Electroacupuncture for pain relief in labour inhibits spinal p38 MAPK-mediated prostaglandin E2 release and uterine prostaglandin E2 receptor expression in rats

Gui-Xiu Jiang1, Qiu-Yan Jiang2, Hai-Xia Mo2, Li Li2 and Meng-Ying Wang2

Abstract

Background: p38 mitogen-activated protein kinase (p38 MAPK) activation involves the release of prostaglandin E2 (PGE2) and hyperalgesia. We have previously reported that electroacupuncture (EA) relieves labour pain, but the potential mechanisms remain unclear.
Objective: To observe the effects of EA on labour pain intensity, serum PGE2 levels and the p38 MAPK signalling pathway in rats during labour.
Methods: Female rats copulated with male rats to induce pregnancy, and then received castor oil to trigger labour. During labour, rats remained untreated (Control group, n=30) or were treated with remifentanil (n=30) or EA at Jiaji (n=30) or SP6+LI4 (n=30), respectively. The warm water tail-flick test was used to assess labour pain. Serum PGE2 levels were measured by ELISA. Protein expression of prostaglandin E2 receptor (PGER2), p38 MAPK and phospholipase A2 (PLA2) were analysed by Western blotting, and mRNA levels were measured by real-time PCR.
Results: EA treatment at Jiaji or SP6+LI4 significantly relieved labour pain, decreased serum PGE2 levels and inhibited protein and gene expression of PGER2 in the myometrium. Moreover, EA reduced protein expression of PLA2 and p38 MAPK, and inhibited phosphorylation of p38 MAPK in the lumbar spinal cord but not in the cerebral grey matter. Additionally, EA markedly decreased mRNA levels of p38 MAPK in the lumbar spinal cord and significantly reduced PLA2-IV mRNA levels in both the lumbar spinal cord and cerebral grey matter.
Conclusions: This study indicates that EA relieves labour pain through, at least in part, inhibition of spinal p38 MAPKmediated PGE2 release and uterine PGER2 expression in rats.

Keywords
electroacupuncture, labour pain management, prostaglandin E2, p38 mitogen-activated protein kinase, phospholipase A2 Accepted 13 March 2018

Introduction

During the childbirth process, women often suffer from labour pain, a challenging clinical problem. Labour pain causes maternal tension and fear, and even results in vascular spasm and uncoordinated contraction of the uterus, leading to prolonged labour and fetal hypoxia, which is a serious threat to the safety of the mother and fetus. Although pharmacological analgesic methods, such as epidural or spinal injections of analgesics and anaesthetic agents, have achieved beneficial outcomes in clinical practice, these analgesic therapies are not perfect choices for labour pain relief because of potential side effects, such as maternal apnoea and neonatal acidosis.1,2 Therefore, non-pharmacological analgesia is becoming more and more popular during labour, including massage, acupressure and acupuncture.3,4
Acupuncture, a non-pharmacological form of analgesia with few side effects, not only relieves labour pain but also relaxes women in labour and decreases the use of pharmacological analgesia.5,6 Electroacupuncture (EA), a modified form of acupuncture that uses electrical stimulation, is also effective for labour pain and shortens the duration of labour.7 We have already reported that EA produces an analgesic effect on labour pain.8 A further study indicated that EA decreased labour pain, increased serum dynorphin (DYN) levels, and enhanced the protein and gene expression of prodynorphin (PDYN) and κ-opioid receptor for DYN in the lumbar spinal cord of rats in labour.9 Moreover, EA reduced serum norepinephrine (NE) levels, increased protein and gene expression of NE transporter and α2 adrenergic receptor in the grey matter, but decreased their expression in the lumbar spinal cord.10 However, the mechanisms by which EA relieves labour pain remain to be fully elucidated.
Studies have reported that p38 mitogen-activated protein kinase (p38 MAPK) activation is necessary for spinal prostaglandin E2 (PGE2) release and thermal hyperalgesia,11 and that p38 MAPK inhibition attenuates PGE2 release and hyperalgesia11 and contributes to neuropathic pain,12 implying a close relationship between p38 MAPK signalling and pain. The present study aimed to observe the effects of EA on labour pain, serum PGE2 levels and the p38 MAPK signalling pathway in rats in labour.

