WRW4

Electroacupuncture decreases inflammatory pain through a pro-resolving mechanism involving the peripheral annexin A1-formyl peptide receptor 2/ALX-opioid receptor pathway

Cintia Vieira 1,2 & Daiana C. Salm 1,2 & Verônica V. Horewicz 1,2 & Daniela D. Ludtke 1,2 & Aline A. Emer 1,2 &
Júlia F. Koerich 1,2 & Gustavo Mazzardo 1 & Sayron Elias 1 & Ari O. O. Moré 4 & Leidiane Mazzardo-Martins 1,3 &
Francisco J. Cidral-Filho 1,2 & William R. Reed 5,6 & Anna Paula Piovezan 1,2 & Daniel F. Martins 1,2

Received: 13 June 2020 /Revised: 28 October 2020 /Accepted: 3 December 2020 / Published online: 20 January 2021 # The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature 2021

Abstract
The pro-resolving mechanism is a recently described endogenous process that controls inflammation. The present study evalu- ated components of this mechanism, including annexin 1 (ANXA1) and the formyl peptide receptor 2/ALX (FPR2/ALX) receptor, in the antihyperalgesic effect induced by electroacupuncture (EA) in an animal model of persistent peripheral inflam- mation. Male Swiss mice underwent intraplantar (i.pl.) injection with complete Freund’s adjuvant (CFA). Mechanical hyperalgesia was assessed with von Frey monofilaments. Animals were treated with EA (2–10 Hz, ST36-SP6) or subcutaneous BML-111 injection (FPR2/ALX agonist) for 5 consecutive days. In a separate set of experiments, on the first and fifth days after CFA injection, animals received i.pl. WRW4 (FPR2/ALX antagonist) or naloxone (non-selective opioid receptor antagonist) before EA or BML-111 injection. Paw protein levels of FPR2/ALX and ANXA1 were evaluated on the second day after CFA injection by western blotting technique. EA and BML-111 reduced mechanical hyperalgesia. I.pl. naloxone or WRW4 prevented the antihyperalgesic effect induced by either EA or BML-111. EA increased ANXA1 but did not alter FPR2/ALX receptor levels in the paw. Furthermore, i.pl. pretreatment with WRW4 prevented the increase of ANXA1 levels induced by EA. This work demonstrates that the EA antihyperalgesic effect on inflammatory pain involves the ANXA1/FPR2/ALX pro-resolution path- way. This effect appears to be triggered by the activation of FPR2/ALX receptors and crosstalk communication with the opioid system.

