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The intraperitoneal administration of MOTS-c produces antinociceptive and anti-inflammatory effects through the activation of AMPK pathway in the mouse formalin test
European Journal of Pharmacology 870 (2020) 172909
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The intraperitoneal administration of MOTS-c produces antinociceptive and anti-inflammatory effects through the activation of AMPK pathway in the mouse formalin test
T
Xinqiang Yina,b, Yuanyuan Jingc, Quan Chena, Abdul Baset Abbasa, Jialiang Hua,d, Hanmei Xua,d,∗ a
The Engineering Research Center of Synthetic Polypeptide Drug Discovery and Evaluation of Jiangsu Province, China Pharmaceutical University, Nanjing, 210009, China School of Basic Medical Sciences, North Sichuan Medical College, Nanchong, 673000, China c Department of Preventive Medicine, North Sichuan Medical College, Nanchong, 637000, China d State Key Laboratory of Natural Medicines, Ministry of Education, China Pharmaceutical University, Nanjing, 210009, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: MOTS-C Formalin test Antinociception Anti-inflammation AMP activated protein kinase
The activation of the AMP activated protein kinase (AMPK) exerts antinociceptive effects in acute and neuropathic pain models. Mitochondrial open-reading-frame of the twelve S rRNA-c (MOTS-c), a mitochondrial-derived peptide, regulates many biological activities via activating AMPK. However, the role of MOTS-c in the formalin-induced inflammatory nociception remains unclear. In this study, we investigated the role of MOTS-c in the formalin-induced inflammatory nociception. The antinociceptive effect of MOTS-c was assessed by recording the time spent licking paw. The anti-inflammatory effect of MOTS-c was evaluated by detecting the inflammatory cytokine level changes in the mouse serum. Western blot was used to detect the changes of protein phosphorylation levels in the mouse spinal cord. Changes of c-fos expression in the spinal cord were assessed by immunohistochemistry. Our results showed that the intraperitoneal administration of MOTS-c reduced the time spent on licking in phase 2 in a dose-dependent manner in the formalin test. The antinociceptive effects of MOTS-c (50 mg/kg, i.p.) were attenuated by the AMPK antagonist compound C (10 mg/kg, i.p.). MOTS-c (50 mg/kg, i.p.) significantly reduced pro-inflammatory cytokine levels and elevated the level of anti-inflammatory cytokine in mouse serum. In addition, MOTS-c treatment significantly increased AMPKα phosphorylation level and suppressed formalin-induced extracellular signal-regulated kinase (ERK), c-Jun aminoterminal kinases (JNK), and P38 activation and c-fos expression in the mouse spinal cord. These results suggest that systemic administration of MOTS-c exerts antinociceptive and anti-inflammatory effects, at least partially, through activating AMPK pathway and inhibiting MAP kinases–c-fos signaling pathway in the mouse formalin test.
1. Introduction Mitochondrial open-reading-frame of the twelve S rRNA-c (MOTSc), a novel bioactive mitochondrial-derived peptide encoded from the 12S rRNA region of mitochondria, was firstly identified in 2015 (Lee et al., 2015). MOTS-c is expressed in various tissues of rodents and humans and can be secreted into blood, which suggests that it plays a role in both a cell-autonomous and hormonal way (Lee et al., 2015). Recent studies have shown that MOTS-c is a versatile peptide. MOTS-c prevented age-dependent or high-fat diet-induced insulin resistance, as well as diet-induced obesity through regulating glucose metabolism by activating AMPK pathway (Kim et al., 2017). MOTS-c suppressed
ovariectomy-induced bone loss via AMPK activation (Ming et al., 2016). In another study, researchers found that MOTS-c exerted an anti-inflammatory effect via suppressing mitogen-activated protein kinases (MAPKs) and increasing aryl hydrocarbon receptor/signal transducer and activator of transcriptional 3 (Ahr/STAT3) signaling pathways (Zhai et al., 2017). Recently, Kim and colleagues revealed that MOTS-c could dynamically translocate to the nucleus in response to metabolic stress and regulate adaptive nuclear gene expression in an AMPK-dependent manner (Kim et al., 2018). The above research results reveal that MOTS-c functions via activating AMPK pathway. Previous studies showed that AMPK, a kinase widely expressed in various tissues, constitutes an important regulator of metabolism
∗ Corresponding author. The Engineering Research Center Synthetic Polypeptide Drug Discovery and Evaluation of Jiangsu Province, China Pharmaceutical University, Nanjing, 210009, China. E-mail addresses: [email protected], [email protected] (H. Xu).
https://doi.org/10.1016/j.ejphar.2020.172909 Received 17 May 2019; Received in revised form 10 December 2019; Accepted 6 January 2020 Available online 08 January 2020 0014-2999/ © 2020 Elsevier B.V. All rights reserved.
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2.3. Formalin test
(Hardie, 2004; Grahame Hardie, 2014). Recent evidence indicated a crucial role of AMPK in the acute and neuropathic pain models (Asiedu et al., 2016; Giri, 2004; Melemedjian et al., 2011; Price et al., 2016; Tillu et al., 2012). AMPK activators, such as AICAR and metformin, exerted anti-inflammatory and antinociceptive effects in formalin- and zymosan-induced paw inflammation models (Russe et al., 2013). Regulation of the AMPKα2 subunit of the kinase played a key role in the antinociceptive effects (Russe et al., 2013). On the molecular level, antinociceptive effects were mediated by reduced activation of different MAP-kinases in the spinal cord and a subsequent decrease in pain-relevant induction of c-fos, a reliable marker of elevated activity in spinal cord neurons following peripheral noxious stimulation (Harris, 1998; Russe et al., 2013). In addition, resveratrol exhibited antinociceptive effects by engaging AMPK to attenuate extracellular signal-regulated kinase (ERK) and the mammalian target of rapamycin (mTOR) signaling in sensory neurons in incision-induced acute and chronic pain (Tillu et al., 2012). MOTS-c targets the methionine-folate cycle, increases 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) levels, and eventually activates AMPK (Lee et al., 2015). The activation of AMPK is responsible for the antinociceptive effects in many models (Asiedu et al., 2016; Giri, 2004; Melemedjian et al., 2011; Price et al., 2016; Tillu et al., 2012). Therefore, we investigated the effects of MOTS-c on the nociceptive response induced by formalin. We found that the intraperitoneal administration of MOTS-c produced antinociceptive and anti-inflammatory effects through the activation of AMPK pathway in the mouse formalin test.
The formalin test procedure was performed as previously described with slight modifications (Sufka et al., 1998). Mice were placed in a plexiglass box (15 cm in diameter and 20 cm in height) with a mirror placed under the floor at a 45° angle to allow an unobstructed view of the paw and were allowed to habituate for 30 min. Then, a 5% formaldehyde solution (formalin, 20 μl) was injected subcutaneously into the dorsal surface of the left hind paw. Right after formalin injection, the time spent licking the formalin-injected paw was recorded at 5-min intervals up to 40 min. The first 10 min were recorded as early nociceptive phase (phase I) and the period from 11 to 40 min was recorded as the inflammatory phase (phase II). 2.4. Protocols for evaluating the effect of compound C on MOTS-c-induced antinociception To evaluate the effect of compound C on MOTS-c-induced antinociception in the mouse formalin test, animals were randomly divided into four groups: Control group (saline + saline group), saline + MOTS-c group, compound C + saline group, and compound C + MOTS-c group. The mice in the control group received two intraperitoneal injections of saline (10 μl/g) 260 min and 240 min before formalin stimulation, respectively. The second group of mice received intraperitoneal administration of saline (10 μl/g) and MOTS-c (50 mg/ kg) 260 min and 240 min before formalin stimulation, respectively. The third group of mice received intraperitoneal administration of compound C (10 mg/kg) and saline (10 μl/g) 260 min and 240 min before formalin stimulation, respectively. The fourth group of mice received intraperitoneal administration of compound C (10 mg/kg) and MOTS-c (50 mg/kg) 260 min and 240 min before formalin stimulation, respectively.
2. Materials and methods 2.1. Animals Male ICR mice (25–30 g) were obtained from the Experimental Animal Center of Qinglong Mountain in Nanjing, China. Animals had free access to food and water and were housed in an animal room that was maintained at 22 ± 2 °C with a 12 h light/dark cycle. All animals were cared for and experiments were carried out in accordance with the European Community guidelines for the use of experimental animals (2010/63/EU). All the protocols in this study were approved by the Ethics Committee of China Pharmaceutical University (SYXK (Su) 2018–0019). Mice were randomly divided into normal group, control group, and drug treatment group. There was no intervention in the normal group. The mice in the control group received intraperitoneal injection of sterile saline before subcutaneous injection of formalin into the dorsal surface of the left hind paw. The mice in the drug treatment group received intraperitoneal administration of drugs before formalin stimulation. The collections and analysis of data were performed by two independent observers blinded to the group assignment.
