.). STING inhibitor C-176 purchased from Selleck (Houston, TX, USA) was dissolved in corn oil for different concentration (2, 4, 8 μg/μl, respectively). Recombinant mice IL-6 from R&D Systems (Minneapolis, MN) was dissolved in Phosphate buffered saline (PBS). For rIL-6 injection, animals received a single injection of 100ng of mice rIL-6 at day 1 following SNI. The drugs were delivered intrathecally (i.t., 5 μl). The STING agonist DMXAA purchased from Selleck (Houston, TX, USA) was dissolved in Dimethyl sulfoxide (DMSO; AppliChem, Darmstadt, Germany), and 100 μg/ml of DMXAA was given in BV-2 cells.
SNI and behavioral tests
The model of SNI was established in this study. Under anesthesia, the left sciatic nerve of mice was exposed at the mid-thigh level, the three peripheral branches of the sciatic nerve (common peroneal, sural, and tibial nerves) were exposed without stretching muscles and nerves. A tight ligation (5-0 silk) and transection of tibial nerves and common peroneal nerves together, removing 2-3 mm length of the nerves distal to the ligation and leaving the sural nerve intact. The animals in the sham group underwent the same procedure without injury of the nerves. For acute or chronic treatment, mice were randomly divided into the following groups: (1) Sham group: sham-operated mice with vehicle injection (corn oil or PBS, 5 μl, i.t.); (2) SNI group: SNI-injured mice with vehicle injection (corn oil or PBS, 5 μl, i.t.); (3) SNI+ C-176 group: SNI-injured mice with C-176 injection (10, 20, 40 μg, i.t.); and (4) SNI+ C-176+ rIL-6 group: SNI-injured mice with C-176 (40 μg, i.t.) combined with rIL-6 (100 ng, i.t.) injection.
Mechanical allodynia was assessed by measurement of paw withdrawal threshold (PWT) using Von Frey filaments (Stoelting, Wood Dale, IL, USA) as previously described [27, 28]. In brief, mice were placed in separate chambers (10×10×15 cm) for 30 minutes before the test. Then, the tip of filaments was placed on the plantar surface of the left hind paw for 5 s per force. Ascending order of forces (0.007, 0.02, 0.04, 0.16, 0.4, 0.6, 1.0, 1.4 and 2g) were used, starting with 0.007 g and ending with 2 g. The duration of each force was maintained for approximately 1s. Quick paw lifting or licking was considered to be positive responses. PWTs were decided as the lowest force required to elicit a positive response [3-5].
Thermal hyperalgesia was determined by measurement of thermal withdrawal latency (TWL) using Hargreaves plantar test (Ugo Basile, Italy). In brief, mice were placed in separate chambers (10×10×15 cm) on a glass plate for 30 minutes before the test. The radiant heat source was applied beneath the plantar surface of the left hind paw and the stimulus shut off when the hind paw moved. Each hind paw was repeated 3 times with a period of 5-6 minutes intervals. The mean TWL was determined from the mean of three measurements. The maximum period was set at 30 seconds to avoid tissue damage. The glass plate was cleaned between each interval.
The Open Field Maze (OFM) was applied to assess movement. Mice were adapted in the laboratory for 30 min before the test. Then the mice were individually placed in the center of open field, which consisted of a plastic base (50×50×50 cm) and divided into 25 grids and the centric 9 grids of equal area were defined as central area. The total distance and speed were recorded.
The murine BV-2 cell lines were cultured in Dulbecco’s modified Eagles medium (DMEM, Gibco, NY, USA) supplemented with 10 % fetal bovine serum (FBS, Yeasen, Uruguay, SA) at 37 ℃ with 100 % humidity in 5 % CO2 for 2 days. Then the cells were put into a 6-well plate or 12-well plate for further experiments. After 24 h, cells were treated with DMXAA (100 μg/ml) with another 24 h . The supernatants were gathered for enzyme linked immunosorbent assay (ELISA) analysis and cells were processed for western blotting and immunostaining.
