Prostaglandin 15d-PGJ₂ targets PPARγ and opioid receptors to prevent muscle hyperalgesia in rats

Abstract

Pharmacological agents directed to either opioid receptors or peroxisome proliferator-activated receptor gamma (PPARγ) at peripheral tissues reduce behavioral signs of persistent pain. Both receptors are expressed in muscle tissue, but the contribution of PPARγ activation to muscle pain and its modulation by opioid receptors remains unknown. To address this question, we first tested whether the endogenous PPARγ ligand 15d-PGJ₂ would decrease mechanical hyperalgesia induced by carrageenan administration into the gastrocnemius muscle of rats. Next, we used receptor antagonists to determine whether the anti-hyperalgesic effect of 15d-PGJ₂ was PPARγ- or opioid receptor-dependent. Three hours after carrageenan, muscle hyperalgesia was quantified with the Randall-Selitto test. 15d-PGJ₂ prevented carrageenan-induced muscle hyperalgesia in a dose-dependent manner. The anti-hyperalgesic effect of 15d-PGJ₂ was dose-dependently inhibited by either the PPARγ antagonist, GW9662, or by the opioid receptor antagonist, naloxone. We conclude that 15d-PGJ₂ targets PPARγ and opioid receptors to prevent muscle hyperalgesia. We suggest that local PPARγ receptors are important pharmacological targets for inflammatory muscle pain.

Introduction

Chronic musculoskeletal pain impacts approximately 5% - 33% of the population (including children)^1,2 and afflicts one third to one-half of multimorbidity patients particularly in the elderly². Chronic musculoskeletal pain limits mobility and is the second most common cause of disability worldwide, carrying a significant socio-economic impact^1,3 and representing a risk factor for the development of depression, obesity, cardiovascular diseases, and earlier mortality⁴. Thus, chronic musculoskeletal pain is among the most prevalent examples of persistent pain that can last throughout life.

Peripheral pro-inflammatory cytokines and nociceptor-sensitizing agents, such as neutrophil migration, bradykinin, sympathetic amines and prostanoids mediate acute muscle hyperalgesia⁵. Intense, long-lasting muscle pain leads to a transition from acute to chronic pain⁶ due to peripheral nociceptor sensitization, central sensitization of dorsal horn neurons, and/or a pathological reorganization of pain modulatory centers in the brain^6,7. Although these mechanisms overlap with other painful conditions, muscle pain has unique characteristics: the quality of muscle pain is often described as aching and cramping; depending on the source of nociceptive input, the clinical presentation of muscle pain is both localized and referred⁷; nerve innervation to muscle is less than the skin, but greater than the visceral structures⁶; and descending inhibition of muscle pain is relatively strong as compared to cutaneous pain⁷.

In this study, we propose the 15-deoxyΔ−12,14-prostaglandin J₂ (15d-PGJ₂) / peroxisome proliferator-activated receptors gamma (PPARγ) system as a new pharmacological target for the control of muscle hyperalgesia. PPARγ is a nuclear receptor established as a primary lipid sensor and a modulator of lipid metabolism⁸. 15d-PGJ₂ is a potent endogenous ligand of PPARγ receptors and produces anti-nociceptive effects in multiple animal models of inflammatory pain including intraplantar carrageenan, formalin and PGE₂⁹, intra-articular formalin¹⁰, collagen-induced arthritis¹¹, gout¹², among others. PPARγ activation by 15d-PGJ₂ induces the local release of opioid peptides from immune cells, leading to a decrease in mechanical hypersensitivity¹³. However, whether 15d-PGJ₂ can also decrease muscle pain remained unknown. Here, we demonstrate for the first time the anti-hyperalgesic effect of 15d-PGJ₂ in an animal model of muscle pain. In addition, using a pharmacological receptor blockade strategy, we show that the anti-hyperalgesic actions of 15d-PGJ₂ are mediated by PPARγ and /or opioid receptors.

Material and methods

Animals

Male Wistar rats (200–250 g) from CEMIB (Multidisciplinary Center for Biological Research) UNICAMP were used under the approval of the ethics committee for animal use of the State University of Campinas (2986–1) and carried out in accordance with IASP guidelines on using laboratory animals. Laboratory animals were housed in plastic cages (five per cage) with soft bedding with food and water ad libitum. 12h light/dark cycles were set in a room temperature of 23°C. Animals were habituated to the test room for 1 h before testing began. Animals were euthanized with CO₂ asphyxiation once experimental procedures were complete.

