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Bexarotent attenuated CCI-induced spinal neuroinflammation and neuropathic pain by targeting MKP-1
Accepted Manuscript
Bexarotent attenuated CCI-induced spinal neuroinflammation and neuropathic pain by targeting MKP-1 Yulong Gui , Shunyuan Duan , Lihong Xiao , Jing Tang , Aiyuan Li PII: DOI: Reference:
S1526-5900(18)30607-2 https://doi.org/10.1016/j.jpain.2019.01.007 YJPAI 3684
To appear in:
Journal of Pain
Received date: Revised date: Accepted date:
19 September 2018 23 December 2018 16 January 2019
Please cite this article as: Yulong Gui , Shunyuan Duan , Lihong Xiao , Jing Tang , Aiyuan Li , Bexarotent attenuated CCI-induced spinal neuroinflammation and neuropathic pain by targeting MKP1, Journal of Pain (2019), doi: https://doi.org/10.1016/j.jpain.2019.01.007
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Highlights Bexarotene reduced CCI-induced neuropathic pain in rats. Bexarotene inhibited CCI-induced neuroinflammation and activation of MAPKs in the spinal cord . Bexarotene induced MKP-1 in the spinal cord. BCI, a MKP-1 inhibitor completely abrogated bexarotene-induced effects in CCI rats. Bexarotene could be a potential therapeutic agent for neuropathic pain
Bexarotent attenuated CCI-induced spinal neuroinflammation and neuropathic pain by
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targeting MKP-1
Yulong Gui1, Shunyuan Duan2, Lihong Xiao1, Jing Tang1, Aiyuan Li1*
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1. Department of Anesthesiology, Maternal and Child Hospital of Hunan Province, Changsha, Hunan 410008, China
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2. Department of Endocrinology, Yongzhou-affiliated Hospital of University of South
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China, Yongzhou 425000, Hunan Province, China
*
To whom correspondence should be addressed: Aiyuan Li Department of Anesthesiology, Maternal and Child Hospital of Hunan Province, 1
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Changsha, Hunan 410008, China
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Email:[email protected]
Abstract
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It is widely accepted that neuroinflammation in the spinal cord contribute to the development of central sensitization in neuropathic pain. MAPKs activation plays an vital role in the development of neuroinflammation in the spinal cord. In this study,
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we investigated the effect of bexarotene, a RXR agonist, on MAPKs activation in
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CCI-induced neuropathic pain. The data showed that daily treatment with bexarotene 50mg/kg significantly alleviated CCI-induced nociceptive hypersensitivity in rats.
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Bexarotene 50mg/kg/day inhibited CCI-induced MAPKs (p38MAPK, ERK1/2, and
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JNK) activation and upregulation of proinflammatory factors(IL-1β, TNF-α and IL-6). Bexarotene also reversed CCI-induced microglia activation in the ipsilateral spinal
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cord. Furthermore, bexarotene treatment significantly upregulated MKP-1 in the spinal cord. These effects were completely abrogated by MKP-1 inhibitor BCI. These results indicated that bexarotene relieved CCI-induced neuroinflammation and neuropathic pain by targeting MKP-1. Therefore, bexarotene might be a potential agent for the treatment of neuropathic pain.
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Keywords: bexarotene, CCI(Chroinc constriction injury), mitogen-activated protein kinases(MAPKs), , mitogen-activated protein kinase phosphatase 1(MKP-1),
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neuropathic pain
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Introduction
It is widely accepted that neuroinflammation in the spinal cord plays an important role in the development of central sensitization in neuropathic pain. Spinal MAPKs
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activation contribute to the development of neurinflammation in neuropathic pain13, 15. However, Ndong C et al reported that mitogen-activated protein kinase phosphatase
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1(MKP-1), a negative feedback of activated MAPKs, was reduced in the development
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of neuropathic pain27. Upregulation of spinal MPK-1 alleviated the behavioral hypersensitivity in neuropathic pain27. MKP-1 might be a potential target for the
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treatment of neuropathic pain. However, selective MKP-1 agonist was not reported,
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yet. However, it was reported that MKP-1 was induced by all-trans retinoic acid28, 37. That pompted us to consider bexarotene, a derivative of all-trans retinoic acid, for the treatment of CCI-induced neuropathic pain. Bexarotene, known as Targretin, was a derivative of all-trans retinoic acid and a selective retinoid X receptor (RXR) agonist. Bexarotene was originally used for treatment of cutaneous T-cell lymphoma. However, recent studies showed bexarotene
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possessed potential neuroprotective and anti-neuroinflammatory effects. Zhong et al. reported that bexarotene protected against traumatic brain injury and inhibited inflammation responses42. In clinic, Cummings et al reported bexarotene alleviated brain amyloid in a proof-of-concept trial5. Smit et al. reported an anti-psoriatic effect
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of bexarotene and no serious adverse events in a phase II multicenter clinical trial31. This prompted us to investigate the effect of bexarotene on CCI-induced neuropathic pain.
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In this study, pain thresholds were measured as the primary outcomes. Expression of proinflammatory factors(IL-1β, TNF-α and IL-6), acitvation of MAPK signals and microglia were measured as the secondary outcomes. BCI, an MKP-1 inhibitor, was
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employed to investigate the role of MKP-1 on bexarotent-induced effects.
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Methods
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Animals and grouping
The current experiments were approved by animal ethical committee of University
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of South China. Young male adult rats (200-250g, 12 weeks old) were used in these
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experiments. All the rats were housed properly for a week adaptation before the experiments began. Three parts were performed. In part one, 84 rats were included into 7 groups through randomization by SPSS 17.0 software: Sham group, Sham+bexarotene (bex) 50mg/kg group, CCI group, CCI+vehicle group, CCI+ bex 12.5 mg/kg group, CCI+ bex 25mg/kg group, CCI+ bex 50mg/kg group (n=12). Ipsilateral behavior tests were performed in 6 rats of each group. The rest rats were
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decapitated in day 7 for ELISA, western blot and immunofluorescence. In part two, 82 rats were treated with intrathecal catheterization before the experiments started. Then, 10 rats with 20% lower pain thresholds than the average were excluded. Then, the rest rats were assigned into 6 groups: CCI+ bex 50mg/kg group, CCI+bex
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50mg/kg+vehicle group(vehicle group), CCI+bex 50mg/kg+BCI group, Sham+BCI group, CCI+BCI group, CCI group (n=12). Behavior tests were operated on 6 rats in each group. The other rats (n=6) were used for western blot and ELISA tests on day 7.
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As was required that both contralateral and ispilateral spinal cord should be shown as a comparison on microglia activation, we did other experiments (Part three). We used aditional 12 rats with CCI (n=6) or CCI+bex 50mg/kg (n=6), respectively. The rats
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immunofluorescence.
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were sacrificed on day 7. The L4-6 spinal cord of both sides was taken for Iba-1
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Drugs and Treatment
Bexarotene (MedChemExpress, USA) was dissolved in 0.5% sodium carboxyl
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methyl cellulose at concentrations of 2.5, 5, 10mg/ml, according to previous study36.
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The oral dosage of bexarotene for the rats was described by Xu L. et al36. Vehicle rats in part one were treated with the same volume of 0.5% sodium carboxyl methyl cellulose without bexarotene. Bexarotene or vehicle administration in part one was operated two hours before behaviour tests from day 1 to day 14. In part two, BCI (Sigma-Aldrich, USA, #Cat:B4313) was primarily dissolved in DMSO at concentrations of 10 mM, respectively. Then, BCI stock solutions were diluted to
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1mM with methylcellulose (0.5%) solution containing 1% Tween. 5μl of 1 mM BCI were intrathecally administrated to rats in BCI groups from day 1 to day 14, according to previous reports26. Here, vehicle rats were treated with 5 μl of methylcellulose (0.5%) solution containing 1% Tween and 10% DMSO. The intrathecal procedure
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was performed under a disinfection condition. BCI or vehicle was intrathecally administrated at a volume of 5μl followed by a 10 µl saline flush. The whole process
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was performed for about 30 seconds.
Surgery procedure
Surgeries were operated under sodium pentobarbital anesthesia (40–60 mg/kg,
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i.p.). CCI model was operated as Bennett et al reported3. Briefly, after skin shaved and sterilized, right sciatic nerve was properly exposed and tied four times. For intrathecal
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catheterization, a PE10 catheter was implanted to the level of the lumbar enlargement
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approximately as described previously8, 38.
