Aprepitant Inhibits JNK and p38/MAPK to Attenuate Inammation in Microglia and Suppresses Inammatory Pain

Background: Substance P (SP) contributes to the pathogenesis of pain by acting on neurokinin ‐ 1 receptor (NK-1R), specialized sensory neurons that detect noxious stimuli. Aprepitant, an antagonist of NK-1R widely used to treat chemotherapy-induced nausea and vomiting. In this study, we assessed aprepitant’s analgesic effect on inammatory pain in mice and its mechanism of action in spinal cord. Methods: The excitability of DRG neurons treatment with aprepitant was measured using whole-cell patch-clamp recordings. The inammatory pain model was induced in adult ICR mice by a single dose intraplantar injection of formalin and carrageenan. The mice were treated with aprepitant, and the behavioral tests were measured after the intraperitoneal injection of aprepitant. The morphological changes on inamed paw tissues were determined by using hematoxylin eosin staining. The mRNA expressions of MCP-1, TNF-α, IL-6, IL-1β, and NF-κBp65 were measured by real-time quantitative PCR analysis. Changes in the protein expression levels were assayed using enzyme linked immunosorbent assay. Western blotting and immunohistochemistry were used to assay the cell signaling. Results: Aprepitant treatment showed a signicantly higher AP threshold in vitro. In vivo, the aprepitant showed a signicant anti-inammatory and analgesia effect in the mice with inammatory pain. After the administration of aprepitant, the paw tissues inammatory damage was signicantly relived. The mRNA levels of MCP-1, TNF-α, IL-6, IL-1β, and NF-κBp65 were down-regulate following aprepitant treatment. Meanwhile, aprepitant signicantly suppressed over-expression of proinammatory cytokines, as well as the phosphorylation of JNK and p38 MAPK in the spinal cord. Conclusions: The present study suggests that the extract of aprepitant attenuates inammatory pain in mice via suppressing the phosphorylation of JNK and p38, and inhibiting the NF-κB signaling pathway.


Introduction
In ammatory pain is a common chronic pain in clinical, which adversely impacts the quality of life [1,2].
Although to relieve in ammatory pain, signi cant progress has been achieved in existing analgesics, such as nonsteroidal anti-in ammatory drugs (NSAIDs), opioid analgesics, the insu cient e cacy and adverse side effects limit the curative effect [1][2][3]. Consequently, promising analgesic agents against in ammatory pain are imminently required.
Mechanisms of in ammatory pain are complex and involve many factors. It has long been generally accepted that microglia in spinal cord plays an important role in the pathogenesis of in ammatory pain.
A previous study has reported that aprepitant could act as an anti-in ammatory agent and attenuate lipopolysaccharide (LPS)-induced in ammatory responses in macrophages [21]. However, there is currently no research on the role of aprepitant in neuroin ammation and in ammatory pain. Therefore, we hypothesized that aprepitant might provide bene t for treatment of in ammatory pain, and interfere with LPS-stimulated microglia activation, as well as the production of in ammatory cytokines in microglia. Hence, this research provides a novel view of the role of aprepitant in anti-in ammatory responses, which in turn provides a potential therapeutic approach for neuroin ammation and in ammatory pain.

Animals
Adult male ICR mice (weighing 18-22 g) were obtained from the Qinglongshan Animal Center of Nanjing, and kept in an animal care facility with controlled temperature, mild humidity, a 12 h natural light-dark cycle, and free access to fresh food and water. Every effort was made to minimize the suffering and number of animals used during the experiments. All experimental procedures were performed in accordance with the guidelines for laboratory animal care of the Nanjing Medical University and China Pharmaceutical University.
Cell culture and viability assay Primary murine microglia BV-2 cells were maintained in Dulbecco's Modi ed Eagle's medium (DMEM, Hyclone/Thermo, Rockford, IL, USA), supplemented with 10% fetal bovine serum (FBS, Gibco, NY, USA), penicillin and streptomycin (Hyclone/Thermo) in 5% CO 2 at 37°C. To assess cell viability, the BV2 cells (5 10 4 cells/mL) were seeded in 96-well plates. After treatment for 24h, 10 μL of MTT solution (5 mg/ml, Sigma-Aldrich, Germany) was added to each well and the cells were incubated for 4 h. The supernatant was removed and 150 μl of dimethylsulfoxide (DMSO, Sigma-Aldrich) was added to dissolve formazan crystals in each well. After 30 min of incubation, absorbance levels for formazan at 490 nm were measured using a microplate reader (Bio-Rad Inc., Hercules, CA, USA).

