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Parameter-specific analgesic effects of electroacupuncture mediated by degree of regulation TRPV1 and P2X3 in inflammatory pain in rats
Life Sciences 200 (2018) 69–80
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Parameter-specific analgesic effects of electroacupuncture mediated by degree of regulation TRPV1 and P2X3 in inflammatory pain in rats
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J.Q. Fang , J.Y. Du, J.F. Fang, T. Xiao, X.Q. Le, N.F. Pan, J. Yu, B.Y. Liu Department of Neurobiology and Acupuncture Research, The Third Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou 310053, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Electroacupuncture Inflammatory pain Frequency-specific P2X3 TRPV1
Aims: Observing the parameter-specific anti-hyperalgesic effects of EA with different stimulation times and frequencies on painful hyperalgesia mediated by the level of TRPV1 and P2X3 expression in DRG after CFA injection. Main methods: The model was induced by the injection of CFA in each rat's right hind paw. EA treatment was applied to the bilateral ST36 and BL60. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were tested with Von Frey filaments and the radiant heat source of the test instrument, respectively. TRPV1 and P2X3 expressions were measured by immunofluorescence and western blot. αβ-meATP and capsaicine combined with EA were further utilized to investigate the change in PWL. Key findings: Different stimulation times (20, 30, 45 min) combined with different frequencies (2 Hz, 100 Hz, 2/ 100 Hz) of EA have analgesic effects on the PWT and PWL; however, the level of the hypoalgesic efficacy of EA was primarily associated with EA frequency. The analgesic effect of EA was better at 100 Hz than at 2 Hz. The level of regulation of 100 Hz EA on TRPV1 and P2X3 in DRG was greater than that of 2 Hz. Furthermore, both TRPV1 agonist and P2X3 agonist may impair the level of EA analgesia. Significance: EA has a parameter-specific effect on chronic inflammatory pain relief, which primarily depend on the stimulation frequency and not on the stimulation time at a certain stimulation time. The parameter-specific analgesic effect of EA is at least partially related to mediation of the protein level of TRPV1 and P2X3 expression in DRG of CFA rats.
1. Introduction Pain that is induced by inflammation remains a serious health problem with significant social and economic consequences [1]. The existing mature treatment method may achieve a certain therapeutic effect but may easy cause adverse side effects. Electroacupuncture (EA), with few side effects displayed potent anti-hyperalgesic effects in several experimental models of chronic pain and in many diseases associated with pain [2]. It is well known that different stimulation times and different frequencies of EA have different analgesic effects [3]; however, the mechanism responsible for those differences remains unclear. Inflammation is primary characteristic of mechanical allodynia and thermal hyperalgesia in addition to activating both mechanical and thermal transducers such as purinergic receptor P2X ligand-gated ion channel 3 (P2X3) and transient receptor potential vanilloid 1 (TRPV1) [4,5]. Accumulated studies have clarified that P2X3 and TRPV1 are associated with pivotal the emergence and development of pain [6,7].
Our previous results and the results of other researchers have indicated that EA may inhibit the up-regulated expression of TRPV1 in DRG and spinal cord in inflammatory pain, neuropathic pain such as chronic constriction injury (CCI) and spinal nerve ligation (SNL), and cancer pain [8–10]. Numerous papers have suggested that suppressing the expression of P2X3 via EA stimulation may reduce many types of experimental pain, such as visceral pain, and neuropathic pain [11,12]. EA, which applies an adequate and continuous electrical current stimulation via needles to acupoints, was recently developed and has become a good complementary and alternative therapy [7]. According to well-established theories, different frequencies of EA have different therapeutic effects on pain relief [13]. Claydon et al. [14] reported that transcutaneous electrical nerve stimulation (TENS), which was similar to EA, had a dose-specific effect; the level of hypoalgesic efficacy of TENS was clearly dependent on the combination of TENS parameters selected (intensity, frequency, and stimulation site) and the experimental pain model. Although, it was also reported that the therapeutic effect of EA had parameter-specific effects on pain relief [15,16], there
⁎ Corresponding author at: Department of Neurobiology and Acupuncture Research, The Third Clinical Medical College, Zhejiang Chinese Medical University, 548 Binwen Road, Binjiang District, Hangzhou, Zhejiang Province 310053, China. E-mail address: [email protected] (J.Q. Fang).
https://doi.org/10.1016/j.lfs.2018.03.028 Received 26 September 2017; Received in revised form 11 December 2017; Accepted 7 March 2018 Available online 14 March 2018 0024-3205/ © 2018 Published by Elsevier Inc.
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2.5. Paw withdrawal latency (PWL)
was few paper discussion the mechanisms underlying the parameterspecific effect of EA. The purpose of this paper is to investigate the parameter-specific anti-hyperalgesic effects of EA by utilizing different stimulation times and frequencies on hyperalgesia mediated by the level of TRPV1 and P2X3 expression in DRG after CFA injection.
PWL was performed according to the methods previously reported [19]. The rats were acclimated to the test environment in individual testing cages for 30 min each day for three consecutive days. On the test day, the rats were allowed a minimum of 20 min to acclimate. After the acclimation period, the radiant heat source of the test instrument (37370, UGO, Italy) was positioned on the central surface of the hind paw, and the elapsed time between the heat application and the withdrawal response was registered as the paw-withdrawal latency (PWL). At each time point, the PWL was repeated three times at 2 min intervals, and the mean of these values was used for further calculations. The radiant heat was set at 35 °C, and the cutoff time was 20 s.
2. Materials and methods 2.1. Animals Two hundred and ten male Sprague–Dawley rats (160–180 g) were purchased through the Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Animal Certificate No. SCXK (沪) 2013-0016) and were fed in the laboratory animal center of Zhejiang (SYXK (浙) 2013–0184). The rats were given minimum of 1 week to adapt to their new environment. The rats were housed six per cage in a temperature-, humidity- and light-controlled environment (25 ± 2 °C, 55% ± 5%, 12-h light/dark cycle). The rats were fed standard rodent food and allowed distilled water ad libitum. All experimental procedures were approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (ZSLL-2015-022).
2.6. EA stimulation The EA stimulation procedure was as reported in our previous study [20]. After the behavioral testing on day 1, the rats were gently immobilized using a special cotton retainer designed by our laboratory (Patent No. ZL 2014 2 0473579.9, State Intellectual Property Office of the People's Republic of China). Four stainless steel acupuncture needles (0.25 mm × 13 mm) were inserted to a depth of 5 mm into the bilateral Zusanli (ST36, 5 mm lateral to the anterior tubercle of the tibia) and Kunlun (BL60, at the ankle joint level and between the tip of the external malleolus and tendo calcaneus) acupoints. The needles were then stimulated by HANS Acupuncture Point Nerve Stimulator (HANS-200A Huawei Co., Ltd., Beijing, China) with intensities ranging from 0.5–1.0–1.5 mA, once per day. EA was performed at different frequencies (2 Hz, 100 Hz, and 2/100 Hz, respectively) and stimulation times (20 min, 30 min, and 45 min, respectively) to compare analgesic effects. The parameters of EA were described below. The 2 Hz EA is a bidirectional rectangular wave with pulse width 0.2 ms; The 100 Hz EA is a bidirectional rectangular wave with pulse width 0.6 ms. The 2/ 100 Hz EA is a combination of 2 Hz EA and 100 Hz EA. When 2/100 Hz EA was administered, 2 Hz and 100 Hz was alternatively administered each 3 s. The frequency and stimulation time that showed the greatest or weakest analgesic effects were selected when conducting experiments on the analgesic mechanism of EA. The CFA + sham EA group animals received needle insertion subcutaneously into ST36 and BL60 (1 mm indepth); the needles were connected to the electrodes, however, there was no electrical stimulation. After finishing the EA or sham EA stimulation, we measure the PWT or PWL immediately.
2.2. Experimental design Four sets of experiments were conducted for this paper. In Experiment 1, the animals were randomly divided into five groups (n = 18): (1) control group, (2) CFA only group, (3) CFA + 2 Hz EA group, (4) CFA + 100 Hz EA group and (5) CFA + 2/100 Hz EA group. Each group was divided into three sub-group according to intervention time (20, 30, and 45 min). In Experiment 2, the animals were randomly divided into five groups (n = 12): (1) control group, (2) CFA only group, (3) CFA + 2 Hz group, (4) CFA + 100 Hz group, and (5) CFA + sham EA group. In Experiment 3, the animals were randomly divided into five groups (n = 6): (1) CFA + vehicle group, (2) CFA + 2 Hz + vehicle group, (3) CFA + 2 Hz + αβ-meATP group, (4) CFA + 100 Hz + vehicle group, and (5) CFA + 100 Hz + αβ-meATP group. In Experiment 4, the animals were randomly divided into five groups (n = 6): (1) CFA + vehicle group, (2) CFA + 2 Hz + vehicle group, (3) CFA + 2 Hz + capsaicine group, (4) CFA + 100 Hz + vehicle group, and (5) CFA + 100 Hz + capsaicine group. 2.3. Model establishment With the exception of the rats in the control group, all of the rats were administered 0.1 mL Freund's Complete Adjuvant (CFA, Sigma USA) by intraplantar injection into the right hind paw to establish the inflammatory pain model [17].
