Cannabinoid CB2 Receptors Contribute to Upregulation of β-endorphin in Inflamed Skin Tissues by Electroacupuncture

The purpose of the following experiments is to determine whether peripheral μ-opioid receptors contribute to the analgesic effect of the CB2R agonist AM1241 or EA on inflammatory pain. First, we examined the antinociceptive effects of AM1241 (using the AM1241 vehicle group as the control) and EA (using the sham EA group as the control) on allodynia and hyperalgesia induced by complete Freund's adjuvant (CFA) injection. Then, we determined whether pretreatment with the μ-opioid receptor antagonist β-funaltrexamine (β-FNA) attenuates the antinociceptive effect of AM1241 or EA.

The baseline withdrawal thresholds in all the experimental groups were similar. CFA injection elicited typical inflammatory responses, including redness, edema, and hypersensitivities to noxious stimuli in the injected paw. These symptoms lasted for about 2 weeks as described before [9]. A large decrease in the thermal withdrawal latency and mechanical threshold was observed 1 day after CFA injection (Figure 1A-D). Compared with the vehicle group, AM1241 treatment (1 mg/kg) from days 2 to 6 significantly increased the thermal withdrawal latency and mechanical threshold in the inflamed hindpaw (Figure 1A, C). Pretreatment with β-FNA (250 μg/kg) on days 1, 3 and 5 significantly attenuated the antinociceptive effect of AM1241 (Figure 1A, C).

EA was applied to GB30 and GB34 for 30 min on days 2, 4 and 6 after CFA injection. EA significantly increased the thermal withdrawal latency and mechanical threshold, compared with those in the sham EA group (Figure 1B, D). Furthermore, pretreatment with β-FNA in the same hindpaw significantly reduced the effect of EA on thermal hyperalgesia and mechanical allodynia (Figure 1B, D).

The objective of the following experiments was to determine (1) the effects of AM1241 (using the AM1241 vehicle group as the control) and EA (using the sham EA group as the control) on the POMC mRNA and β-endorphin protein levels in inflamed tissues; and (2) whether blocking CB2Rs attenuates the potentiating effects of AM1241 or EA (using the AM630 vehicle group as the control) on the POMC mRNA and β-endorphin protein levels in inflamed tissues.

The endogenous ligand of the opioid peptide β-endorphin is derived from the precursor proopiomelanocortin (POMC). The POMC mRNA was detected in the skin tissues of all groups. CFA injection increased the mRNA level of POMC in inflamed skin tissues compared with that in vehicle-injected rats (Figure 2A). AM1241 or EA significantly increased the mRNA level of POMC in inflamed skin tissues compared with that in the vehicle or sham EA group (Figure 2A, B). Pretreatment with AM630 significantly reversed the effects of AM1241 and EA on the mRNA level of POMC in inflamed tissues (Figure 2A, B).

The specific β-endorphin protein band (3.5 kDa) was present in the skin tissues samples obtained from all of the seven groups (Figure 2C). CFA injection increased the protein level of β-endorphin in the skin tissues compared with that in the CFA vehicle group (Figure 2D). Compared with the vehicle group, AM1241 treatment significantly increased the protein level of β-endorphin in inflamed tissues (Figure 2D). Also, the protein level of β-endorphin in inflamed tissues was significantly higher in the EA group than in the sham EA group (Figure 2E). In addition, pretreatment with AM630 in the same hindpaw significantly attenuated the effects of AM1241 and EA on the protein level of β-endorphin in the inflamed skin tissues (Figure 2D, E).

To determine whether CB2R stimulation or EA treatment has any effect on the μ-opioid receptor expression at the inflammatory site, we measured the mRNA and protein amount of μ-opioid receptor-1 in inflamed tissues. Treatment with either AM1241 or EA had no significant effect on the mRNA and protein levels of μ-opioid receptor-1 in inflamed skin tissues on day 6 after CFA injection compared with those in the vehicle group or sham EA group (Figure 3, A-E).

The epidermal layer of the skin tissues obtained from CFA-injected rats became much thicker than that in the vehicle-injected rats (Figure 4A). The β-endorphin immunoreactivity was present in keratinocytes located in the uppermost layer of the epidermis, including the stratum granulosum and the stratum spinosum in the vehicle control group. Intense β-endorphin-immunoreactivity was detected throughout the stratum granulosum and into the stratum spinosum in inflamed skin tissues (Figure 4A). The percentage of β-endorphin-immunoreactive keratinocytes was significantly increased in the skin tissues in CFA-injected rats than that in vehicle-injected rats (P < 0.05, Figure 4B).

