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Research Paper

N-acetyl-cysteine attenuates neuropathic pain by suppressing matrix metalloproteinases

Li, Jiajiea,b; Xu, Lujiea,b; Deng, Xuetinga,b; Jiang, Chunyia,c; Pan, Cailonga; Chen, Lua,b; Han, Yuana,d; Dai, Wenlinga,b; Hu, Lianga; Zhang, Guangqinb; Cheng, Zhixiangc; Liu, Wentaoa,*

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doi: 10.1097/j.pain.0000000000000575
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1. Introduction

Neuropathic pain, defined as “pain caused by a lesion or disease of the somatosensory nervous system,”1 has remained a clinical challenge. Despite decades of extensive studies on its treatment and underlying mechanisms, a safer and more effective solution remains to be found.

Among all the pain-related bioactive substances, matrix metalloproteinase (MMP)-9 and MMP-2 are thought to be critical in the development and maintenance of neuropathic pain.25 MMPs are widely implicated in inflammation and tissue remodeling because of their cleavage of extracellular matrix proteins, cytokines, and chemokines.30 Matrix metalloproteinases can regulate diverse biological processes, including tissue remodeling,10 development, and angiogenesis.44 Specifically, MMP-9/2 may be produced in injured dorsal root ganglion (DRG) neurons and trigger spinal microglial activation and neuropathic pain development.25 Matrix metalloproteinase-9 and -2 also enhance neuronal transmission by phosphorylating the N-methyl-D-aspartate receptor (NR) 1 and NR2B in neurons.29

Based on the information mentioned above, MMP-9 and MMP-2 are considered potential targets for the treatment of neuropathic pain. Unfortunately, a specific, safe, and effective MMP-9/2 inhibitor that can be used in the clinic is not yet available. Given the important roles of MMP-9/2 in neuropathic pain, we were encouraged to examine an “old” drug that has been used clinically in terms of the management of neuropathic pain through the inhibition of MMP-9/2.

The search for a drug that targets MMP-9/2 led us to the question of how MMPs are activated. The “cysteine switch” is a unique structure that is involved in MMP-9/2 activation.43 The cysteine switch contains 2 crucial parts; the first is a critical cysteine residue in the propeptide domain, and the second is a zinc-binding site in the catalytic domain. The modification of the cysteine residues (eg, by S-nitrosylation,14 oxidization,11 and alkylation) and the dissociation of the cysteine residue from the zinc-binding site lead to MMP-9/2 activation.

Because the modification of the cysteine residue is vital to the activation of MMP-9/2, an agent that prevents the cysteine residue on MMP-9/2 from being oxidized would provide a viable solution for the treatment of neuropathic pain. N-acetyl-cysteine (NAC) is a medicine that is commonly used clinically as a mucolytic agent and antidote for acetaminophen overdose.39 There is also good evidence that NAC is effective in the treatment of contrast-induced nephropathy, idiopathic pulmonary fibrosis, influenza, and chronic obstructive pulmonary disease.32 N-acetyl-cysteine has an excellent safety history and contains abundant cysteine residues that are indicative of its potential ability to interfere with the process of the “cysteine switch” during MMP activation. In this study, we provide the first evidence that NAC significantly attenuates chronic constrictive injury (CCI)-induced neuropathic pain through potently inhibiting MMP-9/2 activity.

2. Materials and methods

2.1. Ethics statement

All procedures were strictly performed in accordance with the regulations of the ethics committee of the International Association for the Study of Pain and the Guide for the Care and Use of Laboratory Animals (The Ministry of Science and Technology of China, 2006). All animal experiments were approved by Nanjing Medical University Animal Care and Use Committee and were designed to minimize suffering and the number of animals used.

2.2. Animals and neuropathic pain model

Adult male Sprague-Dawley rats (180-200 g) were provided by the Experimental Animal Center at Nanjing Medical University, Nanjing, China. Animals were housed 5 to 6 per cage under pathogen-free conditions with soft bedding under controlled temperature (22 ± 2°C) and a 12-hour light/dark cycle (lights on at 8:00 am). Behavioral testing was performed during the light cycle (between 9:00 am and 5:00 pm). The animals were allowed to acclimate to these conditions for at least 2 days before starting experiments. Animals were randomly divided into groups (n = 6. The sample size was designed on previous experience28 and to be limited to the minimal as scientifically justified). For each group of experiments, the animals were matched by age and body weight. All surgeries were performed under anesthesia induced by chloral hydrate (300 mg/kg, i.p.). Peripheral nerve injury was imitated by the model of CCI of the sciatic nerve. In brief, the left common sciatic nerve of each rat was exposed at the mid-thigh level. Proximal to the sciatic nerve's trifurcation, approximately 7 mm of nerve was separated from adhering tissue and 4 ligatures (4-0 chronic gut) were tied loosely around it with about 1 mm between ligatures. After surgery, the skin layers and muscle were sutured, and the surgery area was sterilized with iodine.

