|Year : 2020 | Volume
| Issue : 4 | Page : 448-455
Electroacupuncture alleviates neuropathic pain by modulating Th2 infiltration and inhibiting microglial activation in the spinal cord of rats with spared nerve injury
Bin Liu, Wei Long, Ru-Rong Wang
Department of Anesthesiology, West China Hospital of Sichuan University, Chengdu, China
|Date of Submission||20-Mar-2020|
|Date of Acceptance||19-May-2020|
|Date of Web Publication||05-Oct-2020|
Prof. Ru-Rong Wang
Department of Anesthesiology, West China Hospital of Sichuan University, Chengdu 610041, Sichuan
Source of Support: None, Conflict of Interest: None
Objective: The objective is to explore the potential mechanisms mediating the analgesic effect of electroacupuncture (EA) for neuropathic pain. Materials and Methods: Sprague-Dawley (SD) rats were used to establish a spared nerve injury (SNI) model of neuropathic pain. The intensity of neuropathic pain was measured by assessing the mechanical withdrawal threshold and thermal withdrawal latency. Immunofluorescent analysis and western blotting were performed to evaluate the activation of microglia and Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling in the L4-6 spinal cord of SD rats. The infiltration of Th2 and expression of interleukin-10 (IL-10) in the L4-6 spine were analyzed by flow cytometry and enzyme-linked immunosorbent assay. Results: Behavioral test results demonstrated that SNI-induced hyperalgesia was significantly ameliorated by EA stimulation at “Zusanli” (ST36) and “Yanglingquan” (GB34) points. In addition, EA therapy significantly suppressed microglia activation and phosphorylation of JAK2/STAT3 in the L4-6 spine of rats with SNI. Moreover, EA promoted the infiltration of Th2 and secretion of IL-10 in the L4-6 spine of rats with SNI. Conclusions: The study findings indicate that EA stimulation mediated the infiltration of Th2 in the spinal cord of rats with SNI. In addition, EA inhibited microglial activation in the L4-6 spine of rats with SNI.
Keywords: Electroacupuncture, Janus kinase/signal transducer and activator of transcription signaling, microglia, neuropathic pain, Th2
|How to cite this article:|
Liu B, Long W, Wang RR. Electroacupuncture alleviates neuropathic pain by modulating Th2 infiltration and inhibiting microglial activation in the spinal cord of rats with spared nerve injury. World J Tradit Chin Med 2020;6:448-55
|How to cite this URL:|
Liu B, Long W, Wang RR. Electroacupuncture alleviates neuropathic pain by modulating Th2 infiltration and inhibiting microglial activation in the spinal cord of rats with spared nerve injury. World J Tradit Chin Med [serial online] 2020 [cited 2021 Jan 25];6:448-55. Available from: https://www.wjtcm.net/text.asp?2020/6/4/448/303577
| Introduction|| |
Neuropathic pain refers to the intractable pain induced by pathological lesions or dysfunction of the somatosensory nervous system. At present, the specific mechanism underlying neuropathic pain is unknown and requires additional investigation. Patients with neuropathic pain typically experience a set of persistent symptoms, such as pain induced by nonnociceptive stimulations, accompanied by burning and electrical shock-like sensations. Neuropathic pain is usually refractory to analgesics and affects approximately 7%–10% of the general population. Therefore, effective therapy is urgently needed for neuropathic pain.
Microglia, also known as the resident macrophage-like cells in the central nervous system (CNS), monitors the homeostasis of the neural microenvironment., In pathophysiologic conditions, microglia rapidly respond to numerous stimuli that alter the physiological homeostasis, including peripheral nerve injury., Notably, peripheral nerve injury leads to the reactive microgliosis in the spinal dorsal horn. The overexpressed and hyperactivated microglial cells secrete various inflammatory cytokines, which contribute to the development of neuropathic pain., Pertinently, the activity of microglia was therapeutically targeted in many basic and preclinical studies, and this strategy has always been considered useful in the treatment of neuropathic pain., Indeed, accumulating evidence shows that the activation of microglia can be ameliorated by anti-inflammatory factors, such as interleukin-10 (IL-10), transforming growth factor–β, and triggering receptor expressed on myeloid cell-2.,, The Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling pathway has been considered as the downstream mediator of various cytokines; activation of the JAK-STAT pathway triggers a series of events, including microglial activation., However, results of previous vitro study have demonstrated that lipopolysaccharide (LPS)-induced microglial activation was inhibited by IL-10 through the activation of JAK-STAT signaling pathway. Therefore, modulating the level of IL-10 in the spinal dorsal horn might be a promising approach to suppress reactive microgliosis occurring after a nerve injury.
