Clarice C.V. Moura, Rafaela S. dos Santos, Lucienir P. Duarte, Giovane Galdino

1Pharmaceutical Sciences Faculty, Federal University of Amazonas, Manaus, Amazonas, Brazil

2Institute of Motricity Science, Federal University of Alfenas, Alfenas, Minas Gerais, Brazil

3Department of Chemistry, Institute of Exact Sciences, UFMG, Belo Horizonte, Minas Gerais, Brazil

ABSTRACT

KEYWORDS: Tingenone; Antinociception; CB2; Cannabinoid receptor; Endocannabinoids; Microglia

1. Introduction

Pain is generally defined as an ‘unpleasant sensory and emotional experience associated with actual or potential tissue damage'[1].Natural products such as herbal medicines have been used as effective and safe alternatives for pain control[2]. Studies have demonstrated that herbal medicines produce fewer complications and fewer side effects when compared with conventional treatments. Among herbal medicines, pentacyclic triterpenoids have been reported for their analgesic and anti-inflammatory effects[3]. Pentacyclic triterpenoids such as tingenone (Tg), which is isolated from roots of Maytenus imbricata (M. imbricata), have been shown to produce antinociception in mice, in a process that involves opioidergic and nitrergic systems at peripheral level[4-6].

Cannabinoid receptors type 2 (CB) have also been shown to participate in the Tg-induced antinociception[7], while other studies have demonstrated the involvement of CBreceptors in peripheral antinociception[8,9]. Another study found that the CBreceptor agonist palmitoylethanolamide-an endocannabinoid-inhibited the formalininduced nociception and mechanical hyperalgesia induced by intraplantar injection of prostaglandin E[10]. This effect was reversed by intraplantar pretreatment with CBreceptor antagonists[10-12].Although studies have demonstrated that both Tg and CBreceptors are involved in antinociception, no study has investigated a possible antinociceptive effect of Tg at spinal level with participation of CBreceptors.

Studies have shown that glial cells are involved in the genesis of nociception, especially in chronic conditions[13,14]. This population of cells is composed of astrocytes essential for the formation and function of synapses by releasing molecular signals[15], and microglia, a specific population of macrophages found in the central nervous system (CNS),which is quiescent when inactivated but after some injuries becomes activated and hypertrophic[16]. Loggia et al.[17]described that this cell population is responsible for the release of pro-inflammatory proteins that are crucial in nociceptive impulse transmission[13,18,19], leading to the response of allodynia and hyperalgesia.

Given this context, the endocannabinoid system may be an important modulatory pathway of crosstalk between microglia-astrocytesneurons. Endocannabinoids are released ‘on demand' by neurons,as well as by astrocytes and microglia[20]. Thus, this study aimed to investigate the influence of CBreceptors on the spinal microglia during Tg-induced antinociception.

2. Materials and methods

2.1. Animals

This study used male Swiss mice (25—30 g) provided by the Central Vivarium of the Federal University of Alfenas, MG. All mice were housed in a climate-controlled room [(23 ± 2) ℃, (50 ± 10)% relative humidity]under a 12 h light/dark cycle.

2.2. Drugs

The following drugs were used: carrageenan (2%, intraplantar(i.pl.)]; 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl(4 methoxyphenyl) methanone [AM630, 4 μg, intrathecal(i.t.)], a CBreceptor antagonist; (5Z, 8Z, 11Z, 14Z)-5,8,11,14-eicosatetraenyl-methylester phosphonofluoridic acid (MAFP, 2 μg,i.t.), an irreversible fatty acid amide hydrolase (FAAH) inhibitor and minocycline hydrochloride (30 μg, i.t.), a microglial inhibitor.Carrageenan and minocycline were purchased from Sigma (MO,USA) and AM630 and MAFP were purchased from Tocris Bioscience (Ellisville, MO). Carrageenan and minocycline were dissolved in sterile saline, MAFP was dissolved in saline and ethanol(2%, Merck, NJ, USA), and AM630 was dissolved in sterile saline,DMSO (2%, Sigma, USA) and cremophor (5%, Sigma, USA).