Keywords Annexin A1 . Electroacupuncture . FPR2/ALX . Mice . Pain

Introduction
* Daniel F. Martins
[email protected]; [email protected] Experimental Neuroscience Laboratory (LaNEx), University of
Southern Santa Catarina, Palhoça, Santa Catarina, Brazil Postgraduate Program in Health Sciences, University of Southern
Santa Catarina, Palhoça, Santa Catarina, Brazil
Postgraduate Program in Neuroscience, Center of Biological Sciences, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil
Integrative Medicine and Acupuncture Division, University Hospital, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil
Department of Physical Therapy, University of Alabama at Birmingham, Birmingham, AL, USA
Rehabilitation Science Program, University of Alabama at Birmingham, Birmingham, AL, USA
Unlike traditional anti-inflammatory agents that only inhibit the production of key pro-inflammatory substances, pro- resolving mediators promote anti-inflammatory and pro- resolving activities [50]. Lipoxins (i.e., LXA4) [58], resolvins (i.e., RvD1) [51], and annexin 1 (i.e., ANXA1) [8] are among the most studied molecules possessing anti-inflammatory and pro-resolving activities. Persistent inflammation may be a re- sult of the inability of an organism to produce adequate amounts of these pro-resolving mediators or the inability of these mediators to bind to their respective receptors [57].
ANXA1 is widely distributed throughout the body. It is found in white blood cells, the mononuclear phagocyte sys- tem, and neuronal cells, as well as in biological fluids [21, 43]. ANXA1 (a 37-kDa protein) and its fragments or derived pep- tides have been demonstrated to induce analgesic effects in inflammatory pain models [8, 19, 43]. A role for ANXA1 in nociceptive pathways is indicated by ANXA1 knockout(ANXA1) mice being found to be more sensitive to noxious stimuli compared with wild-type mice (ANXA1+/+) [3]. ANXA1 along with its peptidomimetics can activate the G- protein-coupled receptor (GPCR), formyl peptide receptor 2 (FPR2) (also known as FPRL-1) [10]. In addition to being expressed in neutrophils and monocytes [20], FPR2 is also found in the nervous system [17, 23, 39]. Moreover, inhibition of formalin-induced nociception by ANXA1 peptidomimetics was found to rely on activation of FPR family members [43]. Taken together, these studies suggest that FPR2/ALX recep- tors mediate essential components of ANXA1-related pain modulation.
Neutrophils and monocytes/macrophages produce and re- lease endogenous opioids, like Met-enkephalin and beta- endorphin [40, 45], and these molecules bind to opioid recep- tors expressed in the peripheral nociceptive neurons [12, 33, 54]. FPR2/ALX activation has been shown to be involved in molecular pathways related to tonic opioid release from neu- trophils in inflammatory pain [46]. Activation of human neu- trophils by ANXA1 induced a more than fivefold increase in Met-enkephalin secretion. Moreover, FPR-specific antago- nists block ANXA1-induced opioid peptide release [15]. Local injection of naloxone, a non-selective opioid receptor antagonist, prevents formyl peptide–induced antinociception [46]. Complete Freund’s adjuvant (CFA) is a well- characterized experimental inflammatory chronic pain model having two distinct inflammatory pain stages. In the early inflammatory phase, neutrophils are the predominant leuko- cyte population while macrophages become more prevalent during the late phase [5].
Phytotherapy [44] and stretching [4] are integrative thera- pies that are known to release molecules or activate receptors with pro-resolving functions. Among non-pharmacological integrative approaches used to reduce inflammation, acupunc- ture has been used clinically for over 3000 years and has been extensively studied over the last 30 years. Acupuncture’s ef- fect upon endogenous opioid release is well characterized, and its analgesic efficacy is recognized worldwide [1, 29]. Electroacupuncture (EA) is a more recent variation of manual acupuncture (MA) treatment in which, after the needles are inserted into acupoints, electrodes are attached, and a small electric current is applied [63]. Like acupuncture, EA has also been extensively studied and is currently recognized by the National Institutes of Health (NIH)-National Center for Complementary and Integrative Health (NCCIH) and the World Health Organization (WHO) [11, 18, 27] for the treat- ment of pain.
The neuromodulatory effects of EA hold great potential to control pain and impact inflammation [59]. The beneficial effects of EA on inflammation, and consequently on inflam- matory pain, likely involve the release of pro-resolving
mediators with possible activation of FPR2/ALX receptors. Similar to the effects induced by pro-resolving mediators (LXA4, AnxA1, and RvD1), acupuncture (both MA and EA) has been shown to reduce COX-2 expression in an animal model of chronic pain [26], to stimulate neutrophil apoptosis [60], and to regulate the immune system by promoting humor- al and cellular immunity, as well as the activity of NK cells in humans [26]. In the present study, we investigated the periph- eral antinociceptive properties of EA in mice, in both early and late phases of CFA-induced inflammation, as well as ex- amined a potential local mechanism of EA. We designed this study to address the hypothesis that EA induces the release of ANXA1 that subsequently would interact with FPR2/ALX receptors leading to the release of opioid peptides, which, in turn, would activate opioid receptors on peripheral nerve ter- minals to decrease mechanical pain.

Materials and methods

Animals

All experimental procedures were carried out in accordance with the National Institutes of Health Animal Care Guidelines (NIH publications No. 80-23). All experiments were conduct- ed using male Swiss mice (25–35 g), obtained from the vivar- ium of the Federal University of Santa Catarina (UFSC, Florianopolis, Santa Catarina, Brazil). Animals were housed at 22 ± 2 °C under a 12-h light/12-h dark cycle (lights on at 6:00 a.m.), with access to food and water ad libitum. All ex- perimental testing was carried out between 11:00 am and 3:00 pm. The number of animals and the intensity of noxious stimuli used were the minimum necessary to demonstrate the consistent effects of treatments. Experiments were performed after approval of the protocol (17.002.2.07.IV.) by the University of Southern Santa Catarina (UNISUL) Ethics Committee.

Drugs

The following substances were experimentally used: CFA, Griess reagent, sulfanilamide, and anti-beta actin antibody (HRP) from Sigma-Aldrich, (MO, USA); Tween®, PMSF, EDTA, aprotinin A, and benzammonium chloride (BioLegend, San Diego, CA, USA); naloxone hydrochloride, BML-111, and WRW4 (Trp-Arg-Trp-Trp-Trp-NH2) from Tocris Cookson, Inc. (Ellisville, MO, USA); anti-FPR2/ALX receptor antibody from Santa Cruz Biotechnology (TX, USA); and annexin A1 polyclonal antibody from Thermo Fisher Scientific (Waltham, MA, USA). All drugs were dissolved, just before use, in saline. Control animals received the same volume of physiological saline used to di- lute the compounds and were assessed simultaneously with
the experimental groups. When drugs were delivered by sub- cutaneous (s.c.) route, a constant volume of 10 μl/animal was injected. When drugs were administered by intraplantar (i.pl.) route, volumes of 20 μl were injected.

CFA-induced persistent peripheral inflammation

To induce persistent peripheral inflammation, mice in both control (non-treatment) and EA treatment groups were injected with 20 μl of an 80% CFA (Mycobacterium tuberculosis) solution (diluted in phosphate-buffered saline) as described by Meotti and colleagues [36].

