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Methotrexate prevents epidural fibrosis through endoplasmic reticulum stress signalling pathway
Author’s Accepted Manuscript Methotrexate prevents epidural fibrosis through endoplasmic reticulum stress signalling pathway Hui Chen, Lianqi Yan, Jingcheng Wang, Yu Sun, Xiaolei Li, Shuai Zhao, Daxin Wang, Gengyao Zhu, Yuan Liang www.elsevier.com/locate/ejphar
PII: DOI: Reference:
S0014-2999(16)30815-9 http://dx.doi.org/10.1016/j.ejphar.2016.12.032 EJP70994
To appear in: European Journal of Pharmacology Received date: 14 November 2016 Revised date: 12 December 2016 Accepted date: 20 December 2016 Cite this article as: Hui Chen, Lianqi Yan, Jingcheng Wang, Yu Sun, Xiaolei Li, Shuai Zhao, Daxin Wang, Gengyao Zhu and Yuan Liang, Methotrexate prevents epidural fibrosis through endoplasmic reticulum stress signalling pathway, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2016.12.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Methotrexate prevents epidural fibrosis through endoplasmic reticulum stress signalling pathway Hui Chen1, Lianqi Yan1, Jingcheng Wang*, Yu Sun*, Xiaolei Li, Shuai Zhao, Daxin Wang, Gengyao Zhu, Yuan Liang Department of Orthopedics, Clinical medical college of Yangzhou University, Subei People's Hospital of Jiangsu Province, Yangzhou, 225001, China [email protected] (JC Wang) [email protected] (Y Sun) *
Corresponding authors: Jingcheng Wang and, Address: Tel: +86 13301456789 (Pro.
Wang), Fax: +86 514 87937406 *
Corresponding author. Yu Sun. Tel: +86 18051060619 (Yu Sun M.D); Fax: +86 514
87937406 ABSTRACT Lumbar laminectomy is one of the most common treatments for lumbar disc herniation and other lumbar disorders with serious complications, such as failed back surgery syndrome, mainly caused by epidural fibrosis (EF). The developing fibrosis causes radicular pain after the laminectomy or discectomy. Methotrexate (MTX) is a folic acid antagonist that has shown anti-proliferative effects in previous studies. The aim of our experiment is to study whether MTX has positive effects on the outcome of the laminectomy in rats. Our finding first demonstrated the beneficial effect of topical application of MTX in laminectomy models. As the results of a macroscopic scoring system, hydroxyproline content analysis, histological evaluation, the number of fibroblasts and immunohistochemistry showed that MTX suppressed the EF compared with the control group, and the inhibiting effect was in a dose-dependent manner. Furthermore, we hypothesized that the endoplasmic reticulum (ER) stress mediated the suppression effect of the EF. To verify this point of view, fibroblast cells cultured from epidural scar tissues of rats were used. CCK-8 assay, Western blot (for apoptotic genes, such as cleaved PARP) and annexin V-FITC/PI double-labelling 1
Hui Chen and Lianqi Yan equally contributed to paper.
