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MiR-34c Ameliorates Neuropathic Pain by Targeting NLRP3 in a Mouse Model of Chronic Constriction Injury
NEUROSCIENCE RESEARCH ARTICLE L. Xu et al. / Neuroscience 399 (2019) 125–134
MiR-34c Ameliorates Neuropathic Pain by Targeting NLRP3 in a Mouse Model of Chronic Constriction Injury Lijuan Xu, a Qixing Wang, a Wei Jiang, a Shunzhi Yu b and Shouqin Zhang a* a
Department of Critical Care Medicine, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, No. 301 Middle Yanchang Road, Jing’an District, Shanghai 200072, China b Department of Orthopedics, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, No. 301 Middle Yanchang Road, Jing’an District, Shanghai 200072, China
Abstract—MicroRNAs have been reported to be an important pathophysiological factor in neuropathic pain. However, the potential mechanism through which miRNAs function in neuropathic pain remains unclear. The purpose of this study was to explore the potential role of mir-34c in neuropathic pain in a mouse model of chronic constriction injury (CCI). We found that overexpression of miR-34c greatly alleviated CCI-induced neuropathic pain and spinal cord infarction, and reduced cell apoptotic and inflammatory cytokine expression in CCI mice. We also demonstrated that miR-34c suppressed the expression of NLRP3 by directly binding the 30 -untranslated region. Overexpression of miR-34c decreased the protein levels of NLRP3, ASC, caspase-1, IL-1b, and IL-18 in the spinal cord in CCI mice. Together, our results indicated that miR-34c may inhibit neuropathic pain development in CCI mice through inhibiting NLRP3-mediated neuroinflammation. Ó 2018 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: neuropathic pain, chronic constriction injury, miR-34c, NLRP3 inflammasome.
molecular mechanisms and to identify effective methods for treating neuropathic pain. MicroRNAs (miRNAs) are endogenous non-coding small RNAs of approximately 18–25 nucleotides that directly regulate gene expression through binding to the 30 untranslated region (30 -UTR) of their target messenger RNAs (mRNAs) (Hammond, 2015). MiRNAs are involved in early development, cell proliferation, apoptosis, and cell differentiation (Kloosterman and Plasterk, 2006). Many miRNAs, including miR-145 (Shi et al., 2017), miR-223 (Xie et al., 2017b), miR-23a (Pan et al., 2018), miR-183 (Xie et al., 2017a), and miR-150 (Ji et al., 2018), have been shown to contribute to the pathogenesis of neuropathic pain. TaqMan Low Density Array (TLDA) assays showed that miR-34c was downregulated in the L4 ipsilateral dorsal root ganglion (DRG) of the spinal nerve ligation (SNL) cohort compared with their contralateral counterpart (von Schack et al., 2011). The miR-34c is a potential tumor suppressor that has been implicated in tumorigenesis of human malignancies, such as osteosarcoma (Hu et al., 2018), non-small cell lung cancer (Zhao et al., 2017), and ovarian cancer (Tung et al., 2017). The miR-34c has also been reported to regulate the intestinal dysfunction in inflammatory bowel disease (Chen et al., 2017). However, whether miR-34c contributes to the neuroinflammation in neuropathic pain has not been studied.
INTRODUCTION Neuropathic pain, one of the most challenging neurological diseases to treat, results from damage to or dysfunction of the peripheral or central nervous system rather than stimulation of pain receptors (van Hecke et al., 2014). Neuropathic pain is commonly associated with allodynia, pain from a stimulus that does not usually provoke pain and hyperalgesia, and increased pain from a stimulus that usually provokes pain (Jensen and Finnerup, 2014). Nearly 70% of individuals with spinal cord injury develop some degree of central neuropathic pain, which severely affects their emotional well-being and overall quality of life (Yezierski, 2005). Nerve injury triggers an inflammatory response that involves the activation of immune cells, microglia, and astrocytes, and the release of several proinflammatory cytokines plays crucial roles in the development of neuropathic pain (Schomberg et al., 2012). However, the pathogenesis of neuropathic pain is not fully understood. Therefore, it is very important to understand the specific underlying
*Corresponding author. E-mail address: [email protected] (S. Zhang). Abbreviations: ASC, apoptosis-associated speck-like protein; CCI, chronic constriction injury; ELISA, enzyme-linked immunosorbent assay; miRNAs, microRNAs; MMP, matrix metalloproteinase; mRNAs, messenger RNAs; mut, mutant; NLRP3, nucleotide binding domain-like receptor protein 3. https://doi.org/10.1016/j.neuroscience.2018.12.030 0306-4522/Ó 2018 IBRO. Published by Elsevier Ltd. All rights reserved. 125
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The inflammatory response occurs during cell damage and is one of the important physiological mechanisms of neuropathic pain (Abbaszadeh et al., 2018, Xu et al., 2018). The inflammatory response can be triggered by the inflammasome, a polyprotein complex (Zambetti et al., 2012). The most representative inflammasome is nucleotide binding domain-like receptor protein 3 (NLRP3). NLRP3 is activated by various endogenous and exogenous stimulation, and it recruits apoptosis-associated speck-like protein (ASC), which in turn activates caspase 1, thus stimulating the release of the proinflammatory cytokines interleukin (IL)-1b and IL18, which play key roles in innate immunity (Lamkanfi and Dixit, 2014). Caspase-1 activation can also lead to cell pyroptosis, an inflammatory form of programmed cell death distinct from apoptosis (Fink and Cookson, 2005). In addition to defending against pathogens, inflammasomes have been implicated in the pathogenesis of various inflammatory and metabolic diseases (Masters, 2013). In the current study, we investigated the roles of miR34c in chronic constriction injury (CCI)-induced neuropathic pain processing in the central nervous system. We also examined whether miR-34c interacts directly with and regulates the expression of the NLRP3 inflammasome, with the aim of providing novel insights into the molecular mechanism for neuropathic pain.