Electroacupuncture (EA), a modified technique that combines traditional acupuncture with modern electrical stimulation therapy, applies different levels of stimulating currents to acupoints through acupuncture needles. EA has been successfully used for analgesic and physical therapies for centuries, but the mechanisms underlying EA-induced analgesia remain unclear.,,, However, accumulating evidence shows that the therapeutic function of EA is closely associated with its immunoregulatory effect., EA stimulation reportedly modulates the Th1/Th2 imbalance in the CNS. IL-10, mainly released by the Th2 cells, binds to its receptors and triggers a series of internal signaling cascades mediated by JAK/STAT, particularly by STAT3. The signaling initiated by IL-10 regulates microglial activities, protecting the nervous system from excessive inflammation. Collectively, results from a previous study indicate that EA therapy promotes the release of IL-10 to inhibit microglial activation in the treatment of neuropathic pain.
In this study, we selected “Zusanli” (ST36) and “Yanglingquan” (GB34) as the acupoints for EA therapy. Behavioral test, flow cytometry, immunofluorescent staining, western blotting, and enzyme-linked immunosorbent assay (ELISA) were performed to verify the hypothesis that EA therapy could enhance the activity of Th2 cells and suppress the overexpression of microglia in a rat model of neuropathic pain.
| Materials and Methods|| |
Adult, male Sprague-Dawley (SD) rats (200–220 g, 6–8 weeks old) were obtained from Experimental Animals Center of Southwest Medical University (Luzhou, China). All rats were housed in a temperature-controlled (21°C–25°C) room under 12-h light/dark cycle. Food and water were available ad libitum. All experimental procedures were approved by the Animal Care and Use Committee of Southwest Medical University and conform to the guidelines for laboratory animal use promulgated by the Ministry of Science and Technology of the People's Republic of China. All rats were randomly assigned to four groups (n = 6 rats per group): Sham group (rats with exposed sciatic nerve but without any injury), spared nerve injury (SNI; rats subjected to SNI of the L4-6 spinal cord), EA (rats with SNI that received EA treatment), and non-acupoint EA (NEA; rats with SNI that received non-acupoints EA treatment).
Spared nerve injury model of neuropathic pain
The well-established rat model of SNI was used, which induces stable and persistent neuropathic pain behaviors. Rats models of SNI were generated as described previously. After induction of deep anesthesia in rats by inhalation of isoflurane (3%–4%, induction; 2%, maintenance), the right sciatic nerve was exposed and followed. Three peripheral branches were identified on following the sciatic nerve trunk. Then, the tibial and common peroneal nerves were freed from their sheath and tightly ligatured by two knots using a 4-0 silk suture. Subsequently, the nerve segments between the knots were severed completely. Finally, the wound was closed layer by layer and disinfected with povidone-iodine solution. For rats in the Sham group, the three peripheral branches of the sciatic nerve were exposed, but the tibial and common peroneal nerves were not ligatured or severed.
Hwato brand disposable filiform needles (size, 0.30 mm × 25 mm), and HANS EA apparatus (Hans-200A, Jisheng Medical Technology, Co., Ltd., Nanjing, China) were used for EA. “Zusanli” (ST36) and “Yanglingquan” (GB34) were chosen as the acupoints for EA. In rats, ST36 is located proximally, 5 mm underneath the capitulum fibulae and posterolateral point of the knee joint, while GB34 is located in the depression, anterior and inferior to the fibular head. The “Zusanli” (ST36) and “Yanglingquan” (GB34) acupoints were selected as the center. Accordingly, the non-acupoints were located to the left of the center at a distance of 0.5 cm. EA at the respective acupoints and non-acupoints were performed after the SNI operation. The rats with SNI were immobilized in the prone position with a self-made cloth bag, and acupuncture needles were inserted into the acupoints or their corresponding non-acupoints at a depth of 0.5 cm. EA was performed using the HANS electrical apparatus (2 mA/2 Hz) for 30 min each day, for a total of 2 weeks. In this study, both acupoint and nonacupoint EA were performed on the rats with SNI with the same electrical stimulation.
Rats were placed in a Plexiglas chamber and permitted to adapt to the environment for 30 min before the behavioral testing. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured 1 day before the surgery and on days 3, 7, and 14 postoperatively.
MWT was determined for the plantar surface of the ipsilateral paw using von Frey filaments (North Coast Medical, San Jose, CA) with forces ranging from 2 g to 26 g, following the up-down method. In brief, the rats were placed in transparent plastic chambers with wire mesh floor for 30 min. Pressure was applied from below to the ipsilateral mid-plantar surface of the right hind paw with a series of von Frey filaments in ascending order, beginning with the filament having the lowest force (2 g). If withdrawal responses were observed three times with a particular filament, the value of that filament (in grams) was recorded as the MWT.
For the TWL test, the rats were placed in transparent plastic chambers with a glass plate for 30 min. A radiant heat source (Tes7370, Ugo Basile, Comerio, Italy) was placed underneath the glass plate and heat was focused onto the plantar surface of the ipsilateral paw. TWL was determined as the time from the beginning of the thermal stimulation to paw withdrawal.