2.7. Drug intervention The chemicals used in this paper were α,β-methyleneadenosine 5′triphosphate lithium salt (αβ-meATP, P2X3 agonist) and capsaicine (TRPV1 agonist), both from Sigma-Aldrich (St. Louis, MO). αβ-meATP was dissolved in 0.9%NaCl. Capsaicine was dissolved in 100% DMSO and further diluted in saline containing 2% Tween 80. The final concentration of dimethylsulfoxide (DMSO) and Tween 80 was 2%. αβmeATP (600 nmol, 25 μL) and capsaicine (100 μg, 25 μL) were subcutaneously injected into the dorsum of the foot one time before EA stimulation on Day 14 after the model.
2.4. Paw withdrawal threshold (PWT) The rats were acclimated to the test environment in individual testing cages for 30 min each day for three consecutive days. On the test day, rats were allowed a minimum of 20 min to acclimate. The PWT was measured as the hind paw withdrawal response to von Frey hair monofilaments (Stoelting, IL, USA). The stimulus was applied in a consecutive ascending order (0.4, 0.6, 1, 2, 4, 6, 8, 15, 26 g) perpendicular to the central surface of the hind paw to determine the withdrawal threshold. The first filament applied corresponded to a force of 2 g. If a negative response (no movement) was observed, expressed as O, a filament exerting greater force was then applied; and if a positive response (paw withdrawal) was observed, expressed as X, a filament of lesser force was next applied. Counting of the critical 6 data points did not begin until the response threshold was first crossed, at which time the 2 responses straddling the threshold were retrospectively designated the first 2 responses in the series of 6. The PWT was calculated according to the response pattern observed described by Chaplan et al. [18].
2.8. Immunofluorescence Immunofluorescence analysis was performed according to the methods previously reported [21]. After the behavioral testing on day 14, rats were anesthetized using 10% choral hydrate at 0.35 mL/0.1 kg body weight. Once the rats were deeply anesthetized, the animals were quickly perfused with 150 mL 0.9% NaCl (4 °C) followed by 400 mL fresh 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS).
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3. Results
The L4–6 segments of the dorsal root ganglion (DRG) were removed and postfixed in the same fixative for 3 h at 4 °C before transfer to 15%, 30% sucrose for dehydration. Tissue was then embedded in an optimal cutting temperature compound (SAKURA, USA) and cut using a cryostat at a thickness of 16 μm to make the slide. All of the slides were blocked with 5% normal donkey serum in TBST (0.1%Tween-20) for 1 h at 37 °C. The slides were then incubated with rabbit anti-TRPV1 (1:1000 in 5% normal donkey serum, abcam, USA) and rabbit anti-P2X3 (1:1000 in 5% normal donkey serum, abcam, USA) for one night at 4 °C. The slides were then incubated in fluorescein (FITC) AffiniPure donkey anti-rat IgG (H + L) (Jackson, USA) for 1 h at 37 °C. Images were taken using an A1R confocal microscope (Nikon, Tokyo, Japan). We used Image-Pro Plus 6.0 to calculate positive cells. Specific methods are as follows: image format was converted RGB to Gray Scale 8. Using the negative image as the floor to determine the threshold value. The positive cell should meet the following conditions: the gray value was > 63, area was > 100, Area/Box ratio was 0.5–2. The specific area of each positive cell was obtained. Five random sections determined the average for each rat, and 3–5 rats included in each group.
3.1. Effects of EA at different frequencies and different stimulation times on PWT in CFA rats After CFA was injected, the PWT of rats decreased significantly on Day 1 and Day 3 (P = 0.005, P = 0.000, Fig. 1). EA may increase the PWT; however, different frequencies and different stimulation times of EA have different effects on the PWT. With EA stimulation for 20 min each time (Fig. 1A), only 100 Hz EA had a beneficial analgesic effect on the PWT after one or three stimulations (P = 0.021, P = 0.011). The PWT of other frequencies of EA was not different from the model group (P > 0.05). With EA stimulation for 30 min each time (Fig. 1B), 100 Hz and 2/100 Hz may significantly enhance the reduced PWT induced by CFA injection after one stimulation (P = 0.001, P = 0.019). All frequencies of EA may increase the PWT after three stimulations (P = 0.047, P = 0.000, P = 0.013). The analgesic effect of 100 Hz was significantly better than the effect of 2 Hz EA after three stimulations (P = 0.031). With EA stimulation for 45 min each time (Fig. 1C), all frequencies of EA may enhance the PWT after one or three stimulations (P = 0.016, P = 0.000, P = 0.003, P = 0.001, P = 0.000, P = 0.000). The analgesic effect of 100 Hz was significantly better than the effect of 2 Hz EA after one stimulation (P = 0.041).
2.9. Western blot Western blot analysis was performed according to the methods previously reported [20]. Animals were sacrificed after the behavioral testing on day 14. Rats were anesthetized 10% choral hydrate at 0.35 mL/100 g body weight. Once deeply anesthetized, the rats were quickly perfused with 150 mL cold sterilized saline. The L4–6 segments of the DRG were removed and stored at −80 °C. Tissues were homogenized in strong RIPA buffer (50 mM Tris[pH 7.4], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, sodium orthovanadate, 0.1% SDS, EDTA, sodium fluoride, leupeptin, and 1 nM PMSF). The homogenate was allowed to rest on ice for 30 min and then centrifuged at 15,000 rpm for 15 min at 4 °C, at this point, the supernatant was collected. The protein concentration of tissue lysates was determined with a BCA protein assay kit, and 20 μg of protein were loaded in each lane. Protein samples were separated on 5–10% SDS-PAGE gels and electrophoretically transferred to polyvinyl difluoride (PVDF) membranes (Bio-rad, USA). The membranes were blocked with 5% low-fat milk in TBST for 1 h at room temperature. We used anti-TRPV1 (1:1000 in 5% normal donkey serum, abcam, USA) and rabbit anti-P2X3 (1:1000 in 5% normal donkey serum, abcam, USA) as primary antibodies and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG as the secondary antibody (1:10,000). Rabbit anti-GAPDH (HRP Conjugate) (1:1000, CST, USA) was used as the internal control. The membranes were developed with an ECL kit (Pierce, USA), and the signals were captured with an Image Quant LAS 4000 (EG, USA). The density of each band was measured using Image Quant TL 7.0 analysis software (GE, USA). The mean expression level of the target protein in the animals in the control group was considered to be 1, and the relative expression level of the target protein in all animals was adjusted as a ratio to the level of the control group.
3.2. Effects of EA using different frequencies and different stimulation times on PWL in CFA rats After CFA was injected, the PWL of rats was significantly decreased on Day 1 and Day 3 (P = 0.000, P = 0.000, Fig. 2). Like the effect of EA on the PWT, EA may also increase the PWL; however, different frequencies and different stimulation times of EA have different effects on the PWL. With EA stimulation for 20 min each time (Fig. 2A), all frequencies of EA have a beneficial analgesic effect on PWL after stimulation one time or three times (P = 0.004, P = 0.000, P = 0.000, P = 0.000, P = 0.000, P = 0.005). With EA stimulation for 30 min each time (Fig. 2B), only 100 Hz EA significantly enhanced the reduced PWL induced by CFA injection after one stimulation. 100 Hz EA and 2/ 100 Hz EA significantly the PWL after three stimulations (P = 0.010, P = 0.039). The analgesic effect of 100 Hz was significantly better than the effect of 2 Hz EA after one stimulation and three stimulations (P = 0.006, P = 0.018). With EA stimulation for 45 min each time (Fig. 2C), only 100 Hz and 2/100 Hz EA significantly enhanced the reduced PWL induced by CFA injection after one stimulation (P = 0.000, P = 0.003). The analgesic effect of 100 Hz was significantly better than the effect of 2 Hz EA after one stimulation (P = 0.018). All frequencies of EA may enhance the PWL after three stimulations (P = 0.041, P = 0.009, P = 0.012). 3.3. Effects of EA on the PWT and PWL in CFA rats Figs. 1 and 2 indicated that different frequencies and different stimulation times of EA had different effects on the PWT and PWL. Whether stimulation lasted 20 min, 30 min or 45 min, 100 Hz EA had the greatest analgesic effect on the PWT and PWL. The weakest analgesic effect on the PWT and PWL was 2 Hz. Therefore, we chose 2 Hz and 100 Hz EA for 30 min stimulation, the common clinical stimulation time, for further research. The degree of the analgesic effect of EA depends on the stimulation frequency (Fig. 3). As shown in Fig. 3A, both 2 Hz and 100 Hz EA significantly increased the PWT after Day 1, 3, 7 and 14 post CFA injection (P = 0.031, P = 0.000, P = 0.047, P = 0.000, P = 0.024, P = 0.000, P = 0.037, P = 0.000), and maintained higher thresholds than the PWT in the sham EA; however, only the PWT at 100 Hz showed a significant increase compared with the PWT in the sham EA group (P = 0.002, P = 0.009, P = 0.003, P = 0.004). The PWT of the rats in the 100 Hz group was significantly higher compared with 2 Hz group on Day 3 post
2.10. Statistical analyses All data were expressed as the means ± standard error (SEM). The PWT and PWL 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 among groups at each time point. For immunofluorescence and western blot analysis, comparisons between groups were performed using oneway analysis of variance followed by Fisher's Protected Least Significant Difference posthoc tests. P < 0.05 was considered statistically significant.
