Background
Morphine is the most commonly used drug for the treatment of severe pain. However, long-term morphine treatment leads to tolerance which greatly attenuates analgesic effect and diminishes clinical utilization. Therefore, investigating mechanisms of morphine tolerance and identification of solutions are of clinical significance.
Among the previous studies, mechanisms of morphine tolerance are complex and involve many factors, such as ion channels, receptors, cells, and neural networks [
1‐
3]. For a long time, extensive studies suggested that neurons participate in the development of morphine tolerance [
3,
4]. However, compelling evidences recently show that glia cells, especially microglia, play a pivotal role in the initiation and maintenance of morphine tolerance [
5,
6].
Microglia in the spinal cord are significantly activated by chronic morphine treatment. Several studies showed that morphine induces a proinflammatory response through binding to Toll-like receptor 4 (TLR4), leading to initiation of the TLR4 signaling cascade, as do direct modulators of p38 and nuclear factor-κB (NF-κB), subsequently regulating the expression of multiple inflammation factors [
7,
8]. Other studies established that activated microglia secrete large amounts of proinflammatory cytokines including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α), which could enhance the hyperactivity of dorsal horn neurons, induce central sensitization, and reduce the antinociceptive effect of morphine [
9,
10]. Thus, suppression of neuroinflammation by inhibiting microglial activation and proinflammatory cytokines could be a worthwhile strategy for enhancing morphine analgesic efficacy and attenuating morphine tolerance.
5′-Adenosine monophosphate-activated protein kinase (AMPK), a sensor of cellular energy change, regulates energy homeostasis and metabolic stress. Recent studies show that AMPK regulates both energy homeostasis and inflammatory defense [
11‐
13]. Activation of AMPK inhibits ATP-consuming anabolic processes (such as protein translation) mainly via inhibiting mammalian target of rapamycin (mTOR) signaling [
14]. AMPK activation also inhibits mitogen-activated protein kinase (MAPK) signaling. MAPK family, especially p38 mitogen-activated protein kinase (p38 MAPK) in activated microglia, have been shown to play an important role in morphine-induced neuroinflammation and tolerance [
15]. In the brain, AMPK activation inhibits lipopolysaccharide (LPS)-induced pro-inflammatory cytokines expression by modulating NF-κB in primary rat microglia [
16]. AMPK activation also inhibits the expression of pro-inflammatory mediators in the cerebral cortex of LPS-injected rats [
11]. Thus, AMPK may be an interesting target for neuroprotective drugs in inflammatory conditions, such as morphine tolerance. We hypothesized that AMPK activation may represent a novel pharmacological treatment to reduce morphine tolerance by suppressing morphine-induced neuroinflammation through attenuating microglial activation.
To test our hypothesis, we used metformin, a potent antihyperglycaemic agent that has previously been shown to active AMPK, to assess the effect of AMPK activation on morphine-induced microglial activation and tolerance.
Methods
Animals
Adult CD-1 mice (18–22 g) were purchased from the Experimental Animal Center at Nanjing Medical University, Nanjing, China. Five to six mice per cage were housed under pathogen-free conditions with soft bedding under controlled temperature (22 ± 2 °C) and a 12-h light/dark cycle (lights on at 8:00 a.m.). For each group of experiments, the animals were matched by age and body weight. Behavioral testing was performed during the light cycle (between 9:00 a.m. and 5:00 p.m.). Mice were allowed to acclimate to these conditions for at least 2 days before inclusion in experiments.
Reagents
All antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) unless stated otherwise. IL-1β was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ionized calcium binding adapter molecule 1 (IBA-1) were from Sigma-Aldrich (St. Louis, MO, USA) and Wako Pure Chemical Industries (Osaka, Japan). The p65/RelA and immunofluorescence IBA-1 antibodies were from Abcam (Cambridge, MA, USA). Immunofluorescence c-fos and calcitonin gene-related peptide (CGRP) antibodies were from Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Morphine hydrochloride was purchased from Shenyang First Pharmaceutical Factory, Northeast Pharmaceutical Group Company (Shenyang, China). Fetal bovine serum (FBS) and other cell culture media and supplements were purchased from Hyclone (Logan, UT, USA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Sunshine Biotechnology (Nanjing, China).
Cell preparation and stimulation
BV-2 cells mouse brain endothelial cells bEND3 were cultured in humidified 5% CO2 at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) FBS, penicillin (100 U/ml), and streptomycin (100 U/ml) (KeyGEN). For inducing inflammasome activation, 105 cells were plated in 6-well plate overnight, and the medium were changed to serum-free medium next morning and then the cells were treated with morphine (200 μM) with or without metformin for 6 h. Metformin (4, 20, or 100 μM) was administrated 15 min before morphine treatment. Cell extracts and precipitated supernatants were analyzed by immunoblotting.