2.3. Drugs and reagents

N-acetyl-cysteine and sulfasalazine were from Sigma (St. Louis, MO). LY341495 was from MedChem Express (Monmouth Junction, NJ). LY341495 and sulfasalazine were both dissolved in 0.1M NaOH and the pH were adjusted to 7.5 before injection. Antibody for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was from Sigma (St. Louis, MO). Antibodies for ionized calcium-binding adapter molecule 1 (IBA-1), glial fibrillary acidic protein (GFAP), phosphorylated NR1 subunit (Ser896), phosphorylated protein kinase C (PKC) (pan) (gamma Thr514), phosphorylated p38 (Tyr182), phosphorylated extracellular regulated protein kinase (ERK) (Thr202/Tyr204), phosphorylated c-Jun N-terminal kinase (JNK) (Thr183/Tyr185), and calcitonin gene-related peptide (CGRP) were from Cell Signaling Technology (Beverly, MA). Secondary antibodies were from Cell Signaling Technology. Polyclonal antibody for interleukin (IL)-1β was from Santa Cruz (Dallas, TX). All other chemicals were purchased from Sigma Chemical Co (St. Louis, MO).

2.4. Gelatin zymography

Animals were anesthetized deeply with chloral hydrate (300 mg/kg, intraperitoneally), and spinal cord segments were rapidly dissected and homogenized in 1% NP40 lysis. Three hundred to 500 μg of protein per lane was loaded into the wells of precast gels (8% polyacrylamide gels containing 0.1% gelatin). After electrophoresis, each gel was incubated with 50 mL of developing buffer for 48 hours (37.5°C) in shaking bath. Then the gels were stained with Coomassie brilliant blue (1%, with 10% acetic acid, 10% isopropyl alcohol, diluted with dd H2O).

2.5. Western blotting

The spinal cord segments at L1-L6 were rapidly removed and homogenized in RIPA Lysis Buffer after the animals' deep anesthesia with chloral hydrate. The protein concentrations were determined by BCA Protein Assay (Thermo Fisher, Waltham, MA), and 40 to 80 µg of proteins were loaded and separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore Corp, Bedford, MA). The membranes were blocked with 5% bovine serum albumin for 1 hour at room temperature, probed with antibodies overnight at 4°C with the primary antibodies, and then incubated with Horseradish Peroxidase (HRP)-coupled secondary antibodies. The primary antibodies used included IL-1β (1:500), p-NR1 (1:1000), p-PKCγ (1:1000), p-p38 (1:1000), p-JNK (1:1000), p-ERK (1:1000), IBA-1 (1:1000), and GFAP (1:1000). For loading control, the blots were probed with antibody for GAPDH (1:8000). The filters were then developed by enhanced chemiluminescence reagents (PerkinElmer) with secondary antibodies (Chemicon). Data were analyzed with the Molecular Imager (Gel DocTM XR, 170-8170) and the associated software Quantity One-4.6.5 (Bio-Rad Laboratories, Berkeley, CA).

2.6. Behavioral analysis

Animals were habituated to the testing environment daily for at least 2 days before baseline testing. Mechanical sensitivity was detected by Von Frey hairs (Woodland Hills, Los Angeles, CA) test. Animals were placed in boxes, set on an elevated metal mesh floor, and were allowed 30 minutes for habituation before testing. The plantar surface of each hind paw was stimulated with a series of Von Frey hairs with logarithmically incrementing stiffness perpendicularly to the plantar surface. Each rat was tested for 3 times and the average of the threshold was measured.

For testing thermal hyperalgesia, rats' foot withdrawal latency to heat stimulation was measured. An analgesia meter (UGO Basile, Gemonio, Varese, Italy) was used to provide a heat source. In brief, each rat was placed in a box on a smooth, temperature-controlled glass floor. The heat source was focused on a portion of the hind paw, which was flushed toward the glass, so that a radiant thermal stimulus was delivered to that site. The stimulus shuts off when the hind paw withdrew (or the stimulus was removed after 20 seconds to prevent tissue damage). The intensity of the heat stimulus was maintained constant throughout all experiments. The elicited paw movement occurred at latency between 9 and 14 seconds in control animals. Thermal stimuli were delivered 3 times to each hind paw at 5- to 6-minute intervals. Behavioral tests were performed blindly.

2.7. Immunofluorescence

After deep anesthesia by intraperitoneal injection of chloral hydrate (300 mg/kg), the animal was perfused transcardially with normal saline followed by 4% paraformaldehyde in 0.1 M PB, pH 7.4, each for 20 minutes. Then, L4 and/or L5 lumbar segment was dissected out and post-fixed in 4% paraformaldehyde. The embedded blocks were sectioned as 25 μm thick. Sections from each group (5 animals in each group) were incubated with rabbit antibodies for IBA-1 (1:200), GFAP (1:200) or CRGP (1:200). Then, the free-floating sections were washed with PBS (0.01 M) and incubated with the secondary antibody for 2 hours. After washing out 3 times with PBS, the samples were studied under an immunofluorescence microscope (Zeiss AX10; Carl Zeiss, Oberkochen, Germany) for morphologic details of the immunofluorescence staining. Examination was blindly performed. Images were randomly coded and the fluorescence intensities were analyzed by Image-Pro Plus 6.0 software (Media Cybernetics Inc, Rockville, MD). The average green fluorescence intensity of each pixel was normalized to the background intensity in the same image.