Under deep anesthesia, spinal cords of mice (L4-L5) were quickly removed for analysis. Total RNA was isolated with RNAiso Plus (Takara, Kyoto, Japan), and MCP-1, IL-1β and TNF-α mRNA expression were normalized to the β-actin. cDNA was then synthesized using HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China):
MCP-1- 5-CCACTACCTTTTCCACAACCA-3 (sense),
5 -TGTGCTGCTGCGAGATTTG-3 (antisense);
TNF-α- 5 -GTCTACTGAACTTCGGGGTGAT-3 (sense),
5 -TGCTACGACGTGGGCTACA-3 (antisense);
β-Actin- 5-CTGAGAGGGAAATCGTGCGT-3 (sense),
Under deeply anesthetized, the L4 -L5 spinal cord were quickly removed and homogenized on ice using lysis buffer containing a cocktail of protease inhibitors and protein inhibitors. The homogenates or cell lysates were separated by 10% SDS-PAGE and transferred to 0.45 μm PVDF membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked with 5% skim milk or BSA at RT (room temperature) for 2 h. Then the primary antibodies were used: anti-STING (A3262; ABclonal, Wuhan, China), anti- Phospho-TBK1 (AP1026; ABclonal, Wuhan, China), anti-TBK1 (A2573; ABclonal, Wuhan, China), anti-Phospho-NF-κB (AP0123; Abcam, Cambridge, UK), anti-NF-κB (A19653; ABclonal, Wuhan, China), anti-iNOS (A0312; ABclonal, Wuhan, China), anti-CD68 (A13286; ABclonal, Wuhan, China), anti-CD86 (A19026; ABclonal, Wuhan, China), anti-Phospho-JAK2 (#3776; Cell Signaling Tech, MA, USA), anti-JAK2 (A11497; ABclonal, Wuhan, China), anti- Phospho-STAT3 (#9145; Cell Signaling Tech, MA, USA), anti-STAT3 (A11216; ABclonal, Wuhan, China), and anti-β-actin (AC026; ABclonal, Wuhan, China). The membranes were incubated with HRP-conjugated goat anti- rabbit IgG (H+L) (A21020, Abbkine, Wuhan, China) at RT for 2 h. The protein expression was detected using chemiluminescence (Bio-Rad, Hercules, CA) and quantified using System with a Molecular Imager (Bio-Rad, Hercules, CA).
Under deep anesthesia, blood samples were collected from the inferior vena cava of mice and centrifuged for serum collection, dsDNA ELISA kit (ELK8414, ELK Biotechnology CO., LTD, Wuhan, China) were used for measuring the levels of dsDNA concentration in sera according to the manufacturer's instruction. Further, the L4-L5 spinal cord of mice were quickly removed and homogenized on ice-cold 0.01 mol/L PBS. The concentrations of interleukin-6 (IL-6) were measured in BV-2 cells culture supernatants and in the L4-L5 spinal cord of mice using IL-6 ELISA kit (RK00008, Abclonal, Wuhan, China). The concentrations of IFN-β were measured in the L4-L5 spinal cord of mice using IFN-β ELISA kit (ELK8414, ELK Biotechnology CO., LTD, Wuhan, China).
Immunofluorescence staining was conducted as previously described . Under deep anesthesia, mice having been intracardially perfused with 50 ml cold-PBS followed by 50 ml 4% cold-paraformaldehyde (PFA). The L4-5 segment of the spinal cord were collected and post-fixed in 4 % PFA overnight at 4 ℃. 20 μm sections of spinal cord were cut on a cryostat (CM1900, Leica, Heidelberg, Germany). The sections were penetrated with 0.3% TritonX-100 for 10 min and blocked with 5% donkey serum for 45 min at RT. Then the following primary antibodies were used: anti-STING (A3262; ABclonal, Wuhan, China.), anti-Phospho-TBK1 (AP1026; ABclonal, Wuhan, China); anti-neuronal nuclei antibody (NeuN, ab104224, Abcam, Cambridge, UK); anti-glial fibrillary acidic protein antibody (GFAP; 3670; Cell Signaling Technology, Danvers, MA, USA) and anti- Iba1 antibody (ab5076; Abcam, Cambridge, UK). After washing 6 times with PBS, the sections were incubated with donkey anti-rabbit secondary antibody (711-547-003; Jackson ImmunoResearch, PA, USA), donkey anti-mouse secondary antibody (715-585-150; Jackson ImmunoResearch, PA, USA), and donkey anti-goat secondary antibody (705-585-003; Jackson ImmunoResearch, PA, USA) for 2 h at RT. The sections were captured by using a fluorescence microscope (DP70, Olympus, Japan).