2.3. Plant

Tg was isolated from roots of M. imbricata (Celastraceae) plant.The plant's roots were carefully collected, dried, and powdered in a mill. The powder (1.5 kg) was submitted to extractions in a Soxhlet extractor with the following organic solvents: hexane/ethyl ether(1:1), ethyl acetate, and methanol (2 L). Afterwards, the solvents were removed from the extracts in a rotator evaporator. During this phase, the following quantities of each filtrate were obtained: 16.1 g from the hexane/ethyl ether extract, 21.2 g from the ethyl acetate extract, and 176.7 g from the methanol extract. From the hexane/ethyl ether extract, 1.5 g of Tg was isolated and characterized following a previous study[21]. The hexane/ethyl ether extract (3.0 g) was subjected to silica gel (300.8 g) column chromatography and eluted with hexane-ethyl acetate[22]. Three hundred and fifty fractions of 100 mL each were obtained, and the fractions 66 to 85 produced an orange solid (314.0 mg), yielding 15.7% and 145.0—147.9 ℃ m.p., being identified as Tg.

2.4. Injections

2.4.1. I.pl. injection

For i.pl. injections, the animals were lightly anaesthetised with 2% isoflurane and O(2 L/min) by an inhalation analgesia system using a calibrated vaporiser. The mice were positioned flat to allow an unobstructed view of the paws, and the right hind paws of the animals were cleaned with ethyl alcohol (70%) and carefully positioned for the subcutaneous injection of the test agents using a hypodermic syringe with a 24-gauge needle[23]. I.pl. injections of carrageenan were given 60 min after Tg administration.

2.4.2. I.t. injection

Before i.t. injections, the dorsal (lumbar) regions of the animals were shaved, and then they were lightly anaesthetised with 2% isoflurane and O(2 L/min) via an inhalation analgesia system using a calibrated vaporiser and positioned to facilitate the palpation of the L5—L6 intervertebral spaces in the lumbar spine. The i.t. injections were performed in this intervertebral space using a 10-μL Hamilton syringe connected to a 28-gauge needle[24]. A flick of the tail was considered as an indicator of successful i.t. administration. I.t.injections of each drug were given 20 min before Tg administration.

2.4.3. Gavage administration

The administration of Tg was by oral gavage. The animals were gently restrained by the experimenter and maintained in an upright(vertical) position and the gavage needle (18 gauge) passed went through the roof of the mouth until reaching the oesophagus. The volume administered was equivalent to 1% of the weight of each animal. Tg was given 60 min before carrageenan injection.

2.5. Nociceptive test

To measure the mechanical nociceptive threshold (g), the von Frey filament test was used (Stoeling, Wood Dale, IL, USA). For this test,the mice were allocated in small boxes on a metal mesh floor, on an elevated wire mesh platform in individual glass compartments and acclimated for at least 30 min[25]. A mechanical stimulus was applied to the middle of the plantar surface using a series of von Frey filaments with increasing bending forces (0.07, 0.16, 0.4, 0.6,1.0, 1.4, and 2.0 g) and the pressure value recorded at right paw withdrawal. Mean values of three consecutive tests applied at 3 min intervals were recorded.

A total of 102 mice were divided into the following groups (n=6):Cg + Veh, animals that received i.pl. injection of the carrageenan and oral pretreatment with Tg vehicle; Cg + Tg, animals that received i.pl. injection of the carrageenan and oral pretreatment with Tg at the 5.3, 15.9 and 53.0 mg/kg doses; and Veh + Veh, animals that received i.pl. injection of the carrageenan vehicle and oral pretreatment of Tg vehicle. In addition, to investigate the participation of the endocannabinoid system in the Tg-induced antinociception, similar groups were used to those previously described with the preadministration of AM630 (a CBreceptor antagonist) and MAFP(a FAAH inhibitor). To investigate the involvement of microglia in the Tg-induced antinociception, similar groups were used with intrathecal pre-administration of minocycline. The doses of Tg used in this study were chosen following a previous study[4].