Mice lumbar dorsal root ganglion cell culture preparation
Primary DRG neurons were isolated from male ICR mice lumbar DRGs, as previously described. Brie y, DRG neurons were quickly removed and placed into ice-cold DMEM, then digested in collagenase (1 mg/ml, type II; Worthington, USA), and 0.25% trypsin, followed by addition of 0.25% trypsin inhibitor. Then the DRG neurons were triturated with Pasteur pipettes and centrifuged through 15% BSA. The cell pellet was resuspended in 1 mL of Neurobasal media. Isolated neurons were plated onto poly-D-lysine (100 g/mL) and laminin (1 mg/mL)-coated 35-mm tissue culture dishes containing B27 supplement (Invitrogen, Carlsbad, CA) and 1% penicillin-streptomycin, in the incubator at 37°C with 5% carbon dioxide.
Preparation of DRG neurons DRG were isolated from spine and then were enzymolyzed in normal Tyrode solution containing 1 mg/ml collagenase II (Worthington, USA) for 60 min at 37 ℃. DRG were plated onto poly-D-lysine coated glass coverslips.Single cells were grown in DMEM (high glucose) supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg /ml streptomycin. Neurons were cultured at 37 ℃ in 5% CO 2 overnight.Cells were used for patch clamp recordings at 24 h post dissociation.

Electrophysiology
The excitability of DRG neurons were measured using whole-cell patch-clamp recordings. Small DRG neurons (<25μm) were chosen in our study. Voltages were recorded by using a HEKA EPC 10 ampli er (HEKA Instruments, Germany). The data were obtained and analyzed by Pulse software (HEKA Instruments, Germany). Signals were ltered at 3 KHz and sampled at 10 KHz. Electrodes were drawn from borosilicate capillary glass (Institute of Biophysics, Chinese Academy of Sciences, China) using a P-97 puller (Sutter Instrument, USA). Electrodes had a resistance of 2-5 MW when lled with intracellular solution. The pipette solution contained (in mM) KCl 140, MgCl 2 1, EGTA 5, Na 2 ATP 2, and HEPES 5 (pH =7.2, adjusted with KOH). The external solution contained (in mM): NaCl 137, MgCl 2 1.2, KCl 5.4, NaH 2 PO 4 1.2, CaCl 2 1, HEPES 10, and glucose 10(pH =7.4, adjusted with NaOH). The action potentials (Aps) was recorded under the current-clamp mode. APs were evoked by 1 s depolarizing current pulses from -10 up to 100 pA at 5 pA steps. The minimum current that triggered the rst AP was de ned as rheobase. To observe the excitability, DRG neuron was stimulated by 2×rheobase.
Formalin Test 20 microliter of saline or 2% formalin (in saline) were subcutaneously injected into the hind paw. Mice were randomly divided into 5 experimental groups: control (Ctrl), formalin, formalin + 5 mg/kg aprepitant, formalin + 10 mg/kg aprepitant, formalin + 20 mg/kg aprepatant. Mice were intraperitoneal injected aprepitant, followed by a subcutaneous injection of 2% formalin. Mice were monitored for time spent licking paw, and number of lick bouts, for 60 min post-injection by researchers blinded to experimental condition. All sessions were video-recorded. The time spent paw licking and biting was calculated in 5minute and recorded for 60 min. Additionally, acute (0-5 min) and in ammatory (15-45 min) phase pain responses were quanti ed.