MWT and TWL tests were repeated three times with 15 min intervals between consecutive tests and the average values were considered as the final data.
Rats were induced with deep anesthesia and sacrificed on postoperative day 14 after performing the last behavioral test. For western blotting and ELISA experiments, the euthanized rats were perfused transcardially with PBS and the L4-L5 spinal cord was quickly exposed by tracking the right sciatic nerve. Then, ipsilateral L4-L5 spinal cord segments were dissected and stored at − 80°C for subsequent usage. To obtain samples for immunofluorescent staining, the rats were perfused transcardially first with PBS and then with 4% paraformaldehyde. The L4–L5 spinal cord segments were quickly dissected and dehydrated in 30% sucrose solution. After dehydration, the segments were postfixed at 4°C overnight with 4% paraformaldehyde and stored at −80°C for subsequent usage.
The spinal cord segments were sectioned into 20-μm slices with a cryomicrotome. After blocking the sections with 10% goat serum containing 0.3% Triton X-100 for 1 h at 37°C, the slices were incubated with anti-Iba-1 antibody (rabbit, 1:500, Abcam) in 10% goat serum overnight at 4°C. After overnight incubation, the sections were rinsed three times with PBS, and the slices were incubated with FITC-conjugated secondary antibody (1:1000, Abcam) for 2 h at 37°C. Then, all sections were washed for three times again and counterstained with diamidino-2-phenylindole dihydrochloride (1:1000; Beyotime Corp., Shanghai, China) for 5 min. Finally, the sections were mounted and imaged under a fluorescence microscope (Leica, Germany).
Total protein was extracted from the dissected L4-5 spinal cord segments using RIPA lisate (Beyotime Corp., Shanghai, China). After centrifugation, the supernatant was collected and the protein concentration was determined using the bicinchoninic acid (BCA) method (Thermo Scientific). Then, the proteins in the lysate samples were separated using SDS–polyacrylamide gels (Beyotime, Shanghai, China) and electrically transferred onto nitrocellulose membranes. After blockage, the membranes with 5% milk for 2 h at 37°C, the membranes were incubated with anti-Iba-1 antibody (rabbit, 1:1000, Abcam) and anti-β-actin monoclonal primary antibody (rabbit, 1:2000, Abcam) overnight at 4°C. After overnight incubation, the blots were washed three times with TBST for 5 min each; then, the membranes were further incubated with a horseradish peroxidase-conjugated secondary antibody (1:2000, Beyotime Corp., Shanghai, China) at 37°C for 2 h. The desired proteins were visualized using ECL PLUS reagent and the chemiluminescence signals were captured on Hyperfilm (Millipore). The integrated optical density of the bands was analyzed by a Gel-Pol analyzer (National Institutes of Health).
Rats were induced with deep anesthesia on postoperative day 14 after performing the last behavioral test. The anesthetized rats were perfused transcardially with PBS. Ipsilateral L4-5 spinal cord segments were immediately dissected and diced into small pieces with ophthalmic scissors. After digestion of the minced tissue with 2 mg/ml of collagenase D (Roche, Basel, Switzerland) for 15 min and blocking with 10% fetal bovine serum (Beyotime, Shanghai, China) at room temperature, single cell suspension of the spinal cord was prepared using the Percoll gradient centrifugation method. For Th2 analysis, the cells were resuspended in DEME containing 10% fetal bovine serum. Then, the cells were stimulated for 4–6 h using a leukocyte activation cocktail (eBioscience) according to the manufacturer's protocol. After staining with anti-rat CD4 antibody for 30 min at 4°C, the cells were fixed and permeabilized using the Fixation/Permeabilization Kit (eBioscience) for intracellular staining with anti-rat IL-4 antibody (eBioscience). Th2 cell distribution was measured by fluorescent-activated cell sorting analysis of the cytometer (eBioscience).
Enzyme-linked immunosorbent assay
The ipsilateral L4-5 spinal cords were lysed and homogenized with RIPA lysis buffer containing phenylmethylsulfonyl fluoride at 4°C. After centrifugation, the supernatant was collected and protein concentration of the supernatant was determined using the BCA method (Thermo Scientific). The concentration of IL-10 protein was detected using ELISA kits (Shanghai bridge club, Shanghai, China) according to the manufacturer's instructions.