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Fig. 1. Analgesic effects of EA with different frequencies and different times on the PWT of CFA rats. A. Analgesic effect of 20 min EA with 2 Hz, 100 Hz and 2/100 Hz stimulation on PWT. B. Analgesic effect of 30 min EA with 2 Hz, 100 Hz and 2/100 Hz stimulation on PWT. C. Analgesic effect of 45 min EA with 2 Hz, 100 Hz and 2/100 Hz stimulation on PWT. Data are presented as the mean ± SEM, n = 6. *P < 0.05, **P < 0.01, compared with control group; △P < 0.05, △△P < 0.01, compared with CFA only group; ○P < 0.05, ○○P < 0.01, compared with CFA + 2 Hz group.
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Fig. 2. Analgesic effects of EA with different frequencies and different times on PWL of CFA rats. A. Analgesic effect of 20 min EA with 2 Hz, 100 Hz and 2/100 Hz stimulation on PWL. B. Analgesic effect of 30 min EA with 2 Hz, 100 Hz and 2/100 Hz stimulation on PWL. C. Analgesic effect of 45 min EA with 2 Hz, 100 Hz and 2/100 Hz stimulation on PWL. Data are presented as the mean ± SEM, n = 6. *P < 0.05, **P < 0.01, compared with control group; △P < 0.05, △△P < 0.01, compared with CFA only group; ○P < 0.05, ○○P < 0.01, compared with CFA + 2 Hz group.
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Fig. 3. Analgesic effects of EA stimulation on PWT and PWL of CFA rats for 14 days. A. Analgesic effect of EA with 2 Hz and 100 Hz stimulation on PWT. B. Analgesic effect of EA with 2 Hz and 100 Hz stimulation on PWL. Data are presented as the mean ± SEM, n = 6. **P < 0.01, compared with control group; △P < 0.05, △△P < 0.01, compared with CFA only group; ○P < 0.05, ○○P < 0.01, compared with CFA + 2 Hz group. ●P < 0.05, ●●P < 0.01, compared with CFA + 100 Hz group.
2 Hz group were significantly decreased (P = 0.019). However, TRPV1 positive neurons in L4, L5 DRG in the 100 Hz group were markedly decreased compared with the model group (P = 0.007, P = 0.021). Sham EA had no effect on inhibiting the TRPV1 positive neurons in L4–6 DRG. We also observed the size-distribution of TRPV1 in L4–6 DRG of all the group. As shown in Fig. 4C, TRPV1 is mainly expression in the small diameter DRG neurons (areas less of 200 pixel). Furthermore, EA did not regulate the distribution of TRPV1 expression. We also used western blot to measure the TRPV1 protein expression in L4–6 DGR. As shown in Fig. 5, compared with the control group, TRPV1 protein expression in L4, L6 DRG in the model group increased significantly (P = 0.025, P = 0.007); TRPV1 protein expression in L5 DRG in the model group was not different from the control group. Compared with the model group, TRPV1 protein expression in L4, L6 DRG in the 2 Hz group was significantly decreased (P = 0.049, P = 0.046). However, TRPV1 protein expression in L4, L5, L6 DRG in the 100 Hz group was markedly decreased compared with the model
CFA injection (P = 0.047), and at other time points, the PWT of rats in the 100 Hz group trended toward an increase in the PWT of rats in the 2 Hz group. As shown in Fig. 3B, 100 Hz of EA significantly increased PWL after Day 1, 3, 7, and 14 post CFA injection (P = 0.000, P = 0.025, P = 0.003, P = 0.000); the PWT of rats in the 2 Hz EA group was significantly higher than the PWT of the model group only on Day 14 after CFA injection (P = 0.012). Sham EA had no analgesic effect on PWL at any time point (P > 0.05). 3.4. Effects of EA on the expression of TRPV1 in L4–6 DRG The TRPV1 receptor is primarily restricted to the small- and medium-sized DRG neurons. As shown in Fig. 4, compared with the control group, TRPV1 positive neurons in L4 DRG in the model group were significantly increased (P = 0.018); TRPV1 positive neurons in L5, L6 DRG increased gradually but without a statistical difference. Compared with the model group, TRPV1 positive neurons in L4 DRG in the
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Fig. 4. Effect of EA through 2 Hz, 100 Hz on TRPV1 positive neurons measured by immunofluorescence. A. Immunofluorescence confocal micrographs of L4–6 DRG in control group, CFA only group, CFA + 2 Hz group, CFA + 100 Hz group, and CFA + sham EA group. Sections show immunohistochemical green labeling for TRPV1 positive neurons. Scale bars = 100 μm. B. Quantification analysis of TRPV1 positive neurons in L4–6 DRG in control group, CFA only group, CFA + 2 Hz group, CFA + 100 Hz group, and CFA + sham EA group. C. Size distribution of TRPV1 positive neurons in L4–6 DRG in different groups. Data are presented as the mean ± SEM, n = 3–5. *P < 0.05, compared with control group; △P < 0.05, △△P < 0.01, compared with CFA only group; ○P < 0.05, compared with CFA + 2 Hz group. ●P < 0.05, compared with CFA + 100 Hz group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
We also used western blot to measure the P2X3 protein expression in L4–6DGR. As shown in Fig. 7, compared with the control group, P2X3 protein expression in L6 DRG in the model group was significantly increased (P = 0.020); P2X3 protein expression in L4, L5 DRG in the model group was not different from the control group. Compared with the model group, P2X3 protein expression in L6 DRG in the 2 Hz and 100 Hz group was significantly decreased (P = 0.042, P = 0.010). P2X3 protein expression in L4–6 DRG in the 100 Hz group was reduced compared with the 2 Hz group; however, there were not significant differences. Sham EA had no effect on inhibiting the P2X3 protein expression in L4–6 DRG.
group (P = 0.009, P = 0.028, P = 0.029). Sham EA had no effect on inhibiting the TRPV1 protein expression in L4–6 DRG. 3.5. Effects of EA on the expression of P2X3 in L4–6 DRG As shown in Fig. 6, compared with the control group, P2X3 positive neurons in L4, L5, L6 DRG in the model group increased significantly (P = 0.023, P = 0.029, P = 0.001). Compared with the model group, P2X3 positive neurons in L6 DRG in the 2 Hz group were significantly decreased (P = 0.004). However, P2X3 positive neurons in L4, L5, L6 DRG in the 100 Hz group were markedly decreased compared with the model group (P = 0.041, P = 0.013, P = 0.001). Sham EA had no effect on inhibiting the P2X3 positive neurons in L4–6 DRG. We also observed the size-distribution of P2X3 in L4–6 DRG of all the group. As shown in Fig. 6C, P2X3 is mainly expression in the small and media diameter DRG neurons (areas less of 300 pixel). EA did not regulate the distribution of expression P2X3.
3.6. TRPV1 or P2X3 agonist attenuated EA analgesia We further used specific agonist combined with EA to measure the change in pain behavior. As shown in Fig. 8A, compared with the CFA + vehicle group, PWL of CFA + 2 Hz + vehicle group,
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Fig. 5. Effect of EA with 2 Hz and 100 Hz on TRPV1 protein measured by western blot. A. Western blot bands of TPRV1 protein in L4–6 DRG. B. Optical band density analysis. Data are presented as the mean ± SEM, n = 6–7. *P < 0.05, **P < 0.01, compared with control group; △P < 0.05, △△P < 0.01, compared with CFA only group.
45 min) combined with different frequencies (2 Hz, 100 Hz, 2/100 Hz) EA had different analgesic effects on the PWT and PWL of inflammatory pain; however, the level of hypoanalgesic efficacy of EA was primarily associated with EA frequency. The analgesic effect of EA at 100 Hz was better than that at 2 Hz. The results indicate that 2 Hz EA may significantly reduce the positive neuron of TRPV1 in L4 DRG and decrease protein expression of TRPV1 in L4, L6 DRG. However, 100 Hz EA resulted in a higher level of regulation of TRPV1 in DRG than 2 Hz EA, which may significantly reduce the positive neuron of TRPV1 in L4, L5 DRG and down-regulate the protein expression of TRPV1 in L4–6 DRG. With regard to P2X3, 100 Hz could markedly reduce the positive neuron of P2X3 in L4–6 DRG and decrease the protein expression of P2X3 in L6 DRG; however, 2 Hz may only reduce positive neuron and the protein expression of P2X3 in L6 DRG. Furthermore, we used a TRPV1 agonist and a P2X3 agonist in combination with EA to investigate the anti-hyperanalgesic effect of EA. It was observed that both the TRPV1 agonist and the P2X3 agonist may impair the level of EA analgesia. There is both theoretical and empirical support for the hypothesis that alterations in acupuncture (dose) parameter affect therapeutic outcomes. The Delphi process identifies treatment components that contribute to the “dose” of acupuncture, including the number of
CFA + 100 Hz + vehicle group, and CFA + 100 Hz capsaicine group were significantly increased after Day 14 post CFA injection (P = 0.004, P = 0.000, P = 0.012). PWL of CFA + 2 Hz + capsaicine group were lower than that of CFA + 100 Hz + vehicle group (P = 0.037), PWL of CFA + 100 Hz + capsaicine group were lower than that of CFA + 100 Hz + vehicle group (P = 0.016). As shown in Fig. 8B, compared with the CFA + vehicle group, PWL of CFA + 2 Hz + vehicle group, CFA + 100 Hz + vehicle group, and CFA + 100 Hz αβ-meATP group were significantly increased after Day 14 post CFA injection (P = 0.001, P = 0.000, P = 0.023). PWL of CFA + 2 Hz + αβ-meATP group were lower than that of CFA + 100 Hz + vehicle group (P = 0.006), PWL of CFA + 100 Hz + αβ-meATP group were lower than that of CFA + 100 Hz + vehicle group (P = 0.002).