2.8. Statistical analyses

SPSS Rel 15 (SPSS Inc, Chicago, IL) was used to conduct all the statistical analyses. Alteration of expression of the proteins detected and the behavioral responses were tested with 1-way analysis of variance (ANOVA), and the differences in latency over time among groups were tested with 2-way ANOVA. Bonferroni post hoc tests were conducted for all ANOVA models. Results are expressed as mean ± SEM of 3 independent experiments. Results described as significant are based on a criterion of P < 0.05.

3. Results

3.1. N-acetyl-cysteine directly inhibited the activities of matrix metalloproteinase-9 and matrix metalloproteinase-2 in vitro

First, we measured the mechanical thresholds of CCI-treated rats using the Von Frey test. Fourteen days after CCI surgery, the mechanical thresholds were reduced to 5.2 g (Fig. 1A). We then evaluated CCI-induced MMP-9 and MMP-2 activations with gelatin zymography. Matrix metalloproteinase-9 and MMP-2 were significantly activated in the spinal cords of the CCI-treated rats (Fig. 1B–D). N-acetyl-cysteine was administered to evaluate its ability to inhibit the activities of MMP-9 and MMP-2 in vitro. Spinal cord samples from normal (Fig. 1H and I) and CCI-treated rats (Fig. 1E–G) were loaded into each well for electrophoresis. Next, the gel pieces loaded with the proteins were incubated with different concentrations of NAC. N-acetyl-cysteine significantly inhibited the activities of MMP-9 and MMP-2 in vitro (Fig. 1E–G). Moreover, NAC also inhibited the activities of MMP-9 and MMP-2 in the basal condition (Fig. 1H–J).

F1
Figure 1.:
N-acetyl-cysteine (NAC) directly inhibits the chronic constrictive injury (CCI)–induced activations of matrix metalloproteinase (MMP)-9 and MMP-2. (A) Mechanical allodynia was significantly induced after CCI surgery. Mechanical pain threshold was tested by Von Frey hairs. (B–D) MMP-9 and MMP-2 were significantly activated on CCI 14 days. The lumbar spinal cords (L1-L6) were collected and analyzed 14 days after the CCI operation. Gelatin zymography was performed to determine MMPs' activity. Representative bands and a data summary (n = 4) are shown. (E–G) Representative bands and data summary showed the effect of NAC on MMPs' activity in the spinal cords from CCI rats. The lumbar spinal cords (L1-L6) from rats treated with CCI operation for 14 days were separated and homogenized. The same sample was loaded into each well of precast gels for gelatin zymography. After electrophoresis, the gels were cut into pieces. Each piece has 3 electrophoresis lanes on it and was then incubated in buffer with or without NAC (10−6, 10−5, or 10−4 mol/L). Then the gel pieces were stained with 1% Coomassie brilliant blue in the same box and captured in the same image. (H–J) Representative bands and a data summary showed the effect of NAC on MMPs' activity in the spinal cords from normal rats. The same sample was loaded into each well of precast gels for gelatin zymography. After electrophoresis, the gel pieces were incubated in buffer with or without NAC (10−7, 10−6, 10−5, or 10−4 mol/L). Significant difference was revealed after one-way analysis of variance (*P < 0.05, **P < 0.01; Bonferroni post hoc tests).

3.2. Single administrations of N-acetyl-cysteine attenuated chronic constrictive injury–induced neuropathic pain and suppressed the chronic constrictive injury–induced activations of matrix metalloproteinase-9 and matrix metalloproteinase-2 in vivo

To investigate the effects of NAC on MMP-9 and MMP-2 in vivo, single doses of NAC were orally administered to CCI rats. Von Frey hair tests and thermal withdrawal latency tests were performed. Fourteen days after CCI surgery, the mechanical thresholds were decreased by 6.59 g, and the thermal thresholds were reduced by 6.99 seconds in the CCI-treated rats. N-acetyl-cysteine (100, 200 mg/kg, orally) increased the mechanical threshold to 10.21 g after 2 hours. Similarly, the thermal withdrawal threshold increased to 11.52 seconds after 2 hours (Fig. 2A and B). Gelatin zymography results revealed that NAC (200 mg/kg, orally) inhibited the activities of MMP-9 and MMP-2 in the rats' spinal cords in vivo (Fig. 2C–E), which agreed with our in vitro data.

F2
Figure 2.:
Single administration of N-acetyl-cysteine (NAC) attenuates chronic constrictive injury (CCI)-induced neuropathic pain and suppresses the CCI-induced activation of matrix metalloproteinases (MMPs) in vivo. (A) Single administration of NAC (25, 50, 100, 200 mg/kg, orally) significantly attenuated CCI-induced mechanical allodynia (n = 6). (B) Single administration of NAC (25, 50, 100, 200 mg/kg, orally) could significantly attenuate CCI-induced thermal hyperalgesia (n = 6). (C–E) Effects of various doses of NAC (50, 100, 200 mg/kg, orally) on MMPs' activity in the spinal cords. Representative bands and a data summary (n = 4) were shown. Significant difference was revealed after one-way or 2-way analysis of variance (**P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs CCI group; Bonferroni post hoc tests).