EXPERIMENTAL PROCEDURES Animals and ethic statements Adult male C57BL/6 mice (8–10 weeks of age) were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). The mice were housed in individual cages and given free access to water and food. All animal experiments were performed in strict accordance with the American Animal Protection Legislation. All study protocols were approved by the Institutional Animal Care and Use Committee of the Shanghai Tenth People’s Hospital of Tongji University. Establishment of a neuropathic pain animal model The mouse model of neuropathic pain was established through the CCI method, as previously described (Zhang et al., 2013). Briefly, the mice were anesthetized through intraperitoneal injection with 4% chloral hydrate. The right sciatic nerve was exposed at the mid-thigh level by blunt dissection. Three ligatures (4/0 silk) were tied loosely around the sciatic nerve 1.0–1.5 mm apart. The muscle and skin were sutured in layers with 4–0 Ethicon silk sutures. The same procedure was used for sham surgeries except that the sciatic nerve was exposed but not ligated. Plasmid construction To construct the miR-34c overexpression vector, oligonucleotides were designed and synthesized by Hanbio company (Shanghai, China). The primer sequences were as follows: forward, 50 -GGA AGU CUA
GUU ACU GCU CG-30 and reverse, 50 -GCC AGG UAA AAG ACU ACG GGU-30 . Purified PCR products were cloned into the vector pCDH-CMV (System Biosciences) at the EcoRI/NotI sites. All constructs were confirmed by DNA sequencing. Lentivirus packaging and transduction The constructed plasmid together with two envelope plasmids, PSPAX2 and PMD2G, were transfected into HEK293T cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Lentiviral supernatants were collected at 48 h after transfection and cleared with a Centricon Plus-70 filter unit (Millipore). Lentivirus particles with titers 108 TU/mL were used in the experiment. The lentivirus in vivo infections were performed according to a previous study (Pan et al., 2014). Briefly, 1 lL lentivirus or vector was administered to mice through intrathecal injections daily for 3 consecutive days 2 weeks before CCI. Thermal hyperalgesia and mechanical allodynia evaluation Thermal hyperalgesia was measured through paw withdrawal thermal latency, as described previously (Sacerdote et al., 2013). Briefly, the mice were placed in small clear Plexiglas cubicles and allowed to acclimate. Radiant heat with constant intensity (beam diameter 0.5 cm and intensity 20 I.R.) was applied to the midplantar area of the hind paw until the animal lifted its paw from the floor. The time (in seconds) from initial heat application to paw withdrawal was recorded. Mechanical allodynia was assessed through the paw mechanical withdrawal threshold measured with a dynamic plantar esthesiometer (Ugo Basile, Comerio, Italy). Briefly, the mice were placed in a test cage with a wire mesh floor and allowed to acclimate. The rigid tip of a von Frey filament was applied to the skin of the midplantar area of the hind paw with increasing force (up to 5 g over 20 s) until the animal removed its paw. The average values of the withdrawal threshold force (in grams) were calculated from four independent experiments. Tissue sample collection After the thermal hyperalgesia and mechanical allodynia evaluation, the mice in each group were sacrificed by decapitation under pentobarbital anesthesia (50 mg/kg by intraperitoneal injection). For histopathological analysis, the mice were subjected to cardiac perfusion with 4% paraformaldehyde. Segments of spinal cords at the lumbar dorsal L4–L6 were rapidly dissected from the mice, fixed with 4% paraformaldehyde and stored at 4 °C. For biochemical analysis, the L4–L6 dorsal spinal cords were collected, snap-frozen in liquid nitrogen and stored at 80 °C. Histopathology and scoring Paraformaldehyde-fixed and paraffin-embedded transverse sections (8 lm) of spinal cords at the lumbar
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dorsal L4–L6 level from sham mice (n = 6), CCI mice (n = 6), and CCI mice infected with Lenti-miR-34c (n = 6) or Lenti-vector (n = 6) at postoperative day 7 were stained with hematoxylin and eosin (HE). Spinal cords stained with HE were used to assess histopathologic changes in the spinal dorsal horn of gray matter scores (Wu et al., 2014, Sun et al., 2016). The gray matter was scored from 0 to 6 as follows: 0 = no lesion observed; 1 = gray matter contained 1–5 eosinophilic neurons; 2 = gray matter contained 5–10 eosinophilic neurons; 3 = gray matter contained more than 10 eosinophilic neurons; 4 = infarction of less than 1/3 of the gray matter area; 5 = infarction of 1/3 to 1/2 of the gray matter area; 6 = infarction of more than 1/2 of the gray matter area). Scores were generated by one well-trained, board-certified histopathologist who was blinded to the groups of mice. Enzyme-linked immunosorbent assay (ELISA) Tissues in the spinal cord from sham mice (n = 6), CCI mice (n = 6), and CCI mice infected with Lenti-miR-34c (n = 6) or Lenti-vector (n = 6) at postoperative day 7 were removed from the 80 °C refrigerator and ground to extract proteins. The protein expression of TNF-a and IL-1b was measured with a commercially available ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Immunofluorescence Transverse sections of spinal cords from sham mice (n = 6), CCI mice (n = 6), and CCI mice infected with Lenti-miR-34c (n = 6) or Lenti-vector (n = 6) at postoperative day 7 were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked in 1% BSA. For double immunofluorescence staining, the sections were incubated with a mixture of anti-NLRP3 and anti-GFAP, or anti-CD11b (all from Abcam, Cambridge, UK) at 4 °C overnight. After being washed, the sections were incubated with fluorescently conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) at 37 °C for 1 h. Then, the sections were counterstained with DAPI (Beyotime, Nantong, China) and examined under a confocal microscope (7CS SP5, Leica, Germany). Five randomly selected microscopic fields were analyzed per slide by a blinded investigator, and the number of NLRP3 and GFAP or CD11b-positive cells in lamina I–III of the L4–L6 spinal dorsal horn of each mouse was recorded. Quantitative real-time PCR Total RNA of lumbar spinal cord segments (L4–L6) of the indicated mice (n = 6 per group) was extracted with TRIzol reagent (Invitrogen). MiRNA cDNA was synthesized with a Mir-X miRNA First-Strand Synthesis Kit (Qiagen, Dusseldorf, Germany) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed with SYBR Green PCR mix in an ABI Prism 7900HT instrument (Applied Biosystems, Foster City, CA, USA). The primer
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sequences were as follows: miR-34c: forward, 50 -TGT CAC CAA GTC ATC TCT CCA G-30 and reverse, 50 CAG GTC CTC CTC ATA ATT TGG CT-30 ; U6: forward, 50 -GAC CTA CAA CGG GCA TCT GG-30 and reverse, 50 -GAC CTA CAA CGG GCA TCT GG-30 . qRT-PCR was performed for 40 cycles at 95 °C for 15 s and 60 °C for 1 min after an initial 15-min incubation at 95 °C. The fold changes in expression were obtained with the 2DDCt method (Cikos et al., 2007), and the miR-34c expression was normalized to that of U6. TUNEL staining Paraformaldehyde-fixed and paraffin-embedded sections from sham mice (n = 6), CCI mice (n = 6), and CCI mice infected with Lenti-miR-34c (n = 6) or Lenti-vector (n = 6) at postoperative day 7 were used to detect cell death through TUNEL staining according to the manufacturer’s instructions (Roche Applied Science, Indianapolis, IN, USA). Briefly, fixed frozen sections were rehydrated in PBS for 5 min. Slides were then incubated with 20 lg/mL proteinase K solution for 15 min at 25 °C, treated with 3% H2O2 for 5 min, and incubated in equilibrium buffer for 10 min. The slides were then incubated with terminal deoxynucleotidyl transferase for 1 h before being washed and treated with anti-digoxigenin for 30 min. Signal was visualized with DAB. The number of apoptotic cells and the total number of cells were counted in five randomly selected microscopic fields, and the percentage of TUNELpositive cells in spinal dorsal horn was calculated from 6 spinal cord sections from different mice. Luciferase reporter assay To confirm the interaction between the 30 -UTR of NLRP3 mRNA and miR-34c, HEK293T cells were seeded in 12well plates at 1 105 cells/mL and co-transfected with wild-type (wt) or mutant (mut) pGL3-NLRP3 30 -UTR and miR-34c mimics or a control construct (Genepharma, Shanghai, China) with Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. Luciferase activity was calculated as the ratio of firefly to Renilla luciferase activity, as measured with a luciferase assay system (Promega, Madison, WI, USA) after 48-h transfection. The relative luciferase activity was expressed as the ratio of the measured luciferase activity to the control. Western blot analysis Spinal cord tissues in the lumbar spinal cord from sham mice (n = 6), CCI mice (n = 6), and CCI mice infected with Lenti-miR-34c (n = 6) or Lenti-vector (n = 6) at postoperative day 7 were removed from the 80 °C refrigerator and lysed, and proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA). After being blocked in 5% nonfat dry milk containing 0.1% Tween 20 for 2 h, the membranes were incubated at 4 °C overnight with primary antibodies (diluted 1000-fold) that recognized NLRP3, ASC, Caspase-1, IL-1b, IL-18, and
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b-actin (all from Abcam). After being washed in PBS, the membranes were incubated at room temperature with horseradish peroxidase-conjugated secondary antibody diluted 5000-fold for 1 h and visualized with a SuperSignal West Dura extended duration substrate detection system (Thermo Scientific, Rockford, IL, USA). Protein expression was normalized to that of bactin. Statistical analysis Data are represented as mean ± standard deviation (SD) from at least triplicate experiments. Results were analyzed in GraphPad Prism 5 software (GraphPad, San Diego, CA, USA). A one-way analysis of variance (ANOVA) was used to determine significant differences between groups, and the individual comparisons were performed with Tukey’s post hoc test. The threshold for statistical significance was set at p < 0.05.