4. Discussion In the present study, we investigated whether EA with different stimulation times and frequencies had different analgesic effects on inflammatory pain, and we examined whether EA-induced dose-specific anti-hyperalgesia involved mediating the level of P2X3 and TRPV1 in DRG. We observed that different stimulation time (20 min, 30 min,
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Fig. 6. Effect of EA with 2 Hz and 100 Hz on P2X3 positive neurons measured by immunofluorescence. A. Immunofluorescence confocal micrographs of L4–6 DRG in control group, CFA only group, CFA + 2 Hz group, CFA + 100 Hz group, and CFA + sham EA group. Sections show immunohistochemical green labeling for P2X3 positive neurons. Scale bars = 100 μm. B. Quantification analysis of P2X3 positive neurons in L4–6 DRG in control group, CFA only group, CFA + 2 Hz group, CFA + 100 Hz group, and CFA + sham EA group. C. Size distribution of P2X3 positive neuros in L4–6DRG in different groups. Data are presented as the mean ± SEM, n = 3–5. *P < 0.05, **P < 0.01, compared with control group; △P < 0.05, △△P < 0.01, compared with CFA only group; ○○P < 0.01, compared with CFA + 2 Hz group. ●P < 0.05, ●●P < 0.01, compared with CFA + 100 Hz group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
relief that primarily depended on stimulation frequency and less on stimulation time at a certain stimulation time. It has been reported that TRPV1 is selective primarily expressed in small- and medium-diameter sensory neurons, primarily in the peptidergic neurons, DRG, trigeminal ganglia, and nodose ganglia [25], and it is important in the development and maintenance of pain [26]. Preclinical and clinical studies have suggested that TRPV1 plays a pivotal role in pain (inflammatory, visceral, cancer, and neuropathic) [27]. Experimental studies also concluded that TRPV1 in DRG expression and function increased in certain types of pain models such as inflammatory pain (CFA model [28], carrageenan model, capsaicin model [29]), neuropathic pain (CCI model [30]), visceral pain [31], and bone cancer pain [32]. TRPV1−/− mice have shown markedly decreased thermal hyperalgesia in inflammatory pain models [33]. In this paper, TRPV1 positive neurons in L4 DRG were significantly increased; TRPV1 positive neurons in L5, L6 DRG showed a rising trend, but did not differ from the control group. The markedly high protein
needles, type of stimulation, retention time, and number of treatments [22]. Hao et al observed that the components of an acupuncture parameter that affected the outcomes for tension-type headaches were mode of stimulation, needle retention time and frequency of treatment [23]. There is also evidence of a parameter-response relation when acupuncture and related techniques are used for non-gynecological pain conditions [24]. According to well-established theories, different frequencies of EA have different therapeutic effects on pain relief [13]. In this paper, we examined whether the therapeutic effects of EA had parameter-specific effects on chronic inflammatory pain relief, and this treatment was related to stimulation frequency and stimulation time. In this study, stimulation time has a positive relationship with EA analgesia, similarly with previous study [15,16]. However, increasing stimulation time did not change the different effect between 2 Hz and 100 Hz EA. The effect of 100 Hz EA stimulation always > 2 Hz EA, no matter the stimulation time is 20 min, 30 min or 45 min. So we believed that EA had parameter-specific effects on chronic inflammatory pain
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Fig. 7. Effects of EA with 2 Hz and 100 Hz on P2X3 protein measured by western blot. A. Western blot bands of P2X3 protein in L4–6 DRG. B. Optical band density analysis. Data are presented as the mean ± SEM, n = 6–7. **P < 0.05, compared with control group; △P < 0.05, compared with CFA only group; ●P < 0.05, ●●P < 0.01, compared with CFA + 100 Hz group.
examined how the inhibition of TRPV1 up-regulation in ipsilateral adjacent undamaged DRGs contributed to low-frequency EA analgesia for mechanical allodynia induced by spinal nerve ligation [40]. EA may down-regulate the expression of P2X3 in the peripheral enteric nervous system in visceral hyperalgesia associated irritable bowel syndrome [12,41]. In this paper, we also determined that EA may down-regulate the TRPV1 and P2X3 expression in DRG. Xin Jet al. reported that segmental analgesia induced by low-intensity EA was partially mediated by ASIC3 receptors on Aβ-fibers, whereas systemic analgesia induced by higher intensity EA was more likely induced by TRPV1 receptors on Aδ- and C-fibers [42]. In our study, we determined that a high frequency of EA had a stronger effect on reducing TRPV1 and P2X3 expression, which was consistent with its antinociceptive effect. Thus we speculated that level of dose-specific analgesic effects of EA are mediated by regulation degree of TRPV1 and P2X3 in rats with inflammatory pain. Furthermore, we used TRPV1- and P2X3-specific agonists to research the attenuated analgesic effect of EA and determined that both the TRPV agonist and the P2X3 agonist can attenuate the antinociceptive effect of EA but cannot be completely reversed. Other substance participants in EA analgesia may exist, and these should be topics for future research. However, Hsu HC et al clarified that the 2 Hz EA and 15 Hz EA groups exhibited reduced cerebral TRPV4 expressions although the authors did not observe a similar effect for cerebral TRPV1
expressions of TRPV1 in L4 and L6 DRG were not observed in L5 DRG. Similar to TRPV1, P2X3 also displayed a high expression of sensory neurons, and played an important role in many forms of pain, such as inflammatory pain [34], bone cancer pain [35], neuropathic pain [36], and migraine pain [37]. A potent and selective antagonist of P2X3 effectively reduced both nerve injury and chronic inflammatory nociception, but not acute inflammation, or visceral pain [38]. Our research measured the P2X3 positive neurons and protein expression in L4–6 DRG of CFA-induced inflammatory pain and discovered that the P2X3 positive neurons in L4, L5, L6 DRG were significantly increased; however, only L6 DRG was identified with high protein expressions of P2X3, which in L4–5 DRG showed an increasing trend. Although EA has been widely used in clinics and the mechanism of its analgesic effect has been the subject of much research, the mechanism of the dose-specific effects of EA remains unclear. In this paper, we primarily investigated EA with regard to whether regulating the expression of P2X3 and TRPV1 in DRG was related to its dose-specific effect. Previous studies determined that both ipsilateral and contralateral EA may alter TRPV1 to reduce inflammatory pain [39], and both contralateral and ipsilateral EA may inhibit the primary afferent transmission of neuropathic pain induced by the P2X3 receptor. In addition, EA and A-317491 may have an additive effect in inhibiting the transmission of pain mediated by the P2X3 receptor [11]. We also
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Fig. 8. Effects of TRPV1 agonist or P2X3 agonist combined with EA on PWL. A. TRPV1 agonist combined with EA may reduce the analgesic effect of EA on PWL induced by CFA. Data are presented as the mean ± SEM, n = 6. △P < 0.05, △△P < 0.01, compared with CFA + vehicle group; ○P < 0.05, CFA + 2 Hz + vehicle group vs. CFA + 2 Hz + capsaicine group; ●●P < 0.01, CFA + 100 Hz + vehicle group vs. CFA + 100 Hz + capsaicine group. B. P2X3 agonist combined with EA may down-regulate the antinociceptive of EA on PWL induced by CFA. Data are presented as the mean ± SEM, n = 6. △P < 0.05, △△P < 0.01, compared with CFA + vehicle group; ○○P < 0.01, CFA + 2 Hz + vehicle group vs. CFA + 2 Hz + αβ-meATP group; ●●P < 0.01, CFA + 100 Hz + vehicle group vs. CFA + 100 Hz + αβ-meATP group.
immunohistochemistry and Western blotting, tissue fractionation and associated analyses. Ting Xiao, Xiaoqin Le, Ningfang Pan, Jie Yu and Boyi Liu provided supervision for data analysis, study direction, image acquisition, manuscript design and revisions, performed experiments, contributed to the design, data analysis and writing of the manuscript. All of the authors have read and approved the final manuscript.
or spinal TRPV4/TRPV1 expressions in the chronic constriction injury (CCI) model [10]. In the present study, we observed EA on TRPV1 and P2X3 in DRG but not in the spinal cord or cerebra. 5. Conclusions These data demonstrate that EA has parameter-specific effect on chronic inflammatory pain relief; these effects depend primarily on the stimulation frequency rather than stimulation time at a certain stimulation time. The parameter-specific analgesic effect of EA, at least in part, is associated with mediating the protein level of TRPV1 and P2X3 expression in DRG of CFA rats.
Acknowledgments Funding: This work was supported by the National Natural Science Fund of China (grant number 81473772, 81603690), the Major Medical and Health Science and Technology Project of Zhejiang Province (grant number WKJ-ZJ-1419), the Zhejiang Provincial Natural Science Found of China (grant number LQ15H270003), and the Key Science and Technology Innovation Team of Zhejiang Province (grant number 2013TD15), the National Natural Science Fund of China (grant number 81603676), Zhejiang Provincial Natural Science Funds for Distinguished Young Scholars (grant number LR17H270001).
