Intervention of electroacupuncture on spinal p38 MAPK/ATF-2/VR-1 pathway in treating inflammatory pain induced by CFA in rats
© Fang et al.; licensee BioMed Central Ltd. 2013
Received: 29 August 2012
Accepted: 18 March 2013
Published: 22 March 2013
Previous studies have demonstrated that p38 MAPK signal transduction pathway plays an important role in the development and maintenance of inflammatory pain. Electroacupuncture (EA) can suppress the inflammatory pain. However, the relationship between EA effect and p38 MAPK signal transduction pathway in inflammatory pain remains poorly understood. It is our hypothesis that p38 MAPK/ATF-2/VR-1 and/or p38 MAPK/ATF-2/COX-2 signal transduction pathway should be activated by inflammatory pain in CFA-injected model. Meanwhile, EA may inhibit the activation of p38 MAPK signal transduction pathway. The present study aims to investigate that anti-inflammatory and analgesic effect of EA and its intervention on the p38 MAPK signal transduction pathway in a rat model of inflammatory pain.
EA had a pronounced anti-inflammatory and analgesic effect on CFA-induced chronic inflammatory pain in rats. EA could quickly raise CFA-rat’s paw withdrawal thresholds (PWTs) and maintain good and long analgesic effect, while it subdued the ankle swelling of CFA rats only at postinjection day 14. EA could down-regulate the protein expressions of p-p38 MAPK and p-ATF-2, reduced the numbers of p-p38 MAPK-IR cells and p-ATF-2-IR cells in spinal dorsal horn in CFA rats, inhibited the expressions of both protein and mRNA of VR-1, but had no effect on the COX-2 mRNA expression.
The present study indicates that inhibiting the activation of spinal p38 MAPK/ATF-2/VR-1 pathway may be one of the main mechanisms via central signal transduction pathway in the process of anti-inflammatory pain by EA in CFA rats.
Inflammatory pain is a very common symptom in clinical practice. Patients mainly suffer from ongoing pain (spontaneous pain), evoked pain, and hyperalgesia. Among several types of chronic pains, chronic inflammatory pain has been recognized to be most common and difficult to treat. Therapy for inflammatory pain has been composed of symptomatic treatment with nonsteroidal anti-inflammatory drugs (NSAIDs), but long-term uses of this agent has shown side effects such as gastrointestinal toxicity and cardiovascular toxicity [1, 2]. Thus, there is a need for anti-inflammatory pain treatment with less side effects.
P38 mitogen-activated protein kinase (MAPK) signal transduction pathway is typically activated by cellular stress and pro-inflammatory cytokines and plays a critical role in inflammatory responses. Systematic or intrathecal administration of p38 MAPK inhibitor has been shown to effectively alleviate inflammation and arthritis [3, 4]. The activated p38 MAPK is translocated to the nucleus, where it can phosphorylate transcriptional factors such as ATF-2 . The synthesis of several pro-inflammatory mediators such as cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), vanilloid receptor-1 (VR-1) in the spinal cord are up-regulated by p38 MAPK [6–8].
Electroacupuncture (EA), a Chinese medical therapy, is a modified acupuncture method which utilizes electrical stimulation and has been used to reveal the analgesic effects on acute pain and chronic inflammatory pain [9–11]. Clinical trials show that EA has beneficial effects in patients with various inflammatory diseases [12–14]. Experimental studies demonstrate that EA significantly inhibits complete Freund’s adjuvant- (CFA) or collagen-induced hind paw inflammation and hyperalgesia in a rat model [15–17]. In relation with EA analgesia and its mechanism in the inflammatory pain, there were lots of studies focusing on the analgesic effect of EA via opioid system and inflammatory mediators in CFA-induced and carrageenan-induced inflammatory pain models [18–21]. However, the underlying mechanisms of EA for inflammatory pain are still not completely understood. We previously reported that EA could attenuate CFA-induced inflammatory pain, at least in part, through suppressing numbers of p-p38 MAPK IR-cells in spinal cord . In this study, we first used a rat CFA model to observe whether p38 MAPK signal transduction pathway in spinal cord plays an important role in inflammatory pain, and then to investigate whether EA may relieve inflammatory pain by counteracting the activation of p38 MAPK signal transduction pathway.
Anti-inflammatory activity of EA
For comparisons among groups at all time points, the results by One-way ANOVA for independent sample identified that the percentage swelling of the paw in three CFA injected groups were all markedly higher than that in control group at corresponding time points (P<0.01). However, CFA+EA group showed a significant lower score compared with the CFA and CFA+Sham EA groups at day 14 and day 21 after CFA injection.
Analgesic activity of EA
Mean paw withdrawal thresholds (PWTs) for all experimental groups were shown in Figure 1B. The repeated-measures ANOVA with between-subjects indicated differences over days (P<0.01) and between groups (P<0.01). There was a significant interactive effect between days and groups (P<0.01). Post-hoc LSD tests indicated no significant difference in PWTs between CFA+Sham EA group and CFA group. However, the CFA+EA group showed an available hypoalgesic effect when compared with CFA group and CFA+Sham EA group (P<0.01). A significant difference PWTs was also found between control group and CFA+EA group (P<0.05).
To compare four groups at all time points, as shown in Figure 1B, no difference in basal PWTs among groups was observed before saline or CFA injection. PWTs in CFA group and CFA+Sham EA group were lower obviously than those in control group at corresponding time points (P<0.01), and reached its lowest at post-injection day 7. After EA stimulation, the rats’ PWTs were increased rapidly and significantly higher than those in CFA group and CFA+Sham EA group at day 3, 7, 14, 21 after CFA injection (P<0.01), and showed no difference with those in control group (P>0.05).