Hot-plate test
The hot plate test was done at 55.0 ± 0.1℃. Mice were placed separately on the heated surface, and the latency between placement and the rst withdrawal response was recorded. A cut-off time of 25 s was set to avoid tissue damage. All testing was conducted blindly with respect to group assignment.

Mechanical allodynia
Mechanical allodynia was indicated by von Frey laments ranging from 0.02 to 6 g. After 30 min of adaptation, laments were used to stimulate the plantar surface of each hindpaw. The laments were applied to six designated loci distributed over the plantar surface of the hindpaw. The minimal value that induced licking, paw withdrawal, and trembling was recorded as the threshold of mechanical withdrawal.

RNA extraction and real-time PCR
Under deep anesthesia, the L4-L5 spinal cord segments of mice were quickly removed and analyzed.
Total RNA was extracted from tissues or cells with TRIzol reagent and reversely transcribed using a reverse transcription kit (Thermo Fisher Scienti c, MA, USA) according to the manufacturer's instructions. The subsequent real-time PCR was performed with the SYBR® Green qPCR Kit (Vazyme Biotech Co., Ltd. Nanjing, China). Differential expression was calculated according to the 2−ΔΔCT method and statistically evaluated.

Western blotting
Total proteins were isolated from the spinal cord at L4-L5 segments or the cell lines. Protein lysates were separated by SDS PAGE gels and then transferred to PVDF membrane. The membrane was incubated in 5% nonfat milk or 3% bovine serum albumin and then blotted with speci c primary antibody. After incubation in the horseradish peroxidase-conjugated secondary antibody, the membrane was detected with chemiluminescence western blot detection system (Bio-Rad Laboratories, CA, USA).

ELISA
The spinal cord at L4-L5 segments and the paw tissue samples were quickly removed from deeply anesthetized mice. Cell supernatants were collected after treatment with reagents. TNF-α, IL-1β, IL-6 and MCP-1 were analyzed by enzyme linked immunosorbent assay (ELISA) kits (Neobiocisence, Shenzheng, China) following the manufacturer's instructions. Concentrations were determined by measuring absorbance at 450 nm (Bio-Rad Inc., Hercules, CA, USA).

Immunohistochemistry and in ammation scoring
Paw tissues were quickly excised, and then xed in 10% formalin at 4℃. Brie y, sections were cut using a microtome, stained with HE, and visualized by using a light microscope. Immunohistochemistry was performed using immunohistochemistry kit (Key-GEN, Nanjing, China). The degree of in ammation was quanti ed using a 0 to 5 scoring system. The scores were de ned as follows: 0 = no in ammation, 1 = mild in ammation, 2 = mild/moderate in ammation, 3 = moderate in ammation, 4 = moderate/severe in ammation and 5 = severe in ammation.

Statistical analysis
The results were analyzed by GraphPad Prism and expressed as means ± SEM. Alteration of expression of the proteins or mRNA detected and the behavioral responses to mechanical stimuli over time among groups were tested with 1-way and 2-way ANOVA followed by Bonferroni post hoc tests, respectively. Statistical results are considered signi cant if p < 0.05.

Results
Aprepitant inhibits formalin-induced nociceptor-like cultured dorsal root ganglion neurons and suppresses pain behavior when injected intraplantarly in vivo Substance P has been shown to participate in the in ammatory process [22,23], and NK-1R is espressed in DRG neurons [24]. Therefore, we thought to determine whether aprepitant, one of the NK-1R inhibitors, modulated the activity of dissociated nociceptive neurons (about 25 µm diameter) in vitro. To investigate the effect of aprepitant on the excitability of DRG neurons, we observed the characteristics of APs. As shown in Fig. 1A, aprepitant treatment showed a signi cantly higher AP threshold compared with control group. There were no signi cant diferences in resting membrane potential, rheobase, and overshoot.
These results demonstrated that aprepitant could decrease the excitability of DRG neurons. Formalin injection into the mouse's hind paw produces a reliable and widely used model of in ammatory pain when tested with analgesic drugs. Intraplantar injection of formalin induces distinct acute (0-5 min) and long-term in ammatory (15-45 min) phases in nocifensive behavior, while responses to a hotplate or von Frey laments are acute and transient. We discovered that aprepitant treatment attenuated the pain behavior ( Fig. 1B-D, p < 0.01) of in ammatory paw licking/biting after injection of aprepitant. Conversely, aprepitant had no effect on the acute phase response to formalin injection (Fig. 1E, p > 0.05). In addition, aprepitant alleviated the response to thermal (Fig. 1F, p < 0.05) or mechanical (Fig. 1G, p < 0.01) pain.

