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6 mouse model of cuprizone-induced demyelination
Accepted Manuscript Research report Prednisone alleviates demyelination through regulation of the NLRP3 inflammasome in a C57BL/6 mouse model of cuprizone-induced demyelination Hao Yu, Mingfeng Wu, Geng Lu, Tingting Cao, Nan Chen, Yijia Zhang, higuo Jiang, Hongbin Fan, Ruiqin Yao PII: DOI: Reference:
S0006-8993(17)30440-7 https://doi.org/10.1016/j.brainres.2017.09.034 BRES 45511
To appear in:
Brain Research
Received Date: Revised Date: Accepted Date:
12 July 2017 27 September 2017 29 September 2017
Please cite this article as: H. Yu, M. Wu, G. Lu, T. Cao, N. Chen, Y. Zhang, h. Jiang, H. Fan, R. Yao, Prednisone alleviates demyelination through regulation of the NLRP3 inflammasome in a C57BL/6 mouse model of cuprizoneinduced demyelination, Brain Research (2017), doi: https://doi.org/10.1016/j.brainres.2017.09.034
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Prednisone alleviates demyelination through regulation of the NLRP3 inflammasome in a C57BL/6 mouse model of cuprizone-induced demyelination Hao Yu1*, Mingfeng Wu1*, Geng Lu2, Tingting Cao2, Nan Chen2, Yijia Zhang2, Zhiguo Jiang 2, Hongbin Fan3, Ruiqin Yao1# 1 Neurobiological Research Center, Xuzhou Medical University, 209# Tongshan Road, Yunlong District, Xuzhou 221000, China 2 Department of Clinical Medicine, Xuzhou Medical University, 209# Tongshan Road, Yunlong District, Xuzhou 221000, China 3 Department of Neurology, Affiliated Hospital of Xuzhou Medical University, 99# Huaihai Road, Quanshan District, Xuzhou 221009, China
*These authors contributed equally to this work. #
Corresponding Author: Ruiqin Yao E-mail: [email protected]
Abstract Myelin abnormalities, oligodendrocyte damage, and concomitant glia activation are common in demyelinating diseases of the central nervous system (CNS). Increasing evidence has demonstrated that the inflammatory response triggers demyelination and gliosis in demyelinating disorders. Numerous clinical interventions, including those used to treat multiple sclerosis (MS), have confirmed prednisone (PDN) as a powerful anti-inflammatory drug that reduces the inflammatory response and promotes tissue repair in multiple inflammation sites. However, the underlying mechanism of PDN in ameliorating myelin damage is not well understood. In our study, a cuprizone (CPZ)-induced demyelinated mouse model was used to explore the mechanism of the protection provided by PDN. Open-field tests showed that CPZ-treated mice exhibited significantly increased anxiety and decreased exploration. However, PDN improved emotional behavior, as evidenced by an increase in the total distance traveled, and central distance traveled as well as the mean amount of time spent in the central area. CPZ-induced demyelination was observed to be alleviated in PDN-treated mice based on luxol fast blue (LFB) staining and myelin basic protein (MBP) expression analyses. In addition, PDN reduced astrocyte and microglia activation in the corpus callosum. Furthermore, we demonstrated that PDN inhibited the Nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome signaling pathway and related inflammatory cytokines and chemokines, including TNF-α, CCL8, CXCL10 and CXCL16. PDN also reduced the serum corticosterone levels in the CPZ-treated mice. Taken together, these results suggest that inhibition of the NLRP3 signaling pathway may be a novel mechanism by which PDN exerts its protective actions in demyelinating diseases.
Key Words Demyelination; Inflammation; Prednisone; NLRP3; Chemokine Abbreviations CPZ, cuprizone; GFAP, glial fibrillary acidic protein; Iba-1,Ionized calcium binding adaptor molecule-1; LFB, luxol fast blue; NLRP3, Nod-like receptor pyrin domain 1
containing 3; MBP, myelin basic protein; MS, multiple sclerosis; PDN, prednisone.
1. Introduction The inhibition of demyelination is a critical challenge in the treatment of many central nervous system (CNS) degenerative diseases, especially multiple sclerosis (MS)[1-3]. The poor prognosis of this disease may in part due to the fact that neuroinflammation results in the loss of the myelin sheath, which cannot regenerate effectively, ultimately leading to the poor conduction of nerve impulses. Previous studies have reported lymphocyte/macrophage infiltration, astrocyte and microglial activation, and massive cytokine/chemokine secretion in progressive demyelinating diseases[4-6]. Furthermore, the inflammasome which consists of Nod-like receptor pyrin domain containing 3 (NLRP3), the adaptor protein apoptotic speck-containing protein with a CARD (ASC) and pro-caspase-1, has been reported to have a significant role in long-term inflammation responses[7-9]. Other studies have tested the hypothesis that active NLRP3 inflammasome aggravates inflammation by activating pro-IL-1β and pro-IL-18, which then mature to IL-1β and IL-18, via the P2X7 receptor in murine lupus nephritis[10]. Worse still, activated IL-1β can stimulate downstream cytokines and certain chemokine receptors, which interact with abundant cytokines and chemokines secreted by inflammatory cells to trigger inflammatory responses[7]. In addition, chemokines and their receptors have been reported to recruit inflammatory cells, including microglia and leucocytes, to expand the inflammatory region and further exacerbate myelin injury[11]. Therefore, elucidating the mechanisms by which neuroinflammation induces demyelination and blocking this inflammation process is a crucial step for the development of therapeutics and improving patient prognoses. Prednisone (PDN) is representative of glucocorticoid compounds, which have powerful anti-inflammatory activity and neuroprotective characteristics and are widely prescribed drugs[12, 13] . For instance, low-dose PDN treatment gradually benefits patients with rheumatoid arthritis (RA) by reducing chronic inflammation[14]. However, the potential for PDN to improve myelin loss by reducing the inflammatory response is unclear. Cuprizone (CPZ), as a copper chelator, specifically damages oligodendrocytes, without directly affecting other glial cells, and induces consistent demyelination in the C57BL/6 mouse brain. This compound is therefore generally acknowledged as an inducer of demyelination and myelin regeneration in animal models[15, 16]. Thus, we generated a CPZ-induced mouse model of demyelination to examine the function of PDN on the activation of the NLRP3 inflammasome and glia, as well as the level of related cytokines and chemokines. Neurodegeneration is a strong determinant of hypothalamus-pituitary-adrenal (HPA) axis activity. HPA system dysregulation has been shown in MS patients, and many studies showing high levels of cortisol in parts of sufferers of MS[17, 18]. Researchers proposed that the suppressive effects of some medicine on mood disorders such as depression- and anxiety-related behaviors might be primarily mediated by inhibition of corticosterone production[19, 20]. In our study, corticosterone level in serum was obviously enhanced in samples from the cuprizone group, suggesting possible HPA activation[21]. PDN significantly reduced the corticosterone level suggests that the PDN might exhibit the feedback effect on HPA axis. Despite all this, it can not be concluded the direct relationship between demyelination and corticosterone level, further evidence is needed to support the interpretation. We first carried out open field tests to evaluate improvements in the physical condition and 2
behavior of PDN-treated mice. Secondly, we assessed myelination and glial activation to study the role of PDN in ameliorating demyelination in CPZ-treated mice. We lastly explored the related mechanism of PDN mediated changes by detecting the expression levels of NLRP3, caspase-1 and IL-1β, as well as the secretion of TNF-α, CCL8, CXCL10, and CXCL16. These data are the first to indicate that the inhibition of NLRP3 inflammasome activation may be mechanism by which PDN protects the myelin sheath from degeneration in a CPZ-induced demyelinated model.