CFA-induced p38 MAPK activation in the spinal cord
CFA-induced ATF-2 activation in the spinal cord
Effect of EA on phosphor-p38 MAPK
Effect of EA on phosphor-ATF-2
Effect of EA on COX-2 mRNA and protein expressions
Effect of EA on VR-1 mRNA and protein expressions
As a complementary and alternative medicine, EA has been accepted worldwidely, mainly for the treatment of acute and chronic pains. It is well know that EA has a good effect of anti-inflammation and analgesia. From this study, we found that the analgesic effect of EA had the property of a rapid onset, long duration and being strengthened along with EA treatment times, which was consistent with our previous report . Interestingly, it was also found that the role of anti-inflammatory and analgesic effect of EA was not synchronistic. After repeated EA administration, quite a strong difference in the PWTs were found on day 3 between CFA+EA group and CFA group, and statistically significant difference was observed from day 3 to day 21. However, the onset of anti-inflammatory effect of EA appeared later than its analgesic effect. It was not until 14 treatments with EA, we firstly observed the difference in the paw edema of rats in CFA+EA group and that of CFA group. It could be predicted that EA got quicker analgesic effect, maybe through mediating some other way, not or not only by intervening on pro-inflammatory mediators in the treatment of inflammatory pain. Although EA analgesia is well documented, its mechanisms have not been thoroughly clarified. The present study demonstrates, for the first time, that EA suppressed the expression of p-p38 MAPK and its downstream p-ATF-2 as well as gene and protein expressions of VR-1 in the spinal dorsal horn, but had no inhibitory effect on gene and protein expressions of COX-2. Therefore, inhibition of the p38 MAPK activation may be the one of central pain sensitization mechanisms of EA analgesia for inflammatory pain.
The MAPK family includes p38 MAPK, extracellular signal regulated kinase (ERK1/2), c-jun N-terminal kinase (JNK), ERK7/8, ERK3/4 and ERK5 . Initiation of the p38 MAPK cascade involves activation through a classic MAPK kinase kinase (MAP3K) – MAP kinase kinase (MKK) pathway. The p38 MAPK pathway is activated by a wide range of environmental or cellular stresses and proinflammatory cytokines and plays important roles in cell death, neurodegeneration, and inflammation . Recently, p38 MAPK activation was shown to contribute to nociceptive responses in the spinal dorsal horn and dosal root ganglion (DRG) following inflammation and/or nerve injury. Following nerve injury, p-p38 MAPK levels sequentially increased in neurons, microglia, and astrocytes of the spinal dorsal horn; In addition, nerve injury-induced p-p38 MAPK occurred early and was long lasting [24–27]. Moreover, intrathecal injection of p38 MAPK inhibitors attenuated nociceptive response in different neuropathic pain animal models [25, 28–31]. Several reports also indicated that a very robust increase of p-p38 MAPK in spinal dorsal horn induced hindpaw inflammation [4, 6, 32–34]. For example, bee-venom induced rapid increase in p-p38 MAPK in the spinal: p-p38 MAPK began to increase at 1 hour, reached a peak on day 3, and then decreased to normal level on day 7 . In particular, different laboratories have shown that p-p38 MAPK was increased in spinal cord by CFA , formalin , capsaicin , or carrageenan . Besides, intrathecal administration of the p38 inhibitor prevented inflammation-induced thermal and mechanical hypersensitivity [4, 32]. In present study, we demontrated that the numbers of p-p38 MAPK-IR cells in spinal dorsal horn increased and reached at peak at day 3 after CFA injection, maintained for 14 days. The level of p-p38 MAPK protein expression in CFA group have returned to the base at day 21 after CFA-injection, however, inflammation and pain were still existed at that moment. The reason for this is unclear and is worthy of further study.
It is indicated that p38 MAPK is involved in the control of VR-1 mediated glioma apoptotic cell death . Another study reports that p38 MAPK activated in the DRG following peripheral inflammation, by increasing VR-1 levels in nociceptive peripheral terminals, contributes to the maintenance of inflammatory pain . P38 MAPK can potentially regulate protein expression in several different ways, activating transcription factors such as eukaryotic translation initiation factor 4E (eIF4E), Ets likely kinase (ELK-1), and cAMP response element binding protein (CREB) to increase transcription [36–38], increasing mRNA stability  or increasing translation. One major target of p38 MAPK is the translation factor ATF-2. ATF-2 (originally called CRE-BP1) is a member of the ATF/CREB family of transcription factors and is characterized by a basic zipper domain that consists of basic amino acids and a leucine zipper region, which acts as a DNA-binding region. The ATF-2 subfamily contains three members: ATF-2, CRE-BPa, and ATF-7 (originally known as ATF-a) . ATF-2 regulates gene expression via the binding of ATF-2, as a homodimer, to the recognition sequences of the cAMP-response element (CRE) or via formation of a heterodimer with activating protein-1 (AP-1) that binds to CRE sequences. Thus, ATF-2 can interact with other members of the ATF family and with members of the AP-1 family . It has been shown that p-p38 MAPK is involved in regulation of ATF-2 , which then binds to DNA and turns on the target gene . In current report, we found that the numbers of p-p38 MAPK-IR cells at the same time coincided with the numbers of p-ATF-2-IR cells in spinal dorsal horn. So we presumed that ATF-2 as the downstream of p38 MAPK may play an important role in inflammatory pain.