Aprepitant relieved formalin-induced in ammation in vivo
To investigate the effect of aprepitant on proin ammatory cytokines, we measured the levels of MCP-1, IL-1β, TNF-α and IL-6 following formalin injection in mice. Administration of aprepitant (10 mg/kg, and 20 mg/kg) signi cantly inhibited the formalin-induced expression of MCP-1, IL-1β, TNF-α and IL-6 in the in amed paw ( Fig. 2A, p < 0.05). Histopathological examination showed that the control group fed saline had normal paw tissue (Fig. 2BI). In contrast, compared to the control group, the left hind paws of mice that received formalin injections showed massive accumulation of in ltrated cells (Fig. 2BII). However, treatment with aprepitant (10 mg/kg, and 20 mg/kg) remarkably decreased in ammatory cell in ltration ( Fig. 2BIII-V). The degree of in ammation was evaluated by scoring of in ammation from 0 to 5. The in ammation scores indicated that co-treatment with aprepitant (10 mg/kg, and 20 mg/kg) signi cantly reduced formalin-induced in ammation (Fig. 2C, p < 0.05). To further explore the anti-in ammatory effects of aprepitant, the levels of MCP-1, IL-1β, TNF-α and IL-6 in spinal cord were evaluated using ELISA and qRT-PCR. Compared with the control group, the various cytokines, MCP-1, IL-1β, TNF-α and IL-6 protein and mRNA expressions were signi cantly increased in the formalin group ( Fig. 2D and E, p < 0.05). However, aprepitant reduced the expressions of these cytokines ( Fig. 2D and E, p < 0.05).

Aprepitant suppresses LPS-induced microglia activation and in ammtion in BV-2 microglial cells
It has been indicated that activation of microglia is crucial for the in ammatory stimuli, and involved in pain facilitation [5,8,25]. To investigate the effects of aprepitant on LPS-induced microglia activation in vitro, we used the immortalized murine microglial cell line BV-2, which were derived from primary mouse microglial cells. We investigated the effects of aprepitant on the viability of BV-2 microglia. Aprepitant showed little cytotoxic effect at concentration ranging from 1 to 8 µM (Fig. 3A, p > 0.05). Subsequently, we studied the effect of aprepitant on LPS-induced microglia activation in BV-2 cells. BV-2 cells were cocultured with LPS (1 µg/ml) and various concentrations of aprepitant for 16 h. Pretreatment with aprepitant signi cantly suppressed LPS-induced over-expression of IBA-1 (Fig. 3B, p < 0.01), in BV-2 microglia. We investigated the effect of aprepitant on the LPS-caused expression and secretion of proin ammatory cytokines by BV-2 microglia. In contrast with treatment with LPS alone, aprepitant signi cantly suppressed LPS-induced MCP-1, IL-1β, TNF-α and IL-6 mRNA expression in BV-2 microglial cells (Fig. 3C, p < 0.01). In addition, aprepitant decreased the LPS-induced secretion of MCP-1, IL-1β, TNFα and IL-6 in BV-2 (Fig. 3D, p < 0.01). However, aprepitant (2 µM) alone showed no marked effects on the mRNA and protein expression of MCP-1, IL-1β, TNF-α and IL-6 in BV-2 cells (Fig. 3C and D, p > 0.05). These results demonstrate that aprepitant inhibits LPS-induced neuroin ammation.