2. Results 2.1 PDN ameliorates CPZ-induced body weight loss The body weights of the mice were recorded weekly to observe the effect of PDN on mice with CPZ-induced demyelination. In the Ctrl group, the average body weight in normal fed-mice increased gradually, while CPZ treatment decreased body weight dramatically from the fourth to fifth week. There was no obvious difference in the average body weight between the CPZ+NS and CPZ groups. However, PDN treatment alleviated the body weight loss induced by CPZ, and a significant difference was found in this metric between the CPZ+PDN group and the CPZ group at the fourth (P<0.05) and fifth week (P<0.01) (Fig. 1A). 2.2 PDN improved the anxiety and exploratory ability of CPZ-induced mice Nervous system dysfunction is consistently observed in demyelination diseases, including abnormalities in motor function, spatial working memory, social interaction, etc.[22, 23]. Here, we performed an open-field test to evaluate the anxiety and exploration ability of the mice. The CPZ group mice traveled a shorter distance than the control mice in the center area and in the overall area of the apparatus (Fig. 1B, C). Moreover, the time spent in the central area was significantly lower in the CPZ-treated mice than that in the control mice (Fig. 1B, E), suggesting that mice with CPZ-induced demyelination are hypoactive. However, the center distance, total distance and time spent in the central area were all significantly increased in the PDN-treated mice compared with the CPZ group (Fig. 1B-D). No significant changes were observed in the central distance, total distance or time spent in the center zone between the CPZ and CPZ-NS groups. These data suggest that PDN decreases anxiety-like behaviors and increases exploratory behavior ability in mice with CPZ-induced demyelination. 2.3 PDN alleviated demyelination induced by CPZ LFB staining showed that myelination was complete in the corpus callosum in the Ctrl group, whereas most of the myelin sheath was absent from the corpus callosum in the CPZ and CPZ+NS groups. The myelin score was significantly decreased in the CPZ group mice compared with the Ctrl mice, indicating that the myelin sheath was severely damaged by CPZ treatment. No significant differences were found in this metric between the CPZ and CPZ+NS groups. In contrast, PDN-treated mice displayed higher myelin scores than those of CPZ-treated mice (Fig. 2A-B). MBP immunohistochemistry staining in the corpus callosum showed that the mice exposed to CPZ exhibited few MBP-positive oligodendrocytes. However, more MBP-positive oligodendrocytes were found in the PDN-treated mice (Fig. 2C). The IOD value of the MBP-positive cells was significantly decreased in CPZ-treated mice compared with the Ctrl group; however, PDN treatment reversed the decrease in the MBP decrease in IOD value induced by CPZ 3
(Fig. 2D). The levels of MBP protein in the corpus callosum in the CPZ group were also remarkably lower than those in the control group (Fig. 3E, F). However, the expression of MBP was clearly increased by PDN treatment when compared with the CPZ group (Fig. 3E, F). No significant differences were found in the IOD value or the protein levels of MBP between the CPZ and CPZ+NS groups. 2.4 PDN inhibited glial activation in CPZ-treated mice It has been reported that white matter astrocytes and microglia are activated in CPZ-treated mice[24]. To observe whether PDN alleviated demyelination by inhibiting glial activation, GFAP and Iba-1 immunohistochemistry staining were used to detect glial activation. The results showed that the number of GFAP-positive cells (Fig. 3A) and Iba-l-positive cells (Fig. 3B) were both increased in the corpus callosum of mice treated with CPZ, but PDN treatment significantly decreased the number of GFAP- and Iba-l-positive cells when treated by PDN in CPZ-induced model mice (Fig. 3C-D). The data suggested that PDN could inhibit the CPZ-induced activation of astrocytes and microglia. 2.5 PDN suppressed the activation of the NLRP3 inflammasome and down-regulated the secretion of inflammatory factors and chemokines It was recently verified that the NLRP3 inflammasome is activated and involved in demyelination diseases[25]. To explore how PDN protects the myelin sheath against inflammation, we detected the protein levels of NLRP3, cleaved-caspase-1 and the downstream protein cleaved-IL-1β in the corpus callosum. NLRP3 protein expression was dramatically increased in the corpus callosum of CPZ-treated mice, while treatment with PDN clearly reduced the level of NLRP3 (Fig. 4A-B). Moreover, the levels of cleaved-caspase-1 and cleaved-IL-1β were decreased (Fig. 4A, C, D), implying that PDN might alleviate demyelination by suppressing activation of the NLRP3 signaling pathway in mice with CPZ-induced demyelination. Cytokines and chemokines, along with their receptors, have been reported to exacerbate inflammation and the immune reaction in inflammatory CNS diseases[26, 27]. We therefore measured the messenger RNA (mRNA) expression levels of TNF-α, CCL8, CXCL10 and CXCL16 by rtRT-PCR. As shown in Fig. 5A, C-E, the levels of TNF-α, CCL8, CXCL10 and CXCL16 mRNA were higher than that those in Ctrl mice. PDN treatment significantly decreased the levels of these mRNAs, suggesting that PDN might further relieve inflammatory injury by down-regulating the secretion of inflammatory factors and chemokines in a CPZ-induced demyelination model. We also detected the protein levels mentioned above by western blot. The protein expression of TNF-α is consistent with the results of rtRT-PCR (Fig.5B). Corticosterone levels were analyzed by ELISA at the end of experimental period. As expected, serum corticosterone level was significantly higher in the cuprizone-treated mice than that in the control mice (p< 0.01). PDN at 10 mg/kg reduced corticosterone levels, there was significantly difference between the CPZ and PDN groups (p < 0.05, Fig.5F).
3. Discussion
4
CNS demyelination is characterized by myelin sheath damage, oligodendrocyte death, glial activation and remyelination failure [16]. Previous studies have confirmed that PDN has significant anti-inflammatory activity and promotes both brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) expression in the cerebral cortex of EAE mice following minocycline treatment [28]. Here, for the first time, we report that PDN alleviates demyelination by inhibiting the activation of the NLRP3 inflammasome and suppressing the secretion of related inflammatory factors and chemokines in mice with CPZ-induced demyelination. We thereby revealed a new mechanism for PDN in the treatment of for demyelinating diseases. Feeding mice with the copper chelator CPZ is designed to damage mitochondrial function and further induce CNS demyelination [29]. Prior studies have shown that mice fed with 0.2-0.3% CPZ begin to exhibit demyelination from the second week of treatment. Demyelination gradually continues and is complete at the fifth or sixth week in the corpus callosum. When CPZ treatment is stopped, the process of demyelination ceases, allowing myelination to return to a somewhat normal level[4, 30]. In our study, to examine the effect and the mechanisms of PDN on demyelination, PDN was administered intragastrically every day after 3 weeks of CPZ exposure. After 5 weeks, we performed LFB staining to detect the extent of demyelination and MBP expression in the corpus callosum. In our research, we found that PDN protected myelin against CPZ-induced toxicity, improving the myelin score and up-regulating the expression of MBP. Furthermore, consistent with previous studies, C57BL/6 mice exposed to CPZ for 5 weeks exhibited obvious body weight loss. However, PDN treatment alleviated this body weight loss, suggesting that the protective effect of PDN on CPZ-induced body weight loss might be related to improvement in myelination. Limb paralysis and abnormal behaviors are widely demonstrated in MS patients and CPZ model mice exhibit similar symptoms [31-33]. Recently, an increasing number of studies have shown that motor dysfunction and behavioral disorders might be related to white matter damage induced by myelin sheath loss, as well as by the limited regenerative abilities of oligodendrocytes and their precursors [34]. The open-field test is used widely to evaluate motor activity, exploration ability and anxious behavior in vivo in CPZ model mice and other animal models [32, 35]. In our experiment, we used the open-field test to test whether PDN treatment improves the motor difficulties caused by CPZ exposure. The CPZ+PDN group mice showed a longer traveled distance and more time in the center area of open field apparatus than the CPZ group mice, indicating that PDN alleviates behavioral disorders by improving demyelination. Astrocytes are an important type of glial cell in the CNS and play dual roles in demyelinating diseases [36]. On the one hand, astrocytes secretes an abundance of neurotrophic factors to promote the regeneration of the myelin sheath; however, these cells also promote the immune reaction and form glial scars, thereby accelerating demyelination[37, 38]. Microglia act as macrophages in the CNS and promote demyelination recovery by clearing cell debris. However, this cell type can also accelerates inflammatory injury by secreting pro-inflammatory factors, such as TNF-α[39, 40]. In pathologically demyelinated zones in the CNS, astrocyte hyperplasia and microglia activation are consistently observed, as is oligodendrocyte damage. Here, we found that PDN administration for 2 weeks significantly decreased the numbers of GFAP-positive astrocytes and Iba-1-positive microglia, suggesting that PDN could suppress glial activation in mice with CPZ-induced demyelination. 5
