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Spexin/NPQ Induces FBJ Osteosarcoma Oncogene (Fos) and Produces Antinociceptive Effect against Inflammatory Pain in the Mouse Model

Open AccessPublished:January 18, 2019DOI:https://doi.org/10.1016/j.ajpath.2018.12.009
      Spexin/NPQ is a novel highly conserved neuropeptide. It has a widespread expression in the periphery and central nervous system. However, the effects of central spexin on acute inflammatory pain are still unknown. This study explored the mechanisms and effects of supraspinal spexin on inflammatory pain. The results from the mouse formalin test show that i.c.v. administration of spexin decreased licking/biting time during the late and early phases. The nonamidated spexin had no effect on pain response. The antinociception of spexin was blocked by galanin receptor 3 antagonist SNAP 37889. The Galr3 and Adcy4 mRNA levels in the brain were increased after injection with spexin. The antinociceptive effects of spexin were completely reversed by opioid receptor antagonist naloxone and κ-opioid receptor antagonist nor-binaltorphimine dihydrochloride. Spexin up-regulated the dynorphin and κ-opioid receptor gene and protein expression. PCR array assay and real-time PCR analysis show that spexin up-regulated the mRNA level of the FBJ osteosarcoma oncogene (Fos). T-5224, the inhibitor of c FBJ osteosarcoma oncogene (c-Fos)/activator protein 1 (AP-1), blocked the increased mRNA level of Pdyn and Oprk1 induced by spexin. I.C.V. spexin (2.43 mg/kg) increased the number of c-Fos–positive neurons in most subsections of periaqueductal gray. In addition, in the acetic acid–induced writhing test, i.c.v. spexin produced an antinociceptive effect. Our results indicate that spexin might be a novel neuropeptide with an antinociceptive effect against acute inflammatory pain.
      The novel peptide spexin was first identified as a prohormone containing an amidated 14–amino acid peptide using a hidden Markov model.
      • Mirabeau O.
      • Severini C.
      • Audero E.
      • Gascuel O.
      • Possenti R.
      • Birney E.
      • Rosenthal N.
      • Gross C.
      Identification of novel peptide hormones in the human proteome by hidden Markov model screening.
      The precursor of spexin gene (Spx) has been found through a bioinformatics method by Sonmez et al.
      • Sonmez K.
      • Zaveri N.T.
      • Kerman I.A.
      • Burke S.
      • Neal C.R.
      • Xie X.
      • Watson S.J.
      • Toll L.
      Evolutionary sequence modeling for discovery of peptide hormones.
      The spexin prepropeptide contains mature peptides, cleavage sites, and a signal peptide sequence. And, for the record, a small amino acid region, NWTPQAMLYLKGAQ-NH2 (named NPQ peptide or spexin), is substantially conserved in different vertebrates during biological evolution,
      • Mirabeau O.
      • Severini C.
      • Audero E.
      • Gascuel O.
      • Possenti R.
      • Birney E.
      • Rosenthal N.
      • Gross C.
      Identification of novel peptide hormones in the human proteome by hidden Markov model screening.
      • Sonmez K.
      • Zaveri N.T.
      • Kerman I.A.
      • Burke S.
      • Neal C.R.
      • Xie X.
      • Watson S.J.
      • Toll L.
      Evolutionary sequence modeling for discovery of peptide hormones.
      • Wong M.K.
      • Sze K.H.
      • Chen T.
      • Cho C.K.
      • Law H.C.
      • Chu I.K.
      • Wong A.O.
      Goldfish spexin: solution structure and novel function as a satiety factor in feeding control.
      and it is considered most likely active neuropeptide.
      • Porzionato A.
      • Rucinski M.
      • Macchi V.
      • Stecco C.
      • Malendowicz L.K.
      • De Caro R.
      Spexin expression in normal rat tissues.
      Spexin could activate human galanin receptor types 2 and 3 (GALR2/3) in ligand-receptor interaction assay in vitro, suggesting that spexin may be a natural ligand for GALR2/3.
      • Kim D.-K.
      • Yun S.
      • Son G.H.
      • Hwang J.-I.
      • Park C.R.
      • Kim J.I.
      • Kim K.
      • Vaudry H.
      • Seong J.Y.
      Coevolution of the spexin/galanin/kisspeptin family: spexin activates galanin receptor type II and III.
      Recently, some physiological functions of spexin have been found, such as appetite, bowel movement, energy metabolism, cardiovascular function, and hyperoxia. Spexin was involved in the regulation of rat adrenocortical cell proliferation.
      • Rucinski M.
      • Porzionato A.
      • Ziolkowska A.
      • Szyszka M.
      • Macchi V.
      • De Caro R.
      • Malendowicz L.K.
      Expression of the spexin gene in the rat adrenal gland and evidences suggesting that spexin inhibits adrenocortical cell proliferation.
      Under conditions of hyperoxia exposure, spexin expression is up-regulated in postnatal rats.
      • Porzionato A.
      • Rucinski M.
      • Macchi V.
      • Stecco C.
      • Sarasin G.
      • Sfriso M.M.
      • Di Giulio C.
      • Malendowicz L.K.
      • De Caro R.
      Spexin is expressed in the carotid body and is upregulated by postnatal hyperoxia exposure.
      Toll et al
      • Toll L.
      • Khroyan T.V.
      • Sonmez K.
      • Ozawa A.
      • Lindberg I.
      • McLaughlin J.P.
      • Eans S.O.
      • Shahien A.A.
      • Kapusta D.R.
      Peptides derived from the prohormone proNPQ/spexin are potent central modulators of cardiovascular and renal function and nociception.
      shows that spexin contributes to an increase of arterial blood pressure and a decrease of urine flow rate. Central administration of spexin can inhibit appetite in goldfish.
      • Wong M.K.
      • Sze K.H.
      • Chen T.
      • Cho C.K.
      • Law H.C.
      • Chu I.K.
      • Wong A.O.
      Goldfish spexin: solution structure and novel function as a satiety factor in feeding control.
      Spexin suppressed the release of luteinizing hormone in pituitary cells in vitro and decreased the serum luteinizing hormone levels of goldfish.
      • Liu Y.
      • Li S.
      • Qi X.
      • Zhou W.
      • Liu X.
      • Lin H.
      • Zhang Y.
      • Cheng C.H.
      A novel neuropeptide in suppressing luteinizing hormone release in goldfish, Carassius auratus.
      In Ya-fish, the gene expression of Spx was affected by different feeding conditions or metabolic status.
      • Wu H.
      • Lin F.
      • Chen H.
      • Liu J.
      • Gao Y.
      • Zhang X.
      • Hao J.
      • Chen D.
      • Yuan D.
      • Wang T.
      • Li Z.
      Ya-fish (Schizothorax prenanti) spexin: identification, tissue distribution and mRNA expression responses to periprandial and fasting.
      In addition, spexin causes weight loss in diet-induced obesity mice
      • Walewski J.L.
      • Ge F.
      • Lobdell H.
      • Levin N.
      • Schwartz G.J.
      • Vasselli J.R.
      • Pomp A.
      • Dakin G.
      • Berk P.D.
      Spexin is a novel human peptide that reduces adipocyte uptake of long chain fatty acids and causes weight loss in rodents with diet-induced obesity.
      and could enhance bowel movement in mice.
      • Lin C.-Y.
      • Zhang M.
      • Huang T.
      • Yang L.-L.
      • Fu H.-B.
      • Zhao L.
      • Zhong L.L.
      • Mu H.-X.
      • Shi X.-K.
      • Leung C.F.
      Spexin enhances bowel movement through activating L-type voltage-dependent calcium channel via galanin receptor 2 in mice.
      Spexin mRNA and protein were abundantly distributed in both the central nervous system (CNS) and the periphery in humans,
      • Gu L.
      • Ma Y.
      • Gu M.
      • Zhang Y.
      • Yan S.
      • Li N.
      • Wang Y.
      • Ding X.
      • Yin J.
      • Fan N.
      Spexin peptide is expressed in human endocrine and epithelial tissues and reduced after glucose load in type 2 diabetes.
      rodents,
      • Porzionato A.
      • Rucinski M.
      • Macchi V.
      • Stecco C.
      • Malendowicz L.K.
      • De Caro R.
      Spexin expression in normal rat tissues.
      and fish.
      • Wong M.K.
      • Sze K.H.
      • Chen T.
      • Cho C.K.
      • Law H.C.
      • Chu I.K.
      • Wong A.O.
      Goldfish spexin: solution structure and novel function as a satiety factor in feeding control.
      • Wu H.
      • Lin F.
      • Chen H.
      • Liu J.
      • Gao Y.
      • Zhang X.
      • Hao J.
      • Chen D.
      • Yuan D.
      • Wang T.
      • Li Z.
      Ya-fish (Schizothorax prenanti) spexin: identification, tissue distribution and mRNA expression responses to periprandial and fasting.
      In the periphery, spexin mRNA and protein have been detected in various organs, such as liver, kidney, thyroid, ovary, lung, stomach, and small intestine.
      • Porzionato A.
      • Rucinski M.
      • Macchi V.
      • Stecco C.
      • Malendowicz L.K.
      • De Caro R.
      Spexin expression in normal rat tissues.
      • Gu L.
      • Ma Y.
      • Gu M.
      • Zhang Y.
      • Yan S.
      • Li N.
      • Wang Y.
      • Ding X.
      • Yin J.
      • Fan N.
      Spexin peptide is expressed in human endocrine and epithelial tissues and reduced after glucose load in type 2 diabetes.
      In the CNS of rodents, immunohistochemical analysis shows that spexin-like immunoreactivity was detected in brainstem, trigeminal ganglia, and brain cortex,
      • Porzionato A.
      • Rucinski M.
      • Macchi V.
      • Stecco C.
      • Malendowicz L.K.
      • De Caro R.
      Spexin expression in normal rat tissues.
      which is known to be closely related to nociceptive transmission. It indicates that spexin potentially plays a role in modulating nociceptive behavior.
      The formalin test is used as a continuous tonic pain animal model, with the persistent rather than transient nociceptive stimulus and response,
      • Le Bars D.
      • Gozariu M.
      • Cadden S.W.
      Animal models of nociception.
      • Bannon A.W.
      • Malmberg A.B.
      Models of nociception: hot-plate, tail-flick, and formalin tests in rodents.
      which differs from the tail immersion test. The formalin test presents a biphasic behavioral reaction. The early phase lasts 10 minutes, starting from injection with formalin instantly, and it is induced by a bursting activity from pain fibers, such as C fibers. The late phase is commonly between the 10th and 30th minute after treatment and is triggered by inflammation and central sensitization.
      • Tjølsen A.
      • Berge O.-G.
      • Hunskaar S.
      • Rosland J.H.
      • Hole K.
      The formalin test: an evaluation of the method.
      • McNamara C.R.
      • Mandel-Brehm J.
      • Bautista D.M.
      • Siemens J.
      • Deranian K.L.
      • Zhao M.
      • Hayward N.J.
      • Chong J.A.
      • Julius D.
      • Moran M.M.
      TRPA1 mediates formalin-induced pain.
      The formalin test is a valid model for evaluating the antinociceptive effect of different potential therapeutic agents. The nociceptive response in the formalin test is quantifiable as well as reproducible.
      • Reeta K.
      • Mediratta P.
      • Rathi N.
      • Jain H.
      • Chugh C.
      • Sharma K.
      Role of κ- and δ-opioid receptors in the antinociceptive effect of oxytocin in formalin-induced pain response in mice.
      • Padi S.
      • Kulkarni S.K.
      Role of cyclooxygenase-2 in lipopolysaccharide-induced hyperalgesia in formalin test.
      The acetic acid–induced writhing test is commonly used for evaluating and comparing the efficacy of new drugs in visceral pain treatment.
      • Shamsi Meymandi M.
      • Keyhanfar F.
      Assessment of the antinociceptive effects of pregabalin alone or in combination with morphine during acetic acid-induced writhing in mice.
      This test is used to model tonic pain because of its longer duration.
      • Lv S.-Y.
      • Qin Y.-J.
      • Wang N.-B.
      • Yang Y.-J.
      • Chen Q.
      Supraspinal antinociceptive effect of apelin-13 in a mouse visceral pain model.
      Toll et al
      • Toll L.
      • Khroyan T.V.
      • Sonmez K.
      • Ozawa A.
      • Lindberg I.
      • McLaughlin J.P.
      • Eans S.O.
      • Shahien A.A.
      • Kapusta D.R.
      Peptides derived from the prohormone proNPQ/spexin are potent central modulators of cardiovascular and renal function and nociception.
      showed that spexin exerted antinociceptive activity in the tail-flick test, an acute pain model. However, as far as we know, there are no published articles that have reported the effect of spexin on tonic inflammatory pain or visceral pain.
      Considering the distribution of spexin in the CNS, which is known to be closely related to nociceptive transmission, and the lack of research on spexin in tonic pain, it is necessary to determine the effect of spexin on inflammatory pain and visceral pain. The current investigation was designed to explore the antinociceptive effect of central spexin against tonic inflammatory pain using the mouse formalin test and against visceral pain using the writhing test. The expression levels of the related genes and proteins in brain were determined using real-time PCR, PCR array, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry.

