Osthole alleviates neuropathic pain in mice by inhibiting the P2Y₁-receptor-dependent JNK signaling pathway

Osthole inhibits mechanical and thermal hyperalgesia in neuralgia mice

A CCI-induced pain model was used to explore the effect of osthole or morphine on neurodynia and behavior was assessed on POD 0, 1, 3, 5, 7, 10, 14 and 21. Behavioral research revealed that CCI induced a seriously pain hypersensitivity with a characteristic of mechanical allodynia and heat hyperalgesia compared to sham group, maintaining for over 3 weeks (Figs. 1A, B). In sham mice, the ipsilateral hind paws showed no difference in PWT and PWL at all the time point. In CCI mice, PWT was significantly decreased on POD 3, maintained on POD 7 and decreased from 0.90 ± 0.12g on POD 0 to 0.09 ± 0.07g on POD 21(Figure 1A). PWL of CCI mice changed in a similar way and decreased from 12.50 ± 1.21s on POD 0 to 5.13 ± 0.92s on POD 21(Figure 1B). We detected the influence of osthole and morphine on CCI-induced pain. Osthole (5, 10 or 20 mg/kg) severely reduced mechanical allodynia and heat hyperalgesia continued for at least 3 hours with the best repression 1-hour post osthole treatment compared to CCI group. The pain threshold values of osthole treatment and morphine treatment groups were raised day by day, reaching the level of the sham group on POD 10. Although high dose osthole obviously cut down mechanical allodynia and heat hyperalgesia in CCI-mice, it did not change the baseline thresholds in sham group, indicating osthole has an important role for neuropathic pain conditions. Moreover, high dose osthole showed an equal pain threshold compared with that of the morphine treatment group after POD 10.

Figure 1. Osthole attenuates CCI-induced mechanical and thermal hyperalgesia. (A, B) Paw withdrawal threshold and Paw withdrawal latency were both significantly decreased on POD 3, maintained on POD 7. The pain threshold values of osthole-treated and morphine-treated groups were increased day by day, approaching the value of the sham group on POD 10. ###p<0.01, compared with sham group; **p<0.05, ***p<0.01, compared with CCI group, ANOVA followed by Bonferroni post-hoc test, n = 6 mice/group. All data are means ± SD.

CCI induced astrocytic P2Y₁R up-regulation and JNK activation in the spinal cord

Astrocyte activation is a common outcome of neuropathic pain. P2Y receptor family up-regulated and MAPKs activation in astrocytes are involved in neuropathic pain [27]. As shown in Figure 2A, CCI injury induced GFAP, astrocytic marker increased in spinal cord on POD 14. Meanwhile, P2Y₁R up-regulation and JNK phosphorylation occurred accompanied with astrocyte activation. Double staining showed that P2Y₁R and p-JNK in astrocytes promote the development of neuropathic pain. The western blotting results further reflect that GFAP, P2Y₁R and p-JNK protein expression were increased in CCI group compared to sham group (Figure 2B), indicating that a P2Y₁R-dependent JNK pathway was drawn into CCI-induced neuralgia.

Figure 2. CCI induced astrocytic P2Y₁R up-regulation and JNK activation in the spinal cord. (A) Immunostaining showing that CCI injury induced astrocytic marker GFAP expression in the ipsilateral spinal cord on POD 14. JNK phosphorylation and P2Y₁R up-regulation occurred accompanied with astrocyte activation. Scale bar=200μm. (B) Western blot showing that GFAP, p-JNK and P2Y₁R were increased in CCI group compared with sham group. All data are means ± S.D. ###p<0.01, compared with sham group; n=3 mice/group. The differences between groups were analyzed by ANOVA followed by Bonferroni post-hoc test.