NLRP3 inflammasome activation is suggested to be a principal underlying mechanism in experimentally induced autoimmune encephalomyelitis (EAE), another mouse model of demyelination [41, 42]. Recently, it was reported that NLRP3 inflammasome activation is involved in corpus callosum demyelination progression in CPZ-treated mice and that this effect is mediated via the IL-1β pathway[7]. Previous studies have demonstrated that both mature oligodendrocytes and their precursor cells express glucocorticoid receptors (GRs)[43, 44], and glucocorticoids are involved in promoting myelination through GRs[45, 46]. Treatment with high-dose glucocorticoids is still regarded as the most important and efficient therapeutic method to promote the recovery of neurological function in some demyelination diseases, including in acute MS relapses [47]. In our present study, we found that PDN treatment may protect the myelin sheath against CPZ damage by inhibiting the NLRP3 inflammasome signaling pathway. It was also reported that NLRP3 activation could result in the secretion of inflammatory cytokines and chemokines [42]. We also tested the expression levels of inflammatory cytokines and chemokines and verified that PDN inhibited the secretion of TNF-α, CCL8, CXCL10 and CXCL16. Chemokines represent a group of chemotactic proteins with molecular weights ranging from 8 to 14 kDa. These proteins modulate leukocyte attraction and glial activation under inflammatory conditions[27, 48]. Makoto Inoue, et al. reported that the chemotaxis-related proteins CCL8/MCP-2 and its receptor CCR2, as well as CXCL16 and its receptor CXCR6, are up-regulated in the presence of the NLRP3 inflammasome. These authors inferred that leukocyte-attracting chemokines could be novel molecular targets in treating MS patients in the context of NLRP3 inflammasome activation[41]. Moreover, CXCL16 is activated by cytokines and can induce the migration and invasion of glial precursor cells by up-regulating its receptor CXCR6 [49]. CXCL10 acts as a key microglia-attracting chemokine and is secreted at a high level in CPZ-treated wild-type mice [50]. Our study indicates that PDN might alleviate CPZ-induced demyelination by suppressing the activation of NLRP3 signaling and further down-regulating the secretion of inflammatory factors and chemokines, thereby reducing glial activation. In summary, our results demonstrated that the NLRP3 inflammasome signaling pathway is involved in CPZ-induced demyelination. PDN, as an anti-inflammatory drug, may target the NLRP3 inflammasome complex and reduce inflammation through a cascade of reactions, thereby alleviating CPZ-induced myelination and improving behavioral function. Therefore, we demonstrate a novel mechanism by which PDN therapy ameliorates the symptoms of inflammatory demyelinating diseases.
4. Materials & Methods 4.1 Animals Adult wild-type C57BL/6 male mice (8 weeks old, 23-25 g) were obtained from the Xuzhou Medical Universal Experimental Laboratory Animal Center and housed in a particular feeding room with a 12:12-hour light-dark cycle. Food pellets and drinking water were provided ad libitum. All of the experiments in this study were performed in keeping with the guidelines set by the National Institutes of Health guide (NIH) for the care and use of Laboratory animals and were also approved institutionally by both the utilization of laboratory animals of Xuzhou Medical University and Chinese Laws. 6
4.2 Prednisone and Cuprizone Treatment The mice were randomly divided into four groups: Control group (Ctrl), CPZ-treated group (CPZ), CPZ- and normal saline-treated group (CPZ+NS), and CPZ- and PDN-treated group (CPZ+PDN). Experiments were optimally designed to minimize the number of animals required. Ctrl mice were fed a standard rodent chow, while the others were fed diets containing 0.3% (w/w) CPZ (Sigma-Aldrich, USA) to induce demyelination[34]. PDN was dissolved in normal saline at a dose of 10 mg/kg [28]. Normal saline was used as the vehicle. Normal saline and the PDN solution were administered by gavage to the CPZ+NS and CPZ+PDN groups once daily for 2 weeks after three weeks of CPZ treatment
4.3 Body Weight Recording The body weight of every experimental mouse was measured at 8:00-9:00 in the morning every three or four days throughout the testing period.
4.4 Open Field Test Silence should be maintained during this experimental period. The open field apparatus (Huaibei Zhenghua, biological Equipment Co., Ltd., China) consisted of a 25 cm×25 cm black plastic square surrounded by a 35 cm-high wall. After 5 weeks treatment, every single mouse in each group was placed into the center of apparatus. The timing was immediately begun and continued for 5 minutes. Upon completion of the test, the mice were returned to their cages. A video camera (Digital CCD Camera, Sony, China) above the square automatically recorded the paths of the tested mice. The related parameters (total distance moved, center area distance traveled and time in the central area) were recorded by a tracking program above the square (Biowill Co., Ltd., China) using an automated assessment.
4.5 Tissue preparation After the open-field test, the animals (n=20 in total, 5 for each sub-group) were anesthetized intraperitoneally with 10% chloral hydrate (4 ml/kg, Sigma-Aldrich, Inc., USA) and then perfused intracardially with PBS and fixed with cold 4% paraformaldehyde (PFA). The brains were isolated to post-fix in 4% PFA and incubated overnight in 30% sucrose dissolved in a 100 mM sodium phosphate buffer (pH 7.4) at 4°C. The brains were then embedded in Optimal Cutting Temperature medium (Leica Microsystems, Germany) for sectioning. Coronal sections (10 µm or 20 µm) from bregma +1.0 to -1.0 (anterior-posterior) were cut sequentially and placed on 3-aminopropyltriethoxysilane-coated slides (Sigma-Aldrich, Inc., USA) and stored at -80°C. Ten-micron composite sections were prepared for Luxol Fast Blue staining (LFB, Sigma-Aldrich, Inc., USA), while 20 µ m sections were used for immunohistochemistry. For protein and RNA level analyses, the fresh corpus callosum of sacrificed mice (total n=16, 4 from each sub-group) were isolated and stored at -80°C.
7
4.6 LFB Staining LFB staining was performed as previously reported [51]. Briefly, 10 µm coronal brain tissue slides were rehydrated and incubated at 60°C in 0.1% LFB solution for 2 hours. The sections were processed using 95% and 70% ethanol to remove excess stain and to observe myelin staining. To increase the color contrast of the myelin, white matter was distinguished from the grey matter using 0.05% lithium carbonate. The sections were then washed with distilled water, dehydrated quickly with 100% ethanol, cleared in dimethyl benzene (Thermo Fisher Scientific, USA), covered and observed under a microscope (Olympus, Japan). LFB myelination scores were examined by three blinded observers. Scores between zero and three were used to evaluate demyelination. A score of 3 was defined as a totally normal myelin sheath, whereas 0 was defined as complete demyelination. Scores of 1 or 2 were defined as one-third or two-thirds myelination of the corpus callosum, respectively.
4.7 Immunohistochemistry The above-described sections of brain tissue (20 µm) were used for immunofluorescence staining. Briefly, sections were rewarmed at room temperature, and heat-based antigen retrieval was performed if necessary. The sections were blocked in PBS containing 5% bovine serum albumin (BSA) and 0.3% Triton X-100 for 1 h at 37℃. The sections were then incubated overnight with the following primary antibodies: anti-myelin basic protein antibody (MBP; rabbit IgG, 1:1000, Abcam, USA), anti- Glial fibrillary acidic protein antibody (GFAP; rabbit IgG, 1:500, Santa Cruz, USA) or anti-Ionized calcium binding adaptor molecule-1 (Iba-1; mouse IgG, 1:1000, Wako, Japan). The specimens were incubated with goat anti-mouse or goat anti-rabbit fluorescein isothiocyanate (FITC, 1:200, Santa Cruz, USA) or tetramethyl rhodamine isocyanate (TRITC, 1:200, Santa Cruz, USA) for 2 h, after which they were stained with DAPI (Vicmed, China). The slides were covered with 90% glycerol and observed under a Zeiss Axioskop 40 microscope (Carl Zeiss, Oberkochen, Germany). The fluorescence images from the same levels in three randomly selected sections per animal were analyzed for their respective integral optical densities (IODs) with Image-Pro Plus 6.0 software.