Drugs

15d-PGJ₂ was obtained from (Calbiochem, San Diego, CA, USA). The selective PPARγ antagonist GW9662 (2-Chloro-5-nitro-N-phenylbenzamide), the non-selective opioid antagonist naloxone and the inflammatory agent λ-Carrageenan were obtained from Sigma-Aldrich (St. Louis, MO, USA). Naloxone and λ-Carrageenan were diluted in saline, while 15d-PGJ₂ and GW9662 were diluted in 100% dimethyl sulfoxide (DMSO) from Sigma-Aldrich (St. Louis, MO, USA) before dilution in 0.9% NaCl (saline) to a final concentration of 5% DMSO.

Intramuscular injections

All drugs or their vehicles were injected in a total volume of 50 μL using a 30-gauge needle into the left gastrocnemius muscle of briefly restrained, awake rats. As a control for systemic or central actions, 15d-PGJ₂ was also administered into the right gastrocnemius.

Carrageenan-induced inflammatory pain model

Muscle hyperalgesia was induced by single administration of carrageenan in a dose of 100 μg/25μl into the left gastrocnemius muscle.

Behavioral testing

Experiments were conducted between 9:00 a.m. to 5:00 p.m. in a quiet room at a controlled temperature of 23°C. Muscle hyperalgesia was evaluated with a Randall–Selitto analgesiometer (Insight, Ribeirao Preto, SP, Brazil) test of mechanical nociceptive threshold. This method applies a linearly increasing force (g) to the gastrocnemius muscle until a reflex withdrawal of the hindlimb was observed. The average of three measures were collected at 5 minutes intervals. Mechanical muscle hyperalgesia, represented by the y-axis, reflects the difference in threshold between measurements collected before (baseline) versus after carrageenan injection. Measurements were taken three hours after carrageenan administration, at the peak of nociceptive behaviors. Time points represent the time elapsed since carrageenan injection and are referred to as post injection.

Statistical analysis

The groups sizes per experiment was determined by a power analysis (α, 0.05; power, 0.99) using the effect size and variance estimated from previous studies or preliminary data. G*Power 3 was used for the power analysis. Data were analyzed with GraphPad Prism 7.0 by one-way ANOVA followed by Tukey’s post hoc test. Statistical significance was set at P < 0.05. Dose-response curves were calculated via nonlinear regression analysis (Y=Bottom + (Top-Bottom)/(1+10^((X-LogED50))). Data are expressed as a decrease in paw-withdrawal threshold in grams (g) and presented as mean ± SEM.

Results

Intra-muscular administration of 15d-PGJ₂ inhibits carrageenan-induced mechanical muscle hyperalgesia in a dose-dependent manner

We first tested the hypothesis that local administration of 15d-PGJ₂ would reduce carrageenan-induced mechanical muscle hyperalgesia. To this end, animals were pre-treated with 15d-PGJ₂ 15 minutes before induction of muscle hyperalgesia (Fig 1 A). As illustrated in Fig. 1B, carrageenan (100 μg), but not saline vehicle, induced mechanical hyperalgesia (F=52.31; P < 0.0001; n = 5). Pretreatment with 15d-PGJ₂ (100 and 10, but not 1 ng), prevented carrageenan-induced mechanical hyperalgesia when administered in the ipsilateral, but not the contralateral gastrocnemius muscle (F=49.6; P < 0.0001). The anti-hyperalgesic effect of 15d-PGJ₂ occurred in a dose-dependent manner with an ED50 of 5.8 ng (fig 1C).

Figure 1.

Experimental timeline and the effect of the PPARγ receptor agonist 15d-PGJ₂ in carrageenan-induced mechanical muscle hyperalgesia. (A) 15d-PGJ₂ was administered 15 minutes before carrageenan injection. Behavior was quantified 3h after carrageenan. (B) Pre-treatment with 15d-PGJ₂ (10 and 100, but not 1 ng) 15 min before carrageenan (Cg) injection significantly prevented hyperalgesia when administered into the ipsilateral, but not contralateral (CL) gastrocnemius muscle (Tukey post-tests: * P < 0.05 n = 5). (C) Pre-treatment with 15d-PGJ₂ prevented mechanical muscle hyperalgesia in a dose-dependent manner (nonlinear regression analysis; ED₅₀ = 5.8 ng; n = 3–5).

The local anti-hyperalgesic effect of 15d-PGJ₂ in the carrageenan model requires PPARγ activation

Next, we evaluated whether the anti-hyperalgesic effect of 15d-PGJ₂ on carrageenan-induced mechanical muscle hyperalgesia was dependent on PPARγ receptor activation. To this end, animals were pre-treated 15 minutes before 15d-PGJ₂ with the PPARγ antagonist, GW9962 (Fig 2 A). As shown in Fig. 2B, intramuscular administration of GW9662 prevented the anti-hyperalgesic effect of 15d-PGJ₂ (F=114.2 P < 0.0001 in a dose-dependent manner with an ED50 of 4.1 ng (fig 2C). Administration of GW9662 had no effect by itself (p>0.05).