      Materials and Methods

      Animals

      Kunming mice (male, aged 6 to 8 weeks) were obtained from the Animal Center of Henan Province (Zhengzhou, China). The mice were acclimated to the controlled animal room with standard room temperature, humidity, and regular lighting. The animals with free access to dietary food and water were accommodated in five mice per cage for at least 1 week before the behavioral test. All animal experimental protocols were approved by the Committee of Medical Ethics and Welfare for Experimental Animals, Henan University School of Medicine.

      Chemicals

      The peptides used in this study [spexin, nonamidated spexin, and galanin (1–29)] were supplied by GL Biochem (Shanghai) Ltd (Shanghai, China). Bicuculline methiodide was supplied by Tokyo Chemical Industry Co Ltd (Tokyo, Japan). Naloxone, nor-binaltorphimine dihydrochloride, and β-funaltrexamine hydrochloride were supplied by Sigma-Aldrich (St. Louis, MO). M871 was supplied by Abcam Inc. (Burlingame, CA). SNAP 37889 (1-phenyl-3-[[3-(trifluoromethyl)phenyl]imino]-1H-indol-2-one) was obtained from MedChemExpress (Princeton, NJ). T-5224 was provided by APExBIO (Houston, TX). The peptides were dissolved and diluted in sterilized saline at a working concentration before the experiment.

      Drug Administration

      I.C.V. injection was performed according to the protocols shown by Haley and McCormick
      • Haley T.
      • McCormick W.
      Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse.
      and the previous work.
      • Lv S.-Y.
      • Qin Y.-J.
      • Wang H.-T.
      • Xu N.
      • Yang Y.-J.
      • Chen Q.
      Centrally administered apelin-13 induces depression-like behavior in mice.
      The injected point was 1.5 mm lateral, 1.0 mm posterior to bregma, and 3.0 mm below the skull surface. Chemicals or normal saline was treated in a volume of 4 μL (10 μL/minute) by a 10-μL Hamilton microsyringe. The animals in the control group were treated with the same volume of sterilized normal saline. The correctness of the i.c.v. injection was confirmed by checking brain sections for the site of injection after treatment with methylene blue dye.

      Formalin Test

      The tonic inflammatory pain was assessed using the formalin test. This test was performed according to the previous reports.
      • Tjølsen A.
      • Berge O.-G.
      • Hunskaar S.
      • Rosland J.H.
      • Hole K.
      The formalin test: an evaluation of the method.
      • Lv S.-Y.
      • Yang Y.J.
      • Hong S.
      • Wang N.-B.
      • Qin Y.-J.
      • Li W.-X.
      • Chen Q.
      Intrathecal apelin-13 produced different actions in formalin test and tail-flick test in mice.
      In brief, before testing, the animals were put singly into a transparent glass cylinder (height, 20 cm; diameter, 15 cm). A mirror was mounted at 45 degrees beneath the cylinder, allowing unobstructed observation of the hind paws. After acclimation for 30 minutes, the mice were taken out of the chamber and given an intraplantar injection of 20 μL of 1.0% formalin solution into the dorsal surface of the right hind paw. Thereafter, the animals were immediately put back to the observation glass cylinder and time (seconds) spent licking or biting the formalin-injected paw was counted by the timer for 30 minutes in a blinded manner (B.C. and X.Z.). The mouse hind paw exhibited a biphasic pain response: the first 10 minutes (0 to 10 minutes) was regarded as the acute phase (early phase), and the second 20 minutes (10 to 30 minutes) was known as the longer-lasting tonic phase (late phase).

      Acetic Acid–Induced Writhing Test

      The writhing response induced by acetic acid was used to evaluate visceral pain. This model of pain was based on the methods previously described.
      • Lv S.-Y.
      • Qin Y.-J.
      • Wang N.-B.
      • Yang Y.-J.
      • Chen Q.
      Supraspinal antinociceptive effect of apelin-13 in a mouse visceral pain model.
      • Collier H.O.
      • Dinneen L.C.
      • Johnson C.A.
      • Schneider C.
      The abdominal constriction response and its suppression by analgesic drugs in the mouse.
      Each mouse was intraperitoneally treated with acetic acid (1.0%, 10 mL/kg of body weight), and then the animals were put in single transparent cages immediately. The number of writhes (contractions of the abdominal muscles) was counted for 30 minutes in a blinded manner (X.Z. and Y.Z.). The writhing behavior was defined as outstretching of the hind limbs and extension of the whole body. The antinociception was presented as the reduction of the number of writhes.

      The Radiant Heat Paw Withdrawal Test

      The nociceptive response was assessed by the radiant heat paw withdrawal test using PL-200 radiant heat apparatus (Chengdu Taimeng Technology & Market Corp., Chengdu, China), according to the previous report.
      • Wang C.L.
      • Yang D.J.
      • Yuan B.Y.
      • Wang Y.
      C-terminal hydrazide modification changes the spinal antinociceptive profiles of endomorphins in mice.
      Each mouse was adapted to the testing environment for at least 30 minutes before experiments. The radiant heat source was positioned under the transparent floor directly beneath the hind paw. The light beam focused on the plantar of the hind draw, and the latency for paw withdrawal response against radiant heat stimulation was measured. The intensity of radiant heat was adjusted so that the control latency for paw withdrawal response was 5 to 10 seconds before drug administration. The mice were tested again after i.c.v. administration of drugs at different times. The paw withdrawal latency was then recorded, with a maximum cutoff score of 15 seconds to minimize the tissue damage.

      Total RNA Isolation

      Total RNA was extracted from brain tissue by TRIzol Reagent (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA), following the manufacturer's instruction. In brief, no more than 100 mg of tissue was mixed with 1 mL TRIzol Reagent by a homogenizer (PowerGen 125; Fisher Scientific, Pittsburgh, PA). The amount of the purified RNAs and their quality were assessed by NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE).