2.5. Cytokine detection The mice were deeply anesthetized with trichloroacetaldehyde hydrate (350 mg/kg, i.p.) 2 h after formalin injection and euthanized by exanguination after blood collection via cardiac puncture. The blood was centrifuged at 800×g for 15 min at 4 °C to obtain serum. The levels of tumor necrosis factor-α (TNF-α), interleukine-1β (IL-1β), interleukine-10 (IL-10), and interleukine-6 (IL-6) in mouse serum were measured using Enzyme-linked immunosorbent assays (ELISA) kits (Nanjing Jin Yibai Biological Technology Co. Ltd., Nanjing, China) according to the manufacturer's instructions. 2.6. Western blot For Western blot analysis, the mice were deeply anesthetized with trichloroacetaldehyde hydrate (350 mg/kg, i.p.) and then euthanized by exsanguination 2 h after formalin injection. Lumbar spinal cords (L3L5) were dissected out for downstream analysis. Total proteins in tissues were extracted immediately after preparation using Whole Cell Lysis Assay kits (Wanleibio). The protein concentrations were quantified using BCA protein assay kits (Wanleibio). Proteins in tissue homogenates (20 μg) were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes by wet-blotting. Membranes were blocked for 1 h at room temperature with 5% bovine serum albumin (BSA). Then the blots were incubated overnight at 4 °C with primary antibodies against AMPKα (Thr172), p-AMPKα (Thr172), P38, p-P38 (Thr180/Tyr182), c-Jun amino-terminal kinases (JNK), p-JNK1/2 (Thr183/Tyr185), ERK, and p-ERK1/2 (Thr202/Tyr204) (1:500 in blocking buffer, Affinity Biosciences) in blocking buffer. The blots were incubated with a secondary antibody (1:5000 in blocking buffer, Lianke) for 60 min after washing 3 times with TBST. Then, the filters were developed by enhanced chemiluminescence reagents (Wanleibio). Glyceraldehyde-3-
2.2. Drugs MOTS-c peptide (sequence: Met-Arg-Trp-Gln-Glu-Met-Gly-Tyr-IlePhe-Tyr-Pro-Arg-Lys-Leu-Arg) with a purity of more than 95% was synthesized by GL Biochem Co., Ltd. (Shanghai, China). Morphine hydrochloride was purchased from Shenyang First Pharmaceutical Factory (China). Formaldehyde was purchased from Sigma-Aldrich (St. Louis, MO, USA). The rabbit anti-c-fos antibody was obtained from Santa Cruz. Compound C 2HCl, which was used as an AMPK inhibitor, was purchased from Selleck. Trichloroacetaldehyde hydrate was obtained from Sinopharm Chemical Reagent Co., Ltd. MOTS-c, compound C, morphine, and trichloroacetaldehyde hydrate were dissolved in sterile saline (0.9% NaCl) at a concentration of 10 mg/ml, 1 mg/ml, 1 mg/ml, and 100 mg/ml, respectively. The rabbit anti-c-Fos antibody was diluted with PBS (1:500). MOTS-c and morphine were intraperitoneally injected 4 h and 20 min prior to formalin injection, respectively. 2
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phosphate dehydrogenase (GAPDH) (Santa Cruz) was used as the loading control. Densitometries of the blots were analyzed using the Image J2x software. 2.7. c-fos immunohistochemistry Mice were deeply anesthetized with trichloroacetaldehyde hydrate (350 mg/kg, i.p.) 2 h after the formalin injection. Then mice were perfused transcardially with 20 ml saline followed by 20 ml 4% paraformaldehyde in 0.1 M phosphate buffer. L4 and/or L5 lumbar segment was dissected out and post-fixed in the same fixative overnight at 4 °C. The segments were processed for c-fos immunoreactivity as described previously (Singewald et al., 2003). The number of c-fos-positive cells in the ipsilateral dorsal spinal cord was counted manually by an observer blind to the treatment using ImageJ2x software. 2.8. Statistical analysis The results were expressed as mean ± S.E.M. Statistical analysis was performed using IBM SPSS statistics 22 software. In the formalin test, for statistical analysis, data from the first phase and the second phase were considered independently. One-way ANOVA with Bonferroni test post hoc test was used to evaluate the dose-dependent effect of MOTS-c on formalin-induced licking and the effect of compound C on MOTS-c induced antinociception. One-way ANOVA with Bonferroni test post hoc test was also used to examine the effect of MOTS-c on the levels of the serum inflammatory cytokines in the mice of formalin test. Student's t-test was used to compare the number of cfos-like immunoreactivity positive neurons between the MOTS-c treatment group and the control group. In all statistical comparisons, differences with P < 0.05 were considered significant differences. 3. Results Fig. 1. MOTS-c and morphine attenuated inflammatory nociception in the formalin test. (A) Time course of the licking behavior in mice after injection of formalin (5%, 20 μl) into the left hind paw with pretreatment with saline or MOTS-c. The effects of intraperitoneally administrated saline, MOTS-c (12.5, 25, 50, and 100 mg/kg) (B), and morphine (5 mg/kg, 10 mg/kg) (C) on the licking time in phase 1 (0–10 min) and phase 2 (11–40 min) in the mouse formalin test. Formalin was injected at time 0. Saline or MOTS-c was intraperitoneally administrated 4 h prior to formalin. Morphine was intraperitoneally administrated 20 min prior to formalin.The time spent licking the injected paw was measured in 5-min intervals for 40 min. Data are presented as the mean number of licking time ± S.E.M. (n = 6–8 for each group). *P < 0.05, **P < 0.01, ***P < 0.001 versus saline according to one-way ANOVA followed by Bonferroni test.
3.1. Intraperitoneal pretreatment with MOTS-c produced antinociceptive effect in a dose-dependent manner in the mouse formalin test In the formalin test, the typical biphasic pain-like behavior with a first licking phase from 1 to 10 min and a second phase from 11 to 40 min was found after formalin injection (Fig. 1A). Treatment of mice with MOTS-c (12.5, 25, 50, and 100 mg/kg, i.p.) decreased the time spent on licking in phase 2 in a dose-dependent manner (Fig. 1B). However, after MOTS-c (12.5, 25, 50, and 100 mg/kg, i.p.) treatment, the time spent on licking in the first phase did not show a significant difference in comparison with the control group (Fig. 1B). The Bonferroni's test revealed that MOTS-c (25, 50, and 100 mg/kg, i.p.) significantly inhibited the second phase of formalin-induced pain behaviors in comparison with the control group (Fig. 1B). However, 12.5 mg/kg MOTS-c failed to produce a significant antinociceptive effect in comparison with the control group. Under the same condition, morphine (5, 10 mg/kg, i.p) significantly attenuated the nociceptive response induced by formalin in both phases (Fig. 1C). The time spent on licking in phase 2 between the MOTS-c (50 mg/kg) group and the morphine (5 mg/kg) group had no significant difference (Fig. 1C).
3.3. MOTS-c suppressed pro-inflammatory cytokine production, and promoted anti-inflammatory cytokine secretion in serum To assess the effect of MOTS-c on the formalin-induced acute inflammation in the mouse formalin test, the concentrations of the serum inflammatory cytokines were measured by ELISA. Compared to the normal group, production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the control group were significantly increased. Moreover, administration of MOTS-c (50 mg/kg, i.p.) significantly reduced the inflammatory cytokine levels (TNF-α, IL-1β and IL-6) in the mouse serum in comparison with the control group (Fig. 3A–C). In addition, the concentration of IL-10 in serum was significantly elevated after MOTS-c treatment (Fig. 3D). However, morphine did not change the effect induced by formalin on cytokine concentrations (Fig. 3A–D).