Disclosures We declare no disclosures or conflicts of interest for any author. Author contributions
Conflict of interest statement
Jianqiao Fang designed and performed experimental protocols described in this manuscript as well as the writing of the initial draft of the manuscript. Junying Du, Junfan Fang performed the
The authors declare that there are no conflicts of interest. 79
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Parameter-specific analgesic effects of electroacupuncture mediated by degree of regulation TRPV1 and P2X3 in inflammatory pain in rats
T
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J.Q. Fang , J.Y. Du, J.F. Fang, T. Xiao, X.Q. Le, N.F. Pan, J. Yu, B.Y. Liu Department of Neurobiology and Acupuncture Research, The Third Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou 310053, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Electroacupuncture Inflammatory pain Frequency-specific P2X3 TRPV1
Aims: Observing the parameter-specific anti-hyperalgesic effects of EA with different stimulation times and frequencies on painful hyperalgesia mediated by the level of TRPV1 and P2X3 expression in DRG after CFA injection. Main methods: The model was induced by the injection of CFA in each rat's right hind paw. EA treatment was applied to the bilateral ST36 and BL60. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were tested with Von Frey filaments and the radiant heat source of the test instrument, respectively. TRPV1 and P2X3 expressions were measured by immunofluorescence and western blot. αβ-meATP and capsaicine combined with EA were further utilized to investigate the change in PWL. Key findings: Different stimulation times (20, 30, 45 min) combined with different frequencies (2 Hz, 100 Hz, 2/ 100 Hz) of EA have analgesic effects on the PWT and PWL; however, the level of the hypoalgesic efficacy of EA was primarily associated with EA frequency. The analgesic effect of EA was better at 100 Hz than at 2 Hz. The level of regulation of 100 Hz EA on TRPV1 and P2X3 in DRG was greater than that of 2 Hz. Furthermore, both TRPV1 agonist and P2X3 agonist may impair the level of EA analgesia. Significance: EA has a parameter-specific effect on chronic inflammatory pain relief, which primarily depend on the stimulation frequency and not on the stimulation time at a certain stimulation time. The parameter-specific analgesic effect of EA is at least partially related to mediation of the protein level of TRPV1 and P2X3 expression in DRG of CFA rats.
1. Introduction Pain that is induced by inflammation remains a serious health problem with significant social and economic consequences [1]. The existing mature treatment method may achieve a certain therapeutic effect but may easy cause adverse side effects. Electroacupuncture (EA), with few side effects displayed potent anti-hyperalgesic effects in several experimental models of chronic pain and in many diseases associated with pain [2]. It is well known that different stimulation times and different frequencies of EA have different analgesic effects [3]; however, the mechanism responsible for those differences remains unclear. Inflammation is primary characteristic of mechanical allodynia and thermal hyperalgesia in addition to activating both mechanical and thermal transducers such as purinergic receptor P2X ligand-gated ion channel 3 (P2X3) and transient receptor potential vanilloid 1 (TRPV1) [4,5]. Accumulated studies have clarified that P2X3 and TRPV1 are associated with pivotal the emergence and development of pain [6,7].
Our previous results and the results of other researchers have indicated that EA may inhibit the up-regulated expression of TRPV1 in DRG and spinal cord in inflammatory pain, neuropathic pain such as chronic constriction injury (CCI) and spinal nerve ligation (SNL), and cancer pain [8–10]. Numerous papers have suggested that suppressing the expression of P2X3 via EA stimulation may reduce many types of experimental pain, such as visceral pain, and neuropathic pain [11,12]. EA, which applies an adequate and continuous electrical current stimulation via needles to acupoints, was recently developed and has become a good complementary and alternative therapy [7]. According to well-established theories, different frequencies of EA have different therapeutic effects on pain relief [13]. Claydon et al. [14] reported that transcutaneous electrical nerve stimulation (TENS), which was similar to EA, had a dose-specific effect; the level of hypoalgesic efficacy of TENS was clearly dependent on the combination of TENS parameters selected (intensity, frequency, and stimulation site) and the experimental pain model. Although, it was also reported that the therapeutic effect of EA had parameter-specific effects on pain relief [15,16], there
⁎ Corresponding author at: Department of Neurobiology and Acupuncture Research, The Third Clinical Medical College, Zhejiang Chinese Medical University, 548 Binwen Road, Binjiang District, Hangzhou, Zhejiang Province 310053, China. E-mail address: [email protected] (J.Q. Fang).
https://doi.org/10.1016/j.lfs.2018.03.028 Received 26 September 2017; Received in revised form 11 December 2017; Accepted 7 March 2018 Available online 14 March 2018 0024-3205/ © 2018 Published by Elsevier Inc.
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2.5. Paw withdrawal latency (PWL)
was few paper discussion the mechanisms underlying the parameterspecific effect of EA. The purpose of this paper is to investigate the parameter-specific anti-hyperalgesic effects of EA by utilizing different stimulation times and frequencies on hyperalgesia mediated by the level of TRPV1 and P2X3 expression in DRG after CFA injection.
PWL was performed according to the methods previously reported [19]. The rats were acclimated to the test environment in individual testing cages for 30 min each day for three consecutive days. On the test day, the rats were allowed a minimum of 20 min to acclimate. After the acclimation period, the radiant heat source of the test instrument (37370, UGO, Italy) was positioned on the central surface of the hind paw, and the elapsed time between the heat application and the withdrawal response was registered as the paw-withdrawal latency (PWL). At each time point, the PWL was repeated three times at 2 min intervals, and the mean of these values was used for further calculations. The radiant heat was set at 35 °C, and the cutoff time was 20 s.
2. Materials and methods 2.1. Animals Two hundred and ten male Sprague–Dawley rats (160–180 g) were purchased through the Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Animal Certificate No. SCXK (沪) 2013-0016) and were fed in the laboratory animal center of Zhejiang (SYXK (浙) 2013–0184). The rats were given minimum of 1 week to adapt to their new environment. The rats were housed six per cage in a temperature-, humidity- and light-controlled environment (25 ± 2 °C, 55% ± 5%, 12-h light/dark cycle). The rats were fed standard rodent food and allowed distilled water ad libitum. All experimental procedures were approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (ZSLL-2015-022).
2.6. EA stimulation The EA stimulation procedure was as reported in our previous study [20]. After the behavioral testing on day 1, the rats were gently immobilized using a special cotton retainer designed by our laboratory (Patent No. ZL 2014 2 0473579.9, State Intellectual Property Office of the People's Republic of China). Four stainless steel acupuncture needles (0.25 mm × 13 mm) were inserted to a depth of 5 mm into the bilateral Zusanli (ST36, 5 mm lateral to the anterior tubercle of the tibia) and Kunlun (BL60, at the ankle joint level and between the tip of the external malleolus and tendo calcaneus) acupoints. The needles were then stimulated by HANS Acupuncture Point Nerve Stimulator (HANS-200A Huawei Co., Ltd., Beijing, China) with intensities ranging from 0.5–1.0–1.5 mA, once per day. EA was performed at different frequencies (2 Hz, 100 Hz, and 2/100 Hz, respectively) and stimulation times (20 min, 30 min, and 45 min, respectively) to compare analgesic effects. The parameters of EA were described below. The 2 Hz EA is a bidirectional rectangular wave with pulse width 0.2 ms; The 100 Hz EA is a bidirectional rectangular wave with pulse width 0.6 ms. The 2/ 100 Hz EA is a combination of 2 Hz EA and 100 Hz EA. When 2/100 Hz EA was administered, 2 Hz and 100 Hz was alternatively administered each 3 s. The frequency and stimulation time that showed the greatest or weakest analgesic effects were selected when conducting experiments on the analgesic mechanism of EA. The CFA + sham EA group animals received needle insertion subcutaneously into ST36 and BL60 (1 mm indepth); the needles were connected to the electrodes, however, there was no electrical stimulation. After finishing the EA or sham EA stimulation, we measure the PWT or PWL immediately.
2.2. Experimental design Four sets of experiments were conducted for this paper. In Experiment 1, the animals were randomly divided into five groups (n = 18): (1) control group, (2) CFA only group, (3) CFA + 2 Hz EA group, (4) CFA + 100 Hz EA group and (5) CFA + 2/100 Hz EA group. Each group was divided into three sub-group according to intervention time (20, 30, and 45 min). In Experiment 2, the animals were randomly divided into five groups (n = 12): (1) control group, (2) CFA only group, (3) CFA + 2 Hz group, (4) CFA + 100 Hz group, and (5) CFA + sham EA group. In Experiment 3, the animals were randomly divided into five groups (n = 6): (1) CFA + vehicle group, (2) CFA + 2 Hz + vehicle group, (3) CFA + 2 Hz + αβ-meATP group, (4) CFA + 100 Hz + vehicle group, and (5) CFA + 100 Hz + αβ-meATP group. In Experiment 4, the animals were randomly divided into five groups (n = 6): (1) CFA + vehicle group, (2) CFA + 2 Hz + vehicle group, (3) CFA + 2 Hz + capsaicine group, (4) CFA + 100 Hz + vehicle group, and (5) CFA + 100 Hz + capsaicine group. 2.3. Model establishment With the exception of the rats in the control group, all of the rats were administered 0.1 mL Freund's Complete Adjuvant (CFA, Sigma USA) by intraplantar injection into the right hind paw to establish the inflammatory pain model [17].