Data were processed using the SPSS 17.0 software. The quantitative data are presented as mean ± standard deviation. Data of behavioral tests from the four groups were analyzed using two-way repeated measures analysis of variance (ANOVA), followed by Holm-Sidak post hoc analysis. Other data were analyzed using one-way ANOVA followed by the LSD or SNK post hoc test. P < 0.05 was considered statistically significant.
| Results|| |
Electroacupuncture alleviated neuropathic pain caused by spared nerve injury
The MWT and TWL were used to determine mechanical allodynia and thermal hyperalgesia of the experimental rats after SNI surgery. For MWT and TWL, an analysis by two-way ANOVA indicated a significant effect of group (P < 0.05), time (P < 0.05), and time × group (P < 0.05). Moreover, the MWT in the SNI and NEA groups evidently decreased with time as compared with the Sham group. However, MWT in the EA group was significantly higher than that in the SNI and NEA groups [Figure 1]a, P < 0.05]. Correspondingly, the TWL in the SNI, NEA, and EA groups exhibited a remarkable reduction on postoperative day 3. In addition, the SNI and NEA groups maintained a downward trend for TWL with time prolonged, while the EA group exhibited a slightly upward trend from postoperative day 3 to day 14 [Figure 1]b, P < 0.05]. These results revealed that rats with SNI developed mechanical allodynia and thermal hyperalgesia postoperatively. EA at ST36 and GB34 induced an analgesia in rats with SNI, but EA at the non-acupoint did not show any anti-nociceptive effect.
|Figure 1: Effects of EA on allodynia and hyperalgesia in SNI-induced neuropathic pain. (a) MWT and (b) TWL were examined at 1 d before SNI and 3 d, 7 d, 14 d post SNI. MWT: Mechanical withdrawal threshold, TWL: Thermal withdrawal latency, SNI: Spared nerve injury, EA: Electroacupuncture, NEA: Non-acupoint electroacupuncture. Data are presented as the mean ± Sprague-Dawley Significance of MWT and TWL were analyzed with two-way analysis of variance followed by Holm-Sidak post hoc analysis. *P < 0.05 versus sham group, #P < 0.05 versus SNI and NEA groups (n = 6/group)|
Click here to view
Electroacupuncture suppressed the expression of microglia in the spinal cord of rats with spared nerve injury
The central sensitization of neuropathic pain is closely associated with the activation and overexpression of microglia in the spinal cord. Accordingly, immunofluorescent staining and western blotting of the microglial marker (Iba-1) was used to evaluate the microglial activation at the L4-5 spinal cord. As shown in [Figure 2], the fluorescence density and relative expression of Iba-1 in the SNI and NEA groups were obviously increased on postoperative day 14 as compared with the Sham group (P < 0.05). Moreover, the fluorescence density and relative expression of Iba-1 significantly decreased in the EA group as compared with the SNI and NEA groups on postoperative day 14 (P < 0.05). Our data revealed that persistent EA treatment of rats with SNI could suppress microglial activation in the spinal cord, EA of the non-acupoint conferred marginal effects.
|Figure 2: Effects of EA treatment on activation of microglia. (a) Representative immunofluorescence staining pictures showed the expression of Iba-1 in the ipsilateral L4-6 spinal dorsal horn at postoperative day 14 in Sham, SNI, EA, and NEA group, bar = 50 μ. (b) The western blot bands and integrated optical density of Iba1/β-actin in the L4-6 spinal cord at postoperative day 14 in Sham, SNI, EA, and NEA group. SNI: Spared nerve injury, EA: Electroacupuncture, NEA: Non-acupoint electroacupuncture. Data were presented as the mean ± Sprague-Dawley Significance of relative Iba1 levels were analyzed with one-way analysis of variance followed by Student–Newman–Keuls post hoc test. *P < 0.05 versus Sham group, #P = 0.05 versus SNI group and NEA groups (n = 4/group)|
Click here to view
EA suppressed the activity of Janus kinase 2/signal transducer and activator of transcription 3 signaling
A previous study has demonstrated that JAK2 and STAT3 phosphorylation, but not expression, is dramatically upregulated in rat models of neuropathic pain. In addition, the upregulation of p-JAK2 and p-STAT3 were regarded as the activation of JAK2/STAT3 signaling pathway. Therefore, to explore the involvement of JAK2/STAT3 signaling pathway in EA treatment, we investigated the phosphorylation levels of JAK2/STAT3 by western blotting [Figure 3]. The data showed that the levels of p-JAK2 and p-STAT3 were significantly increased in the SNI and NEA groups on postoperative day 14 as compared with the Sham group (P < 0.05). After EA treatment of rats with SNI, the phosphorylation levels of JAK2/STAT3 demonstrated a significant decrease (P < 0.05). Thus, our results revealed that EA could restrain the activation of JAK2/STAT3 signaling pathway in rats with SNI. The data also indicated that EA treatment of the non-acupoint did not affect the phosphorylation status of JAK2 and STAT3.