4.8 Western Blot The isolated corpus callosa were immersed in ice-cold lysis buffer containing certain protease inhibitors and phosphatase inhibitors (Beyotime Institute of Biotechnology, China). A tissue homogenizer was used to homogenize the ground tissue and then centrifuged at 7000×ｇ for 20 min. The supernatant was collected to detect the protein concentration for the subsequent experiments. An equal amount of proteins (100 µ g/lane) were fractionated by 10% or 15% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose (NC) filter membranes. After blocking in 5% nonfat dry milk buffer (dissolved in PBS), the membranes were incubated with following primary antibodies: MBP (rabbit IgG2b, 1:1000, Santa Cruz, USA), NLRP3 (rabbit IgG, 1:500, Abcam, UK), Caspase-1 (rabbit IgG,1:1000, Santa Cruz, USA), IL-1β (rabbit IgG,1:500, Santa Cruz, USA), TNF-α (rabbit IgG,1:500, Santa Cruz, USA) and β-actin (mouse IgG1, 1:1000; 8
Abcam, UK). The membranes were then incubated with the corresponding secondary antibodies and scanned with an Odyssey Western Blot Detection System (LI-COR Biosciences, USA). The gray value of every band was analyzed using Image-Pro Plus 6.0 software, and the data are expressed as the ratio of target protein expression to β-actin, which acted as the loading control.
4.9 Real-time Reverse Transcriptase Polymerase Chain Reaction (rtRT-PCR) Total RNA were extracted from the corpus callosa of different groups using TRIzol reagent (Invitrogen, CA, USA) and resuspended in a certain volume of ribozyme-free water. Complementary DNA (cDNA) was obtained via reverse transcription with HiScript®ǁQRT SuperMix (Vazyme biotech co., Itd., China). The quantitative one-step RT-PCR kit (Vazyme biotech co., Itd., China) and appropriate primers (synthesized by Sangon Biotech, China) were then used to detect the mRNA expression levels of TNF-α, CCL8, CXCL10 and CXCL16 according to a standardized protocol, as described in the Roche Applied Science LightCycler™480 manual (Roche Science, Switzerland). The primer sequences are listed in table 1. The expression level of ribosomal RNA 18s (18sRNA) was used as a reference for the samples. The PCR amplification products were then analyzed by agarose gel-electrophoresis to assess amplification specificity (not shown).
4.10 Blood sample preparation and corticosterone assays One day after the behavioral tests finished, the mice were anesthetized with ketamine and the blood was collected from the hearts of sacrificed mice. Trunk blood was allowed to coagulate at room temperature in serum tubes for 1 hour. Samples were centrifuged in an Eppendorf 5415R centrifuge at 2000rpm for 15 minutes at 4℃. Serum was aliquoted and frozen at −20 °C until analysis. Corticosterone levels in serum were measured using a competitive enzyme-linked immunosorbent assay (ELISA) kit (ADI-900-907, Enzo life sciences, NY, USA) according to the manufacturer's protocols. The absorbance of each sample was measured at a wave length of 450nm and the results are presented as ng/mL.
4.11 Statistical Analysis All of the statistical data were analyzed using SPSS 19.0. Quantitative differences between groups were evaluated using an independent sample t-test or one-way analysis of variance (ANOVA) followed by LSD or Tukey’s HSD post-test. All optical intensity analyses were performed by individuals who were blind to the experimental treatment. All of the statistical results are given as arithmetic means±SD, and p less than 0.05 was considered to be statistically significant. Table 1 List of primers used for rtRT-PCR Name
Forward sequence5’-3’
Reverse sequence 5’-3’
18sRNA
cct gga tac cgc agc tag ga
gcg gcg caa tac gaa tgc ccc
TNF-α
gca cag aaa gca tga ccc g
gcc ccc cat ctt ttg gg
CCL8
taa ggc tcc agt cac ctg ct
ata ccc tgc ttg gtc tgg aa
CXCL10
gcc gtc att ttc tgc ctc a
cgt cct tgc gag agg gat c 9
CXCL16
cct caa gcc agt acc cag ac
Gct cct gat gga aga gtg ga
Acknowledgments This work was supported by the National Natural Science Foundation of China grants (No. 81271345, 81302519) and the Natural Science Foundation of Jiangsu Province (No. BK20131132, BK20130221, BK20161174).
Conflict of interest statement None of the authors have financial conflicts of interest.
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Figure legends Figure 1. PDN ameliorates the body weight loss and improves behavioral functions in mice with CPZ-induced demyelinated. (A) The average body weight in Ctrl, CPZ, CPZ+NS and CPZ+PDN groups during the 5-week trial. (B) Representative images showing typical examples of exploratory behavior in the open-field test in the Ctrl, CPZ, CPZ+NS and CPZ+PDN groups. (C) The central distance traveled and (D) the total traveled distance in the open field. (E) Time spent in the center area. All of the values are expressed as the mean±SD. (n=12 per group) * P<0.05, **P < 0.01, ***P < 0.01versus the Ctrl group; #P < 0.05, ##P < 0.01 versus the CPZ group. Figure 2. PDN alleviated myelin sheath loss induced by consumption of the CPZ diet. (A) Representative images of LFB-stained sections in the corpus callosum of the Ctrl, CPZ, CPZ+NS and CPZ+PDN groups. Scale bar, 20 µm. (B) Scores of myelination obtained from the assessment of LFB-stained images. The data are expressed as the mean±SD. (n=4 per group). (C) Representative images of immunofluorescence staining for MBP in the corpus callosum are displayed for the Ctrl, CPZ, CPZ+NS and CPZ+PDN groups. Scale bar, 20 µm.（D）An analysis of the mean optical density values of MBP-positive cells. (E, F) The quantitative analysis of MBP protein levels as measured via western blot. The values are expressed as the mean±SD. (n=4 per group). *P<0.05, ** P< 0.01 versus the Ctrl group; #P< 0.05 versus the CPZ group. Figure 3. PDN inhibits astrocyte and microglial activation in the CPZ-induced demyelination model. (A, B) Representative images of immunofluorescence staining for GFAP and Iba-1 in the corpus callosum are displayed for the Ctrl, CPZ, CPZ+NS and CPZ+PDN groups. (C, D) Analysis of the numbers of GFAP- and Iba-1positive cells in the corpus callosum in different groups. Scale bar, 15 µm. The values are expressed as the mean±SD. (n = 4 per group). ** P< 0.01 versus the Ctrl group; #P< 0.05 versus the CPZ group. Figure 4. PDN inhibits NLRP3 inflammasome activation and IL-1β secretion induced by CPZ. (A) Detection of the protein levels of NLRP3, cleaved caspase-1 and cleaved IL-1β in the corpus callosum in the Ctrl, CPZ, CPZ+NS and CPZ+PDN groups. (B-D) Quantitative analysis of NLRP3, cleaved caspase-1 and cleaved IL-1β levels, as measured via western blot. The values are expressed as the mean ±SD. (n = 5 per group). *P< 0.05 versus the Ctrl group; # P< 0.05 versus the CPZ group. Figure 5. PDN down-regulates the secretion of inflammatory factors and chemokines in CPZ-treated mice. The levels of TNF-α (A), CCL8 (C), CXCL10 (D) and CXCL16 (E) mRNA 13
were detected by rtRT-PCR in the Ctrl, CPZ, CPZ+NS and CPZ+PDN groups(n = 5 per group). The protein level of TNF-α was dectected by western blot (B).The serum corticosterone was tested by ELISA (F). The values are expressed as the mean±SD. (n =8 per group). *P<0.05, **P < 0.01, *** P < 0.001 versus the Ctrl group; #P< 0.05 versus the CPZ group.