Osthole cut down P2Y₁R up-regulation and p-JNK activation on a concentration-dependent manner

We harvested the spinal cords of mice treated with osthole (5, 10 or 20 mg/kg) on POD 14. Immunostaining showed that P2Y₁R immunoreactivity was decreased by osthole. In particular, P2Y₁R immunoreactivity treated with 20 mg/kg osthole was similar to those observed for sham group (Figure 3A). Western blot analysis showed that osthole decreased P2Y₁R of CCI mice on a concentration dependent manner (Figure 3B). Osthole treatment also attenuated P2Y₁R mRNA compared to CCI group (Figure 3C). Meanwhile, Immunostaining showed that osthole could decrease the immune response of p-JNK. In particular, p-JNK immunoreactivity treated with 20 mg/kg osthole was nearby to that seen in sham group (Figure 3D). Western blot analysis showed that osthole decreased p-JNK of CCI mice on a concentration dependent manner (Figure 3E). Furthermore, our results showed that osthole treatment attenuated TNF-α, IL-1β, and IL-6 expression on a dose dependent manner (Figure 3F) in the spinal cord. The results indicated that P2Y₁R and p-JNK participated in CCI-induced pain, osthole attenuated P2Y₁R up-regulation and p-JNK activation on a concentration-dependent manner.

Figure 3. Osthole cut down P2Y₁R up-regulation and p-JNK activation on a concentration-dependent manner. (A–C) Osthole decreased P2Y₁R of CCI mice on a concentration dependent manner. ###p<0.01, compared with sham group; **p<0.05, ***p<0.01, compared with CCI group, ANOVA followed by Bonferroni post-hoc test, n=6 mice/group. (D, E) Osthole reduced p-JNK expression after CCI in the spinal cord on POD 14. ###p<0.01, compared with sham group; **p<0.05, ***p<0.01, compared with CCI group, ANOVA followed by Bonferroni post-hoc test, n=6 mice/group. (F) Osthole reduced inflammatory factors after CCI in the spinal cord on POD 14. ###p<0.01, compared with sham group; ***p<0.01, compared with CCI group, ANOVA followed by Bonferroni post-hoc test, n=6 mice/group. All data are means ± S.D.

Osthole inhibited neuronal excitability and excitatory synaptic transmission

Signals deliver of nervous system is inseparable with synaptic transmission. Neuronal excitability represents the severity of neuralgia. In order to explore the effect of osthole on neuronal excitability, mEPSPs of lamina I-II neurons in CCI mice were detected with or without osthole perfusion. As shown in Figure 4A, mEPSPs frequency was depressed by 100 μmol/L osthole, but mEPSPs amplitude did not show significant changes. Furthermore, we checked mEPSPs of lamina I-II neurons of normal mice with or without osthole treatment. Figure 4B showed that osthole did not alter mEPSPs frequency and amplitude of normal mice. These findings suggest that osthole effectively inhibit excitatory synaptic transmission. We also tested the actions of osthole on excitatory synaptic inputs to neurons in lamina I-II from C fibers. As shown in Figure 5, a reproducible monosynaptic eEPSPs in the lamina I-II neurons was evoked. Osthole bath-applied for 10 minutes at a dose of 100 μmol/L. The amplitude of C fiber-evoked monosynaptic eEPSPs were significantly decreased by osthole compared with CCI group.

Figure 4. Osthole induced synaptic inhibitory effects. (A) mEPSPs frequency was depressed by 100 μmol/L osthole, but mEPSPs amplitude did not show significant changes. (B) Osthole did not alter mEPSPs frequency and amplitude of normal mice. pCLAMP 10 software (Axon Instruments) were used to acquire and analyze the potential data. **p<0.05, compared with CCI group, ANOVA followed by Bonferroni post-hoc test, n = 6 mice/group. All data are means ± SD.

Figure 5. Osthole inhibits excitatory synaptic transmission. Traces of eEPSPs in lamina 1-II neurons in the presence of osthole. The histogram indicates the monosynaptic eEPSP peak amplitude compared with control. ***p<0.01, compared with CCI group, ANOVA followed by Bonferroni post-hoc test, n = 6 mice/group. All data are means ± SD.

