Inhibition of the spinal astrocytic JNK/MCP-1 pathway activation correlates with the analgesic effects of tanshinone IIA sulfonate in neuropathic pain

Male Sprague–Dawley rats (220 to 250 g) were housed in a temperature-controlled room in plastic cages (6 animals per cage) with free access to food and water at 22°C to 25°C on a 12-h light/dark cycle. All of the experiments reported in this study were conducted according to an experimental protocol approved by the Animal Use and Care Committee for Research and Education of The Fourth Military Medical University (Xi’an, China). All efforts were made to minimize the animals’ suffering and the number of animals used [21].

The experimental design was as follows: Firstly, to verify the analgesic effects of TIIAS in neuropathic pain and explore the related mechanisms, 144 rats were divided into 6 groups in the experiments: sham-vehicle group, sham-TIIAS (50 mg/kg) group, sham-vehicle group, SNL-TIIAS (2 mg/kg) group, SNL-TIIAS (10 mg/kg) group, and SNL-TIIAS (50 mg/kg) group (n = 24 in each group). In each group, 12 rats were used for the tests of mechanical hypersensitivity and the other 12 rats were used for immunofluorescence, Western blot, and enzyme-linked immunosorbent assay (ELISA), 4 rats respectively. Secondly, to assess whether the intraperitoneal administration of TIIAS influenced motor function, another 24 rats were divided into 4 groups for rotarod test: naïve group, TIIAS (2 mg/kg) group, TIIAS (10 mg/kg) group, and TIIAS (50 mg/kg) group (n = 6 in each group). Thirdly, to further verify the role of JNK in the analgesic effects of TIIAS, specific JNK inhibitor (SP600125) was administrated and another 80 rats were divided into 5 groups: sham group, SNL-vehicle group, SNL-TIIAS (ED₅₀) group, SNL-SP600125 group, and SNL-TIIAS (ED₅₀)-SP600125 group (n = 16 in each group). In each group, 12 rats were used for the test of mechanical hypersensitivity and the other 4 rats were used for ELISA test of MCP-1 expression on post-operative day (POD) 10 of SNL injury.

SNL was performed according to our previous protocols [22,23]. Briefly, rats were anesthetised with chloral hydrate (300 mg/kg, i.p.). A midline incision was then made at the L3 to S2 level, and the dorsal vertebral column from L4 to S2 was exposed. After a portion of the L6 transverse process was carefully removed, the left L5 spinal nerve was carefully isolated and tightly ligated distal to the dorsal root ganglion (DRG) with 6–0 silk thread. Sham-operated animals were subjected to a similar surgical procedure in which the isolated spinal nerves were not ligated.

TIIAS (99% purity) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP; Beijing, China). TIIAS was dissolved in physiological saline for the in vivo experiments. And the doses for the intraperitoneal administration of TIIAS (three different doses: 2, 10, and 50 mg/kg, i.p.) were selected according to previous studies [19,20]. Considering that astrocytes are activated from POD 3 of SNL, TIIAS or saline was administrated intraperitoneally 30 min prior to behavioral experiments from POD 3 to POD 10 once a day. To further verify the role of JNK in the analgesic effects of TIIAS, a specific JNK inhibitor SP600125 (5 μg/rat, in 5 ul normal saline containing 1% DMSO) or vehicle (normal saline containing 1% DMSO) was intrathecally administrated 15 min prior to the measurement of mechanical pain behaviors from POD 3 to POD 10 after SNL. Intrathecal implantation was performed by inserting polyethylene (PE) tubing to inject the drug directly into the subarachnoid space of the lumbar enlargement. After surgery, the neurologically normal rats were injected with 2% lidocaine (10 μl) through the intrathecal catheter to confirm whether the PE tubing was in the intrathecal space. Only the rats showing complete paralysis of both hind legs and tail after administration of lidocaine were used for the subsequent experiments. At the end of each experiment, the position of the PE tubing in the intrathecal space at the lumbar enlargement was verified visually by exposing the lumbar spinal cord. Data from rats with incorrect PE tubing position were abandoned from the study.

Motor dysfunction can have evident effects on nociceptive behavioral tests [24]. To assess whether the intraperitoneal administration of TIIAS influenced motor function, another 24 rats were used in rotarod tests via an accelerating rotating rod (Ugo Basile 7650, Varese, Italy). After 1 min of training, rats were placed on the rotarod, which was linearly accelerated from 4 to 40 rpm over 5 min. The elapsed time before the rat fell on each of the three runs with 10-min intervals between runs was recorded for each rat. The test was repeated 30 min after the intraperitoneal administration of TIIAS or saline once per day for 7 days, and the time that the rat remained on the rotarod was recorded. Final results are expressed as a percentage of baseline value of each rat.

Rats were placed on an elevated mesh grid that completely exposed the middle of the hind paw. Mechanical hypersensitivity was tested using von Frey filaments (Stoelting, Kiel, WI, USA) by experimenters who were blinded to group assignment as described previously [25]. The stiffness of the von Frey filaments was 2, 4, 6, 8, 10, 15, and 26 g. The hind paw was pressed with each filament, in order of increasing stiffness, for 5 s. Rapid pulling back, biting, or shaking of the hind limb within 5 s of the hind limb being pricked by one of the von Frey filaments was taken as a positive sign of withdrawal. The interval between trials was at least 5 min. For each trial, the same hind limb was stimulated ten times by a single von Frey filament before being stimulated by the next larger filament. The minimal value that resulted in at least six responses to ten stimulations was recorded as the paw withdrawal thresholds (PWTs). The formula for calculating the percentage change was 100–100 × (baseline of SNL-TIIAS − post-SNL-TIIAS)/(baseline of SNL-vehicle − post-SNL-TIIAS).