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Highlights 1. We showed that prednisone could inhibit the activation of Nod like receptor pyrin domain containing 3 (NLRP3) inflammasome in cuprizone induced C57BL/6 mice. 2. The level of TNF-a, CCL8, CXCL10 and CXCL16 were also down-regulated to reduce the inflammation and even alleviate myelin loss. 3. NLRP3 signaling pathway may be a novel mechanism for prednisone in demyelinated diseases treatment.
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S0006-8993(17)30440-7 https://doi.org/10.1016/j.brainres.2017.09.034 BRES 45511
To appear in:
Brain Research
Received Date: Revised Date: Accepted Date:
12 July 2017 27 September 2017 29 September 2017
Please cite this article as: H. Yu, M. Wu, G. Lu, T. Cao, N. Chen, Y. Zhang, h. Jiang, H. Fan, R. Yao, Prednisone alleviates demyelination through regulation of the NLRP3 inflammasome in a C57BL/6 mouse model of cuprizoneinduced demyelination, Brain Research (2017), doi: https://doi.org/10.1016/j.brainres.2017.09.034
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Prednisone alleviates demyelination through regulation of the NLRP3 inflammasome in a C57BL/6 mouse model of cuprizone-induced demyelination Hao Yu1*, Mingfeng Wu1*, Geng Lu2, Tingting Cao2, Nan Chen2, Yijia Zhang2, Zhiguo Jiang 2, Hongbin Fan3, Ruiqin Yao1# 1 Neurobiological Research Center, Xuzhou Medical University, 209# Tongshan Road, Yunlong District, Xuzhou 221000, China 2 Department of Clinical Medicine, Xuzhou Medical University, 209# Tongshan Road, Yunlong District, Xuzhou 221000, China 3 Department of Neurology, Affiliated Hospital of Xuzhou Medical University, 99# Huaihai Road, Quanshan District, Xuzhou 221009, China
*These authors contributed equally to this work. #
Corresponding Author: Ruiqin Yao E-mail: [email protected]
Abstract Myelin abnormalities, oligodendrocyte damage, and concomitant glia activation are common in demyelinating diseases of the central nervous system (CNS). Increasing evidence has demonstrated that the inflammatory response triggers demyelination and gliosis in demyelinating disorders. Numerous clinical interventions, including those used to treat multiple sclerosis (MS), have confirmed prednisone (PDN) as a powerful anti-inflammatory drug that reduces the inflammatory response and promotes tissue repair in multiple inflammation sites. However, the underlying mechanism of PDN in ameliorating myelin damage is not well understood. In our study, a cuprizone (CPZ)-induced demyelinated mouse model was used to explore the mechanism of the protection provided by PDN. Open-field tests showed that CPZ-treated mice exhibited significantly increased anxiety and decreased exploration. However, PDN improved emotional behavior, as evidenced by an increase in the total distance traveled, and central distance traveled as well as the mean amount of time spent in the central area. CPZ-induced demyelination was observed to be alleviated in PDN-treated mice based on luxol fast blue (LFB) staining and myelin basic protein (MBP) expression analyses. In addition, PDN reduced astrocyte and microglia activation in the corpus callosum. Furthermore, we demonstrated that PDN inhibited the Nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome signaling pathway and related inflammatory cytokines and chemokines, including TNF-α, CCL8, CXCL10 and CXCL16. PDN also reduced the serum corticosterone levels in the CPZ-treated mice. Taken together, these results suggest that inhibition of the NLRP3 signaling pathway may be a novel mechanism by which PDN exerts its protective actions in demyelinating diseases.
Key Words Demyelination; Inflammation; Prednisone; NLRP3; Chemokine Abbreviations CPZ, cuprizone; GFAP, glial fibrillary acidic protein; Iba-1,Ionized calcium binding adaptor molecule-1; LFB, luxol fast blue; NLRP3, Nod-like receptor pyrin domain 1
containing 3; MBP, myelin basic protein; MS, multiple sclerosis; PDN, prednisone.
1. Introduction The inhibition of demyelination is a critical challenge in the treatment of many central nervous system (CNS) degenerative diseases, especially multiple sclerosis (MS)[1-3]. The poor prognosis of this disease may in part due to the fact that neuroinflammation results in the loss of the myelin sheath, which cannot regenerate effectively, ultimately leading to the poor conduction of nerve impulses. Previous studies have reported lymphocyte/macrophage infiltration, astrocyte and microglial activation, and massive cytokine/chemokine secretion in progressive demyelinating diseases[4-6]. Furthermore, the inflammasome which consists of Nod-like receptor pyrin domain containing 3 (NLRP3), the adaptor protein apoptotic speck-containing protein with a CARD (ASC) and pro-caspase-1, has been reported to have a significant role in long-term inflammation responses[7-9]. Other studies have tested the hypothesis that active NLRP3 inflammasome aggravates inflammation by activating pro-IL-1β and pro-IL-18, which then mature to IL-1β and IL-18, via the P2X7 receptor in murine lupus nephritis[10]. Worse still, activated IL-1β can stimulate downstream cytokines and certain chemokine receptors, which interact with abundant cytokines and chemokines secreted by inflammatory cells to trigger inflammatory responses[7]. In addition, chemokines and their receptors have been reported to recruit inflammatory cells, including microglia and leucocytes, to expand the inflammatory region and further exacerbate myelin injury[11]. Therefore, elucidating the mechanisms by which neuroinflammation induces demyelination and blocking this inflammation process is a crucial step for the development of therapeutics and improving patient prognoses. Prednisone (PDN) is representative of glucocorticoid compounds, which have powerful anti-inflammatory activity and neuroprotective characteristics and are widely prescribed drugs[12, 13] . For instance, low-dose PDN treatment gradually benefits patients with rheumatoid arthritis (RA) by reducing chronic inflammation[14]. However, the potential for PDN to improve myelin loss by reducing the inflammatory response is unclear. Cuprizone (CPZ), as a copper chelator, specifically damages oligodendrocytes, without directly affecting other glial cells, and induces consistent demyelination in the C57BL/6 mouse brain. This compound is therefore generally acknowledged as an inducer of demyelination and myelin regeneration in animal models[15, 16]. Thus, we generated a CPZ-induced mouse model of demyelination to examine the function of PDN on the activation of the NLRP3 inflammasome and glia, as well as the level of related cytokines and chemokines. Neurodegeneration is a strong determinant of hypothalamus-pituitary-adrenal (HPA) axis activity. HPA system dysregulation has been shown in MS patients, and many studies showing high levels of cortisol in parts of sufferers of MS[17, 18]. Researchers proposed that the suppressive effects of some medicine on mood disorders such as depression- and anxiety-related behaviors might be primarily mediated by inhibition of corticosterone production[19, 20]. In our study, corticosterone level in serum was obviously enhanced in samples from the cuprizone group, suggesting possible HPA activation[21]. PDN significantly reduced the corticosterone level suggests that the PDN might exhibit the feedback effect on HPA axis. Despite all this, it can not be concluded the direct relationship between demyelination and corticosterone level, further evidence is needed to support the interpretation. We first carried out open field tests to evaluate improvements in the physical condition and 2
behavior of PDN-treated mice. Secondly, we assessed myelination and glial activation to study the role of PDN in ameliorating demyelination in CPZ-treated mice. We lastly explored the related mechanism of PDN mediated changes by detecting the expression levels of NLRP3, caspase-1 and IL-1β, as well as the secretion of TNF-α, CCL8, CXCL10, and CXCL16. These data are the first to indicate that the inhibition of NLRP3 inflammasome activation may be mechanism by which PDN protects the myelin sheath from degeneration in a CPZ-induced demyelinated model.