3.3. Continuous administration of N-acetyl-cysteine significantly alleviated chronic constrictive injury–induced neuropathic pain and suppressed the chronic constrictive injury–induced activation of matrix metalloproteinase-9 and matrix metalloproteinase-2

Furthermore, the action of NAC was studied through continuous administration 14 days after CCI surgery. In the CCI group, the rats' mechanical thresholds were reduced from 12.36 to 5.18 g, and the thermal thresholds were reduced from 12.36 to 6.30 seconds. After continuous administration of NAC (100 mg/kg, orally) for 7 days, the mechanical thresholds were elevated to 9.88 g, and the thermal thresholds increased to 9.67 seconds (Fig. 3A and B). Moreover, NAC (100 mg/kg, orally) significantly inhibited the activity of MMP-9 in the spinal cords of the CCI-treated rats in vivo (Fig. 3C and D).

F3
Figure 3.:
Chronic administration of N-acetyl-cysteine (NAC) significantly alleviates chronic constrictive injury (CCI)-induced neuropathic pain and suppresses the CCI-induced activations of matrix metalloproteinases (MMPs) in vivo. (A) Consecutive administration of NAC (50, 100 mg/kg, orally) for 7 days significantly attenuated CCI-induced mechanical allodynia (n = 6). (B) Consecutive administration of NAC (50, 100 mg/kg, orally) for 7 days significantly attenuated CCI-induced thermal hyperalgesia (n = 6). Behavior tests were performed at 30 minutes after NAC treatment. (C and D) Effects of NAC (100 mg/kg, orally) on MMPs' activity in the spinal cords. Representative bands and a data summary (n = 4) were shown. Significant difference was revealed after one-way or 2-way analysis of variance (**P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs CCI group; Bonferroni post hoc tests).

3.4. Preadministration of N-acetyl-cysteine dramatically delayed and attenuated chronic constrictive injury–induced neuropathic pain

Previous reports have demonstrated that MMP-9 induces neuropathic pain primarily in the early stages, whereas MMP-2 may maintain neuropathic pain in the later stage.25 Therefore, we detected the effects of NAC in the early stage of neuropathic pain. N-acetyl-cysteine (100 mg/kg, orally) was administered consecutively for 7 days before neuropathic pain was elicited. Interestingly, NAC significantly delayed the formation of neuropathic pain. N-acetyl-cysteine administration increased the mechanical threshold and the thermal threshold by 38.5% and 51.4%, respectively, compared with the CCI group on the 14th day (Fig. 4A and B).

F4
Figure 4.:
Pretreatment with N-acetyl-cysteine (NAC) significantly delays the occurrence of chronic constrictive injury (CCI)-induced neuropathic pain and attenuates the pain behaviors. (A) Consecutive administration of NAC (100 mg/kg, p.o.) for 7 days right after CCI surgery showed a delay and attenuation of CCI-induced mechanical allodynia (n = 6). (B) Consecutive administration of NAC (100 mg/kg, orally) for 7 days right after CCI surgery showed a delay and attenuation of CCI-induced thermal hyperalgesia (n = 6). Significant difference was revealed after 2-way analysis of variance (*P < 0.05, **P < 0.01 vs control; ##P < 0.01 vs CCI group; Bonferroni post hoc tests).

3.5. Readministration of N-acetyl-cysteine effectively attenuated chronic constrictive injury–induced neuropathic pain

Because the neuropathic pain reoccurred 28 days after CCI, in addition to the administration of NAC from days 1 to 7, NAC (100 mg/kg, orally) was again administered to the rats for another 7 continuous days, beginning on the 28th day. N-acetyl-cysteine also obviously effectively alleviated mechanical allodynia and thermal hyperalgesia at this time, and excitingly, the effects of NAC were not reduced compared with the first treatment; the mechanical threshold rebounded from 1.95 g to 7.38 g, and the thermal threshold rebounded from 6.49 to 8.7 seconds (Fig. 5A and B). Together, these results demonstrated that NAC effectively alleviated CCI-induced mechanical allodynia and thermal hyperalgesia while exhibiting no indications of drug resistance.

F5
Figure 5.:
Retreatment with N-acetyl-cysteine (NAC) after withdrawal effectively attenuates chronic constrictive injury (CCI)-induced neuropathic pain. N-acetyl-cysteine effectively alleviated CCI-induced mechanical allodynia (A) and thermal hyperalgesia (B). After NAC withdrawal for 21 days, CCI-induced neuropathic pain formed again. The rats were then retreated with NAC (100 mg/kg, orally) consecutively for 7 days (n = 6). Significant difference was revealed after 2-way analysis of variance (**P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs CCI group; Bonferroni post hoc tests).

3.6. Single administrations of N-acetyl-cysteine significantly alleviated matrix metalloproteinase-9 or matrix metalloproteinase-2-induced mechanical allodynia

To uncover the direct correlations between NAC and MMPs, MMP-9 (1.2 pmol/15 μL) or MMP-2 (1.2 pmol/15 μL) was intrathecally administered. The mechanical thresholds of the MMP-9 and MMP-2 groups were reduced from 12.27 to 6.05 g but the thermal thresholds did not change after administration. Moreover, NAC (100 mg/kg, orally) administered 15 minutes after MMP-9 or MMP-2 blocked the MMP-9-induced and or MMP-2-induced mechanical allodynia (Fig. 6).