|Figure 3: Effects of EA on the activation of JAK2/STAT3 signaling after SNI. (a) Representative bands of p JAK2 and p STAT3 in the L4 6 spinal cord at postoperative day 14 in Sham, SNI, EA, and NEA group. (b, c) The integrated optical density of p JAK2/β actin and p STAT3/β actin in the L4 6 spinal cord at postoperative day 14 in Sham, SNI, EA, and NEA group. SNI: Spared nerve injury, EA: Electroacupuncture, NEA: Non-acupoint electroacupuncture. Data were presented as the mean ± Sprague-Dawley Significance of relative p-JAK2 and p-STAT3 levels were analyzed with one-way analysis of variance followed by Student–Newman–Keuls post hoc test. *P < 0.05 versus Sham group, #P < 0.05 versus SNI group and NEA groups (n = 4/group)|
Click here to view
EA promoted the infiltration of Th2 and upregulated the secretion of interleukin-10 in the spinal cord after spared nerve injury
To determine the effect of EA on the infiltration of Th2 in the spinal cord of rats with SNI, flow cytometry was performed. An evidently upregulated infiltration of Th2 into the spinal cord was observed in the EA group as compared with that in the SNI and NEA groups on postoperative day 14 [Figure 4]a and [Figure 4]b; P < 0.05]. In addition, the level of IL-10 in the L4-5 spinal cord of the EA-treated rats was significantly upregulated as compared with the SNI and NEA groups [Figure 4]c and [Figure 4]d; P < 0.05]. These results reveal the anti-inflammatory effects of EA therapy in rats with SNI. However, the Th2 cells and IL-10 levels in the L4-5 spinal cord were not significantly different from those in the SNI and NEA groups, which indicated that the non-acupoint EA had little effects on anti-inflammation in rats with SNI.
|Figure 4: The anti-inflammatory effects of EA treatment on rats after SNI. (a) Representative flow cytometry pictures of Th2 in the L4-6 spinal cord in Sham, SNI, EA, and NEA group at postoperative day 14. (b) The percentage of Th2 cells in the L4-6 spinal cord at postoperative day 14 in Sham, SNI, EA, and NEA group. (c) The protein levels of interleukin-10 in L4-6 spinal cord were examined by ELISA at postoperative day 14. SNI: Spared nerve injury, EA: Electroacupuncture, NEA: Non-acupoint electroacupuncture, ELISA: Enzyme-linked immunosorbent assay. Data were presented as the mean ± Sprague-Dawley Significance of Th2% and interleukin-10 levels were analyzed with one-way analysis of variance followed by Student–Newman–Keuls post hoc test. *P < 0.05 versus Sham group, #P < 0.05 versus SNI group and NEA groups (n = 4/group)|
Click here to view
| Discussion|| |
In the present study, we have demonstrated the anti-nociceptive effects of EA stimulation at GB34 and ST36 in a rat model of SNI. Infiltration of Th2 cells and secretion of IL-10 in the spinal cord is involved in its analgesic activity. In addition, the increased activation of microglia, p-JAK2, and p-STAT3, were suppressed by persistent EA therapy in rats with SNI.
Electroacupuncture alleviated neuropathic pain in rats with spared nerve injury
The pain threshold of rats with SNI significantly decreased after the SNI operation and sustained until postoperative day 14, which was similar to the results reported previously. Following cumulative EA treatment, the MWT and TWL dramatically increased as compared with that in the SNI group, suggesting that EA stimulation conferred an analgesic effect, which was identical to the outcomes of other recent studies., However, the therapeutic effects of EA therapy are determined by various factors, including the current intensity, stimulating frequency and duration, and even the location of stimulation. Our data revealed that EA at the non-acupoint did not confer any apparent anti-nociceptive effect in rats with SNI. Therefore, it is important to accurately locate the acupoint for efficacious EA treatment. In addition, previous studies have verified that 2-Hz EA offered better analgesic outcome compared to that with 100 Hz., Furthermore, EA with 2 Hz had analgesic effects in this study. Therefore, different manual manipulations of EA should be adopted according to specific circumstances and are very important in EA treatment.
Electroacupuncture promoted Th2 infiltration in the spinal cord
Cytokines and neutrophils are indispensable in the early stage of acute pain, whereas subsets of T cells seem to play a key role in neuropathic pain. Recently, the imbalance of Th1/Th2 in the spinal cord was demonstrated in an animal model of neuropathic pain. An autoimmune reaction characterized by a Th1 profile has been consistently reported in the process of aggravation from acute to chronic pain state. Therefore, modulating the immunological balance between T helper subsets has been demonstrated as a potential strategy for inducing analgesia. Consistently, we have demonstrated that EA is involved in promoting Th2 infiltration in the spinal cords of rats with SNI. Accumulating evidence has verified that EA stimulation improves inflammatory responses through restoration of the Th1/Th2 balance, promotion of Th2 differentiation, and curbing Th1 differentiation., Regarding T help cells as the fundamental elements of the adaptive immune system, a relative lack of Th2 cells can induce neuroinflammation. In fact, the anti-inflammatory effect of Th2 is dependent on the release of cytokines such as IL-10. In this experiment, the concentration of IL-10 in the spinal cord was obviously different among the four experimental groups, and the difference in IL-10 concentration was strongly associated with the levels of Th2 cells. It is well-established that IL-10 acts as an anti-inflammatory cytokine, controlling microglial activation by influencing its innate activities. As aforementioned, the current results of this study indicate that the analgesic effects of EA might be attributed to its anti-inflammatory role in the spinal cord via the promotion of Th2 infiltration and IL-10 secretion.