Treatment with AM1241 or EA significantly increased the percentage of keratinocytes labeled with β-endorphin compared with that in the vehicle group or sham EA group (P < 0.05, Figure 4B, C). Furthermore, pretreatment with AM630 in the same hindpaw significantly attenuated the effect of AM1241 or EA treatment on the percentage of keratinocytes immunoreactive to β-endorphin in the inflamed skin tissues (P < 0.05, Figure 4B, C).

In vehicle-injected rats, only a few ED1-positive macrophages were found in the dermis of the skin, and the sizes of these cells were much smaller than those in the CFA-treated group. Also, ED1-positive macrophages were rarely labeled with β-endorphin in the skin tissues of vehicle-injected rats. In contrast, there were large numbers of infiltrating macrophages in the inflamed skin of CFA-injected rats. These cells were generally large in sizes and multivacuolated, and many of them were immunoreactive to β-endorphin (Figure 5A). The percentage of macrophages labeled with β-endorphin was also significantly increased in the inflamed skin tissues than that in vehicle-injected rats (P < 0.05, Figure 5B).

AM1241 or EA treatment significantly increased the percentage of macrophages labeled with β-endorphin in the inflamed skin tissues compared with the vehicle group or sham EA group (P < 0.05, Figure 5B, C). Furthermore, pretreatment with AM630 in the same hindpaw significantly blocked the effects of AM1241 and EA on the percentage of macrophages immunoreactive to β-endorphin in the inflamed skin tissues (P < 0.05, Figure 5B, C).

TCR-positive T-lymphocytes were present in the dermis of the skin sections obtained from CFA-injected rats and had the size and oval shape consistent with the typical characteristics of T-lymphocytes [10]. However, these cells were rarely present in the rat skin tissues in the vehicle-injected rats, and only few of them were immunoreactive to β-endorphin. In the skin tissues from CFA-treated rats, there was a marked increase in infiltrating TCR-positive T-lymphocytes and some of them were labeled with β-endorphin (Figure 6A). Also, the percentage of T-lymphocytes labeled with β-endorphin in the total of T-lymphocytes was significantly increased in the inflamed skin tissues compared with that in the control skin tissues (P < 0.05, Figure 6B).

Treatment with AM1241 or EA significantly increased the percentage of T-lymphocytes immunoreactive to β-endorphin in the inflamed skin tissues compared with that in the vehicle group or sham EA group (P < 0.05, Figure 6B, C). Pretreatment with AM630 in the same hindpaw significantly reversed the effect of AM1241 or EA treatment on the percentage of T-lymphocytes labeled with β-endorphin in the inflamed skin tissues (P < 0.05, Figure 6B, C).

Experiments were carried out on male adult Sprague-Dawley rats (180-200 g) purchased from Experimental Animal Center of Tongji Medical College of Huazhong University of Science and Technology. All procedures were approved by the Animal Care Committee at Huazhong University of Science and Technology and conformed to the ethical guidelines of the International Association for the Study of Pain [37]. The rats were individually housed in cages with a 12-hr light/dark cycle and had free access to food and water.

Inflammation was induced by injecting 50 μL of complete Freund's adjuvant (CFA; Sigma, St. Louis, MO) subcutaneously into the dorsal surface of left hindpaw of rats using a 25-gauge hypodermic needle [38]. The injections were carried out under light anesthesia by means of ether inhalation. We selected the dorsal surface of the hindpaw as the injection site in order to produce an inflammatory pain focus in Gallbladder Channel of Foot Shaoyang, where GB30 and GB34 are located, according to the meridian theory of traditional Chinese medicine [4, 39]. In separate rats, mineral oil was injected into the dorsal surface of the hindpaw and was used as the vehicle control.

In the EA treatment group, the rats received EA on the ipsilateral "Huantiao" (GB30) and "Yanglingquan" (GB34) once every other day, starting at the second day after CFA injection. EA (1 mA and 0.1 ms) was administered at 2 Hz for 30 min. Current was delivered with a modified current-constant Han's Acupoint Nerve Stimulator (LH202, Huawei Co.Ltd., Beijing, China). GB30 and GB34 were chosen based on their effective use in reducing inflammatory pain in rats [4, 9].

Two acupuncture needles were inserted into two acupoints corresponding to GB30 and GB34 in humans. GB30 is located at the junction of the lateral 1/3 and medial 2/3 of the distance between the greater trochanter and the hiatus of the sacrum; and GB34 lies on the lateral aspect of the leg in the depression anterior and inferior to the head of the fibula in rats [40]. During EA treatment, each rat was placed in an inverted clear plastic chamber (approximately 4 cm × 4 cm × 11 cm) but was not restrained. The animals remained still during EA treatment and showed no evident signs of distress. For sham control, acupuncture needles were inserted ipsilaterally into GB30 and GB34 without electrical stimulation or manual needle manipulation.