EA and BML-111 treatments

Experimental procedures used for EA treatment have been previously described in earlier studies [2, 28, 35, 47]. To min- imize restraint-induced stress, mice were slightly sedated with 1–2% isoflurane and stainless acupuncture needles (Dong Bang, 0.18 mm/diameter and 8 mm/length) with electrodes soldered to their handles were inserted into the acupoints SP6 and ST36 ipsilateral to the CFA-injected paw [24, 35]. The needles were positioned approximately 3 mm deep in each acupoint, and the intensity of electrostimulation was in- creased until a mild twitch was observed at the tibialis anterior muscle (ST36) and flexor digitorum muscle (SP6). Mild mus- cle twitch was usually obtained at an intensity of 2 to 3 mA. In total, this needling procedure typically required 20 s to complete.
The tibial nerve (SP6) and the fibular nerve (ST36) acupoints were specifically selected for this study because they are related to the segmental innervation of the animals’ hind paw [35, 47, 48]. Moreover, other studies using different animal models have demonstrated positive effects of these particular acupoints in controlling pain and inflammation [2, 57]. Electrostimulation parameters used included a dense-disperse asymmetric balanced wave (F1 = 2 Hz, 0.7-ms pulse with 5 s of stimulation; F2 = 10 Hz, 0.2-ms pulse with 5 s of stimulation) with alternating polarities, using the NKL EL- 608 electrostimulator (NKL Electronic Products, Brusque, SC). Previous EA-related studies have established that 2 to 10 Hz frequencies are effective at inhibiting inflammatory and neuropathic pain [24, 35, 53]. To determine the most effective EA treatment duration in our CFA-induced periph- eral pain model, 5 min, 10 min, and 20 min of electrical stim- ulation were tested using different groups of animals.
In order to determine whether electrostimulation is essen- tial and whether the needle position was relevant to treatment outcomes, two different needling control groups were includ- ed. In control group 1, the same procedures of the active EA group were performed, but no electrical current was delivered to the acupoints (i.e., ST36 and SP6). Other groups of animals were treated by administration of BML-111 (0.3 μg/100 μl, 1 μg/100 μl, and 3 μg/100 μl, s.c.) and were evaluated at the same experimental times as the animals treated with EA. Additional groups of animals (BML-111 doses of 0.3 μg/
100 μl and 1 μg/100 μl, s.c.) were treated only on the first and fifth days, in experiments designed to investigate EA- related mechanisms of action.

Study outline

The primary outcome of this study was mechanical hyperalgesia as evaluated by the von Frey test (Fig. 1). Twenty-four hours after the induction of the inflammatory procedure, different groups of animals were treated at different times (5 min, 10 min, and 20 min) with either EA or BML-111 (0.3–3 μg/100 μl/s.c.). Animals were evaluated following dif- ferent treatment durations or dosages to determine the specific dosage that produced optimal antihyperalgesic effects (Fig. 1). After determining the most effective EA treatment duration and BML-111 dose, subsequent experiments used these optimal treatment parameters. Edema was assessed on the fourth day after CFA. Additionally, a second experiment was performed to determine the possible summation effect of daily EA or BML-111 (i.e., on the first and fifth days post CFA injection).
After characterizing the antihyperalgesic effect of EA and BML-111, the next step was to analyze the involvement of opioid receptors in this antihyperalgesic effect. To this end, on the first and fifth days after CFA i.pl. injection, mice were pre- administered (i.pl.) saline or naloxone (i.pl.) and then treated with BML-111 or EA. Thirty minutes later, mechanical hyperalgesia was evaluated. Respective control groups were also used. The involvement of FPR2/ALX receptors in the antihyperalgesic effect of EA and BML-111 was also investi- gated on the first and fifth days after CFA i.pl. injection. In these experiments, mice were pre-administered saline or WRW4 (i.pl.) and then treated with BML-111 or EA. Thirty minutes later, mechanical hyperalgesia was evaluated. Respective control groups were also used. Finally, the expres- sion of FPR2/ALX receptors and AnxA1 in paw tissue of animals treated with EA or BML-111 was determined on the second day after CFA injection.

Evaluation of mechanical hyperalgesia

After acclimation, mechanical stimulus withdrawal thresholds were determined using von Frey filaments applied to the gla- brous skin of the hind paw with mice placed on an elevated wire mesh platform in a clear plexiglass box (9 × 7 × 11 cm3) [34]. The right hind paw was stimulated with a constant pres- sure of a 0.6 g von Frey filament (VFF) (Stoelting, Chicago, IL, USA). The withdrawal response frequency (expressed as a percentage) to 10 von Frey filament applications was interpreted as nociceptive stimulus–induced behavior.
To assess the effects of EA and BML-111 on CFA-induced persistent inflammatory pain, animals were treated with EA or BML-111, on the 1st or 5th day after CFA i.pl. injection. Development of mechanical hyperalgesia was evaluated 0.5 h, 1 h, and 2 h after treatment to verify the time course of EA or BML-111 in reducing mechanical hyperalgesia. To investigate the effects of repeated treatments, EA or BML-111 treatments were conducted once a day. Mechanical hyperalgesia was evaluated 0.5 h after treatment (time of max- imal inhibition observed in the acute treatment) for five con- secutive days.