2.7. Drug intervention The chemicals used in this paper were α,β-methyleneadenosine 5′triphosphate lithium salt (αβ-meATP, P2X3 agonist) and capsaicine (TRPV1 agonist), both from Sigma-Aldrich (St. Louis, MO). αβ-meATP was dissolved in 0.9%NaCl. Capsaicine was dissolved in 100% DMSO and further diluted in saline containing 2% Tween 80. The final concentration of dimethylsulfoxide (DMSO) and Tween 80 was 2%. αβmeATP (600 nmol, 25 μL) and capsaicine (100 μg, 25 μL) were subcutaneously injected into the dorsum of the foot one time before EA stimulation on Day 14 after the model.
2.4. Paw withdrawal threshold (PWT) The rats were acclimated to the test environment in individual testing cages for 30 min each day for three consecutive days. On the test day, rats were allowed a minimum of 20 min to acclimate. The PWT was measured as the hind paw withdrawal response to von Frey hair monofilaments (Stoelting, IL, USA). The stimulus was applied in a consecutive ascending order (0.4, 0.6, 1, 2, 4, 6, 8, 15, 26 g) perpendicular to the central surface of the hind paw to determine the withdrawal threshold. The first filament applied corresponded to a force of 2 g. If a negative response (no movement) was observed, expressed as O, a filament exerting greater force was then applied; and if a positive response (paw withdrawal) was observed, expressed as X, a filament of lesser force was next applied. Counting of the critical 6 data points did not begin until the response threshold was first crossed, at which time the 2 responses straddling the threshold were retrospectively designated the first 2 responses in the series of 6. The PWT was calculated according to the response pattern observed described by Chaplan et al. [18].
2.8. Immunofluorescence Immunofluorescence analysis was performed according to the methods previously reported [21]. After the behavioral testing on day 14, rats were anesthetized using 10% choral hydrate at 0.35 mL/0.1 kg body weight. Once the rats were deeply anesthetized, the animals were quickly perfused with 150 mL 0.9% NaCl (4 °C) followed by 400 mL fresh 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS).
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3. Results
The L4–6 segments of the dorsal root ganglion (DRG) were removed and postfixed in the same fixative for 3 h at 4 °C before transfer to 15%, 30% sucrose for dehydration. Tissue was then embedded in an optimal cutting temperature compound (SAKURA, USA) and cut using a cryostat at a thickness of 16 μm to make the slide. All of the slides were blocked with 5% normal donkey serum in TBST (0.1%Tween-20) for 1 h at 37 °C. The slides were then incubated with rabbit anti-TRPV1 (1:1000 in 5% normal donkey serum, abcam, USA) and rabbit anti-P2X3 (1:1000 in 5% normal donkey serum, abcam, USA) for one night at 4 °C. The slides were then incubated in fluorescein (FITC) AffiniPure donkey anti-rat IgG (H + L) (Jackson, USA) for 1 h at 37 °C. Images were taken using an A1R confocal microscope (Nikon, Tokyo, Japan). We used Image-Pro Plus 6.0 to calculate positive cells. Specific methods are as follows: image format was converted RGB to Gray Scale 8. Using the negative image as the floor to determine the threshold value. The positive cell should meet the following conditions: the gray value was > 63, area was > 100, Area/Box ratio was 0.5–2. The specific area of each positive cell was obtained. Five random sections determined the average for each rat, and 3–5 rats included in each group.
3.1. Effects of EA at different frequencies and different stimulation times on PWT in CFA rats After CFA was injected, the PWT of rats decreased significantly on Day 1 and Day 3 (P = 0.005, P = 0.000, Fig. 1). EA may increase the PWT; however, different frequencies and different stimulation times of EA have different effects on the PWT. With EA stimulation for 20 min each time (Fig. 1A), only 100 Hz EA had a beneficial analgesic effect on the PWT after one or three stimulations (P = 0.021, P = 0.011). The PWT of other frequencies of EA was not different from the model group (P > 0.05). With EA stimulation for 30 min each time (Fig. 1B), 100 Hz and 2/100 Hz may significantly enhance the reduced PWT induced by CFA injection after one stimulation (P = 0.001, P = 0.019). All frequencies of EA may increase the PWT after three stimulations (P = 0.047, P = 0.000, P = 0.013). The analgesic effect of 100 Hz was significantly better than the effect of 2 Hz EA after three stimulations (P = 0.031). With EA stimulation for 45 min each time (Fig. 1C), all frequencies of EA may enhance the PWT after one or three stimulations (P = 0.016, P = 0.000, P = 0.003, P = 0.001, P = 0.000, P = 0.000). The analgesic effect of 100 Hz was significantly better than the effect of 2 Hz EA after one stimulation (P = 0.041).
2.9. Western blot Western blot analysis was performed according to the methods previously reported [20]. Animals were sacrificed after the behavioral testing on day 14. Rats were anesthetized 10% choral hydrate at 0.35 mL/100 g body weight. Once deeply anesthetized, the rats were quickly perfused with 150 mL cold sterilized saline. The L4–6 segments of the DRG were removed and stored at −80 °C. Tissues were homogenized in strong RIPA buffer (50 mM Tris[pH 7.4], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, sodium orthovanadate, 0.1% SDS, EDTA, sodium fluoride, leupeptin, and 1 nM PMSF). The homogenate was allowed to rest on ice for 30 min and then centrifuged at 15,000 rpm for 15 min at 4 °C, at this point, the supernatant was collected. The protein concentration of tissue lysates was determined with a BCA protein assay kit, and 20 μg of protein were loaded in each lane. Protein samples were separated on 5–10% SDS-PAGE gels and electrophoretically transferred to polyvinyl difluoride (PVDF) membranes (Bio-rad, USA). The membranes were blocked with 5% low-fat milk in TBST for 1 h at room temperature. We used anti-TRPV1 (1:1000 in 5% normal donkey serum, abcam, USA) and rabbit anti-P2X3 (1:1000 in 5% normal donkey serum, abcam, USA) as primary antibodies and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG as the secondary antibody (1:10,000). Rabbit anti-GAPDH (HRP Conjugate) (1:1000, CST, USA) was used as the internal control. The membranes were developed with an ECL kit (Pierce, USA), and the signals were captured with an Image Quant LAS 4000 (EG, USA). The density of each band was measured using Image Quant TL 7.0 analysis software (GE, USA). The mean expression level of the target protein in the animals in the control group was considered to be 1, and the relative expression level of the target protein in all animals was adjusted as a ratio to the level of the control group.
3.2. Effects of EA using different frequencies and different stimulation times on PWL in CFA rats After CFA was injected, the PWL of rats was significantly decreased on Day 1 and Day 3 (P = 0.000, P = 0.000, Fig. 2). Like the effect of EA on the PWT, EA may also increase the PWL; however, different frequencies and different stimulation times of EA have different effects on the PWL. With EA stimulation for 20 min each time (Fig. 2A), all frequencies of EA have a beneficial analgesic effect on PWL after stimulation one time or three times (P = 0.004, P = 0.000, P = 0.000, P = 0.000, P = 0.000, P = 0.005). With EA stimulation for 30 min each time (Fig. 2B), only 100 Hz EA significantly enhanced the reduced PWL induced by CFA injection after one stimulation. 100 Hz EA and 2/ 100 Hz EA significantly the PWL after three stimulations (P = 0.010, P = 0.039). The analgesic effect of 100 Hz was significantly better than the effect of 2 Hz EA after one stimulation and three stimulations (P = 0.006, P = 0.018). With EA stimulation for 45 min each time (Fig. 2C), only 100 Hz and 2/100 Hz EA significantly enhanced the reduced PWL induced by CFA injection after one stimulation (P = 0.000, P = 0.003). The analgesic effect of 100 Hz was significantly better than the effect of 2 Hz EA after one stimulation (P = 0.018). All frequencies of EA may enhance the PWL after three stimulations (P = 0.041, P = 0.009, P = 0.012). 3.3. Effects of EA on the PWT and PWL in CFA rats Figs. 1 and 2 indicated that different frequencies and different stimulation times of EA had different effects on the PWT and PWL. Whether stimulation lasted 20 min, 30 min or 45 min, 100 Hz EA had the greatest analgesic effect on the PWT and PWL. The weakest analgesic effect on the PWT and PWL was 2 Hz. Therefore, we chose 2 Hz and 100 Hz EA for 30 min stimulation, the common clinical stimulation time, for further research. The degree of the analgesic effect of EA depends on the stimulation frequency (Fig. 3). As shown in Fig. 3A, both 2 Hz and 100 Hz EA significantly increased the PWT after Day 1, 3, 7 and 14 post CFA injection (P = 0.031, P = 0.000, P = 0.047, P = 0.000, P = 0.024, P = 0.000, P = 0.037, P = 0.000), and maintained higher thresholds than the PWT in the sham EA; however, only the PWT at 100 Hz showed a significant increase compared with the PWT in the sham EA group (P = 0.002, P = 0.009, P = 0.003, P = 0.004). The PWT of the rats in the 100 Hz group was significantly higher compared with 2 Hz group on Day 3 post
2.10. Statistical analyses All data were expressed as the means ± standard error (SEM). The PWT and PWL 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 among groups at each time point. For immunofluorescence and western blot analysis, comparisons between groups were performed using oneway analysis of variance followed by Fisher's Protected Least Significant Difference posthoc tests. P < 0.05 was considered statistically significant.