2.6. Immunoreactivity of cannabinoid CB2 receptor and microglia

To evaluate the expression and co-localisation of CBreceptors and microglia in the spinal cord, double stain immunofluorescence was performed in sections of the spinal cord (L4—L6 segments). For that,the animals were euthanised and transcardially perfused with 0.01 M phosphate buffer solution, pH 7.4, followed by paraformaldehyde(4%) and 0.1 M phosphate buffer solution (pH 7.4). After that,the samples were fixed and stored in sucrose for cryoprotection.Afterwards, spinal cord sections (20 μm) were washed (0.1 M PBS) and incubated in 0.5% triton X glycine (0.1 M), then washed and incubated again for 1 h in 3% bovine serum albumin (Sigma-Aldrich). The sections were incubated overnight at 4 ℃ in anti-CBreceptor antibody (1:100; Santa Cruz, USA) and anti-CD11B (1:500;Millipore, USA). After incubation in the primary antibody, sections of tissue were washed and incubated in a 488 anti-rabbit goat IgG(1:200, Santa Cruz, USA) and Alexa Fluor 594 anti-rat goat IgG(1:400; ThermoFisher, USA) for 2 h. After that, the sections were assembled using fluoromount and analysed by epifluorescence acquired using a confocal microscope (K2, Nikon, Japan). Sections were visually chosen using a 10× objective. Images were analysed using ImageJ software (NIH, Bethesda, USA).

2.7. Experimental protocol

2.7.1. Protocol 1

To evaluate the antinociceptive effect of Tg, the animals orally received three doses of Tg (5.3, 15.9, and 53.0 mg/kg), 1 h before carrageenan injection (Supplementary Figure 1). The mechanical nociceptive threshold was measured before (baseline) and at 1, 3, 4,5, 6, and 7 h after carrageenan injection (Supplementary Figure 1).

Figure 1. Effect of tingenone on hyperalgesia induced by carrageenan. Data are expressed as mean ± SEM of the nociceptive threshold of animals per group. ***P ＜ 0.001 versus the control group (Veh+Veh), which received only vehicles; ###P ＜ 0.001 between the group that received carrageenan and groups that received carrageenan plus tingenone (Cg+Tg). Two-way ANOVA followed by Bonferroni's multiple comparison test. BL: Baseline latency, Tg: tingenone; Cg: carrageenan; Veh: vehicle.

2.7.2. Protocol 2

To investigate the involvement of the endocannabinoid system in this Tg-induced antinociception, the baseline latency of nociceptive threshold was firstly measured and AM630 (4 μg/5 μL) and MAFP(2 μg/5 μL) were i.t. injected 20 min before administration of Tg(Supplementary Figure 1). Then, measurements of nociceptive mechanical threshold were performed 20, 60, 120, 180, 240,300, 360, 420, 480 and 1 440 min after carrageenan injection(Supplementary Figure 1).

2.7.3. Protocol 3

To investigate microglial involvement in Tg-induced antinociception, minocycline (30 μg/5 μL i.t.) was administered 20 min before Tg (Supplementary Figure 1). The nociceptive mechanical threshold was evaluated 20, 60, 180, 240, 300, 360, 420,480, 540, and 1 440 min after carrageenan injection (Supplementary Figure 1).

The doses of AM630, MAFP and minocycline used in this study were according to previous study conducted by Dos Santos et al.[25].

2.8. Statistical analysis

The data were presented as mean ± SEM of the evaluated parameter and were analysed for statistical significance as follows: two-way ANOVA for repeated measurements, followed by Bonferroni's post hoc test for multiple comparisons of nociceptive threshold measurements; and by one-way ANOVA followed by Bonferroni's post hoc test for analysis of immunofluorescence results. Significance was considered when P ＜ 0.05. Statistical analyses and preparation of figures were done using the GraphPad Prism 5.0 software(GraphPad Software, La Jolla, CA).

2.9. Ethical statement

This study was previously approved by the Animal Care and Use Committee at the Federal University of Alfenas (protocol number:683/2015). Experiments were carried out following the International Association for the Study of Pain guidelines for animal studies[26].