The rats were deeply anesthetised by injection of pentobarbital (60 mg/kg, i.p.) and transcardially perfused with 200 ml of 5 mM sodium phosphate-buffered 0.9% (w/v) saline (PBS; pH 7.3), followed by 500 ml of 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The L5 spinal cord segments were harvested and immersed in 30% (w/v) sucrose in 0.1 M PB overnight at 4°C. Transverse spinal sections (25-μm thickness) were then cut on a cryostat (Leica CM1800; Heidelberg, Germany). After been rinsed in PBS (pH 7.2 to 7.4) 3 times (10 min each), the sections were incubated for 2 h at room temperature and then for 48 h at 4°C with anti-glial fibrillary acidic protein (GFAP) mouse IgG (1:5,000; Chemicon, Temecula, CA, USA) or anti-GFAP mouse IgG (1:5,000; Millipore, Billerica, MA, USA) mixed with anti-pJNK rabbit IgG (1:100; Cell Signalling, Danvers, MA, USA) in PBS containing 0.3% (v/v) Triton X-100, 0.25% (w/v) λ-carrageenan and 5% (v/v) donkey serum (PBS-XCD). After being washed three times in 0.01 M PBS (10 min each), the sections were incubated for 4 h at room temperature with Alexa 594-conjugated donkey anti-mouse IgG (1:500; Millipore, Billerica, MA, USA) or a mixture of Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 594-conjugated donkey anti-mouse IgG. After staining, all of the sections were mounted onto glass slides and cover-slipped with 50% (v/v) glycerol and 2.5% (w/v) triethylenediamine (anti-fading agent) in 0.05 M PBS. Using a confocal laser-scanning microscope (FV1000; Olympus, Tokyo, Japan), the sections were observed with the appropriate laser beams and filter settings for green-emitting Alexa 488 (excitation 488 nm; emission 530 nm) or red-emitting Alexa 594 (excitation, 543 nm; emission, 590 to 615 nm). The digital images were captured using FV10-ASW-1.6 software (Olympus, Tokyo, Japan), modified (15% to 20% contrast enhancement) in Photoshop CS4 (Adobe Systems, San Jose, CA, USA) and then saved as TIFF files.

Animals were deeply anesthetised by injection of pentobarbital (60 mg/kg, i.p.) and then rapidly sacrificed. The L5 spinal cord segments were dissected on ice according to the termination of the L4 and L5 dorsal roots. The left dorsal part of spinal cord was further split and then homogenized with a hand-held pestle in sodium dodecyl sulphate (SDS) sample buffer (10 ml/mg tissue) containing a mixture of proteinase and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). The protein concentrations were estimated using the bicinchoninic acid (BCA) method. The samples were heated in boiling water for 8 min, loaded onto 10% SDS-polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to polyvinylidene difluoride membranes (PVDF; Immobilon-P, Millipore, Billerica, MA, USA). Membranes were blocked in a 5% BSA solution for 2 h and probed with the following primary antibodies overnight at 4°C: anti-GFAP mouse IgG (1:5,000; Chemicon, Temecula, CA, USA), anti-pJNK rabbit IgG (1:300; Cell Signalling, Danvers, MA, USA), anti-JNK rabbit IgG (1:300; Cell Signalling, Danvers, MA, USA), and anti-β-actin mouse IgG (1:3,000, Sigma-Aldrich, St. Louis, MO, USA). The membranes were then incubated with the following secondary antibodies for 2 h: horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (1:5000; Zhongshan, Beijing, China) or HRP-conjugated donkey anti-mouse IgG (1:5,000; Zhongshan, Beijing, China). The membranes were rinsed three times (10 min each) with Tris-buffered saline with Tween-20 (TBST) between each step. All reactions were detected by the enhanced chemiluminescence (ECL) detection method (Amersham Corporation, Arlington Heights, IL, USA). Restore Western blot stripping buffer (Pierce Biotechnology, Inc., Rockford, IL, USA) was used to detect the different proteins with close molecular weight in the same PVDF membranes. Briefly, after the antibodies for detecting pJNK were stripped, the same PVDF membrane was used for the detection of β-actin. The detailed steps were carried out according to the manufacturer’s instructions. The densities of protein blots were analyzed using Labworks Software (Ultra-Violet Products, Cambridge, UK). The densities of target proteins and β-actin immunoreactive bands were quantified with background subtraction. The same size of square was drawn around each band to measure the density, and the background near that band was subtracted. Target protein levels were normalized against β-actin levels and expressed as relative fold changes compared to the sham-vehicle group. The intensity of blots was quantified with densitometry by the persons who were blinded to the treatments.

The left dorsal horns of the L5 spinal segments of animals in different groups were split according to the same method used for Western blot analysis. Spinal cord tissues were homogenized in a lysis buffer containing protease and phosphatase inhibitors. The amounts of IL-6, TNF-α, IL-1β, and MCP-1 were measured by enzyme-linked immunosorbent assays using corresponding ELISA kits (R&D Systems, Minneapolis, MN, USA).

The TIIAS dosages were transformed into logarithm dose with Prism, and the non-line fit was performed so as to build the dose-effect curve. Based on the dose-effect curve, the ED₅₀ of the effects of TIIAS on analgesia was calculated. The reliability of the ED₅₀ calculated from a specific dose-effect curve was evaluated using the slope factor generated by the GraphPad Prism version 5.01 for Windows (San Diego, CA, USA, www.​graphpad.​com).