AM1241 is a potent and selective CB2R agonist [41]. AM630 is a highly specific CB2R antagonist with a 70-165-fold selectivity for the CB2R in vitro [42]. AM1241 (1 mg/kg, Enzo Life Sciences, PA, USA) and AM630 (150 μg/kg, Enzo Life Sciences, PA, USA) were dissolved in the vehicle solution containing 5% Tween-80 and 5% DMSO in normal saline. β-FNA is a selective and irreversible μ-opioid receptor antagonist [19]. β-FNA (250 μg/kg, Sigma, St. Louis, MO, USA) was dissolved in methanol. The dose of AM1241, AM630 and β-FNA was selected based on the previous studies [4, 41, 43] and our preliminary dose-response study and was the lowest that produced reproducible behavioral effects. AM1241 or its vehicle (50 μL) was injected subcutaneously into the dorsal surface of the left hindpaw of rats once every other day, starting at the second day after CFA injection. AM630 or the vehicle (50 μL) was injected subcutaneously into the dorsal surface of the left hindpaw at the same injection time of AM1241 or 5 min before EA or sham EA treatment each time. β-FNA or the vehicle (50 μL) was injected subcutaneously into the dorsal surface of the left hindpaw 24 h before AM1241, EA or sham EA treatment each time. The investigators involved in behavioral tests and biochemical assays were blinded to the drug injection throughout the study.

The behavioral tests were performed 3 times before CFA injection and once every day, starting from the first day after CFA injection. The animals were habituated to the testing environment for 30 min. Thermal hyperalgesia was assessed by exposing the mid-plantar surface of the hindpaw to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter (Ugo Basile, Italy), as previously described [44]. The withdrawal latency was recorded for both left and right hindpaws as the time taken from the onset of radiant heat stimulation to withdrawal of the hindpaw.

Mechanical allodynia was assessed by placing rats on an elevated mesh floor, and the tactile threshold was measured by using an electronic von Frey anesthesiometer (Ugo Basile, Italy) applied to the plantar surface of the left hindpaw. The force (g) needed to produce a paw withdrawal response was tested 4 times separated by 2- to 3-min intervals. A mean value of 4 consecutive measurements was used.

The skin tissues (5 mm × 5 mm × 2 mm) at the site of CFA or vehicle injection were removed on day 6 after CFA injection (i.e., 25 min after the third EA treatment or 60 min after the third AM1241 treatment). The skin tissues were excised from rats immediately after the animals were anesthetized with an overdose of sodium pentobarbitone (120 mg ⁄kg, i.p.) and decapitated. Total RNA was isolated from the skin specimens using Trizol reagent (Invitrogen, CA, USA). Aliquots of 3 μg total RNA were reverse transcribed into cDNA using ReverTra Ace-α-TM (Toyobo, Osaka, Japan). The 20 μL (total volume) of the PCR mixture consisted of 1 μL diluted cDNA, 10 μL SYBR green-PCR master mixture (2×) (Toyobo, Osaka, Japan), and 0.3 μM of each primer. Real-time PCR was performed using the Stratagene Mx3005P. Sequence-specific primers were listed in Table 1.

The expression level of each gene was determined by the threshold cycle (CT). Samples that had larger amounts of a particular gene had correspondingly lower CT values. For each sample, a CT value was obtained for POMC, μ-opioid receptor-1, and β-actin. The CT value of β-actin was subtracted from that of POMC or μ-opioid receptor-1 to obtain a ΔCT value. The mRNA amount, normalized to the endogenous control (β-actin), was given by 2^-ΔΔCt. Results of six independent experiments were expressed as the % change over relative mRNA level of the vehicle control group.

The plantar skin of the left hindpaw (5 mm × 5 mm × 2 mm) was removed as described above, minced with scissors, and homogenized in 300 μL RIPA Lysis Buffer (Beyotime Biotechnology, Nanjing, China) with 2 mM phenylmethylsulfonyl fluoride and the protease inhibitor cocktail (Roche Applied Science, CT, USA), and centrifuged at 12,000 × g for 10 min. The pellet was discarded and protein concentrations from the supernatant were determined using the Enhanced BCA Protein Assay Kit (Beyotime Biotechnology, Nanjing, China). To quantify β-endorphin protein level, 150 μg total protein of each tissue was processed with 2 × Tricine-SDS-PAGE loading buffer (Tiandz Biotech, Beijing, China), and then separated on SDS-PAGE gel as previously described [45]. To determine the μ-opioid receptor-1 protein level, 30 μg total protein of each tissue was processed with 5 × SDS-PAGE loading buffer at 95°C for 5 min, and then separated on a 12% glycine-SDS-PAGE gel. The proteins were transferred onto a PVDF membrane, blocked for 1 hr in 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween-20. The membrane was incubated with the rabbit anti-β-endorphin antibody (1:20,000; Abcam, San Francisco, CA, USA) and rabbit anti-μ-opioid receptor-1 antibody (1:200; Santa Cruz, CA, USA) at 4°C overnight, respectively. The molecular weight of the protein band detected matched with that reported in previous studies. The specificity of the anti-β-endorphin antibody has been shown by detecting the specific β-endorphin band at 3.5 kDa [22]. Blotting using the μ-opioid receptor-1 antibody resulted in two protein bands at ~50 and ~70 kDa [46, 47]. After washes in 0.1% TBS-Tween 20, the membranes were then incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:20000; Jackson ImmunoResearch, MD, USA) for 1 h at room temperature, and then washed three times. The enhanced chemiluminescence method (ECL Plus Western blotting detection reagents; Pierce, IL, USA) was used to reveal the protein bands according to the manufacturer's protocol. The optical density of each band was then measured with a computer-assisted imaging analysis system (Quantity one, Bio-Rad, Hemel Hempstead, UK) and normalized with β-actin. Results of six independent experiments were expressed as the % change over the protein amount in the CFA vehicle control group.