3. Results

3.1. Effect of Tg on inflammatory pain induced by carrageenan

Figure 1 shows that i.pl. administration of carrageenan induced significant mechanical hyperalgesia (P ＜ 0.001, F= 17.4),which lasted from 1 to 7 h. This effect was reversed (P ＜ 0.001,F= 17.4) by Tg at 5.3, 15.9 and 53 mg/kg doses, characterising an antinociceptive effect. Tg vehicle did not alter the nociceptive threshold.

3.2. Spinal participation of CB2 receptor in Tg-induced antinociception

After we found the antinociceptive effect induced by Tg, the next step was to investigate the involvement of the CBreceptor in this effect at spinal level. The result showed that Tg-induced antinociception was significantly reversed (P ＜ 0.001, F= 32.89)by the CBreceptor antagonist AM630 at the 4 μg dose (Figure 2),suggesting the participation of this receptor in this effect. The vehicle did not alter the nociceptive threshold.

Figure 2. Participation of the cannabinoid CB2 receptor in the tingenoneinduced antinociception. Data are expressed as mean ± SEM of the nociceptive threshold of six animals per group. ###P ＜ 0.001, versus the carrageenan plus tingenone group (Cg+Tg). Two-way ANOVA followed by Bonferroni's multiple comparison test.

3.3. Involvement of the endocannabinoids in the Tg-induced antinociception

In addition to participation of the CBreceptor, we investigated the involvement of the endocannabinoid in the antinociceptive effect induced by Tg. The pretreatment with MAFP (2 μg, i.t.), an inhibitor of the FAAH enzyme, potentiated and prolonged the antinociception induced by Tg (P ＜ 0.001, F= 48.42; Figure 3). The vehicle did not alter the nociceptive threshold.

Figure 3. Involvement of endocannabinoids in the tingenone-induced antinociception. Data are expressed as mean ± SEM of the nociceptive threshold of six animals per group. ###P ＜ 0.001, versus the carrageenan plus tingenone group (Cg+Tg). Two-way ANOVA followed by Bonferroni's multiple comparison test.

3.4. Spinal involvement of microglia in the antinociception induced by Tg

As the CBreceptor is present in microglia, we assume that it may interfere in the pain process by inhibiting these cells. Thus, to demonstrate the involvement of spinal microglia in the genesis of inflammatory pain, minocycline was i.t. administered-a selective microglial inhibitor drug.

We thus verified that minocycline prolonged and potentiated the antinociceptive effect of Tg (P ＜ 0.001, F= 60.62) after 420 min in the inflammatory pain group (Figure 4).

Figure 4. Participation of the spinal microglia in the antinociceptive effect induced by tingenone. Data are expressed as mean ± SEM of the nociceptive threshold of six animals per group. ###P ＜ 0.001, versus the carrageenan plus tingenone group (Cg+Tg). Two-way ANOVA followed by Bonferroni's multiple comparison test. Mino: minocycline.

3.5. Effect of Tg on the CB2 receptor and microglia immunoreactivity in spinal cord

After we demonstrated that the spinal CBreceptor participates in the Tg-induced antinociception, and that spinal microglia are involved in inflammatory pain, we evaluated the effect of Tg on the expression of this receptor and microglial cells in the spinal cord.Figure 5 shows that the group received carrageenan and Tg induced a significant increase (P ＜ 0.001) of the microglia and CBreceptor expression in the spinal cord. Furthermore, the increased spinal microglia expression was reduced (P ＜ 0.001) in mice that received carrageenan and pretreatment with minocycline, MAFP, and AM630.Moreover, the CBexpression was inhibited in mice pre-treated with AM630 (Figure 5).

Figure 5. Microglia and cannabinoid CB2 receptor immunoreactivity in mice with inflammatory pain pretreated with tingenone. Representative immunofluorescence staining of microglia (red) and cannabinoid CB2 receptor (green) in the spinal cord dorsal horn. Scale bars, 20 μm. Data are expressed as mean ± SEM of 4-5 animals per group. ***P ＜ 0.001, versus the control group (Co). One-way ANOVA followed by the Bonferroni test.

4. Discussion

This study demonstrated that Tg inhibits inflammatory pain and that this effect involves the participation of the spinal CBreceptor.This study also verified that spinal microglia may participate in the hyperalgesia induced by carrageenan, suggesting that the inhibition of these cells potentiated the Tg-induced antinociception.