showed that MTX could induce cell apoptosis. The expression of CHOP and GRP78 and the activation of ER stress-associated genes strongly suggested that ER stress mediated the apoptotic signalling pathway; immunohistochemistry of GRP78 and CHOP further verified this. Our findings indicate that topical application of MTX could indeed reduce EF, and the application of MTX could induce apoptosis through ER stress in rats. Keywords: Methotrexate, endoplasmic reticulum stress, fibroblast apoptosis, epidural fibrosis
1. Introduction Lumbar laminectomy is one of the most common treatments for lumbar disc herniation and other lumbar disorders; however, it may cause a series of symptoms, including “failed back surgery syndrome” (FBSS)(GuyerPatterson and Ohnmeiss 2006, Emmez et al. 2008), which could result in poor clinical outcomes. These clinical outcomes include recurrent, persistent low back pain and disability(Xu et al. 2012). There are many etiologic reasons for FBSS, but epidural fibrosis (EF) after laminectomy is implicated as the main contributing factor(SiqueiraKranzler and Dharkar 1983, Ross et al. 1996). The developing fibrosis causes radicular pain by leading compression or tethering the nerve roots and impeding their normal motion, then these constrained nerve roots were stretched by the movement of the vertebral column(Andrychowski et al. 2013, Yang et al. 2011). Thus, preventing EF formation is believed to be the best approach to manage this problem. A number of methods have been studied to prevent EF, including surgical methods and material agents(Abitbol et al. 1994); however, effects of these treatments are not satisfactory, so their clinical applications are still limited. Therefore, preventing the occurrence of EF is still a great challenge to surgeons. Many drugs, including some anti-cancer agents, such as mitomycin C (MMC), 5fluorouracil (5-FU) and hydroxycamptothecin (HCPT)(Lee et al. 2004, Yildiz et al. 2007, Sun et al. 2008), and some immunosuppressive agents, such as tacrolimus
(FK506) and pimecrolimus(Cemil et al. 2009, Yan et al. 2013), have been studied to prevent this condition, but experiments showed that they still have quite a lot of limitations before clinical trials. Methotrexate (MTX) is a folic acid antagonist(Belinsky et al. 2007), which is used in the treatment of cancer chemotherapy, rheumatoid arthritis and psoriasis(Arena et al. 2012a). Recently, it was reported that MTX induces apoptosis in CCRF-CEM and Nalm6 cells through the activation of endoplasmic reticulum (ER) stress(Kuznetsov et al. 2011). The ER is the place where protein synthesis, folding, assembly and transport occurs(Marciniak and Ron 2006). When the unfolded/misfolded proteins assemble under a certain degree, the unfolded protein response (UPR) provides a protective effect to the cells from the injury and then restore regular functions(Lenna and Trojanowska 2012). However, chronic stress or a failed adaptive response in the ER causes apoptosis by triggering the accumulation of glucose-regulated protein 78 (GRP78/BiP) and activation of the dsRNA-activated protein kinase (PKR)-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and transcription factor 6 (ATF6). Then the PERK phosphorylates eukaryotic initiation factor-2 (eIF2α), regulates the expression of ER stress target genes, including CCAAT/enhancer binding protein homologous protein (CHOP)(Gotoh et al. 2004, Tajiri et al. 2004). The Bcl-2 family is the downstream of CHOP, which plays a vital role in the ER stress-mediated cell death pathways(Hetz 2007). Therefore, we are interested in whether MTX induces fibroblast apoptosis by ER stress and whether it could reduce and prevent EF, in the hopes of treating EF in the future.
2. Materials and Methods 2.1. Fibroblast culture and treatment Fibroblast cells were obtained from epidural scar tissue isolated from rats that underwent laminectomies. Fibroblasts were cultured at 37°C under 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY),
supplemented with 20% foetal bovine serum (FBS; Gibco), 100 U/ml penicillin and 100 mg/ml streptomycin (PS; Thermo, Rockford, IL). Cells in exponential growth phase between passages 3 and 6 were used for the experiments. Fibroblast cell monolayers were seeded into 96-well plates, 6-well plates, or 10-cm dishes overnight until reaching approximately 50–80% density and then were washed with phosphatebuffered saline (PBS; pH 7.4) and treated with MTX purchased from Sigma (St. Louis, MO, USA) in various concentrations of reagent and in various times. The controls were treated with 5-min applications of PBS only. After treatment, the cells were immediately washed three times with PBS and were maintained in the growth medium for subsequent experiments. 2.2. Cell Viability Cell viability was measured using a Cell Counting Kit-8 (CCK-8;Dojindo, Tokyo, Japan). The cells were cultured in triplicate in 96-well plates and were treated with 105