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Determination of nociceptive hypersensitivity
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In this study, Electronic Von Frey unit EVF (Bioseb, USA) was used. Briefly,
ipsi-injured hind paws of the rats were treated five times with filament (up to 90 gram probe). Mechanical thresholds were determinated as the average of the final readings. Ipsilateral thermal thresholds (TTs) were tested as follow. The rats were placed into a chamber with a light source. Ipsilateral paws were heated to withdrawal latency or a 20 s cut off as previously reported3, 36. The withdrawal latency was considered as
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lifting paws and/or licking in response to at least 3 out of 5 tests. The progresses were repeated three times with a 5-min interval. Measurement of IL-1β, TNF-α and IL-6 by enzyme-linked immunosorbent assay (ELISA)
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On day 7, 6 rats in each group were sacrificed by cervical dislocation. Half of ipsilateral spinal cords (L5-6) were dissected, lysed, and centrifugated at 20,000×g, 4°C for 30 minutes. Then, the supernatant of each samples was collected seperately
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for use. Rat ELISA kits (Kangwei Bio-tech., China) were used to detect the IL-1β, TNF-α and IL-6 in the samples according to the instructions.
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Western blot
After being lysed and centrifugated, the supernatant was determined by BCA kit.
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After being normalized and boiled with loading buffer, the proteins were separated
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and transformed into PVDF membrane(Millipore, USA). After being treated with 5% BSA for one hour, membranes were incubated with primary antibodies in 1% BSA. In
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this study, rabbit anti-p-p38MAPK, p38MAPK, p-ERK1/2, ERK1/2, RXRα (Cell
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Signaling Technology, USA,1:1000), rabbit anti-p-JNK1/2(Abcam, UK,1:2000), JNK1/2 (Abcam, UK,1:5000), mouse anti-MKP-1 (Santa Cruz Technology, USA,1:500), goat anti-iba-1 antibody(Abcam, UK, 1:1000), rabbit GAPDH and anti-β-actin (ABclonal Technology, USA,1:5000) were used. After being washed, membranes were treated with HRP-linked anti-rabbit or mouse secondary antibodies
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(Cell Signaling Technology, USA,1:1000) for one hour. Signal were visualized by HRP substrate (Millipore,USA, Luminata™ Forte).
Immunofluorescence
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The ispilateral L4-5 spinal cords on day 7 were treated with 4% paraformaldehyde for 8 hours and 30% sucrose overnight at 4°C. Then, samples were embedded and sliced. After being washed and treated with 0.3% Triton X-100 for 20
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minutes, they were incubated with 5% donkey serum (DS) in PBS for an hour and primary antibody in 1% DS overnight. Goat anti-iba-1 antibody(Abcam, UK, 1:200) were used. After being washed again, sections were treated with donkey anti-goat IgG
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Dylight594 (1:200, Jackson ImmunoResearch Laboratories, USA). Images were taken at 100×magnification (Leica DM500B, Wetzlar, Germany).
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To detect colocalization of RXRα/MKP-1 and RXRα/Iba-1, rabbit anti-RXRα
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(1:200, Cell Signaling Technology, USA), goat anti-iba-1 antibody(Abcam, UK, 1:200) and mouse anti-MKP-1 (1:50, Santa Cruz Technology, USA) were used as
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primary antibodies in 1% DS. Secondary antibodies were donkey anti-rabbit IgG
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Dylight488 (1:200, Jackson ImmunoResearch Laboratories, USA), donkey anti-goat IgG Dylight594 (1:200, Jackson ImmunoResearch Laboratories, USA) and donkey anti-mouse IgG Dylight594 (1:200, Jackson ImmunoResearch Laboratories, USA).
Statistical analysis
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In this study, SPSS version 17.0 was used for data analysis. Data in this study were presented as Mean ± S.E.M. Tow-way repeated measure ANOVA (group×time) with correction for multiple comparisons by LSD-t analysis was used to evaluate the difference in MTs and TTs among the groups. One-way ANOVA was used to evaluate
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the difference in behavior baseline, western blot and ELISA results. P < 0.05 was considered as significant difference.
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Results Bexarotene attenuated CCI-induced allodynia in rats
Firstly, effect of bexarotene on CCI-induced pain behaviors was measured. The
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baselines of mechanical and thermal thresholds showed no significant difference among the groups (Fig.1, PMT=0.920,PTT=0.880). Sham+bex 50mg/kg group showed
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no significant difference compared with sham group on pain thresholds (PMT= 0.579,
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PTT=0.856). CCI surgery significantly decreased ipsilateral mechanical and thermal thresholds in rats . (PMT＜0.001, PTT＜0.001). However, this effect was relieved by
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daily oral bexarotene in a dosage-dependent manner (MTs, Fgroup(36,180)=92.464,
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PMT＜0.001; TTs, Fgroup(36,180)=71.815, PTT＜0.001).
Bexarotene suppressed CCI-induced expressions of IL-1β, TNF-α and IL-6 in the spinal cord. Effect of bexarotene on expressions of IL-1β, TNF-α and IL-6 in the spinal cord was detected by ELISA (Fig.2). Results showed that CCI surgery significantly
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induced upregulation of IL-1β, TNF-α and IL-6(P＜0.001). Vehicle treatment did not affect the production of IL-1β, TNF-α and IL-6 in CCI rats in Part one (PIL-1β= 0.527, PIL-6=0.493, PTNF-α=0.676). Daily treatment with bexarotene 50mg/kg relieved CCI-induced upregulation of IL-1β, TNF-α and IL-6 in the spinal cord (P＜0.001).
in sham rats (PIL-1β= 0.615, PIL-6=0.640, PTNF-α=0.781).
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However, bex 50mg/kg treatment did not affect the produce of IL-1β, TNF-α and IL-6
Microglia
activation
in
the
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Bexarotene suppressed CCI-induced microglia activation in the spinal cord ipsilateral
spinal
cord
was
detected
by
immunofluorescence and westernblot of Iba-1. Immunofluorescence results showed
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that CCI increased the number of detectable microglia in the ipsilateral dorsal horn compared with sham or sham+bex 50 mg/kg group (Fig.3). However, this effect was
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significantly inhibited by daily oral bexarotene 50 mg/kg. To reduce the
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microtomy-caused bias in immunofluorescence and better qualification, Iba-1 expression was detected by western blot. Results showed that CCI significantly
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increased ipsilateral spinal Iba-1 expression compared to sham group (P=0.002). No
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significant difference existed between CCI group and CCI+vehicle group (P=0.835). Neither significant difference existed between sham group and sham+bex 50mg/kg group on the Iba-1 expression (P=0.712). Dietary bexarotene 50mg/kg significantly inhibited the effect of CCI-induced Iba-1 upregulation (P=0.006), which meant bexarotene 50mg/kg inhibited CCI-induced microglia activation. As was required that both contralateral and ispilateral spinal cord should be shown as a comparison on
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microglia activation, we did the Iba-1 immunofluorescence of both contralateral and ispilateral spinal cord (Fig.3D). Result showed that CCI sugery induced microglia activation in the ispilateral but not contralateral spinal dorsal horn. However, this
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effect was significantly alleviated by bexarotene 50mg/kg treatment.
Bexarotene suppressed CCI-induced MAPKs activation in the spinal cord
MAPKs activation in the ipsilateral spinal cord were dectected by western blot.
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Results indicated that no significant difference existed among the groups on total MAPKs expression(Pp38/β-actin=0.328, PERK/β-actin=0.728, PJNK/β-actin=0.285). Bexarotene 50mg/kg treatment did not alter spinal MAPKs phosphorylation in sham groups
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(Fig.4, Pp-p38/p38= 0.195, PpERK/ERK= 0.500, PpJNK/JNK= 0.195). Vehicle treatment did not affect MAPKs phosphorylation in CCI rats, neither (P PpJNK/JNK=
0.897).
CCI
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PpERK/ERK=0.722,
surgery
p-p38/p38=0.810,
significantly
induced
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phosphorylation of p38MAPK, ERK1/2 and JNK ,compared to sham group (P p-p38/p38 ＜0.001, PpERK/ERK＜0.001, PpJNK/JNK＜0.001). However, dietary bexarotene 50mg/kg
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abrogated CCI-induced phosphorylation of p38MAPK, ERK1/2 and JNK ( P p-p38/p38=
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0.001, PpERK/ERK ＜ 0.001, PpJNK/JNK= 0.001). This meant that dietary bexarotene 50mg/kg suppressed CCI -induced MAPKs activation.
Bexarotene induced MKP-1 and RXRα expression in the ispilateral spinal cord. Spinal expression and colocation of MKP-1 and RXRα were detected by westernblot and immunofluorescence (Fig.5). Western blot results showed that no
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difference existed on spinal MKP-1expression between sham group and sham+bex 50mg/kg group(PMPK-1= 0.469). Bexarotene 50mg/kg did not alter RXRα expression in sham rats (PRXRα=0.088). CCI surgery significantly decreased spinal MKP-1 expression, compared to sham group(PMPK-1＜0.001). No difference was found on
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RXRα expression between sham and CCI group (PRXRα=0.789). No difference existed between CCI and CCI+vehicle group on MKP-1 and RXRα expression (PMPK-1= 0.195, PRXRα=0.299). Dietary bexarotene 50 mg/kg significantly upregulated spinal
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MKP-1 and RXRα expression in CCI rats (PMPK-1＜0.001, PRXRα=0.012). Through the double stain of RXRα and MKP-1 in the ipsilateral spinal dorsal horn of Sham, Sham+bex 50mg/kg, CCI and CCI+bex 50 mg/kg rats, we found that RXRα(green) and MKP-1(red) co-located in the nucleus (Fig.5B). CCI surgery induced decrease of
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RXRα and MKP-1 in the nucleus. However, this effect was alleivated by bexarotene
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treatment. To further conform that this colocation exist in the microglia, we
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double-stained RXRα(green) and Iba-1(red) in the ipsilateral spinal dorsal horn of CCI+bex 50mg/kg rats (Fig. 5C). Results showed that about 63.2% of RXRα was
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shown in microglia nucleus. This meant that the RXRα-MKP-1 colocation existed in
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microglia.