Figure 2.

The anti-nociceptive effect of 15d-PGJ₂ in carrageenan-induced mechanical muscle hyperalgesia is PPARγ receptor-dependent. (A) PPARγ receptor antagonist, GW9662 was administered 15 minutes before 15d-PGJ₂ and behavior was quantified 3h after carrageenan (B) Pre-treatment with GW9662 (3 and 9, but not 1 ng or vehicle) significantly prevented the anti-nociceptive effect of 15d-PGJ_2, when tested 3h after carrageenan (Cg) injection (Tukey post-tests: * P < 0.05 n = 5). (C) Pre-treatment with GW9662 prevented the anti-hyperalgesic effect of 15d-PGJ₂ in a dose-dependent manner (nonlinear regression analysis, p<0.05; ED₅₀ = 4.1ng n = 4–5).

The local anti-hyperalgesic effect of 15d-PGJ₂ in the carrageenan model requires opioid receptor activation

To evaluate whether the anti-hyperalgesic effect of 15d-PGJ₂ requires opioid receptor activation, the animals were pre-treated 15 minutes before 15d-PGJ₂ with naloxone (Fig 3 A). As illustrated in Fig. 3B, intramuscular naloxone prevented the anti-hyperalgesic effect of 15d-PGJ₂ (fig 3B, F=55.06; P < 0.0001; n = 5) in a dose-dependent manner with an ED50 of 0.2 μg (fig 3C).

Figure 3.

The anti-nociceptive effect of 15d-PGJ₂ in carrageenan-induced mechanical muscle hyperalgesia is opioid receptor-dependent. (A) The non-specific opioid receptor antagonist, Naloxone was administered 15 minutes before 15d-PGJ₂ and behavior was quantified 3h after carrageenan (B) Pre-treatment with naloxone (0.1 and 1, but not 0.01 μg or vehicle) significantly prevented the anti-hyperalgesic effect of 15d-PGJ₂ when tested 3h after carrageenan (Cg) (Tukey post-tests: * P < 0.05 n = 5). (C) Naloxone prevented the anti-hyperalgesia effect of 15d-PGJ₂ in a dose-dependent manner (nonlinear regression analysis, p<0.05; ED₅₀ = 0.2μg n = 4–5).

Discussion

In the present study, we show for the first time that intra-muscular administration of 15d-PGJ₂ prevents mechanical hyperalgesia in an inflammatory model of muscle pain. Moreover, the selective PPARγ receptor antagonist GW9662 and the non-selective opioid receptor antagonist naloxone prevented the anti-hyperalgesic effect of the 15d-PGJ₂, indicating a PPARγ- and opioid receptor-dependent mechanism.

Relying on muscle hyperalgesia models to test a potential analgesic is important due to their unique characteristics that differs from other types of pain regarding the neural mechanisms⁶. Different muscle skeletal pain conditions are related to markers of inflammation such as cytokines and prostaglandins that can lead to peripheral and central sensitization⁷. For example, carrageenan-induced muscle hyperalgesia is associated with the local release of pro-inflammatory cytokines such as IL-6, CINC-1 and IL-1β¹⁴.

The potent antinociceptive effect of 15d-PGJ₂ is at least in part due to downregulation of proinflammatory cytokines¹⁵. 15d-PGJ₂ can inhibit NF-κB¹² first leading to downregulation of TNFα¹¹, IL-6, IL-12, IL-18 and/or the chemokine CINC-1¹⁵, and then leading decreases in nociceptive neuronal excitability and hypersensitivity⁹. Furthermore, chronic administration of 15d-PGJ₂ decreases mechanical hypersensitivity by inhibition of T cells¹¹, while single injection of 15d-PGJ₂ decreases mechanical hypersensitivity in multiple models including PGE2-induced hyperalgesia and hyperalgesia induced by MSU crystals (gout pain model) by decreasing local neutrophil and leucocyte migration, respectively^10,12. Although we did not measure the expression of pro-nociceptive signaling molecules or cell types involved in the anti-hyperalgesic effect of 15d-PGJ₂, given that muscle hyperalgesia is modulated by polymorphonuclear cells⁵, we hypothesize that 15d-PGJ₂ prevented carrageenan-induced muscle hyperalgesia due its ability to modulate peripheral immune cells leading to the downregulation of pro-nociceptive mediators.