to 10-9 M MTX. In another group, the cells were maintained in DMEM for 0, 12, 24,
36, 48 or 72 h, as described previously, and the cells were further incubated with 10 μl WST-8 (2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfonyl]-2Htetrazolium; Dojindo Laboratories, Kumamoto, Japan) for 1 h at 37°C. Cells that stained positively with WST-8 were considered viable cells and were expressed as a percentage compared with the control cells. 2.3. Western Blot Analysis Treated cells were lysed on ice in lysis buffer (Beyotime, Hangzhou, China), according to the manufacturer’s instructions. The protein concentration was determined by the BCA Protein Assay Kit (Beyotime, Hangzhou, China). Equal amounts (60 μg/lane) of total proteins were subjected to electrophoresis on a 6%, 10% or 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 5% skim milk in tris-buffered saline and Tween 20 (TBST) for 2 h at room temperature and then incubated with the primary antibodies. Anti-78-kDa glucose-regulated protein (GRP78), anti-CHOP, anti-phosphoIRE1α (S724) antibodies, anti-activating transcription factor 6 (ATF6), anti- phospho-eIF2α,
anti- eIF2α, anti-Bax and anti-Bcl-2 antibodies were obtained from Abcam (Abcam, Hong Kong, China). Anti-phospho-PERK and anti-inositol-requiring enzyme 1α (IRE1α) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antipoly ADP-ribose polymerase (PARP), anti-PERK and anti-β-actin antibody were purchased from Cell Signalling Technology (Cell Signaling Technology, Beverly, MA, USA). The membranes were next washed three times in TBST and incubated with horseradish peroxidase-conjugated goat antimouse or anti-rabbit IgG (Santa Cruz Biotechnology) (diluted 1:5,000) for 2 h and then washed with TBST three times. The immune complexes were visualised via fluorography enhanced by the electrochemiluminescence system (Millipore, Bedford, USA). 2.4. Flow cytometry analysis of cell apoptosis The cells were plated in 6-well plates and incubated overnight at 37°C. After treatment with 1 μM MTX for 24 h, the detached and adherent cells were collected and washed three times with ice-cold PBS buffer. Cells were then resuspended in binding buffer at 1 × 106 cells/well and incubated with annexin V-FITC and PI (BD Biosciences, Singapore) for double-staining, according to the manufacturer’s protocol. Before the analysis, the mixture was incubated in the dark for 15 min at room temperature. 2.5. Animals A total of 48 Sprague-Dawley, young-adult, male rats (purchased from the experimental animal centre of Yangzhou university, China) with weights of 250 ± 20 g were used for this study. All animals received care in compliance with the principles of Laboratory Animal Care of international recommendations, and the experimental protocol was approved by the Animal Care and Research Committee of the Yangzhou University, China. The rats were randomly divided into four groups (12 rats per group): MTX (0.5 mg/ml), MTX (1 mg/ml), MTX (2 mg/ml) or control (saline). The rats were acclimated to the environment for 1 week before the experiment. 2.6. Animal mode Laminectomy models were performed on rats according to the procedure in the previous study(Sun et al. 2007). After anaesthesia by intraperitoneal injection of
ketamine (100 mg/kg body weight), the fur around the location of L1 and L2 were shaved, then antisepsis of the exposed skin was performed with iodophor. The L1 vertebral plate was exposed by a midline skin incision and separation of the paraspinal muscles. The dura mater was exposed after removing the spinous process and vertebral plate of L1 by rongeur forceps. A total laminectomy of L1 was performed. 2.7. Topical application of drugs After depilation, disinfection and hemostasis of the lumbar region, MTX in various concentrations of 0.5, 1 and 2 mg/ml or saline were administered to the laminectomy areas with cotton pads (4 × 4 mm) for 5 min. The surrounding tissues were covered by wet gauzes to avoid touching the agent. After the cotton pads were removed, the decorticated areas of laminectomy were irrigated with saline to get rid of the remaining MTX immediately. The wounds were then sutured to close. After the operations, an intramuscular injection of Cefazolin sodium was administered for 3 days to the rats to prevent infection after the operation. 2.8. Macroscopic assessment of EF Six rats were randomly selected from each group after 4 weeks for macroscopic evaluation. The surgical sites were reopened, and epidural scar adhesion was evaluated under double-blind trials to prevent bias, and the amount of adhesion was judged based on the Rydell classification(Rydell 1970): grade 0, epidural scar tissue was not adherent to the dura mater; grade 1, epidural scar tissue was adherent to the dura mater but easily dissected; grade 2, epidural scar tissue was adherent to the dura mater and dissected with difficulty without disrupting the dura matter; grade 3, epidural scar tissue was firmly adherent to the dura mater and could not be dissected. 2.9. Determination of hydroxyproline content in epidural scar tissue After macroscopic evaluation, approximately 5 mg of wet-weight scar tissue was obtained from the decorticated areas for hydroxyproline content (HPC) analysis, according to a previous study(WOESSNER 1961). Briefly, the samples were lyophilised, ground and hydrolysed with 6 mol/l HCl at 110°C for 24 h. Then 1 ml hydroxyproline developer (β–dimethylaminobenzaldehyde solution) was added to the