It is now realized that a certain members of the transient receptor potential (TRP) receptors are key molecular integrators of the initiation and maintenance of pain. Vanilloid receptor-1 (VR-1), known as the transient receptor potential ion channel TRPV1, is essential for generating and transmitting pain [44, 45]. It has been found to be expressed within those components of the peripheral and central nervous systems involved in pain detection, transmission and regulation . It is all known that VR-1 can be found in a large population of primary sensory neurons, within the spinal cord itself, and within the brain. It has been reported that deletion of VR-1 is important for mediating thermal hyperalgesia and inflammatory swelling after CFA-induced inflammation . This study was supported by other complementary studies which showed that the severity of arthritis in rodent models was reduced when VR-1 was either blocked or deleted . VR-1 also plays an important role in mechanical hyperalgesia of inflammatory pain. When wild-type and VR-1 null mice are injected in one knee joint with CFA, the VR-1 null mice develops mild joint swelling (evidencing edema) and reduced mechanical hypersensitivity as compared with wild-type animals . In present study, periphery inflammation induced a high expression of VR-1 in ipsilateral spinal dorsal horn on 14 day. EA could down-regulate the expressions of both VR-1mRNA and protein in spinal dorsal horn with increased PWTs.
Several factors have verified that p-p38 MAPK could up-regulate many proinflammatory mediators such as COX-2, IL-1β and VR-1 [6–8]. It has been demonstrated that local induction of COX-2 at the site of peripheral and the subsequent release of prostaglandin E2 (PGE2) has major roles in peripheral pain sensitization, which alters the threshold and excitability of the nociceptive peripheral terminal . In addition to this peripheral action, COX-2 and prostaglandin also have a central function. The central PGE2 produced by COX-2 induced an electrophysiological transmitter release, direct depolarization of postsynaptic membranes and inhibiting glycine receptor activation . The upregulation of central COX-2 level, induced by peripheral inflammatory stimuli such as CFA  and carrageenan , was linked to inflammatory allodynia and hyperalgesia. However, COX-2 just plays an important role in the development, but not maintenance of inflammation. Spinal COX-2 mRNA increased to a peak at 4 h after a intraspinal injection with IL-1α  and similarly increased to a peak at 4 h  or 8 h  after mechanical injury to the spinal cord. The expression of COX-2 protein also increased in the spinal cord during hindpaw inflammation at 4–6 h induced by CFA, paralleled the COX-2mRNA, and returned to baseline within 3 d after induction of inflammation . According to another report, the expression of COX-2 mRNA on the ipsilateral side of spinal cord obtained from CFA injection was significantly increased at both 6 hour and 3 day after CFA injection compared with that from the saline-treated mice . In agreement with above studies, we detected that the level of COX-2 mRNA in ipsilateral spinal dorsal horn was increased respectively at 5 hour and 3 day (Additional file 1: Figure S5), and returned to baseline within 14 day after CFA-induction of inflammation. Whereas, EA treatment did not suppress the increase of COX-2 mRNA expression in spinal dorsal horn at 5 hour, 3 day and 14 day.
As we mentioned above, robust p38 MAPK activation was essential for VR-1 and COX-2 expression in SCDH after peripheral inflammation, and it could regulate ATF-2 phosphorylation in SCDH of CFA-induced inflammation. Our previous report has demonstrated that EA analgesia and anti-inflammation were associated with its inhibition of spinal p38 MAPK activation . In agreement with our previous study, pre-eletroacupuncture treatment has prophylactic analgesic effects on rats suffering from visceral pain by suppressing the local and spinal p38 MAPK . The p38 MAPK signal transduction pathway is partly involved in the regulatory mechanism of this analgesic effect. Another study has reported that EA could improve immune suppression involving in the signaling pathway of p38 MAPK . In this study, we first systemically investigated whether EA treatment might regulate the downstream materials of p38 MAPK. We found that EA did suppress the activation of the p38 MAPK and its downstream p-ATF-2 as well as VR-1mRNA and protein, but had no influence on gene and protein expressions of COX-2 at day 14 after CFA injection. Similarly, this study showed significant analgesic effect of EA at day 14 after CFA-injection, as well as a relative weak anti-inflammatory effect. PWTs of CFA+EA group had no difference with that of control group on day 14 in CFA-induced inflammatory pain, but the percent paw swelling of CFA+EA group had remarkable difference with that of control group at the same time. It could be suggested that both COX-2 and VR-1 play essential roles in inflammatory pain, however, EA just regulates the expressions of VR-1 mRNA and protein, and does not have obvious effect on COX-2 mRNA and protein expressions. It is well known that COX-2 mainly regulates the release of inflammatory mediators, leading to the generation of inflammatory pain, and VR-1 enhances the excitability of nerve resulting in pain [59, 60]. Therefore, we speculate that the analgesic effect of EA, different from the mechanism of NASIDs, is not reached by inhibiting the release of inflammatory cytokines but rather by regulating pain sensitization, because we have not observed a suppressing effect of EA on COX-2 mRNA and protein, but an inhibitory effect on p38 MAPK, ATF-2 and VR-1 expressions.
It is the feature of EA treatment that the desynchronized effect of analgesia and anti-inflammation exists when applied to alleviate inflammatory pain. EA can significantly attenuate CFA-induced inflammatory pain, which, at least in part, is contributed to its regulation of p38 MAPK/ATF-2/VR-1 pathway but not of p38 MAPK/ATF-2/COX-2 pathway in the persistent phase of inflammatory pain. In addition, EA could be a good complementary and alternative medicinal therapy in clinic for treatment of chronic inflammatroy pain.
In total, 126 male Sprague–Dawley rats (Department of Animal Sciences China), weighing between 220–240 g, were used for this study. They were housed six per cage on a 12-h light/dark cycle with controlled temperature and free accessed to food and water. Efforts were made to minimize animal discomfort and reduced numbers of animals used. All rats were used strictly in accordance with the National Institutions of Health Guide for the Care and Use of Laboratory Animals.
Model establishment and experimental groups
Inflammatory pain model was established by injecting subcutaneously with 0.1 ml Complete Freund’s Adjuvant (CFA, Sigma, USA) into right hindpaw of rats.
The rats were separated randomly into 4 groups: 1) control group, 0.1 ml saline injection as used for CFA injection; 2) CFA group, 0.1 ml CFA injection, immobilization; 3) CFA+EA group, 0.1 ml CFA injection, immobilization and with EA stimulation; 4) CFA+ Sham EA group, 0.1 ml CFA injection, immobilization and with Sham EA stimulation.