      Reverse Transcription and RT-qPCR

      Total RNA (0.5 μg) from each brain was reverse transcribed to synthesize cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). The expression of Galr2, Galr3, adenylate cyclase 1 (Adcy1)∼Adcy9, proopiomelanocortin (Pomc), prodynorphin (Pdyn), proenkephalin (Penk), μ-opioid receptor (Oprm1), κ-opioid receptor (Oprk1), δ-opioid receptor (Oprd1), Gabra1Gabra5, FBJ osteosarcoma oncogene (Fos), Egr1, and Jun1 mRNAs was assessed using quantitative real-time RT-PCR (RT-qPCR). Real-time PCR was performed by the 7500HT thermal cycler and SYBR Green master mix (Applied Biosystems). Primer pairs used are shown in Table 1, which were designed following the previous reports.
      • Chadzinska M.
      • Starowicz K.
      • Scislowska-Czarnecka A.
      • Bilecki W.
      • Pierzchala-Koziec K.
      • Przewlocki R.
      • Przewlocka B.
      • Plytycz B.
      Morphine-induced changes in the activity of proopiomelanocortin and prodynorphin systems in zymosan-induced peritonitis in mice.
      • Wang H.B.
      • Laverghetta A.V.
      • Foehring R.
      • Deng Y.P.
      • Sun Z.
      • Yamamoto K.
      • Lei W.L.
      • Jiao Y.
      • Reiner A.
      Single-cell RT-PCR, in situ hybridization histochemical, and immunohistochemical studies of substance P and enkephalin co-occurrence in striatal projection neurons in rats.
      • Gaveriaux-Ruff C.
      • Nozaki C.
      • Nadal X.
      • Hever X.C.
      • Weibel R.
      • Matifas A.
      • Reiss D.
      • Filliol D.
      • Nassar M.A.
      • Wood J.N.
      Genetic ablation of delta opioid receptors in nociceptive sensory neurons increases chronic pain and abolishes opioid analgesia.
      • Gabrilovac J.
      • Čupić B.
      • Zapletal E.
      • Brozovic A.
      IFN-γ up-regulates kappa opioid receptors (KOR) on murine macrophage cell line J774.
      • Hashikawa-Hobara N.
      • Ogawa T.
      • Sakamoto Y.
      • Matsuo Y.
      • Ogawa M.
      • Zamami Y.
      • Hashikawa N.
      Calcitonin gene-related peptide pre-administration acts as a novel antidepressant in stressed mice.
      • Simpson D.A.
      • Feeney S.
      • Boyle C.
      • Stitt A.W.
      Technical brief: retinal VEGF mRNA measured by SYBR green I fluorescence: a versatile approach to quantitative PCR.
      All samples were analyzed in duplicate, measuring both the gene of interest and 36B4 as an internal control. After each RT-qPCR, the dissociation curve analysis was performed. The normalized expression of the target genes was analyzed by the 2−ΔΔCt method,
      • Livak K.J.
      • Schmittgen T.D.
      Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method.
      where ΔΔCt = (Ct,Target − Ct,36B4)drug − (Ct,Target − Ct,36B4)control.
      Table 1Primer Sequence Used for RT-qPCR
      Primer namePrimer sequenceSize, bp
      Galr2-F5′-TGGACGATTGGGTGTTTGCG-3′130
      Galr2-R5′-AGCGGGTAGCGGATGGC-3′
      Galr3-F5′-GGCCGTCTCAGTGGATAGGT-3′137
      Galr3-R5′-AGCTTAGGTAGGGCGCGGA-3′
      Adcy1-F5′-CCGGAACATGGACCTCTACTAC-3′284
      Adcy1-R5′-ATAGGTGGGAGGAGATGGACTG-3′
      Adcy2-F5′-CCTGGGACCAGGTGTCATTC-3′412
      Adcy2-R5′-CCTGCTTTGGGTCCCTGTAG-3′
      Adcy3-F5′-TACTTCAAAAGGCAGCGCCA-3′482
      Adcy3-R5′-TTGGCCAGGATCTCCCTCAG-3′
      Adcy4-F5′-TTGACCCAAAGCGGGCAG-3′248
      Adcy4-R5′-GCACACAGCACAGTTGTCAG-3′
      Adcy5-F5′-ACTTGGCCATCTCTCTGCAC-3′445
      Adcy5-R5′-TGATTCTCCGCAGCCAACTT-3′
      Adcy6-F5′-GCGGTGAGGGAGAATCACTG-3′163
      Adcy6-R5′-TCACACCTGTTACCTCACGC-3′
      Adcy7-F5′-GCAGGTAACAGGGTCGGAG-3′392
      Adcy7-R5′-AGGTCCTCAGCTCTTTGCAC-3′
      Adcy8-F5′-TTGCGGAGTGGCGATAAGTT-3′482
      Adcy8-R5′-ACAAAGTACTCTGGGTAGGAGC-3′
      Adcy9-F5′-AAGACCAGCACCAAGGCTTC-3′183
      Adcy9-R5′-GTTCTTGAACCTGAGCGGGA-3′
      Pomc-F5′-AGATTCAAGAGGGAGCTGGA-3′159
      Pomc-R5′-CTTCTCGGAGGTCATGAAGC-3′
      Pdyn-F5′-CGGAACTCCTCTTGGGGTAT-3′154
      Pdyn-R5′-TTTGGCAACGGAAAAGAATC-3′
      Penk-F5′-AACAGGATGAGAGCCACTTGC-3′474
      Penk-R5′-CTTCATCGGAGGGCAGAGACT-3′
      Oprm1-F5′-ATCCTCTCTTCTGCCATTGGT-3′127
      Oprm1-R5′-TGAAGGCGAAGATGAAGACA-3′
      Oprd1-F5′-AAGTACTTGGCGCTCTGGAA-3′125
      Oprd1-R5′-GCTCGTCATGTTTGGCATC-3′
      Oprk1-F5′-CCGATACACGAAGATGAAGAC-3′341
      Oprk1-R5′-GTGCCTCCAAGGACTATCGC-3′
      Gabra1-F5′-ATGGGGATTAGGGCCAGAGT-3′432
      Gabra1-R5′-TTGCACAGCTCGGGGC-3′
      Gabra2-F5′-CCACGACTCCTCAAGCTCTC-3′322
      Gabra2-R5′-TTAGCCAGCACCAACCTGAC-3′
      Gabra3-F5′-GTGACACTCGATCTCACAGGT-3′133
      Gabra3-R5′-CTTGGCTAGTGGTTCCAGGG-3′
      Gabra4-F5′-AAGCTTGACAAGGTGCGGAG-3′499
      Gabra4-R5′-TCTGTAACAGGACCCCCAAAT-3′
      Gabra5-F5′-CGCGTAGGCGTCAAGATCAA-3′340
      Gabra5-R5′-TGGGCCAAAGCTGGTAACAT-3′
      Fos-F5′-GGTGAAGACCGTGTCAGGAGG-CAG-3′117
      Fos-R5′-GCCATCTTATTCCGTTCCCTT-CGG-3′
      Jun-F5′-CTCATACCAGTTCGCACAGGCGGC-3′298
      Jun-R5′-CCGCTAGCACTCACGTTGGTA-GCG-3′
      Egr1-F5′-GAGCACCTGACCACAGAGTC-3′172
      Egr1-R5′-AAAGGGGTTCAGGCCACAAA-3′
      36B4-F5′-CGACCTGGAAGTCCAACTAC-3′109
      36B4-R5′-ATCTGCTGCATCTGCTTG-3′
      F, forward; R, reverse; RT-qPCR, real-time quantitative RT-PCR.

      PCR Array

      Pathway-focused gene expression profiling was performed through a 96-well mouse PCR array, RT2 Profiler PCR Array (PAMM-075Z, Mouse Transcription Factors; Qiagen, Hilden, Germany). This RT2 Profiler PCR Array contains a total of 96 wells with 84 transcription factor genes and other internal and technical controls (positive, negative, and DNA contamination controls). All of the 84 genes in the array participate in various processes of transcription factor signaling. The RT2 Profiler PCR Array and data analysis were done following the manufacturer's instruction (Qiagen).

      ELISA

      Mice in each group were sacrificed under ether anesthesia, and their brains were immediately taken out after 15 minutes of i.c.v. injection with spexin. Thereafter, each mouse brain was weighed, homogenized with 10% w/v phosphate buffer (0.1 mol/L, pH 7.4), and centrifuged for 6714 × g at 4°C for 15 minutes. Then, the supernatants obtained were used for ELISA. The mouse dynorphin was quantified using the Mouse Dynorphin ELISA Kit (Shanghai Fusheng Shiye Co Ltd, Shanghai, China), following the manufacturer's instruction.

      Western Blot Analysis

      The mouse brain tissue samples were homogenized with radioimmunoprecipitation assay lysis buffer containing protease inhibitor. Then, the homogenate was centrifuged at 14,000 × g for 10 minutes at 4°C to obtain the tissue protein. The quantitation of protein concentration was measured by bicinchoninic acid method. Equivalent amounts of protein were loaded into each lane of a gel, resolved by SDS-PAGE gel, and then blotted onto polyvinylidene difluoride membranes. After blocking with 5% milk, the blots were incubated with the first antibodies, κ-opioid receptor (KOR; 1:1000; Abcam, Cambridge, MA) or β-actin (1:1000; Beyotime, Shanghai, China), at 4°C overnight. Then, the blots were incubated with the proper horseradish peroxidase–labeled secondary antibodies for 1 hour. The membrane was washed, and the immunoreactive proteins were visualized by electrochemiluminescence.

      Preparation of Brain Sections and c-Fos Immunohistochemistry

      Fifteen minutes after i.c.v. injection with spexin or normal saline, mice were anesthetized with 100 mg/kg (intraperitoneally) of pentobarbital sodium, then perfused transcardially with 25 mL normal saline, followed by 25 mL of 4% paraformaldehyde in phosphate-buffered saline. Brains were taken out and fixed in fixation fluid overnight at 4°C and then embedded in paraffin. Coronal sections (5 μm thick) of each example were stained by immunohistochemistry, according to the previous reports.
      • Singewald N.
      • Salchner P.
      • Sharp T.
      Induction of c-Fos expression in specific areas of the fear circuitry in rat forebrain by anxiogenic drugs.
      • Saito Y.
      • Miyasaka T.
      • Hatsuta H.
      • Takahashi-Niki K.
      • Hayashi K.
      • Mita Y.
      • Kusano-Arai O.
      • Iwanari H.
      • Ariga H.
      • Hamakubo T.
      • Yoshida Y.
      • Niki E.
      • Murayama S.
      • Ihara Y.
      • Noguchi N.
      Immunostaining of oxidized DJ-1 in human and mouse brains.
      After deparaffinization, the sections were incubated with 10% normal goat serum and incubated 12 hours with the polyclonal anti–c FBJ osteosarcoma oncogene (c-Fos) antibody (1:100; Abcam Inc., Burlingame, CA). The sections were incubated for 2 hours with the biotinylated secondary antibody and then incubated for 30 minutes at room temperature with the avidin-biotin-peroxidase complex (Corning Inc., Corning, NY). The sections of the examples were incubated with 3,3-diaminobenzidine containing hydrogen peroxide (ZSGB-bio, Beijing, China) to examine the antibody binding. The sections were then lightly counterstained with hematoxylin. To analyze the results, the periaqueductal gray (PAG) was divided into four areas, including dorsomedial, dorsolateral, lateral, and ventrolateral parts. Three sections (at -4.1, -4.5, and -4.9 mm from bregma) in rostral, intermediate, and caudal PAG, respectively, were determined in each brain. The number of c-Fos–like immunoreactive (FLI) neurons was calculated using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD) in a blinded manner (Y.Z.).