Aprepitant inhibits LPS-induced p38 and JNK phosphorylation, but not ERK1/2 phosphorylation in BV2 Microglia
To clarify the mechanisms underlying the anti-in ammatory effects of aprepitant, we assessed the effects of aprepitant on LPS-induced phosphorylation of MAPK in BV-2 cells. The results revealed that aprepitant signi cantly suppressed LPS-induced up-regulated of phosphorylation of p38 mitogenactivated protein kinase (p38) and c-Jun NH2 terminal protein kinase (JNK) (Fig. 4A, p < 0.05), in BV-2 microglia. However, we did not con rm that treatment with aprepitant reduces LPS-induced up-regulated of phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) in BV-2 (Fig. 4A, p > 0.05). Furthermore, LPS treatment induced the translocation from the cytoplasm to the nucleus (Fig. 4B) and increased of nuclear factor κB p65 (NF-κBp65) mRNA expression (Fig. 4C, p < 0.05), compared with the LPS-treated group, co-incubation with aprepitant signi cantly reduced the effect ( Fig. 4B and C, p < 0.01).
The JNK, p38/MAPK and NF-κBp65 signal pathways involved in the analgesic mechanisms of aprepitant To determine whether the analgesia effects of aprepitant on mouse paw injected with formalin were associated with inhibition of the MAPK and NF-κBp65 signaling pathway, we evaluated phosphorylation of ERK1/2, JNK, p38 MAPK and the expression of NF-kBp65 in mouse spinal cord. Compared to the control group, the phosphorylation of ERK1/2, JNK, p38 MAPK and the activation of microglia in the formalin group were signi cantly increased (Fig. 5A, p < 0.05). Aprepitant effectively inhibited the formalin-induced phosphorylation of JNK, p38 MAPK and over-activation of microglia ( Fig. 5A-C, p < 0.05). Simultaneously, the administration of aprepitant suppressed the mRNA expression of NF-kBp65 (Fig. 5D, p < 0.01).
We also evaluated the effects of aprepitant on pain behavior, phosphorylation of p38 MAPK and the mRNA expression of NF-kBp65 in mice with carrageenan-induced in ammatory pain (Fig. 6). As shown in Fig. 6A, the pain behavioral test demonstrated that treatment with aprepitant via the intrathecal (2 nmol, i.t.) and intraperitoneal (i.p.) route remarkably alleviated the mechanical withdrawal threshold induced by carrageenan (p < 0.05). To verify the suppressive effect of aprepitant on mechanical allodynia, we evaluated whether the p38 inhibitor (SB203580) and JNK inhibitor (SP600125) showed similar effects to that of aprepitant in carrageenan-induced in ammatory pain. The results showed that SB203580 (10 nmol, i.t.) and SP600125 (5 µg, i.t.) reduced carrageenan-induced decreased of mechanical withdrawal threshold (Fig. 6A, p < 0.01). Aprepitant markedly reduced phosphorylation of JNK and p38 MAPK and the mRNA expression of NF-kBp65 in the spinal cord in mice with carrageenan-induced in ammatory pain ( Fig. 6B-D, p < 0.01). In addition, the administration of aprepitant suppressed the expression of proin ammatory cytokines induced by carrageenan in the spinal cord (Fig. 6E, p < 0.05).