The EA treatment procedure was the same as reported in our previous study . Rats were loosely immobilized by assistants’ hands. Four stainless steel acupuncture needles of 0.25 mm in diameter were inserted a depth of 5 mm into bilateral “Zusanli” (ST36, 5 mm lateral to the anterior tubercule of the tibia) and “Kunlun” (BL60, at the ankle joint level and between the tip of the external malleolus and tendo calcaneus) acupoints. It has been showed that these two acupoints were good at treat inflammatory pain . The two ipsilateral needles were connected with the output terminals of the HANS Acupuncture point Nerve Stimulator (LH-202H, Huawei Co., Ltd., Beijing, China). The EA parameters were set as follows: square wave current output (pulse width: 0.2 ms); intensities ranging from 1–2 mA (each intensity for 15 min, totaling 30 min); at a 100 Hz and 2 Hz alternating frequencies (automatically shifting between 100 Hz and 2 Hz stimulation for three seconds each) . The stimulation was given for 30 min, once per day, and started on day 1 after CFA injection when the assessment of behavioral test has been finished. Sham EA group animals received needle insertion subcutaneously into ST36 and BL60, but without electrical stimulation.
Twelve rats of each group were included for assessment of behavioral test, which consist of paw volume and paw withdrawal thresholds (PWTs). Paw volume and PWTs were measured before CFA/saline injection (as Base) and 1 day after CFA/saline injection (before EA stimulation), 3, 7, 14 and 21 day after CFA/saline injection (after EA stimulation).
Paw volume was measured by a water displacement plethysmometer (7140, UGO Basile, Italy). The right hindpaw was immersed in a chamber containing electrolyte solution up to the boundary between hairy and nonhairy skin, and the volume displacement was determined electronically. The percentage swelling of the paw was calculated by the following formula: % swelling= (Vt-V0)/V0×100, where Vt is the paw volume after CFA/saline injection and V0 is basal paw volume.
Rats were placed on a metal mesh table and adapted to the new environment. The mechanical stimulus was delivered to the plantar surface of right hind paw from below the floor of the test chamber by an automated testing device (dynamic plantar aesthesiometer 37450, UGO Basile, Italy). A steel rod (diameter of 0.5 mm) was pushed against the hind paw with ascending force. The force went from 0 to 50 g over a 20 s period. When the animal withdrew its hind paw, the mechanical stimulus was automatically stopped, and the force at which the animal withdrew its paw was recorded as PWTs. Withdrawal responses were taken from four consecutive trials with at least 3min between trials and averaged.
Perfusion and tissue harvest
Animals were sacrificed after 3, 7, 14 and 21 days post-model respectively. Rats were administered 10% choral hydrate in a volume of 0.35 mL, i.p., per 100 g body weight. Five animals of each group were perfused for immunohistochemical analysis. Once animals were deeply anesthetized, they were quickly perfused with 150 ml cold sterilized saline followed by 500 ml cold, fresh 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). The L4-6 segments of spinal cord were removed and postfixed in the same fixative for 6 h at 4°C before transfer to 15%, 30% sucrose for cytoprotection. Tissue was then embedded in an optimum cutting temperature medium, cut using a cryostat at a thickness of 30 μm and processed as free floating sections. Thirteen animals of each group were perfused with ice cold saline to clear contaminating blood. The same region of spinal cord as that used for western blot and qPCR snap frozen in liquid nitrogen and stored at −80°C.
All of the sections were blocked with 5% goat serum in TBST (0.1%Tween-20) for 1 h at 37°C and incubated over one nights at 4°C with primary antibodies (diluted in 5% BSA/TBST), The following primary antibodies were used: rabbit anti-rat p-p38MAPK (1:200, CST, USA), rabbit anti-rat p-ATF-2 (1:200, CST, USA). The sections were then incubated for 1 h at 37°C with HRP-conjugated-avidin (1:400). Following incubation with HRP-conjugated-avidin (1:400) for 1 h at 37°C, sections were incubated with DAB substrate for 30 s. The stained sections were examined with a Leica microscope, and images were captured with a CCD camera. The number of p-p38 MAPK and p-ATF-2 positive cells was counted automatically five regions in a given area of upper left, lower left, upper right, lower right and middle using Image Pro Plus 6.0 under blinded conditions and averaged. A minimum of five tissue sections/animal with a minimum of three-five animals/group were counted.
Samples were kept at −80°C until manually homogenized with liquid nitrogen and added to RIPA buffer (1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin, 150 mM NaCl,1% Triton X-100,50 mM Tris, pH 7.4) containing protease inhibitors, phosphatase inhibitor. The homogenate was allowed to rest on ice for 30 min and was then centrifuged at 15,000 rcf for 15 min at 4°C, and the supernatant collected. The protein concentration of tissue lysates was determined with a BCA protein Assay Kit, and 30 μg of protein was loaded in each lane. Protein samples were separated on 5%-10% SDS-PAGE gel and electrophoretically transferred to nitrocellulose membranes (Millipore, USA). The membranes were blocked with 5% low-fat milk in TBST for 1 h at room temperature. We used rabbit anti-rat p-p38 MAPK (1:1000, CST, USA), rabbit anti-rat p-ATF-2 (1:1000, CST, USA), rabbit anti-rat COX-2 (1:300, Cayman, USA), rabbit anti-rat VR-1 (1:200, Santa Cruz, USA) as primary antibodies and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG as secondary antibody (1:10000). Rabbit anti-rat β-actin (1:1000, CST, USA) was used as internal control. The membranes were developed with ECL kit (Pierce, USA) and the signals were captured with Image Quant LAS 4000 (GE, USA). Scanned images were analyzed by Image Quant TL7.0 Analysis Software (GE, USA).