Osthole inhibited neuronal cell signaling molecules in a dose dependent manner

When inflammation cytokines and chemokines bind to their receptor, what happens in neuron as follows? Glutamate in presynaptic element liberation increased, causing glutamic acid receptor in postsynaptic element activated, such as AMPA and NMDA. Excitatory receptors in neurons leads to increased calcium influx [28], initiating a series of downstream signal pathways, including p-ERK, p-CREB and c-Fos, which are closely related with pain sensitivity [3]. Postsynaptic receptor was also detected to further prove the influence of osthole on neuronal excitability. The results showed that pGluA1 (ser845) and pGluN2B (S1303) were decreased after osthole treatment compared to CCI mice (Figure 6A). At the same time, pain-related signal molecules in neurons of CCI mice were checked. We found that CCI induced p-ERK, p-CREB and c-Fos increased compared to sham group, while osthole dose-dependently inhibited expression of p-ERK, p-CREB and c-Fos protein compared to CCI group (Figure 6B). In summary, osthole induced synaptic inhibitory effects and reduced neuronal excitability.

Figure 6. The effect of osthole on Postsynaptic receptor pain-related signal molecules in neurons of CCI mice. (A) pGluA1 (ser845) and pGluN2B (S1303) were decreased after osthole treatment compared to CCI mice. (B) Osthole dose-dependently inhibited expression of p-ERK, p-CREB and c-Fos protein compared to CCI group. ###p<0.01, compared with sham group; **p<0.05, ***p<0.01, compared with CCI group, ANOVA followed by Bonferroni post-hoc test, n = 6 mice/group. All data are means ± SD.

P2Y₁ receptor-dependent JNK signaling pathway in spinal astrocyte was involved in pain relief after osthole treatment

Research shows that when neuropathic pain occurred, c-Fos expression increased. c-Fos can be used as a marker of neuropathic pain. A dose of 4 μg P2Y₁R inhibitor MRS2179, 5 μg p-JNK inhibitor SP600125 or 10mg/kg osthole were used to explore molecular mechanism of osthole in pain relief and clarify the relationship between P2Y₁R, osthole and c-Fos. Behavior was assessed four hours after MRS2179, SP600125 or osthole was injected on POD 14. As shown in Figure 7A, single injection MRS2179, SP600125 and osthole all seriously changed the CCI-induced mechanical allodynia and heat hyperalgesia. The pain threshold values of MRS2179, SP600125 and osthole treated group were increased compared to CCI group. We harvested the spinal cords of mice treated with MRS2179, SP600125 or osthole on POD 14. Western blot analysis demonstrated that MRS2179 and osthole significantly depressed P2Y₁R, p-JNK and c-Fos expression compared to CCI group. Interestingly, SP600125 significantly depressed p-JNK and c-Fos expression, while has no effect on P2Y₁R (Figure 7B). These results show that JNK signaling pathway adjusted by P2Y₁R, osthole exerts analgesic effect through P2Y₁R.

Figure 7. P2Y₁ receptor-dependent JNK signaling pathway in spinal astrocyte was involved in pain relief after osthole treatment. (A) Single injection MRS2179, SP600125 and osthole all seriously changed the CCI-induced mechanical allodynia and heat hyperalgesia. (B) The effect of MRS2179, SP600125 and osthole on P2Y₁R, p-JNK and c-Fos expression compared to CCI group. ###p<0.01, compared with sham group; ***p<0.01, compared with CCI group, ANOVA followed by Bonferroni post-hoc test, n=6 mice/group. All data are means ± S.D.