3.2. Compound C effectively reversed the antinociceptive effect of MOTS-c in the mouse formalin test The effect of compound C, an AMPK inhibitor, on the time spent on licking was examined. Compared to the control group, injection of compound C (10 mg/kg, i.p.) followed with saline (10 μl/g, i.p.) had no effect on pain behavior response induced by formalin (Fig. 2). However, intraperitoneal injection of compound C (10 mg/kg) 20 min before MOTS-c treatment (50 mg/kg, i.p.), significantly reversed the antinociceptive effect of MOTS-c (Fig. 2). 3
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3.4. MOTS-c promoted AMPK activation and inhibited MAPKs activation in the spinal cord 3.4.1. Administration of MOTS-c increased AMPKα phosphorylation level in the spinal cord It has been reported that the activation of AMPK played crucial roles in models of acute and neuropathic nociception and inflammatory nociception. Therefore, we investigated the effects of MOTS-c (50 mg/kg, i.p.) on AMPK activation in the mouse spinal cord 2 h after formalin injection by Western blot analysis. Our results showed that administration of MOTS-c (50 mg/kg, i.p.) significantly increased the AMPKα phosphorylation level in the spinal cord in comparison with the control group (Fig. 4A). In addition, compared to the MOTS-c group, AMPKα phosphorylation in the spinal cord was significantly inhibited by the AMPK inhibitor compound C (10 mg/kg, i.p.) (Fig. 4A). 3.4.2. MOTS-c suppressed fomalin-induced ERK, JNK, and P38 phosphorylation in the spinal cord To investigate AMPK-activated signal transduction mechanisms that might be involved in the antinociceptive effects, we analyzed the impact of MOTS-c-mediated AMPK activation on the activation of ERK, JNK, and P38 MAP-kinases in the formalin assay. These kinases have already been described as potential AMPK targets and important mediators of inflammatory nociception. As expected, formalin induced a significantly increased phosphorylation and thereby activation of ERK (Fig. 4B), JNK (Fig. 4C), and P38 (Fig. 4D) in comparison with the normal group. Compared to the control group, MOTS-c (50 mg/kg, i.p.) treatment significantly suppressed phosphorylation of ERK, JNK, and P38 in the spinal cord, which indicated that these effects may contribute to the antinociceptive effects observed after MOTS-c (50 mg/kg, i.p.) treatment (Fig. 4B–D). Furthermore, AMPK inhibitor compound C treatment (10 mg/kg, i.p.) significantly reversed the inhibitory effects of MOTS-c (50 mg/kg, i.p.) on formalin-induced increases of ERK, JNK, and P38 phosphorylation in the spinal cord in comparison with the
Fig. 2. The impact of compound C (10 mg/kg, i.p.)) on the antinociceptive effect of MOTS-c (50 mg/kg, i.p.) in the mouse formalin test. MOTS-c was intraperitoneally administrated 4 h prior to formalin. Compound C was intraperitoneally administrated 20 min prior to MOTS-c. Saline was intraperitoneally injected in the corresponding time. The time spent licking the injected paw was measured in 5-min intervals for 40 min. Data are presented as the mean number of licking time ± S.E.M. (n = 7–8 for each group). **P < 0.01 versus control according to one-way ANOVA followed by Bonferroni test.
Fig. 3. MOTS-c treatment reduced the levels of inflammatory cytokines in the mice serum in the formalin test. 2 h after formalin injection, mice were killed and blood samples were collected and the concentrations of TNF-α (A), IL-1β (B), IL-6 (C), and IL-10 (D) were measureded by ELISA in the normal, control (10 μl/g, i.p.), MOTSc (50 mg/kg, i.p.), and Morphine (5 mg/kg, i.p.) group. Results are expressed as mean ± S.E.M. (n = 6–8 for each group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus control according to one-way ANOVA followed by Bonferroni test. 4
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Fig. 4. MOTS-c activated AMPK and suppressed formalin-induced phosphorylation of ERK, JNK, and P38 in the spinal cord. The spinal cord was dissected out 2 h after formalin injection and the levels of phosphorylated AMPK and MAP kinases were evaluated by densitometric analysis of the blots. (A) AMPK activation, (B) ERK activation, (C) JNK activation, and (D) P38 activation. The blots showed representative samples. All samples derived from the same experiment and that blots were processed in parallel. AMPK and p-AMPK were normalized to the expression of GAPDH on the same membrane. Each phosphoprotein was normalized to the expression of the corresponding total protein of the same sample in Fig. 5B–D. Results are expressed as mean ± S.E.M. (n = 6–8 for each group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 according to one-way ANOVA followed by Bonferroni test.
in the formalin-injected mice. Compared to the control group, MOTS-c (50 mg/kg, i.p.) significantly suppressed the expression of c-fos protein in the ipsilateral dorsal horn of the spinal cord (Fig. 5).
MOTS-c treatment group (Fig. 4B–D). 3.5. MOTS-c suppressed formalin-induced c-fos protein expression in the spinal cord
4. Discussion c-fos is a downstream effector of MAP-kinases and a well-accepted marker for inflammatory and nociceptive response. We investigated the effect of MOTS-c (50 mg/kg, i.p.) on spinal cord c-fos protein expression
MOTS-c, a mitochondria-derived peptide identified in 2015, has been detected in various tissues, as well as in circulation of human and 5
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Fig. 5. The number of c-fos-like immunoreactivity positive cells between the control group and the MOTS-c (i.p, 50 mg/kg) treatment group. Data are shown as mean ± S.E.M. (n = 7 for control and n = 8 for 50 mg/kg MOTS-c, respectively). **P < 0.01 versus control according to Student's t-test.
We also found that the antinociceptive effects of AMPK activation were accompanied by anti-inflammatory effects, which was in good agreement with several previous studies showing anti-inflammatory effects in LPS-induced acute lung injury models (Wang et al., 2016; Hoogendijk et al., 2013) as well as in macrophages (Jeong et al., 2009), the adipose tissue of patients and skeletal muscles of rats (Lihn et al., 2008) after AMPK stimulation. Formalin induced paw edema and increased pro-inflammatory cytokine levels such as TNF-α, IL-1β, and IL-6. The release of pro-inflammatory cytokines is an important cause of inflammatory nociception (Cunha et al., 2005; Xiang et al., 2019). TNF-α, induced by carrageenan in carrageenan-induced inflammatory hypernociception in mice, acts on tumor necrosis factor receptor 1 (TNF-R1) to stimulate the release of IL-1β and IL-6 (Cunha et al., 2005). The mature fragment of IL-1β, which is expressed by activated macrophages as proprotein and proteolytically processed by Caspase 1, induces sensitization of nerve fibers of primary sensory neurons to cause pain hypersensitivity (Xiang et al., 2019). IL-6 is an intermediate in TNF-α-induced IL-1β release and induces the production of prostanoids (Cunha et al., 2005). In the mouse formalin test, formalin-induced production of pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) may be one of the causes of the inflammatory nociception. Furthermore, it has been reported that AMPK activation inhibits pro-inflammatory processes via inhibition of nuclear factor-κB (NF-κB) signaling, inhibition of MAPK pathways, regulation of reactive oxygen species, inhibition of the Janus kinase/signal transducer and activator of transcriptions (JAK-STAT) signaling, inhibition of leukocyte infiltration, regulation of cytokine synthesis, and regulation of lipid metabolism (Salt and Palmer, 2012). The current study found that intraperitoneal administration of MOTS-c produced antinociceptive and anti-inflammatory effects in the mouse formalin test. The anti-inflammatory effects may also contribute to the antinociceptive effect. With regard to the downstream effector of AMPK, it has been reported that AMPK activation inhibits the activation of different MAP kinases (Lee et al., 2010; Petti et al., 2012). Furthermore, several lines
rodent plasma (Lee et al., 2015). The wide distribution of MOTS-c indicates its key roles in organisms. It has been reported that MOTS-c might be involved in many physiological and pathological functions via activation of AMPK (Lee et al., 2015; Ming et al., 2016; Zhai et al., 2017; Kim et al., 2018; Lu et al., 2019). Recent studies suggested that the activation of AMPK might have an impact on pain in animal models of neuropathy and acute nociception (Asiedu et al., 2016; Giri, 2004; Melemedjian et al., 2011; Price et al., 2016; Tillu et al., 2012). In this study, we explored the role of MOTS-c in formalin-induced inflammatory nociception and found that intraperitoneal administration of MOTS-c produced antinociceptive effect in the mouse formalin test. We hypothesized that the antinociceptive effect of MOTS-c was through activating AMPK signaling. Therefore, compound C, a wellknown AMPK inhibitor, was used in this study to test whether the inhibitory effect of MOTS-c on formalin-induced nociception behaviors was mediated through the activation of AMPK. The results showed that intraperitoneal injection of compound C (10 mg/kg) alone had no effect on the formalin-induced licking response. However, administration of compound C 20 min prior to MOTS-c significantly attenuated the antinociceptive effect induced by MOTS-c (50 mg/kg, i.p.) treatment. Furthermore, Western blot results showed that the phosphorylation level of AMPKα was significantly improved in the mouse spinal cord after MOTS-c (50 mg/kg, i.p.) treatment and compound C significantly reversed MOTS-c induced AMPKα phosphorylation. All these results indicated that the antinociceptive effect of MOTS-c was due to the activation of AMPK. MOTS-c targets the methionine-folate cycle, increases AICAR levels, and eventually activates AMPK (Lee et al., 2015). Through its active metabolite ZMP, AICAR binds to the AMP-binding site of the γ subunit of AMPK to activate it. It has been considered that other AMP-regulated molecules, such as adenosine receptors, glycogen phosphorylase, and fructose-1, 6-bisphosphatase (FBPase), which are also involved in the regulation of nociceptive processing, might be interfered by AICAR (Russe et al., 2013). Therefore, it cannot be excluded that the observed antinociceptive effects are mediated via one or more of these targets. 6