The majority of the studies using inflammatory pain models have described a potent antinociceptive effect of 15d-PGJ₂ when locally administered^9,13. Intraplantar administration of 15d-PGJ₂ decreased mechanical hypersensitivity induced by the plantar injection of either carrageenan or prostaglandin E2⁹. This effect lasted for 3 hours; a time frame similar with the present study. A rapid effect of 15d-PGJ₂ can be also found in neuropathic pain models: 15d-PGJ₂ dose-dependently decreased mechanical hypersensitivity within 45 minutes, a time course that suggests a transcription-independent mechanism of action¹⁰. In fact, PPARγ agonists can inhibit hyperalgesia by both genomic and non-genomic receptor-dependent mechanisms¹⁶. Therefore, considering the very short half-life of 15d-PGJ₂, the antihyperalgesic effect 3 hours after carrageenan administration, as shown in the present study, may indicate a receptor-dependent genomic or translation mechanism.

We used a receptor antagonist strategy to ask whether the inhibitory effect of 15d-PGJ₂ on muscle hyperalgesia was dependent on PPARγ receptors. We found that the selective PPARγ antagonist GW9662 prevented the antihyperalgesic effect of 15d-PGJ₂ in a dose dependent manner, suggesting a local receptor dependent-mechanism. 15d-PGJ₂ is endogenously synthesized and therefore might activate PPARγ and thus tonically inhibit pain. To test this hypothesis, we asked whether GW9662 given by itself would increase carrageenan-induced mechanical hyperalgesia. We found that GW9662 did not change hyperalgesia. This result is consistent with the literature indicating that baseline or inflammation-evoked concentrations of 15d-PGJ₂ are insufficient to tonically activate PPARγ receptors⁸. Furthermore, lipopolysaccharide-induced COX activation in vitro by LPS did not increase the biosynthesis of 15d-PGJ₂, and the same study reported similar 15d-PGJ₂ concentrations in the joint fluid of patients with or without arthritis¹⁷. It is likely that a 1000 -fold increase in 15d-PGJ₂ concentrations is required to sufficiently activate PPARγ so as to downregulate NF-κB signaling^17,18.

In the present study, the non-selective opioid receptor antagonist, naloxone, prevented the anti-hyperalgesic effect of 15d-PGJ₂ in a dose dependent manner. These results are consistent with previous data showing that the anti-hyperalgesic effect of 15d-PGJ₂ was prevented by naloxone in the intra-plantar PGE2 model of hyperalgesia⁹. Further studies using selective opioid antagonists showed that kappa (κ) and delta (δ), but not mu (μ) receptors¹⁰, were involved in the anti-hyperalgesic effect of 15d-PGJ₂ via a peripheral site of action¹³. Other evidence for opioid-dependent effects of PPARγ agonists include studies of morphine tolerance. For example, pioglitazone, a PPARγ receptors agonist, negatively modulated morphine tolerance, while a PPARγ antagonist facilitated signs of opioid withdrawal¹⁹.

Based on published preclinical and clinical studies, we do not believe that pharmacological inhibition of peripheral or central opioid receptors with naloxone would increase muscle hyperalgesia in our carrageenan model. Although tonic descending inhibition of dorsal horn nociceptive neurons requires opioidergic signaling at the supraspinal level²⁰, pre-treatment with systemic naloxone does not further increase mechanical or thermal hyperalgesia induced by intraplantar carrageenan²¹. Naloxone does not to increase hyperalgesia in other models of pain either. For example, neither naloxone (crosses blood–brain barrier), nor the peripherally restricted opioid antagonist naloxone methiodide, changed the intensity of nociceptive responses induced by a 5% hypertonic saline injection in the gastrocnemius, triceps or masseter muscles of rats²². Also, continuous intravenous infusion of naloxone did not change intraplantar formalin-evoked nociceptive responses²³. Furthermore, naloxone does not change muscle pain induced by maximum voluntary contraction and post-exercise muscle ischemia in humans²⁴. However, it must be kept in mind that we cannot completely exclude the possibility that intra-muscular carrageenan produces a latent sensitization that is tonically inhibited by mu opioid receptor constitutive activity, whereby naloxone would unmask hyperalgesia²⁵.

15d-PGJ₂-induced activation of PPARγ can increase the expression and likely the release of β-endorphin and dynorphin in leucocytes. Subsequent binding to κ and δ opioid receptors in primary sensory neurons may then lead to anti-nociception¹³. We conclude that 15d-PGJ₂ -mediated anti-hyperalgesia engages endogenous opioid systems. If this occurs without producing adverse effects such as withdrawal or tolerance (as occurs with opioid receptors agonists), then 15d-PGJ₂-like compounds may be useful as a new class of analgesics for muscle pain.

Therefore, we conclude that intramuscular administration of 15d-PGJ₂ prevented mechanical muscle hyperalgesia induced by carrageenan. This effect is mediated by both PPARγ and opioid receptors. We propose future investigations to clarify the mechanism of action, aiming to establish 15d-PGJ₂ activation of PPARγ as an important pharmacological strategy for the treatment of muscle pain.