Several studies have demonstrated that CBreceptor activation inhibits pain at spinal level[25,27,28]; however, none have investigated this effect induced by herbal medicines such as Tg. A previous study showed that Tg promoted antinociception via CBreceptor activation, but this effect was evaluated peripherally[7]. Type 2 cannabinoid receptors are widely found in immune cells[29], although studies have described these receptors in the CNS[30]. Once activated by endocannabinoids, the firing rate of excitable cells is reduced,leading to the inhibition of neurotransmitter release and reduction in the nociceptive response[31]. Other authors reported an association between increased CBreceptor expression and reduced spinal microglia activation in rodents with inflammatory and neuropathic pain[32,33]. According to these findings, we suggest that Tg may activate the CBreceptors on the spinal microglia, facilitating the inhibition mechanism of hyperalgesia.

This activation is likely to occur directly at the receptors, or indirectly by increasing levels of endocannabinoids. We found that MAFF-a FAAH inhibitor-is an enzyme that hydrolyses endocannabinoids anandamide and that 2-AG, potentiated and prolonged the Tg-induced antinociception after 360 to 480 min of its administration. In a previous study conducted by Veloso et al.[7],it was verified that MAFP injected peripherally did not alter the antinociceptive effect of Tg. Thus, the effect produced by MAFP and Tg in the present study may be restricted to the spinal level and involve the release and increase of endocannabinoids. Furthermore,i.t. injection of MAFP also potentiated the antinociception induced by exercise in mice with muscle inflammatory pain[25],reinforcing the hypothesis that this inhibitor increased the supply of endocannabinoids during Tg-induced antinociception.

Moreover, the present study suggests that all these events occur together with spinal microglia involvement. Microglia are macrophage-like cells in the CNS that regulate homeostasis in the brain and spinal cord[34]. After tissue or nerve injury, synaptic plasticity in the brain and spinal cord increases, an event known as central sensitisation; microglia are the main cells involved in this response[34]. Once activated, a signaling cascade occurs in the microglia, which results in the release of pronociceptive molecules responsible for sensitising neurons in the dorsal horn of the spinal cord and facilitating the transmission of the nociceptive impulse[34].Thus, we suggest that Tg may activate CBreceptors on the spinal microglia, reducing the nociceptive impulse.

Studies also found an association between the increase in CBreceptor expression and the reduction of spinal microglia activation in animals with paw incision and peripheral nerve injury[28,32,33,35,36].This hypothesis was supported by different studies that used different CBreceptor agonists (HU-210, WIN55, 212—2, and JWH-133)and found that nociceptive molecules, such as tumour necrosis factor and nitric oxide were reduced, as well as increases in antiinflammatory cytokines caused by microglia[32,33,37-39]. In this study, MAFP, AM630, and minocycline reduced the spinal microglia immunoreactivity in mice that received i.pl. injection of carrageenan,supporting the hypothesis that these cells are involved in the inflammatory pain model used and that the endocannabinoid system may inhibit these cells.

The main limitations of the present study were that it is not yet possible to carry out the antinociceptive effect in humans, mainly due to ethical issues for conducting controlled trials, and in addition,Tg is not commercially available.

Thus, the present study suggests that spinal microglia participate in the inflammatory pain induced by carrageenan and that Tg may inhibit this process by activation of the endocannabinoid system.These findings may be important for future studies that investigate the effect of herbal medicines in the control of pain.

Conflict of interest statement

We declare that there is no conflict of interest.

Funding

This work was supported by the Coordena??o de Aperfei?oamento de Pessoal de Nível Superior - Brasil (CAPES) (Finance Code 001).

Authors' contributions

CCVM developed theoretical formalism, contributed to the original, revision, and final version of the manuscript, as well as supervised the project. GG performed the analytic calculation and formal analysis, and contributed to the original, revision, and final version of the manuscript. RSS contributed to the original version of the manuscript, as well as performed the analytic calculation and formal analysis. LPD ceded tingenone for pharmacological studies.