Methods

Animals and treatments

The Animal Experimental Centre of Guangxi Medical University (Nanning, China) provided Sprague-Dawley rats, aged 3 months and weighing 250–350 g. After 7 days of environmental adaption, the female rats were maintained with male rats (1:1 ratio) to induce copulation indicated by a sperm-positive vaginal smear, and then given castor oil to trigger labour as previously described.9 The rats in labour were randomly assigned to three groups according to the different treatments: the control group (n=30), which did not received any treatment; the remifentanil group (n=30), which received remifentanil at a dose of 5 µg/kg/min (Yichang Humanwell Pharmaceutical Co, Ltd, Yichang, China) by injection into the caudal vein; and the EA group (n=60), which was randomly divided into two subgroups: the Jiaji group (n=30) and the SP6+LI4 group (n=30). The rats in the Jiaji group were given EA at bilateral Jiaji, and the rats in the SP6+LI4 group received EA at SP6 (Sanyinjiao) and LI4 (Hegu). Acupuncture needles (0.17 mm in diameter, 7 mm in length, Tianjin Xinglin College Medical Instrument Co, Ltd, Tianjin, China) were first inserted at Jiaji (6 mm depth), SP6 (3 mm depth) and LI4 (1 mm depth), respectively. Then, the needle handles were connected to a HANS EA therapeutic instrument (LH202H, Beijing Huawei Co, Ltd, Beijing, China) and stimulated. The frequency of the EA therapeutic instrument was adjusted to 2/100 Hz, and the intensity was 0.1 mA. The stimulation could induce slight shaking of the rat limb muscles and was performed for 20 min. The stimulation was repeated every 2 hours until the birth of the last pup. The Ethics Committee of Guangxi University of Chinese Medicine approved this study (reference no. 201302026). All animal experiments were conducted in accordance with the National Institutes of Health guidelines for the use and care of animals.

Labour pain evaluation
Before and after treatment, the labour pain of the rats in each group was assessed by the warm water tail-flick test as previously described.9 In brief, the distal part of the rats’ tails (4 cm length) were dipped into water at a temperature of 50±0.5°C. The time interval after which the tail withdrew from the water was recorded and taken as a measure of pain threshold. A 40 s cut-off was set to avoid tail damage. The procedure was repeated three times on each rat, and the average value was used for statistical analysis. The value was positively correlated with the ability of rats to tolerate pain.

Serum PGE2 analysis

After treatment and subsequent labour pain evaluation, the rats were anaesthetised with chloral hydrate (400 mg/kg) by intraperitoneal injection, and then blood was collected and allowed to clot for 2 hours at room temperature. Blood was centrifuged (1000 g for 15 min, 4°C) and the serum (supernatant) collected to analyse PGE2 using a rat PGE2 ELISA kit (CusaBio, Wuhan, China) according to the manufacturer’s instructions.

Western blotting

After treatment and subsequent blood collection, the rats were decapitated and the cerebral grey matter, lumbar spinal cord and myometrium were dissected out and stored in a fridge at −80°C. Protein expression in the aforementioned tissues was measured by Western blotting as previously described with certain modifications.9 Briefly, total protein was extracted from the tissues using a Tissue Total Protein Extraction kit (Sangon Biotech Co, Ltd, Shanghai, China) according to the manufacturer’s instructions. The protein concentrations were determined by the BCA (bicinchoninic acid) method. After denaturation by boiling for 4 min, the protein was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride membranes (Sangon Biotech Co, Ltd, Shanghai, China). The membranes were blocked with TBST (tris-buffered saline and Polysorbate 20) containing 5% non-fat milk for 1–2 hours at room temperature and then incubated with primary antibodies to p38 MAPK, phospholipase A2 (PLA2) (Cell Signalling Technology, Danvers, MA, USA), p-p38 MAPK (phosphorylated Thr180/pTYr182, MyBiosourse, San Diego, CA, USA) and prostaglandin E2 receptor (PGER2, R&D Systems, Inc, Minneapolis, MN, USA) overnight at 4°C. After washing with TBST the next day, the membranes were incubated with appropriate secondary antibodies (Thermo Fisher Scientific, Rockford, IL, USA) coupled to horseradish peroxidase (HRP) for 1 hour at room temperature. The immunoreactive bands were visualised by ECL detection reagents (Thermo Fisher Scientific, Rockford, IL, USA).