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Fig. 1. Analgesic effects of EA with different frequencies and different times on the PWT of CFA rats. A. Analgesic effect of 20 min EA with 2 Hz, 100 Hz and 2/100 Hz stimulation on PWT. B. Analgesic effect of 30 min EA with 2 Hz, 100 Hz and 2/100 Hz stimulation on PWT. C. Analgesic effect of 45 min EA with 2 Hz, 100 Hz and 2/100 Hz stimulation on PWT. Data are presented as the mean ± SEM, n = 6. *P < 0.05, **P < 0.01, compared with control group; △P < 0.05, △△P < 0.01, compared with CFA only group; ○P < 0.05, ○○P < 0.01, compared with CFA + 2 Hz group.
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Fig. 2. Analgesic effects of EA with different frequencies and different times on PWL of CFA rats. A. Analgesic effect of 20 min EA with 2 Hz, 100 Hz and 2/100 Hz stimulation on PWL. B. Analgesic effect of 30 min EA with 2 Hz, 100 Hz and 2/100 Hz stimulation on PWL. C. Analgesic effect of 45 min EA with 2 Hz, 100 Hz and 2/100 Hz stimulation on PWL. Data are presented as the mean ± SEM, n = 6. *P < 0.05, **P < 0.01, compared with control group; △P < 0.05, △△P < 0.01, compared with CFA only group; ○P < 0.05, ○○P < 0.01, compared with CFA + 2 Hz group.
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Fig. 3. Analgesic effects of EA stimulation on PWT and PWL of CFA rats for 14 days. A. Analgesic effect of EA with 2 Hz and 100 Hz stimulation on PWT. B. Analgesic effect of EA with 2 Hz and 100 Hz stimulation on PWL. Data are presented as the mean ± SEM, n = 6. **P < 0.01, compared with control group; △P < 0.05, △△P < 0.01, compared with CFA only group; ○P < 0.05, ○○P < 0.01, compared with CFA + 2 Hz group. ●P < 0.05, ●●P < 0.01, compared with CFA + 100 Hz group.
2 Hz group were significantly decreased (P = 0.019). However, TRPV1 positive neurons in L4, L5 DRG in the 100 Hz group were markedly decreased compared with the model group (P = 0.007, P = 0.021). Sham EA had no effect on inhibiting the TRPV1 positive neurons in L4–6 DRG. We also observed the size-distribution of TRPV1 in L4–6 DRG of all the group. As shown in Fig. 4C, TRPV1 is mainly expression in the small diameter DRG neurons (areas less of 200 pixel). Furthermore, EA did not regulate the distribution of TRPV1 expression. We also used western blot to measure the TRPV1 protein expression in L4–6 DGR. As shown in Fig. 5, compared with the control group, TRPV1 protein expression in L4, L6 DRG in the model group increased significantly (P = 0.025, P = 0.007); TRPV1 protein expression in L5 DRG in the model group was not different from the control group. Compared with the model group, TRPV1 protein expression in L4, L6 DRG in the 2 Hz group was significantly decreased (P = 0.049, P = 0.046). However, TRPV1 protein expression in L4, L5, L6 DRG in the 100 Hz group was markedly decreased compared with the model
CFA injection (P = 0.047), and at other time points, the PWT of rats in the 100 Hz group trended toward an increase in the PWT of rats in the 2 Hz group. As shown in Fig. 3B, 100 Hz of EA significantly increased PWL after Day 1, 3, 7, and 14 post CFA injection (P = 0.000, P = 0.025, P = 0.003, P = 0.000); the PWT of rats in the 2 Hz EA group was significantly higher than the PWT of the model group only on Day 14 after CFA injection (P = 0.012). Sham EA had no analgesic effect on PWL at any time point (P > 0.05). 3.4. Effects of EA on the expression of TRPV1 in L4–6 DRG The TRPV1 receptor is primarily restricted to the small- and medium-sized DRG neurons. As shown in Fig. 4, compared with the control group, TRPV1 positive neurons in L4 DRG in the model group were significantly increased (P = 0.018); TRPV1 positive neurons in L5, L6 DRG increased gradually but without a statistical difference. Compared with the model group, TRPV1 positive neurons in L4 DRG in the
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Fig. 4. Effect of EA through 2 Hz, 100 Hz on TRPV1 positive neurons measured by immunofluorescence. A. Immunofluorescence confocal micrographs of L4–6 DRG in control group, CFA only group, CFA + 2 Hz group, CFA + 100 Hz group, and CFA + sham EA group. Sections show immunohistochemical green labeling for TRPV1 positive neurons. Scale bars = 100 μm. B. Quantification analysis of TRPV1 positive neurons in L4–6 DRG in control group, CFA only group, CFA + 2 Hz group, CFA + 100 Hz group, and CFA + sham EA group. C. Size distribution of TRPV1 positive neurons in L4–6 DRG in different groups. Data are presented as the mean ± SEM, n = 3–5. *P < 0.05, compared with control group; △P < 0.05, △△P < 0.01, compared with CFA only group; ○P < 0.05, compared with CFA + 2 Hz group. ●P < 0.05, compared with CFA + 100 Hz group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
We also used western blot to measure the P2X3 protein expression in L4–6DGR. As shown in Fig. 7, compared with the control group, P2X3 protein expression in L6 DRG in the model group was significantly increased (P = 0.020); P2X3 protein expression in L4, L5 DRG in the model group was not different from the control group. Compared with the model group, P2X3 protein expression in L6 DRG in the 2 Hz and 100 Hz group was significantly decreased (P = 0.042, P = 0.010). P2X3 protein expression in L4–6 DRG in the 100 Hz group was reduced compared with the 2 Hz group; however, there were not significant differences. Sham EA had no effect on inhibiting the P2X3 protein expression in L4–6 DRG.
group (P = 0.009, P = 0.028, P = 0.029). Sham EA had no effect on inhibiting the TRPV1 protein expression in L4–6 DRG. 3.5. Effects of EA on the expression of P2X3 in L4–6 DRG As shown in Fig. 6, compared with the control group, P2X3 positive neurons in L4, L5, L6 DRG in the model group increased significantly (P = 0.023, P = 0.029, P = 0.001). Compared with the model group, P2X3 positive neurons in L6 DRG in the 2 Hz group were significantly decreased (P = 0.004). However, P2X3 positive neurons in L4, L5, L6 DRG in the 100 Hz group were markedly decreased compared with the model group (P = 0.041, P = 0.013, P = 0.001). Sham EA had no effect on inhibiting the P2X3 positive neurons in L4–6 DRG. We also observed the size-distribution of P2X3 in L4–6 DRG of all the group. As shown in Fig. 6C, P2X3 is mainly expression in the small and media diameter DRG neurons (areas less of 300 pixel). EA did not regulate the distribution of expression P2X3.
3.6. TRPV1 or P2X3 agonist attenuated EA analgesia We further used specific agonist combined with EA to measure the change in pain behavior. As shown in Fig. 8A, compared with the CFA + vehicle group, PWL of CFA + 2 Hz + vehicle group,
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Fig. 5. Effect of EA with 2 Hz and 100 Hz on TRPV1 protein measured by western blot. A. Western blot bands of TPRV1 protein in L4–6 DRG. B. Optical band density analysis. Data are presented as the mean ± SEM, n = 6–7. *P < 0.05, **P < 0.01, compared with control group; △P < 0.05, △△P < 0.01, compared with CFA only group.
45 min) combined with different frequencies (2 Hz, 100 Hz, 2/100 Hz) EA had different analgesic effects on the PWT and PWL of inflammatory pain; however, the level of hypoanalgesic efficacy of EA was primarily associated with EA frequency. The analgesic effect of EA at 100 Hz was better than that at 2 Hz. The results indicate that 2 Hz EA may significantly reduce the positive neuron of TRPV1 in L4 DRG and decrease protein expression of TRPV1 in L4, L6 DRG. However, 100 Hz EA resulted in a higher level of regulation of TRPV1 in DRG than 2 Hz EA, which may significantly reduce the positive neuron of TRPV1 in L4, L5 DRG and down-regulate the protein expression of TRPV1 in L4–6 DRG. With regard to P2X3, 100 Hz could markedly reduce the positive neuron of P2X3 in L4–6 DRG and decrease the protein expression of P2X3 in L6 DRG; however, 2 Hz may only reduce positive neuron and the protein expression of P2X3 in L6 DRG. Furthermore, we used a TRPV1 agonist and a P2X3 agonist in combination with EA to investigate the anti-hyperanalgesic effect of EA. It was observed that both the TRPV1 agonist and the P2X3 agonist may impair the level of EA analgesia. There is both theoretical and empirical support for the hypothesis that alterations in acupuncture (dose) parameter affect therapeutic outcomes. The Delphi process identifies treatment components that contribute to the “dose” of acupuncture, including the number of
CFA + 100 Hz + vehicle group, and CFA + 100 Hz capsaicine group were significantly increased after Day 14 post CFA injection (P = 0.004, P = 0.000, P = 0.012). PWL of CFA + 2 Hz + capsaicine group were lower than that of CFA + 100 Hz + vehicle group (P = 0.037), PWL of CFA + 100 Hz + capsaicine group were lower than that of CFA + 100 Hz + vehicle group (P = 0.016). As shown in Fig. 8B, compared with the CFA + vehicle group, PWL of CFA + 2 Hz + vehicle group, CFA + 100 Hz + vehicle group, and CFA + 100 Hz αβ-meATP group were significantly increased after Day 14 post CFA injection (P = 0.001, P = 0.000, P = 0.023). PWL of CFA + 2 Hz + αβ-meATP group were lower than that of CFA + 100 Hz + vehicle group (P = 0.006), PWL of CFA + 100 Hz + αβ-meATP group were lower than that of CFA + 100 Hz + vehicle group (P = 0.002).