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Authors’ contributions
of studies have shown that AMPK activation negatively regulates aberrant translation control after nerve injury, resolves neuropathic allodynia and decreases sensory neuron excitability through suppressing mTOR and MAPKs (Price and Dussor, 2013). Accumulating evidence showed that MAP kinase family members, particularly ERK, p38, and JNK, have been associated with the development of pain hypersensitivity during inflammation and neuropathy through their effects on different signaling mechanisms (Ji et al., 2009). ERK activation in spinal dorsal horn neurons by nociceptive activity plays an important role in central sensitization by regulating the activity of glutamate receptors and potassium channels and inducing gene transcription. By inhibiting the spinal interleukine-33/the signal transduction society (IL-33/STS) signaling and the downstream ERK and JNK pathway, electroacupuncture alleviated formalin-induced inflammatory pain (Han et al., 2015). Thalidomide repressed inflammatory response and reduced radiculopathic pain by inhibiting interleukin-1 receptor-associated kinase 1 (IRAK-1) and NF-κB/p38/ JNK signaling (Song, 2016). ERK, P38, and JNK are differentially activated in spinal glial cells after nerve injury, leading to the synthesis of pro-inflammatory/pronociceptive mediators, thereby enhancing and prolonging pain. Inhibition of all three MAPK pathways has been shown to attenuate inflammatory and neuropathic pain in different animal models (Zhang et al., 2018). Therefore, we further investigated the regulation of these kinases after AMPK stimulation in vivo in the spinal cord. Formalin-induced activation of ERK, p38, and JNK was significantly inhibited by treating mice with MOTS-c, possibly contributing to the antinociceptive effects in the inflammatory models. However, it must be pointed out that distinct AMPK target proteins might also contribute to the effects on nociception and that an interplay of different AMPK downstream effectors might lead to the drastic improvement of the nociceptive behavior. The transcription factor c-fos is a well-known downstream effector of MAP-kinases and a well-accepted marker for inflammatory and nociceptive response in the spinal cord neurons following peripheral stimulation (Bullitt, 1990; Harris, 1998; Zhang et al., 2002). Thus, the strength of nociceptive signaling can be correlated with the number of c-fos positive neurons. MOTS-c treatment significantly reduced the formalin-induced c-fos positive cell number in the ipsilateral dorsal horn of the spinal cord, indicating that inhibition of the MAPKs–c-fos signaling pathway by MOTS-c-activated AMPK plays a crucial role in mediating antinociceptive and anti-inflammatory effects. MOTS-c exerted antinociceptive and anti-inflammatory effects in this study. The unique involvement of the interplay between the folate cycle, AICAR, and AMPK signaling in MOTS-c action provide an interesting opportunity to expand our understanding of the role of MOTS-c. However, no specific signal receptor has been identified for MOTS-c yet, and AMPK is the only molecule it is known to functionally interact with. The identification of MOTS-c-specific binding partners may provide further insight into understanding the action mechanism of MOTSc. Taken together, our results indicated that systemic administration of MOTS-c exerted antinociceptive and anti-inflammatory effects, at least partially, by activating AMPK pathway and inhibiting ERK, JNK, and P38 MAP kinases–c-fos signaling pathways. Therefore, MOTS-c is a novel and potential therapeutic agent against inflammatory pain.
Xinqiang Yin: Conceptualization, Methodology, Investigation, Formal analysis, Writing- Original draft preparation. Yuanyuan Jing: Formal analysis, Writing- Reviewing and Editing. Quan Chen: Investigation, Validation. Abdul Baset Abbas: Methodology, WritingReviewing and Editing. Jialiang Hu: Writing- Reviewing and Editing, Supervision. Hanmei Xu: Supervision, Project administration, Funding acquisition. All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the Project Program of State Key Laboratory of Natural Medicines (no. SKLNMBZ201403), and the National Science and Technology Major Projects of New Drugs (2018ZX 09201001004001, 2018ZX09301039002, 2018ZX 09301053001) in China. This project was also funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Nanchong City-North Sichuan Medical College Cooperative Scientific Research Project (18SXHZ0292). References Asiedu, M.N., Dussor, G., Price, T.J., 2016. Targeting AMPK for the alleviation of pathological pain. Exp. Suppl. 107, 257–285. Bullitt, E., 1990. Expression of c-fos-like protein as a marker for neuronal activity following noxious stimulation in the rat. J. Comp. Neurol. 296, 517–530. Cunha, T.M., Verri Jr., W.A., Silva, J.S., Poole, S., Cunha, F.Q., Ferreira, S.H., 2005. A cascade of cytokines mediates mechanical inflammatory hypernociception in mice. Proc. Natl. Acad. Sci. U. S. A. 102, 1755–1760. Giri, S., 2004. 5-aminoimidazole-4-carboxamide-1- -4-ribofuranoside inhibits proinflammatory response in glial cells: a possible role of AMP-activated protein kinase. J. Neurosci. 24, 479–487. Grahame Hardie, D., 2014. AMP-activated protein kinase: a key regulator of energy balance with many roles in human disease. J. Intern. Med. 276, 543–559. Han, P., Liu, S., Zhang, M., Zhao, J., Wang, Y., Wu, G., et al., 2015. Inhibition of spinal interlukin-33/ST2 signaling and downstream ERK and JNK pathways in electroacupuncture analgesia in formalin mice. PLoS One 10, e0129576. Hardie, D.G., 2004. The AMP-activated protein kinase pathway – new players upstream and downstream. J. Cell Sci. 117, 5479–5487. Harris, J.A., 1998. Using c-fos as a neural marker of pain. Brain Res. Bull. 45, 1–8. Hoogendijk, A.J., Pinhanços, S.S., van der Poll, T., Wieland, C.W., 2013. AMP- activated protein kinase activation by 5-aminoimidazole-4-carbox-amide-1-β-D-ribofuranoside (AICAR) reduces lipoteichoic acid-induced lung inflammation. J. Biol. Chem. 288, 7047–7052. Jeong, H.W., Hsu, K.C., Lee, J.W., Ham, M., Huh, J.Y., Shin, H.J., Kim, W.S., Kim, J.B., 2009. Berberine suppresses proinflammatory responses through AMPK activation in macrophages. Am. J. Physiol. Endocrinol. Metab. 296, E955–E964. Ji, R.-R., Gereau, R.W., Malcangio, M., Strichartz, G.R., 2009. MAP kinase and pain. Brain Res. Rev. 60, 135–148. Kim, K.H., Son, J.M., Benayoun, B.A., Lee, C., 2018. The mitochondrial-encoded peptide MOTS-c translocates to the nucleus to regulate nuclear gene expression in response to metabolic stress. Cell Metabol. 28, 516–524 e7. Kim, S.-J., Xiao, J., Wan, J., Cohen, P., Yen, K., 2017. Mitochondrially derived peptides as novel regulators of metabolism. J. Physiol. 595, 6613–6621. Lee, C., Zeng, J., Drew, Brian G., Sallam, T., Martin-Montalvo, A., Wan, J., Kim, S.-J., Mehta, H., Hevener, Andrea L., de Cabo, R., Cohen, P., 2015. The mitochondrialderived peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin resistance. Cell Metabol. 21, 443–454. Lee, Y.S., Kim, Y.S., Lee, S.Y., Kim, G.H., Kim, B.J., Lee, S.H., Lee, K.U., Kim, G.S., Kim, S.W., Koh, J.M., 2010. AMP kinase acts as a negative regulator of RANKL in the differentiation of osteoclasts. Bone 47, 926–937. Lihn, A.S., Pedersen, S.B., Lund, S., Richelsen, B., 2008. The anti-diabetic AMPK activator AICAR reduces IL-6 and IL-8 in human adipose tissue and skeletal muscle cells. Mol. Cell. Endocrinol. 292, 36–41. Lu, H., Wei, M., Zhai, Y., Li, Q., Ye, Z., Wang, L., Luo, W., Chen, J., Lu, Z., 2019. MOTS-c peptide regulates adipose homeostasis to prevent ovariectomy-induced metabolicdysfunction. J. Mol. Med. (Berl.) 97, 473–485. Melemedjian, O.K., Asiedu, M.N., Tillu, D.V., Sanoja, R., Yan, J., Lark, A., Khoutorsky, A., Johnson, J., Peebles, K.A., Lepow, T., Sonenberg, N., Dussor, G., Price, T.J., 2011. Targeting adenosine monophosphate-activated protein kinase (AMPK) in preclinical
5. Conclusion The present study demonstrated that the intraperitoneal administration of MOTS-c produced antinociceptive and anti-inflammatory effects in the mouse formalin test. The antinociceptive and anti-inflammatory effects of MOTS-c, at least partially, depended on activation of AMPK and inhibition of MAP kinases–c-fos signaling pathway.