Measurement of mRNA

Total RNA was isolated from the cerebral grey matter, lumbar spinal cord and myometrium with TRIzol reagent (Invitrogen Life Technologies Carlsbad, CA, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesised from total RNA and then kept in a fridge at −20°C until determination of mRNA levels. Real-time PCR was performed to quantify the relative gene abundance with cDNA samples using a real-time PCR system (LightCycler 480, Roche). The reaction conditions were: holding stage, 95°C for 10 min; cycling stage, denaturation, 94°C for 15 s, anneal, 58°C for 10 s, and extension, 72°C for 10 s. The primer pairs used were as follows: p38 MAPK, forward, 5’-GAT TGG TCT GTT GGA TGT GTT CAC-3’, reverse, 5’-GCT TCT GGC ACT TCA CGA TGT-3’; PLA2-IV, forward, 5’-GTC TCC TCA CAC TGG CTT GGT-3’, reverse, 5’-CAT TCT TCC TGG TCA CCT TCT CAA T-3’; PGER2, forward, 5’-GCG GAT TGT CTG GCA GTA GC-3’, reverse, 5’-TGG CGA AGG TGA TGG TCA TAA TG-3’; GAPDH, forward, 5’-AAG TTC AAC GGC ACA GTC AAG G-3’, reverse, 5’-GAC ATA CTC AGC ACC AGC ATC AC-3’.

Statistical analysis

The data were analysed with SPSS 20.0 for Windows and presented as mean±SD. The difference in pain threshold before and after treatment was analysed using the paired sample t-test. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test when there were more than two groups. A value of p<0.05 was considered statistically significant.

Results

EA relieves labour pain

Before treatment, the pain thresholds of the rats in labour in the four groups were similar to each other (Figure 1, all P>0.05). There was no significant difference in pain threshold in the control group before and after treatment (P>0.05). After treatment, however, the pain threshold was markedly increased in the Jiaji and SP6+LI4 groups when compared with before treatment (both P<0.01). The pain threshold after treatment in the SP6+LI4 group did not differ significantly from that in the Jiaji group (P>0.05). In this study, remifentanil, a synthetic μ opioid receptor agonist for labour pain, was taken as a positive control for EA and significantly increased the pain threshold (P<0.01), which was higher than that in each EA group (both P<0.01). Together, the results indicate that EA relieves labour pain in rats.

EA decreases serum PGE2 levels

As shown in Figure 2, EA treatment at Jiaji or SP6+LI4 dramatically reduced serum PGE2 levels in rats in labour when compared with the control group (both P<0.01). Furthermore, there was no significant difference in serum PGE2 levels between the two EA groups (P>0.05). Additionally, serum PGE2 levels in the remifentanil group were much lower than that in the control group (P<0.01) or either EA group (P<0.01). The data suggest that EA decreases serum PGE2 levels in rats in labour.

EA inhibits protein and gene expression of PGER2 in the myometrium

Figure 3 shows protein and gene expression of PGER2 in the myometrium. PGER2 protein expression and mRNA levels in each EA group were much lower than those in the control group (all P<0.01). Remifentanil also decreased PGER2 protein and gene expression (P<0.01), both of which were lower than those in the Jiaji or SP6+LI4 group (all P<0.01), respectively. Additionally, there were similar levels of PGER2 expression between the Jiaji and the SP6+LI4 groups (P>0.05).

EA decreases protein expression of p38 MAPK and PLA2, and inhibits phosphorylation of p38 MAPK in the lumbar spinal cord

EA treatment at Jiaji or SP6+LI4 significantly reduced protein expression of p38 MAPK (Figure 4A) and PLA2 (Figure 4C), and decreased phosphorylation of p38 MAPK (Figure 4A) in the lumbar spinal cord of rats in labour when compared with the control group (all P<0.05 or P<0.01). Also, the level of expression of each protein in the two EA groups was higher than that in the remifentanil group (all P<0.05 or P<0.01). Remifentanil also notably reduced protein expression of p38 MAPK and PLA2, and inhibited p38 MAPK phosphorylation in the lumbar spinal cord (all P<0.01). Similarly, there were no significant differences in expression of either protein in the lumbar spinal cord between the two EA groups (all P>0.05). Additionally, neither EA nor remifentanil significantly changed protein expression of p38 MAPK (Figure 4B) or PLA2 (Figure 4D), or phosphorylation of p38 MAPK (Figure 4B) in the cerebral grey matter when compared with the control group (all P>0.05).

Effects of EA on mRNA levels of p38 MAPK and PLA2-IV

Compared with the control group, EA treatment at Jiaji or SP6+LI4 markedly decreased p38 MAPK mRNA levels in the lumbar spinal cord but not in the cerebral grey matter (Figure 5A, P<0.01), where levels were higher than those in the remifentanil group (P<0.01). Furthermore, EA significantly decreased PLA2-IV mRNA levels not only in the lumbar spinal cord but also in the cerebral grey matter (Figure 5B, P<0.01). Interestingly, administration of remifentanil markedly inhibited mRNA levels of p38 MAPK in the lumbar spinal cord, and PLA2-IV in the lumbar spinal cord and cerebral grey matter (all P<0.01).