Osthole exerts analgesic effect by directly acting on P2Y₁ receptor of astrocyte

To prove the effect of osthole on P2Y₁R, primary astrocytes transfect with a pcDNA3.1 vector contained P2Y₁R sequence. Our results showed that P2Y₁R sequence was successfully transfected into astrocyte, P2Y₁R expression in transfected cells markedly increase compared to normal astrocyte. Meanwhile, osthole obviously decreased P2Y₁R protein and gene levels (Figure 8A). We also detected c-Fos expression in primary neurons dealt with astrocyte-conditioned medium. Our research showed that astrocyte-conditioned medium induced the change trend of c-Fos consistent with that of P2Y₁R. In astrocyte-conditioned medium pretreated with P2Y₁R (+), expression of c-Fos significant increase and attenuated in osthole group. Meanwhile, c-Fos expression decreased further in osthole (+) group (Figure 8B). Data above indicated that the effect of osthole on P2Y₁R in astrocyte leads to decrease of c-Fos expression in neurons and produces analgesic effect. Perhaps, osthole has some direct effect on neurons.

Figure 8. Osthole exerts analgesic effect by directly acting on P2Y₁ receptor of astrocyte. (A) P2Y₁R expression in transfected cells markedly increase compared to normal astrocyte. (B) Astrocyte-conditioned medium induced the change trend of c-Fos consistent with that of P2Y₁R. ###p<0.01, compared with sham group; ***p<0.01, compared with P2Y₁R overexpression group, ANOVA followed by Bonferroni post-hoc test, n=6 mice/group. All data are means ± S.D.

Drug and reagents

Osthole (>98%, CAS: 484-12-8) was obtained from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Mouse Anti-GFAP (GA5), Rabbit Anti-SAPK/JNK, Rabbit Anti-phospho-ERK (1/2) (Thr202/Tyr204), Rabbit Anti-ERK (1/2), Rabbit Anti-phospho-CREB, Rabbit Anti-CREB and Rabbit Anti-c-Fos (9F6) were obtained from Cell Signaling Technology (Danvers, United States). Rabbit Anti-phospho-JNK (Thr183/Tyr185), Mouse Anti-phospho-Glutamate Receptor 1(AMPA subtype) (S845), Rabbit Anti-Glutamate Receptor 1(AMPA subtype), Rabbit Anti- phospho-NMDAR2B (s1303), Rabbit Anti-NMDAR2B and Rabbit Anti-GAPDH were obtained from Abcam technology (Cambridge, United Kingdom). Rabbit Anti-P2Y₁R for immunofluorescence was obtained from Yansheng technology (Shanghai, China). Goat Anti-Rabbit IgG and Goat Anti-Mouse IgG for western blots and Immunofluorescence were all purchased from Cell Signaling Technology (Danvers, United States). TNF-α, IL-1β and IL-6 ELISA kits were provided by Abcam technology (Cambridge, The United Kingdom). DAPI dihydrochioride was purchased from Diyi technology (Shanghai, China). MRS2179 and SP600125 were purchased from Abcam technology (Cambridge, The United Kingdom). DMEM medium is got from HyClone (Logan, USA). Lipofectamine 2000 was purchased from Thermo Fisher Scientific (Waltham, USA). Trizol reagent and SYBR Premix Ex TaqTM II kit were purchased from TaKaRa (Shiga, Japan). All other reagents are analytical reagents and are available on the market.

Animals and experimental design

C57BL/6 mice, 18~22 g, were obtained from the Experimental Animal Center of Fourth Military Medical University. Under a 12/12 h light/dark cycle, mice were placed in controlled indoor humidity (45~75%) and temperature (22±2°C) and provided water and food at will. Mice were acclimatized for 4 days to the laboratory conditions and for 3 days to manipulations and devices prior to behavioral studies [42]. This experiment was carried out with consent of Chinese Food and Drug Administration (cFDA). Best effort was contributed to minimize the number of animals used and their suffering.