Evaluation of paw edema

Post-CFA edema was determined by comparing differences between the thicknesses of the right hind paw with baseline measurements (taken before CFA injection). A digital microm- eter (Insize®, SP, Brazil) was used to obtain hind paw measure- ments, and results were expressed as percent differences.

Evaluations were conducted on the 4th day after CFA injection, 0.5 h, 1 h, 2 h, 3 h, and 24 h after treatments [34].

Peripheral administration of opioid receptor antagonist

To assess the possible contribution of peripheral opioid recep- tors on the antihyperalgesic action of EA, mice were admin- istered saline (20 μl/i.pl.) or naloxone (5 μg/i.pl.) 15 min be- fore saline injection (10 ml/kg, s.c.; control group) and 20 min EA (EA group) or BML-111 (0.3 μg/100 μl, s.c.). Mechanical hyperalgesia was evaluated 0.5 h after treatments.

Peripheral administration of FPR2/ALX antagonist

In a separate set of experiments designed to evaluate the in- volvement of peripheral FPR2/ALX in the antihyperalgesia induced by EA, animals received an intraplantar injection (i.pl.) with 20 μl of saline or WRW4 (10 μg/paw) [44] in the right hind paw. After 15 min, the animals were treated with EA for 20 min or BML-111 (0.3 μg/100 μl, s.c.). Mechanical hyperalgesia was evaluated using the von Frey filament 0.5 h after EA.

Western blotting: quantification of ANXA1 and FPR2/ALX receptor in skin paw

Right hind paw tissue samples were collected on the 2nd day after CFA injection, 0.5 h after the daily treatment had ended (animals had been treated once a day for two consecutive days). Hind paw tissue samples were stored in the freezer (- 80 °C). Skin paws were immersed in liquid nitrogen, pulver- ized, and immediately placed in tubes containing RIPA lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and PBS) plus 100 mM sodium orthovanadate, 100 mM PMSF, and a cocktail of 1% protease inhibitors (Sigma- Aldrich, St. Louis, MO, USA), and then incubated on ice for 30 min. The tubes containing the lysates were centrifuged at 10,000 rpm for 20 min at 4 °C. Supernatants were collected, and the protein concentration was determined using the Bradford method.
Electrophoretic separation was accomplished using 60 μg of protein per well in 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), running in a Mini-PROTEAN® Tetra cell apparatus under a PowerPac™ HC power supply (Bio-Rad, CA, USA). The proteins were transferred onto a PVDF membrane (Bio-Rad Laboratories Inc., Hercules, CA, USA), blocked in 5% BSA (prepared in TBS-T buffer, pH 7.4; concentration in mmol/l: 20 Tris-HCl, 137 NaCl, 0.1% Tween 20), and incubated overnight at 4 °C with primary antibodies to FPR2/ALX receptor (dilution 1:500) and ANXA1 (dilution 1:1000). Peroxidase- conjugated monoclonal antibody against β-actin (dilution

1:45,000) was used as a loading control for all samples tested. After primary antibody incubation, the membranes were washed three times (10 min each) with TBS-T solution and incubated at room temperature (1 h) with the specific second- ary antibody conjugated to horseradish peroxidase (HRP). The membranes were again washed three times (10 min each) with TBS-T solution and exposed to HRP substrate (Pierce Biotechnology, Rockford, IL, USA). Immune complexes were visualized by chemiluminescence using ChemiDoc MP System (Bio-Rad Laboratories). Bands were quantified by densitometry using the manufacturer’s software (Image Lab; version 4.1; Bio-Rad Laboratories, Hercules, CA, USA). Values were normalized using the data obtained for β-actin and expressed as arbitrary units. In these experiments, the following groups (n = 5–8) were analyzed: saline + saline, CFA + saline, CFA + EA 20 min, CFA + BML-111 (0.3 μg/100 μl, s.c.), CFA + WRW4 (10 μg/i.pl.), CFA + WRW4 (10 μg/i.pl.) + EA 20 min, and CFA + WRW4 (10 μg/i.pl.) + BML-111 (0.3 μg/100 μl, s.c.).