After tissue homogenization in ice-cold tubes, total RNA was extracted using Trizol (Invitrogen, France). Reverse transcription was performed using 1 μg total RNA using the PrimeScript® RT reagent Kit With gDNA Eraser (TaKaRa, Japan). Expression of COX-2 and VR-1 genes was analyzed by real-time quantitative polymerase chain reactions (qPCR) using the CFX96™ real-time PCR detection system (Bio-Rad, USA). The glyceraldehyde-3- phosphate dehydrogenase (GAPDH) gene served as an internal control for expression levels of target genes. Primers were synthesized to amplify a gene segment of GAPDH of 288 base pairs (bp): 5′-TGCTGAGTATGTCGTGGAG-3′(sense), 5′-GTCTTCTGAGTGGCAGTGAT-3′(anti-sense). A 161-bp segment of the COX-2 gene was amplified with 5′-CACGGACTTGCTCACTTTGTT-3′ (sense), and 5′-AAGCGTTTGCGGTACTCATT-3′ (anti-sense). A 129-bp segment of the VR-1 gene was amplified with 5′-GAAAGGATGGAACAACGGGCGC-3′ (sense), and 5′-GAAGATGTGGGGCTTGACTGG-3′ (anti-sense). The primers were designed based on sequences from the Genebank database. EvaGreen (Bio-Rad, USA) served as a dye that binds to amplified DNA to emit fluorescence during reactions. EvaGreen recently emerged as an optimal green fluorescent DNA dye for qPCR, it has equal or better sensitivity compared with SYBR Green I. The reaction mixture of 20 μl contained 10 μl SsoFast EvaGreen supermix (Bio-Rad, USA), o.4 μl sense and anti-sense primers (400 nM), 1.5 μl template complementary DNA, 7.7 μl Rnase/Dnase-free water. Reactions (total volume, 20 μl) were incubated at 95°C for 30 m, followed by 40 cycles of 10 s at 95°C and 30 s at 55.9°C (COX-2 mRNA) / 59.0°C (VR-1 mRNA). Water controls were included to ensure specificity. Each sample was measured in triplicate, and data points were examined for integrity by analysis of the amplification plot. Adding the melting curve analysis in the reaction condition, the analytical model: 65°C-95°C, an increase of 0.5°C every 10 s. The comparative cycle threshold Cq method was used for relative quantification of gene expression. The amount of COX-2 mRNA and VR-1 mRNA, normalized to the GAPDH and relative to a calibrator, was given by 2-ΔΔCq, with Cq indicating the cycle number at which the fluorescence signal of the PCR product crosses an arbitrary threshold set with the exponential phase of the PCR.
All data were expressed as means ± standard error mean (SEM). The PWTs and the percentage swelling of paw change data were normally distributed, and were therefore analyzed using repeated-measures ANOVA with between-subjects factors. A one-way ANOVA for independent samples compared differences between groups at each time. Post hoc testing was performed with a Fisher’s PLSD test for differences between groups. The criterion for statistical significance was P<0.05.
This work was supported by the National Natural Science Fund of China (No. 30873305), Research Fund of Zhejiang Provincial First-foremost Key Subject-Acupuncture & Moxibustion (No. 255), Zhejiang Provincial Natural Science Found of China (No. Y2091151).
- Bombardier C, Laine L, Reicin A, Shapiro D, Burgos-Vargas R, Davis B, Day R, Ferraz MB, Hawkey CJ, Hochberg MC, Kvien TK, Schnitzer TJ, VIGOR StudyGroup: Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. VIGOR Study Group. N Engl J Med 2000, 343: 1520–1528. 1522 p following 1528 10.1056/NEJM200011233432103View ArticlePubMedGoogle Scholar
- Zhang B, He XL, Ding Y, Du GH: Gaultherin, a natural salicylate derivative from Gaultheria yunnanensis: towards a better non-steroidal anti-inflammatory drug. Eur J Pharmacol 2006, 530: 166–171. 10.1016/j.ejphar.2005.11.030View ArticlePubMedGoogle Scholar
- Kumar S, Boehm J, Lee JC: p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases. Nat Rev Drug Discov 2003, 2: 717–726. 10.1038/nrd1177View ArticlePubMedGoogle Scholar
- Boyle DL, Jones TL, Hammaker D, Svensson CI, Rosengren S, Albani S, Sorkin L, Firestein GS: Regulation of peripheral inflammation by spinal p38 MAP kinase in rats. PLoS Med 2006, 3: e338. 10.1371/journal.pmed.0030338PubMed CentralView ArticlePubMedGoogle Scholar
- Ji RR, Gereau RW 4th, Malcangio M, Strichartz GR: MAP kinase and pain. Brain Res Rev 2009, 60: 135–148. 10.1016/j.brainresrev.2008.12.011PubMed CentralView ArticlePubMedGoogle Scholar
- Svensson CI, Marsala M, Westerlund A, Calcutt NA, Campana WM, Freshwater JD, Catalano R, Feng Y, Protter AA, Scott B, Yaksh TL: Activation of p38 mitogen-activated protein kinase in spinal microglia is a critical link in inflammation-induced spinal pain processing. J Neurochem 2003, 86: 1534–1544. 10.1046/j.1471-4159.2003.01969.xView ArticlePubMedGoogle Scholar