      Experimental Design

      To study the role of spexin in tonic inflammatory pain, spexin (0.081, 0.81, and 2.43 mg/kg) or galanin (0.32 mg/kg) was intracerebroventricularly administrated at 5 minutes before the formalin injection. After that, the formalin-induced pain behavior was detected. In addition, to compare the analgesic effect of spexin with nonamidated spexin, the nonamidated spexin (2.43 mg/kg) was intracerebroventricularly administrated with the identical protocol. To evaluate the analgesic effect of spexin after the induction of the pain state, spexin (0.81 and 2.43 mg/kg) was intracerebroventricularly injected at 5 minutes after formalin treatment.
      To further explore the mechanism of the antinociception caused by spexin, the GALR2 antagonist M871 (0.11 mg/kg) and GALR3 antagonist SNAP 37889 (0.018 mg/kg) were co-administrated with 2.43 mg/kg spexin (ie, each antagonist was mixed with spexin and given in one i.c.v. administration of 4 μL). And the Galr2, Galr3, and Adcy1Adcy9 mRNAs were determined by RT-qPCR. The opioid receptor antagonist naloxone (0.2 mg/kg) and γ-aminobutyric acid A (GABAA) receptor antagonist bicuculline (20 μg/kg) were co-administered with spexin (2.43 mg/kg). The doses of these antagonists were used following the previous reports.
      • Lv S.-Y.
      • Qin Y.-J.
      • Wang N.-B.
      • Yang Y.-J.
      • Chen Q.
      Supraspinal antinociceptive effect of apelin-13 in a mouse visceral pain model.
      • Mahmoudi M.
      • Zarrindast M.-R.
      Effect of intracerebroventricular injection of GABA receptor agents on morphine-induced antinociception in the formalin test.
      • de Souza M.M.
      • Silote G.P.
      • Herbst L.S.
      • Funck V.R.
      • Joca S.R.L.
      • Beijamini V.
      The antidepressant-like effect of galanin in the dorsal raphe nucleus of rats involves GAL2 receptors.
      • Swanson C.J.
      • Blackburn T.P.
      • Zhang X.
      • Zheng K.
      • Xu Z.-Q.D.
      • Hokfelt T.
      • Wolinsky T.D.
      • Konkel M.J.
      • Chen H.
      • Zhong H.
      • Walker M.W.
      • Craig D.A.
      • Gerald C.P.G.
      • Branchek T.A.
      Anxiolytic- and antidepressant-like profiles of the galanin-3 receptor (Gal(3)) antagonists SNAP 37889 and SNAP 398299.
      To investigate which subtypes of opioid peptide(s), opioid receptor(s), or transcription factor(s) were involved in the antinociceptive effect of spexin in the formalin test, after 15 minutes of administration with spexin or normal saline (control), brains were quickly removed, frozen in liquid nitrogen, and preserved at −80°C for further studies (RT-qPCR, PCR array, ELISA, or Western blot analysis). And the related mRNA levels were determined. The κ-opioid receptor antagonist nor-binaltorphimine dihydrochloride (73.47 μg/kg) was co-administered with spexin (2.43 mg/kg), and the nociceptive response was evaluated. T-5224 (100 mg/kg), the inhibitor of c-Fos/activator protein 1 (AP-1), was administered orally to block the Fos activity, and then the Pdyn and Oprk1 mRNA levels were detected. The doses of the antagonists were chosen following the previous reports.
      • Lv S.-Y.
      • Qin Y.-J.
      • Wang H.-T.
      • Xu N.
      • Yang Y.-J.
      • Chen Q.
      Centrally administered apelin-13 induces depression-like behavior in mice.
      • Miyazaki H.
      • Morishita J.
      • Ueki M.
      • Nishina K.
      • Shiozawa S.
      • Maekawa N.
      The effects of a selective inhibitor of c-Fos/activator protein-1 on endotoxin-induced acute kidney injury in mice.
      To further determine the sites of the up-regulated Fos induced by spexin in the mouse brain, the c-Fos neuronal activity in the PAG, the key component of descending inhibitory pain circuitry, was detected by immunohistochemistry method.
      To further study whether spexin could produce an antinociceptive effect against visceral pain, spexin (0.081, 0.81, and 2.43 mg/kg) or galanin (0.32 mg/kg) was intracerebroventricularly administrated at 5 minutes before acetic acid injection. The brain tissue was quickly collected at 15 minutes after spexin treatment, then frozen in liquid nitrogen, and stored at −80°C for further gene expression analysis.

      Statistical Analysis

      Statistical results were presented as means ± SEM. Analysis was performed by one-way analysis of variance, followed by Dunnett's test for multiple comparisons. The unpaired t-test was used to test the difference between the two groups. P < 0.05 was considered statistically significant.

      Results

      The Effect of I.C.V. Spexin on Nociceptive Response in the Mouse Formalin Test

      I.C.V. spexin induced a dose-related decrease in licking/biting time in the early phase [F(3, 31) = 8.090, P < 0.001] (Figure 1A). Spexin markedly decreased the licking/biting time at the doses of 0.81 and 2.43 mg/kg (P < 0.01 and P < 0.001, respectively), whereas no obvious difference was found at the lower dose of 0.081 mg/kg (P = 0.80) compared with the control group. In the late phase (Figure 1B), spexin only at the dose of 2.43 mg/kg significantly decreased the time spent on licking/biting, but not the doses of 0.081 or 0.81 mg/kg (0.081 mg/kg, P = 0.97; 0.81 mg/kg, P = 0.36; 2.43 mg/kg, P < 0.05), compared with the control group. The nonselective galanin receptor agonist galanin (0.32 mg/kg), as a positive control, exhibited a significant reduction in licking/biting time in both early (P < 0.05) and late (P < 0.01) phases. The antinociceptive effect of 0.32 mg/kg of galanin is equivalent to 2.43 mg/kg of spexin (Figure 1, A and B).
      Figure thumbnail gr1
      Figure 1The antinociceptive effect of spexin in the formalin test in mice. A and B: Normal saline (control), spexin (0.081, 0.81, and 2.43 mg/kg), or galanin (GAL; 0.32 mg/kg) was intracerebroventricularly administered to mice at 5 minutes before the intraplantar (i.pl.) injection of formalin. C and D: Normal saline or nonamidated spexin (2.43 mg/kg) was intracerebroventricularly administered to mice at 5 minutes before the i.pl. injection of formalin. Antinociception was recorded at 0 to 10 minutes (early phase) and 10 to 30 minutes (late phase) after formalin injection. E and F: Normal saline or spexin (0.81 and 2.43 mg/kg) was intracerebroventricularly administered at 5 minutes after formalin treatment. Each point is the mean licking/biting time. The unpaired t-test was performed to test the difference between nonamidated spexin and the control group. Data are expressed as means ± SEM (AF). n = 7 to 10 per group (A–F). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus control.
      To further determine whether the C-terminal amidation is necessary for spexin to exert its activity, the nonamidated spexin was used. I.C.V. nonamidated spexin (2.43 mg/kg) had no influence on paw licking/biting time in either the early phase (P = 0.30) or the late phase (P = 0.12) (Figure 1, C and D). Spexin at 2.43 mg/kg produced an analgesic effect in the late phase (P < 0.01) (Figure 1F), not the early phase (P > 0.05) (Figure 1E), after the induction of the pain state in the formalin test.
      The nociceptive response was also assessed by the radiant heat paw withdrawal test. Compared with the control group, i.c.v. administration of spexin (0.81 mg/kg) significantly increased the paw withdrawal latency at 15 minutes after treatment in the radiant heat paw withdrawal test (P < 0.05), although it did not reach a statistically significant level at 30 or 45 minutes after injection. The result indicates that i.c.v. spexin (0.81 mg/kg) produced an antinociceptive effect in the radiant heat paw withdrawal test in mice (Supplemental Figure S1).

      The Effect of I.C.V. Spexin on GALR2/3

      A previous report shows that spexin activates GALR2/3.
      • Kim D.-K.
      • Yun S.
      • Son G.H.
      • Hwang J.-I.
      • Park C.R.
      • Kim J.I.
      • Kim K.
      • Vaudry H.
      • Seong J.Y.
      Coevolution of the spexin/galanin/kisspeptin family: spexin activates galanin receptor type II and III.
      To identify which type of galanin receptor mediated the antinociception of spexin in the formalin test, the GALR2 antagonist M871 and the GALR3 antagonist SNAP 37889 were used, and the Galr2 and Galr3 gene expression in the whole brain was detected at 15 minutes after spexin injection. M871 (0.11 mg/kg) could not block the decreasing licking/biting time caused by spexin in either the early phase (Figure 2A) or the late phase (Figure 2B) of the formalin test. However, SNAP 37889 (0.018 mg/kg) significantly antagonized the antinociceptive effect of spexin in the early phase (P < 0.01) (Figure 2C) and late phase (P < 0.05) (Figure 2D) of the formalin test. I.C.V. spexin significantly increased the mRNA level of Galr3, but not Galr2 (Figure 2E). These results indicate that Galr3 may be involved in the antinociception of spexin. The gene expression of Adcy (from type 1 to type 9), as GALR2/3's downstream signal, was also analyzed. Only Adcy4 mRNA levels were markedly increased (Figure 2F).
      Figure thumbnail gr2
      Figure 2The effects of GALR2/3 antagonists on the antinociception of spexin and the influence of spexin on Galr2, Galr3, and Adcy1Adcy9 gene expression. The effects of GALR2 antagonist M871 (0.11 mg/kg; A and B) and GALR3 antagonist SNAP 37889 (0.018 mg/kg; C and D) on the antinociception induced by spexin (2.43 mg/kg) in the formalin test. Formalin was injected at 5 minutes after spexin or normal saline treatment. Data are presented as mean licking/biting time. Histogram showing the relative mRNA levels of the Galr2 and Galr3 (E) and Adcy1, Adcy2, Adcy3, Adcy4, Adcy5, Adcy6, Adcy7, Adcy8, and Adcy9 (F) normalized with the housekeeping gene 36B4 mRNA expression. The gene expression was detected at 15 minutes after spexin (2.43 mg/kg) or normal saline (control) treatment. Formalin was injected at 5 minutes after spexin or normal saline administration. Data are expressed as means ± SEM (A–F). n = 9 to 10 per group (A–D); n = 6 to 8 per group (E and F). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

      The Effects of Naloxone and Bicuculline on the Antinociception of Spexin in the Formalin Test

      To explore whether the opioid receptor was involved in the antinociceptive effect of spexin, the opioid receptor antagonist naloxone was selected. Compared with the control group, i.c.v. naloxone (0.2 mg/kg) alone did not influence the nociceptive behavior induced by formalin in either the early phase (P = 0.94) or the late phase (P = 0.79) (Figure 3, A and B ). However, i.c.v. co-administration of naloxone significantly reversed the antinociception of spexin in the early phase (Figure 3A) and late phase (Figure 3B).
      Figure thumbnail gr3
      Figure 3The effect of i.c.v. administration of naloxone (NLX) and bicuculline (Bic) on spexin-induced antinociception in the mouse formalin test. The opioid receptor antagonist naloxone (0.2 mg/kg; A and B) or GABAA receptor antagonist bicuculline (20 μg/kg; C and D) was co-administrated (intracerebroventricularly) with spexin (2.43 mg/kg). Antinociception was recorded at 0 to 10 minutes (early phase) and 10 to 30 minutes (late phase) after formalin injection. Data are presented as mean licking/biting time. Data are expressed as means ± SEM (A–D). n = 7 to 10 per group (A–D). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
      To ascertain whether the antinociceptive effect of spexin was related to GABAA receptor, bicuculline was used. The licking/biting responses in the two phases were not affected by i.c.v. administration of bicuculline compared with the control group (Figure 3, C and D). I.C.V. bicuculline (20 μg/kg) failed to block the antinociceptive response induced by 2.43 mg/kg of spexin in the early phase (Figure 3C) and late phase (Figure 3D) in the mouse formalin test.