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models reveals a potential mechanism for the treatment of neuropathic pain. Mol. Pain 7, 70. Ming, W., Lu, G., Xin, S., Huanyu, L., Yinghao, J., Xiaoying, L., Chengming, X., Banjun, R., Li, W., Zifan, L., 2016. Mitochondria related peptide MOTS-c suppresses ovariectomyinduced bone loss via AMPK activation. Biochem. Bioph. Res. Co. 476, 412–419. Petti, C., Vegetti, C., Molla, A., Bersani, I., Cleris, L., Mustard, K.J., Formelli, F., Hardie, G.D., Sensi, M., Anichini, A., 2012. AMPK activators inhibit the proliferation of human melanomas bearing the activated MAPK pathway. Melanoma Res. 22, 341–350. Price, T.J., Das, V., Dussor, G., 2016. Adenosine monophosphate-activated protein kinase (AMPK) activators for the prevention, treatment and potential reversal of pathological pain. Curr. Drug Targets 17, 908–920. Price, T.J., Dussor, G., 2013. AMPK: an emerging target for modification of injury-induced pain plasticity. Neurosci. Lett. 557 Pt A, 9–18. Russe, O.Q., Möser, C.V., Kynast, K.L., King, T.S., Stephan, H., Geisslinger, G., Niederberger, E., 2013. Activation of the AMP-activated protein kinase reduces inflammatory nociception. J. Pain 14, 1330–1340. Salt, I.P., Palmer, T.M., 2012. Exploiting the anti-inflammatory effects of AMP-activated protein Kinase activation. Expert Opin. Investig. Drugs 21, 1155–1167. Singewald, N., Salchner, P., Sharp, T., 2003. Induction of c-Fos expression in specific areas of the fear circuitry in rat forebrain by anxiogenic drugs. Biol. Psychiatry 53, 275–283. Song, T., Ma X, Gu K, Yang Y, Yang L, Ma P, et al., 2016. Thalidomide represses inflammatory response and reduces radiculopathic pain by inhibiting IRAK-1 and NF-
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Full length article
The intraperitoneal administration of MOTS-c produces antinociceptive and anti-inflammatory effects through the activation of AMPK pathway in the mouse formalin test
T
Xinqiang Yina,b, Yuanyuan Jingc, Quan Chena, Abdul Baset Abbasa, Jialiang Hua,d, Hanmei Xua,d,∗ a
The Engineering Research Center of Synthetic Polypeptide Drug Discovery and Evaluation of Jiangsu Province, China Pharmaceutical University, Nanjing, 210009, China School of Basic Medical Sciences, North Sichuan Medical College, Nanchong, 673000, China c Department of Preventive Medicine, North Sichuan Medical College, Nanchong, 637000, China d State Key Laboratory of Natural Medicines, Ministry of Education, China Pharmaceutical University, Nanjing, 210009, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: MOTS-C Formalin test Antinociception Anti-inflammation AMP activated protein kinase
The activation of the AMP activated protein kinase (AMPK) exerts antinociceptive effects in acute and neuropathic pain models. Mitochondrial open-reading-frame of the twelve S rRNA-c (MOTS-c), a mitochondrial-derived peptide, regulates many biological activities via activating AMPK. However, the role of MOTS-c in the formalin-induced inflammatory nociception remains unclear. In this study, we investigated the role of MOTS-c in the formalin-induced inflammatory nociception. The antinociceptive effect of MOTS-c was assessed by recording the time spent licking paw. The anti-inflammatory effect of MOTS-c was evaluated by detecting the inflammatory cytokine level changes in the mouse serum. Western blot was used to detect the changes of protein phosphorylation levels in the mouse spinal cord. Changes of c-fos expression in the spinal cord were assessed by immunohistochemistry. Our results showed that the intraperitoneal administration of MOTS-c reduced the time spent on licking in phase 2 in a dose-dependent manner in the formalin test. The antinociceptive effects of MOTS-c (50 mg/kg, i.p.) were attenuated by the AMPK antagonist compound C (10 mg/kg, i.p.). MOTS-c (50 mg/kg, i.p.) significantly reduced pro-inflammatory cytokine levels and elevated the level of anti-inflammatory cytokine in mouse serum. In addition, MOTS-c treatment significantly increased AMPKα phosphorylation level and suppressed formalin-induced extracellular signal-regulated kinase (ERK), c-Jun aminoterminal kinases (JNK), and P38 activation and c-fos expression in the mouse spinal cord. These results suggest that systemic administration of MOTS-c exerts antinociceptive and anti-inflammatory effects, at least partially, through activating AMPK pathway and inhibiting MAP kinases–c-fos signaling pathway in the mouse formalin test.
1. Introduction Mitochondrial open-reading-frame of the twelve S rRNA-c (MOTSc), a novel bioactive mitochondrial-derived peptide encoded from the 12S rRNA region of mitochondria, was firstly identified in 2015 (Lee et al., 2015). MOTS-c is expressed in various tissues of rodents and humans and can be secreted into blood, which suggests that it plays a role in both a cell-autonomous and hormonal way (Lee et al., 2015). Recent studies have shown that MOTS-c is a versatile peptide. MOTS-c prevented age-dependent or high-fat diet-induced insulin resistance, as well as diet-induced obesity through regulating glucose metabolism by activating AMPK pathway (Kim et al., 2017). MOTS-c suppressed
ovariectomy-induced bone loss via AMPK activation (Ming et al., 2016). In another study, researchers found that MOTS-c exerted an anti-inflammatory effect via suppressing mitogen-activated protein kinases (MAPKs) and increasing aryl hydrocarbon receptor/signal transducer and activator of transcriptional 3 (Ahr/STAT3) signaling pathways (Zhai et al., 2017). Recently, Kim and colleagues revealed that MOTS-c could dynamically translocate to the nucleus in response to metabolic stress and regulate adaptive nuclear gene expression in an AMPK-dependent manner (Kim et al., 2018). The above research results reveal that MOTS-c functions via activating AMPK pathway. Previous studies showed that AMPK, a kinase widely expressed in various tissues, constitutes an important regulator of metabolism
∗ Corresponding author. The Engineering Research Center Synthetic Polypeptide Drug Discovery and Evaluation of Jiangsu Province, China Pharmaceutical University, Nanjing, 210009, China. E-mail addresses: [email protected], [email protected] (H. Xu).
https://doi.org/10.1016/j.ejphar.2020.172909 Received 17 May 2019; Received in revised form 10 December 2019; Accepted 6 January 2020 Available online 08 January 2020 0014-2999/ © 2020 Elsevier B.V. All rights reserved.
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2.3. Formalin test
(Hardie, 2004; Grahame Hardie, 2014). Recent evidence indicated a crucial role of AMPK in the acute and neuropathic pain models (Asiedu et al., 2016; Giri, 2004; Melemedjian et al., 2011; Price et al., 2016; Tillu et al., 2012). AMPK activators, such as AICAR and metformin, exerted anti-inflammatory and antinociceptive effects in formalin- and zymosan-induced paw inflammation models (Russe et al., 2013). Regulation of the AMPKα2 subunit of the kinase played a key role in the antinociceptive effects (Russe et al., 2013). On the molecular level, antinociceptive effects were mediated by reduced activation of different MAP-kinases in the spinal cord and a subsequent decrease in pain-relevant induction of c-fos, a reliable marker of elevated activity in spinal cord neurons following peripheral noxious stimulation (Harris, 1998; Russe et al., 2013). In addition, resveratrol exhibited antinociceptive effects by engaging AMPK to attenuate extracellular signal-regulated kinase (ERK) and the mammalian target of rapamycin (mTOR) signaling in sensory neurons in incision-induced acute and chronic pain (Tillu et al., 2012). MOTS-c targets the methionine-folate cycle, increases 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) levels, and eventually activates AMPK (Lee et al., 2015). The activation of AMPK is responsible for the antinociceptive effects in many models (Asiedu et al., 2016; Giri, 2004; Melemedjian et al., 2011; Price et al., 2016; Tillu et al., 2012). Therefore, we investigated the effects of MOTS-c on the nociceptive response induced by formalin. We found that the intraperitoneal administration of MOTS-c produced antinociceptive and anti-inflammatory effects through the activation of AMPK pathway in the mouse formalin test.
The formalin test procedure was performed as previously described with slight modifications (Sufka et al., 1998). Mice were placed in a plexiglass box (15 cm in diameter and 20 cm in height) with a mirror placed under the floor at a 45° angle to allow an unobstructed view of the paw and were allowed to habituate for 30 min. Then, a 5% formaldehyde solution (formalin, 20 μl) was injected subcutaneously into the dorsal surface of the left hind paw. Right after formalin injection, the time spent licking the formalin-injected paw was recorded at 5-min intervals up to 40 min. The first 10 min were recorded as early nociceptive phase (phase I) and the period from 11 to 40 min was recorded as the inflammatory phase (phase II). 2.4. Protocols for evaluating the effect of compound C on MOTS-c-induced antinociception To evaluate the effect of compound C on MOTS-c-induced antinociception in the mouse formalin test, animals were randomly divided into four groups: Control group (saline + saline group), saline + MOTS-c group, compound C + saline group, and compound C + MOTS-c group. The mice in the control group received two intraperitoneal injections of saline (10 μl/g) 260 min and 240 min before formalin stimulation, respectively. The second group of mice received intraperitoneal administration of saline (10 μl/g) and MOTS-c (50 mg/ kg) 260 min and 240 min before formalin stimulation, respectively. The third group of mice received intraperitoneal administration of compound C (10 mg/kg) and saline (10 μl/g) 260 min and 240 min before formalin stimulation, respectively. The fourth group of mice received intraperitoneal administration of compound C (10 mg/kg) and MOTS-c (50 mg/kg) 260 min and 240 min before formalin stimulation, respectively.