Statistical analysis

The results were analyzed using GraphPad Prism software (version 6.0; La Jolla, CA, USA). The Shapiro-Wilk normal- ity test was initially applied to evaluate the normality of the data. Results are expressed as means ± standard deviation (SD) for continuous variables. Data was analyzed using one- way analysis of variance (ANOVA) with the Student- Newman-Keuls post hoc test or two-way ANOVA with re- peated measures followed by the Bonferroni post hoc test as appropriate. Differences with a value of P < .05 were consid- ered significant. Results EA and BML-111 reduce mechanical hyperalgesia Results presented in Fig. 2a demonstrate that on the first day post CFA i.pl. injection, acute treatment with EA for 20 min produced a significant reduction of mechanical hyperalgesia 0.5 h (P < .001) and 1 h (P < .001) post treatment, with max- imum inhibition (MI) of 64.0 ± 5%, 0.5 h post treatment. EA for 10 min reduced mechanical hyperalgesia only 0.5 h after treatment with MI of 35.0 ± 10% (Fig. 2a). EA for 5 min, on the other hand, did not affect paw withdrawal frequency (Fig. 2a). Besides that, daily treatment with EA for 20 min for 5 consecutive days reduced (P < .001) mechanical hyperalgesia on all evaluations (Fig. 2b). Figure 2c demon- strates that on the fifth day after CFA i.pl. injection, treatment with EA for 20 min significantly reduced mechanical hyperalgesia up to 2 h with MI of 65.0 ± 13%, 1 h after treatment. Results depicted in Fig. 2d demonstrate that, on the first day post CFA i.pl. injection, acute treatment with BML-111 (0.3–3 μg/100 μl), administered by subcutaneous route, pro- moted a significant reduction of mechanical hyperalgesia (P < .001) with MI of 59.0 ± 8% in the dose of 0.3 μg/ 100 μl, 30 min after treatment. Daily treatment with BML- 111 (0.3 μg/100 μl, s.c.) reduced paw withdrawal frequency on the first (P < .001) and third (P < .05) days (Fig. 2e). Figure 2f demonstrates that on the fifth day post CFA i.pl. injection, only BML-111 (0.3 μg/100 μl, s.c.) significantly reduced mechanical hyperalgesia with MI of 34.0 ± 11%, 0.5 h after treatment. EA and BML-111 did not alter paw edema Daily treatment for 4 consecutive days with either EA or BML-111 (0.3 μg/100 μl, s.c.) did not reduce paw edema (P > .05, data not shown).

Peripheral opioid receptors mediate the antihyperalgesic effect of EA and BML-111

In Fig. 3a, two-way ANOVA revealed significant main effects of naloxone pretreatment (F(1,21) = 8.519; P = .0082), and a naloxone pretreatment × BML-111 interaction (F(1,21) = 4.982; P = .0366), but not main effects of BML-111 (F(1,21) = 2.948; P = .1007). Post hoc analyses indicated that the treatment of mice with BML-111 reduced the mechanical hyperalgesia (P = .0424), and the pretreatment with naloxone prevented (P = .0314) the antihyperalgesic effect elicited by BML-111. In addition, two-way ANOVA revealed significant main effects of naloxone pretreatment (F(1,26) = 5.501; P = .0269) and EA 20 min (F(1,26) = 5.905; P = .0223), but not a naloxone pretreatment × EA 20-min interaction (F(1,26) = 4.201; P = .0506). Post hoc analyses indicated that the pretreatment of mice with naloxone prevented (P = .0345) the antihyperalgesic effect elicited by EA 20 min (Fig. 3b) on the first day after CFA i.pl. injection.
The results illustrated in Fig. 3c and d demonstrate that on day 5 after CFA i.pl. injection, two-way ANOVA revealed significant main effects of naloxone pretreatment (F(1,26) = 5.864; P = .0227), and a naloxone pretreatment × BML-111 interaction (F(1,26) = 5.864; P = .0227), but not main effects of BML-111 (F(1,21) = 2.772; P = .1079). Post hoc analyses indicated that the pretreatment of mice with naloxone prevented the antihyperalgesic effect elicited by BML-111 (P = .0162). Furthermore, two-way ANOVA revealed signif- icant main effects of naloxone pretreatment (F(1,25) = 5.324; P = .0296) and EA 20 min (F(1,25) = 7.445; P = .0115), and a naloxone pretreatment × EA 20-min interaction (F(1,25) = 7.161; P = .0130). Post hoc analyses indicated that the pre- treatment of mice with naloxone prevented (P = .0154) the antihyperalgesic effect elicited by EA 20 min (Fig. 3d).

Peripheral FPR2/ALX receptors participate in the antihyperalgesic effect of EA and BML-111