      The mRNA and Protein Levels of the Endogenous Opioid Peptides and Opioid Receptors after Spexin Administration in Mice

      To understand which kind of endogenous opioid peptides were involved in the analgesia of spexin, the mRNA levels of the endogenous ligand for Oprm1 Pomc, the endogenous ligand for Oprd1 Penk, and the endogenous ligand for Oprk1 Pdyn were determined. Spexin did not affect the Pomc or Penk mRNA levels compared with the control group (Figure 4A). However, a significant increase in Pdyn mRNA level was demonstrated in the spexin-treated group (Figure 4A).
      Figure thumbnail gr4
      Figure 4The gene and protein expression levels of endogenous opioid peptides and opioid receptors in the mouse brain after administration of spexin and the effect of KOR antagonist on the antinociception of spexin. A and B: Histogram showing the relative mRNA expression levels of Pomc, Penk, Pdyn, Oprm1, Oprd1, and Oprk1 normalized with the housekeeping gene 36B4 mRNA expression using real-time PCR. C: The concentration of dynorphin in brain was determined using enzyme-linked immunosorbent assay. D and E: Western blot analysis and densitometric determination of the protein levels of KOR. The brain tissues were removed at 15 minutes after spexin (2.43 mg/kg) or normal saline (control) treatment. Formalin was injected at 5 minutes after spexin or normal saline administration. F and G: The effect of KOR antagonist nor-binaltorphimine dihydrochloride (nor-BNI; 73.47 μg/kg) on the antinociception caused by spexin (2.43 mg/kg) in the formalin test. Formalin was injected at 5 minutes after spexin or normal saline treatment. Data are presented as mean of licking/biting time. Data are expressed as means ± SEM (A–C and E–G). n = 4 to 6 per group (A–C and E); n = 9 to 10 per group (F and G). P < 0.05, ∗∗∗P < 0.001 versus control; P < 0.05, ††P < 0.01.
      Furthermore, it was examined which subtype of opioid receptors was involved in the analgesic effect of spexin. The mRNA levels of three classic subtypes of opioid receptors, including Oprm1, Oprd1, and Oprk1, were detected. The expression levels of Oprm1 or Oprd1 mRNA did not change markedly compared with the control group (Figure 4B). However, the Oprk1 mRNA levels significantly increased (Figure 4B), which is consistent with the previous result (Figure 4A) that the mRNA levels of Pdyn, the endogenous ligand for KOR, were up-regulated by central spexin.
      The ELISA test result shows that the level of dynorphin was increased compared with the control group (Figure 4C). Western blot analysis demonstrates that the protein level of KOR was up-regulated compared with the control group (Figure 4, D and E). The KOR antagonist nor-binaltorphimine dihydrochloride (73.47 μg/kg) blocked the analgesic response of spexin (2.43 mg/kg) in the early phase (P < 0.01) (Figure 4F) and late phase (P < 0.01) (Figure 4G) of the formalin test. The μ-opioid receptor (MOR) antagonist β-funaltrexamine hydrochloride (49 μg/kg) could not reverse the antinociception of spexin (2.43 mg/kg) in either the early or the late phase of the formalin test (Supplemental Figure S2).

      Transcription Factor–Linked Gene Assay and Real-Time PCR Analysis

      To further explore the mechanism by which spexin up-regulates PDYN and KOR expression, a Mouse Transcription Factors RT2 Profiler PCR Array was performed to determine which genes in transcription factors were regulated by spexin. The results show that Fos gene expression was increased, with approximately 2.3-fold in brain after spexin treatment (Figure 5). Moreover, the result from real-time PCR showed that spexin significantly up-regulated the Fos gene expression (Figure 6A), which was consistent with the result of PCR array. Spexin did not change the expression of Jun or Egr1 (Figure 6A). T-5224 (100 mg/kg), an inhibitor of c-Fos/AP-1, obviously blocked the up-regulated Pdyn and Oprk1 mRNA levels caused by 2.43 mg/kg of spexin (Figure 6, B and C). This suggests that spexin may induce Fos expression, resulting in stimulation of opioid signaling.
      Figure thumbnail gr5
      Figure 5Gene analysis using transcription factor PCR array. A: Spexin treatment increases Fos expression in mouse brain. Mice were treated with spexin (2.43 mg/kg). After 5 minutes, mice were intraplantarly injected with formalin. After 10 minutes of injection, total RNAs were prepared for PCR array analysis. B: Scatter plot between spexin-treated group and nontreated control group. n = 3 per group (A).
      Figure thumbnail gr6
      Figure 6Quantitative determination of the effects of spexin on the mRNA levels of Fos, Jun, and Egr1 and the effects of the T-5224 on the increased Pdyn and Oprk1 mRNA levels induced by spexin. Histogram showing the relative mRNA expression levels of Fos, Jun, and Egr1 (A), Pdyn (B), and Oprk1 (C) normalized with the housekeeping gene 36B4 mRNA expression. The gene expression levels were detected at 15 minutes after spexin (2.43 mg/kg) or normal saline (control) treatment. T-5224 (100 mg/kg), the inhibitor of c-Fos/AP-1, was administered orally at 2 hours before spexin treatment. Formalin was injected at 5 minutes after spexin or normal saline administration. Data are expressed as means ± SEM (AC). n = 4 to 6 per group (A–C). P < 0.05.

      Effect of Spexin on c-Fos Protein Expression in the PAG

      The number of FLI neurons was calculated in up to three sections, which include rostral, intermediate, and caudal parts. Each part contains dorsomedial, dorsolateral, lateral, and ventrolateral columns of PAG. The numbers of c-Fos–positive cells were compared within each subdivision at each bregma level. Spexin at 2.43 mg/kg markedly increased the number of FLI neurons in the caudal dorsomedial PAG (P < 0.05), the caudal lateral PAG (P < 0.05), and caudal ventrolateral PAG (P < 0.05), compared with saline control (Figure 7, A–D ). Figure 7, E and F, illustrate the examples of FLI neurons in the caudal PAG (at -4.9 mm from bregma).
      Figure thumbnail gr7
      Figure 7The effect of spexin on c-Fos protein expression in the periaqueductal gray (PAG) in mice. A–D: Comparison of the number of c-Fos–like immunoreactive (FLI) cells between the two groups (normal saline and 2.43 mg/kg spexin) within the four columns of the PAG dorsomedial (A), dorsolateral (B), lateral (C), and ventrolateral (D) areas. The three sections are in the rostral, the intermediate, and the caudal PAG, which are correspondingly located at bregma -4.1, -4.5, and -4.9 mm. Formalin was treated at 5 minutes after spexin injection. The values are expressed as the mean number of FLI neurons. E and F: Photomicrographs showing c-Fos–labeled neurons in the PAG (at bregma -4.9 mm) in representative sections from the two groups separately intracerebroventricularly injected with normal saline (E) and 2.43 mg/kg spexin (F). Data are expressed as means ± SEM (A–D). n = 7 to 8 per group (A–D). P < 0.05. Scale bars = 200 μm (E and F).

      The Role of Central Spexin on Nociceptive Response in the Acetic Acid–Induced Writhing Test

      I.C.V. spexin dose relatedly decreased the number of writhes [F(3, 34) = 4.578, P < 0.01] (Figure 8A). The decrease on the number of writhes caused by i.c.v. spexin reached statistical significance at the doses of 0.81 and 2.43 mg/kg (P < 0.05 and P < 0.01, respectively). Galanin (0.32 mg/kg, the positive control) markedly reduced the number of writhes.
      Figure thumbnail gr8
      Figure 8The antinociceptive effects of spexin on the acetic acid–induced mouse visceral pain model and the related gene expression analysis. A: The effects of i.c.v. administration of spexin (0.081, 0.81, and 2.43 mg/kg) and galanin (GAL; 0.32 mg/kg) on the number of writhes in the acetic acid–induced writhing test. Normal saline (control), spexin, or GAL was administrated at 5 minutes before i.p. injection of acetic acid. B and C: Quantitative analysis of Galr2, Galr3, Adcy1, Adcy2, Adcy3, Adcy4, Adcy5, Adcy6, Adcy7, Adcy8, and Adcy9 mRNA expression in the brain of mice at 15 minutes after spexin or normal saline treatment. Acetic acid was injected at 5 minutes after spexin administration. D and E: Quantitative analysis of Pomc, Penk, Pdyn, Oprm1, Oprd1, and Oprk1 mRNA expression in brain of mice at 15 minutes after spexin or normal saline treatment. Acetic acid was injected at 5 minutes after spexin administration. F: Quantitative analysis of Gabra1, Gabra2, Gabra3, Gabra4, and Gabra5 mRNA expression in brain of mice at 15 minutes after spexin or normal saline treatment. Acetic acid was injected at 5 minutes after spexin administration. G: Quantitative analysis of Fos, Jun, and Egr1 mRNA expression in brain of mice at 15 minutes after spexin or normal saline treatment. Acetic acid was injected at 5 minutes after spexin administration. Data are expressed as means ± SEM (A–G). n = 8 to 10 animals per group (A); n = 4 to 6 in each group (B–E); n = 6 in each group (F); n = 5 to 6 in each group (G). P < 0.05, ∗∗P < 0.01 versus control.
      In the writhing test, i.c.v. spexin up-regulated the Galr2 mRNA levels, but not Galr3 (Figure 8B). All of the nine types of Adcy mRNA were detected, and Adcy1 gene levels were obviously increased (Figure 8C). To understand whether opioid receptor or GABAA was involved in the antinociception of spexin in the acetic acid–induced visceral pain model, the mRNA levels of endogenous opioid peptides (Pomc, Penk, and Pdyn), opioid receptors (Oprm1, Oprk1, and Oprd1), and GABAA receptors (Gabra1, Gabra2, Gabra3, Gabra4, and Gabra5) were determined. The mRNA levels of Pomc and Oprm1 were markedly elevated (Figure 8, D and E), whereas the mRNA levels of each GABAA receptor were not changed (Figure 8F). To determine which signal molecule was involved in the effect of spexin on POMC/MOR, the Fos, Jun, and Egr1 gene expression levels were measured. The Fos mRNA levels were markedly increased (Figure 8G).