- Ji RR, Suter MR: p38 MAPK, microglial signaling, and neuropathic pain. Mol Pain 2007, 3: 33. 10.1186/1744-8069-3-33PubMed CentralView ArticlePubMedGoogle Scholar
- Amantini C, Mosca M, Nabissi M, Lucciarini R, Caprodossi S, Arcella A, Giangaspero F, Santoni G: Capsaicin-induced apoptosis of glioma cells is mediated by TRPV1 vanilloid receptor and requires p38 MAPK activation. J Neurochem 2007, 102: 977–990. 10.1111/j.1471-4159.2007.04582.xView ArticlePubMedGoogle Scholar
- Baek YH, Choi DY, Yang HI, Park DS: Analgesic effect of electroacupuncture on inflammatory pain in the rat model of collagen-induced arthritis: mediation by cholinergic and serotonergic receptors. Brain Res 2005, 1057: 181–185. 10.1016/j.brainres.2005.07.014View ArticlePubMedGoogle Scholar
- Kim JH, Min BI, Schmidt D, Lee HJ, Park DS: The difference between electroacupuncture only and electroacupuncture with manipulation on analgesia in rats. Neurosci Lett 2000, 279: 149–152. 10.1016/S0304-3940(99)00994-5View ArticlePubMedGoogle Scholar
- Li A, Wang Y, Xin J, Lao L, Ren K, Berman BM, Zhang RX: Electroacupuncture suppresses hyperalgesia and spinal Fos expression by activating the descending inhibitory system. Brain Res 2007, 1186: 171–179.PubMed CentralView ArticlePubMedGoogle Scholar
- Zijlstra FJ, van den Berg-de LI, Huygen FJ, Klein J: Anti-inflammatory actions of acupuncture. Mediators Inflamm 2003, 12: 59–69. 10.1080/0962935031000114943PubMed CentralView ArticlePubMedGoogle Scholar
- Chen R, Nickel JC: Acupuncture ameliorates symptoms in men with chronic prostatitis/chronic pelvic pain syndrome. Urology 2003, 61: 1156–1159. discussion 1159 10.1016/S0090-4295(03)00141-9View ArticlePubMedGoogle Scholar
- Woźniak PR, Stachowiak GP, Pieta-Dolińska AK, Oszukowski PJ: Anti-phlogistic and immunocompetent effects of acupuncture treatment in women suffering from chronic pelvic inflammatory diseases. Am J Chin Med 2003, 31: 315–320. 10.1142/S0192415X03000990View ArticlePubMedGoogle Scholar
- Lao L, Zhang RX, Zhang G, Wang X, Berman BM, Ren K: A parametric study of electroacupuncture on persistent hyperalgesia and Fos protein expression in rats. Brain Res 2004, 1020: 18–29. 10.1016/j.brainres.2004.01.092View ArticlePubMedGoogle Scholar
- Zhang RX, Lao L, Wang X, Fan A, Wang L, Ren K, Berman BM: Electroacupuncture attenuates inflammation in a rat model. J Altern Complement Med 2005, 11: 135–142. 10.1089/acm.2005.11.135View ArticlePubMedGoogle Scholar
- Park DS, Seo BK, Baek YH: Analgesic effect of electroacupuncture on inflammatory pain in collagen-induced arthritis rats: mediation by alpha2- and beta-adrenoceptors. Rheumatol Int 2013, 33: 309–314. 10.1007/s00296-012-2369-5View ArticlePubMedGoogle Scholar
- Sekido R, Ishimaru K, Sakita M: Differences of electroacupuncture-induced analgesic effect in normal and inflammatory conditions in rats. Am J Chin Med 2003, 31: 955–965. 10.1142/S0192415X03001491View ArticlePubMedGoogle Scholar
- Huang C, Hu ZP, Long H, Shi YS, Han JS, Wan Y: Attenuation of mechanical but not thermal hyperalgesia by electroacupuncture with the involvement of opioids in rat model of chronic inflammatory pain. Brain Res Bull 2004, 63: 99–103. 10.1016/j.brainresbull.2004.01.006View ArticlePubMedGoogle Scholar
- Lee JH, Jang KJ, Lee YT, Choi YH, Choi BT: Electroacupuncture inhibits inflammatory edema and hyperalgesia through regulation of cyclooxygenase synthesis in both peripheral and central nociceptive sites. Am J Chin Med 2006, 34: 981–988. 10.1142/S0192415X06004454View ArticlePubMedGoogle Scholar
- Su TF, Zhao YQ, Zhang LH, Peng M, Wu CH, Pei L, Tian B, Zhang J, Shi J, Pan HL, Li M: Electroacupuncture reduces the expression of proinflammatory cytokines in inflamed skin tissues through activation of cannabinoid CB2 receptors. Eur J Pain 2012, 16: 624–635. 10.1002/j.1532-2149.2011.00055.xView ArticlePubMedGoogle Scholar
- Liang Y, Fang JQ, Du JY, Fang JF: Effect of Electroacupuncture on Activation of p38MAPK in Spinal Dorsal Horn in Rats with Complete Freund’s Adjuvant-Induced Inflammatory Pain. Evid Based Complementary Alternat Med 2012, 568273.Google Scholar
- Krishna M, Narang H: The complexity of mitogen-activated protein kinases (MAPKs) made simple. Cell Mol Life Sci 2008, 65: 3525–3544. 10.1007/s00018-008-8170-7View ArticlePubMedGoogle Scholar
- Clark AK, Yip PK, Grist J, Gentry C, Staniland AA, Marchand F, Dehvari M, Wotherspoon G, Winter J, Ullah J, Bevan S, Malcangio M: Inhibition of spinal microglial cathepsin S for the reversal of neuropathic pain. Proc Natl Acad Sci USA 2007, 104: 10655–10660. 10.1073/pnas.0610811104PubMed CentralView ArticlePubMedGoogle Scholar
- Milligan ED, Twining C, Chacur M, Biedenkapp J, O’Connor K, Poole S, Tracey K, Martin D, Maier SF, Watkins LR: Spinal glia and proinflammatory cytokines mediate mirror-image neuropathic pain in rats. J Neurosci 2003, 23: 1026–1040.PubMedGoogle Scholar
