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Research Article | Open Access
Electroacupuncture-Induced Dynamic Processes of Gene Expression Levels of Endogenous Opioid Peptide Precursors and Opioid Receptors in the CNS of Goats
In order to investigate the dynamic processes of mRNA levels of proenkephalin, proopiomelanocortin, prodynorphin, and opioid receptors (-, -, and -receptor) induced by electroacupuncture (EA) in the central nerve system, goats were stimulated by EA of 60 Hz for 0.5 h at a set of Baihui, Santai, Ergen, and Sanyangluo points. The pain threshold was measured using the method of potassium iontophoresis. The mRNA levels of the three opioid peptide precursors and three opioid receptors were determined with quantitative real-time PCR and the levels of Met-enkephalin with SABC immunohistochemistry at 0.5 h before and at 0, 2, 4, 6, 8, 12, and 24 h after EA. The results showed that the pain threshold correlated () with Met-enkephalin immunoactivities in the measured nuclei and areas of goats. The analgesic aftereffect lasted for 12 h at least. The mRNA levels of the three opioid peptide precursors and three opioid receptors began to increase at 0 h, reached the peak during the time from 4 h to 6 h or at 12 h, and remained higher at 24 h after EA was discontinued. These results suggested that the initiation of gene expression of opioid peptides and the three receptors may be associated with EA-induced analgesic aftereffect.
Electroacupuncture (EA) is a modern version of acupuncture and extensively used in the clinic practice because it has better analgesic effect and its stimulation can be objectively quantified and controlled . EA has been used not only for effective treatment of painful diseases, but also for successful relief of pain in various operations, such as cesarean section, gastrectomy, enterectomy, and castration in human or animals [2–4]. Since the 1960s, many scientists have investigated the mechanism by which electroacupuncture induces analgesic effect. Early studies showed that analgesia induced by EA was involved in modulations of neurotransmitters (serotonin, acetylcholine, catecholamine, etc.) in the central nerve system (CNS) . Latter, studies verified that neuromodulators, especially some endogenous opioid peptides (enkephalin, β-endorphin, and dynorphin), played a more important role in EA-induced analgesia. EA can promote the release of different endogenous opioid peptides (EOPs) which act on their corresponding receptors (δ-, μ-, or κ-receptor) to exert analgesic effect [6–9].
Previous studies indicated that EA not only induces an “immediate analgesia,” but also causes an analgesic aftereffect (analgesia lasts for a while after EA is discontinued). This aftereffect plays an important role in the treatment of painful diseases and is conducive to the recovery from the surgery. So far the mechanisms by which acupuncture induces analgesic aftereffect have not been fully studied. Some reports showed that EA induced the gene expression of opioid peptide precursors, such as proopiomelanocortin (POMC, precursor of endorphin), proenkephalin (PENK, precursor of enkephalin), and prodynorphin (PDYN, precursor of dynorphin) in the CNS of rats [10–12]. The initiation of EOP gene expressions is inferred to replenish the consumed opioid peptides because opioid peptides are immediately decomposed by some specific enzymes after they are induced to release and act on their corresponding receptors. However, the roles of the gene expressions of endogenous opioid peptides and their receptors in EA-induced aftereffect are not completely confirmed.
Analgesia induced by EA has been proven to vary in animal species . In order to quantitatively estimate the degree of acupuncture-induced analgesia, some researchers compared the dosage of some anesthetic in the anesthetic group with its dosage in the EA plus anesthetic group with the same complete analgesia. They found that EA resulted in the reduction of the dosage of anesthetics in the EA plus anesthetic group in human, rat, and goat by 45%–55%, 50%–60%, and over 75%, respectively [13, 14]. It is clear that the analgesic effect induced by EA in goats (a ruminant) is superior to that in rats or human. Ruminants should be optimal model animals for researches on the mechanisms of EA-induced analgesia. In the present study, goats were stimulated with EA for 30 min to determine the relationships of the gene expression of Met-enkephalin (M-ENK), beta-endorphin (β-EP), and dynorphin (DYN-A) and their receptors (δ-, μ-, and κ-receptor) with pain threshold and levels of enkephalin (a representative of opioid peptides) in order to probe into the mechanisms of EA-induced aftereffect.