F6
Figure 6.:
Single administration of N-acetyl-cysteine (NAC) significantly alleviates matrix metalloproteinase (MMP)-9 and MMP-2–induced mechanical allodynia. (A) N-acetyl-cysteine significantly attenuated MMP-9–induced mechanical allodynia. (B) Matrix metalloproteinase-9 did not cause thermal hyperalgesia. (C) N-acetyl-cysteine significantly attenuated MMP-2–induced mechanical allodynia. (D) Matrix metalloproteinase-2 did not cause thermal hyperalgesia. N-acetyl-cysteine (100 mg/kg, orally) was administrated 15 minutes after MMP-9 (1.2 pmol/15 μL, i.t.) or MMP-2 (1.2 pmol/15 μL, i.t.) given (n = 6). Significant difference was revealed after 2-way analysis of variance (**P < 0.01 vs control; ##P < 0.01 vs CCI group; Bonferroni post hoc tests).

3.7. N-acetyl-cysteine significantly inhibited chronic constrictive injury–induced interleukin-1β cleavage, protein kinase Cγ phosphorylation, and NR1 phosphorylation

The above results proved that NAC significantly inhibited the activation of MMP-9/2 and attenuated the CCI-induced neuropathic pain. A critical substrate of MMP-9 might be IL-1β, which is essential for pain generation. In this study, we report that the continuous administration of NAC blocked the production of mature IL-1β induced by CCI but elicited no marked effect on the level of pro-IL-1β (Fig. 7A and B).

F7
Figure 7.:
N-acetyl-cysteine (NAC) significantly attenuates chronic constrictive injury (CCI)-induced neuron activation and interleukin (IL)-1β production in the spinal cord. (A and B) Consecutive administration of NAC significantly inhibited the cleavage of IL-1β but did not affect the production of pro-IL-1β. N-acetyl-cysteine (100 mg/kg, orally) was administered daily from day 14 to day 28 after CCI operation. (C–F) Single administration of NAC inhibited CCI-induced phosphorylation of protein kinase C (PKC) γ and N-methyl-D-aspartate receptor (NR) 1. N-acetyl-cysteine (50, 100, or 200 mg/kg, orally) was administered at day 14 after CCI operation. (G–J) Consecutive administration of NAC inhibited CCI-induced phosphorylation of PKCγ and NR1. (K and L) Consecutive administration of NAC significantly inhibited CCI-induced activation of calcitonin gene-related peptide in the dorsal horns. Quantification of immunofluorescence was represented as mean fluorescence pixels in the superficial dorsal horns (n = 6 images per animal). N-acetyl-cysteine (100 mg/kg, orally) was administered daily from day 14 to day 28 after CCI operation. The lumbar spines (L1-L6) were collected and analyzed 120 minutes after the last administration. Significant difference was revealed after 1-way or 2-way analysis of variance (*P < 0.05, **P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs CCI group; Bonferroni post hoc tests).

Interleukin-1β is expressed in DRG neurons and some satellite cells. We next evaluated NAC's influence on the neuronal activation induced by the CCI operation. N-acetyl-cysteine dramatically decreased the CCI-induced phosphorylation of PKCγ and NR1 after both single administration (50, 100, 200 mg/kg, orally; Fig. 7C–F) and continuous administration (100 mg/kg, orally; Fig. 7G–J). Our immunofluorescence results also revealed a suppressive effect of NAC on the nociceptive-related activation of CGRP in the dorsal horn of the spinal cord in the CCI model (Fig. 7K and L).

3.8. N-acetyl-cysteine significantly inhibited chronic constrictive injury–induced MAPK family phosphorylation

Accumulating evidence indicates that all 3 MAPK pathways contribute to pain sensitization after tissue and nerve injury through distinct molecular and cellular mechanisms.22 The activation (phosphorylation) of MAPKs under different persistent pain conditions results in the induction and maintenance of pain hypersensitivity. Therefore, we tested the effects of NAC on the MAPK family using Western blot. Single administrations of NAC (50-200 mg/kg) significantly reduced the CCI-induced phosphorylation of the 3 major members of the MAPK family, ie, p38, JNK, and ERK, in the spinal cords (Fig. 8A–D).

F8
Figure 8.:
N-acetyl-cysteine (NAC) significantly inhibits chronic constrictive injury (CCI)–induced phosphorylation of mitogen-activated protein kinase in the spinal cord. (A) Single administration of NAC inhibited CCI-induced phosphorylation of p38, JNK, and ERK MAPKs. N-acetyl-cysteine (50, 100, or 200 mg/kg, orally) was administered at day 14 after CCI operation. (B–D) Data summary (n = 4) was shown. Significant difference was revealed after one-way analysis of variance (*P < 0.05, **P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs CCI group; Bonferroni post hoc tests).