2. Results 2.1 PDN ameliorates CPZ-induced body weight loss The body weights of the mice were recorded weekly to observe the effect of PDN on mice with CPZ-induced demyelination. In the Ctrl group, the average body weight in normal fed-mice increased gradually, while CPZ treatment decreased body weight dramatically from the fourth to fifth week. There was no obvious difference in the average body weight between the CPZ+NS and CPZ groups. However, PDN treatment alleviated the body weight loss induced by CPZ, and a significant difference was found in this metric between the CPZ+PDN group and the CPZ group at the fourth (P<0.05) and fifth week (P<0.01) (Fig. 1A). 2.2 PDN improved the anxiety and exploratory ability of CPZ-induced mice Nervous system dysfunction is consistently observed in demyelination diseases, including abnormalities in motor function, spatial working memory, social interaction, etc.[22, 23]. Here, we performed an open-field test to evaluate the anxiety and exploration ability of the mice. The CPZ group mice traveled a shorter distance than the control mice in the center area and in the overall area of the apparatus (Fig. 1B, C). Moreover, the time spent in the central area was significantly lower in the CPZ-treated mice than that in the control mice (Fig. 1B, E), suggesting that mice with CPZ-induced demyelination are hypoactive. However, the center distance, total distance and time spent in the central area were all significantly increased in the PDN-treated mice compared with the CPZ group (Fig. 1B-D). No significant changes were observed in the central distance, total distance or time spent in the center zone between the CPZ and CPZ-NS groups. These data suggest that PDN decreases anxiety-like behaviors and increases exploratory behavior ability in mice with CPZ-induced demyelination. 2.3 PDN alleviated demyelination induced by CPZ LFB staining showed that myelination was complete in the corpus callosum in the Ctrl group, whereas most of the myelin sheath was absent from the corpus callosum in the CPZ and CPZ+NS groups. The myelin score was significantly decreased in the CPZ group mice compared with the Ctrl mice, indicating that the myelin sheath was severely damaged by CPZ treatment. No significant differences were found in this metric between the CPZ and CPZ+NS groups. In contrast, PDN-treated mice displayed higher myelin scores than those of CPZ-treated mice (Fig. 2A-B). MBP immunohistochemistry staining in the corpus callosum showed that the mice exposed to CPZ exhibited few MBP-positive oligodendrocytes. However, more MBP-positive oligodendrocytes were found in the PDN-treated mice (Fig. 2C). The IOD value of the MBP-positive cells was significantly decreased in CPZ-treated mice compared with the Ctrl group; however, PDN treatment reversed the decrease in the MBP decrease in IOD value induced by CPZ 3
(Fig. 2D). The levels of MBP protein in the corpus callosum in the CPZ group were also remarkably lower than those in the control group (Fig. 3E, F). However, the expression of MBP was clearly increased by PDN treatment when compared with the CPZ group (Fig. 3E, F). No significant differences were found in the IOD value or the protein levels of MBP between the CPZ and CPZ+NS groups. 2.4 PDN inhibited glial activation in CPZ-treated mice It has been reported that white matter astrocytes and microglia are activated in CPZ-treated mice[24]. To observe whether PDN alleviated demyelination by inhibiting glial activation, GFAP and Iba-1 immunohistochemistry staining were used to detect glial activation. The results showed that the number of GFAP-positive cells (Fig. 3A) and Iba-l-positive cells (Fig. 3B) were both increased in the corpus callosum of mice treated with CPZ, but PDN treatment significantly decreased the number of GFAP- and Iba-l-positive cells when treated by PDN in CPZ-induced model mice (Fig. 3C-D). The data suggested that PDN could inhibit the CPZ-induced activation of astrocytes and microglia. 2.5 PDN suppressed the activation of the NLRP3 inflammasome and down-regulated the secretion of inflammatory factors and chemokines It was recently verified that the NLRP3 inflammasome is activated and involved in demyelination diseases[25]. To explore how PDN protects the myelin sheath against inflammation, we detected the protein levels of NLRP3, cleaved-caspase-1 and the downstream protein cleaved-IL-1β in the corpus callosum. NLRP3 protein expression was dramatically increased in the corpus callosum of CPZ-treated mice, while treatment with PDN clearly reduced the level of NLRP3 (Fig. 4A-B). Moreover, the levels of cleaved-caspase-1 and cleaved-IL-1β were decreased (Fig. 4A, C, D), implying that PDN might alleviate demyelination by suppressing activation of the NLRP3 signaling pathway in mice with CPZ-induced demyelination. Cytokines and chemokines, along with their receptors, have been reported to exacerbate inflammation and the immune reaction in inflammatory CNS diseases[26, 27]. We therefore measured the messenger RNA (mRNA) expression levels of TNF-α, CCL8, CXCL10 and CXCL16 by rtRT-PCR. As shown in Fig. 5A, C-E, the levels of TNF-α, CCL8, CXCL10 and CXCL16 mRNA were higher than that those in Ctrl mice. PDN treatment significantly decreased the levels of these mRNAs, suggesting that PDN might further relieve inflammatory injury by down-regulating the secretion of inflammatory factors and chemokines in a CPZ-induced demyelination model. We also detected the protein levels mentioned above by western blot. The protein expression of TNF-α is consistent with the results of rtRT-PCR (Fig.5B). Corticosterone levels were analyzed by ELISA at the end of experimental period. As expected, serum corticosterone level was significantly higher in the cuprizone-treated mice than that in the control mice (p< 0.01). PDN at 10 mg/kg reduced corticosterone levels, there was significantly difference between the CPZ and PDN groups (p < 0.05, Fig.5F).
3. Discussion
4
CNS demyelination is characterized by myelin sheath damage, oligodendrocyte death, glial activation and remyelination failure [16]. Previous studies have confirmed that PDN has significant anti-inflammatory activity and promotes both brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) expression in the cerebral cortex of EAE mice following minocycline treatment [28]. Here, for the first time, we report that PDN alleviates demyelination by inhibiting the activation of the NLRP3 inflammasome and suppressing the secretion of related inflammatory factors and chemokines in mice with CPZ-induced demyelination. We thereby revealed a new mechanism for PDN in the treatment of for demyelinating diseases. Feeding mice with the copper chelator CPZ is designed to damage mitochondrial function and further induce CNS demyelination [29]. Prior studies have shown that mice fed with 0.2-0.3% CPZ begin to exhibit demyelination from the second week of treatment. Demyelination gradually continues and is complete at the fifth or sixth week in the corpus callosum. When CPZ treatment is stopped, the process of demyelination ceases, allowing myelination to return to a somewhat normal level[4, 30]. In our study, to examine the effect and the mechanisms of PDN on demyelination, PDN was administered intragastrically every day after 3 weeks of CPZ exposure. After 5 weeks, we performed LFB staining to detect the extent of demyelination and MBP expression in the corpus callosum. In our research, we found that PDN protected myelin against CPZ-induced toxicity, improving the myelin score and up-regulating the expression of MBP. Furthermore, consistent with previous studies, C57BL/6 mice exposed to CPZ for 5 weeks exhibited obvious body weight loss. However, PDN treatment alleviated this body weight loss, suggesting that the protective effect of PDN on CPZ-induced body weight loss might be related to improvement in myelination. Limb paralysis and abnormal behaviors are widely demonstrated in MS patients and CPZ model mice exhibit similar symptoms [31-33]. Recently, an increasing number of studies have shown that motor dysfunction and behavioral disorders might be related to white matter damage induced by myelin sheath loss, as well as by the limited regenerative abilities of oligodendrocytes and their precursors [34]. The open-field test is used widely to evaluate motor activity, exploration ability and anxious behavior in vivo in CPZ model mice and other animal models [32, 35]. In our experiment, we used the open-field test to test whether PDN treatment improves the motor difficulties caused by CPZ exposure. The CPZ+PDN group mice showed a longer traveled distance and more time in the center area of open field apparatus than the CPZ group mice, indicating that PDN alleviates behavioral disorders by improving demyelination. Astrocytes are an important type of glial cell in the CNS and play dual roles in demyelinating diseases [36]. On the one hand, astrocytes secretes an abundance of neurotrophic factors to promote the regeneration of the myelin sheath; however, these cells also promote the immune reaction and form glial scars, thereby accelerating demyelination[37, 38]. Microglia act as macrophages in the CNS and promote demyelination recovery by clearing cell debris. However, this cell type can also accelerates inflammatory injury by secreting pro-inflammatory factors, such as TNF-α[39, 40]. In pathologically demyelinated zones in the CNS, astrocyte hyperplasia and microglia activation are consistently observed, as is oligodendrocyte damage. Here, we found that PDN administration for 2 weeks significantly decreased the numbers of GFAP-positive astrocytes and Iba-1-positive microglia, suggesting that PDN could suppress glial activation in mice with CPZ-induced demyelination. 5