RESULTS Overexpressed miR-34c alleviates CCI-induced neuropathic pain and tissue damage To investigate the relationship between miR-34c and neuropathic pain, mice with chronic constriction of the sciatic nerve were generated via surgical ligation, and the expression of miR-34c in the spinal cord was then measured. At 0, 3, 7, 14, and 21 days after CCI, the spinal cords of the mice were collected, and the miR34c levels in the spinal cords were analyzed with qRTPCR. In the CCI mice, compared with mice receiving sham surgery, miR-34c was significantly downregulated in a time-dependent manner (Fig. 1A). To investigate the biological role of miR-34c in neuropathic pain, miR34c was stably overexpressed in mice through intrathecal injection of lentivirus for expression of miR34c (Lenti-miR-34c). In the injected mice, compared with the CCI control mice, miR-34c was significantly overexpressed (Fig. 1B). Furthermore, our results showed that miR-34c overexpression attenuated CCIinduced mechanical allodynia (Fig. 1C) and thermal hyperalgesia (Fig. 1D). Finally, after each treatment at 7 days after CCI, the spinal cords were stained with HE and were assigned histopathology scores by an experienced pathologist according to changes in the gray matter (Fig. 1E, F). A higher score indicated greater tissue damage. Overexpression of miR-34c, compared with CCI control treatment, resulted in significantly lower histopathology scores, thus indicating less tissue infarction after CCI. Together, these findings indicated that CCI induced pain hypersensitivity and increased tissue damage, which could be reversed by upregulation of miR-34c expression. Overexpression of miR-34c suppresses cell apoptosis and inflammation in CCI mice To further explore the biological effects of miR-34c on apoptosis and inflammation, a TUNEL assay was performed on sections of the spinal cord collected at day 7 after CCI, and the levels of TNF-a and IL-1b were
measured by ELISA in the spinal cords of mice at 7 days after CCI. The proportion of apoptotic cells was significantly higher in the CCI mice than in the sham operated mice (Fig. 2A, B), and TNF-a (Fig. 2C) and IL1b (Fig. 2D) levels were elevated in all CCI treatment groups. Overexpression of miR-34c, compared with CCI control treatment, significantly decreased the number of apoptotic cells and the levels of TNF-a and IL-1b, thus indicating that miR-34c decreased the cell apoptotic and inflammatory response in CCI mice. MiR-34c binds the 30 -UTR of NLRP3 and affects NLRP3 inflammasomes To explore the potential molecular mechanism through which miR-34c regulates neuropathic pain, we performed bioinformatic analyses to determine the direct target gene of miR-34c. We found that NLRP3 30 -UTR contained a conserved binding site for miR-34c, and the sequence of an RNA mutant of the 30 -UTR of NLRP3 was also present in the alignment (Fig. 3A). To confirm the interaction between the 30 -UTR of NLRP3 and miR34c, we co-transfected HEK293T cells with the miR-34 mimics or the control construct (miR-34 NC) and the dual-luciferase vector containing wt or mut pGL3-NLRP3 30 -UTR. Luciferase reporter assays showed that miR34c significantly decreased the luciferase activity of the wt 30 -UTR but not that of the mut 30 -UTR of NLRP3, thus confirming that NLRP3 is a target gene of miR-34c (Fig. 3B). Next, the relative protein levels of NLRP3, ASC, and activated caspase-1 were examined by western blot analysis in the spinal cords of CCI model mice infected with Lenti-miR-34c or control Lenti-vector at postoperative day 7 (Fig. 3C). The results revealed significantly higher NLRP3, ASC, and activated caspase-1 protein levels in the spinal cord in CCI mice than in sham mice. Moreover, overexpression of miR34c, compared with control CCI treatment, significantly inhibited the expression levels of NLRP3, ASC, and activated caspase-1 in the spinal cord (Fig. 3D). The protein levels of mature IL-1b and IL-18 in the spinal cord were also measured by western blot analysis (Fig. 3E). As shown in Fig. 3F, the protein levels of both cytokines were higher in CCI mice than sham mice, and overexpression of miR-34c decreased cytokine maturation. These findings further confirmed the interaction between miR-34c and NLRP3 and suggested that miR-34c negatively regulate the expression of NLRP3 and subsequently inhibit inflammasome activity (NLRP3, ASC, and caspase-1) and cytokine maturation. Overexpression of miR-34c inhibits CCI induced NLRP3 inflammasome activation in spinal cord astrocytes microglia and neurons To provide direct evidence of the involvement of miR-34c in CCI-induced pain behavior, we examined NLRP3 expression in the spinal cord in CCI model mice infected with Lenti-miR-34c or control Lenti-vector at postoperative day 7. The protein expression and cellular distribution of NLRP3 in the spinal cord were probed with double immunofluorescence staining with
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Fig. 2. Overexpression of miR-34c suppresses cell apoptotic and inflammation in CCI mice. (A) TUNEL assays were performed on the spinal cord sections from each group at day 7 after CCI, and arrows show apoptotic cells. Scale bars = 20 lm. (B) The percentage of apoptotic cells in the spinal cord tissue of each group was calculated. The spinal cord tissue levels of TNF-a (C) and IL-1b (D) were measured by ELISA in all treatment groups and the sham group. n = 6 per group. *p < 0.05, **p < 0.01 vs. sham group. #p < 0.05 vs. CCI group.
antibodies against GFAP (an astrocyte marker), CD11b (a microglia marker) and NeuN (a neuron marker), respectively. We detected higher NLRP3 expression in astrocytes (Fig. 4A) microglia (Fig. 4B) and neurons (Fig. 4C) in the spinal cord in the CCI mice than in the sham operated controls. Moreover, significant inhibition of NLRP3 expression in spinal cord astrocytes, microglia and neurons was observed in the Lenti-miR34c infected mice compared with CCI control mice. Therefore, NLRP3 inflammasome activation in spinal cord astrocytes microglia and neurons contributes to the processing of CCI-induced neuropathic pain, and overexpression of miR-34c inhibited NLRP3 inflammasome assembly.
DISCUSSION The mechanisms underlying neuropathic pain remain poorly understood despite the enormous efforts that have been made in basic and clinical research. The prototypical opioid morphine is commonly used to control severe pain; however, tolerance to the drug’s analgesic effects develops after repeated or continuous use and requires an increase in dosage to maintain the same efficacy. In addition, morphine is not always sufficient in neuropathic pain, and patients receiving morphine are particularly susceptible to abuse and addiction (Berrios et al., 2008). Therefore, novel targets that may lead to the development of promising analgesics are eagerly anticipated for improved pain management.
