In the current study, we investigated the possibility that EP may be potential analgesic for formalin-induced inflammatory nociception. When EP was administrated intraperitoneally 1 hour before formalin injection into the plantar surface of the hind paw, it attenuated nociceptive behavior, the size of hind paw edema (Figure 1), and the activation of c-Fos and ERK in the neurons of L4-L5 spinal DH (Figures 2,3,4), which is considered a consequence of its central and peripheral pharmacological actions. In addition, the i.t. introduction of the MEK inhibitor, PD-98059, reduced formalin-induced inflammatory nociception (Figure 5). These data indicate that neuronal ERK phosphorylation is involved in the acute inflammatory nociceptive mechanism, and the EP can attenuate acute inflammatory nociception by inhibiting neuronal ERK activation in spinal DH.
Subcutaneous hind paw injection of formalin elicits two-phase nociceptive responses. While phase I is considered to reflect acute nociceptive pain by a direct stimulation of the nerve by the formalin, phase II is attributed to the combination of ongoing inflammatory-related afferent input from peripheral tissue and functional changes in the spinal DH (central sensitization) [29, 41]. In the current study, administration of EP clearly reduced the size of hind paw edema by formalin stimulation and nociceptive behavior during phase II, but not during phase I (Figure 1A). And it has been demonstrated that most peripheral inflammation is often accompanied by a variety of pain , and that EP seems to exert pharmacological effects, such as suppression of inflammation (i.e., severe sepsis, acute pancreatitis) . It also has been reported that EP has an anti-inflammatory effect in the nervous system by inhibiting microglial activation in models of stroke and neural damage [6, 7, 17, 18, 43]. Based on these collective findings, we suggest that EP could produce anti-nociceptive effect by regulating peripheral and/or central mechanisms underlying formalin-induced inflammatory nociception.
Intraplantar injection of formalin produces a massive inflammatory response at the injection site , thereby causing paw edema . To verify the peripheral effect of EP, we examined the changes of hind paw edema 1 hour following formalin injection. When rats were given EP injection 1 hour before formalin injection, the thickness of hind paw edema was significantly decreased (12.5 ~ 25.7%) compared to that of animals treated with formalin alone (Figure 5). Reduction of the formalin-induced paw edema by EP (Figure 1C) suggests its clear anti- edematous effects in the inflammatory site. Several mechanisms could explain the anti-edematous effects of EP for formalin-induced inflammation. First, EP may inhibit the activation and recruitment of peripheral immune cells to formalin-induced inflammatory site. Jang et al.  recently demonstrated that EP has the ability to inhibit neutrophil activation, inflammatory cytokine (TNF-α, IL-1β) release, and nuclear factor κB (NF-kB) translocation in ischemia/reperfusion-induced heart injury. Second, EP may inhibit peripheral inflammation such as adenosine. It has been demonstrated that the i.t. administration of the adenosine receptor agonist, cyclohexyladenosine (5 μg/kg), suppresses peripheral inflammation by decreasing neutrophil infiltration into skin lesions . Third, like botulinum toxin A, EP may reduce neurogenic inflammation in the inflamed skin by reducing the releasing of neurotransmitters such SP, CGRP and glutamate from peripheral sensory nerve terminals by formalin injection. Released neurotransmitters contribute to the formalin-induced edema . Peptide-mediated transdermal delivery of botulinum neurotoxin type. A reduces neurogenic inflammation in the skin . The detailed cellular and molecular mechanisms underlying the anti-edematous effects of EP in the periphery remain to be elucidated. The detailed cellular and molecular mechanisms underlying the anti-edematous effects of EP in the periphery remain to be elucidated.
To verify the possible central mechanism of EP, we examined the changes in c-Fos expression in the spinal DH during phase II (the peak time point of nociception; 36–40 minutes after formalin injection) of formalin-induced nociception. In agreement with our previous report , the increase in formalin-induced c-Fos expression was mainly observed in the L4-L5 superficial and deep laminae where the primary nociceptive afferents from spinal nerve terminate (Figure 2). However, the upregulation of c-Fos expression by formalin stimulation was clearly inhibited by EP (Figure 2). Because c-Fos is expressed in the spinal cord subjected to many kinds of peripheral noxious stimulation [31–33], the reduction of c-Fos expression in the spinal DH clearly indicates an anti-nociceptive role of EP.
Accumulating evidence shows that MAPKs (ERK, p38, and JNK) pathways contribute to pain sensitization after tissue/nerve injury via distinct molecular/cellular mechanisms [24–27]. In particular, ERK mediates intracellular signal transduction in response to a variety of stimuli. The phosphorylation of ERK in the nociceptive neurons of spinal DH occurs in response to axotomy, electrical stimulation to the peripheral nerve, noxious stimulation of the peripheral tissue, and peripheral inflammation [20, 27]. The phosphorylation of ERK plays a critical role in central sensitization by regulating the activity of glutamate receptors and potassium channels, and inducing gene transcription, and thereby contributes to persistent inflammatory and neuropathic pain . These reports suggest that the materials regulating the phosphorylation of ERK could control nociceptive mechanism. Presently, ERK was phosphorylated in mainly neurons of L4-L5 spinal DH by formalin-injection; 83.1% of p-ERK IR cells were NeuN-IR cells (Figure 4). However, the elevated p-ERK expression by formalin injection was clearly attenuated by EP administration (Figure 3). In accordance with these results, it was recently reported that EP can suppress the phosphorylation of ERK in LPS-stimulated BV2 cells . These results indicate that EP could inhibit inflammatory nociception by regulating the phosphorylation of ERK in neurons of spinal DH after formalin injection.