For immunocytochemistry, BV-2 cells were washed with cold-PBS for 3 times and then fixed with 4 % PFA at RT for 15min. The following primary antibodies were used after treatment with 0.3 % Triton X-100 followed by 5 % donkey serum: anti-STING (A3262; ABclonal, Wuhan, China); anti-Phospho-TBK1 (AP1026; ABclonal, Wuhan, China); anti-Phospho-NF-κB (AP0123; Abcam, Cambridge, UK); anti-neuronal nuclei antibody (ab104224; Abcam, Cambridge, UK); anti-glial fibrillary acidic protein antibody (3670; Cell Signaling Technology, Danvers, MA, USA) and anti-Iba1 antibody (ab5076; Abcam, Cambridge, UK). After washing 3 times with PBS, cells were then processed with the secondary antibody (Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody; 711-547-003; Jackson ImmunoResearch, PA, USA) for 2 h at RT. After washing 4 times with PBS, the cells were then counterstained with DAPI for 7 min. Fluorescent images were then captured by using a fluorescence microscope (DP70, Olympus, Japan).
Experimental designs and animal groups
The experimental designs and animal groups have been exhibited as Fig. 1.
Experiment 1: Time course of pain behaviors and STING signaling expression following SNI.
Fifty mice were randomly assigned into sham or SNI group. The pain behaviors were measured at days 1, 3, 7, 14 after SNI surgery, and then, the L4-5 spinal cord was removed for Elisa, western blot, and immunofluorescent analysis.
Experiment 2: The effects of DMXAA on microglia cells.
After SNI surgery, STING and p-TBK1 was mainly colocalized with Iba1(microglia cells). Thus, the cultured BV2 cell was stimulated with DMXAA. Cells were assigned into control and DMXAA group. 24 h later, cells were obtained for real-time PCR, Elisa, western blot, and immunofluorescent analysis.
Experiment 3: The effects of C-176 on pain hypersensitivity and microglia activation following SNI.
After SNI surgery, fifty mice were divided into SNI+Vehicle and SNI+C-176 group. For early injection, C-176 was injected once daily from day 1 to day 5, and the pain behaviors were assessed 0.5h before injection and from day 1 to day 5; for late injection, C-176 was injected once daily from day 7 to day 11, and the pain behaviors were assessed 0.5h before injection and from day 7 to day 11. We found that early but not late C-176 injection could inhibit pain development following SNI. Thus, the L4-5 spinal cord was removed for further analysis following SNI at day 7 with or without C-176 injection by real time PCR, Elisa, western blot, and immunofluorescent analysis. Correspondingly, the L4-5 spinal cord of sham operated group was collected at day 7.
Experiment 4: The analgesic effects of C-176 was abolished by mice rIL-6 following SNI.
After SNI surgery, forty rats were divided into SNI+Vehicle, SNI+C-176 and SNI+ C-176+rIL-6 group. C-176 was given from day 1 to day 5 following SNI, and mice rIL-6 was simultaneously injected with C-176 at day 1, 3 and 5 in SNI+ C-176+rIL-6 groups. Mechanical allodynia was assessed 0.5h before injection and at day 7.
All data are presented as means ± SEM and analyzed by GraphPad Prism version 6.0. Student’s t test (two-tailed) was used for differences between two groups, one-way ANOVA followed by Bonferroni post hoc test was used for differences between multiple groups. Behavior results (such as PWT and TWL) were analyzed by two-way ANOVA with repeated measures, followed by Bonferroni post hoc test. p < 0.05 was indicated statistically significant.