3 Fig. 1. Overexpressed miR-34c alleviates CCI-induced neuropathic pain and tissue damage. (A) miR-34c levels in the spinal cords of mice were measured by qRT-PCR at postoperative days 0, 3, 7, 14, and 21. Sham operated mice were used for comparison. n = 6 for each time point. * p < 0.05, **p < 0.01 vs. sham group. (B) The expression of miR-34c in the spinal cords of CCI model mice infected with lentiviral vectors carrying miR-34c (Lenti-miR-34c) or lentiviral vector (Lenti-vector) at postoperative day 7. n = 6 per group. *p < 0.05, **p < 0.01 vs. sham group. #p < 0.05 vs. CCI group. (C) The effect of upregulated miR-34c on thermal hyperalgesia was assessed by paw withdrawal thermal latency (PWTL). (D) The effect of miR-34c overexpression on mechanical allodynia was evaluated according to the paw mechanical withdrawal threshold (PMWT). n = 6 per group. *p < 0.05, **p < 0.01. (E) Spinal cord sections collected at day 7 after CCI were stained with HE, and arrows represent lesions. Scale bars = 20 lm. (F) Histopathology scores were assigned by an experienced pathologist. n = 6 per group. *p < 0.05, **p < 0.01 vs. sham group. # p < 0.05 vs. CCI group.
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Fig. 3. MiR-34c binds the 30 -UTR of NLRP3 and affects NLRP3 inflammasomes. (A) Schematic diagram of the putative binding sites between miR34c and 30 -UTR of NLRP3. The paired miR-34c seed sequence and the seed-recognizing site in the wild-type (wt) and mutant (mut) 30 -UTR of NLRP3 are indicated. (B) Dual-luciferase reporter assays of miR-34c and NLRP3 30 -UTR. The miR-34 mimics or the control (miR-34 NC) was cotransfected with the dual-luciferase vector containing wt or mut pGL3-NLRP3 30 -UTR into HEK293T cells and incubated for 48 h. The relative luciferase activity was calculated as the ratio of firefly to Renilla luciferase activity, as measured with the Dual-Luciferase Assay System. **p < 0.01 vs. miR-34c NC group. n = 3 per group. Three independent experiments were performed. (C) Western blot analysis of NLRP3, ASC, and activated (cleaved) caspase-1 in the spinal cords of CCI model mice infected with Lenti-miR-34c or Lenti-vector at postoperative day 7. (D) Quantification of NLRP3, ASC, and activated (cleaved) caspase-1 protein levels relative to that of b-actin. (E) Representative western blotting results for mature IL1b and IL-18. (F) The intensity of each protein was quantified and normalized to the signal intensity of the corresponding pro-IL-1b or pro-IL-18 band present on the same membrane. n = 6 per group. *p < 0.05, **p < 0.01 vs. sham group. #p < 0.05 vs. CCI group.
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Fig. 4. Overexpression of miR-34c inhibits CCI-induced NLRP3 inflammasome activation in spinal cord astrocyte and microglia. (A) Double immunohistochemical staining of spinal cord sections from CCI model mice infected with Lenti-miR-34c or Lenti-vector at postoperative day 7, combining NLRP3 with (A) GFAP, (B) CD11b or (C) NeuN labeling. Scale bar = 50 lm. From the stained sections, the numbers of NLRP3 and GFAP, CD11b or NeuN double-labeled cells were counted. n = 6 per group. *p < 0.05, **p < 0.01 vs. sham group. #p < 0.05 vs. CCI group.
Many relevant studies have shown that miRNAs have a regulatory role in neuropathic pain (Shi et al., 2013, Lopez-Gonzalez et al., 2017, Wang et al., 2018). MiR34c has been widely investigated, and it plays an important role in many diseases, including cancer (Tung et al., 2017, Zhao et al., 2017, Hu et al., 2018) and inflammatory bowel disease (Chen et al., 2017). In addition, an important role of miR-34c in the nervous system has also been demonstrated. Kabaria et al. (2015) have found that downregulation of miR-34c in the brain contributes to Parkinson’s disease pathogenesis. However, the relationship between miR-34c and neuropathic pain remains unclear. In the current study, we found that miR-34c plays a suppressive role by targeting NLRP3 in neuropathic pain development in CCI mice. MiR-34c was downregulated in CCI mouse models, and it may alleviate neuropathic pain development. Overexpression of miR-34c greatly attenuated neuropathic pain and neuroinflammation in vivo. Our data showed that miR-34c inhibited neuropathic pain, possibly through inhibiting NLRP3-triggered neuroinflammation, thus providing novel insights into the molecular pathogenesis of neuropathic pain.
Among the NLR inflammasomes, NLRP3 has been the most intensively studied, given its role in inherited autoinflammatory syndromes (Hoffman et al., 2001) and several metabolic diseases including obesity, diabetes, atherosclerosis, and gout (Wen et al., 2012). In the central nervous system, the NLRP3 inflammasome plays an important role in the development of Alzheimer’s disease and bacterial meningitis (Liu et al., 2013). CCI mice with thermal hyperalgesia and mechanical allodynia have elevated levels of the NLRP3 inflammasome proteins NLRP3 and ASC and downstream caspase-1, IL-1b, and IL-18 in spinal cord tissues. These results suggest that CCI induces the NLRP3 inflammasome activation and the subsequent cleavage of TNF-a and IL-1b. Intrathecal injections of lentiviral vectors bearing miR-34c in mice 2 weeks before CCI ameliorated CCI-induced pain behaviors. These analgesic effects of miR-34c overexpression were accompanied by decreased NLRP3, ASC, caspase-1, IL-1b, and IL-18 protein levels, and diminished TNF-a and IL-1b production. Overexpression of miR-34c also decreased CCI-induced spinal cord tissue infarction, and spinal cord cell apoptosis; this may suggest a
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mechanism of action by which miR-34c protects against infarction via inhibition of NLRP inflammasome activation, thus resulting in the inhibition of cell apoptosis and the downregulation of proinflammatory cytokines. In addition, CCI induces activation of microglia and astrocytes, and neuron damage, whereas intrathecal injection of miR34c lentiviral vector reverses this phenomenon, possibly because the function of the miR-34c lentiviral vector is mediated by primary sensory neurons, the injured neurons that activate spinal astrocytes and microglia. Collectively, these findings provide evidence that the miR-34c and NLRP3 inflammasome is involved in the pathogenesis of neuropathic pain. IL-1b is produced as a precursor, pro-IL-1b, which must be cleaved either by caspase-1 or by matrix metalloproteinase (MMP)-9 or MMP-2 to become biologically active (Schonbeck et al., 1998, Martinon and Tschopp, 2007). MMP-9 and MMP-2, which are overexpressed in injured spinal cord tissues, are key mediators of neuropathic pain through IL-1b cleavage (Schomberg et al., 2012). Our study demonstrated that the NLRP3 inflammasome, the best-characterized inflammasome activated by cellular infection or stress, contributed to the development of neuropathic pain through induction of IL-1b cleavage and spinal cord cell apoptosis. Overexpression of miR-34c inhibited this pathway and may provide effective analgesics for pain management. In summary, the experimental results in this study showed that miR-34c suppressed the activity of the NLRP3 inflammasome (NLRP3, ASC, and Caspase-1), thereby alleviating CCI-induced neuropathic pain in mice through downregulation of NLRP3. However, the mechanisms of miR-34c and NLRP3 related to the occurrence, development, and prognosis of neuropathic pain require further confirmation, because the expression of miR-34c at the cellular level was not determined in our study, owing to limitations in funding, and further studies are needed.