Rats were deeply anesthetized with an overdose of sodium pentobarbitone (120 mg/kg, i.p.) and were transcardially perfused with normal saline and 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The skin tissues with the injection point in the middle was harvested. Tissues were postfixed at 4°C in the perfusion fixative for 8 h, cryoprotected in 30% sucrose overnight, and sectioned at 15 μm on a cryostat in a plane perpendicular to the skin surface and parallel to the long axis of the foot. The sections were mounted onto gelatin-coated slides, air-dried overnight, and used for double-immunofluorescence labeling of β-endorphin with markers of keratinocytes, macrophages, or T-lymphocytes [14, 25].

Double immunolabeling was performed with rabbit anti-β-endorphin (1:500; Abcam, Cambridge, UK) and mouse monoclonal anti-pan cytokeratin antibody (1:100; Abcam, Cambridge, UK) for identification of keratinocytes, mouse monoclonal anti-CD68 antibody (Clone ED1, 1:200; Serotec, Oxford, UK) for detection of macrophages, or mouse monoclonal anti-αβ T cell receptor (TCR) antibody (Clone R73, 1:200; BD Biosciences-PharMingen, San Diego, CA, USA) for identification of T-lymphocytes. The specificity of the β-endorphin antibody has been demonstrated by antigen preabsorption with the corresponding blocking peptides. The anti-pan cytokeratin antibody reacts specifically with the skin epidermal keratinocytes [48]. The anti-CD68 antibody specifically labels macrophages in the tissues [49]. The anti-TCR antibody is a specific marker for peripheral T-lymphocytes and does not react with γδ TCR-bearing T cells [50].

All sections were blocked for 30 min with 5% donkey serum and 0.2% Tween-20 in PBS, followed by incubation at 37°C for 1 hr then at 4°C overnight with the primary antibody diluted in PBS containing 5% bovine serum albumin (BSA). The sections were washed 4 times with 0.05% Tween-20 in PBS for 5 min, and incubated with a mixture of secondary antibodies: donkey anti-mouse IgG conjugated with Dynight 488 (1:400; Jackson ImmunoResearch) and donkey anti-rabbit IgG conjugated with Dynight 594 (1:500; Jackson ImmunoResearch). Sections were washed 4 times with 0.05% Tween-20 in PBS for 5 min, and then treated with the fluorescence-mounting medium to inhibit quenching of fluorescence before being coverslipped. Negative controls were included by omitting the primary antibodies and with primary antibodies preabsorbed with their specific blocking peptides in the above procedures, which resulted in no positive labeling in the skin tissues. Six rats per group were used for the double-immunolabeling and cell counting.

Digital confocal images were acquired using a laser scanning confocal microscope (FV500-IX71, Olympus, Japan). The sections were scanned using excitation at 488 nm (argon laser) for Dylight 488 and at 594 nm (helium neon laser) for Dylight 594. A total of 3-4 sections were imaged from the same skin tissues in each rat, and counting of single- and double-labeled cells was done on confocal images randomly taken from three view fields in each section. Cell counting was performed by an investigator in a blind fashion using NIH Image J software (Bethesda, MD, USA). The percentage of double-labeled cells in the total of single-labeled cells was used for statistical analysis.

Data are presented as means ± SEM. We used one-way ANOVA (mRNA and protein levels and the percentage of double-labeled cells) or two-way ANOVA (behavioral data) to determine the overall effect of interventions. Tukey's post hoc test was then used to determine the statistical difference in the mRNA and protein levels and the percentage of double-labeled cells between individual groups. To determine the statistical difference in the withdrawal thresholds between different groups and time points, we used Bonferroni's post hoc test. A P value of less than 0.05 was considered statistically significant.