samples and standards. The absorbances were evaluated at 558 nm using a spectrophotometer. In the end, the HPC/mg scar tissue was calculated according to the standard curve constructed with serial concentrations of commercial hydroxyproline. 2.10. Histological analysis Six remaining rats were picked, and the histological analysis was performed after 4 weeks. The rats were anaesthetised and underwent intracardial perfusion with 4% paraformaldehyde. The whole L1 spinal column, including the paraspinal muscles and epidural fibrotic tissue, was removed and immersed in 10% buffered formalin. The specimens were decalcified and then embedded in paraffin. Successive 4-μm transverse sections were made through the L1 vertebra from the top to bottom. The sections were stained with hematoxylin–eosin (H&E) and Masson’s trichrome stains. The EF and epidural collagen tissues were evaluated by light microscopy using ×40 and ×200 magnifications. 2.11. Immunohistochemistry Immunohistochemistry for GRP78 and CHOP proteins was performed using the avidin-biotin-peroxidase complex method. For antigen retrieval, the sections for GRP78 were treated with 0.5% trypsin at 37°C for 10 min. After blocking with 1% skim milk, tissue sections were incubated with anti-grp78/BiP polyclonal antibody and anti-CHOP antibody overnight at 4°C and then washed in PBS solution. Biotinylated anti-rabbit IgG was used as the secondary antibody, and the sections were visualised as previously described. 2.12. Statistical analysis The statistical analysis was performed by SPSS 19.0 statistical software. Significant differences between the treatment groups were analyzed via one-way ANOVA. All of our data were expressed as mean ± standard deviation (S.D.) values. Statistical significance was defined as a P value < 0.05. 3. Results 3.1 MTX induces apoptotic cell death in fibroblasts To study whether MTX could induce cell death in fibroblasts, we used different concentrations of MTX to treat the cells for 24 h, then cell viability was determined
by CCK-8 assay. According to the results of the CCK-8 assay, the cell viability was reduced sharply as the concentration increased. In addition, cells were treated with MTX (1 μM) for various durations (0–60 h) and then examined by CCK-8 assay. These data showed that MTX reduced cell viability in a dose- and time-dependent manner in fibroblast (Fig. 1A and B). Similar results were observed using the annexin V-FITC/PI double labelling (Fig. 1C, and D), which demonstrated that the apoptosis rate was significantly increased. Furthermore, we examined cleaved-PARP after different durations of incubation with 1 μM MTX because it is regarded as a marker of apoptosis(Oliver et al. 1998). The result showed that the cleaved-PARP was increased which confirmed our hypothesis (Fig. 1E). Collectively, these results suggest that MTX induces apoptotic cell death in fibroblasts. 3.2. MTX induces ER stress-mediated apoptosis We determined whether ER stress is induced by MTX. According to previous studies, GRP78 and CHOP are markers of ER stress. When treated with MTX for various concentrations, the anti-apoptogenic Bcl-2 expression decreased significantly, whereas the apoptogenic Bax and CHOP expressions increased. The ratio of Bcl2/Bax was decreased markedly (Fig. 2A). After treatment with different concentrations of MTX overnight, cells were collected; as shown in Fig. 2B and 2C the GRP78 expression was markedly increased. MTX also induced the activation of IRE-1, PERK and ATF-6, which are the three sensors of the ER stress response, the eIF2α, which is the downstream protein of PERK was also being phosphorylated. These data demonstrated that MTX induced ER stress, which eventually decreased the Bcl-2/Bax ratio in the apoptosis signalling pathways of fibroblasts. 3.3. Macroscopic evaluation of epidural scar adhesion To study the effect of MTX on EF, laminectomy models were established. The results of macroscopic observation, classified according to Rydell’s classification, suggested that grade 3 epidural adhesions existed in all of the control group rats. Grade 0, 1 and 2 were found in the MTX group (Table 1). 3.4. Histological evaluation In Fig. 3A, the H&E images show that topical application of MTX could reduce