The results illustrated in Fig. 4a and b demonstrate that on day 1after CFA i.pl. injection, two-way ANOVA revealed signif- icant main effects of BML-111 treatment (F(1,23) = 16.97; P = .0004), and a WRW4 (10 μg/i.pl.) pretreatment × BML- 111 interaction (F(1,23) = 10.48; P = .0036), but not main ef- fects of WRW4 (10 μg/i.pl.) pretreatment (F(1,23) = 4.243; P = .0509). Post hoc analyses indicated that the pretreatment of mice with WRW4 (10 μ g/i.pl.) prevented the antihyperalgesic effect elicited by BML-111 (P = .0045, Fig. 4a).
In Fig. 4b, two-way ANOVA revealed significant main effects of WRW4 (10 μg/i.pl.) pretreatment (F(1,24) = 6.584; P = .0170), and EA 20 min (F(1,24) = 8.766; P = .0068), and a WRW4 (10 μg/i.pl.) pretreatment × EA 20-min interaction (F(1,24) = 6.584; P = .0170). Post hoc analyses indicated that the pretreatment of mice with
WRW4 (10 μg/i.pl.) prevented the antihyperalgesic effect elicited by EA 20 min (P = .0068).
The results illustrated in Fig. 4c and d demonstrate that on day 5 after CFA i.pl. injection, two-way ANOVA revealed significant main effects of WRW4 (10 μg/i.pl.) pretreatment (F(1,23) = 6.169; P = .0207), and BML-111 (F(1,23) = 7.599; P = .0112), and a WRW4 (10 μg/i.pl.) pretreatment × BML- 111 interaction (F(1,23) = 7.599; P = .0112). Post hoc analy- ses indicated that the pretreatment of mice with WRW4 (10 μg/i.pl.) prevented the antihyperalgesic effect elicited by BML-111 (P = .0050, Fig. 4c).
In Fig. 4d, two-way ANOVA revealed significant main effects of WRW4 (10 μg/i.pl.) pretreatment (F(1,23) = 8.946; P = .0065), and EA 20 min (F(1,23) = 11.65; P = .0024), and a WRW4 (10 μg/i.pl.) pretreatment × EA 20-min interaction (F(1,23) = 11.65; P = .0024). Post hoc analyses indicated that the pretreatment of mice with WRW4 (10 μg/i.pl.) prevented the antihyperalgesic effect elicited by EA 20 min (P = .0010).