NLRP3 inflammasome activation is suggested to be a principal underlying mechanism in experimentally induced autoimmune encephalomyelitis (EAE), another mouse model of demyelination [41, 42]. Recently, it was reported that NLRP3 inflammasome activation is involved in corpus callosum demyelination progression in CPZ-treated mice and that this effect is mediated via the IL-1β pathway[7]. Previous studies have demonstrated that both mature oligodendrocytes and their precursor cells express glucocorticoid receptors (GRs)[43, 44], and glucocorticoids are involved in promoting myelination through GRs[45, 46]. Treatment with high-dose glucocorticoids is still regarded as the most important and efficient therapeutic method to promote the recovery of neurological function in some demyelination diseases, including in acute MS relapses [47]. In our present study, we found that PDN treatment may protect the myelin sheath against CPZ damage by inhibiting the NLRP3 inflammasome signaling pathway. It was also reported that NLRP3 activation could result in the secretion of inflammatory cytokines and chemokines [42]. We also tested the expression levels of inflammatory cytokines and chemokines and verified that PDN inhibited the secretion of TNF-α, CCL8, CXCL10 and CXCL16. Chemokines represent a group of chemotactic proteins with molecular weights ranging from 8 to 14 kDa. These proteins modulate leukocyte attraction and glial activation under inflammatory conditions[27, 48]. Makoto Inoue, et al. reported that the chemotaxis-related proteins CCL8/MCP-2 and its receptor CCR2, as well as CXCL16 and its receptor CXCR6, are up-regulated in the presence of the NLRP3 inflammasome. These authors inferred that leukocyte-attracting chemokines could be novel molecular targets in treating MS patients in the context of NLRP3 inflammasome activation[41]. Moreover, CXCL16 is activated by cytokines and can induce the migration and invasion of glial precursor cells by up-regulating its receptor CXCR6 [49]. CXCL10 acts as a key microglia-attracting chemokine and is secreted at a high level in CPZ-treated wild-type mice [50]. Our study indicates that PDN might alleviate CPZ-induced demyelination by suppressing the activation of NLRP3 signaling and further down-regulating the secretion of inflammatory factors and chemokines, thereby reducing glial activation. In summary, our results demonstrated that the NLRP3 inflammasome signaling pathway is involved in CPZ-induced demyelination. PDN, as an anti-inflammatory drug, may target the NLRP3 inflammasome complex and reduce inflammation through a cascade of reactions, thereby alleviating CPZ-induced myelination and improving behavioral function. Therefore, we demonstrate a novel mechanism by which PDN therapy ameliorates the symptoms of inflammatory demyelinating diseases.
4. Materials & Methods 4.1 Animals Adult wild-type C57BL/6 male mice (8 weeks old, 23-25 g) were obtained from the Xuzhou Medical Universal Experimental Laboratory Animal Center and housed in a particular feeding room with a 12:12-hour light-dark cycle. Food pellets and drinking water were provided ad libitum. All of the experiments in this study were performed in keeping with the guidelines set by the National Institutes of Health guide (NIH) for the care and use of Laboratory animals and were also approved institutionally by both the utilization of laboratory animals of Xuzhou Medical University and Chinese Laws. 6
4.2 Prednisone and Cuprizone Treatment The mice were randomly divided into four groups: Control group (Ctrl), CPZ-treated group (CPZ), CPZ- and normal saline-treated group (CPZ+NS), and CPZ- and PDN-treated group (CPZ+PDN). Experiments were optimally designed to minimize the number of animals required. Ctrl mice were fed a standard rodent chow, while the others were fed diets containing 0.3% (w/w) CPZ (Sigma-Aldrich, USA) to induce demyelination[34]. PDN was dissolved in normal saline at a dose of 10 mg/kg [28]. Normal saline was used as the vehicle. Normal saline and the PDN solution were administered by gavage to the CPZ+NS and CPZ+PDN groups once daily for 2 weeks after three weeks of CPZ treatment
4.3 Body Weight Recording The body weight of every experimental mouse was measured at 8:00-9:00 in the morning every three or four days throughout the testing period.
4.4 Open Field Test Silence should be maintained during this experimental period. The open field apparatus (Huaibei Zhenghua, biological Equipment Co., Ltd., China) consisted of a 25 cm×25 cm black plastic square surrounded by a 35 cm-high wall. After 5 weeks treatment, every single mouse in each group was placed into the center of apparatus. The timing was immediately begun and continued for 5 minutes. Upon completion of the test, the mice were returned to their cages. A video camera (Digital CCD Camera, Sony, China) above the square automatically recorded the paths of the tested mice. The related parameters (total distance moved, center area distance traveled and time in the central area) were recorded by a tracking program above the square (Biowill Co., Ltd., China) using an automated assessment.
4.5 Tissue preparation After the open-field test, the animals (n=20 in total, 5 for each sub-group) were anesthetized intraperitoneally with 10% chloral hydrate (4 ml/kg, Sigma-Aldrich, Inc., USA) and then perfused intracardially with PBS and fixed with cold 4% paraformaldehyde (PFA). The brains were isolated to post-fix in 4% PFA and incubated overnight in 30% sucrose dissolved in a 100 mM sodium phosphate buffer (pH 7.4) at 4°C. The brains were then embedded in Optimal Cutting Temperature medium (Leica Microsystems, Germany) for sectioning. Coronal sections (10 µm or 20 µm) from bregma +1.0 to -1.0 (anterior-posterior) were cut sequentially and placed on 3-aminopropyltriethoxysilane-coated slides (Sigma-Aldrich, Inc., USA) and stored at -80°C. Ten-micron composite sections were prepared for Luxol Fast Blue staining (LFB, Sigma-Aldrich, Inc., USA), while 20 µ m sections were used for immunohistochemistry. For protein and RNA level analyses, the fresh corpus callosum of sacrificed mice (total n=16, 4 from each sub-group) were isolated and stored at -80°C.
7
4.6 LFB Staining LFB staining was performed as previously reported [51]. Briefly, 10 µm coronal brain tissue slides were rehydrated and incubated at 60°C in 0.1% LFB solution for 2 hours. The sections were processed using 95% and 70% ethanol to remove excess stain and to observe myelin staining. To increase the color contrast of the myelin, white matter was distinguished from the grey matter using 0.05% lithium carbonate. The sections were then washed with distilled water, dehydrated quickly with 100% ethanol, cleared in dimethyl benzene (Thermo Fisher Scientific, USA), covered and observed under a microscope (Olympus, Japan). LFB myelination scores were examined by three blinded observers. Scores between zero and three were used to evaluate demyelination. A score of 3 was defined as a totally normal myelin sheath, whereas 0 was defined as complete demyelination. Scores of 1 or 2 were defined as one-third or two-thirds myelination of the corpus callosum, respectively.
4.7 Immunohistochemistry The above-described sections of brain tissue (20 µm) were used for immunofluorescence staining. Briefly, sections were rewarmed at room temperature, and heat-based antigen retrieval was performed if necessary. The sections were blocked in PBS containing 5% bovine serum albumin (BSA) and 0.3% Triton X-100 for 1 h at 37℃. The sections were then incubated overnight with the following primary antibodies: anti-myelin basic protein antibody (MBP; rabbit IgG, 1:1000, Abcam, USA), anti- Glial fibrillary acidic protein antibody (GFAP; rabbit IgG, 1:500, Santa Cruz, USA) or anti-Ionized calcium binding adaptor molecule-1 (Iba-1; mouse IgG, 1:1000, Wako, Japan). The specimens were incubated with goat anti-mouse or goat anti-rabbit fluorescein isothiocyanate (FITC, 1:200, Santa Cruz, USA) or tetramethyl rhodamine isocyanate (TRITC, 1:200, Santa Cruz, USA) for 2 h, after which they were stained with DAPI (Vicmed, China). The slides were covered with 90% glycerol and observed under a Zeiss Axioskop 40 microscope (Carl Zeiss, Oberkochen, Germany). The fluorescence images from the same levels in three randomly selected sections per animal were analyzed for their respective integral optical densities (IODs) with Image-Pro Plus 6.0 software.