To investigate the analgesic effect of osthole in neuropathic pain, seven groups of animals were used: sham group, treatment of sham with osthole (20 mg/kg), CCI model treated with osthole (0, 5, 10, or 20 mg/kg) and morphine (3 mg/kg) groups. The sham group and CCI group received a same volume of vehicle. Conduct a behavioral experiment on post-operative day (POD) 0, 1, 3, 5, 7, 10, 14 and 21 every day after surgery. Osthole was administered intraperitoneally after surgery from POD 0 to POD 14. On POD 14, mice were killed for experiment. MRS2179 and SP600125 were injected to mice intrathecal pathway and transported to the cerebral spinal fluid in a 30G needle with a total volume of 20 μl between the L5 and L6 intervertebral. To investigate the mechanism of osthole in cultured cells, the treatment of osthole (100 μmol) was started 24 h. MRS2179 and SP600125 were started 1 h before cells collected.

CCI induction in mice

The well-established L5-L6 CCI model of neuropathy was used in this study. Briefly, mice were anesthetized with 1% pentobarbital (10 mL/kg) intraperitoneally (i.p.), and the left common sciatic nerve of each mouse was exposed at the mid-thigh level. Close to the end of the sciatic trigeminal nerve, the nerve was separated from adhered tissue, and 4 ligatures (5-0 chronic gut) are loosely tied around it, about 1 mm between ligatures. After operation, the skin layers and muscle were sutured and iodine was used to disinfect the operation area [43].

Behavioral assessments of mechanical allodynia and heat hyperalgesia in mice

All manipulations were performed under quiet conditions by the same experimenter in a test room to avoid stress. Use the calibrated von Frey filaments (Stoelting, Kiel, WI, USA) to assess claw contraction for mechanical stimulation. Animals were placed on an elevated mesh grid and habituated to the testing environment for at least 0.5 h before examination. A series of von Frey filament with logarithmical incremental stiffness (0.016~1g, Woodland Hills, Los Angeles) were used to stimulated the plantar surface of each hind in an order of increasing stiffness for 5 s. A positive response was indicated by rapid pulling back, biting, or shaking of the hind limb within 5 s of the application of von Frey filament. The interval between different filaments was at least 5 minutes. A single filament stimulated the same hind limb for 10 times. The minimum value that resulted in at least six responses to ten stimulations was recorded as the paw withdrawal threshold [44].

Paw withdrawal in response to noxious thermal stimuli was assessed using a Full-Automatic Plantar Analgesia Tester (BME-410C, Institute of biomedical engineering, CAMS) that produces radiant heat by directing a beam of light to the plantar surface of hind paw. The mice were placed in plastic boxes on a glass plate for at least 0.5 h. Six trials on the ipsilateral paw were performed with an interval of at least 5 min. To prevent tissue damage, radiant heat was administered for a maximum of 30 s.

Spinal cord slices preparation and patch-clamp recording

Mice were anesthetized with 1% pentobarbital (10 mL/kg) intraperitoneally (i.p.) and perfused through the aorta with sucrose- substituted artificial cerebral spinal fluid (mmol/L: sucrose, 75.00; NaCl, 80; KCl, 2.5; CaCl₂, 2.5; MgCl₂, 1.2; NaH₂PO₄, 1.25; NaHCO₃, 25; ascorbate, 1.3; and pyruvate, 3) aerated with 95% O₂ and 5% CO₂ for 30 minutes and stored at 4°C). The lumbar segment was dissected and fixed in sucrose ACSF quickly. The parasagittal spinal slice (450-500 μm thick) with an attached dorsal root (0.8-1.0 cm long) was cut with a vibrating microtome and was incubated with Glucose ACSF (mmol/L: NaCl, 125; KCl, 2.5; CaCl₂, 2; MgCl₂, 1; NaH₂PO₄, 1.25; NaHCO₃, 26; d-glucose, 25; ascorbate, 1.3; and pyruvate, 3;) aerated with 95% O₂ and 5% CO₂) at room temperature (22°C-25°C, 3.5 mL/min) [45]. The procedures for patch-clamp recordings was as follows [46]. A pipette electrode (5-10MΩ), with Axon 200B enlarger was used to record mEPSPs of C fibers in lamina I-II neurons of spinal cord. In current-clamped mode, the potential was maintained at -50 mV to show mixed excitation and inhibitory synaptic events. For eEPSPs, the average amplitude was obtained before 5 minutes, during 10 minutes and after 20 minutes osthole application. In an intro hyper-excitability model (mmol/L: bicuculline, 10; strychnine, 2; Tetrodotoxin, 5), mEPSPs were recorded before 5 minutes, during 10 minutes and after 20 minutes osthole application.