Discussion
In the present study, we found that aprepitant suppressed formalin-induced nociceptor-like cultured dorsal root ganglion neurons and alleviated formalin-induced pain behavior. Furthermore, aprepitant application suppressed the LPS-induced in ammatory responses and proin ammatory cytokines by inhibiting p38/MAPK and NF-kBp65 activation in BV-2 microglial cells. Simultaneously, the administration of aprepitant inhibited microglial activation and proin ammatory cytokines in mice with in ammatory pain. Therefore, aprepitant is a potential therapeutic strategy to alleviate the neuroin ammation and in ammatory pain mediated by overactivation of microglia. Model of the mechanism underlying in ammatory pain and aprepitant-induced inhibition on in ammatory pain is illustrated in Fig. 7.
In ammatory pain refers to pain associated with tissue damage and in ammation of any cause, such as trauma, infection, heat, and so on. The most common clinical treatments for in ammatory pain include NSAIDs and opioids [1,2]. Although these drugs are therapeutically effective at some time, they are associated with a number of side effects, such as liver damage, kidney damage and severe gastrointestinal reactions [2,26]. Moreover, treatment of pain is often a long-term and complex process. Aprepitant is widely used for the prevention of chemotherapy-induced nausea and vomiting in clinic. Evidence revealed that, in the electrical hyperalgesia model in human, aprepitant has no therapeutical effects on measures of central sensitization [27]. However, a growing number of studies indicated that aprepitant has analgesic, anti-in ammatory effects [16,19,20]. It is a controversial issue, meanwhile, there is little research on the in uences of aprepitant in in ammatory pain. Thus, we studied the effects of aprepitant on in ammatory pain, and explored the underlying mechanisms.
The data we presented from dissociated DRG neurons show that application of aprepitant leads to inhibition of the neuronal excitatory effects. Using the mice model with formalin-induced in ammatory pain, we found that the time of licking/biting was signi cantly decreased in formalin mice with aprepitant administration compared with the mice with formalin application alone. In addition, the threshold of mechanical allodynia, was markedly reduced in formalin mice, but increased with aprepitant administration, indicating that aprepitant treatment relieves pain. We observed that mice treated with formalin had high level of IL-1β, IL-6, and TNFα in the injected paw, while after aprepitant administration, the levels of them were down-regulated. Based on the potent anti-in ammatory effects of NK-1R antagonist, aprepitant, on peripheral in ammatory conditions [28,29], and its remarkable ability to inhibit mechanical allodynia, it is reasonable to believe that aprepitant may also alleviate in ammatory pain through reduction of central in ammation. Then, we used the hotplate test, which is a useful tool for screening analgesic drugs that producing central effects, to explore this hypothesis. The data showed that the latency to paw withdrawal from hotplate markedly reduced in formalin mice, but increased with aprepitant treatment. Studies have shown that central changes in in ammatory pain are key contributors to the initiation and maintenance of chronic pain.
Microglia is an important component that plays a critical role in the initiation, maintenance, and resolution of chronic pain in the central nervous system [4,5,25]. Microglia activation is initiated in response to various extracellular stimuli such as LPS and in ammatory cytokines [30]. Activated microglia cells release abundant in ammatory mediators, including IL-1β, IL-6, TNFα, and MCP-1, which can activate nociceptors directly or increase the excitability of neurons [25,31,32]. Neuroin ammation mediated by microglia involves in the pathogenesis of in ammatory pain[8, 25,33]. We observed that mice treated with formalin had high level of IL-1β, IL-6, TNFα, and MCP-1 in the injected paw and in the spinal cord, while after aprepitant administration, the levels of IL-1β, IL-6, TNFα, and MCP-1 were downregulated. We also observed the local in ammatory cell in ltration of the hind paw following formalin injection by histopathological examination, the results showed that treatment with aprepitant remarkably decreased formalin-induced in ammatory cell in ltration.