BCI, an MKP-1 inhibitor, reversed bexarotene-induced upregulation of MKP-1 in CCI rats. Western blot results showed that BCI abrogated the effect of bexarotene 50 mg/kg on spinal MKP-1 expression in CCI rats (Fig.6, PMPK-1= 0.003). CCI+bex 50mg/kg
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group showed no difference compared with vehicle group (PMPK-1=0.622). CCI+BCI group showed no significant difference compared with CCI group(PMPK-1= 0.397). Sham+BCI group showed no sginficant difference compared with the samples of
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Sham group in part 1(data not shown).
BCI abrogated bexarotene-induced anti-neuroinflammatory effect in CCI rats.
Effect of BCI on spinal MAPKs activation and Iba-1 expression in
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bexarotene-treated CCI rats was detected by western blot. Results showed that BCI abolished the effect of bexarotene on MAPKs activation in CCI rats (Fig.7A-D, Pp-p38/p38 ＜ 0.001, Pp-ERK/ERK ＜ 0.001,Pp-JNK/JNK ＜ 0.001). BCI also abrogated bexarotene-induced downregulation of Iba-1 in the ispilateral spinal cord (Fig.7A and
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E, PIba-1//β-actin ＜0.001). No significant difference existed between CCI+bex and
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vehicle group on MAPK activation and Iba-1 expression (P
p-p38/p38=
0.650,
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Pp-ERK/ERK= 0.272, Pp-JNK/JNK= 0.594, PIba-1//β-actin= 0.702). Neither significant difference existed between CCI+BCI and CCI group on MAPKs activation and Iba-1
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expression( Pp-p38/p38=0.748, Pp-ERK/ERK=0.556, Pp-JNK/JNK=0.711, PIba-1//β-actin=0.580).
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Sham+BCI group also showed no signficant differences on spinal MAPKs activation and Iba-1 expression compared with the samples of Sham grooup in part 1(data not shown).
Secondly, we measured the effect of BCI on the production of IL-1β, TNF-α and IL-6 in the spinal cords by ELISA(Fig.7F-H). Results indicated that BCI abolished the effect of bexarotene on expressions of IL-1β, TNF-α and IL-6 in CCI rats ( P IL-1β＜
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0.001, PTNF-α ＜ 0.001, PIL-6 ＜ 0.001). No significant difference existed between CCI+bex and vehicle group on IL-1β, TNF-α and IL-6 expression(P
IL-1β=0.748,
PTNF-α=0.248, PIL-6=0.818). CCI+BCI group showed no difference with CCI group on the production of IL-1β, TNF-α and IL-6(P IL-1β=0.080, PTNF-α=0.387, PIL-6=0.452).
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Sham+BCI group showed no signficant difference on spinal expressions of IL-1β, TNF-α and IL-6 with the samples of Sham group in part 1(data not shown). All above indicated that BCI abrogated bexarotene - induced anti-neuroinflammatory effect in
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CCI rats.
BCI inhibited bexarotene - induced anti-allodynia effect in CCI rats.
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Finally, effect of BCI on neuropathic pain-like behaviors in bexarotene- treated CCI rats was measured (Fig.8). Results indicated that no significant difference existed
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between CCI+bex 50mg/kg group and vehicle group (PMT=0.624, PTT=0.877). BCI
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completely reversed bexarotene-induced anti-allodynia effect in CCI rats (effect vs group × time interaction, Fgroup, MT(30, 150)= 15.084, Fgroup, TT(30,150)=17.832, PMT＜
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0.001, PTT＜0.001). CCI+BCI showed no significant difference on pain thresholds
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compared with CCI group (P MT=0.0.401, PTT=0.777). Sham+BCI group showed no significant difference compared with the pain thresholds of Sham group in Part 1(data not shown).
Discussion
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Various
studies
reported
the
neuroprotective
effect
of
bexarotene
in
neurodegenerative diseases and brain injury. In this study, the effect and the possible mechanism of oral bexarotene on CCI-induced neuropathic pain were investigated. Results indicated that bexarotene mitigated the generation of CCI-induced
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neuropathic pain and spinal neuroinflammation by targeting MKP-1.
Bexarotene, a RXRα agonist, upregulated spinal RXRα expression and
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allievated CCI-induced neuropathic pain
RXRs were a member of a orphan nucleus receptor family activated by 9-cis RA and non-retinoid lipid ligands such as docosahexaenoic acid7. RXRs played an
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important role in multiple physiological and pathological processes in the nerve system. Huang et al. reported that RXR agonist 9-cis-retinoic acid accelerates
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remyelination and inhibited neuroinflammation in the central nervous system under
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multiple sclerosis lesions12. Heras-Sandoval et al reviewed that docosahexaenoic acid, a RXR ligand, inhibited activation of glia cell and alleviated inflammation response in
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Alzheimer’s disease9. Dheer et al reported that a decreased RXRα expression was
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identified in Alzheimer's disease mouse and cell model in vitro6. However, bexarotene rescued the RXR expression loss and protected the cells against Aβ-induced ER stress and pro-apoptotic BAD protein activation6. In our study, immunofluorescence results showed that CCI significantly decreased nucleus RXRα expression in the ipsilateral spinal dorsal horn. However, CCI sugery did not signficantly decrease the total RXRα expression in the ispilateral spinal cord. This might be the result of small sample size
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(n=6). Since RXR alpha was not just expressed in the spinal dorsal horn. And only the ispilateral spinal dorsal horn was involved in the development of neuropathic pain. Unlike pro-inflammatory factors or MAPKs that were strongly expressed in the spinal cord, the changes of RXR alpha might be extenuated in western blot results.
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Enlargement of sample size would make the changes more accurate. Besides, Liu Y et al also reported that Aβ25 ‑ 35 treatment did not alter RXRα mRNA and protein expression in hippocampal tissue and N2a cells23. However, bexarotene induced
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spinal RXR alpha up-regulation in both westerblot and immunofluorescence results in our study. This was consistent with previous reports6.
It was reported that RXRα was expressed in microglia and bexarotene inhibited
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microglia activation after experimental subarachnoid hemorrhage18, 34. To conform whether bexarotene directly targeted microglia activation in the spinal cord, we
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double-stained RXRα/Iba-1 in ispilateral spinal dorsal horn. Results showed that
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about 63.2% of RXRα colocated with Iba-1 staining in ispilateral spinal dorsal horn. This meant that RXRα directly induced microglia inhibition in CCI rats. This was
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consistent with previous studies18.
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Yin et al. reported that intraplantar injection of bexarotene at 30μM or above
induced TRPV1 activation and sensory hypersensitivity39. However, Shibahara et al reported that more than 95% of bexarotene were presented in the liver after intravenous administration, and the contraction of bexarotene in central and peripheral nervous system was about 1%30. This meant that oral bexarotene treatment could not induced high plasma concentration of bexarotene to produce sensory hypersensitivity.
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In our study, oral bexarotene 50mg/kg in sham rats did not produced mechanical allodynia or thermal hyperalgesia. Mechanical allodynia or thermal hyperalgesia were neither shown in naive rats in our study (data not shown).This was consistent with Shibahara’s report30.
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Tachibana et al. reported that long-term bexarotene administration in aged mice causes some side effects such as body weight loss and liver dysfunction32. However, this was not shown in our experiments (data not shown). We proposed that different
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objects resulted in this consequence.
Spinal neuroinflammation was involved in the anti-allodynic effects of
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bexarotene in CCI-induced neuropathic pain
Activation of MAPKs played an important role in neuroinflammation in the
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generation of neuropathic pain. IL-1β, TNF-α and IL-6 were three of the downstream
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proinflammatory factors which could lead to hypersensitivity. In our study, CCI surgery significantly induced activation of p-p38MAPK, pERK1/2 and pJNK. CCI
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surgery also upregulated expression of IL-1β, TNF-α and IL-6. However, dietary
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bexarotene 50mg/kg alleviated CCI-induced MAPKs activation and upregulation of IL-1β, TNF-α and IL-6. This was consistent with previous studies9, 12. Glia activation also played a vital role in neuroinflammation, central sensitization
in neuropathic pain25. Microglia was activated since 4 hours till day 14 after surgery33. Though both microglia and astrocytes could be activated by factors such as ATP, TNF-α and CXCL13, astrocyte activation peaked at day 14 and lasted at least by day
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2814, 33. In this study, CCI surgery significantly induced activation of microglia in the spinal cord on day 7. However, microglia activation was signficantly inhibited by bexarotene 50mg/kg treatment. This was consistent with previous researches4. We also did another experiment for the effect of bexarotene on CCI-induced astrocyte
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activation on day 14. However, bexarotene 50mg/kg treatment did not alter astrocyte activation on day 14(data not shown). This was interesting since increased MAP kinases pERK and later pJNK were reported in acitvated astrocytes24. As was reported 19, 35
.