4.8 Western Blot The isolated corpus callosa were immersed in ice-cold lysis buffer containing certain protease inhibitors and phosphatase inhibitors (Beyotime Institute of Biotechnology, China). A tissue homogenizer was used to homogenize the ground tissue and then centrifuged at 7000×ｇ for 20 min. The supernatant was collected to detect the protein concentration for the subsequent experiments. An equal amount of proteins (100 µ g/lane) were fractionated by 10% or 15% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose (NC) filter membranes. After blocking in 5% nonfat dry milk buffer (dissolved in PBS), the membranes were incubated with following primary antibodies: MBP (rabbit IgG2b, 1:1000, Santa Cruz, USA), NLRP3 (rabbit IgG, 1:500, Abcam, UK), Caspase-1 (rabbit IgG,1:1000, Santa Cruz, USA), IL-1β (rabbit IgG,1:500, Santa Cruz, USA), TNF-α (rabbit IgG,1:500, Santa Cruz, USA) and β-actin (mouse IgG1, 1:1000; 8
Abcam, UK). The membranes were then incubated with the corresponding secondary antibodies and scanned with an Odyssey Western Blot Detection System (LI-COR Biosciences, USA). The gray value of every band was analyzed using Image-Pro Plus 6.0 software, and the data are expressed as the ratio of target protein expression to β-actin, which acted as the loading control.
4.9 Real-time Reverse Transcriptase Polymerase Chain Reaction (rtRT-PCR) Total RNA were extracted from the corpus callosa of different groups using TRIzol reagent (Invitrogen, CA, USA) and resuspended in a certain volume of ribozyme-free water. Complementary DNA (cDNA) was obtained via reverse transcription with HiScript®ǁQRT SuperMix (Vazyme biotech co., Itd., China). The quantitative one-step RT-PCR kit (Vazyme biotech co., Itd., China) and appropriate primers (synthesized by Sangon Biotech, China) were then used to detect the mRNA expression levels of TNF-α, CCL8, CXCL10 and CXCL16 according to a standardized protocol, as described in the Roche Applied Science LightCycler™480 manual (Roche Science, Switzerland). The primer sequences are listed in table 1. The expression level of ribosomal RNA 18s (18sRNA) was used as a reference for the samples. The PCR amplification products were then analyzed by agarose gel-electrophoresis to assess amplification specificity (not shown).
4.10 Blood sample preparation and corticosterone assays One day after the behavioral tests finished, the mice were anesthetized with ketamine and the blood was collected from the hearts of sacrificed mice. Trunk blood was allowed to coagulate at room temperature in serum tubes for 1 hour. Samples were centrifuged in an Eppendorf 5415R centrifuge at 2000rpm for 15 minutes at 4℃. Serum was aliquoted and frozen at −20 °C until analysis. Corticosterone levels in serum were measured using a competitive enzyme-linked immunosorbent assay (ELISA) kit (ADI-900-907, Enzo life sciences, NY, USA) according to the manufacturer's protocols. The absorbance of each sample was measured at a wave length of 450nm and the results are presented as ng/mL.
4.11 Statistical Analysis All of the statistical data were analyzed using SPSS 19.0. Quantitative differences between groups were evaluated using an independent sample t-test or one-way analysis of variance (ANOVA) followed by LSD or Tukey’s HSD post-test. All optical intensity analyses were performed by individuals who were blind to the experimental treatment. All of the statistical results are given as arithmetic means±SD, and p less than 0.05 was considered to be statistically significant. Table 1 List of primers used for rtRT-PCR Name
Forward sequence5’-3’
Reverse sequence 5’-3’
18sRNA
cct gga tac cgc agc tag ga
gcg gcg caa tac gaa tgc ccc
TNF-α
gca cag aaa gca tga ccc g
gcc ccc cat ctt ttg gg
CCL8
taa ggc tcc agt cac ctg ct
ata ccc tgc ttg gtc tgg aa
CXCL10
gcc gtc att ttc tgc ctc a
cgt cct tgc gag agg gat c 9
CXCL16
cct caa gcc agt acc cag ac
Gct cct gat gga aga gtg ga
Acknowledgments This work was supported by the National Natural Science Foundation of China grants (No. 81271345, 81302519) and the Natural Science Foundation of Jiangsu Province (No. BK20131132, BK20130221, BK20161174).
Conflict of interest statement None of the authors have financial conflicts of interest.
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Figure legends Figure 1. PDN ameliorates the body weight loss and improves behavioral functions in mice with CPZ-induced demyelinated. (A) The average body weight in Ctrl, CPZ, CPZ+NS and CPZ+PDN groups during the 5-week trial. (B) Representative images showing typical examples of exploratory behavior in the open-field test in the Ctrl, CPZ, CPZ+NS and CPZ+PDN groups. (C) The central distance traveled and (D) the total traveled distance in the open field. (E) Time spent in the center area. All of the values are expressed as the mean±SD. (n=12 per group) * P<0.05, **P < 0.01, ***P < 0.01versus the Ctrl group; #P < 0.05, ##P < 0.01 versus the CPZ group. Figure 2. PDN alleviated myelin sheath loss induced by consumption of the CPZ diet. (A) Representative images of LFB-stained sections in the corpus callosum of the Ctrl, CPZ, CPZ+NS and CPZ+PDN groups. Scale bar, 20 µm. (B) Scores of myelination obtained from the assessment of LFB-stained images. The data are expressed as the mean±SD. (n=4 per group). (C) Representative images of immunofluorescence staining for MBP in the corpus callosum are displayed for the Ctrl, CPZ, CPZ+NS and CPZ+PDN groups. Scale bar, 20 µm.（D）An analysis of the mean optical density values of MBP-positive cells. (E, F) The quantitative analysis of MBP protein levels as measured via western blot. The values are expressed as the mean±SD. (n=4 per group). *P<0.05, ** P< 0.01 versus the Ctrl group; #P< 0.05 versus the CPZ group. Figure 3. PDN inhibits astrocyte and microglial activation in the CPZ-induced demyelination model. (A, B) Representative images of immunofluorescence staining for GFAP and Iba-1 in the corpus callosum are displayed for the Ctrl, CPZ, CPZ+NS and CPZ+PDN groups. (C, D) Analysis of the numbers of GFAP- and Iba-1positive cells in the corpus callosum in different groups. Scale bar, 15 µm. The values are expressed as the mean±SD. (n = 4 per group). ** P< 0.01 versus the Ctrl group; #P< 0.05 versus the CPZ group. Figure 4. PDN inhibits NLRP3 inflammasome activation and IL-1β secretion induced by CPZ. (A) Detection of the protein levels of NLRP3, cleaved caspase-1 and cleaved IL-1β in the corpus callosum in the Ctrl, CPZ, CPZ+NS and CPZ+PDN groups. (B-D) Quantitative analysis of NLRP3, cleaved caspase-1 and cleaved IL-1β levels, as measured via western blot. The values are expressed as the mean ±SD. (n = 5 per group). *P< 0.05 versus the Ctrl group; # P< 0.05 versus the CPZ group. Figure 5. PDN down-regulates the secretion of inflammatory factors and chemokines in CPZ-treated mice. The levels of TNF-α (A), CCL8 (C), CXCL10 (D) and CXCL16 (E) mRNA 13
were detected by rtRT-PCR in the Ctrl, CPZ, CPZ+NS and CPZ+PDN groups(n = 5 per group). The protein level of TNF-α was dectected by western blot (B).The serum corticosterone was tested by ELISA (F). The values are expressed as the mean±SD. (n =8 per group). *P<0.05, **P < 0.01, *** P < 0.001 versus the Ctrl group; #P< 0.05 versus the CPZ group.
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Highlights 1. We showed that prednisone could inhibit the activation of Nod like receptor pyrin domain containing 3 (NLRP3) inflammasome in cuprizone induced C57BL/6 mice. 2. The level of TNF-a, CCL8, CXCL10 and CXCL16 were also down-regulated to reduce the inflammation and even alleviate myelin loss. 3. NLRP3 signaling pathway may be a novel mechanism for prednisone in demyelinated diseases treatment.
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