We then explored the mechanisms underlying the analgesic effect of aprepitant and the suppressive effect of aprepitant in microglial activation. As all known, exposure to LPS led to the activation of BV-2 microglial cells and induced in ammatory responses [34][35][36]. In this study, aprepitant suppressed the LPS-induced expression of IBA1. Consistently, aprepitant also inhibited LPS-induced production and expression of IL-1β, IL-6, TNFα, and MCP-1 in BV-2. These results demonstrate that aprepitant inhibited LPS-induced activation of microglia and neuroin ammation in BV-2 microglial cells. MAPK signaling pathway is activated to induce in ammation and the expression of proin ammatory mediators [8,37]. The MAPK family consists of p38 mitogen-activated protein kinase (p38), c-Jun NH2 terminal protein kinase (JNK), extracellular signal-regulated kinase-1/2 (ERK1/2) and ERK5 [5], participates in chronic pain. Interestingly, MAPK subtype activation has a cell-type speci c distribution in the spinal cord of rodents with chronic pain. Microglial p38 MAPK is primarily and dramatically activated in the spinal dorsal horn [38]. In addition, SB203580, a speci c p38 MAPK inhibitor, signi cantly attenuates pain behaviors and proin ammatory cytokines synthesis [38][39][40]. Meanwhile, JNK MAPK plays an important role in in ammatory response in microglia [41,42]. It has been reported that JNK inhibitor can signi cantly alleviate persistent in ammatory pain and microglial activation [42][43][44]. We found that aprepitant treatment alone did not in uence the phosphorylation of JNK and p38 MAPK in BV-2 cells, but remarkably down-regulated the LPS-induced increased phosphorylation of JNK and p38 MAPK. These data suggest that the p38 MAPK is involved in the effects of aprepitant. Many studies have proved that the activation of p38 MAPK can initiate the NF-κBp65 signaling. Once activated, NF-κBp65 translocates into the nucleus from cytoplasm to trigger the transcripition of various mediators, which are involved in proin ammatory responses [40,45,46]. In addition, the inhibition of NF-κBp65 can reverse the production and expression of pro-in ammatory mediators following carrageenan or LPS treatment [47,48]. These data suggest that the activation of NF-κBp65 may play an important role in the expressions of proin ammatory cytokine in in ammatory pain. Thus, based on our results demonstrating that aprepitant inhibits LPS-induced JNK and p38 MAPK phosphorylation in BV-2, we further explored the effect of aprepitant on NF-κBp65 activation. Our results revealed that aprepitant effectively reversed the LPSinduced over-translocation into nucleus of NF-κBp65. Hence, it appears that inhibition of NF-κBp65 activation by aprepitant may also be a possible mechanism underlying its inhibitory effect in microglia, which is critical for the suppression of the production of proin ammatory cytokine.
We further investigated whether aprepitant could inhibit microglial activation in in ammatory pain in vivo. We detected the IBA1 and the phosphorylation of p38 MAPK in the spinal cord of mice with formalin-induced in ammatory pain. The results showed that IBA1, and the JNK phosphorylation, as well as the p38 MAPK phosphorylation were signi cantly increased in the spinal cord of formalin model mice. In addition, aprepitant could effectively inhibit the up-regulation of IBA1 and the p38 MAPK phosphorylation in the spinal cord. As established previously, the protein levels of IL-1β, IL-6, TNFα, and MCP-1 and the mRNA expressions of IL-1β, IL-6, TNFα, MCP-1, and NF-κBp65 in mice spinal cord were considerably suppressed by aprepitant. Consistently, aprepitant markedly alleviated the pain behavior and reduced phosphorylation of p38 MAPK and the mRNA expression of NF-kBp65 in the spinal cord in mice with carrageenan-induced in ammatory pain, further con rming that the activation of p38 MAPK and NF-κBp65 in the spinal cord were involved in the pain regulation of aprepitant in in ammatory pain.

Conclusions
In conclusion, this study demonstrated a novel role for aprepitant in the therapy of in ammatory pain through mechanisms associated with the inhibition of multiple targets in activated microglia in the spinal cord. Our results support the potential for repurposing this agent for the treatment of in ammatory pain. The datasets used and/or analyzed during the current study are available through the corresponding author on reasonable request. to >50% of trials) in aprepitant treatment groups versus control mice. Eleven to twelve mice were included in each of the groups. Data are expressed as mean ± SEM. Two-way ANOVA, *p < 0.05, **p < 0.01 versus vehicle or control, #p < 0.05, ##p < 0.01 versus formalin. Figure 2 relative expression of IBA-1. (C) The mRNA expression of MCP-1, IL-1β, TNF-α and IL-6 in BV-2 microglia. (D) ELISA showed that aprepitant decreased the LPS-induced secretion of MCP-1, IL-1β, TNF-α and IL-6 in BV-2 microglia. Data are expressed as mean ± SEM. One-way ANOVA, *p < 0.05, **p < 0.01 versus vehicle or control, #p < 0.05, ##p < 0.01 versus LPS.