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that demyelination was involved in the development of neuropathic pain17,
Though both RXRα and RXRβ were expressed in astrocyte. Nucleus expression of retinoid X receptor β was induced in astrocytes during demyelination16. And nucleus RXRα expression was downregulated under neuroinflammation23. However, Xu et al.
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reported that RXRβ was not involved in the induction of MKP-137. So, bexarotene did
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not lead to an alteration in astrocyte activation. This was consistent with our results.
Spinal MKP-1 played a vital role in bexarotene-induced anti-allodynic
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effects in the development of neuropathic pain.
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MKP-1, a archetypal member of MAPK phosphatases, was widely expressed in
the central nervous system10. The targets of MKP-1 were activated MAPKs. Various studies indicated that MKP-1 had anti-inflammatory effects on multiple conditions22, 40
. MPK-1 was induced immediately in response to stressors such as inflammation10.
However, Ndong et al reported that MKP-1 was decreased in a rodent model of neuropathic pain and MKP-1 cDNA in vivo nanoparticle transfection in the spinal
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cord prevented the development of tactile sensitivity27. In our study, CCI sugery signficantly decreased spinal MKP-1 expression in day 7. This was consistent with Ndong’s report27. Bexarotene 50mg/kg per day treatment significantly relieved CCI-induced decrease of MKP-1，which was consistent with previous study27. Further,
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MKP-1 inhibition by BCI abrogate the anti-neuroinflammatory and anti-allodynic effect induced by bexarotene. This meant the vital role of MKP-1 in bexarotene-induced effects in CCI rats.
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Then, how did bexarotene induced MKP-1 upregulation under CCI-induced neuroinflammation in the spinal cord? It was known that mTOR activation contributed to the development of neuropathic pain2. However, inhibition of mTOR pathway induced MKP-1 upregulation through phosphorylation of three transcription
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factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter29.
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. This was consistent with previous study37. However, the detail
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pathway1,
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Bexarotene, on the other hand, induced mTOR inhibition though RXRα/PTEN/AKT
examination of PTEN/AKT signaling in the ispilateral spinal cord was not included in
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this study.
Limitations and conclusion Through the data proved that bexarotene significantly inhibited CCI-induced
neuropathic pain through spinal RXR/MKP-1 signaling, there were still some limitations in the study. Firstly, the data indicated upregulation of RXRα by bexarotene, but the mechanism was unclear. Secondly, both RXRα and RXRβ were
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expressed in the spinal cord41. However, Xu et al. reported that RXRβ was not involved in the induction of MKP-137. In this study, spinal RXRβ was not detected. Besides, studies were reported that T cells infiltration were involved in the formation of CCI-induced neuropathic pain11, 21. Though bexarotene was reported to regulate
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T-cell activation in CTCL patients, the effects of bexarotene on normal mammals were still unknown. And the effects of bexarotene on peripheral T cells were not included in this study. Whether bexarotene-induced T cells regulation was also
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involved in the anti-allodynic effects remained unknown.
In summary, daily treatment bexarotene 50mg/kg significantly inhibited CCI-induced upregulation of IL-1β, TNF-α and IL-6, activation of MAPKs and
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microglia through RXR/MKP-1 signaling. Bexarotene may be a potential choice for
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the treatment of neuropathic pain.
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Competing Interests
The authors declare that they have no competing interests.
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Acknowledgments This work was supported by a grant from the Natural Science Foundation of Hunan
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Province (2017JJ3146) .
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Figure legends
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Fig.1 Daily treatment with bexarotene dosage-dependently alleviated CCI-induced mechanical hyperalgesia and thermal allodynia. Data were expressed as mean±S.E.M. n=6. ***P＜0.001,Sham vs CCI group; $P＜0.05, $$P＜0.01, CCI vs CCI+bex 25mg/kg; #P ＜0.05, ##P ＜0.01, ###P ＜0.001, CCI vs CCI+bex 50mg/kg group. Two-way ANOVA (effect vs. group × time interaction) followed by post hoc LSD-t test, mechanical thresholds, Fgroup(36,180)=92.464, PMT＜0.001; thermal thresholds , TTs, Fgroup(36,180)=71.815, PTT＜0.001).
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Fig.2 Daily treatment with bexarotene dosage-dependently reversed CCI- induced expression of IL-1β (A), TNF-α (B) and IL-6 (C). Data were expressed as mean±S.E.M. n=6. ***P＜0.001, vs sham group; #P ＜0.05, ##P ＜0.01, ###P ＜ 0.001, vs CCI group.
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Fig.3 Daily treatment with bexarotene dosage-dependently reversed this effect of CCI-induced micrglia activation(A). Wester blot results were consistene with Iba-1 immunofluorescence in the spinal dorsal horn (B,C). Data were expressed as mean±S.E.M. n=6. **P＜0.01, vs sham group; ##P ＜0.01, vs CCI group. Fig.3D showed a comparison of contralateral and ispilateral spinal cord in CCI and CCI+bex 50mg/kg rats. Results showed CCI sugery induced microglia activation in the ispilateral but not contralateral spinal dorsal horn. However, this effect was significantly alleviated by bexarotene 50mg/kg treatment.
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Fig.4 Daily treatment with bexarotene dosage-dependently inhibited phosphorylation of MAPKs. Data were expressed as mean±S.E.M. n=6. ***P＜0.001, vs sham group; # P ＜0.05, ##P ＜0.01, ###P ＜0.001, vs CCI group.
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Fig.5 CCI surgery significantly decreased MKP-1 expression in day 7 (***P＜0.001, vs sham group).Data were expressed as mean±S.E.M. n=6. (A).Daily treatment with bexarotene dosage-dependently increased spinal RXRα and MKP-1 expression in day 7.( #P ＜0.05, ##P ＜0.01, vs CCI group. (B).According to western blot data, we detected colocalization of spinal RXRα and MKP-1 by double-stain immunofluorescence at 400×magnification. Results showed that spinal RXRα (green) and MKP-1(red) were colocated in the nucleus in sham, sham+bex 50mg/kg and CCI+ bex 50mg/kg group. However, RXRα and MKP-1 expression was much lower and less was shown in the nucleus. This meant that RXRα was deactivated in CCI rats. This was consistent with western blot results. To further conform that this colocation exist in the microglia, we double-stained RXRα(green) and Iba-1(red) in the ipsilateral spinal dorsal horn of CCI+bex 50mg/kg rats (Fig. 5C). Results showed that about 63.2% of RXRα was shown in microglia nucleus. This meant that the RXRα-MKP-1 colocation existed in microglia.
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Fig.6 BCI, a MKP-1 inhibitor, significantly reversed upregulation of spinal MKP-1 by bexarotene. Data were expressed as mean±S.E.M. n=6. BCI **P ＜0.01, ***P ＜ 0.001, CCI+bex vs CCI+bex +BCI group; No difference existed in CCI+bex and CCI+bex+vehicle group(P=0.622). Neither difference existed in CCI+BCI and CCI group (P=0.380).
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Fig.7 BCI significantly reversed the effect of bexarotene on spinal neuroinflammation in CCI rats. Westernblot results showed that BCI abrogated bexarotene-induced MAPK deactivation and Iba-1 downregulation in CCI rats (A, B, C, D, E). ELISA results showed that BCI abolished the effect of bexarotene on expressions of IL-1β, TNF-α and IL-6 in CCI rats(F, G, H). Data were expressed as mean±S.E.M. n=6.(***P ＜0.001, CCI+bex vs CCI+bex +BCI group). No difference existed in CCI+bex 50mg/kg and CCI+bex 50mg/kg+vehicle group(P≥0.05). Neither difference existed in CCI+BCI and CCI group (P≥0.05).
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Fig.8 BCI treatment abrogated the anti-allodynic effect of bexarotene in CCI-induced neuropathic pain. Data were expressed as mean±S.E.M. n=6.*P ＜0.05, ** P ＜0.01, CCI+bex 50mg/kg vs CCI+bex 50mg/kg+BCI group). Two-way ANOVA (effect vs. group × time interaction) followed by post hoc LSD-t test, mechanical thresholds, Fgroup, MT(30, 180)= 15.084, P＜0.001; thermal thresholds, Fgroup, (30,180)= 17.832, P＜0.001). CCI+BCI showed no significant difference on pain thresholds compared with CCI group (P MT=0.0.401, PTT=0.777).