3.9. N-acetyl-cysteine significantly inhibited chronic constrictive injury–induced ionized calcium-binding adapter molecule 1 activation without influencing glial fibrillary acidic protein

Neuronal synaptic plasticity and central sensitization result in the excessive activation of neurons. Overactivated neurons release numerous substances, such as glutamate, ATP, and CCL2, which activate glial cells.33 Once activated, glial cells, including microglia and astrocytes,21,33 synthesize and release inflammatory factors (eg, IL-1β and TNF-α),33 proteinases (eg, MMPs),25 and chemokines (eg, CCL2),21 which can further enhance neuronal signaling.13 This crosstalk between glia and neurons causes central sensitization and therefore aggravates neuropathic pain. Glial activation and the crosstalk between glia and neurons also enhance neuronal transmission. We next examined whether NAC affected the activation of glia induced by the CCI operations. Our results revealed that NAC (100 mg/kg, orally) significantly suppressed the upregulation of the microglia marker IBA-1 in the spinal cord after the CCI operation (Fig. 9A and B). Immunofluorescence for IBA1 in the dorsal horn also revealed a significant inhibition of CCI-induced activation of the microglia by NAC (Fig. 9C and D). However, NAC did not exhibit a notable influence on the astrocytic marker GFAP (Fig. 9E–H).

F9
Figure 9.:
N-acetyl-cysteine (NAC) significantly inhibits chronic constrictive injury (CCI)–induced activation of the microglial marker ionized calcium-binding adapter molecule 1 (IBA-1), while exhibiting no notable effect on the astrocyte marker glial fibrillary acidic protein (GFAP). (A and B) Effects of NAC (100 mg/kg, orally) on the expression of IBA-1 in the spinal cords. (C and D) Images and quantification of immunofluorescence showing IBA-1 in the dorsal horns. (E and F) Effects of NAC (100 mg/kg, orally) on the expression of GFAP in the spinal cords. Representative bands and a data summary (n = 4) were shown. (G and H) Images and quantification of immunofluorescence showing GFAP in the dorsal horns. Quantification of immunofluorescence was represented as mean fluorescence pixels in the superficial dorsal horns (n = 6 images per animal). N-acetyl-cysteine (100 mg/kg, orally) was consecutively administrated daily from day 14th to day 28th after CCI operation. The lumbar spines (L1-L6) were collected and analyzed 120 minutes after the last drug administration. Significant difference was revealed after one-way analysis of variance (**P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs CCI group; Bonferroni post hoc tests).

3.10. Coadministration of LY341495 and sulfasalazine with N-acetyl-cysteine did not abrogate its inhibition on the chronic constrictive injury–induced activation of matrix metalloproteinases in vivo

Previous work indicates that NAC may cause analgesia by enhancing the endogenous activation of type-2 metabotropic glutamate (mGlu) receptor.4 Single administration of NAC could attenuate CCI-induced neuropathic pain, and this effect could be abrogated by LY341495 (an mGlu 2/3 antagonist, 1 mg/kg, intraperitoneally) or sulfasalazine (an L-cystine/L-glutamate membrane exchanger inhibitor, 8 mg/kg, intraperitoneally).4 These findings pose a problem that whether inhibition of MMPs by immediate administration of NAC in vivo could be abrogated by blocking mGlu2 signaling. Therefore, the animals with neuropathic pain were cotreated with NAC and LY341495 or sulfasalazine. Single administration of NAC (200 mg/kg, orally) with or without 15-minutes pretreatment of LY341495 (1 mg/kg, intraperitoneally) or sulfasalazine (8 mg/kg, intraperitoneally) both showed a significant inhibition on CCI-induced mechanical allodynia and MMP-9 activation in vivo. Mechanical allodynia was attenuated by 74.9% after NAC single administration, whereas by 52.1% (NAC + LY341495) and 43.6% (NAC + sulfasalazine), respectively. These 2 compounds demonstrated a mild inhibition on the analgesia effect of NAC and delayed the peak time of NAC's effect (Fig. 10A). Activity of MMP-9 was reduced by 84.6% (NAC), 68.5% (NAC + sulfasalazine), and 61.76% (NAC + LY341495), respectively (Fig. 10B–D), which showed no significant difference (P > 0.05).

F10
Figure 10.:
LY341495 and sulfasalazine mildly suppressed the effects of chronic constrictive injury (CCI)-induced mechanical allodynia and matrix metalloproteinase-9 activity by N-acetyl-cysteine (NAC). (A) Behavioral test of mechanical allodynia. Single dose of NAC (200 mg/kg, orally) with 15-minutes pretreatment of LY341495 (1 mg/kg, intraperitoneally) or sulfasalazine (8 mg/kg, intraperitoneally) or saline (3 mL/kg, intraperitoneally) was administered at day 14th after CCI operation. (B) Representative band and (C and D) data summary (n = 4) of matrix metalloproteinase 9/2 with gelatin zymography. The lumbar spines (L1-L6) were collected 120 minutes after the last drug administration and analyzed. Significant difference was revealed after one-way analysis of variance (**P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs CCI group; Bonferroni post hoc tests).