2. Materials and Methods
2.1. Animal Preparation
One hundred and eight healthy 1- to 2-year-old hybrid male goats, weighing 23–28 kg, purchased from the goat farm of Hubei Agricultural Academy of Science, were used in this experiment (54 goats for the measurement of gene expression levels of endogenous opioid peptides and opioid receptors, another 54 goats for the measurement of M-ENK levels). All goats drunk freely and were maintained on a dry grass diet which was supplemented with a cereal-based concentrate. They were dewormed and accustomed to being approached. Feed was withheld for 24 h before the start of the experiments. The experiments were performed in a quiet environment, and the ambient temperature fluctuated between 23°C and 24°C. The experimental protocol was approved by the Animal Care Center, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
A set of Baihui (hundred meetings), Santai (three platforms), Ergen (ear base), and Sanyangluo (three Yang communication) points was selected for EA. The anatomic location of these points has been described in detail in veterinary medicine [13, 15]. The Baihui and Santai points on the dorsal midline and the Ergen and Sanyangluo points on the left side of the body were chosen in this study. Needle insertion and EA were conducted with the method reported by Liu et al. . Experimental animals were restrained in right recumbency and stimulated with EA at 60 Hz for 0.5 h via WQ-6F Electronic Acupunctoscope (Beijing Xindonghua Electronic Instrument Co., Ltd., Beijing, China). The sham control goats which were only dealt with needles left in the acupoints for 0.5 h without electricity were restrained as the experimental goats.
2.3. Determination of Pain Threshold
At 0.5 h before and at 0, 2, 4, 6, 8, 12, and 24 h after EA, the pain threshold was measured on the center of the left flank using the method of potassium iontophoresis as described in detail by Cheng et al. . The pain threshold in the sham control was measured at 0.5 h before needle insertion and at 0 h after needle withdrawal.
2.4. Measurement of Gene Expression Levels of Endogenous Opioid Peptides and Opioid Receptors
Six goats were taken from the experimental goats at 0.5 h before and at 0, 2, 4, 6, 8, 12, and 24 h after EA, respectively, anesthetized with intravenous administration of xylidinothiazoline at 3 mg/kg, and slaughtered for the measurement of gene expression levels of endogenous opioid peptides and opioid receptors. According to the results of repeated pretest, gene expression of endogenous opioid peptides and opioid receptors in most nuclei and areas in the CNS reached a higher level at about 4 h after EA. Therefore, six sham control goats in the present study were euthanized for brain sampling at 4 h after needle withdrawal. The goat’s brain, hypophysis, and a part of the adjacent spinal cord were immediately taken out of the skull and cervical vertebral canal. The brain was transected into 17 sections quickly with the method described by Cheng et al. . The nuclei and areas were identified according to the photographic atlas of the goat brain and the morphological characteristics of the neurons [17–19]. The analgesia-related nuclei and areas were obtained with 4–8 mm diameter plastic tubes dealt with 1% DEPC solution and then put into RNAstore solution (Beijing Tiangen Biological Technology Ltd., Beijing, China) to prevent RNA degradation. The mRNA levels of EOP precursors and the three opioid receptors were examined in the nuclei or areas in bilateral brain regions of goats.