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Bexarotent attenuated CCI-induced spinal neuroinflammation and neuropathic pain by targeting MKP-1 Yulong Gui , Shunyuan Duan , Lihong Xiao , Jing Tang , Aiyuan Li PII: DOI: Reference:
S1526-5900(18)30607-2 https://doi.org/10.1016/j.jpain.2019.01.007 YJPAI 3684
To appear in:
Journal of Pain
Received date: Revised date: Accepted date:
19 September 2018 23 December 2018 16 January 2019
Please cite this article as: Yulong Gui , Shunyuan Duan , Lihong Xiao , Jing Tang , Aiyuan Li , Bexarotent attenuated CCI-induced spinal neuroinflammation and neuropathic pain by targeting MKP1, Journal of Pain (2019), doi: https://doi.org/10.1016/j.jpain.2019.01.007
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Highlights Bexarotene reduced CCI-induced neuropathic pain in rats. Bexarotene inhibited CCI-induced neuroinflammation and activation of MAPKs in the spinal cord . Bexarotene induced MKP-1 in the spinal cord. BCI, a MKP-1 inhibitor completely abrogated bexarotene-induced effects in CCI rats. Bexarotene could be a potential therapeutic agent for neuropathic pain
Bexarotent attenuated CCI-induced spinal neuroinflammation and neuropathic pain by
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targeting MKP-1
Yulong Gui1, Shunyuan Duan2, Lihong Xiao1, Jing Tang1, Aiyuan Li1*
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1. Department of Anesthesiology, Maternal and Child Hospital of Hunan Province, Changsha, Hunan 410008, China
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2. Department of Endocrinology, Yongzhou-affiliated Hospital of University of South
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China, Yongzhou 425000, Hunan Province, China
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To whom correspondence should be addressed: Aiyuan Li Department of Anesthesiology, Maternal and Child Hospital of Hunan Province, 1
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Changsha, Hunan 410008, China
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Email:[email protected]
Abstract
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It is widely accepted that neuroinflammation in the spinal cord contribute to the development of central sensitization in neuropathic pain. MAPKs activation plays an vital role in the development of neuroinflammation in the spinal cord. In this study,
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we investigated the effect of bexarotene, a RXR agonist, on MAPKs activation in
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CCI-induced neuropathic pain. The data showed that daily treatment with bexarotene 50mg/kg significantly alleviated CCI-induced nociceptive hypersensitivity in rats.
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Bexarotene 50mg/kg/day inhibited CCI-induced MAPKs (p38MAPK, ERK1/2, and
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JNK) activation and upregulation of proinflammatory factors(IL-1β, TNF-α and IL-6). Bexarotene also reversed CCI-induced microglia activation in the ipsilateral spinal
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cord. Furthermore, bexarotene treatment significantly upregulated MKP-1 in the spinal cord. These effects were completely abrogated by MKP-1 inhibitor BCI. These results indicated that bexarotene relieved CCI-induced neuroinflammation and neuropathic pain by targeting MKP-1. Therefore, bexarotene might be a potential agent for the treatment of neuropathic pain.
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Keywords: bexarotene, CCI(Chroinc constriction injury), mitogen-activated protein kinases(MAPKs), , mitogen-activated protein kinase phosphatase 1(MKP-1),
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neuropathic pain
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Introduction
It is widely accepted that neuroinflammation in the spinal cord plays an important role in the development of central sensitization in neuropathic pain. Spinal MAPKs
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activation contribute to the development of neurinflammation in neuropathic pain13, 15. However, Ndong C et al reported that mitogen-activated protein kinase phosphatase
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1(MKP-1), a negative feedback of activated MAPKs, was reduced in the development
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of neuropathic pain27. Upregulation of spinal MPK-1 alleviated the behavioral hypersensitivity in neuropathic pain27. MKP-1 might be a potential target for the
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treatment of neuropathic pain. However, selective MKP-1 agonist was not reported,
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yet. However, it was reported that MKP-1 was induced by all-trans retinoic acid28, 37. That pompted us to consider bexarotene, a derivative of all-trans retinoic acid, for the treatment of CCI-induced neuropathic pain. Bexarotene, known as Targretin, was a derivative of all-trans retinoic acid and a selective retinoid X receptor (RXR) agonist. Bexarotene was originally used for treatment of cutaneous T-cell lymphoma. However, recent studies showed bexarotene
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possessed potential neuroprotective and anti-neuroinflammatory effects. Zhong et al. reported that bexarotene protected against traumatic brain injury and inhibited inflammation responses42. In clinic, Cummings et al reported bexarotene alleviated brain amyloid in a proof-of-concept trial5. Smit et al. reported an anti-psoriatic effect
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of bexarotene and no serious adverse events in a phase II multicenter clinical trial31. This prompted us to investigate the effect of bexarotene on CCI-induced neuropathic pain.
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In this study, pain thresholds were measured as the primary outcomes. Expression of proinflammatory factors(IL-1β, TNF-α and IL-6), acitvation of MAPK signals and microglia were measured as the secondary outcomes. BCI, an MKP-1 inhibitor, was
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employed to investigate the role of MKP-1 on bexarotent-induced effects.
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Methods
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Animals and grouping
The current experiments were approved by animal ethical committee of University
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of South China. Young male adult rats (200-250g, 12 weeks old) were used in these
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experiments. All the rats were housed properly for a week adaptation before the experiments began. Three parts were performed. In part one, 84 rats were included into 7 groups through randomization by SPSS 17.0 software: Sham group, Sham+bexarotene (bex) 50mg/kg group, CCI group, CCI+vehicle group, CCI+ bex 12.5 mg/kg group, CCI+ bex 25mg/kg group, CCI+ bex 50mg/kg group (n=12). Ipsilateral behavior tests were performed in 6 rats of each group. The rest rats were
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decapitated in day 7 for ELISA, western blot and immunofluorescence. In part two, 82 rats were treated with intrathecal catheterization before the experiments started. Then, 10 rats with 20% lower pain thresholds than the average were excluded. Then, the rest rats were assigned into 6 groups: CCI+ bex 50mg/kg group, CCI+bex
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50mg/kg+vehicle group(vehicle group), CCI+bex 50mg/kg+BCI group, Sham+BCI group, CCI+BCI group, CCI group (n=12). Behavior tests were operated on 6 rats in each group. The other rats (n=6) were used for western blot and ELISA tests on day 7.
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As was required that both contralateral and ispilateral spinal cord should be shown as a comparison on microglia activation, we did other experiments (Part three). We used aditional 12 rats with CCI (n=6) or CCI+bex 50mg/kg (n=6), respectively. The rats
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immunofluorescence.
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were sacrificed on day 7. The L4-6 spinal cord of both sides was taken for Iba-1
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Drugs and Treatment
Bexarotene (MedChemExpress, USA) was dissolved in 0.5% sodium carboxyl
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methyl cellulose at concentrations of 2.5, 5, 10mg/ml, according to previous study36.
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The oral dosage of bexarotene for the rats was described by Xu L. et al36. Vehicle rats in part one were treated with the same volume of 0.5% sodium carboxyl methyl cellulose without bexarotene. Bexarotene or vehicle administration in part one was operated two hours before behaviour tests from day 1 to day 14. In part two, BCI (Sigma-Aldrich, USA, #Cat:B4313) was primarily dissolved in DMSO at concentrations of 10 mM, respectively. Then, BCI stock solutions were diluted to
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1mM with methylcellulose (0.5%) solution containing 1% Tween. 5μl of 1 mM BCI were intrathecally administrated to rats in BCI groups from day 1 to day 14, according to previous reports26. Here, vehicle rats were treated with 5 μl of methylcellulose (0.5%) solution containing 1% Tween and 10% DMSO. The intrathecal procedure
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was performed under a disinfection condition. BCI or vehicle was intrathecally administrated at a volume of 5μl followed by a 10 µl saline flush. The whole process
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was performed for about 30 seconds.
Surgery procedure
Surgeries were operated under sodium pentobarbital anesthesia (40–60 mg/kg,
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i.p.). CCI model was operated as Bennett et al reported3. Briefly, after skin shaved and sterilized, right sciatic nerve was properly exposed and tied four times. For intrathecal
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catheterization, a PE10 catheter was implanted to the level of the lumbar enlargement
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approximately as described previously8, 38.