- Katsura H, Obata K, Mizushima T, Sakurai J, Kobayashi K, Yamanaka H, Dai Y, Fukuoka T, Sakagami M, Noguchi K: Activation of Src-family kinases in spinal microglia contributes to mechanical hypersensitivity after nerve injury. J Neurosci 2006, 26: 8680–8690. 10.1523/JNEUROSCI.1771-06.2006View ArticlePubMedGoogle Scholar
- Wen YR, Suter MR, Kawasaki Y, Huang J, Pertin M, Kohno T, Berde CB, Decosterd I, Ji RR: Nerve conduction blockade in the sciatic nerve prevents but does not reverse the activation of p38 mitogen-activated protein kinase in spinal microglia in the rat spared nerve injury model. Anesthesiology 2007, 107: 312–321. 10.1097/01.anes.0000270759.11086.e7View ArticlePubMedGoogle Scholar
- Jin SX, Zhuang ZY, Woolf CJ, Ji RR: p38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. J Neurosci 2003, 23: 4017–4022.PubMedGoogle Scholar
- Schäfers M, Svensson CI, Sommer C, Sorkin LS: Tumor necrosis factor-α induces mechanical allodynia after spinal nerve ligation by activation of p38 MAPK in primary sensory neurons. J Neurosci 2003, 23: 2517–2521.PubMedGoogle Scholar
- Tsuda M, Mizokoshi A, Shigemoto-Mogami Y, Koizumi S, Inoue K: Activation of p38 mitogen-activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury. Glia 2004, 45: 89–95. 10.1002/glia.10308View ArticlePubMedGoogle Scholar
- Hains BC, Waxman SG: Activated microglia contribute to the maintenance of chronic pain after spinal cord injury. J Neurosci 2006, 26: 4308–4317. 10.1523/JNEUROSCI.0003-06.2006View ArticlePubMedGoogle Scholar
- Cui XY, Dai Y, Wang SL, Yamanaka H, Kobayashi K, Obata K, Chen J, Noguchi K: Differential activation of p38 and extracellular signal-regulated kinase in spinal cord in a model of bee venom-induced inflammation and hyperalgesia. Mol Pain 2008, 4: 17. 10.1186/1744-8069-4-17PubMed CentralView ArticlePubMedGoogle Scholar
- Mizushima T, Obata K, Yamanaka H, Dai Y, Fukuoka T, Tokunaga A, Mashimo T, Noguchi K: Activation of p38 MAPK in primary afferent neurons by noxious stimulation and its involvement in the development of thermal hyperalgesia. Pain 2005, 113: 51–60. 10.1016/j.pain.2004.09.038View ArticlePubMedGoogle Scholar
- Fitzsimmons BL, Zattoni M, Svensson CI, Steinauer J, Hua XY, Yaksh TL: Role of spinal p38alpha and beta MAPK in inflammatory hyperalgesia and spinal COX-2 expression. Neuroreport 2010, 21: 313–317. 10.1097/WNR.0b013e32833774bfPubMed CentralView ArticlePubMedGoogle Scholar
- Ji RR, Samad TA, Jin SX, Schmoll R, Woolf CJ: p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron 2002, 36: 57–68. 10.1016/S0896-6273(02)00908-XView ArticlePubMedGoogle Scholar
- Xing J, Kornhauser JM, Xia Z, Thiele EA, Greenberg ME: Nerve growth factor activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphorylation. Mol Cell Biol 1998, 18: 1946–1955.PubMed CentralPubMedGoogle Scholar
- Gingras AC, Raught B, Sonenberg N: eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 1999, 68: 913–963. 10.1146/annurev.biochem.68.1.913View ArticlePubMedGoogle Scholar
- Ji RR, Woolf CJ: Neuronal plasticity and signal transduction in nociceptive neurons: implications for the initiation and maintenance of pathological pain. Neurobiol Dis 2001, 8: 1–10. 10.1006/nbdi.2000.0360View ArticlePubMedGoogle Scholar
- Faour WH, He Y, He QW, de Ladurantaye M, Quintero M, Mancini A, Di Battista JA: Prostaglandin E(2) regulates the level and stability of cyclooxygenase-2 mRNA through activation of p38 mitogen-activated protein kinase in interleukin-1 beta-treated human synovial fibroblasts. J Biol Chem 2001, 276: 31720–31731. 10.1074/jbc.M104036200View ArticlePubMedGoogle Scholar
- Vlahopoulos SA, Logotheti S, Mikas D, Giarika A, Gorgoulis V, Zoumpourlis V: The role of ATF‐2 in oncogenesis. Bioessays 2008, 30: 314–327. 10.1002/bies.20734View ArticlePubMedGoogle Scholar
- Eferl R, Wagner EF: AP-1: a double-edged sword in tumorigenesis. Nat Rev Cancer 2003, 3: 859–868.View ArticlePubMedGoogle Scholar
- Maekawa T, Jin W, Ishii S: The role of ATF-2 family transcription factors in adipocyte differentiation: antiobesity effects of p38 inhibitors. Mol Cell Biol 2010, 30: 613–625. 10.1128/MCB.00685-09PubMed CentralView ArticlePubMedGoogle Scholar
- Johnson GL, Lapadat R: Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002, 298: 1911–1912. 10.1126/science.1072682View ArticlePubMedGoogle Scholar
- Davis JB, Gray J, Gunthorpe MJ, Hatcher JP, Davey PT, Overend P, Harries MH, Latcham J, Clapham C, Atkinson K, Hughes SA, Rance K, Grau E, Harper AJ, Pugh PL, Rogers DC, Bingham S, Randall A, Sheardown SA: Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 2000, 405: 183–187. 10.1038/35012076View ArticlePubMedGoogle Scholar