4. Discussion In the present study, we investigated whether EA with different stimulation times and frequencies had different analgesic effects on inflammatory pain, and we examined whether EA-induced dose-specific anti-hyperalgesia involved mediating the level of P2X3 and TRPV1 in DRG. We observed that different stimulation time (20 min, 30 min,
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Fig. 6. Effect of EA with 2 Hz and 100 Hz on P2X3 positive neurons measured by immunofluorescence. A. Immunofluorescence confocal micrographs of L4–6 DRG in control group, CFA only group, CFA + 2 Hz group, CFA + 100 Hz group, and CFA + sham EA group. Sections show immunohistochemical green labeling for P2X3 positive neurons. Scale bars = 100 μm. B. Quantification analysis of P2X3 positive neurons in L4–6 DRG in control group, CFA only group, CFA + 2 Hz group, CFA + 100 Hz group, and CFA + sham EA group. C. Size distribution of P2X3 positive neuros in L4–6DRG in different groups. Data are presented as the mean ± SEM, n = 3–5. *P < 0.05, **P < 0.01, compared with control group; △P < 0.05, △△P < 0.01, compared with CFA only group; ○○P < 0.01, compared with CFA + 2 Hz group. ●P < 0.05, ●●P < 0.01, compared with CFA + 100 Hz group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
relief that primarily depended on stimulation frequency and less on stimulation time at a certain stimulation time. It has been reported that TRPV1 is selective primarily expressed in small- and medium-diameter sensory neurons, primarily in the peptidergic neurons, DRG, trigeminal ganglia, and nodose ganglia [25], and it is important in the development and maintenance of pain [26]. Preclinical and clinical studies have suggested that TRPV1 plays a pivotal role in pain (inflammatory, visceral, cancer, and neuropathic) [27]. Experimental studies also concluded that TRPV1 in DRG expression and function increased in certain types of pain models such as inflammatory pain (CFA model [28], carrageenan model, capsaicin model [29]), neuropathic pain (CCI model [30]), visceral pain [31], and bone cancer pain [32]. TRPV1−/− mice have shown markedly decreased thermal hyperalgesia in inflammatory pain models [33]. In this paper, TRPV1 positive neurons in L4 DRG were significantly increased; TRPV1 positive neurons in L5, L6 DRG showed a rising trend, but did not differ from the control group. The markedly high protein
needles, type of stimulation, retention time, and number of treatments [22]. Hao et al observed that the components of an acupuncture parameter that affected the outcomes for tension-type headaches were mode of stimulation, needle retention time and frequency of treatment [23]. There is also evidence of a parameter-response relation when acupuncture and related techniques are used for non-gynecological pain conditions [24]. According to well-established theories, different frequencies of EA have different therapeutic effects on pain relief [13]. In this paper, we examined whether the therapeutic effects of EA had parameter-specific effects on chronic inflammatory pain relief, and this treatment was related to stimulation frequency and stimulation time. In this study, stimulation time has a positive relationship with EA analgesia, similarly with previous study [15,16]. However, increasing stimulation time did not change the different effect between 2 Hz and 100 Hz EA. The effect of 100 Hz EA stimulation always > 2 Hz EA, no matter the stimulation time is 20 min, 30 min or 45 min. So we believed that EA had parameter-specific effects on chronic inflammatory pain
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Fig. 7. Effects of EA with 2 Hz and 100 Hz on P2X3 protein measured by western blot. A. Western blot bands of P2X3 protein in L4–6 DRG. B. Optical band density analysis. Data are presented as the mean ± SEM, n = 6–7. **P < 0.05, compared with control group; △P < 0.05, compared with CFA only group; ●P < 0.05, ●●P < 0.01, compared with CFA + 100 Hz group.
examined how the inhibition of TRPV1 up-regulation in ipsilateral adjacent undamaged DRGs contributed to low-frequency EA analgesia for mechanical allodynia induced by spinal nerve ligation [40]. EA may down-regulate the expression of P2X3 in the peripheral enteric nervous system in visceral hyperalgesia associated irritable bowel syndrome [12,41]. In this paper, we also determined that EA may down-regulate the TRPV1 and P2X3 expression in DRG. Xin Jet al. reported that segmental analgesia induced by low-intensity EA was partially mediated by ASIC3 receptors on Aβ-fibers, whereas systemic analgesia induced by higher intensity EA was more likely induced by TRPV1 receptors on Aδ- and C-fibers [42]. In our study, we determined that a high frequency of EA had a stronger effect on reducing TRPV1 and P2X3 expression, which was consistent with its antinociceptive effect. Thus we speculated that level of dose-specific analgesic effects of EA are mediated by regulation degree of TRPV1 and P2X3 in rats with inflammatory pain. Furthermore, we used TRPV1- and P2X3-specific agonists to research the attenuated analgesic effect of EA and determined that both the TRPV agonist and the P2X3 agonist can attenuate the antinociceptive effect of EA but cannot be completely reversed. Other substance participants in EA analgesia may exist, and these should be topics for future research. However, Hsu HC et al clarified that the 2 Hz EA and 15 Hz EA groups exhibited reduced cerebral TRPV4 expressions although the authors did not observe a similar effect for cerebral TRPV1
expressions of TRPV1 in L4 and L6 DRG were not observed in L5 DRG. Similar to TRPV1, P2X3 also displayed a high expression of sensory neurons, and played an important role in many forms of pain, such as inflammatory pain [34], bone cancer pain [35], neuropathic pain [36], and migraine pain [37]. A potent and selective antagonist of P2X3 effectively reduced both nerve injury and chronic inflammatory nociception, but not acute inflammation, or visceral pain [38]. Our research measured the P2X3 positive neurons and protein expression in L4–6 DRG of CFA-induced inflammatory pain and discovered that the P2X3 positive neurons in L4, L5, L6 DRG were significantly increased; however, only L6 DRG was identified with high protein expressions of P2X3, which in L4–5 DRG showed an increasing trend. Although EA has been widely used in clinics and the mechanism of its analgesic effect has been the subject of much research, the mechanism of the dose-specific effects of EA remains unclear. In this paper, we primarily investigated EA with regard to whether regulating the expression of P2X3 and TRPV1 in DRG was related to its dose-specific effect. Previous studies determined that both ipsilateral and contralateral EA may alter TRPV1 to reduce inflammatory pain [39], and both contralateral and ipsilateral EA may inhibit the primary afferent transmission of neuropathic pain induced by the P2X3 receptor. In addition, EA and A-317491 may have an additive effect in inhibiting the transmission of pain mediated by the P2X3 receptor [11]. We also
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Fig. 8. Effects of TRPV1 agonist or P2X3 agonist combined with EA on PWL. A. TRPV1 agonist combined with EA may reduce the analgesic effect of EA on PWL induced by CFA. Data are presented as the mean ± SEM, n = 6. △P < 0.05, △△P < 0.01, compared with CFA + vehicle group; ○P < 0.05, CFA + 2 Hz + vehicle group vs. CFA + 2 Hz + capsaicine group; ●●P < 0.01, CFA + 100 Hz + vehicle group vs. CFA + 100 Hz + capsaicine group. B. P2X3 agonist combined with EA may down-regulate the antinociceptive of EA on PWL induced by CFA. Data are presented as the mean ± SEM, n = 6. △P < 0.05, △△P < 0.01, compared with CFA + vehicle group; ○○P < 0.01, CFA + 2 Hz + vehicle group vs. CFA + 2 Hz + αβ-meATP group; ●●P < 0.01, CFA + 100 Hz + vehicle group vs. CFA + 100 Hz + αβ-meATP group.
immunohistochemistry and Western blotting, tissue fractionation and associated analyses. Ting Xiao, Xiaoqin Le, Ningfang Pan, Jie Yu and Boyi Liu provided supervision for data analysis, study direction, image acquisition, manuscript design and revisions, performed experiments, contributed to the design, data analysis and writing of the manuscript. All of the authors have read and approved the final manuscript.
or spinal TRPV4/TRPV1 expressions in the chronic constriction injury (CCI) model [10]. In the present study, we observed EA on TRPV1 and P2X3 in DRG but not in the spinal cord or cerebra. 5. Conclusions These data demonstrate that EA has parameter-specific effect on chronic inflammatory pain relief; these effects depend primarily on the stimulation frequency rather than stimulation time at a certain stimulation time. The parameter-specific analgesic effect of EA, at least in part, is associated with mediating the protein level of TRPV1 and P2X3 expression in DRG of CFA rats.
Acknowledgments Funding: This work was supported by the National Natural Science Fund of China (grant number 81473772, 81603690), the Major Medical and Health Science and Technology Project of Zhejiang Province (grant number WKJ-ZJ-1419), the Zhejiang Provincial Natural Science Found of China (grant number LQ15H270003), and the Key Science and Technology Innovation Team of Zhejiang Province (grant number 2013TD15), the National Natural Science Fund of China (grant number 81603676), Zhejiang Provincial Natural Science Funds for Distinguished Young Scholars (grant number LR17H270001).
Disclosures We declare no disclosures or conflicts of interest for any author. Author contributions
Conflict of interest statement
Jianqiao Fang designed and performed experimental protocols described in this manuscript as well as the writing of the initial draft of the manuscript. Junying Du, Junfan Fang performed the
The authors declare that there are no conflicts of interest. 79
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J.Q. Fang et al.
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