Electroacupuncture inhibited the activation of spinal cord microglia and overexpression of Janus kinase 2/signal transducer and activator of transcription 3in rats with spared nerve injury
The over-activation of microglia has been implicated in the pathogenesis of neuropathic pain. In this process, M1 polarization of microglia correlates with the induction of neuroinflammation through the secretion of pro-inflammatory cytokines. Moreover, the M1 phenotype microglia exhibited remarkably increased expression of Iba-1. Therefore, detecting the expression level of Iba-1 might be a potential way to evaluate the activation of M1 microglia in the spinal cord in rats with SNI. As expected, we found upregulation of Iba-1 (microglial marker) in the L4-5 spinal cord segments of rats with SNI. However, this upregulation was reversed after repeated EA stimulation. Indeed, our results revealed that EA treatment might hinder microglial activation. Consistent with our study, Xia et al. reported that 2-Hz EA relieved SNI-induced neuropathic pain, and blockade of HMGB1/NF-κB signaling in the spinal cord was involved in mediating the analgesic effects of EA. The activation of JAK2/STAT3 signaling in the spinal cord was closely associated with the development of neuropathic pain. Previous studies have demonstrated that JAK2/STAT3 signaling is involved in the activation of astrocyte in the spinal cord of mice with SNI., In our experiment, we found the upregulation of p-JAK2 and p-STAT3 in the spinal cord of rats with SNI, indicating that JAK2/STAT3 signaling in the spinal cord plays a vital role in neuropathic pain. However, our study did not explore the correlation between JAK2/STAT3 signaling and astrocyte. Instead, the similarities between p-JAK2/p-STAT3 and microglia led us to hypothesize that the phosphorylation of JAK2/STAT3 also associated with the increased expression of microglia in the spinal cord of rats with SNI. Interestingly, the previous research has demonstrated that the JAK-STAT pathway serves as an indispensable innate signaling cascade, which leads to the microglial activation. Silencing the expression of STAT3 could inhibit LPS-induced microglial activation, thus reducing the release of harmful factors. Thus, the activation of JAK2/STAT3 signaling might promote microglial activation. In addition, the phosphorylation levels of JAK2 and STAT3 in rats with SNI were significantly suppressed by repeated EA stimulation in the present study. Therefore, we speculated that blocking JAK2/STAT3 signaling in the spinal cord may be the innate mechanism underlying the anti-nociceptive effect of EA in SNI-induced neuropathic pain.
| Conclusions|| |
In summary, the findings from this study revealed that sustained stimulation of EA at ST36 and GB43 could relieve neuropathic pain in rats with SNI. The potential mechanisms underlying this analgesic effect may be as follows: suppressing the activation of microglia and JAK2/STAT3 signaling, promoting the infiltration of Th2, and preventing the release of IL-10.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Treede RD, Jensen TS, Campbell JN, Cruccu G, Dostrovsky JO, Griffin JW, et al
. Neuropathic pain: Redefinition and a grading system for clinical and research purposes. Neurology 2008;70:1630-5.
Colloca L, Ludman T, Bouhassira D, Baron R, Dickenson AH, Yarnitsky D, et al
. Neuropathic pain. Nat Rev Dis Primers 2017;3:17002.
van Hecke O, Austin SK, Khan RA, Smith BH, Torrance N. Neuropathic pain in the general population: A systematic review of epidemiological studies. Pain 2014;155:654-62.
Zhao H, Alam A, Chen Q, A Eusman M, Pal A, Eguchi S, et al
. The role of microglia in the pathobiology of neuropathic pain development: What do we know? Br J Anaesth 2017;118:504-16.
Chen G, Zhang YQ, Qadri YJ, Serhan CN, Ji RR. Microglia in pain: Detrimental and protective roles in pathogenesis and resolution of pain. Neuron 2018;100:1292-311.
Hilla AM, Diekmann H, Fischer D. Microglia are irrelevant for neuronal degeneration and axon regeneration after acute injury. J Neurosci 2017;37:6113-24.
Chen Y, Shi Y, Wang G, Li Y, Cheng L, Wang Y. Memantine selectively prevented the induction of dynamic allodynia by blocking Kir2.1 channel and inhibiting the activation of microglia in spinal dorsal horn of mice in spared nerve injury model. Mol Pain 2019;15:1744806919838947.