2. Materials and methods 2.1. Animals Male ICR mice (25–30 g) were obtained from the Experimental Animal Center of Qinglong Mountain in Nanjing, China. Animals had free access to food and water and were housed in an animal room that was maintained at 22 ± 2 °C with a 12 h light/dark cycle. All animals were cared for and experiments were carried out in accordance with the European Community guidelines for the use of experimental animals (2010/63/EU). All the protocols in this study were approved by the Ethics Committee of China Pharmaceutical University (SYXK (Su) 2018–0019). Mice were randomly divided into normal group, control group, and drug treatment group. There was no intervention in the normal group. The mice in the control group received intraperitoneal injection of sterile saline before subcutaneous injection of formalin into the dorsal surface of the left hind paw. The mice in the drug treatment group received intraperitoneal administration of drugs before formalin stimulation. The collections and analysis of data were performed by two independent observers blinded to the group assignment.
2.5. Cytokine detection The mice were deeply anesthetized with trichloroacetaldehyde hydrate (350 mg/kg, i.p.) 2 h after formalin injection and euthanized by exanguination after blood collection via cardiac puncture. The blood was centrifuged at 800×g for 15 min at 4 °C to obtain serum. The levels of tumor necrosis factor-α (TNF-α), interleukine-1β (IL-1β), interleukine-10 (IL-10), and interleukine-6 (IL-6) in mouse serum were measured using Enzyme-linked immunosorbent assays (ELISA) kits (Nanjing Jin Yibai Biological Technology Co. Ltd., Nanjing, China) according to the manufacturer's instructions. 2.6. Western blot For Western blot analysis, the mice were deeply anesthetized with trichloroacetaldehyde hydrate (350 mg/kg, i.p.) and then euthanized by exsanguination 2 h after formalin injection. Lumbar spinal cords (L3L5) were dissected out for downstream analysis. Total proteins in tissues were extracted immediately after preparation using Whole Cell Lysis Assay kits (Wanleibio). The protein concentrations were quantified using BCA protein assay kits (Wanleibio). Proteins in tissue homogenates (20 μg) were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes by wet-blotting. Membranes were blocked for 1 h at room temperature with 5% bovine serum albumin (BSA). Then the blots were incubated overnight at 4 °C with primary antibodies against AMPKα (Thr172), p-AMPKα (Thr172), P38, p-P38 (Thr180/Tyr182), c-Jun amino-terminal kinases (JNK), p-JNK1/2 (Thr183/Tyr185), ERK, and p-ERK1/2 (Thr202/Tyr204) (1:500 in blocking buffer, Affinity Biosciences) in blocking buffer. The blots were incubated with a secondary antibody (1:5000 in blocking buffer, Lianke) for 60 min after washing 3 times with TBST. Then, the filters were developed by enhanced chemiluminescence reagents (Wanleibio). Glyceraldehyde-3-
2.2. Drugs MOTS-c peptide (sequence: Met-Arg-Trp-Gln-Glu-Met-Gly-Tyr-IlePhe-Tyr-Pro-Arg-Lys-Leu-Arg) with a purity of more than 95% was synthesized by GL Biochem Co., Ltd. (Shanghai, China). Morphine hydrochloride was purchased from Shenyang First Pharmaceutical Factory (China). Formaldehyde was purchased from Sigma-Aldrich (St. Louis, MO, USA). The rabbit anti-c-fos antibody was obtained from Santa Cruz. Compound C 2HCl, which was used as an AMPK inhibitor, was purchased from Selleck. Trichloroacetaldehyde hydrate was obtained from Sinopharm Chemical Reagent Co., Ltd. MOTS-c, compound C, morphine, and trichloroacetaldehyde hydrate were dissolved in sterile saline (0.9% NaCl) at a concentration of 10 mg/ml, 1 mg/ml, 1 mg/ml, and 100 mg/ml, respectively. The rabbit anti-c-Fos antibody was diluted with PBS (1:500). MOTS-c and morphine were intraperitoneally injected 4 h and 20 min prior to formalin injection, respectively. 2
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phosphate dehydrogenase (GAPDH) (Santa Cruz) was used as the loading control. Densitometries of the blots were analyzed using the Image J2x software. 2.7. c-fos immunohistochemistry Mice were deeply anesthetized with trichloroacetaldehyde hydrate (350 mg/kg, i.p.) 2 h after the formalin injection. Then mice were perfused transcardially with 20 ml saline followed by 20 ml 4% paraformaldehyde in 0.1 M phosphate buffer. L4 and/or L5 lumbar segment was dissected out and post-fixed in the same fixative overnight at 4 °C. The segments were processed for c-fos immunoreactivity as described previously (Singewald et al., 2003). The number of c-fos-positive cells in the ipsilateral dorsal spinal cord was counted manually by an observer blind to the treatment using ImageJ2x software. 2.8. Statistical analysis The results were expressed as mean ± S.E.M. Statistical analysis was performed using IBM SPSS statistics 22 software. In the formalin test, for statistical analysis, data from the first phase and the second phase were considered independently. One-way ANOVA with Bonferroni test post hoc test was used to evaluate the dose-dependent effect of MOTS-c on formalin-induced licking and the effect of compound C on MOTS-c induced antinociception. One-way ANOVA with Bonferroni test post hoc test was also used to examine the effect of MOTS-c on the levels of the serum inflammatory cytokines in the mice of formalin test. Student's t-test was used to compare the number of cfos-like immunoreactivity positive neurons between the MOTS-c treatment group and the control group. In all statistical comparisons, differences with P < 0.05 were considered significant differences. 3. Results Fig. 1. MOTS-c and morphine attenuated inflammatory nociception in the formalin test. (A) Time course of the licking behavior in mice after injection of formalin (5%, 20 μl) into the left hind paw with pretreatment with saline or MOTS-c. The effects of intraperitoneally administrated saline, MOTS-c (12.5, 25, 50, and 100 mg/kg) (B), and morphine (5 mg/kg, 10 mg/kg) (C) on the licking time in phase 1 (0–10 min) and phase 2 (11–40 min) in the mouse formalin test. Formalin was injected at time 0. Saline or MOTS-c was intraperitoneally administrated 4 h prior to formalin. Morphine was intraperitoneally administrated 20 min prior to formalin.The time spent licking the injected paw was measured in 5-min intervals for 40 min. Data are presented as the mean number of licking time ± S.E.M. (n = 6–8 for each group). *P < 0.05, **P < 0.01, ***P < 0.001 versus saline according to one-way ANOVA followed by Bonferroni test.
3.1. Intraperitoneal pretreatment with MOTS-c produced antinociceptive effect in a dose-dependent manner in the mouse formalin test In the formalin test, the typical biphasic pain-like behavior with a first licking phase from 1 to 10 min and a second phase from 11 to 40 min was found after formalin injection (Fig. 1A). Treatment of mice with MOTS-c (12.5, 25, 50, and 100 mg/kg, i.p.) decreased the time spent on licking in phase 2 in a dose-dependent manner (Fig. 1B). However, after MOTS-c (12.5, 25, 50, and 100 mg/kg, i.p.) treatment, the time spent on licking in the first phase did not show a significant difference in comparison with the control group (Fig. 1B). The Bonferroni's test revealed that MOTS-c (25, 50, and 100 mg/kg, i.p.) significantly inhibited the second phase of formalin-induced pain behaviors in comparison with the control group (Fig. 1B). However, 12.5 mg/kg MOTS-c failed to produce a significant antinociceptive effect in comparison with the control group. Under the same condition, morphine (5, 10 mg/kg, i.p) significantly attenuated the nociceptive response induced by formalin in both phases (Fig. 1C). The time spent on licking in phase 2 between the MOTS-c (50 mg/kg) group and the morphine (5 mg/kg) group had no significant difference (Fig. 1C).
3.3. MOTS-c suppressed pro-inflammatory cytokine production, and promoted anti-inflammatory cytokine secretion in serum To assess the effect of MOTS-c on the formalin-induced acute inflammation in the mouse formalin test, the concentrations of the serum inflammatory cytokines were measured by ELISA. Compared to the normal group, production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the control group were significantly increased. Moreover, administration of MOTS-c (50 mg/kg, i.p.) significantly reduced the inflammatory cytokine levels (TNF-α, IL-1β and IL-6) in the mouse serum in comparison with the control group (Fig. 3A–C). In addition, the concentration of IL-10 in serum was significantly elevated after MOTS-c treatment (Fig. 3D). However, morphine did not change the effect induced by formalin on cytokine concentrations (Fig. 3A–D).