Discussion

Labour pain is a challenging clinical problem. Currently, more and more parturients hope to receive non-pharmacological analgesia because of the potential side effects of pharmacological analgesia including maternal apnoea and neonatal acidosis.1,2 In this study, EA at Jiaji or SP6+LI4 significantly increased the pain threshold of rats in labour, which was consistent with past reports and our previous studies,7–10 implying an analgesic effect of EA at Jiaji and SP6+LI4 on labour pain. It is well known that PGE2 is a key mediator of inflammation and contributes to pain secondary to inflammation and mechanical injury.13–15 Intrathecal administration of PGE2 induces hyperalgesia and allodynia.16,17 Elevated PGE2 levels increase binding to the receptor PGER2, and then the message is passed to the spinal cord and brain, thus promoting sensory neuron hyperexcitability leading to hyperalgesia and the maintenance of pain. Additionally, PGE2 can be released from cells of the lumbar spinal cord and amniotic membrane.18,19 Inhibition of PGE2 production relieves pain.20 It has been reported that EA alleviates the hyperalgesic state by inhibiting local PGE2 secretion.21 Therefore, we inferred that EA reduced labour pain due to PGE2. In this study, EA decreased serum PGE2 levels in rats in labour. Moreover, EA reduced protein and gene expression of PGER2 in the myometrium, potentially inhibiting the formation and transmission of the pain signal. This suggests the analgesic effect of EA on labour pain is closely related to a reduction in serum PGE2 levels and PGER2 expression in the myometrium.
Studies have indicated that p38 MAPK signalling plays a key role in controlling PGE2 release and generating pain hypersensitivity.11,22 Activation of p38 MAPK through phosphorylation leads to the activation of cytosolic PLA2,23,24 the enzymes of which hydrolyse fatty acid from the sn-2 position of glycerolipids to produce lysophospholipid, thereby regulating the release of arachidonic acid.25 Just as well known, arachidonic acid is the precursor of PGE2 through cyclooxygenase (COX) catalysis.26 In fact, PLA2 has been directly linked to the release of PGE2 and is associated with inflammatory hyperalgesia.27 Inhibition of p38 MAPK signalling by a p38 MAPK inhibitor blocks COX-2 induction, and abolishes spinal release of PGE2 and pain facilitation.28,29 Likewise, pre-treatment with a PLA2 inhibitor also decreased interleukin 6 (IL-6)-induced muscle hyperalgesia in mice.30 Therefore, p38 MAPK phosphorylation induces PLA2 activation and subsequent PGE2 release.31
In order to further reveal the mechanisms of action underlying the effects of EA on labour pain, we observed the impact of EA on expression of p38 MAPK and PLA2 in the central nervous system. In this study, EA not only inhibited protein expression of p38 MAPK, but also decreased phosphorylation of p38 MAPK in the lumbar spinal cord, but not in the cerebral grey matter. Similarly, EA reduced protein expression of PLA2 in the lumbar spinal cord, but had no effects in the cerebral grey matter. Moreover, EA inhibited mRNA levels of p38 MAPK in the lumbar spinal cord but not in the cerebral grey matter, while gene expression of PLA2-IV in the lumbar spinal cord and the cerebral grey matter was reduced. Liang et al. and Xu et al. have reported that EA exerts anti-hyperalgesia by inhibiting phosphorylation of p38 MAPK in the spinal cord,31,32 which is in line with the findings of our study.
Remifentanil, a synthetic μ opioid receptor agonist used for labour pain relief,33 was taken as a positive control for EA in this study. This study indicated that remifentanil relieved labour pain, decreased serum PGE2, and inhibited the protein and gene expression of PGER2 in the myometrium. Moreover, remifentanil reduced protein and gene expression of p38 MAPK and PLA2 in the lumbar spinal cord. These effects were in accordance with EA, and thereby lend further support to proposed mechanisms underlying the analgesic effects of EA during labour. Additionally, there were no differences in pain threshold, serum PGE2 levels or expression of p38 MAPK and PLA2 in the lumbar spinal cord between EA at Jiaji and at SP6+LI4, indicating similar effects and mechanisms of EA at these different sites. We have reported that EA relieves labour pain through the involvement of the spinal dynorphin/κ-opioid receptor system, central nervous norepinephrine transporter and α2 adrenergic receptor expression,9,10 implying that EA relieves labour pain through multiple mechanisms. Moreover, EA regulates the release of opioids that have a close relationship with pain,8,9,34 but the underlying mechanisms remain to be fully elucidated and need further study.
In conclusion, our study indicates that EA relieves labour pain through, at least in part, the inhibition of spinal p38 MAPK-mediated PGE2 release and uterine PGER2 expression in rats.

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