      Discussion

      This study indicates that spexin produced an analgesic effect in the tonic inflammatory pain mouse model using the formalin test and in the visceral pain model using the acetic acid–induced writhing test at the supraspinal level. These results demonstrate that i.c.v. spexin enhanced Galr3 and Adcy4 gene expression and up-regulated the mRNA and protein levels of dynorphin and KOR, which were mediated by Fos (especially in PAG), thereby producing an inhibitory effect against inflammatory pain in the tonic inflammatory pain mouse model. However, in the visceral pain model, the antinociception of spexin was mediated by stimulating Galr2 and Adcy1 gene expression and then up-regulating the mRNA levels of Pomc and Oprm1.
      The formalin test is considered as a valid model of persistent pain and is commonly used to evaluate potential analgesic compounds.
      • Bannon A.W.
      • Malmberg A.B.
      Models of nociception: hot-plate, tail-flick, and formalin tests in rodents.
      In this study, a mouse injected with 1.0% formalin in the paw displays a nociceptive behavior with a biphasic characteristic, as shown in the previous reports.
      • Le Bars D.
      • Gozariu M.
      • Cadden S.W.
      Animal models of nociception.
      • Spooner M.-F.
      • Robichaud P.
      • Carrier J.
      • Marchand S.
      Endogenous pain modulation during the formalin test in estrogen receptor beta knockout mice.
      This result indicates that i.c.v. administration of 0.81 and 2.43 mg/kg spexin significantly decreased the licking/biting time in the early phase, whereas i.c.v. spexin at the dose of 2.43 mg/kg significantly reduced the licking/biting time in the late phase. These results were consistent with the previous report that central spexin produced an analgesic effect in the tail-flick test.
      • Toll L.
      • Khroyan T.V.
      • Sonmez K.
      • Ozawa A.
      • Lindberg I.
      • McLaughlin J.P.
      • Eans S.O.
      • Shahien A.A.
      • Kapusta D.R.
      Peptides derived from the prohormone proNPQ/spexin are potent central modulators of cardiovascular and renal function and nociception.
      The tail-flick test is an acute pain model using a short-lasting thermal stimuli, whereas the formalin test is a tonic pain model with a continuous chemical stimuli.
      • Le Bars D.
      • Gozariu M.
      • Cadden S.W.
      Animal models of nociception.
      The consistent analgesic effects of spexin in both models indicate that spexin is a potent central modulator of pain at the supraspinal level.
      • Porzionato A.
      • Rucinski M.
      • Macchi V.
      • Stecco C.
      • Malendowicz L.K.
      • De Caro R.
      Spexin expression in normal rat tissues.
      • Basbaum A.I.
      • Bráz J.M.
      • kruger L.
      • Light A.
      Transgenic mouse models for the tracing of “pain” pathways.
      The C-terminal residue of spexin is aminated. Our results show that i.c.v. nonamidated spexin could not markedly influence the licking/biting response in the formalin test. In addition, the molecular form of spexin that exerts its effect on obesity,
      • Gu L.
      • Ma Y.
      • Gu M.
      • Zhang Y.
      • Yan S.
      • Li N.
      • Wang Y.
      • Ding X.
      • Yin J.
      • Fan N.
      Spexin peptide is expressed in human endocrine and epithelial tissues and reduced after glucose load in type 2 diabetes.
      bowel movement in mice,
      • Lin C.-Y.
      • Zhang M.
      • Huang T.
      • Yang L.-L.
      • Fu H.-B.
      • Zhao L.
      • Zhong L.L.
      • Mu H.-X.
      • Shi X.-K.
      • Leung C.F.
      Spexin enhances bowel movement through activating L-type voltage-dependent calcium channel via galanin receptor 2 in mice.
      and cardiovascular and renal function
      • Toll L.
      • Khroyan T.V.
      • Sonmez K.
      • Ozawa A.
      • Lindberg I.
      • McLaughlin J.P.
      • Eans S.O.
      • Shahien A.A.
      • Kapusta D.R.
      Peptides derived from the prohormone proNPQ/spexin are potent central modulators of cardiovascular and renal function and nociception.
      is amidated spexin. The results were consistent with the previous study in which most of the prepropeptides were processed through modification after translation, and >50% of the peptides underwent C-terminal amidation.
      • Eipper B.A.
      • Stoffers D.A.
      • Mains R.E.
      The biosynthesis of neuropeptides: peptide alpha-amidation.
      Spexin could activate GALR2/3 in the ligand-receptor interaction study, indicating that spexin is a natural ligand for GALR2/3.
      • Kim D.-K.
      • Yun S.
      • Son G.H.
      • Hwang J.-I.
      • Park C.R.
      • Kim J.I.
      • Kim K.
      • Vaudry H.
      • Seong J.Y.
      Coevolution of the spexin/galanin/kisspeptin family: spexin activates galanin receptor type II and III.
      Our result shows that the antinociceptive response induced by spexin was blocked by GALR3 antagonist SNAP 37889, but not M871, in the mouse formalin test. The real-time PCR result shows that i.c.v. spexin increased Galr3 mRNA expression, but not Galr2. ADCY was GALR2/3's downstream signaling molecule.
      • Lang R.
      • Gundlach A.L.
      • Holmes F.E.
      • Hobson S.A.
      • Wynick D.
      • Hoekfelt T.
      • Kofler B.
      Physiology, signaling, and pharmacology of galanin peptides and receptors: three decades of emerging diversity.
      The Adcy4 mRNA level was increased. These results indicate that central spexin produces an antinociceptive effect by activating GALR3/ADCY4 in the formalin test.
      Opioid receptors are involved in controlling many regions of the nervous system.
      • Dirksen R.
      Opioid receptors and pain.
      Our result shows that naloxone, a classic opioid receptor antagonist, could significantly block the analgesia induced by i.c.v. spexin in both early and late phases in the formalin test. It suggests the involvement of opioid receptor in the analgesia of spexin. However, Toll et al
      • Toll L.
      • Khroyan T.V.
      • Sonmez K.
      • Ozawa A.
      • Lindberg I.
      • McLaughlin J.P.
      • Eans S.O.
      • Shahien A.A.
      • Kapusta D.R.
      Peptides derived from the prohormone proNPQ/spexin are potent central modulators of cardiovascular and renal function and nociception.
      show that naloxone could not antagonize the antinociception of spexin in the tail-flick assay. The tail-flick test is an acute pain test, whereas the formalin test is a persistent pain model.
      • Bannon A.W.
      • Malmberg A.B.
      Models of nociception: hot-plate, tail-flick, and formalin tests in rodents.
      The neurotransmitters and neuromodulators mediating pain responses in each of these models may differ.
      • Bannon A.W.
      • Malmberg A.B.
      Models of nociception: hot-plate, tail-flick, and formalin tests in rodents.
      The inconsistent effects of naloxone may be due to the different mechanisms of the two classes of pain models.
      • Le Bars D.
      • Gozariu M.
      • Cadden S.W.
      Animal models of nociception.
      In addition, it is well known that GABAA receptor participates in pain control in the CNS, essentially through the descending inhibitory system.
      • Mahmoudi M.
      • Zarrindast M.-R.
      Effect of intracerebroventricular injection of GABA receptor agents on morphine-induced antinociception in the formalin test.
      • Millan M.J.
      The induction of pain: an integrative review.
      The GABAA receptor antagonist bicuculline failed to antagonize the antinociceptive effect of spexin, indicating that GABAA receptor is not related to the antinociception of spexin.
      The classic opioid receptor comprises three receptor subtypes: MOR, δ-opioid receptor (DOR), and KOR. Our result shows that spexin did not affect the Pomc, Penk, Oprm1, or Oprd1 mRNA levels, whereas spexin significantly increased the mRNA levels of Pdyn and Oprk1. The ELISA and Western blot analysis show that the protein levels of dynorphin and KOR were increased. The synchronous changes of mRNA and protein levels of dynorphin and KOR suggest that the antinociceptive effect induced by i.c.v. spexin was mediated by the dynorphin/KOR system. Moreover, the KOR antagonist nor-binaltorphimine dihydrochloride antagonized the antinociception of spexin in the formalin test, which further supports the involvement of KOR. KOR is involved in antinociceptive activity in a variety of animal pain models.
      • Kivell B.
      • Prisinzano T.E.
      Kappa opioids and the modulation of pain.
      KOR agonists dynorphin A and ICI 199,441 induce an antinociceptive response in the formalin test,
      • Obara I.
      • Parkitna J.R.
      • Korostynski M.
      • Makuch W.
      • Kaminska D.
      • Przewlocka B.
      • Przewlocki R.
      Local peripheral opioid effects and expression of opioid genes in the spinal cord and dorsal root ganglia in neuropathic and inflammatory pain.
      which is in accordance with the result in this study. However, the activation of the dynorphin/KOR system could induce aversive effects, such as depression,
      • Muschamp J.W.
      • Carlezon Jr., W.A.
      Roles of nucleus accumbens CREB and dynorphin in dysregulation of motivation.
      which would limit the effectiveness of spexin-derived therapies in the inflammatory pain state.
      Fos and pain are closely related, and the Fos expression could be considered as a special factor to map out the effect of antinociceptive drugs in the study of pain.
      • Munglani R.
      • Hunt S.P.
      Molecular biology of pain.
      • Sun W.Z.
      • Shyu B.C.
      • Shieh J.Y.
      Nitrous oxide or halothane, or both, fail to suppress c-fos expression in rat spinal cord dorsal horn neurones after subcutaneous formalin.
      Fos influences the transcription of opioid family genes by acting on activation protein-1.
      • Ahmad A.H.
      • Ismail Z.
      c-fos and its consequences in pain.
      These results show that i.c.v. spexin induced a significant increase of Fos mRNA level. T-5224, an inhibitor of c-Fos/AP-1, notably reversed the increase of Pdyn and Oprk1 mRNA levels caused by spexin. Considering the fact that spexin significantly increased the mRNA and protein levels of dynorphin and KOR, we proposed that central spexin may activate the dynorphin/KOR by the Fos pathway, inducing release of endogenous opioid peptide dynorphin and increase of KOR and producing antinociception in the mouse model of acute inflammatory pain. The result was also supported by the previous report that Fos directly led to dynorphin gene expression.
      • Corvello C.M.
      • Metz R.
      • Bravo R.
      • Armelin M.C.
      Expression and characterization of mouse cFos protein using the baculovirus expression system: ability to form functional AP-1 complex with coexpressed cJun protein.
      • Dubner R.
      • Ruda M.A.
      Activity-dependent neuronal plasticity following tissue injury and inflammation.
      c-Fos protein expression, commonly as a marker of neuronal activity, has generally been used to determine the nervous pathway.
      • Dragunow M.
      • Faull R.
      The use of c-fos as a metabolic marker in neuronal pathway tracing.
      The PAG, as a key position of the pain regulatory system, participated in control of pain signaling in the CNS.
      • Fields H.L.
      • Heinricher M.M.
      • Mason P.
      Neurotransmitters in nociceptive modulatory circuits.
      • Bandler R.
      • Shipley M.T.
      Columnar organization in the midbrain periaqueductal gray: modules for emotional expression?.
      Spexin up-regulated the quantity of FLI cells in almost all subdivisions of the PAG detected, particularly the caudal dorsomedial PAG, the caudal lateral PAG, and the caudal ventrolateral PAG. The result indicated that spexin might activate neurons in the PAG, which integrated the ascending pain signals and information from supraspinal pain circuitry,
      • Maletic V.
      • Raison C.L.
      Neurobiology of depression, fibromyalgia and neuropathic pain.
      subsequently exerting antinociceptive activity.
      In the acetic acid–induced writhing test, i.c.v. spexin produced an antinociceptive effect against visceral pain. This result is consistent with that in the formalin test. Spexin increased Galr2 and Adcy4 gene expression. GABAA receptor (Gabra1Gabra5) mRNA levels were not affected by spexin, whereas Pomc and Oprm1 mRNA levels were elevated, suggesting the involvement of POMC/MOR in the antinociception of spexin in the mouse visceral pain model. The molecules induced by spexin in the acetic acid–induced writhing test differ from those in the formalin test, which may be due to different molecular mechanisms between visceral pain and inflammatory pain.
      • Le Bars D.
      • Gozariu M.
      • Cadden S.W.
      Animal models of nociception.
      Furthermore, i.c.v. spexin up-regulated the Fos gene expression in the visceral pain model, which is consistent with that in the formalin test. This result conformed with the involvement of Fos in the antinociceptive response induced by spexin, and it was supported by the evidence that Fos was involved in transactivation of the opioid gene.
      • Sonnenberg J.L.
      • Rauscher 3rd, F.J.
      • Morgan J.I.
      • Curran T.
      Regulation of proenkephalin by Fos and Jun.
      • Ziolkowska B.
      • Przewlocka B.
      • Mika J.
      • Labuz D.
      • Przewlocki R.
      Evidence for Fos involvement in the regulation of proenkephalin and prodynorphin gene expression in the rat hippocampus.
      In summary, the present study shows that central spexin produced an antinociceptive effect in the formalin test and acetic acid–induced writhing test. In the formalin test, i.c.v. spexin stimulates Galr3 and Adcy4 gene expression, and then activates the endogenous dynorphin/KOR system through Fos, which were particular in PAG, accordingly producing an antinociceptive effect against inflammatory pain. In the writhing test, central spexin up-regulates Galr2 and Adcy1 mRNAs, and then activates POMC/MOR via Fos, accordingly inducing an antinociceptive effect against visceral pain. This study supports a key role for spexin in central nociceptive behavior, and spexin could be considered as a novel target for treatment of inflammatory pain as well as visceral pain.