I.t. injection of the MEK inhibitor, PD-98059, blocks the central sensitization-mediated phase II of the painful response to formalin injection [27, 48]. MEK dominant negative mutant mice in which MEK function is suppressed exclusively in neurons show decreased phase II responses in the formalin-induced nociception . The MEK inhibitor, U0126, also blocks secondary mechanical hypersensitivity from central sensitization following intraplantar injection of capsaicin . In addition, i.t. injection of MEK inhibitors inhibits inflammatory thermal/mechanical hypersensitivity following intraplantar injection of bee venom  and CFA [19, 52], and in a model of monoarthritis and inflammatory visceral pain [26, 53]. I.t. MEK inhibitors can also suppress neuropathic pain by streptozotocin-induced diabetes and spinal cord injury [54, 55]. Presently, we verified that PD-98059 completely blocked formalin-induced nociception during phase II (Figure 6). These results suggest that the phosphorylation of spinal ERK could play a critical role in development and maintenance of formalin-induced inflammatory nociception.
Because EP has an anti-inflammatory effect in the nervous system by inhibiting the microglial activation in a model of stroke and excitotoxic neuronal damage, and in a LPS-stimulated in vitro model [6, 7, 17, 18, 43], we can speculate that central pharmacological roles of EP could target the microglial activation of spinal DH following formalin injection. However, morphological changes of CD11/b-IR microglia was not clearly observed at the peak time of nociception, 36–40 minutes after formalin injection, and the CD11/b-IR microglia was also not affected by EP at the same time, indicating that EP produced its maximal effect. Microglia was sufficiently activated 3 days after formalin injection, and the activated microglia was completely inhibited by EP (Figure 5). These findings were consistent with previous reports that CD11/b- or OX-42-IR microglia are not distinctly activated as early as 1 hour following formalin injection, but only increase after 1 day and peak 7 days following formalin injection [28, 30, 40, 56]. Thus, the collective findings suggest that microglial activation might not directly contribute to the anti-nociceptive effects of EP on the early stage of formalin-induced nociception.
After nerve damage, the three MAPKs are differentially activated in spinal neurons and glial cells by various postsynaptic receptors and multiple protein kinases, and the activated glial cells induces the synthesis of pronociceptive and proinflammatory mediators that act to develop and maintain pain . ERK integrates multiple signaling pathways and regulates the Kv4.2 potassium channel in the spinal cord, and contributes to the induction and maintenance of central sensitization via posttranslational and transcriptional regulation, respectively . ERK is activated in neurons from 10 minutes to 6 hours, in microglia on day 2, in both microglia and astrocytes on day 10, and in astrocytes on day 21 after spinal nerve ligation . In agreement with these reports, we confirmed that ERK was phosphorylated mainly in the spinal neurons, but not microglia and astrocytess at 36–40 minutes after formalin injection, and that the elevated ERK phosphorylation was inhibited by EP. These results suggest that the inhibition of neuronal phosphorylation of ERK in spinal DH might be associated with anti-nociceptive effects produced by EP. Compared to mechanisms of ERK pathway in neuropathic pain, neuronal and glial mechanisms of ERK for inflammatory nociception control remain to be elucidated. p38 and JNK are phosphorylated in primarily spinal microglia and astrocytes, respectively, after peripheral inflammation and peripheral nerve injury [57–59]. The activation of the p38 and JNK in microglia and astrocytes is critical for the maintenance of inflammatory/neuropathic pain. Therefore, we investigated whether the phosphorylation of p38 and JNK were increased in spinal DH at peak time point of nociceptive behavior after formalin injection. Expression of p-p38 and p-JNK was not increased or decreased in spinal DH by formalin injection or EP administration ( Additional file 1: Figure S1). These findings are supported by that p-p38 begins to increase at 12 hours, reaches a peak at 3 days after a spinal nerve ligation, and is maintained at elevated levels even after 3 weeks [24, 57], and that p-JNK is persistently increased in spinal astrocytes at 1, 3, 10, and 21 days after spinal nerve ligation  and partial sciatic nerve injury . Therefore, our findings suggest that p38 and JNK might not directly contribute to the development and maintenance of hypersensitivity in the formalin-induced nociception. Interestingly, recent publications reported contradictory results in the expression time of p-p38; p38 was rapidly activated in the spinal microglia minutes following intrathecal administration of substance P or intradermal injection of formalin and the activation persisted for 1 hour [38, 60]. In addition, induction of a secondary increase of p-p38 expression in spinal microglia occurred and was maximal 3 to 7 days after injection . The exact role of p-p38 and microglia in inflammatory pain are still unclear. Future studies should investigate the clear function of activation of MAPKs signaling pathway in various pains.