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(Received 12 August 2018, Accepted 17 December 2018) (Available online 26 December 2018)
MiR-34c Ameliorates Neuropathic Pain by Targeting NLRP3 in a Mouse Model of Chronic Constriction Injury Lijuan Xu, a Qixing Wang, a Wei Jiang, a Shunzhi Yu b and Shouqin Zhang a* a
Department of Critical Care Medicine, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, No. 301 Middle Yanchang Road, Jing’an District, Shanghai 200072, China b Department of Orthopedics, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, No. 301 Middle Yanchang Road, Jing’an District, Shanghai 200072, China
Abstract—MicroRNAs have been reported to be an important pathophysiological factor in neuropathic pain. However, the potential mechanism through which miRNAs function in neuropathic pain remains unclear. The purpose of this study was to explore the potential role of mir-34c in neuropathic pain in a mouse model of chronic constriction injury (CCI). We found that overexpression of miR-34c greatly alleviated CCI-induced neuropathic pain and spinal cord infarction, and reduced cell apoptotic and inflammatory cytokine expression in CCI mice. We also demonstrated that miR-34c suppressed the expression of NLRP3 by directly binding the 30 -untranslated region. Overexpression of miR-34c decreased the protein levels of NLRP3, ASC, caspase-1, IL-1b, and IL-18 in the spinal cord in CCI mice. Together, our results indicated that miR-34c may inhibit neuropathic pain development in CCI mice through inhibiting NLRP3-mediated neuroinflammation. Ó 2018 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: neuropathic pain, chronic constriction injury, miR-34c, NLRP3 inflammasome.
molecular mechanisms and to identify effective methods for treating neuropathic pain. MicroRNAs (miRNAs) are endogenous non-coding small RNAs of approximately 18–25 nucleotides that directly regulate gene expression through binding to the 30 untranslated region (30 -UTR) of their target messenger RNAs (mRNAs) (Hammond, 2015). MiRNAs are involved in early development, cell proliferation, apoptosis, and cell differentiation (Kloosterman and Plasterk, 2006). Many miRNAs, including miR-145 (Shi et al., 2017), miR-223 (Xie et al., 2017b), miR-23a (Pan et al., 2018), miR-183 (Xie et al., 2017a), and miR-150 (Ji et al., 2018), have been shown to contribute to the pathogenesis of neuropathic pain. TaqMan Low Density Array (TLDA) assays showed that miR-34c was downregulated in the L4 ipsilateral dorsal root ganglion (DRG) of the spinal nerve ligation (SNL) cohort compared with their contralateral counterpart (von Schack et al., 2011). The miR-34c is a potential tumor suppressor that has been implicated in tumorigenesis of human malignancies, such as osteosarcoma (Hu et al., 2018), non-small cell lung cancer (Zhao et al., 2017), and ovarian cancer (Tung et al., 2017). The miR-34c has also been reported to regulate the intestinal dysfunction in inflammatory bowel disease (Chen et al., 2017). However, whether miR-34c contributes to the neuroinflammation in neuropathic pain has not been studied.
INTRODUCTION Neuropathic pain, one of the most challenging neurological diseases to treat, results from damage to or dysfunction of the peripheral or central nervous system rather than stimulation of pain receptors (van Hecke et al., 2014). Neuropathic pain is commonly associated with allodynia, pain from a stimulus that does not usually provoke pain and hyperalgesia, and increased pain from a stimulus that usually provokes pain (Jensen and Finnerup, 2014). Nearly 70% of individuals with spinal cord injury develop some degree of central neuropathic pain, which severely affects their emotional well-being and overall quality of life (Yezierski, 2005). Nerve injury triggers an inflammatory response that involves the activation of immune cells, microglia, and astrocytes, and the release of several proinflammatory cytokines plays crucial roles in the development of neuropathic pain (Schomberg et al., 2012). However, the pathogenesis of neuropathic pain is not fully understood. Therefore, it is very important to understand the specific underlying
*Corresponding author. E-mail address: [email protected] (S. Zhang). Abbreviations: ASC, apoptosis-associated speck-like protein; CCI, chronic constriction injury; ELISA, enzyme-linked immunosorbent assay; miRNAs, microRNAs; MMP, matrix metalloproteinase; mRNAs, messenger RNAs; mut, mutant; NLRP3, nucleotide binding domain-like receptor protein 3. https://doi.org/10.1016/j.neuroscience.2018.12.030 0306-4522/Ó 2018 IBRO. Published by Elsevier Ltd. All rights reserved. 125
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The inflammatory response occurs during cell damage and is one of the important physiological mechanisms of neuropathic pain (Abbaszadeh et al., 2018, Xu et al., 2018). The inflammatory response can be triggered by the inflammasome, a polyprotein complex (Zambetti et al., 2012). The most representative inflammasome is nucleotide binding domain-like receptor protein 3 (NLRP3). NLRP3 is activated by various endogenous and exogenous stimulation, and it recruits apoptosis-associated speck-like protein (ASC), which in turn activates caspase 1, thus stimulating the release of the proinflammatory cytokines interleukin (IL)-1b and IL18, which play key roles in innate immunity (Lamkanfi and Dixit, 2014). Caspase-1 activation can also lead to cell pyroptosis, an inflammatory form of programmed cell death distinct from apoptosis (Fink and Cookson, 2005). In addition to defending against pathogens, inflammasomes have been implicated in the pathogenesis of various inflammatory and metabolic diseases (Masters, 2013). In the current study, we investigated the roles of miR34c in chronic constriction injury (CCI)-induced neuropathic pain processing in the central nervous system. We also examined whether miR-34c interacts directly with and regulates the expression of the NLRP3 inflammasome, with the aim of providing novel insights into the molecular mechanism for neuropathic pain.