The gene expression levels of PENK, POMC, PDYN, and opioid receptors (δ-, μ-, and κ-receptor) were measured through the method of quantitative real-time PCR using ABI Prism 7500 real-time PCR instrument (ABI Co., USA). The PCR primers were designed according to the sequences of β-actin-mRNA (GeneBank accession no. AF481159), PENK-mRNA (GeneBank accession no. NM174141), POMC-mRNA (GeneBank accession no. NM001009266), PDYN-mRNA (GeneBank accession no. NM174139), δ-receptor-mRNA (GeneBank accession no. NM001191148), μ-receptor-mRNA (GeneBank accession no. AF266480), and κ-receptor-mRNA (GeneBank accession no. DQ065757). The PCR products were tested through the method of normal PCR. The homology between the referenced sequences and the products’ sequences was greater than or equal to 95%. The sequences of PCR products had been submitted to GeneBank (accession nos. GU169095 for PENK, GU167924 for POMC, GU169905 for PDYN, JQ756319 for δ-receptor, JQ241177 for μ-receptor, and JQ241178 for κ-receptor). Their upstream and downstream primers were presented in Table 1. All experimental data were analyzed using the method of through SDSShell software (ABI Co., USA).
2.5. Measurement of M-ENK Level
The level of M-ENK was measured through the method of SABC immunohistochemistry. The experimental goats were taken and anesthetized as above. According to the previous research results that opioid peptides in the CNS were released to the higher level at the end of EA , six sham control goats in this study were anesthetized and perfused immediately after needle withdrawal. The goats were fixed with the infusion of 4% paraformaldehyde through bilateral carotid arteries. The goat’s brain was taken out and transected into 17 sections with the method described by Cheng et al. . Each of the subsections was embedded in a paraffin block, sectioned at 5 μm, mounted on polylysine-coated slides, deparaffined, and rehydrated sequentially. Four serial slides were chosen from near the middle of each section for immunohistochemical staining. Of these four slides, the three were incubated with rabbit-anti-M-ENK IgG (1 : 100) (Wuhan Boster Biological Technology Ltd., Wuhan, China), while the rest was incubated with PBS instead of the corresponding antibody as negative control. Experimental procedures of SABC immunohistochemistry followed the instructions provided by the reagent company (Wuhan Boster Biological Technology Ltd., Wuhan, China). The cytoplasm of positive cells was stained as brown yellow. Optical density of the stained nuclei or areas in the CNS was obtained with a light microscope connected to a video-based and computer-linked system (high-resolution pathological image analysis system-1000, Wuhan Qianping Ltd., Wuhan, China). This system was programmed to calculate the mean optical density (MOD) for three fields of each slide examined under 400x magnification. The level of M-ENK in each nucleus or area was represented with the mean value % of the mean optical density from the three slides.
2.6. Statistical Analysis
Statistical analysis was performed using SPSS version 18.0 (SPSS Inc., Chicago, USA). All the data presented as mean ± SD. Pain threshold, mRNA levels of EOP precursors, and opioid receptors were used for ANOVA followed by the Bonferroni post hoc test. The correlation coefficient (Pearson’s) was used to examine the relations between pain threshold and levels of M-ENK. Statistical significance was evaluated by determining if the value was equal to or less than 0.05.
3.1. Pain Threshold Changes Induced by Electroacupuncture
The analgesic effects of experimental goats were expressed as the pain threshold (Figure 1). The pain threshold of experimental goats at 0.5 h before EA was not different () from that of sham control goats. After EA stimulated the goats, the pain threshold increased and reached the peak at 0 h. Then the pain threshold gradually decreased, began to rebound at 6 h, came to the second peak at 8 h, and then fell gradually again. At 0, 2, 4, 6, 8, and 12 h after EA, the pain threshold increased by 88%, 47%, 32%, 24%, 46%, and 21%, respectively. The pain threshold during the time from 0 to 12 h after EA was higher () than that at 0.5 h before EA. The pain threshold value at 8 h was higher () than that at 4 h, 6 h, 12 h, or 24 h although it was lower () than that at 0 h after EA. There was no difference () between pain thresholds at 2 h and 8 h.