Sommer C, Leinders M, Üçeyler N. Inflammation in the pathophysiology of neuropathic pain. Pain 2018;159:595-602.
Kobayashi M, Konishi H, Sayo A, Takai T, Kiyama H. TREM2/DAP12 signal elicits proinflammatory response in microglia and exacerbates neuropathic pain. J Neurosci 2016;36:11138-50.
Bian J, Zhang Y, Liu Y, Li Q, Tang HB, Liu Q. P2Y6 Receptor-mediated spinal microglial activation in neuropathic pain. Pain Res Manag 2019;2019:2612534.
Zhang TT, Xue R, Fan SY, Fan QY, An L, Li J, et al
. Ammoxetine attenuates diabetic neuropathic pain through inhibiting microglial activation and neuroinflammation in the spinal cord. J Neuroinflammation 2018;15:176.
Buttgereit A, Lelios I, Yu X, Vrohlings M, Krakoski NR, Gautier EL, et al
. Sall1 is a transcriptional regulator defining microglia identity and function. Nat Immunol 2016;17:1397-406.
Recasens M, Shrivastava K, Almolda B, González B, Castellano B. Astrocyte-targeted IL-10 production decreases proliferation and induces a downregulation of activated microglia/macrophages after PPT. Glia 2019;67:741-58.
Krasemann S, Madore C, Cialic R, Baufeld C, Calcagno N, El Fatimy R, et al
. The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity 2017;47:566-81.
Qin H, Buckley JA, Li X, Liu Y, Fox TH 3rd
, Meares GP, et al
. Inhibition of the JAK/STAT pathway protects against α-Synuclein-induced neuroinflammation and dopaminergic neurodegeneration. J Neurosci 2016;36:5144-59.
Alhadidi Q, Shah ZA. Cofilin mediates LPS-induced microglial cell activation and associated neurotoxicity through activation of NF-κB and JAK-STAT pathway. Mol Neurobiol 2018;55:1676-91.
Cianciulli A, Dragone T, Calvello R, Porro C, Trotta T, Lofrumento DD, et al
. IL-10 plays a pivotal role in anti-inflammatory effects of resveratrol in activated microglia cells. Int Immunopharmacol 2015;24:369-76.
Chassot M, Dussan-Sarria JA, Sehn FC, Deitos A, de Souza A, Vercelino R, et al
. Electroacupuncture analgesia is associated with increased serum brain-derived neurotrophic factor in chronic tension-type headache: A randomized, sham controlled, crossover trial. BMC Complement Altern Med 2015;15:144.
Ntritsou V, Mavrommatis C, Kostoglou C, Dimitriadis G, Tziris N, Zagka P, et al
. Effect of perioperative electroacupuncture as an adjunctive therapy on postoperative analgesia with tramadol and ketamine in prostatectomy: A randomised sham-controlled single-blind trial. Acupunct Med 2014;32:215-22.
Long Q, Li J, Wen Y, He B, Li YZ, Yue CC, et al
. Effect of electroacupuncture preconditioning combined with induced urination on urinary retention after milligan-morgan hemorrhoidectomy. Zhongguo Zhen Jiu 2019;39:821-4.
Zhang W, Lang S, Zheng Y, Qin X, Chen H, You Y, et al
. The effects of transcranial direct current stimulation versus electroacupuncture on working memory in healthy subjects. J Altern Complement Med 2019;25:637-42.
Lu SF, Yuan J, Ding YJ, Yu ML, Fu SP, Hong H, et al
. Effect of electroacupuncture on myocardial infarct size and expression of inflammatory cytokines and sympathetic-active substances in the myocardial ischemic tissue of rats. Zhen Ci Yan Jiu 2019;44:313-8.
Fang JF, Fang JQ, Shao XM, Du JY, Liang Y, Wang W, et al
. Electroacupuncture treatment partly promotes the recovery time of postoperative ileus by activating the vagus nerve but not regulating local inflammation Sci Rep 2017;7:39801.
Liu YM, Liu XJ, Bai SS, Mu LL, Kong QF, Sun B, et al
. The effect of electroacupuncture on T cell responses in rats with experimental autoimmune encephalitis. J Neuroimmunol 2010;220:25-33.
Murray PJ. Understanding and exploiting the endogenous interleukin-10/STAT3-mediated anti-inflammatory response. Curr Opin Pharmacol 2006;6:379-6.
Li S, Gu P, Tu W, Jiang X, Chen W, Hu Q, et al
. Effects of electroacupuncture on activation of microglia cells in spinal cord in rats with neuropathic pain. Zhongguo Zhen Jiu 2017;37:411-6.
Zhou J, Fan Y, Chen H. Analyses of long non-coding RNA and mRNA profiles in the spinal cord of rats using RNA sequencing during the progression of neuropathic pain in an SNI model. RNA Biol 2017;14:1810-26.
Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994;53:55-63.
Wang Y, Xue M, Xia Y, Jiang Q, Huang Z, Huang C. Electroacupuncture treatment upregulates α7nAChR and inhibits JAK2/STAT3 in dorsal root ganglion of rat with spared nerve injury. J Pain Res 2019;12:1947-5.
Descalzi G, Mitsi V, Purushothaman I, Gaspari S, Avrampou K, Loh YE, et al
. Neuropathic pain promotes adaptive changes in gene expression in brain networks involved in stress and depression. Sci Signal 2017;10:eaaj1549.
Wang JY, Gao YH, Qiao LN, Zhang JL, Duan-Mu CL, Yan YX, et al
. Repeated electroacupuncture treatment attenuated hyperalgesia through suppression of spinal glial activation in chronic neuropathic pain rats. BMC Complement Altern Med 2018;18:74.
Li C, Ji BU, Kim Y, Lee JE, Kim NK, Kim ST, et al
. Electroacupuncture enhances the antiallodynic and antihyperalgesic effects of milnacipran in neuropathic rats. Anesth Analg 2016;122:1654-62.
Yu Z, Luo L, Li Y, Wu Q, Deng S, Lian S, et al
. Different manual manipulations and electrical parameters exert different therapeutic effects of acupuncture. J Tradit Chin Med 2014;34:754-8.
Xing GG, Liu FY, Qu XX, Han JS, Wan Y. Long-term synaptic plasticity in the spinal dorsal horn and its modulation by electroacupuncture in rats with neuropathic pain. Exp Neurol 2007;208:323-32.
Huang C, Li HT, Shi YS, Han JS, Wan Y. Ketamine potentiates the effect of electroacupuncture on mechanical allodynia in a rat model of neuropathic pain. Neurosci Lett 2004;368:327-31.
Perkins NM, Tracey DJ. Hyperalgesia due to nerve injury: Role of neutrophils. Neuroscience 2000;101:745-57.
Tao L, Ding Q, Gao C, Sun X. Resveratrol attenuates neuropathic pain through balancing pro-inflammatory and anti-inflammatory cytokines release in mice. Int Immunopharmacol 2016;34:165-72.
Zhang X, Wu Z, Hayashi Y, Okada R, Nakanishi H. Peripheral role of cathepsin S in Th1 cell-dependent transition of nerve injury-induced acute pain to a chronic pain state. J Neurosci 2014;34:3013-22.
He FY, Feng WZ, Zhong J, Xu W, Shao HY, Zhang YR. Effects of propofol and dexmedetomidine anesthesia on Th1/Th2 of rat spinal cord injury. Eur Rev Med Pharmacol Sci 2017;21:1355-61.
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;109:1666-73.
González H, Pacheco R. T-cell-mediated regulation of neuroinflammation involved in neurodegenerative diseases. J Neuroinflammation 2014;11:201.
Tsuda M. Microglia in the spinal cord and neuropathic pain. J Diabetes Investig 2016;7:17-26.
Jin GL, He SD, Lin SM, Hong LM, Chen WQ, Xu Y, et al
. Koumine Attenuates Neuroglia Activation and Inflammatory Response to Neuropathic Pain. Neural Plast 2018;2018:9347696.
Zhang Q, Lu Y, Bian H, Guo L and Zhu H. Activation of the α7 nicotinic receptor promotes lipopolysaccharide-induced conversion of M1 microglia to M2. Am J Transl Res 2017;9:971-85.
Xia YY, Xue M, Wang Y, Huang ZH, Huang C. Electroacupuncture alleviates spared nerve injury-induced neuropathic pain and modulates HMGB1/NF-κB signaling pathway in the spinal cord. J Pain Res 2019;12:2851-63.
Liu S, Mi WL, Li Q, Zhang MT, Han P, Hu S, et al
. Spinal IL-33/ST2 signaling contributes to neuropathic pain via neuronal CaMKII-CREB and astroglial JAK2-STAT3 cascades in mice. Anesthesiology 2015;123:1154-69.
Liu S, Li Q, Zhang MT, Mao-Ying QL, Hu LY, Wu GC, et al
. Curcumin ameliorates neuropathic pain by down-regulating spinal IL-1β via suppressing astroglial NALP1 inflammasome and JAK2-STAT3 signalling. Sci Rep 2016;6:28956.
Popiolek-Barczyk K, Mika J. Targeting the microglial signaling pathways: New insights in the modulation of neuropathic pain. Curr Med Chem 2016;23:2908-28.
Przanowski P, Dabrowski M, Ellert-Miklaszewska A, Kloss M, Mieczkowski J, Kaza B, et al
. The signal transducers Stat1 and Stat3 and their novel target Jmjd3 drive the expression of inflammatory genes in microglia. J Mol Med (Berl) 2014;92:239-54.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]