EXPERIMENTAL PROCEDURES Animals and ethic statements Adult male C57BL/6 mice (8–10 weeks of age) were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). The mice were housed in individual cages and given free access to water and food. All animal experiments were performed in strict accordance with the American Animal Protection Legislation. All study protocols were approved by the Institutional Animal Care and Use Committee of the Shanghai Tenth People’s Hospital of Tongji University. Establishment of a neuropathic pain animal model The mouse model of neuropathic pain was established through the CCI method, as previously described (Zhang et al., 2013). Briefly, the mice were anesthetized through intraperitoneal injection with 4% chloral hydrate. The right sciatic nerve was exposed at the mid-thigh level by blunt dissection. Three ligatures (4/0 silk) were tied loosely around the sciatic nerve 1.0–1.5 mm apart. The muscle and skin were sutured in layers with 4–0 Ethicon silk sutures. The same procedure was used for sham surgeries except that the sciatic nerve was exposed but not ligated. Plasmid construction To construct the miR-34c overexpression vector, oligonucleotides were designed and synthesized by Hanbio company (Shanghai, China). The primer sequences were as follows: forward, 50 -GGA AGU CUA
GUU ACU GCU CG-30 and reverse, 50 -GCC AGG UAA AAG ACU ACG GGU-30 . Purified PCR products were cloned into the vector pCDH-CMV (System Biosciences) at the EcoRI/NotI sites. All constructs were confirmed by DNA sequencing. Lentivirus packaging and transduction The constructed plasmid together with two envelope plasmids, PSPAX2 and PMD2G, were transfected into HEK293T cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Lentiviral supernatants were collected at 48 h after transfection and cleared with a Centricon Plus-70 filter unit (Millipore). Lentivirus particles with titers 108 TU/mL were used in the experiment. The lentivirus in vivo infections were performed according to a previous study (Pan et al., 2014). Briefly, 1 lL lentivirus or vector was administered to mice through intrathecal injections daily for 3 consecutive days 2 weeks before CCI. Thermal hyperalgesia and mechanical allodynia evaluation Thermal hyperalgesia was measured through paw withdrawal thermal latency, as described previously (Sacerdote et al., 2013). Briefly, the mice were placed in small clear Plexiglas cubicles and allowed to acclimate. Radiant heat with constant intensity (beam diameter 0.5 cm and intensity 20 I.R.) was applied to the midplantar area of the hind paw until the animal lifted its paw from the floor. The time (in seconds) from initial heat application to paw withdrawal was recorded. Mechanical allodynia was assessed through the paw mechanical withdrawal threshold measured with a dynamic plantar esthesiometer (Ugo Basile, Comerio, Italy). Briefly, the mice were placed in a test cage with a wire mesh floor and allowed to acclimate. The rigid tip of a von Frey filament was applied to the skin of the midplantar area of the hind paw with increasing force (up to 5 g over 20 s) until the animal removed its paw. The average values of the withdrawal threshold force (in grams) were calculated from four independent experiments. Tissue sample collection After the thermal hyperalgesia and mechanical allodynia evaluation, the mice in each group were sacrificed by decapitation under pentobarbital anesthesia (50 mg/kg by intraperitoneal injection). For histopathological analysis, the mice were subjected to cardiac perfusion with 4% paraformaldehyde. Segments of spinal cords at the lumbar dorsal L4–L6 were rapidly dissected from the mice, fixed with 4% paraformaldehyde and stored at 4 °C. For biochemical analysis, the L4–L6 dorsal spinal cords were collected, snap-frozen in liquid nitrogen and stored at 80 °C. Histopathology and scoring Paraformaldehyde-fixed and paraffin-embedded transverse sections (8 lm) of spinal cords at the lumbar
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dorsal L4–L6 level from sham mice (n = 6), CCI mice (n = 6), and CCI mice infected with Lenti-miR-34c (n = 6) or Lenti-vector (n = 6) at postoperative day 7 were stained with hematoxylin and eosin (HE). Spinal cords stained with HE were used to assess histopathologic changes in the spinal dorsal horn of gray matter scores (Wu et al., 2014, Sun et al., 2016). The gray matter was scored from 0 to 6 as follows: 0 = no lesion observed; 1 = gray matter contained 1–5 eosinophilic neurons; 2 = gray matter contained 5–10 eosinophilic neurons; 3 = gray matter contained more than 10 eosinophilic neurons; 4 = infarction of less than 1/3 of the gray matter area; 5 = infarction of 1/3 to 1/2 of the gray matter area; 6 = infarction of more than 1/2 of the gray matter area). Scores were generated by one well-trained, board-certified histopathologist who was blinded to the groups of mice. Enzyme-linked immunosorbent assay (ELISA) Tissues in the spinal cord from sham mice (n = 6), CCI mice (n = 6), and CCI mice infected with Lenti-miR-34c (n = 6) or Lenti-vector (n = 6) at postoperative day 7 were removed from the 80 °C refrigerator and ground to extract proteins. The protein expression of TNF-a and IL-1b was measured with a commercially available ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Immunofluorescence Transverse sections of spinal cords from sham mice (n = 6), CCI mice (n = 6), and CCI mice infected with Lenti-miR-34c (n = 6) or Lenti-vector (n = 6) at postoperative day 7 were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked in 1% BSA. For double immunofluorescence staining, the sections were incubated with a mixture of anti-NLRP3 and anti-GFAP, or anti-CD11b (all from Abcam, Cambridge, UK) at 4 °C overnight. After being washed, the sections were incubated with fluorescently conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) at 37 °C for 1 h. Then, the sections were counterstained with DAPI (Beyotime, Nantong, China) and examined under a confocal microscope (7CS SP5, Leica, Germany). Five randomly selected microscopic fields were analyzed per slide by a blinded investigator, and the number of NLRP3 and GFAP or CD11b-positive cells in lamina I–III of the L4–L6 spinal dorsal horn of each mouse was recorded. Quantitative real-time PCR Total RNA of lumbar spinal cord segments (L4–L6) of the indicated mice (n = 6 per group) was extracted with TRIzol reagent (Invitrogen). MiRNA cDNA was synthesized with a Mir-X miRNA First-Strand Synthesis Kit (Qiagen, Dusseldorf, Germany) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed with SYBR Green PCR mix in an ABI Prism 7900HT instrument (Applied Biosystems, Foster City, CA, USA). The primer
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sequences were as follows: miR-34c: forward, 50 -TGT CAC CAA GTC ATC TCT CCA G-30 and reverse, 50 CAG GTC CTC CTC ATA ATT TGG CT-30 ; U6: forward, 50 -GAC CTA CAA CGG GCA TCT GG-30 and reverse, 50 -GAC CTA CAA CGG GCA TCT GG-30 . qRT-PCR was performed for 40 cycles at 95 °C for 15 s and 60 °C for 1 min after an initial 15-min incubation at 95 °C. The fold changes in expression were obtained with the 2DDCt method (Cikos et al., 2007), and the miR-34c expression was normalized to that of U6. TUNEL staining Paraformaldehyde-fixed and paraffin-embedded sections from sham mice (n = 6), CCI mice (n = 6), and CCI mice infected with Lenti-miR-34c (n = 6) or Lenti-vector (n = 6) at postoperative day 7 were used to detect cell death through TUNEL staining according to the manufacturer’s instructions (Roche Applied Science, Indianapolis, IN, USA). Briefly, fixed frozen sections were rehydrated in PBS for 5 min. Slides were then incubated with 20 lg/mL proteinase K solution for 15 min at 25 °C, treated with 3% H2O2 for 5 min, and incubated in equilibrium buffer for 10 min. The slides were then incubated with terminal deoxynucleotidyl transferase for 1 h before being washed and treated with anti-digoxigenin for 30 min. Signal was visualized with DAB. The number of apoptotic cells and the total number of cells were counted in five randomly selected microscopic fields, and the percentage of TUNELpositive cells in spinal dorsal horn was calculated from 6 spinal cord sections from different mice. Luciferase reporter assay To confirm the interaction between the 30 -UTR of NLRP3 mRNA and miR-34c, HEK293T cells were seeded in 12well plates at 1 105 cells/mL and co-transfected with wild-type (wt) or mutant (mut) pGL3-NLRP3 30 -UTR and miR-34c mimics or a control construct (Genepharma, Shanghai, China) with Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. Luciferase activity was calculated as the ratio of firefly to Renilla luciferase activity, as measured with a luciferase assay system (Promega, Madison, WI, USA) after 48-h transfection. The relative luciferase activity was expressed as the ratio of the measured luciferase activity to the control. Western blot analysis Spinal cord tissues in the lumbar spinal cord from sham mice (n = 6), CCI mice (n = 6), and CCI mice infected with Lenti-miR-34c (n = 6) or Lenti-vector (n = 6) at postoperative day 7 were removed from the 80 °C refrigerator and lysed, and proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA). After being blocked in 5% nonfat dry milk containing 0.1% Tween 20 for 2 h, the membranes were incubated at 4 °C overnight with primary antibodies (diluted 1000-fold) that recognized NLRP3, ASC, Caspase-1, IL-1b, IL-18, and
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b-actin (all from Abcam). After being washed in PBS, the membranes were incubated at room temperature with horseradish peroxidase-conjugated secondary antibody diluted 5000-fold for 1 h and visualized with a SuperSignal West Dura extended duration substrate detection system (Thermo Scientific, Rockford, IL, USA). Protein expression was normalized to that of bactin. Statistical analysis Data are represented as mean ± standard deviation (SD) from at least triplicate experiments. Results were analyzed in GraphPad Prism 5 software (GraphPad, San Diego, CA, USA). A one-way analysis of variance (ANOVA) was used to determine significant differences between groups, and the individual comparisons were performed with Tukey’s post hoc test. The threshold for statistical significance was set at p < 0.05.