Primary cell culture

Astrocytes cultures were prepared from cerebral cortex of neonatal C57BL/6 mice. Neonatal mice were decapitated and meninges were carefully removed from isolated cerebral hemispheres in cold PBS. The cerebral hemisphere is separated into a separate cell suspension and collected by dissociation for 5 minutes at 400 g. The dissociated cells were suspended in the DMEM supplemented with 10% (v/v) FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml). After trituration, the cell suspension was plated into 6-well plates at a density of 3×10⁵ cells/cm², and cultured for 10-12 days [47]. The medium was changed twice a week. The fairly pure astrocytes (>90%) were prepared by shaking the flasks 180 rpm overnight and then culturing them with 10 ml of 0.05% trypsin in a cell incubator for 15 min to separate microglial cells and oligodendrocytes. Astrocytes were divided into 4 groups: Sham (Normal cultured cells), P2Y₁R (-) (cells transfected with an empty pcDNA3.1 vector), P2Y₁R (+) (cells transfected with a pcDNA3.1 vector contained P2Y₁R sequence) and osthole (cells transfected with a pcDNA3.1 vector contained P2Y₁R sequence and treated with 100μmol/L osthole for 24 h). Lipofectamine 2000 was used to transfect astrocytes. For neuron culture, the cell suspension was plated into a plate pre-coated with cell adherent reagent. After incubation in DMEM containing 10% FBS for 6 h, the medium was changed to B27 supplement and 0.5 mmol/L glutamine. The experiment started 5 to 6 days after electroplating. Harvested neurons showed dendritic spine morphology and exhibited about 90% purity. Astrocyte-conditioned medium pre-treated with P2Y₁R (-), P2Y₁R (+), osthole and osthole (+) (cells treated with 100μmol/L osthole directly) were used to substitute for half of the neuronal medium.

Immunofluorescence

Mice were deeply anesthetized with 1% pentobarbital (10 mL/kg) intraperitoneally (i.p.), and perfused through the aorta with 20 mL of saline, then there is 50mL 4% paraformaldehyde. The spinal cord segment (L4-L5) was removed and frozen in 30% sucrose solution after being fixed overnight in 4 % formaldehyde. Spinal cord was cut into 20 μm-thick serial sections using a cryostat (Leica CM1950) and mounted on 3-aminopropyl-triethoxysilane-coated glass slides. Spinal cord sections were stored at −20 °C. Immunofluorescence was performed as follows. The sections were air-dried and circled with an Immunity Staining Guard Pen. Firstly, the slices were incubated with 3% bovine serum albumin containing 0.4% Triton X-100 acted at room temperature for 1 h. For dual immunofluorescence, the mixture of polyclonal and monoclonal primary antibodies were incubated overnight at 4 °C as follows: Phospho-JNK antibody (1:100, rabbit), P2Y₁R antibody (1:500, rabbit), and GFAP antibody (1:500, mouse). The sections were washed in PBS and then incubated in the dark for 2 hours at room temperature. The secondary antibodies (1:1000) coupled with FITC were used to incubate sections at room temperature for 2 hours. Sections were washed 3 times for 5 min with PBS and incubated using a PBS-based mounting medium containing 10 μg/mL DAPI for 15 min. Sections were washed 3 times for 5 min with PBS and cover slipped using mounting liquid. Stained sections were visualized, and photographs were obtained under a fluorescent microscope (Leica Microsystems GmbH, Mannheim, Germany) [48].