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Determination of nociceptive hypersensitivity
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In this study, Electronic Von Frey unit EVF (Bioseb, USA) was used. Briefly,
ipsi-injured hind paws of the rats were treated five times with filament (up to 90 gram probe). Mechanical thresholds were determinated as the average of the final readings. Ipsilateral thermal thresholds (TTs) were tested as follow. The rats were placed into a chamber with a light source. Ipsilateral paws were heated to withdrawal latency or a 20 s cut off as previously reported3, 36. The withdrawal latency was considered as
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lifting paws and/or licking in response to at least 3 out of 5 tests. The progresses were repeated three times with a 5-min interval. Measurement of IL-1β, TNF-α and IL-6 by enzyme-linked immunosorbent assay (ELISA)
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On day 7, 6 rats in each group were sacrificed by cervical dislocation. Half of ipsilateral spinal cords (L5-6) were dissected, lysed, and centrifugated at 20,000×g, 4°C for 30 minutes. Then, the supernatant of each samples was collected seperately
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for use. Rat ELISA kits (Kangwei Bio-tech., China) were used to detect the IL-1β, TNF-α and IL-6 in the samples according to the instructions.
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Western blot
After being lysed and centrifugated, the supernatant was determined by BCA kit.
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After being normalized and boiled with loading buffer, the proteins were separated
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and transformed into PVDF membrane(Millipore, USA). After being treated with 5% BSA for one hour, membranes were incubated with primary antibodies in 1% BSA. In
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this study, rabbit anti-p-p38MAPK, p38MAPK, p-ERK1/2, ERK1/2, RXRα (Cell
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Signaling Technology, USA,1:1000), rabbit anti-p-JNK1/2(Abcam, UK,1:2000), JNK1/2 (Abcam, UK,1:5000), mouse anti-MKP-1 (Santa Cruz Technology, USA,1:500), goat anti-iba-1 antibody(Abcam, UK, 1:1000), rabbit GAPDH and anti-β-actin (ABclonal Technology, USA,1:5000) were used. After being washed, membranes were treated with HRP-linked anti-rabbit or mouse secondary antibodies
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(Cell Signaling Technology, USA,1:1000) for one hour. Signal were visualized by HRP substrate (Millipore,USA, Luminata™ Forte).
Immunofluorescence
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The ispilateral L4-5 spinal cords on day 7 were treated with 4% paraformaldehyde for 8 hours and 30% sucrose overnight at 4°C. Then, samples were embedded and sliced. After being washed and treated with 0.3% Triton X-100 for 20
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minutes, they were incubated with 5% donkey serum (DS) in PBS for an hour and primary antibody in 1% DS overnight. Goat anti-iba-1 antibody(Abcam, UK, 1:200) were used. After being washed again, sections were treated with donkey anti-goat IgG
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Dylight594 (1:200, Jackson ImmunoResearch Laboratories, USA). Images were taken at 100×magnification (Leica DM500B, Wetzlar, Germany).
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To detect colocalization of RXRα/MKP-1 and RXRα/Iba-1, rabbit anti-RXRα
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(1:200, Cell Signaling Technology, USA), goat anti-iba-1 antibody(Abcam, UK, 1:200) and mouse anti-MKP-1 (1:50, Santa Cruz Technology, USA) were used as
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primary antibodies in 1% DS. Secondary antibodies were donkey anti-rabbit IgG
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Dylight488 (1:200, Jackson ImmunoResearch Laboratories, USA), donkey anti-goat IgG Dylight594 (1:200, Jackson ImmunoResearch Laboratories, USA) and donkey anti-mouse IgG Dylight594 (1:200, Jackson ImmunoResearch Laboratories, USA).
Statistical analysis
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In this study, SPSS version 17.0 was used for data analysis. Data in this study were presented as Mean ± S.E.M. Tow-way repeated measure ANOVA (group×time) with correction for multiple comparisons by LSD-t analysis was used to evaluate the difference in MTs and TTs among the groups. One-way ANOVA was used to evaluate
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the difference in behavior baseline, western blot and ELISA results. P < 0.05 was considered as significant difference.
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Results Bexarotene attenuated CCI-induced allodynia in rats
Firstly, effect of bexarotene on CCI-induced pain behaviors was measured. The
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baselines of mechanical and thermal thresholds showed no significant difference among the groups (Fig.1, PMT=0.920,PTT=0.880). Sham+bex 50mg/kg group showed
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no significant difference compared with sham group on pain thresholds (PMT= 0.579,
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PTT=0.856). CCI surgery significantly decreased ipsilateral mechanical and thermal thresholds in rats . (PMT＜0.001, PTT＜0.001). However, this effect was relieved by
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daily oral bexarotene in a dosage-dependent manner (MTs, Fgroup(36,180)=92.464,
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PMT＜0.001; TTs, Fgroup(36,180)=71.815, PTT＜0.001).
Bexarotene suppressed CCI-induced expressions of IL-1β, TNF-α and IL-6 in the spinal cord. Effect of bexarotene on expressions of IL-1β, TNF-α and IL-6 in the spinal cord was detected by ELISA (Fig.2). Results showed that CCI surgery significantly
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induced upregulation of IL-1β, TNF-α and IL-6(P＜0.001). Vehicle treatment did not affect the production of IL-1β, TNF-α and IL-6 in CCI rats in Part one (PIL-1β= 0.527, PIL-6=0.493, PTNF-α=0.676). Daily treatment with bexarotene 50mg/kg relieved CCI-induced upregulation of IL-1β, TNF-α and IL-6 in the spinal cord (P＜0.001).
in sham rats (PIL-1β= 0.615, PIL-6=0.640, PTNF-α=0.781).
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However, bex 50mg/kg treatment did not affect the produce of IL-1β, TNF-α and IL-6
Microglia
activation
in
the
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Bexarotene suppressed CCI-induced microglia activation in the spinal cord ipsilateral
spinal
cord
was
detected
by
immunofluorescence and westernblot of Iba-1. Immunofluorescence results showed
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that CCI increased the number of detectable microglia in the ipsilateral dorsal horn compared with sham or sham+bex 50 mg/kg group (Fig.3). However, this effect was
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significantly inhibited by daily oral bexarotene 50 mg/kg. To reduce the
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microtomy-caused bias in immunofluorescence and better qualification, Iba-1 expression was detected by western blot. Results showed that CCI significantly
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increased ipsilateral spinal Iba-1 expression compared to sham group (P=0.002). No
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significant difference existed between CCI group and CCI+vehicle group (P=0.835). Neither significant difference existed between sham group and sham+bex 50mg/kg group on the Iba-1 expression (P=0.712). Dietary bexarotene 50mg/kg significantly inhibited the effect of CCI-induced Iba-1 upregulation (P=0.006), which meant bexarotene 50mg/kg inhibited CCI-induced microglia activation. As was required that both contralateral and ispilateral spinal cord should be shown as a comparison on
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microglia activation, we did the Iba-1 immunofluorescence of both contralateral and ispilateral spinal cord (Fig.3D). Result showed that CCI sugery induced microglia activation in the ispilateral but not contralateral spinal dorsal horn. However, this
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effect was significantly alleviated by bexarotene 50mg/kg treatment.
Bexarotene suppressed CCI-induced MAPKs activation in the spinal cord
MAPKs activation in the ipsilateral spinal cord were dectected by western blot.
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Results indicated that no significant difference existed among the groups on total MAPKs expression(Pp38/β-actin=0.328, PERK/β-actin=0.728, PJNK/β-actin=0.285). Bexarotene 50mg/kg treatment did not alter spinal MAPKs phosphorylation in sham groups
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(Fig.4, Pp-p38/p38= 0.195, PpERK/ERK= 0.500, PpJNK/JNK= 0.195). Vehicle treatment did not affect MAPKs phosphorylation in CCI rats, neither (P PpJNK/JNK=
0.897).
CCI
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PpERK/ERK=0.722,
surgery
p-p38/p38=0.810,
significantly
induced
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phosphorylation of p38MAPK, ERK1/2 and JNK ,compared to sham group (P p-p38/p38 ＜0.001, PpERK/ERK＜0.001, PpJNK/JNK＜0.001). However, dietary bexarotene 50mg/kg
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abrogated CCI-induced phosphorylation of p38MAPK, ERK1/2 and JNK ( P p-p38/p38=
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0.001, PpERK/ERK ＜ 0.001, PpJNK/JNK= 0.001). This meant that dietary bexarotene 50mg/kg suppressed CCI -induced MAPKs activation.
Bexarotene induced MKP-1 and RXRα expression in the ispilateral spinal cord. Spinal expression and colocation of MKP-1 and RXRα were detected by westernblot and immunofluorescence (Fig.5). Western blot results showed that no
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difference existed on spinal MKP-1expression between sham group and sham+bex 50mg/kg group(PMPK-1= 0.469). Bexarotene 50mg/kg did not alter RXRα expression in sham rats (PRXRα=0.088). CCI surgery significantly decreased spinal MKP-1 expression, compared to sham group(PMPK-1＜0.001). No difference was found on
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RXRα expression between sham and CCI group (PRXRα=0.789). No difference existed between CCI and CCI+vehicle group on MKP-1 and RXRα expression (PMPK-1= 0.195, PRXRα=0.299). Dietary bexarotene 50 mg/kg significantly upregulated spinal
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MKP-1 and RXRα expression in CCI rats (PMPK-1＜0.001, PRXRα=0.012). Through the double stain of RXRα and MKP-1 in the ipsilateral spinal dorsal horn of Sham, Sham+bex 50mg/kg, CCI and CCI+bex 50 mg/kg rats, we found that RXRα(green) and MKP-1(red) co-located in the nucleus (Fig.5B). CCI surgery induced decrease of
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RXRα and MKP-1 in the nucleus. However, this effect was alleivated by bexarotene
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treatment. To further conform that this colocation exist in the microglia, we
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double-stained RXRα(green) and Iba-1(red) in the ipsilateral spinal dorsal horn of CCI+bex 50mg/kg rats (Fig. 5C). Results showed that about 63.2% of RXRα was
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shown in microglia nucleus. This meant that the RXRα-MKP-1 colocation existed in
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microglia.