3.2. Compound C effectively reversed the antinociceptive effect of MOTS-c in the mouse formalin test The effect of compound C, an AMPK inhibitor, on the time spent on licking was examined. Compared to the control group, injection of compound C (10 mg/kg, i.p.) followed with saline (10 μl/g, i.p.) had no effect on pain behavior response induced by formalin (Fig. 2). However, intraperitoneal injection of compound C (10 mg/kg) 20 min before MOTS-c treatment (50 mg/kg, i.p.), significantly reversed the antinociceptive effect of MOTS-c (Fig. 2). 3
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3.4. MOTS-c promoted AMPK activation and inhibited MAPKs activation in the spinal cord 3.4.1. Administration of MOTS-c increased AMPKα phosphorylation level in the spinal cord It has been reported that the activation of AMPK played crucial roles in models of acute and neuropathic nociception and inflammatory nociception. Therefore, we investigated the effects of MOTS-c (50 mg/kg, i.p.) on AMPK activation in the mouse spinal cord 2 h after formalin injection by Western blot analysis. Our results showed that administration of MOTS-c (50 mg/kg, i.p.) significantly increased the AMPKα phosphorylation level in the spinal cord in comparison with the control group (Fig. 4A). In addition, compared to the MOTS-c group, AMPKα phosphorylation in the spinal cord was significantly inhibited by the AMPK inhibitor compound C (10 mg/kg, i.p.) (Fig. 4A). 3.4.2. MOTS-c suppressed fomalin-induced ERK, JNK, and P38 phosphorylation in the spinal cord To investigate AMPK-activated signal transduction mechanisms that might be involved in the antinociceptive effects, we analyzed the impact of MOTS-c-mediated AMPK activation on the activation of ERK, JNK, and P38 MAP-kinases in the formalin assay. These kinases have already been described as potential AMPK targets and important mediators of inflammatory nociception. As expected, formalin induced a significantly increased phosphorylation and thereby activation of ERK (Fig. 4B), JNK (Fig. 4C), and P38 (Fig. 4D) in comparison with the normal group. Compared to the control group, MOTS-c (50 mg/kg, i.p.) treatment significantly suppressed phosphorylation of ERK, JNK, and P38 in the spinal cord, which indicated that these effects may contribute to the antinociceptive effects observed after MOTS-c (50 mg/kg, i.p.) treatment (Fig. 4B–D). Furthermore, AMPK inhibitor compound C treatment (10 mg/kg, i.p.) significantly reversed the inhibitory effects of MOTS-c (50 mg/kg, i.p.) on formalin-induced increases of ERK, JNK, and P38 phosphorylation in the spinal cord in comparison with the
Fig. 2. The impact of compound C (10 mg/kg, i.p.)) on the antinociceptive effect of MOTS-c (50 mg/kg, i.p.) in the mouse formalin test. MOTS-c was intraperitoneally administrated 4 h prior to formalin. Compound C was intraperitoneally administrated 20 min prior to MOTS-c. Saline was intraperitoneally injected in the corresponding time. The time spent licking the injected paw was measured in 5-min intervals for 40 min. Data are presented as the mean number of licking time ± S.E.M. (n = 7–8 for each group). **P < 0.01 versus control according to one-way ANOVA followed by Bonferroni test.
Fig. 3. MOTS-c treatment reduced the levels of inflammatory cytokines in the mice serum in the formalin test. 2 h after formalin injection, mice were killed and blood samples were collected and the concentrations of TNF-α (A), IL-1β (B), IL-6 (C), and IL-10 (D) were measureded by ELISA in the normal, control (10 μl/g, i.p.), MOTSc (50 mg/kg, i.p.), and Morphine (5 mg/kg, i.p.) group. Results are expressed as mean ± S.E.M. (n = 6–8 for each group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus control according to one-way ANOVA followed by Bonferroni test. 4
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Fig. 4. MOTS-c activated AMPK and suppressed formalin-induced phosphorylation of ERK, JNK, and P38 in the spinal cord. The spinal cord was dissected out 2 h after formalin injection and the levels of phosphorylated AMPK and MAP kinases were evaluated by densitometric analysis of the blots. (A) AMPK activation, (B) ERK activation, (C) JNK activation, and (D) P38 activation. The blots showed representative samples. All samples derived from the same experiment and that blots were processed in parallel. AMPK and p-AMPK were normalized to the expression of GAPDH on the same membrane. Each phosphoprotein was normalized to the expression of the corresponding total protein of the same sample in Fig. 5B–D. Results are expressed as mean ± S.E.M. (n = 6–8 for each group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 according to one-way ANOVA followed by Bonferroni test.
in the formalin-injected mice. Compared to the control group, MOTS-c (50 mg/kg, i.p.) significantly suppressed the expression of c-fos protein in the ipsilateral dorsal horn of the spinal cord (Fig. 5).
MOTS-c treatment group (Fig. 4B–D). 3.5. MOTS-c suppressed formalin-induced c-fos protein expression in the spinal cord
4. Discussion c-fos is a downstream effector of MAP-kinases and a well-accepted marker for inflammatory and nociceptive response. We investigated the effect of MOTS-c (50 mg/kg, i.p.) on spinal cord c-fos protein expression
MOTS-c, a mitochondria-derived peptide identified in 2015, has been detected in various tissues, as well as in circulation of human and 5
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Fig. 5. The number of c-fos-like immunoreactivity positive cells between the control group and the MOTS-c (i.p, 50 mg/kg) treatment group. Data are shown as mean ± S.E.M. (n = 7 for control and n = 8 for 50 mg/kg MOTS-c, respectively). **P < 0.01 versus control according to Student's t-test.
We also found that the antinociceptive effects of AMPK activation were accompanied by anti-inflammatory effects, which was in good agreement with several previous studies showing anti-inflammatory effects in LPS-induced acute lung injury models (Wang et al., 2016; Hoogendijk et al., 2013) as well as in macrophages (Jeong et al., 2009), the adipose tissue of patients and skeletal muscles of rats (Lihn et al., 2008) after AMPK stimulation. Formalin induced paw edema and increased pro-inflammatory cytokine levels such as TNF-α, IL-1β, and IL-6. The release of pro-inflammatory cytokines is an important cause of inflammatory nociception (Cunha et al., 2005; Xiang et al., 2019). TNF-α, induced by carrageenan in carrageenan-induced inflammatory hypernociception in mice, acts on tumor necrosis factor receptor 1 (TNF-R1) to stimulate the release of IL-1β and IL-6 (Cunha et al., 2005). The mature fragment of IL-1β, which is expressed by activated macrophages as proprotein and proteolytically processed by Caspase 1, induces sensitization of nerve fibers of primary sensory neurons to cause pain hypersensitivity (Xiang et al., 2019). IL-6 is an intermediate in TNF-α-induced IL-1β release and induces the production of prostanoids (Cunha et al., 2005). In the mouse formalin test, formalin-induced production of pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) may be one of the causes of the inflammatory nociception. Furthermore, it has been reported that AMPK activation inhibits pro-inflammatory processes via inhibition of nuclear factor-κB (NF-κB) signaling, inhibition of MAPK pathways, regulation of reactive oxygen species, inhibition of the Janus kinase/signal transducer and activator of transcriptions (JAK-STAT) signaling, inhibition of leukocyte infiltration, regulation of cytokine synthesis, and regulation of lipid metabolism (Salt and Palmer, 2012). The current study found that intraperitoneal administration of MOTS-c produced antinociceptive and anti-inflammatory effects in the mouse formalin test. The anti-inflammatory effects may also contribute to the antinociceptive effect. With regard to the downstream effector of AMPK, it has been reported that AMPK activation inhibits the activation of different MAP kinases (Lee et al., 2010; Petti et al., 2012). Furthermore, several lines