Western blot

Mice were anesthetized with 1% pentobarbital (10 mL/kg) intraperitoneally (i.p.) and the spinal cord segment of L4-L5 was removed. For cultured astrocytes and neurons, after incubation with osthole and other regents, cells were collected. The tissue or cell was homogenized in a radio immunoprecipitation assay lytic buffer containing 0.1 mol/L phenylmethyl sufonyl fluoride on ice for 30 min. Protein concentrations of each samples were determinate by bicinchoninic acid Protein Assay. Equal content of proteins (30 μg) was mixed with a one-fifth volume of 5× loading buffer and heated to 100°C for 5 min. Protein samples and rainbow-colored protein molecular markers were separated by 10% SDS- polyacrylamide gel electrophoresis gel and transferred to polyvinylidene fluoride membranes. In a Tris-buffered saline containing 0.5% Tween-20 solution, these blots were first sealed for 30 min and then incubated overnight at 4°C with antibody against GFAP (1:1000, mouse), P2Y₁R (1:1000, rabbit), p-JNK (1:1000, rabbit), JNK (1:1000, rabbit), pGluA1 (1:1000, rabbit), GluA1 (1:1000, rabbit), p-GluN2B (1:1000, rabbit), GluN2B (1:1000, rabbit), p-ERK (1:1000, rabbit), ERK (1:1000, rabbit), p-CREB (1:1000, rabbit), CREB (1:1000, rabbit), and c-Fos (1:1000, rabbit). In order to control the load, the blots were probed with GAPDH antibody (1:1000, mouse). The Membrane is rinsed with TBST for 30 minutes, then cultured with horseradish peroxidase-conjugated secondary antibody for 2 h, and developed in electrochemiluminescence solution [49]. The intensity of protein bands was quantified using densitometry in Image ProPlus 6.0 software.

Enzyme-linked immunosorbent assay

Mice were decapitated under anesthesia with an over-dose of 1% pentobarbital sodium. The plasma collected before animals sacrificed, homogenized L4-L5 spinal cord tissue, and the supernatant of astrocytes were all used to determine TNF-α, IL-1β, and IL-6 levels by corresponding ELISA kits. Absorbance was read at 450 nm. For each reaction in the 96-well plate, a 100 ml of sample was used and ELISA was performed in a three-link manner according to the manufacturer's instructions. The experiment includes a standard curve.

Real-time quantitative PCR

Mice were deeply anaesthetized with pentobarbital sodium. Trizol reagent was used for the isolation of total RNA from L4-L5 spinal segments. RNA quantity and purity were determined using an ultra micro-volume spectrophotometer (NanoVue, General Electric Company, USA). cDNA is amplified by the following primers: P2Y₁R forward, 5’-GAC TTC TTG TAC GTG CTG ACT CT-3’, P2Y₁R reverse, 5’-GAC CTC TTG TCA CCT GAT ACG TG-3’; product size: 647 bp. All quantitative PCR reactions were performed in a Real-time Detection System (Applied Biosystem, USA) by QuantiTect Rev. Transcription Kit. PCR was amplified at 95°C for 30s, then 45 cycles at 95°C for 5s, 56°C for 30s, and 72°C for 30 s. Step-one Analysis Software is used to analysis data. The melting curves is obtained after the cycle is completed to ensure that there are no non-specific products.

Statistical analysis

The data obtained were recorded as means ± S.D. Adult C57BL/6 mice (weighing 18~22 g) were randomly separated for behavioral studies (n=6) or biochemical studies (n=6), and n refers to the number of animals per group. One-way AVOVA were used to test the changes of values for the verification of significance. The differences between groups were analyzed by one-way variance (ANOVA) followed by a Bonferroni post-hoc test. Data were analyzed by SPSS (IBM SPSS Statistics v19.0), all statistical figures were created using GraphPad Prism 6.0.