      Supplemental Data

      Figure thumbnail figs1
      Supplemental Figure S1The antinociceptive effects of i.c.v. administration of spexin (0.81 mg/kg) in the radiant heat paw withdrawal test. Data points represent means ± SEM. n = 7 to 8 mice per group. P < 0.05.
      Figure thumbnail figs2
      Supplemental Figure S2The effects of MOR antagonist β-funaltrexamine hydrochloride (β-FNA; 49 μg/kg) on the antinociception induced by spexin (2.43 mg/kg) in the formalin test. Formalin was injected at 5 minutes after spexin or normal saline treatment. A: Early phase. B: Late phase. Data are presented as amount of licking/biting time. Data are expressed as means ± SEM (A and B). n = 9 to 10 per group (A and B). P < 0.05, ∗∗∗P < 0.001.

      References

        • Mirabeau O.
        • Severini C.
        • Audero E.
        • Gascuel O.
        • Possenti R.
        • Birney E.
        • Rosenthal N.
        • Gross C.
        Identification of novel peptide hormones in the human proteome by hidden Markov model screening.
        Genome Res. 2007; 17: 320-327
        • Sonmez K.
        • Zaveri N.T.
        • Kerman I.A.
        • Burke S.
        • Neal C.R.
        • Xie X.
        • Watson S.J.
        • Toll L.
        Evolutionary sequence modeling for discovery of peptide hormones.
        PLoS Comput Biol. 2009; 5: e1000258
        • Wong M.K.
        • Sze K.H.
        • Chen T.
        • Cho C.K.
        • Law H.C.
        • Chu I.K.
        • Wong A.O.
        Goldfish spexin: solution structure and novel function as a satiety factor in feeding control.
        Am J Physiol Endocrinol Metab. 2013; 305: E348-E366
        • Porzionato A.
        • Rucinski M.
        • Macchi V.
        • Stecco C.
        • Malendowicz L.K.
        • De Caro R.
        Spexin expression in normal rat tissues.
        J Histochem Cytochem. 2010; 58: 825-837
        • Kim D.-K.
        • Yun S.
        • Son G.H.
        • Hwang J.-I.
        • Park C.R.
        • Kim J.I.
        • Kim K.
        • Vaudry H.
        • Seong J.Y.
        Coevolution of the spexin/galanin/kisspeptin family: spexin activates galanin receptor type II and III.
        Endocrinology. 2014; 155: 1864-1873
        • Rucinski M.
        • Porzionato A.
        • Ziolkowska A.
        • Szyszka M.
        • Macchi V.
        • De Caro R.
        • Malendowicz L.K.
        Expression of the spexin gene in the rat adrenal gland and evidences suggesting that spexin inhibits adrenocortical cell proliferation.
        Peptides. 2010; 31: 676-682
        • Porzionato A.
        • Rucinski M.
        • Macchi V.
        • Stecco C.
        • Sarasin G.
        • Sfriso M.M.
        • Di Giulio C.
        • Malendowicz L.K.
        • De Caro R.
        Spexin is expressed in the carotid body and is upregulated by postnatal hyperoxia exposure.
        Adv Exp Med Biol. 2012; 758: 207-213
        • Toll L.
        • Khroyan T.V.
        • Sonmez K.
        • Ozawa A.
        • Lindberg I.
        • McLaughlin J.P.
        • Eans S.O.
        • Shahien A.A.
        • Kapusta D.R.
        Peptides derived from the prohormone proNPQ/spexin are potent central modulators of cardiovascular and renal function and nociception.
        FASEB J. 2012; 26: 947-954
        • Liu Y.
        • Li S.
        • Qi X.
        • Zhou W.
        • Liu X.
        • Lin H.
        • Zhang Y.
        • Cheng C.H.
        A novel neuropeptide in suppressing luteinizing hormone release in goldfish, Carassius auratus.
        Mol Cel Endocrinol. 2013; 374: 65-72
        • Wu H.
        • Lin F.
        • Chen H.
        • Liu J.
        • Gao Y.
        • Zhang X.
        • Hao J.
        • Chen D.
        • Yuan D.
        • Wang T.
        • Li Z.
        Ya-fish (Schizothorax prenanti) spexin: identification, tissue distribution and mRNA expression responses to periprandial and fasting.
        Fish Physiol Biochem. 2016; 42: 39-49
        • Walewski J.L.
        • Ge F.
        • Lobdell H.
        • Levin N.
        • Schwartz G.J.
        • Vasselli J.R.
        • Pomp A.
        • Dakin G.
        • Berk P.D.
        Spexin is a novel human peptide that reduces adipocyte uptake of long chain fatty acids and causes weight loss in rodents with diet-induced obesity.
        Obesity. 2014; 22: 1643-1652
        • Lin C.-Y.
        • Zhang M.
        • Huang T.
        • Yang L.-L.
        • Fu H.-B.
        • Zhao L.
        • Zhong L.L.
        • Mu H.-X.
        • Shi X.-K.
        • Leung C.F.
        Spexin enhances bowel movement through activating L-type voltage-dependent calcium channel via galanin receptor 2 in mice.
        Sci Rep. 2015; 5: 12095
        • Gu L.
        • Ma Y.
        • Gu M.
        • Zhang Y.
        • Yan S.
        • Li N.
        • Wang Y.
        • Ding X.
        • Yin J.
        • Fan N.
        Spexin peptide is expressed in human endocrine and epithelial tissues and reduced after glucose load in type 2 diabetes.
        Peptides. 2015; 71: 232-239
        • Le Bars D.
        • Gozariu M.
        • Cadden S.W.
        Animal models of nociception.
        Pharmacol Rev. 2001; 53: 597-652
        • Bannon A.W.
        • Malmberg A.B.
        Models of nociception: hot-plate, tail-flick, and formalin tests in rodents.
        Curr Protoc Neurosci. 2007; (Chapter 8:Unit 8.9)
        • Tjølsen A.
        • Berge O.-G.
        • Hunskaar S.
        • Rosland J.H.
        • Hole K.
        The formalin test: an evaluation of the method.
        Pain. 1992; 51: 5-17
        • McNamara C.R.
        • Mandel-Brehm J.
        • Bautista D.M.
        • Siemens J.
        • Deranian K.L.
        • Zhao M.
        • Hayward N.J.
        • Chong J.A.
        • Julius D.
        • Moran M.M.
        TRPA1 mediates formalin-induced pain.
        Proc Natl Acad Sci U S A. 2007; 104: 13525-13530
        • Reeta K.
        • Mediratta P.
        • Rathi N.
        • Jain H.
        • Chugh C.
        • Sharma K.
        Role of κ- and δ-opioid receptors in the antinociceptive effect of oxytocin in formalin-induced pain response in mice.
        Regul Pept. 2006; 135: 85-90
        • Padi S.
        • Kulkarni S.K.
        Role of cyclooxygenase-2 in lipopolysaccharide-induced hyperalgesia in formalin test.
        Indian J Exp Biol. 2005; 43: 53-60
        • Shamsi Meymandi M.
        • Keyhanfar F.
        Assessment of the antinociceptive effects of pregabalin alone or in combination with morphine during acetic acid-induced writhing in mice.
        Pharmacol Biochem Behav. 2013; 110: 249-254
        • Lv S.-Y.
        • Qin Y.-J.
        • Wang N.-B.
        • Yang Y.-J.
        • Chen Q.
        Supraspinal antinociceptive effect of apelin-13 in a mouse visceral pain model.
        Peptides. 2012; 37: 165-170
        • Haley T.
        • McCormick W.
        Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse.
        Br J Pharmacol Chemother. 1957; 12: 12-15
        • Lv S.-Y.
        • Qin Y.-J.
        • Wang H.-T.
        • Xu N.
        • Yang Y.-J.
        • Chen Q.
        Centrally administered apelin-13 induces depression-like behavior in mice.
        Brain Res Bull. 2012; 88: 574-580
        • Lv S.-Y.
        • Yang Y.J.
        • Hong S.
        • Wang N.-B.
        • Qin Y.-J.
        • Li W.-X.
        • Chen Q.
        Intrathecal apelin-13 produced different actions in formalin test and tail-flick test in mice.
        Protein Pept Lett. 2013; 20: 926-931
        • Collier H.O.
        • Dinneen L.C.
        • Johnson C.A.
        • Schneider C.
        The abdominal constriction response and its suppression by analgesic drugs in the mouse.
        Br J Pharmacol Chemother. 1968; 32: 295-310
        • Wang C.L.
        • Yang D.J.
        • Yuan B.Y.
        • Wang Y.
        C-terminal hydrazide modification changes the spinal antinociceptive profiles of endomorphins in mice.
        Peptides. 2018; 99: 128-133
        • Chadzinska M.
        • Starowicz K.