Cell viability assessment
The cell viability was evaluated by CCK-8 assay (Dojindo Molecular Technologies, Inc.). BV-2 cells were plated in the 96-well plates (2.0 × 104 cell per well) and incubated for 24 h before experiments. The cells were washed with D-Hanks buffer solution. Two hundred microliters of CCK-8 solution was added to each well and incubated for an additional 1 h at 37 °C. The optical density (OD) of each well at 450 nm was recorded on a Microplate Reader (Thermo, Varioskan Flash). The cell viability (% of control) is expressed as the percentage of (ODtest − ODblank) / (ODcontrol − ODblank), where ODcontrol is the optical density of the control sample and ODblank is the optical density of the wells without BV-2 cells.
Tolerance models and behavioral analysis
Animals was habituated in the testing environments for 2 days and carried out behavioral testing in a blinded manner. For the test of chronic tolerance, mice were injected with saline or morphine (10 mg/kg) subcutaneously every 12 h for 7 days and analgesia was assessed 30 min later by the tail-flick assay [
17]. The test was performed by gently holding the mouse in a terry cloth towel and immersing between 2 and 3 cm from the tip of the tail into warm water (52 °C). A cutoff time of 10 s was set to avoid tissue damage. Data were calculated as percentage of maximal possible effect (% MPE), which was calculated by the following formula: 100% × [(Drug response time − Basal response time) / (10 s − Basal response time)] = % MPE. The experimenters were blinded to the treatment. Metformin (50, 100, or 200 mg/kg) was dissolved in saline and administered intraperitoneally 15 min before morphine treatment twice a day from day 1 to day 7.
NF-κB activation assay
Cells (BV2) were plated in class bottom cell culture dishes and treated with morphine (200 μM) for 2 h with or without metformin (100 μM). Cells were fixed with ice-cold methanol and were permeabilized with 0.25% Triton X-100/PBST. After blocking with 1% bovine serum albumin (BSA) in PBST for 1 h, the coverslips with BV-2 cells were incubated for 2 h at room temperature with the p65/RelA antibody diluted in 1% BSA (1:50). Then, the coverslips were exposed to the fluorescein isothiocyanate (FITC)-conjugated antirabbit IgG (1:100, at room temperature for 1 h) and then were rinsed three times with PBS. Finally, the coverslips were stained with 1 μg/mL DAPI (4′,6-diamidino-2-phenylindole, a fluorescent DNA dye to mark nucleus) for 1 min. Confocal microscopy analyses were carried out using Olympus FV1000 confocal system.
Analysis of mRNA levels by quantitative real-time polymerase chain reaction (PCR)
Cells samples were homogenized in Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and total RNA was treated by DNaseI and subjected to quantitative PCR, which was performed with ABI Prism 7300 sequence detection system (Applied Biosystems, Foster City, CA, USA) using SYBR Green I dye. The specific primer sequences for IL-1β, IL-6, TNF-α, TLR4, and GAPDH are listed as follows: IL-1β sense 5′-TCATTGTGGCTGTGGAGAAG-3′, antisense 5′-AGGCCACAGGTATTTTGTCG-3′, TNF-α sense 5′-CATCTTCTCAAAATTCGAGTGACAA-3′, antisense 5′-TGGGAGTAGACAAGGTACAACCC-3′, IL-6 sense 5′-ATCCAGTTGCCTTCTTGGGACTGA-3′, antisense 5′-TAAGCCTCCGACTTGTGAAGTGGT-3′, IL-4 sense 5′-CGAGGTCACAGGAGAAGG-3′, antisense 5′-TGAGGACGTTTGGCACAT-3′, TGF-β sense 5′-ATGGTGGACCGCAACAAC-3′, antisense 5′-GCACTGCTTCCCGAATGTC-3′, Toll-like receptor-4 (TLR-4) sense 5′-ACTGTTCTTCTCCTGCCTGACA-3′, antisense 5′-CCTAGTCTTTGAGTCGTTTCAGG-3′, IL-10 sense 5′-AACATACTGCTAACCGACTC-3′, antisense 5′-GGATCATTTCCGATAAGG-3′, GAPDH sense 5′-CAAAAGGGTCATCTCC-3′, and antisense 5′-CCCCAGCATCAAAGGTG-3′ GAPDH. Gene was used as an endogenous control to normalize for differences in the amount of total RNA in each sample.