- Vogel G: Hot pepper receptor could help manage pain. Science 2000, 288: 241–242.View ArticlePubMedGoogle Scholar
- Palazzo E, Luongo L, de Novellis V, Rossi F, Marabese I, Maione S: Transient receptor potential vanilloid type 1 and pain development. Curr Opin Pharmacol 2012, 12: 9–17. 10.1016/j.coph.2011.10.022View ArticlePubMedGoogle Scholar
- Keeble J, Russell F, Curtis B, Starr A, Pinter E, Brain SD: Involvement of transient receptor potential vanilloid 1 in the vascular and hyperalgesic components of joint inflammation. Arthritis Rheum 2005, 52: 3248–3256. 10.1002/art.21297View ArticlePubMedGoogle Scholar
- Brain SD: TRPV1 and TRPA1 channels in inflammatory pain: elucidating mechanisms. Ann N Y Acad Sci 2011, 1245: 36–37. 10.1111/j.1749-6632.2011.06326.xView ArticlePubMedGoogle Scholar
- Barton NJ, McQueen DS, Thomson D, Gauldie SD, Wilson AW, Salter DM, Chessell IP: Attenuation of experimental arthritis in TRPV1R knockout mice. Exp Mol Pathol 2006, 81: 166–170. 10.1016/j.yexmp.2006.04.007View ArticlePubMedGoogle Scholar
- Broom DC, Samad TA, Kohno T, Tegeder I, Geisslinger G, Woolf CJ: Cyclooxygenase 2 expression in the spared nerve injury model of neuropathic pain. Neuroscience 2004, 124: 891–900. 10.1016/j.neuroscience.2004.01.003View ArticlePubMedGoogle Scholar
- Hay CH, Trevethick MA, Wheeldon A, Bowers JS, de Belleroche JS: The potential role of spinal cord cyclooxygenase-2 in the development of Freund’s complete adjuvant-induced changes in hyperalgesia and allodynia. Neuroscience 1997, 78: 843–850. 10.1016/S0306-4522(96)00598-2View ArticlePubMedGoogle Scholar
- Ichitani Y, Shi T, Haeggstrom JZ, Samuelsson B, Hőkfelt T: Increased levels of cyclooxygenase-2 mRNA in the rat spinal cord after peripheral inflammation: an in situ hybridization study. Neuroreport 1997, 8: 2949–2952. 10.1097/00001756-199709080-00028View ArticlePubMedGoogle Scholar
- Tonai T, Taketani Y, Ueda N, Nishisho T, Ohmoto Y, Sakata Y, Muraguchi M, Wada K, Yamamoto S: Possible Involvement of Interleukin‐1 in Cyclooxygenase‐2Induction After Spinal Cord Injury in Rats. J Neurochem 1999, 72: 302–309.View ArticlePubMedGoogle Scholar
- Resnick DK, Graham SH, Dixon CE, Marion DW: Role of cyclooxygenase 2 in acute spinal cord injury. J Neurotrauma 1998, 15: 1005–1013. 10.1089/neu.1998.15.1005View ArticlePubMedGoogle Scholar
- Beiche F, Scheuerer S, Brune K, Geisslinger G, Goppeil-Struebe M: Up-regulation of cyclooxygenase-2 mRNA in the rat spinal cord following peripheral inflammation. FEBS Lett 1996, 390: 165–169. 10.1016/0014-5793(96)00604-7View ArticlePubMedGoogle Scholar
- Narita M, Shimamura M, Imai S, Kubota C, Yajima Y, Takagi T, Shiokawa M, Inoue T, Suzuki M, Suzuki T: Role of interleukin-1β and tumor necrosis factor-α-dependent expression of cyclooxygenase-2 mRNA in thermal hyperalgesia induced by chronic inflammation in mice. Neuroscience 2008, 152: 477–486. 10.1016/j.neuroscience.2007.10.039View ArticlePubMedGoogle Scholar
- Xu KD, Liang T, Wang K, Tian DA: Effect of pre-electroacupuncture on p38 and c-Fos expression in the spinal dorsal horn of rats suffering from visceral pain. Chin Med J (Engl) 2010, 123: 1176–1181.Google Scholar
- Wang K, Wu H, Wang G, Li M, Zhang Z, Gu G: The effects of electroacupuncture on TH1/TH2 cytokine mRNA expression and mitogen-activated protein kinase signaling pathways in the splenic T cells of traumatized rats. Anesth Analg 2009, 109: 1666–1673. 10.1213/ANE.0b013e3181b5a234View ArticlePubMedGoogle Scholar
- Prochazkova M, Dolezal T, Sliva J, Krsiak M: Different Patterns of Spinal Cyclooxygenase-1 and Cyclooxygenase-2 mRNA Expression in Inflammatory and Postoperative Pain. Basic Clin Pharmacol Toxicol 2006, 99: 173–179. 10.1111/j.1742-7843.2006.pto_457.xView ArticlePubMedGoogle Scholar
- Palazzo E, Luongo L, de Novellis V, Berrino L, Rossi F, Maione S: Moving towards supraspinal TRPV1 receptors for chronic pain relief. Mol Pain 2010, 6: 66. 10.1186/1744-8069-6-66PubMed CentralView ArticlePubMedGoogle Scholar
- Li WM, Cui KM, Li N, Gu QB, Schwarz W, Ding GH, Wu GC: Analgesic effect of electroacupuncture on complete Freund’s adjuvant-induced inflammatory pain in mice: a model of antipain treatment by acupuncture in mice. Jpn J Physiol 2005, 55: 339–344. 10.2170/jjphysiol.RP001505View ArticlePubMedGoogle Scholar
- Zhang YQ, Ji GC, Wu GC, Zhao ZQ: Excitatory amino acid receptor antagonists and electroacupuncture synergetically inhibit carrageenan-induced behavioral hyperalgesia and spinal fos expression in rats. Pain 2002, 99: 525–535. 10.1016/S0304-3959(02)00268-3View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.