RESULTS Overexpressed miR-34c alleviates CCI-induced neuropathic pain and tissue damage To investigate the relationship between miR-34c and neuropathic pain, mice with chronic constriction of the sciatic nerve were generated via surgical ligation, and the expression of miR-34c in the spinal cord was then measured. At 0, 3, 7, 14, and 21 days after CCI, the spinal cords of the mice were collected, and the miR34c levels in the spinal cords were analyzed with qRTPCR. In the CCI mice, compared with mice receiving sham surgery, miR-34c was significantly downregulated in a time-dependent manner (Fig. 1A). To investigate the biological role of miR-34c in neuropathic pain, miR34c was stably overexpressed in mice through intrathecal injection of lentivirus for expression of miR34c (Lenti-miR-34c). In the injected mice, compared with the CCI control mice, miR-34c was significantly overexpressed (Fig. 1B). Furthermore, our results showed that miR-34c overexpression attenuated CCIinduced mechanical allodynia (Fig. 1C) and thermal hyperalgesia (Fig. 1D). Finally, after each treatment at 7 days after CCI, the spinal cords were stained with HE and were assigned histopathology scores by an experienced pathologist according to changes in the gray matter (Fig. 1E, F). A higher score indicated greater tissue damage. Overexpression of miR-34c, compared with CCI control treatment, resulted in significantly lower histopathology scores, thus indicating less tissue infarction after CCI. Together, these findings indicated that CCI induced pain hypersensitivity and increased tissue damage, which could be reversed by upregulation of miR-34c expression. Overexpression of miR-34c suppresses cell apoptosis and inflammation in CCI mice To further explore the biological effects of miR-34c on apoptosis and inflammation, a TUNEL assay was performed on sections of the spinal cord collected at day 7 after CCI, and the levels of TNF-a and IL-1b were
measured by ELISA in the spinal cords of mice at 7 days after CCI. The proportion of apoptotic cells was significantly higher in the CCI mice than in the sham operated mice (Fig. 2A, B), and TNF-a (Fig. 2C) and IL1b (Fig. 2D) levels were elevated in all CCI treatment groups. Overexpression of miR-34c, compared with CCI control treatment, significantly decreased the number of apoptotic cells and the levels of TNF-a and IL-1b, thus indicating that miR-34c decreased the cell apoptotic and inflammatory response in CCI mice. MiR-34c binds the 30 -UTR of NLRP3 and affects NLRP3 inflammasomes To explore the potential molecular mechanism through which miR-34c regulates neuropathic pain, we performed bioinformatic analyses to determine the direct target gene of miR-34c. We found that NLRP3 30 -UTR contained a conserved binding site for miR-34c, and the sequence of an RNA mutant of the 30 -UTR of NLRP3 was also present in the alignment (Fig. 3A). To confirm the interaction between the 30 -UTR of NLRP3 and miR34c, we co-transfected HEK293T cells with the miR-34 mimics or the control construct (miR-34 NC) and the dual-luciferase vector containing wt or mut pGL3-NLRP3 30 -UTR. Luciferase reporter assays showed that miR34c significantly decreased the luciferase activity of the wt 30 -UTR but not that of the mut 30 -UTR of NLRP3, thus confirming that NLRP3 is a target gene of miR-34c (Fig. 3B). Next, the relative protein levels of NLRP3, ASC, and activated caspase-1 were examined by western blot analysis in the spinal cords of CCI model mice infected with Lenti-miR-34c or control Lenti-vector at postoperative day 7 (Fig. 3C). The results revealed significantly higher NLRP3, ASC, and activated caspase-1 protein levels in the spinal cord in CCI mice than in sham mice. Moreover, overexpression of miR34c, compared with control CCI treatment, significantly inhibited the expression levels of NLRP3, ASC, and activated caspase-1 in the spinal cord (Fig. 3D). The protein levels of mature IL-1b and IL-18 in the spinal cord were also measured by western blot analysis (Fig. 3E). As shown in Fig. 3F, the protein levels of both cytokines were higher in CCI mice than sham mice, and overexpression of miR-34c decreased cytokine maturation. These findings further confirmed the interaction between miR-34c and NLRP3 and suggested that miR-34c negatively regulate the expression of NLRP3 and subsequently inhibit inflammasome activity (NLRP3, ASC, and caspase-1) and cytokine maturation. Overexpression of miR-34c inhibits CCI induced NLRP3 inflammasome activation in spinal cord astrocytes microglia and neurons To provide direct evidence of the involvement of miR-34c in CCI-induced pain behavior, we examined NLRP3 expression in the spinal cord in CCI model mice infected with Lenti-miR-34c or control Lenti-vector at postoperative day 7. The protein expression and cellular distribution of NLRP3 in the spinal cord were probed with double immunofluorescence staining with
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Fig. 2. Overexpression of miR-34c suppresses cell apoptotic and inflammation in CCI mice. (A) TUNEL assays were performed on the spinal cord sections from each group at day 7 after CCI, and arrows show apoptotic cells. Scale bars = 20 lm. (B) The percentage of apoptotic cells in the spinal cord tissue of each group was calculated. The spinal cord tissue levels of TNF-a (C) and IL-1b (D) were measured by ELISA in all treatment groups and the sham group. n = 6 per group. *p < 0.05, **p < 0.01 vs. sham group. #p < 0.05 vs. CCI group.
antibodies against GFAP (an astrocyte marker), CD11b (a microglia marker) and NeuN (a neuron marker), respectively. We detected higher NLRP3 expression in astrocytes (Fig. 4A) microglia (Fig. 4B) and neurons (Fig. 4C) in the spinal cord in the CCI mice than in the sham operated controls. Moreover, significant inhibition of NLRP3 expression in spinal cord astrocytes, microglia and neurons was observed in the Lenti-miR34c infected mice compared with CCI control mice. Therefore, NLRP3 inflammasome activation in spinal cord astrocytes microglia and neurons contributes to the processing of CCI-induced neuropathic pain, and overexpression of miR-34c inhibited NLRP3 inflammasome assembly.
DISCUSSION The mechanisms underlying neuropathic pain remain poorly understood despite the enormous efforts that have been made in basic and clinical research. The prototypical opioid morphine is commonly used to control severe pain; however, tolerance to the drug’s analgesic effects develops after repeated or continuous use and requires an increase in dosage to maintain the same efficacy. In addition, morphine is not always sufficient in neuropathic pain, and patients receiving morphine are particularly susceptible to abuse and addiction (Berrios et al., 2008). Therefore, novel targets that may lead to the development of promising analgesics are eagerly anticipated for improved pain management.