EA treatment increases ANXA1, but not FPR2/ALX re- ceptor expression in the paw

In Fig. 5a, one-way ANOVA revealed significant main effects of treatments (between columns) (F(4,27) = 5.806;
P = .0017). The results show that animals injected with i.pl. saline express constitutively ANXA1 in paw skin, and Tukey’s post hoc multiple comparisons test demonstrated that
2days after CFA i.pl. injection, there is a decrease (P < .05) in the levels of this protein. This decrease was evidenced in the Fig. 4 Evidence for the involvement of peripheral FPR2/ALX in the antihyperalgesia in- duced by EA or BML-111 on pe- ripheral inflammation induced by CFA injection. Intraplantar pre- treatment with WRW4 (FPR2/ALX-selective antagonist) on day 1 (panels a and b) and day 5 (panels c and d) on the antihyperalgesic effect of BML- 111 (panels a–c) or EA (panels b– d). Vertical bars represent an av- erage of 6–7 animals, and vertical lines show standard deviation. Statistical analyses were per- formed by two-way ANOVA followed by the Bonferroni post hoc test. The symbols denote a significant difference of **P < .01 and ***P < .001 or ##P < .01 and ###P < .001. LXA4 receptor (ALX)/formyl peptide receptor (FPR) 2; i.pl., intraplantar; s.c., subcutaneous groups injected (i.pl.) with CFA that were treated only with WRW4 (P = .0399). The group of animals that were treated only with saline EA for 20 min presented higher values (P = .0474) of ANXA1, when compared to the animals with CFA treated with saline. However, the animals pre- administrated with WRW4 and treated with EA for 20 min presented a lower expression level (P = .0119) of ANXA1 in the hind paw skin, when compared to the animals treated with EA for 20 min. In Fig. 5b, one-way ANOVA revealed significant main effects of treatments (between columns) (F(4,32) = 7.420; P = .0002). Tukey’s post hoc multiple comparisons test indi- cated that the group of animals injected (i.pl.) with CFA and treated with WRW4 (P = .0002) or WRW4 + BML-111 (P = .0015), but not the animals injected with CFA and treated with saline (P = .1611) or BML-111 (P = .0803), presented lower values in the expression of ANXA1. In Fig. 6a, one-way ANOVA revealed significant main effects of treatments (between columns) (F(4,34) = 5.302; P = .0020). Tukey’s post hoc multiple comparisons test indi- cated that the group of animals injected (i.pl.) with saline expresses constitutively the FPR2/ALX receptor in the paw and that 2 days after CFA i.pl. injection, there is an increase (P < .05) in the expression of this receptor. This increase was also evidenced in the groups subjected to CFA i.pl. injection and that were treated only with saline (P = .0362) or EA for 20 min (P = .0025). Animals that received WRW4 and were treated with EA for 20 min presented a lower expression level (P = .0307) of FPR2/ALX receptor in the paw, when com- pared to the animals that only received EA for 20 min. In Fig. 6b, one-way ANOVA revealed significant main effects of treatments (between columns) (F(4,35) = 4.432; P = .0053). Tukey’s post hoc multiple comparisons test indi- cated that the group of animals injected (i.pl.) with CFA and treated with saline (P = .0315) and BML-111 (P = .0048) pre- sented lower values in the expression of FPR2/ALX receptor. In addition, the group of animals pre-administered with WRW4 and treated with BML-111 presented lower values (P = .4690) of FPR2/ALX receptor expression when com- pared to the group treated with only BML-111 (although re- sults were not statistically significant). Discussion The main findings of the present study are as follows: (i) EA reduced mechanical hyperalgesia in different phases (early and late) of CFA-induced inflammation and FPR2/ALX antagonist reduced EA’s effect, (ii) peripheral opioid receptor antagonism prevented the antihyperalgesic effect induced by EA treatment and by systemic injection of FPR2/ALX agonist, and (iii) EA increased ANXA1 levels in the inflamed paw. Using pharmacological approaches, the activation of the FPR2/ALX receptor by LXA4 or ANXA1 has been demon- strated to produce anti-inflammatory and antihyperalgesic ef- fects, including inhibition of leukocyte adhesion to vascular walls [7, 9], induction of apoptosis of tissue neutrophils [7], activation of monocytes to perform efferocytosis [22], inhibi- tion of proinflammatory cytokine release [52], and induction of opioid release by neutrophils [49], among other actions. Considering potential mechanisms of non-pharmacological treatment modalities, it has been demonstrated that EA pro- duces at least some of the abovementioned effects [14, 31, 41]. However, the involvement of the FPR2/ALX receptor in the antihyperalgesic effect of EA is unknown. Thus, in the present study, the effect of EA in early and late phases of inflamma- tory pain induced by CFA i.pl. injection in mice, as well as the participation of peripheral FPR2/ALX receptors in this effect, was investigated. CFA i.pl. injection induces persistent inflammatory pain for weeks [30]. A single treatment with EA produced a short and time-dependent antihyperalgesic effect in the early phase of inflammation. EA daily treatment continued to induce antihyperalgesia. In the late stage of inflammation, EA had a more pronounced long-lasting analgesic effect when com- pared to the early phase. The results of the present study con- firm and extend the current literature data by demonstrating that low-intensity segmental EA in ST36 and SP6 acupoints reduces mechanical hyperalgesia induced by CFA i.pl injec- tion in mice. Corroborating to the results, Huang and colleagues [25] showed that low-frequency EA (2 Hz) only in ST36 acupoint for 20 min, performed immediately after CFA or carrageenan injection, reduces mechanical and ther- mal hyperalgesia (heat and cold) in rats. Chen and colleagues [7] demonstrated in mice with paw inflammation induced by CFA or carrageenan that EA treatment with low frequency (2 Hz) only in acupoint ST36 for 20 min reduced mechanical hyperalgesia on days 1, 2, 4, and 7 after injections of phlogis- tic agents. Finally, Liao and colleagues [29] showed that EA in acupoints ST36 and SP6 for 15 min (2 Hz) reduced me- chanical hyperalgesia for 3 consecutive days in mice subjected to CFA i.pl. injection. Although analgesic effects of EA are well established in the literature [62], the present study adds important new information detailing the time course of EA’s antihyperalgesic effect conducted with different stimulation durations in both the early and late phases of CFA-induced inflammation. The anti-inflammatory role of FPR2/ALX receptors is well known. LXA4 and ANXA1 are the most extensively studied agonists of this G-protein-coupled receptor [16]. Since BML- 111 (an LXA4 analogue) is already well established as an agonist for the FPR2/ALX receptor, the present study used this agonist as a positive control in the analysis of FPR2/ ALX receptor involvement in the antihyperalgesic effect in- duced by EA. We also examined the effect of systemically administered BML-111, in the early and