rodent plasma (Lee et al., 2015). The wide distribution of MOTS-c indicates its key roles in organisms. It has been reported that MOTS-c might be involved in many physiological and pathological functions via activation of AMPK (Lee et al., 2015; Ming et al., 2016; Zhai et al., 2017; Kim et al., 2018; Lu et al., 2019). Recent studies suggested that the activation of AMPK might have an impact on pain in animal models of neuropathy and acute nociception (Asiedu et al., 2016; Giri, 2004; Melemedjian et al., 2011; Price et al., 2016; Tillu et al., 2012). In this study, we explored the role of MOTS-c in formalin-induced inflammatory nociception and found that intraperitoneal administration of MOTS-c produced antinociceptive effect in the mouse formalin test. We hypothesized that the antinociceptive effect of MOTS-c was through activating AMPK signaling. Therefore, compound C, a wellknown AMPK inhibitor, was used in this study to test whether the inhibitory effect of MOTS-c on formalin-induced nociception behaviors was mediated through the activation of AMPK. The results showed that intraperitoneal injection of compound C (10 mg/kg) alone had no effect on the formalin-induced licking response. However, administration of compound C 20 min prior to MOTS-c significantly attenuated the antinociceptive effect induced by MOTS-c (50 mg/kg, i.p.) treatment. Furthermore, Western blot results showed that the phosphorylation level of AMPKα was significantly improved in the mouse spinal cord after MOTS-c (50 mg/kg, i.p.) treatment and compound C significantly reversed MOTS-c induced AMPKα phosphorylation. All these results indicated that the antinociceptive effect of MOTS-c was due to the activation of AMPK. MOTS-c targets the methionine-folate cycle, increases AICAR levels, and eventually activates AMPK (Lee et al., 2015). Through its active metabolite ZMP, AICAR binds to the AMP-binding site of the γ subunit of AMPK to activate it. It has been considered that other AMP-regulated molecules, such as adenosine receptors, glycogen phosphorylase, and fructose-1, 6-bisphosphatase (FBPase), which are also involved in the regulation of nociceptive processing, might be interfered by AICAR (Russe et al., 2013). Therefore, it cannot be excluded that the observed antinociceptive effects are mediated via one or more of these targets. 6
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Authors’ contributions
of studies have shown that AMPK activation negatively regulates aberrant translation control after nerve injury, resolves neuropathic allodynia and decreases sensory neuron excitability through suppressing mTOR and MAPKs (Price and Dussor, 2013). Accumulating evidence showed that MAP kinase family members, particularly ERK, p38, and JNK, have been associated with the development of pain hypersensitivity during inflammation and neuropathy through their effects on different signaling mechanisms (Ji et al., 2009). ERK activation in spinal dorsal horn neurons by nociceptive activity plays an important role in central sensitization by regulating the activity of glutamate receptors and potassium channels and inducing gene transcription. By inhibiting the spinal interleukine-33/the signal transduction society (IL-33/STS) signaling and the downstream ERK and JNK pathway, electroacupuncture alleviated formalin-induced inflammatory pain (Han et al., 2015). Thalidomide repressed inflammatory response and reduced radiculopathic pain by inhibiting interleukin-1 receptor-associated kinase 1 (IRAK-1) and NF-κB/p38/ JNK signaling (Song, 2016). ERK, P38, and JNK are differentially activated in spinal glial cells after nerve injury, leading to the synthesis of pro-inflammatory/pronociceptive mediators, thereby enhancing and prolonging pain. Inhibition of all three MAPK pathways has been shown to attenuate inflammatory and neuropathic pain in different animal models (Zhang et al., 2018). Therefore, we further investigated the regulation of these kinases after AMPK stimulation in vivo in the spinal cord. Formalin-induced activation of ERK, p38, and JNK was significantly inhibited by treating mice with MOTS-c, possibly contributing to the antinociceptive effects in the inflammatory models. However, it must be pointed out that distinct AMPK target proteins might also contribute to the effects on nociception and that an interplay of different AMPK downstream effectors might lead to the drastic improvement of the nociceptive behavior. The transcription factor c-fos is a well-known downstream effector of MAP-kinases and a well-accepted marker for inflammatory and nociceptive response in the spinal cord neurons following peripheral stimulation (Bullitt, 1990; Harris, 1998; Zhang et al., 2002). Thus, the strength of nociceptive signaling can be correlated with the number of c-fos positive neurons. MOTS-c treatment significantly reduced the formalin-induced c-fos positive cell number in the ipsilateral dorsal horn of the spinal cord, indicating that inhibition of the MAPKs–c-fos signaling pathway by MOTS-c-activated AMPK plays a crucial role in mediating antinociceptive and anti-inflammatory effects. MOTS-c exerted antinociceptive and anti-inflammatory effects in this study. The unique involvement of the interplay between the folate cycle, AICAR, and AMPK signaling in MOTS-c action provide an interesting opportunity to expand our understanding of the role of MOTS-c. However, no specific signal receptor has been identified for MOTS-c yet, and AMPK is the only molecule it is known to functionally interact with. The identification of MOTS-c-specific binding partners may provide further insight into understanding the action mechanism of MOTSc. Taken together, our results indicated that systemic administration of MOTS-c exerted antinociceptive and anti-inflammatory effects, at least partially, by activating AMPK pathway and inhibiting ERK, JNK, and P38 MAP kinases–c-fos signaling pathways. Therefore, MOTS-c is a novel and potential therapeutic agent against inflammatory pain.
Xinqiang Yin: Conceptualization, Methodology, Investigation, Formal analysis, Writing- Original draft preparation. Yuanyuan Jing: Formal analysis, Writing- Reviewing and Editing. Quan Chen: Investigation, Validation. Abdul Baset Abbas: Methodology, WritingReviewing and Editing. Jialiang Hu: Writing- Reviewing and Editing, Supervision. Hanmei Xu: Supervision, Project administration, Funding acquisition. All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the Project Program of State Key Laboratory of Natural Medicines (no. SKLNMBZ201403), and the National Science and Technology Major Projects of New Drugs (2018ZX 09201001004001, 2018ZX09301039002, 2018ZX 09301053001) in China. This project was also funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Nanchong City-North Sichuan Medical College Cooperative Scientific Research Project (18SXHZ0292). References Asiedu, M.N., Dussor, G., Price, T.J., 2016. Targeting AMPK for the alleviation of pathological pain. Exp. Suppl. 107, 257–285. Bullitt, E., 1990. Expression of c-fos-like protein as a marker for neuronal activity following noxious stimulation in the rat. J. Comp. Neurol. 296, 517–530. Cunha, T.M., Verri Jr., W.A., Silva, J.S., Poole, S., Cunha, F.Q., Ferreira, S.H., 2005. A cascade of cytokines mediates mechanical inflammatory hypernociception in mice. Proc. Natl. Acad. Sci. U. S. A. 102, 1755–1760. Giri, S., 2004. 5-aminoimidazole-4-carboxamide-1- -4-ribofuranoside inhibits proinflammatory response in glial cells: a possible role of AMP-activated protein kinase. J. Neurosci. 24, 479–487. Grahame Hardie, D., 2014. AMP-activated protein kinase: a key regulator of energy balance with many roles in human disease. J. Intern. Med. 276, 543–559. Han, P., Liu, S., Zhang, M., Zhao, J., Wang, Y., Wu, G., et al., 2015. Inhibition of spinal interlukin-33/ST2 signaling and downstream ERK and JNK pathways in electroacupuncture analgesia in formalin mice. PLoS One 10, e0129576. Hardie, D.G., 2004. The AMP-activated protein kinase pathway – new players upstream and downstream. J. Cell Sci. 117, 5479–5487. Harris, J.A., 1998. Using c-fos as a neural marker of pain. Brain Res. Bull. 45, 1–8. Hoogendijk, A.J., Pinhanços, S.S., van der Poll, T., Wieland, C.W., 2013. AMP- activated protein kinase activation by 5-aminoimidazole-4-carbox-amide-1-β-D-ribofuranoside (AICAR) reduces lipoteichoic acid-induced lung inflammation. J. Biol. Chem. 288, 7047–7052. Jeong, H.W., Hsu, K.C., Lee, J.W., Ham, M., Huh, J.Y., Shin, H.J., Kim, W.S., Kim, J.B., 2009. Berberine suppresses proinflammatory responses through AMPK activation in macrophages. Am. J. Physiol. Endocrinol. Metab. 296, E955–E964. Ji, R.-R., Gereau, R.W., Malcangio, M., Strichartz, G.R., 2009. MAP kinase and pain. Brain Res. Rev. 60, 135–148. Kim, K.H., Son, J.M., Benayoun, B.A., Lee, C., 2018. The mitochondrial-encoded peptide MOTS-c translocates to the nucleus to regulate nuclear gene expression in response to metabolic stress. Cell Metabol. 28, 516–524 e7. Kim, S.-J., Xiao, J., Wan, J., Cohen, P., Yen, K., 2017. Mitochondrially derived peptides as novel regulators of metabolism. J. Physiol. 595, 6613–6621. Lee, C., Zeng, J., Drew, Brian G., Sallam, T., Martin-Montalvo, A., Wan, J., Kim, S.-J., Mehta, H., Hevener, Andrea L., de Cabo, R., Cohen, P., 2015. The mitochondrialderived peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin resistance. Cell Metabol. 21, 443–454. Lee, Y.S., Kim, Y.S., Lee, S.Y., Kim, G.H., Kim, B.J., Lee, S.H., Lee, K.U., Kim, G.S., Kim, S.W., Koh, J.M., 2010. AMP kinase acts as a negative regulator of RANKL in the differentiation of osteoclasts. Bone 47, 926–937. Lihn, A.S., Pedersen, S.B., Lund, S., Richelsen, B., 2008. The anti-diabetic AMPK activator AICAR reduces IL-6 and IL-8 in human adipose tissue and skeletal muscle cells. Mol. Cell. Endocrinol. 292, 36–41. Lu, H., Wei, M., Zhai, Y., Li, Q., Ye, Z., Wang, L., Luo, W., Chen, J., Lu, Z., 2019. MOTS-c peptide regulates adipose homeostasis to prevent ovariectomy-induced metabolicdysfunction. J. Mol. Med. (Berl.) 97, 473–485. Melemedjian, O.K., Asiedu, M.N., Tillu, D.V., Sanoja, R., Yan, J., Lark, A., Khoutorsky, A., Johnson, J., Peebles, K.A., Lepow, T., Sonenberg, N., Dussor, G., Price, T.J., 2011. Targeting adenosine monophosphate-activated protein kinase (AMPK) in preclinical
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