        • Scislowska-Czarnecka A.
        • Bilecki W.
        • Pierzchala-Koziec K.
        • Przewlocki R.
        • Przewlocka B.
        • Plytycz B.
        Morphine-induced changes in the activity of proopiomelanocortin and prodynorphin systems in zymosan-induced peritonitis in mice.
        Immunol Lett. 2005; 101: 185-192
        • Wang H.B.
        • Laverghetta A.V.
        • Foehring R.
        • Deng Y.P.
        • Sun Z.
        • Yamamoto K.
        • Lei W.L.
        • Jiao Y.
        • Reiner A.
        Single-cell RT-PCR, in situ hybridization histochemical, and immunohistochemical studies of substance P and enkephalin co-occurrence in striatal projection neurons in rats.
        J Chem Neuroanat. 2006; 31: 178-199
        • Gaveriaux-Ruff C.
        • Nozaki C.
        • Nadal X.
        • Hever X.C.
        • Weibel R.
        • Matifas A.
        • Reiss D.
        • Filliol D.
        • Nassar M.A.
        • Wood J.N.
        Genetic ablation of delta opioid receptors in nociceptive sensory neurons increases chronic pain and abolishes opioid analgesia.
        Pain. 2011; 152: 1238-1248
        • Gabrilovac J.
        • Čupić B.
        • Zapletal E.
        • Brozovic A.
        IFN-γ up-regulates kappa opioid receptors (KOR) on murine macrophage cell line J774.
        J Neuroimmunol. 2012; 245: 56-65
        • Hashikawa-Hobara N.
        • Ogawa T.
        • Sakamoto Y.
        • Matsuo Y.
        • Ogawa M.
        • Zamami Y.
        • Hashikawa N.
        Calcitonin gene-related peptide pre-administration acts as a novel antidepressant in stressed mice.
        Sci Rep. 2015; 5: 12559
        • Simpson D.A.
        • Feeney S.
        • Boyle C.
        • Stitt A.W.
        Technical brief: retinal VEGF mRNA measured by SYBR green I fluorescence: a versatile approach to quantitative PCR.
        Mol Vis. 2000; 6: 178-183
        • Livak K.J.
        • Schmittgen T.D.
        Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method.
        Methods. 2001; 25: 402-408
        • Singewald N.
        • Salchner P.
        • Sharp T.
        Induction of c-Fos expression in specific areas of the fear circuitry in rat forebrain by anxiogenic drugs.
        Biol Psychiatry. 2003; 53: 275-283
        • Saito Y.
        • Miyasaka T.
        • Hatsuta H.
        • Takahashi-Niki K.
        • Hayashi K.
        • Mita Y.
        • Kusano-Arai O.
        • Iwanari H.
        • Ariga H.
        • Hamakubo T.
        • Yoshida Y.
        • Niki E.
        • Murayama S.
        • Ihara Y.
        • Noguchi N.
        Immunostaining of oxidized DJ-1 in human and mouse brains.
        J Neuropathol Exp Neurol. 2014; 73: 714-728
        • Mahmoudi M.
        • Zarrindast M.-R.
        Effect of intracerebroventricular injection of GABA receptor agents on morphine-induced antinociception in the formalin test.
        J Psychopharmacol. 2002; 16: 85-91
        • de Souza M.M.
        • Silote G.P.
        • Herbst L.S.
        • Funck V.R.
        • Joca S.R.L.
        • Beijamini V.
        The antidepressant-like effect of galanin in the dorsal raphe nucleus of rats involves GAL2 receptors.
        Neurosci Lett. 2018; 681: 26-30
        • Swanson C.J.
        • Blackburn T.P.
        • Zhang X.
        • Zheng K.
        • Xu Z.-Q.D.
        • Hokfelt T.
        • Wolinsky T.D.
        • Konkel M.J.
        • Chen H.
        • Zhong H.
        • Walker M.W.
        • Craig D.A.
        • Gerald C.P.G.
        • Branchek T.A.
        Anxiolytic- and antidepressant-like profiles of the galanin-3 receptor (Gal(3)) antagonists SNAP 37889 and SNAP 398299.
        Proc Natl Acad Sci U S A. 2005; 102: 17489-17494
        • Miyazaki H.
        • Morishita J.
        • Ueki M.
        • Nishina K.
        • Shiozawa S.
        • Maekawa N.
        The effects of a selective inhibitor of c-Fos/activator protein-1 on endotoxin-induced acute kidney injury in mice.
        BMC Nephrol. 2012; 13: 153
        • Spooner M.-F.
        • Robichaud P.
        • Carrier J.
        • Marchand S.
        Endogenous pain modulation during the formalin test in estrogen receptor beta knockout mice.
        Neuroscience. 2007; 150: 675-680
        • Basbaum A.I.
        • Bráz J.M.
        • kruger L.
        • Light A.
        Transgenic mouse models for the tracing of “pain” pathways.
        in: Kruger L. Light A.R. Translational Pain Research: From Mouse to Man. CRC Press/Taylor & Francis, Boca Raton, FL2010: 1-17
        • Eipper B.A.
        • Stoffers D.A.
        • Mains R.E.
        The biosynthesis of neuropeptides: peptide alpha-amidation.
        Annu Rev Neurosci. 1992; 15: 57-85
        • Lang R.
        • Gundlach A.L.
        • Holmes F.E.
        • Hobson S.A.
        • Wynick D.
        • Hoekfelt T.
        • Kofler B.
        Physiology, signaling, and pharmacology of galanin peptides and receptors: three decades of emerging diversity.
        Pharmacol Rev. 2015; 67: 118-175
        • Dirksen R.
        Opioid receptors and pain.
        Pharm Weekbl. 1990; 12: 41-45
        • Millan M.J.
        The induction of pain: an integrative review.
        Prog Neurobiol. 1999; 57: 1-164
        • Kivell B.
        • Prisinzano T.E.
        Kappa opioids and the modulation of pain.
        Psychopharmacology. 2010; 210: 109-119
        • Obara I.
        • Parkitna J.R.
        • Korostynski M.
        • Makuch W.
        • Kaminska D.
        • Przewlocka B.
        • Przewlocki R.
        Local peripheral opioid effects and expression of opioid genes in the spinal cord and dorsal root ganglia in neuropathic and inflammatory pain.
        Pain. 2009; 141: 283-291
        • Muschamp J.W.
        • Carlezon Jr., W.A.
        Roles of nucleus accumbens CREB and dynorphin in dysregulation of motivation.
        Cold Spring Harb Perspect Med. 2013; 3: a012005
        • Munglani R.
        • Hunt S.P.
        Molecular biology of pain.
        Br J Anaesth. 1995; 75: 186-192
        • Sun W.Z.
        • Shyu B.C.
        • Shieh J.Y.
        Nitrous oxide or halothane, or both, fail to suppress c-fos expression in rat spinal cord dorsal horn neurones after subcutaneous formalin.
        Br J Anaesth. 1996; 76: 99-105
        • Ahmad A.H.
        • Ismail Z.
        c-fos and its consequences in pain.
        Malays J Med Sci. 2002; 9: 3-8
        • Corvello C.M.
        • Metz R.
        • Bravo R.
        • Armelin M.C.
        Expression and characterization of mouse cFos protein using the baculovirus expression system: ability to form functional AP-1 complex with coexpressed cJun protein.
        Cell Mol Biol Res. 1995; 41: 527-535
        • Dubner R.
        • Ruda M.A.
        Activity-dependent neuronal plasticity following tissue injury and inflammation.
        Trends Neurosci. 1992; 15: 96-103
        • Dragunow M.
        • Faull R.
        The use of c-fos as a metabolic marker in neuronal pathway tracing.
        J Neurosci Methods. 1989; 29: 261-265
        • Fields H.L.
        • Heinricher M.M.
        • Mason P.
        Neurotransmitters in nociceptive modulatory circuits.
        Annu Rev Neurosci. 1991; 14: 219-245
        • Bandler R.
        • Shipley M.T.
        Columnar organization in the midbrain periaqueductal gray: modules for emotional expression?.
        Trends Neurosci. 1994; 17: 379-389
        • Maletic V.
        • Raison C.L.
        Neurobiology of depression, fibromyalgia and neuropathic pain.
        Front Biosci (Landmark Ed). 2009; 14: 5291-5338
        • Sonnenberg J.L.
        • Rauscher 3rd, F.J.
        • Morgan J.I.
        • Curran T.
        Regulation of proenkephalin by Fos and Jun.
        Science. 1989; 246: 1622-1625
        • Ziolkowska B.
        • Przewlocka B.
        • Mika J.
        • Labuz D.
        • Przewlocki R.
        Evidence for Fos involvement in the regulation of proenkephalin and prodynorphin gene expression in the rat hippocampus.
        Brain Res Mol Brain Res. 1998; 54: 243-251