Western blot
To identify temporal expression level of IBA-1, GAPDH, IL-1β, TNF-α, and the phosphorylated protein levels of p38 MAPK, N-methyl-d-aspartic acid receptor NR1 (NMDAR-NR1), PKCγ, protein samples were analyzed as described before. In brief, samples (cells or spinal cord tissue segments at L1-L6) were collected and washed with ice-cold PBS before being lysed in radio immunoprecipitation assay (RIPA) lysis buffer [Beyotime, Shanghai, China; 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mmol/L phenylmethylsulfonyl fluoride, 0.15 U/mL aprotinin, and 1 mg/mL pepstatin] and then sample lysates were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA, USA). The membranes were blocked with 10% whole milk in TBST (Tris-Hcl, NaCl, Tween 20) for 2 h at room temperature, probed with primary antibodies at 4 °C overnight [GAPDH, 1:8000; IBA-1, 1:1000; IL-1β, 1:1000; TNF-α, 1:1000; p-p38 (Tyr182), 1:1000; p-NR1(Ser896), 1:1000; p-PKCγ, 1:1000] and then incubated with horseradish peroxidase-coupled secondary antibodies from Cell Signaling Technology (Beverly, MA, USA). Data were acquired with the Molecular Imager (Gel DocTM XR, 170–8170) and analyzed with Quantity One-4.6.5 (Bio-Rad Laboratories, Berkeley, CA, USA).
Immunofluorescence assay
After anesthesia by intraperitoneal injection of sodium pentobarbital (100 mg/kg), the animal was perfused with normal saline followed by 4% paraformaldehyde in 0.1 M PBS, pH 7.2–7.4, for 20 min. Then, L4 and/or L5 lumbar segment were dissected out and post-fixed in the same fixative. The embedded blocks were sectioned as 30 μm thick and processed for immunofluorescence assay. Sections from each group (five mice in each group) were incubated with primary antibody (IBA-1, 1:200; c-fos, 1:200; CGRP, 1:200). Then, the free-floating sections were washed with PBS and incubated with the secondary antibody (1:300; Jackson Laboratories, USA) for 2 h at room temperature. After using PBS to wash three times, the samples were investigated with a confocal microscope (Leica TCS SPEII, Leica Biosystems, Wetzlar, Germany) for morphologic details. Images were randomly coded and transferred to a computer for analysis.
Statistical analysis
SPSS Rel 15 (SPSS Inc., Chicago, IL, USA) was used to conduct all the statistical analyses. Data were statistically evaluated by two-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests. The mean fluorescent pixels of IBA-1 and CGRP were measured by Image Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA). Results were represented as mean ± SEM of three independent experiments. Results described as significant were based upon a criterion of p < 0.05.
Discussion
In our study, we found that metformin, a potent antihyperglycaemic agent and AMPK activator, had a significant inhibitory effect on morphine-induced microglial activation. Metformin inhibited the morphine-induced up-regulation of p38 MAPK phosphorylation, NF-κB nuclear translocation, and proinflammatory cytokine expression in microglia, which were abolished by AMPK inhibition. Thus, metformin significantly attenuated the development of chronic morphine tolerance in an efficient manner. The study may provide a new solution by inhibiting microglial activation through increasing AMPK activation to improve clinical analgesic efficacy of morphine.
Non-neuronal cells, especially microglia, are crucial in the pathogenesis of morphine tolerance [
20]. Microglial activation has been shown to play an important role in cytokine release in the CNS [
21]. After chronic or acute exposure to morphine, activated microglia exhibit increased expression of pro-inflammatory cytokines, such as IL-1β, IL-6, TNF-α, and chemokines [
22,
23]. These changes contribute to morphine analgesic tolerance. In the present study, we found that morphine increased the mRNA expression of IL-1β, IL-6, and TNF-α in BV-2 cells (Fig.
1a–c). Interestingly, metformin, a potent antihyperglycemic agent, was found to inhibit cytokines production induced by morphine (Fig.
1), and AMPK inhibition abolished the effects of metformin (Fig.
2a–c). These data suggest that metformin may be beneficial to reducing microglial activation and morphine tolerance.
Previous studies have shown that both pro-inflammatory and anti-inflammatory cytokines are involved in the development and maintenance of morphine tolerance [
22,
24,
25]. IL-4, IL-10, and TGF-β are powerful anti-inflammatory cytokines with a wide spectrum of biological effects [
26‐
28]. Therefore, we tested whether metformin inhibit inflammatory response via regulating anti-inflammatory cytokines production. In accordance with the previous study [
26], we found that morphine did not affect the expression of anti-inflammatory cytokines mRNA level. However, metformin did not increase anti-inflammatory cytokines mRNA level (Fig. S1). These data suggest that the effects of metformin are mainly relates to the regulation of pro-inflammatory cytokines.