4. Discussion

The major findings of this study were as follows: (1) NAC directly suppressed the activation of MMP-9 and MMP-2 both in vitro and in vivo; (2) NAC significantly attenuated the induction and development of CCI-induced mechanical allodynia and thermal hyperalgesia in rats; (3) NAC significantly inhibited the CCI-induced phosphorylations of the MAPK family, PKCγ, and NR1 in the spinal cord; and (4) NAC also inhibited CCI-induced microglia activation and the cleavage of IL-1β.

The “cysteine switch” was first defined in 1990 by Harold E. Van Wart and Henning Birkedal.16 The cysteine switch in MMP-9/2 consists of 2 domains, ie, the critical cysteine residue in the propeptide domain and the zinc-binding site in the catalytic domain.16 The activation of the cysteine switch leads to the exposure of the zinc-binding active site, which results in the activations of MMP-9 and MMP-2. The “cysteine switch” is activated by oxidants, disulfides, alkylating reagents, and Hg(II) and Au(I) compounds.9 Matrix metalloproteinase activation also involves S-nitrosylation in vivo.14 These reagents are related to numerous cytokines that are closely related to neuropathic pain, such as reactive oxygen species (ROS) and nNOS, which produce -ONOO.

As a compound with abundant cysteine residues, NAC significantly reduced the activity of MMP-9/2 both in vitro and in vivo by more than 50% (Fig. 1E and H). Moreover, the NAC-mediated inhibition of MMP-9 was more intense than the inhibition of MMP-2 (Fig. 1E; inhibition efficiencies: MMP-9 86.3% and MMP-2 70.8% in vitro in the CCI rats' spinal cords; and MMP-9 62.0% and MMP-2 47.3% after single administrations of 200 mg/kg, orally; Fig. 2C–E).

In accordance with the gelatin zymography results detailed above, behavioral tests also revealed that NAC very effectively attenuated CCI-induced neuropathic pain (Figs. 2A and B, 3A and B, 4A and B, and 5A and B). Matrix metalloproteinase-9 was demonstrated to contribute to the early stage of neuropathic pain, whereas MMP-2 was more important during the late stage.25 Our results indicated that NAC exhibited strong effects during the entire process of treatment regardless of its administration before or after the establishment of neuropathic pain (Figs. 3A and B, 4A and B). These findings also support our hypothesis that NAC alleviates neuropathic pain through the inhibition of both MMP-9 and MMP-2.

In addition, NAC exhibited stronger MMP-9/2-inhibiting effects in the spinal cords of the CCI-treated rats than in those of the normal rats (Fig. 1E–J). This finding suggests that NAC may selectively affect “the disease-activated MMPs” in a quite beneficial manner because the normal physiological actions of MMPs are also very important during development in humans and in the formation of memories in the hippocampus.19 Moreover, MMP-9 knockout mice exhibit altered repair process in response to injuries of the skin, cornea, and central nervous system, altered bone marrow reconstitution, and altered inflammatory responses. These results indicate that NAC might be a drug with negligible or acceptable side effects and could thus potentially represent a better therapeutic strategy for the treatment of neuropathic pain.

Both neurons and glia have been demonstrated to play vital roles in the induction and development of neuropathic pain. Spinal N-methyl-D-aspartate (NMDA) receptor-dependent synaptic plasticity contributes to enhanced sensory responses after injury.47 The activation of NMDA receptors induces Ca2+ influx, which activates calcium-sensors such as CaMKII.47 These proteins can phosphorylate downstream molecules (eg, PKCγ and ERK), which in turn leads to further activation of NMDA receptors and initiates prolonged enhancement of the excitability of spinal cord neurons.18 This process is considered to be the main neuronal mechanism of neuropathic pain. Previous studies have demonstrated that MMP-9 activates NR1 through integrin β1 or nitric oxide pathways. Protein kinase Cγ is also vital in the development of neuropathic pain.31 Extracellular regulating kinase activation in the spinal dorsal horn neurons has been proven to be nociceptive specific.24 Brain-derived neurotrophic factor (BDNF) is a substrate of MMP-9/2 and has been proven to be the upstream signal of ERK. N-acetyl-cysteine significantly suppressed the phosphorylation levels of NR1, PKC, and ERK (Fig. 7C–J, Fig. 8A and D). The results revealed that NAC inhibited the downstream signals of MMP-9 activation.

Calcitonin gene-related peptide (CGRP) is a peptide that is released by primary afferents and mediates the activation of NMDA receptors in neurons.40 Calcitonin gene-related peptide upregulation is also believed to be a gold standard indicator of nociceptive activation. Our immunofluorescence results indicated that NAC could significantly inhibit CCI-induced CGRP upregulation (Fig. 7K and L).

In addition to the neuronal inhibition, NAC also elicits strong effects in glial cells, particularly microglia. The phosphorylation of p38 MAPK plays an important role in the process of neuropathic pain.25 The activation of most microglial receptors can converge on p38 phosphorylation, which leads to the synthesis and release of multiple inflammatory mediators23 such as IL-1β. Our data revealed that NAC significantly inhibited CCI-induced phosphorylation of p38.