BCI, an MKP-1 inhibitor, reversed bexarotene-induced upregulation of MKP-1 in CCI rats. Western blot results showed that BCI abrogated the effect of bexarotene 50 mg/kg on spinal MKP-1 expression in CCI rats (Fig.6, PMPK-1= 0.003). CCI+bex 50mg/kg
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group showed no difference compared with vehicle group (PMPK-1=0.622). CCI+BCI group showed no significant difference compared with CCI group(PMPK-1= 0.397). Sham+BCI group showed no sginficant difference compared with the samples of
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Sham group in part 1(data not shown).
BCI abrogated bexarotene-induced anti-neuroinflammatory effect in CCI rats.
Effect of BCI on spinal MAPKs activation and Iba-1 expression in
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bexarotene-treated CCI rats was detected by western blot. Results showed that BCI abolished the effect of bexarotene on MAPKs activation in CCI rats (Fig.7A-D, Pp-p38/p38 ＜ 0.001, Pp-ERK/ERK ＜ 0.001,Pp-JNK/JNK ＜ 0.001). BCI also abrogated bexarotene-induced downregulation of Iba-1 in the ispilateral spinal cord (Fig.7A and
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E, PIba-1//β-actin ＜0.001). No significant difference existed between CCI+bex and
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vehicle group on MAPK activation and Iba-1 expression (P
p-p38/p38=
0.650,
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Pp-ERK/ERK= 0.272, Pp-JNK/JNK= 0.594, PIba-1//β-actin= 0.702). Neither significant difference existed between CCI+BCI and CCI group on MAPKs activation and Iba-1
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expression( Pp-p38/p38=0.748, Pp-ERK/ERK=0.556, Pp-JNK/JNK=0.711, PIba-1//β-actin=0.580).
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Sham+BCI group also showed no signficant differences on spinal MAPKs activation and Iba-1 expression compared with the samples of Sham grooup in part 1(data not shown).
Secondly, we measured the effect of BCI on the production of IL-1β, TNF-α and IL-6 in the spinal cords by ELISA(Fig.7F-H). Results indicated that BCI abolished the effect of bexarotene on expressions of IL-1β, TNF-α and IL-6 in CCI rats ( P IL-1β＜
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0.001, PTNF-α ＜ 0.001, PIL-6 ＜ 0.001). No significant difference existed between CCI+bex and vehicle group on IL-1β, TNF-α and IL-6 expression(P
IL-1β=0.748,
PTNF-α=0.248, PIL-6=0.818). CCI+BCI group showed no difference with CCI group on the production of IL-1β, TNF-α and IL-6(P IL-1β=0.080, PTNF-α=0.387, PIL-6=0.452).
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Sham+BCI group showed no signficant difference on spinal expressions of IL-1β, TNF-α and IL-6 with the samples of Sham group in part 1(data not shown). All above indicated that BCI abrogated bexarotene - induced anti-neuroinflammatory effect in
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CCI rats.
BCI inhibited bexarotene - induced anti-allodynia effect in CCI rats.
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Finally, effect of BCI on neuropathic pain-like behaviors in bexarotene- treated CCI rats was measured (Fig.8). Results indicated that no significant difference existed
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between CCI+bex 50mg/kg group and vehicle group (PMT=0.624, PTT=0.877). BCI
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completely reversed bexarotene-induced anti-allodynia effect in CCI rats (effect vs group × time interaction, Fgroup, MT(30, 150)= 15.084, Fgroup, TT(30,150)=17.832, PMT＜
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0.001, PTT＜0.001). CCI+BCI showed no significant difference on pain thresholds
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compared with CCI group (P MT=0.0.401, PTT=0.777). Sham+BCI group showed no significant difference compared with the pain thresholds of Sham group in Part 1(data not shown).
Discussion
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Various
studies
reported
the
neuroprotective
effect
of
bexarotene
in
neurodegenerative diseases and brain injury. In this study, the effect and the possible mechanism of oral bexarotene on CCI-induced neuropathic pain were investigated. Results indicated that bexarotene mitigated the generation of CCI-induced
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neuropathic pain and spinal neuroinflammation by targeting MKP-1.
Bexarotene, a RXRα agonist, upregulated spinal RXRα expression and
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allievated CCI-induced neuropathic pain
RXRs were a member of a orphan nucleus receptor family activated by 9-cis RA and non-retinoid lipid ligands such as docosahexaenoic acid7. RXRs played an
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important role in multiple physiological and pathological processes in the nerve system. Huang et al. reported that RXR agonist 9-cis-retinoic acid accelerates
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remyelination and inhibited neuroinflammation in the central nervous system under
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multiple sclerosis lesions12. Heras-Sandoval et al reviewed that docosahexaenoic acid, a RXR ligand, inhibited activation of glia cell and alleviated inflammation response in
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Alzheimer’s disease9. Dheer et al reported that a decreased RXRα expression was
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identified in Alzheimer's disease mouse and cell model in vitro6. However, bexarotene rescued the RXR expression loss and protected the cells against Aβ-induced ER stress and pro-apoptotic BAD protein activation6. In our study, immunofluorescence results showed that CCI significantly decreased nucleus RXRα expression in the ipsilateral spinal dorsal horn. However, CCI sugery did not signficantly decrease the total RXRα expression in the ispilateral spinal cord. This might be the result of small sample size
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(n=6). Since RXR alpha was not just expressed in the spinal dorsal horn. And only the ispilateral spinal dorsal horn was involved in the development of neuropathic pain. Unlike pro-inflammatory factors or MAPKs that were strongly expressed in the spinal cord, the changes of RXR alpha might be extenuated in western blot results.
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Enlargement of sample size would make the changes more accurate. Besides, Liu Y et al also reported that Aβ25 ‑ 35 treatment did not alter RXRα mRNA and protein expression in hippocampal tissue and N2a cells23. However, bexarotene induced
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spinal RXR alpha up-regulation in both westerblot and immunofluorescence results in our study. This was consistent with previous reports6.
It was reported that RXRα was expressed in microglia and bexarotene inhibited
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microglia activation after experimental subarachnoid hemorrhage18, 34. To conform whether bexarotene directly targeted microglia activation in the spinal cord, we
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double-stained RXRα/Iba-1 in ispilateral spinal dorsal horn. Results showed that
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about 63.2% of RXRα colocated with Iba-1 staining in ispilateral spinal dorsal horn. This meant that RXRα directly induced microglia inhibition in CCI rats. This was
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consistent with previous studies18.
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Yin et al. reported that intraplantar injection of bexarotene at 30μM or above
induced TRPV1 activation and sensory hypersensitivity39. However, Shibahara et al reported that more than 95% of bexarotene were presented in the liver after intravenous administration, and the contraction of bexarotene in central and peripheral nervous system was about 1%30. This meant that oral bexarotene treatment could not induced high plasma concentration of bexarotene to produce sensory hypersensitivity.
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In our study, oral bexarotene 50mg/kg in sham rats did not produced mechanical allodynia or thermal hyperalgesia. Mechanical allodynia or thermal hyperalgesia were neither shown in naive rats in our study (data not shown).This was consistent with Shibahara’s report30.
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Tachibana et al. reported that long-term bexarotene administration in aged mice causes some side effects such as body weight loss and liver dysfunction32. However, this was not shown in our experiments (data not shown). We proposed that different
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objects resulted in this consequence.
Spinal neuroinflammation was involved in the anti-allodynic effects of
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bexarotene in CCI-induced neuropathic pain
Activation of MAPKs played an important role in neuroinflammation in the
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generation of neuropathic pain. IL-1β, TNF-α and IL-6 were three of the downstream
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proinflammatory factors which could lead to hypersensitivity. In our study, CCI surgery significantly induced activation of p-p38MAPK, pERK1/2 and pJNK. CCI
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surgery also upregulated expression of IL-1β, TNF-α and IL-6. However, dietary
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bexarotene 50mg/kg alleviated CCI-induced MAPKs activation and upregulation of IL-1β, TNF-α and IL-6. This was consistent with previous studies9, 12. Glia activation also played a vital role in neuroinflammation, central sensitization
in neuropathic pain25. Microglia was activated since 4 hours till day 14 after surgery33. Though both microglia and astrocytes could be activated by factors such as ATP, TNF-α and CXCL13, astrocyte activation peaked at day 14 and lasted at least by day
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2814, 33. In this study, CCI surgery significantly induced activation of microglia in the spinal cord on day 7. However, microglia activation was signficantly inhibited by bexarotene 50mg/kg treatment. This was consistent with previous researches4. We also did another experiment for the effect of bexarotene on CCI-induced astrocyte
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activation on day 14. However, bexarotene 50mg/kg treatment did not alter astrocyte activation on day 14(data not shown). This was interesting since increased MAP kinases pERK and later pJNK were reported in acitvated astrocytes24. As was reported 19, 35
.