It is well known that NF-κB has diverse and complicated effects on the immune response and nervous systems [
29,
30]. Activation of NF-κB is one of the major events following the onset of an inflammatory response mainly initiated by proinflammatory cytokines [
31]. Activation of NF-κB also induces production of cytokines that activate NF-κB in cancer cells to induce chemokines that attract more inflammatory cells into the tumor [
32]. Morphine can induce the translocation of NF-κB p65 from the cytosol to the nucleus, and NF-κB inhibition can reverse the mRNA expression of IL-1β, IL-6, and TNF-α following morphine treatment [
33]. Therefore, NF-κB is an important transcription factor and plays critical roles in inflammation. Our study showed that morphine markedly increased NF-κB p65 level in the nucleus, which was reversed by metformin (Fig.
3). These data suggest that metformin may decrease the cytokines production via inhibiting activation of NF-κB.
Recent studies have demonstrated that the MAPK family, including p38 MAPK, extracellular signal-regulated protein kinase (ERK), and c-Jun N-terminal kinase, plays important roles in morphine tolerance [
34‐
36]. The expression of pro-inflammatory cytokines and other harmful signaling molecules is regulated by p38 MAPK/NF-κB signaling pathway in the CNS [
37]. Microglia inhibitor minocycline and p38 inhibitor SB203580 markedly attenuate morphine-induced pro-inflammatory cytokines production and inhibit morphine tolerance [
38,
39]. We found that morphine increased p38 MAPK phosphorylation, which was decreased by metformin (Fig.
1d). The effect of metformin was abolished by AMPK inhibitor (Fig.
2d). In addition, compelling evidence has suggested that morphine induces microglial activation through binding with the Toll-like receptor 4 (TLR4) expressed in spinal microglia, activating downstream intracellular signaling pathways, leading to the release of cytokines and suppression of inflammatory response. TLR4 also plays a critical role in p38 phosphorylation induced by morphine. In our study, we found that morphine treatment significantly increased the mRNA expression of TLR4 in BV-2 cells, which was abolished by metformin (Fig.
1e). These findings suggest that TLR4/p38 MAPK signaling pathway was involved in the protection effects of metformin. Metformin inhibit microglial activation via reducing the morphine-induced mRNA expression of TLR 4 and proinflammatory cytokines. Consistent with our findings, Eidson and Murphy reported that blockade of TLR 4 attenuates morphine tolerance and facilitates the pain relieving properties of morphine [
7].
Several studies have indicated that microvascular endothelial cells could be involved in morphine-induced neuroinflammation and play an important role [
40,
41]. We tested whether metformin-attenuated morphine tolerance relate to regulating states of microvascular endothelial cells. We found that metformin did not affect the expression of CCL2, TLR4, and IL-1β. However, metformin significantly decreased the upregulation of IL-6 and TNF-α mRNA level induced by morphine (Fig.
4d, e). These data showed that regulation of IL-6 and TNF-α mRNA level by metformin in microvascular endothelial cells was partially involved in the attenuation of morphine tolerance.
Furthermore, we investigated whether metformin could attenuate morphine tolerance in vivo. In the present study, behavioral tests showed that the mice developed allodynia and hyperalgesia following morphine withdrawal (Fig.
5). Metformin reduced chronic morphine tolerance in a dose-dependent manner. Previous studies have shown that expression of the microglial maker IBA-1 is significantly increased when microglia are activated. Our data showed that resveratrol notably inhibited microglial activation, suppressing the up-regulated IBA-1 expression in the spinal dorsal horn (Fig.
6).
The NMDA receptor, regulating neuronal activity and synaptic efficacy, plays an important role in various inflammation states [
21]. The NMDA receptor 1 (NR1) is preferentially phosphorylated by PKCγ [
42]. PKCγ activation plays well-developed role on central sensitization in morphine tolerance, which may contribute to increasing the excitability of nociceptive neurons [
43]. Our results showed that metformin provide an inhibition in the activation of PKCγ and the phosphorylation of NMDA receptors NR1 in morphine tolerance mice (Fig.
8a). These data suggest that metformin may inhibit microglial activation and further suppress central sensitization occurring in the spinal cord, which contribute to the attenuation of morphine tolerance. In addition, calcitonin gene-related peptide (CGRP) and c-fos have been implicated in pain transmission and morphine tolerance [
44‐
46] and have been considered as the indicators of morphine tolerance. It is widely distributed in central nervous system and peripheral organs in rodents. Our group has demonstrated that the increase of CGRP and c-fos release induced by morphine could be almost completely abolished by metformin (Fig.
8b). These data suggest that metformin could be an operative medicine to attenuate morphine tolerance.