late phases of inflammatory pain induced by CFA i.pl. injection in mice. BML-111 reduced mechanical hyperalgesia for 5 consecutive days of treatment, which indicates that FPR2/ALX is an important component in CFA-induced inflammatory pain. Therefore, the next logical step was to examine the effect of FPR2/ALX blockade on EA- induced antihyperalgesia. Indeed, FPR2/ALX antagonism with WRW4 impaired BML-111 and EA effects both in the early (first day) and in the late (fifth day) phases of CFA- induced inflammation. These results may be related to the population of predominant cells during the evaluation period. FPR2/ALX is expressed in immune cells such as neutrophils and monocytes [37]. In previous studies, neutrophils were shown to be the major opioid peptide containing leukocyte population in early inflammation (within 24 h of injection) while monocytes/macrophages are predominant in later stages [5]. Thus far, an ANXA1-FPR pathway has been shown to induce opioid tonic release from neutrophils in inflammatory pain [40, 45]. It has been reported that both opioid and serotonin recep- tors are involved in the antinociceptive effects elicited by EA stimulation [6]. Low-frequency EA induces the release of en- dorphin and enkephalin, resulting in an analgesic effect of slower onset and longer duration that can be reversed by nal- oxone [61]. However, how EA induces the release of endog- enous opioids has not yet been fully elucidated. Assuming that antihyperalgesic effects of EA could be due to endogenous opioids released from immune cells during inflammation, we locally administered a non-selective opioid receptor antago- nist (naloxone), and our data shows that either EA or BML- 111 antihyperalgesic effects were prevented in the early and late phases of CFA-induced inflammation. Data suggests that release of ANXA1 induced by EA activates the FPR2/ALX receptor, since ANXA1 can induce the release of opioid by neutrophils [19, 43]. Opioid release can induce analgesia in CFA-induced inflammation depending on the mobilization of being present, LPS is known to increase FPR2/ALX expres- sion in microglia [13] and in microvascular endothelial cells [38]. Interestingly, the animal groups that were pretreated with WRW4 and treated with EA or BML-111 had lower FPR2/ALX receptor expression in the paw. Taken together, the re- sults of the pharmacological blockade and protein levels of peripheral FPR2/ALX receptors demonstrate a role of this receptor in the antihyperalgesic effect of EA and BML-111. To our knowledge, the expression of the FPR2/ALX recep- tor at the peripheral terminal of nociceptive primary afferents has not yet been demonstrated. However, it is known that peripheral immune and supportive cells can influence neuro- nal activity (nociception). For example, in injured skin, factors expressed and/or secreted by non-neuronal cells (i.e., fibro- blasts, keratinocytes, as well as infiltrating immune cells) in the proximity of peripheral nociceptive terminals can modu- late nociceptor sensitivity, thereby impacting pro-algesic or analgesic pain sensation. Despite the role of immune cells in antinociception being well established [56], the precise con- tribution of opioid expression by keratinocytes and fibroblasts in peripheral analgesia is poorly understood. Since FPRs have been reported to be involved in keratinocyte activation, further studies exploring the contributions of both cell types are need- ed (for example, establishing a system of co-cultured neutro- phils and keratinocytes). Our findings show a decrease of ANXA1 levels in the hind paw skin after CFA treatment, and that EA treatment acts to prevent this decline. In addition, the i.pl. pretreatment with WRW4 prevented the increase of ANXA1 levels in the paw induced by EA. Thus, our study provides direct evidence that FPR2/ALX mediates the antihyperalgesic effects of ANXA1 on inflammatory pain and by inference that increased levels of ANXA1 might be the result of synthesis and release from neutrophils (the major group of cells 2 days after CFA insult). intracellular Ca2+ and activation of the enzyme phos- Pei and colleagues [42] showed that a high dose of ANXA1 phatidylinositol 3-kinase, both of which are known to be in- tracellular signals of ANXA1 and the FPR2/ALX receptor. These effects are mediated by μ and δ opioid receptors, and when FPR2/ALX antagonist is administered locally, this an- algesic effect can be blocked [15].
Following our initial results with EA and peripheral opi- oid receptors, the next major step became investigating the involvement of peripheral FPR2/ALX receptors in the antihyperalgesic effect of EA, in the early and late phases of CFA-induced inflammation. The results of the current study indicate that peripheral FPR2/ALX receptor-specific antagonist prevents the antihyperalgesic effect induced by EA at the same dose it prevented the antihyperalgesic activ- ity of BML-111.
The present study demonstrated that FPR2/ALX receptor is constitutively expressed in skin paw and that CFA increased its expression. Since CFA contains heat-inactivated mycobacteria in oily solution, with lipopolysaccharide (LPS)
peptide and BML-111 did not increase but, in fact, mildly decreased ANXA1 expression. This data suggests that, as the expression of ANXA1 increases after Anxa12 –26 (ANXA1-derived peptide) and BML-111 administration, the expression of FPR2/ALX might change, too. Studies reported that FPR2/ALX expression rapidly changes in the presence of high or low agonist concentrations [32, 55]. Thus, the current study findings suggest that activation of the FPR2/ALX re- ceptor by ANXA1 may precede release of opioids induced by EA. Future studies should analyze the expression of other mediators that also activate the FPR2/ALX receptor, such as LXA4, as this would be interesting and would expand our current findings.
In conclusion, the main contribution of our current work is to demonstrate for the first time that EA or administration of the analogue of LXA4 (BML-111) reduces mechanical hyperalgesia, but not edema in a CFA-induced inflammatory pain model. This effect is mediated, at least in part, by ANXA1/FPR2/ALX pathway and crosstalk with the opioid system. Thus, our data suggests that a receptor, primarily de- scribed as a mediator of immune responses, may play a rele- vant role in mechanical hyperalgesia observed in inflammato- ry conditions and act as a possible target for EA. Taken to- gether, these findings provide new insights for novel therapeu- tic interventions in the EA treatment of pain.

Funding This work was supported by grants from the National Council for Scientific and Technological Development (CNPq; grant number 430556/2018-7) and Foundation of Support for Research and Innovation of the State of Santa Catarina (FAPESC; grant number 2019TR73) and by the Coordination for the Higher Education (CAPES) and Unisul Scientific Initiation Program (PUIC), Brazil. DFM is supported by research fellowships from CNPq (309407/2017-6).

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