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that demyelination was involved in the development of neuropathic pain17,
Though both RXRα and RXRβ were expressed in astrocyte. Nucleus expression of retinoid X receptor β was induced in astrocytes during demyelination16. And nucleus RXRα expression was downregulated under neuroinflammation23. However, Xu et al.
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reported that RXRβ was not involved in the induction of MKP-137. So, bexarotene did
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not lead to an alteration in astrocyte activation. This was consistent with our results.
Spinal MKP-1 played a vital role in bexarotene-induced anti-allodynic
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effects in the development of neuropathic pain.
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MKP-1, a archetypal member of MAPK phosphatases, was widely expressed in
the central nervous system10. The targets of MKP-1 were activated MAPKs. Various studies indicated that MKP-1 had anti-inflammatory effects on multiple conditions22, 40
. MPK-1 was induced immediately in response to stressors such as inflammation10.
However, Ndong et al reported that MKP-1 was decreased in a rodent model of neuropathic pain and MKP-1 cDNA in vivo nanoparticle transfection in the spinal
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cord prevented the development of tactile sensitivity27. In our study, CCI sugery signficantly decreased spinal MKP-1 expression in day 7. This was consistent with Ndong’s report27. Bexarotene 50mg/kg per day treatment significantly relieved CCI-induced decrease of MKP-1，which was consistent with previous study27. Further,
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MKP-1 inhibition by BCI abrogate the anti-neuroinflammatory and anti-allodynic effect induced by bexarotene. This meant the vital role of MKP-1 in bexarotene-induced effects in CCI rats.
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Then, how did bexarotene induced MKP-1 upregulation under CCI-induced neuroinflammation in the spinal cord? It was known that mTOR activation contributed to the development of neuropathic pain2. However, inhibition of mTOR pathway induced MKP-1 upregulation through phosphorylation of three transcription
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factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter29.
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. This was consistent with previous study37. However, the detail
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pathway1,
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Bexarotene, on the other hand, induced mTOR inhibition though RXRα/PTEN/AKT
examination of PTEN/AKT signaling in the ispilateral spinal cord was not included in
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this study.
Limitations and conclusion Through the data proved that bexarotene significantly inhibited CCI-induced
neuropathic pain through spinal RXR/MKP-1 signaling, there were still some limitations in the study. Firstly, the data indicated upregulation of RXRα by bexarotene, but the mechanism was unclear. Secondly, both RXRα and RXRβ were
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expressed in the spinal cord41. However, Xu et al. reported that RXRβ was not involved in the induction of MKP-137. In this study, spinal RXRβ was not detected. Besides, studies were reported that T cells infiltration were involved in the formation of CCI-induced neuropathic pain11, 21. Though bexarotene was reported to regulate
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T-cell activation in CTCL patients, the effects of bexarotene on normal mammals were still unknown. And the effects of bexarotene on peripheral T cells were not included in this study. Whether bexarotene-induced T cells regulation was also
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involved in the anti-allodynic effects remained unknown.
In summary, daily treatment bexarotene 50mg/kg significantly inhibited CCI-induced upregulation of IL-1β, TNF-α and IL-6, activation of MAPKs and
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microglia through RXR/MKP-1 signaling. Bexarotene may be a potential choice for
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the treatment of neuropathic pain.
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Competing Interests
The authors declare that they have no competing interests.
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Acknowledgments This work was supported by a grant from the Natural Science Foundation of Hunan
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Province (2017JJ3146) .
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Fig.1 Daily treatment with bexarotene dosage-dependently alleviated CCI-induced mechanical hyperalgesia and thermal allodynia. Data were expressed as mean±S.E.M. n=6. ***P＜0.001,Sham vs CCI group; $P＜0.05, $$P＜0.01, CCI vs CCI+bex 25mg/kg; #P ＜0.05, ##P ＜0.01, ###P ＜0.001, CCI vs CCI+bex 50mg/kg group. Two-way ANOVA (effect vs. group × time interaction) followed by post hoc LSD-t test, mechanical thresholds, Fgroup(36,180)=92.464, PMT＜0.001; thermal thresholds , TTs, Fgroup(36,180)=71.815, PTT＜0.001).
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Fig.2 Daily treatment with bexarotene dosage-dependently reversed CCI- induced expression of IL-1β (A), TNF-α (B) and IL-6 (C). Data were expressed as mean±S.E.M. n=6. ***P＜0.001, vs sham group; #P ＜0.05, ##P ＜0.01, ###P ＜ 0.001, vs CCI group.
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Fig.3 Daily treatment with bexarotene dosage-dependently reversed this effect of CCI-induced micrglia activation(A). Wester blot results were consistene with Iba-1 immunofluorescence in the spinal dorsal horn (B,C). Data were expressed as mean±S.E.M. n=6. **P＜0.01, vs sham group; ##P ＜0.01, vs CCI group. Fig.3D showed a comparison of contralateral and ispilateral spinal cord in CCI and CCI+bex 50mg/kg rats. Results showed CCI sugery induced microglia activation in the ispilateral but not contralateral spinal dorsal horn. However, this effect was significantly alleviated by bexarotene 50mg/kg treatment.
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Fig.4 Daily treatment with bexarotene dosage-dependently inhibited phosphorylation of MAPKs. Data were expressed as mean±S.E.M. n=6. ***P＜0.001, vs sham group; # P ＜0.05, ##P ＜0.01, ###P ＜0.001, vs CCI group.
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Fig.5 CCI surgery significantly decreased MKP-1 expression in day 7 (***P＜0.001, vs sham group).Data were expressed as mean±S.E.M. n=6. (A).Daily treatment with bexarotene dosage-dependently increased spinal RXRα and MKP-1 expression in day 7.( #P ＜0.05, ##P ＜0.01, vs CCI group. (B).According to western blot data, we detected colocalization of spinal RXRα and MKP-1 by double-stain immunofluorescence at 400×magnification. Results showed that spinal RXRα (green) and MKP-1(red) were colocated in the nucleus in sham, sham+bex 50mg/kg and CCI+ bex 50mg/kg group. However, RXRα and MKP-1 expression was much lower and less was shown in the nucleus. This meant that RXRα was deactivated in CCI rats. This was consistent with western blot results. To further conform that this colocation exist in the microglia, we double-stained RXRα(green) and Iba-1(red) in the ipsilateral spinal dorsal horn of CCI+bex 50mg/kg rats (Fig. 5C). Results showed that about 63.2% of RXRα was shown in microglia nucleus. This meant that the RXRα-MKP-1 colocation existed in microglia.
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Fig.6 BCI, a MKP-1 inhibitor, significantly reversed upregulation of spinal MKP-1 by bexarotene. Data were expressed as mean±S.E.M. n=6. BCI **P ＜0.01, ***P ＜ 0.001, CCI+bex vs CCI+bex +BCI group; No difference existed in CCI+bex and CCI+bex+vehicle group(P=0.622). Neither difference existed in CCI+BCI and CCI group (P=0.380).
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Fig.7 BCI significantly reversed the effect of bexarotene on spinal neuroinflammation in CCI rats. Westernblot results showed that BCI abrogated bexarotene-induced MAPK deactivation and Iba-1 downregulation in CCI rats (A, B, C, D, E). ELISA results showed that BCI abolished the effect of bexarotene on expressions of IL-1β, TNF-α and IL-6 in CCI rats(F, G, H). Data were expressed as mean±S.E.M. n=6.(***P ＜0.001, CCI+bex vs CCI+bex +BCI group). No difference existed in CCI+bex 50mg/kg and CCI+bex 50mg/kg+vehicle group(P≥0.05). Neither difference existed in CCI+BCI and CCI group (P≥0.05).
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Fig.8 BCI treatment abrogated the anti-allodynic effect of bexarotene in CCI-induced neuropathic pain. Data were expressed as mean±S.E.M. n=6.*P ＜0.05, ** P ＜0.01, CCI+bex 50mg/kg vs CCI+bex 50mg/kg+BCI group). Two-way ANOVA (effect vs. group × time interaction) followed by post hoc LSD-t test, mechanical thresholds, Fgroup, MT(30, 180)= 15.084, P＜0.001; thermal thresholds, Fgroup, (30,180)= 17.832, P＜0.001). CCI+BCI showed no significant difference on pain thresholds compared with CCI group (P MT=0.0.401, PTT=0.777).
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