Matrix metalloproteinase-9/2 has been proven to contribute to the cleavage of IL-1β.25 Our data revealed that the CCI-induced production of active IL-1β in the spinal cord was significantly inhibited by NAC (Fig. 7A and B), whereas NAC had little influence on the level of pro-IL-1β, which indicated the effects of NAC on MMP-9/2 cleavage activity.

However, it should be noted that one of the most important inflammatory cytokines in the central nervous system, ie, IL-1β, is also activated through another mechanism that involves NACHT, LRR, and PYD domains-containing protein 3 (NLRP3). NLRP3 is known for its critical role in caspase-1 cleavage and maturation. Caspase-1 is crucial for the processing of pro-IL-1β to mature IL-1β.20 In this process, the generation of ROS has been reported to induce unprompted NLRP3 inflammasome activation.46 As an ROS scavenger, NAC has also been reported to act as an inhibitor of NLRP3 inflammasome–mediated cleavage of IL-1β.36 Consequently, NAC-mediated inhibition of the cleavage of IL-1β through this mechanism cannot be excluded.

c-Jun N-terminal kinase activation in spinal cord astrocytes contributes to the maintenance of chronic pain.12 In addition, in DRG neurons, JNK can regulate the synthesis of MMP-9.12 In addition, the p38 signaling pathway is involved in the regulation of MMP-9 secretion and in vitro invasion through AP-1–dependent gene promoter activation.38 N-acetyl-cysteine's inhibition of phosphorylated JNK and p38 has also been reported in other studies.15 Our results demonstrated that NAC suppressed the phosphorylation of JNK and p38, which suggests that NAC might downregulate MMP-9/2 at the transcriptional level.

In this study, we also demonstrated that NAC significantly inhibited CCI-activated IBA-1 expression but elicited no notable effect on GFAP expression. Astrocytes have been reported to underlie persistent activation in the maintenance of neuropathic pain with MMP-2 upregulation, and microglia are quickly activated in the early stage of neuropathic pain with the activation of MMP-925; these findings may be partly due to the short half-life (2.27 hour) of NAC6 and NAC's more intense inhibition of MMP-9 (Fig. 3C and D).

In addition, although single administrations of NAC elicited significant inhibition of the activation of MMP-9/2 both in vitro and in vivo, the possibility that sustained NAC administrations reduces MMP protein synthesis through other transcriptional mechanisms that cannot be excluded. The transcriptions of MMP-9 and MMP-2 have been proven to rely on the AP-1 or NF-κB pathway.7,27,38,42 Reactive oxygen species are vital stimulating factors in the activation of AP-1 and NF-κB5 and contribute to the activation of the MAPK/NF-κB signaling pathway.8 Moreover, NAC has been proven to suppress ROS,45 and the abolishment of in situ gelatin lytic activity and MMP-9 expression by NAC in the treatment of explanted lesions is thought to be due to its ROS scavenging function.48

Previous studies have indicated that NAC modulates peripheral neuropathy through the inhibition of ROS.3,35 N-acetyl-cysteine also attenuates CCI-induced neuropathic pain by affecting the spinal-cord glutathione system and nitric oxide metabolites in rats.17 Moreover, NAC has also been found to cause analgesia in inflammatory pain models by enhancing the endogenous activation of type-2 metabotropic glutamate receptors.4 Other reports pointed out that NAC could indirectly activate type 2/3 and type 5 metabotropic glutamate receptors (mGlu 2/3 and mGlu 5) through regulating system Xc26,34 and causing potentiation or depotentiation in prefrontal cortex–nucleus accumbens core synapses26 in rats. These findings are in accord because ROS also interacts with the glutathione system2 and nitric-oxide metabolites.37 We also performed experiments to evaluate whether the coadministration of an mGlu2 inhibitor and NAC could abrogate NAC's inhibition of MMP-9/2. Our results showed that inhibition of mGlu 2/3 receptor or system Xc could mildly suppress NAC's effects on MMP-9 activity in the spinal cords from rats with CCI operation (Fig. 10B–D). It also delayed and partly reduced NAC's inhibition of CCI-induced mechanical allodynia (Fig. 10A). These results indicate the importance of mGlu 2/3 and system Xc in NAC's effects, but these findings also suggest that MMP-9 may be a more vital target of NAC in the treatment of chronic pain.

Most recently, another study provided further insight into the role of NAC in nociceptive transmission in humans.41 These data lay the groundwork for investigations of the therapeutic potential of NAC for patients with chronic pain.

Given the excellent clinical safety history and low cost of NAC, our findings may represent a bright prospect for the treatment of neuropathic pain or provide inspiration for the development of new analgesic agents, which target the “cysteine switch” of MMPs with longer half-life and better blood–brain barrier penetrability.

Conflict of interest statement

The authors declare no conflict of interest.

This work should be attributed equally to the institutions.

This work was supported by National Natural Science Foundation of China (Nos. 81471142, 81200860, 81202513, 81171044).

Acknowledgements

Jiajie Li, Lujie Xu, Xueting Deng and Chunyi Jiang contributed equally to this work. The authors thank Dr Xuefeng Wu and Dr Zikai Zhou for their improving command and proofreading of the article.

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Keywords:

Neuropathic pain; Matrix metalloproteinase; N-acetyl-cysteine

© 2016 International Association for the Study of Pain