3.2. mRNA Levels of Endogenous Opioid Peptide Precursors in the CNS of Goats
PENK, POMC, and PDYN are the precursors of enkephalin, endorphin, and dynorphin, respectively. The mRNA levels of three endogenous opioid peptide precursors were measured in the analgesia- and distribution-related nuclei or areas. These nuclei or areas mainly included nucleus accumbens (ACB), caudate nucleus (CAU), amygdala (AMY), supraoptic nucleus (SON), paraventricular nucleus of hypothalamus (PVH), ventromedial nucleus of hypothalamus (VMH), arcuate nucleus (ARC), paraventricular nucleus of thalamus (PVT), periaqueductal gray (PAG), dorsal raphe nucleus (DR), habenular nucleus (HB), parabrachial nucleus (PBN), nucleus raphe magnus (NRM), gigantocellular reticular nucleus (GI), solitary nucleus (SOL), neurohypophysis (NH), and spinal cord dorsal horn (SCD).
The mRNA levels of PENK changed in the measured nuclei or areas in a similar pattern after EA stimulated the animals; they increased () with the peak at 6 h in ACB, PAG, DR, NH, CAU, VMH, PBN, SOL, and PVH, at 8 h in GI, at 12 h in AMY, or at 24 h in HB (Table 2). PENK mRNAs remained higher levels () at 24 h after EA than at 0.5 h before EA in the measured nuclei or areas except ACB. The nucleus or area sequence of the amplitude by which PENK mRNAs increased were NH > PAG > DR = VMH > GI > PVH > PBN > SOL > AMY > CAU > ACB > HB.
|There was difference () between the values with different letters, and no difference () with the same letters in a line. The letters in the following tables have the same meanings.|
The mRNA levels of POMC increased () with one or two peaks in the measured nuclei or areas after EA (Table 3). The peak of POMC mRNA levels occurred at 2 h in SOL, at 4 h in CAU, PVH, VMH, and PAG, or at 6 h in NH. There were two peaks of POMC mRNA levels which appeared at 2 h and 8 h in ARC and GI, or at 4 h and 12 h in NRM, or at 4 h and 8 h in AMY, respectively. POMC mRNA levels remained higher () at 24 h after EA than at 0.5 h before EA in the measured nuclei or areas.
The mRNA levels of PDYN began to increase () in most of the measured nuclei or areas at 2 h or 4 h (Table 4). They reached the first peaks at 4 h in AMY, PVT, PAG, PBN, SCD, and CAU or at 6 h in PVH, VMH, SON, and SOL. The second peaks of PDYN mRNA levels, occurred at 12 h, were higher () than the first peaks in all the measured nuclei or areas. PDYN mRNA levels at 12 h increased by times from 0.96 to 2.21, compared with those at 0.5 h before EA stimulated the animals. PDYN mRNAs in AMY, PAG, SCD, and SON remained higher levels () at 24 h while mRNA levels of PDYN in PVT, PBN, PVH, VMH, SOL, and CAU returned to the preacupuncture level ().
There were no differences () in mRNA levels of PENK, POMC, or PDYN between sham control goats and experimental goats at 0.5 h before EA in the measured nuclei and areas.
3.3. mRNA Levels of Endogenous Opioid Receptors in the CNS of Goats
The mRNA levels of δ-, μ-, or κ-receptor were measured in the corresponding nuclei or areas where their ligands are distributed (Tables 5, 6, and 7). The mRNA levels of δ-receptor increased () with the peak at 6 h in ACB, PAG, DR, NH, CAU, PVH, VMH, PBN, and SOL, at 8 h in GI, or at 12 h in AMY. δ-receptor mRNA remained higher levels in the measured nuclei or areas except ACB at 24 h and kept uptrend in NH, VMH, PBN, SOL, and HB. The δ-receptor mRNA levels at the peak in NH, PAG, DR, GI, VMH, PVH, PBN, SOL, AMY, HB, ACB, and CAU increased by 3.24, 2.30, 1.44, 1.36, 1.24, 1.22, 1.20, 1.10, 1.10, 0.96, 0.71, and 0.71 times, respectively.