3 Fig. 1. Overexpressed miR-34c alleviates CCI-induced neuropathic pain and tissue damage. (A) miR-34c levels in the spinal cords of mice were measured by qRT-PCR at postoperative days 0, 3, 7, 14, and 21. Sham operated mice were used for comparison. n = 6 for each time point. * p < 0.05, **p < 0.01 vs. sham group. (B) The expression of miR-34c in the spinal cords of CCI model mice infected with lentiviral vectors carrying miR-34c (Lenti-miR-34c) or lentiviral vector (Lenti-vector) at postoperative day 7. n = 6 per group. *p < 0.05, **p < 0.01 vs. sham group. #p < 0.05 vs. CCI group. (C) The effect of upregulated miR-34c on thermal hyperalgesia was assessed by paw withdrawal thermal latency (PWTL). (D) The effect of miR-34c overexpression on mechanical allodynia was evaluated according to the paw mechanical withdrawal threshold (PMWT). n = 6 per group. *p < 0.05, **p < 0.01. (E) Spinal cord sections collected at day 7 after CCI were stained with HE, and arrows represent lesions. Scale bars = 20 lm. (F) Histopathology scores were assigned by an experienced pathologist. n = 6 per group. *p < 0.05, **p < 0.01 vs. sham group. # p < 0.05 vs. CCI group.
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Fig. 3. MiR-34c binds the 30 -UTR of NLRP3 and affects NLRP3 inflammasomes. (A) Schematic diagram of the putative binding sites between miR34c and 30 -UTR of NLRP3. The paired miR-34c seed sequence and the seed-recognizing site in the wild-type (wt) and mutant (mut) 30 -UTR of NLRP3 are indicated. (B) Dual-luciferase reporter assays of miR-34c and NLRP3 30 -UTR. The miR-34 mimics or the control (miR-34 NC) was cotransfected with the dual-luciferase vector containing wt or mut pGL3-NLRP3 30 -UTR into HEK293T cells and incubated for 48 h. The relative luciferase activity was calculated as the ratio of firefly to Renilla luciferase activity, as measured with the Dual-Luciferase Assay System. **p < 0.01 vs. miR-34c NC group. n = 3 per group. Three independent experiments were performed. (C) Western blot analysis of NLRP3, ASC, and activated (cleaved) caspase-1 in the spinal cords of CCI model mice infected with Lenti-miR-34c or Lenti-vector at postoperative day 7. (D) Quantification of NLRP3, ASC, and activated (cleaved) caspase-1 protein levels relative to that of b-actin. (E) Representative western blotting results for mature IL1b and IL-18. (F) The intensity of each protein was quantified and normalized to the signal intensity of the corresponding pro-IL-1b or pro-IL-18 band present on the same membrane. n = 6 per group. *p < 0.05, **p < 0.01 vs. sham group. #p < 0.05 vs. CCI group.
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Fig. 4. Overexpression of miR-34c inhibits CCI-induced NLRP3 inflammasome activation in spinal cord astrocyte and microglia. (A) Double immunohistochemical staining of spinal cord sections from CCI model mice infected with Lenti-miR-34c or Lenti-vector at postoperative day 7, combining NLRP3 with (A) GFAP, (B) CD11b or (C) NeuN labeling. Scale bar = 50 lm. From the stained sections, the numbers of NLRP3 and GFAP, CD11b or NeuN double-labeled cells were counted. n = 6 per group. *p < 0.05, **p < 0.01 vs. sham group. #p < 0.05 vs. CCI group.
Many relevant studies have shown that miRNAs have a regulatory role in neuropathic pain (Shi et al., 2013, Lopez-Gonzalez et al., 2017, Wang et al., 2018). MiR34c has been widely investigated, and it plays an important role in many diseases, including cancer (Tung et al., 2017, Zhao et al., 2017, Hu et al., 2018) and inflammatory bowel disease (Chen et al., 2017). In addition, an important role of miR-34c in the nervous system has also been demonstrated. Kabaria et al. (2015) have found that downregulation of miR-34c in the brain contributes to Parkinson’s disease pathogenesis. However, the relationship between miR-34c and neuropathic pain remains unclear. In the current study, we found that miR-34c plays a suppressive role by targeting NLRP3 in neuropathic pain development in CCI mice. MiR-34c was downregulated in CCI mouse models, and it may alleviate neuropathic pain development. Overexpression of miR-34c greatly attenuated neuropathic pain and neuroinflammation in vivo. Our data showed that miR-34c inhibited neuropathic pain, possibly through inhibiting NLRP3-triggered neuroinflammation, thus providing novel insights into the molecular pathogenesis of neuropathic pain.
Among the NLR inflammasomes, NLRP3 has been the most intensively studied, given its role in inherited autoinflammatory syndromes (Hoffman et al., 2001) and several metabolic diseases including obesity, diabetes, atherosclerosis, and gout (Wen et al., 2012). In the central nervous system, the NLRP3 inflammasome plays an important role in the development of Alzheimer’s disease and bacterial meningitis (Liu et al., 2013). CCI mice with thermal hyperalgesia and mechanical allodynia have elevated levels of the NLRP3 inflammasome proteins NLRP3 and ASC and downstream caspase-1, IL-1b, and IL-18 in spinal cord tissues. These results suggest that CCI induces the NLRP3 inflammasome activation and the subsequent cleavage of TNF-a and IL-1b. Intrathecal injections of lentiviral vectors bearing miR-34c in mice 2 weeks before CCI ameliorated CCI-induced pain behaviors. These analgesic effects of miR-34c overexpression were accompanied by decreased NLRP3, ASC, caspase-1, IL-1b, and IL-18 protein levels, and diminished TNF-a and IL-1b production. Overexpression of miR-34c also decreased CCI-induced spinal cord tissue infarction, and spinal cord cell apoptosis; this may suggest a
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mechanism of action by which miR-34c protects against infarction via inhibition of NLRP inflammasome activation, thus resulting in the inhibition of cell apoptosis and the downregulation of proinflammatory cytokines. In addition, CCI induces activation of microglia and astrocytes, and neuron damage, whereas intrathecal injection of miR34c lentiviral vector reverses this phenomenon, possibly because the function of the miR-34c lentiviral vector is mediated by primary sensory neurons, the injured neurons that activate spinal astrocytes and microglia. Collectively, these findings provide evidence that the miR-34c and NLRP3 inflammasome is involved in the pathogenesis of neuropathic pain. IL-1b is produced as a precursor, pro-IL-1b, which must be cleaved either by caspase-1 or by matrix metalloproteinase (MMP)-9 or MMP-2 to become biologically active (Schonbeck et al., 1998, Martinon and Tschopp, 2007). MMP-9 and MMP-2, which are overexpressed in injured spinal cord tissues, are key mediators of neuropathic pain through IL-1b cleavage (Schomberg et al., 2012). Our study demonstrated that the NLRP3 inflammasome, the best-characterized inflammasome activated by cellular infection or stress, contributed to the development of neuropathic pain through induction of IL-1b cleavage and spinal cord cell apoptosis. Overexpression of miR-34c inhibited this pathway and may provide effective analgesics for pain management. In summary, the experimental results in this study showed that miR-34c suppressed the activity of the NLRP3 inflammasome (NLRP3, ASC, and Caspase-1), thereby alleviating CCI-induced neuropathic pain in mice through downregulation of NLRP3. However, the mechanisms of miR-34c and NLRP3 related to the occurrence, development, and prognosis of neuropathic pain require further confirmation, because the expression of miR-34c at the cellular level was not determined in our study, owing to limitations in funding, and further studies are needed.
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(Received 12 August 2018, Accepted 17 December 2018) (Available online 26 December 2018)