EF, and the loose fibrotic tissues did not adhere to the dura mater in the laminectomy areas. Fibroblast counting showed that the number of fibroblasts was decreased as the concentration of MTX rose (Fig. 3B and C); this means the degree of EF in the MTX group was in a dose-independent manner. These results revealed that MTX could reduce EF in rats. 3.5. Effect of MTX on epidural collagen tissue in rats In Fig. 4A, Masson’s trichrome-stained images show that MTX treatment could reduce the density of epidural collagen tissue, and the results were coincidence with H&E staining. In the control group, the collagen density of epidural tissue was much more than the MTX groups. In addition, we observed that the degree of epidural collagen in the MTX group was in a dose-independent manner. The results of the HPC analysis were in accordance with the Masson’s trichrome staining. The HPCs of the epidural fibrotic tissue from the different groups are shown in Fig. 4B. Compared to the control group, topical application of MTX could markedly decrease HPC, and the tendency of the HPC of the MTX group was also coincidence with the concentrations. The HPC in the 1 mg/ml group was less than that in the control and 0.5 mg/ml group; the 2 mg/ml group remained the least. These results demonstrate that topical application of MTX could reduce collagen density and HPC in epidural fibrotic tissue of rats. 3.6. MTX suppresses the EF through ER stress To determine whether ER stress is involved in MTX-mediated EF suppression, we further assessed the GRP78 and CHOP expression through immunohistochemical analysis. As discussed previously, GRP78 and CHOP are markers of ER stress, when the ER stress response is activated, GRP78 and CHOP are located exclusively in the cytoplasm. Epidural fibrotic tissues were isolated and were 4% paraformaldehyde fixed and paraffin embedded. The results of the immunohistochemical analysis are shown in Fig. 5A and B. In contrast to the control group, the immunostained granules of cytoplasmic GRP78 and CHOP were significantly increased in the MTX-treated groups. The laboratory findings are strongly suggestive that ER stress is activated in MTX-mediated EF suppression.
4. Discussion According to previous studies, EF often results in negative effects on outcome after lumbar laminectomy. To solve the problem, numerous methods have been studied to prevent EF through surgical methods and new materials, including hyaluronic acid and fat grafts(Robertson et al. 1993, Sandoval and HernandezVaquero 2008); however, clinical application of these methods have been limited because the effects were not as satisfactory as expected. Recently, it was reported that many anti-cancer and some immunosuppressive agents have been studied in light of this problem. Though experimental results of these agents demonstrated that they indeed have positive effects on FBSS, toxic and side effects limit their clinical applications. MTX is a folic acid antagonist and it is used in the treatment of cancer chemotherapy, rheumatoid arthritis, and psoriasis. Numerous studies have investigated its properties. The medication time, dosage and route of administration will significantly affect its function. Long-term therapy of MTX orally induces pulmonary toxicity and liver stiffness(Arena et al. 2012b, Ohbayashi et al. 2014a); when taken intraperitoneally for a week, it will induce oxidative tissue damage on spinal tissue(Ayromlou et al. 2011). Recent studies have found that, low-dose MTX has a beneficial effect by reducing early neutrophil infiltration and decreasing lipid peroxidation levels, and has significantly protective effects on injured spinal cord tissue in the first 24 h after spinal cord injury, showing it has a beneficial effect on neurons and glia(Hashizume et al. 2000, Bakar et al. 2013, Sanli et al. 2012). In previous studies, MTX induced apoptosis in CCRF-CEM and Nalm6 cells through the activation of ER stress. It is known that apoptosis is an important way of cell death; it generally occurs via extrinsic and intrinsic pathways, also known as receptor-mediated or mitochondrial-mediated. Recently, it was reported that ER stress also plays an important role in cell apoptosis(Oyadomari and Mori 2004). Thus, it is reasonable to doubt whether topical application of MTX could induce the EF suppression with little side effects.
In this present study, topical application of MTX could effectively prevent EF. The concentration of MTX chosen was based on previous studies(Heenen et al. 1998, Hashizume et al. 2000, Ohbayashi et al. 2014b, Jiang et al. 2011). Our experimental data from CCK-8 assays show that MTX treatment could reduce the cell viability in a dose- and time-dependent manner. Further studies included annexin V-FITC/PI double labelling and Western blotting for cleaved-PARP, demonstrating that the reduction of cell viability was occurring through apoptosis. In bone defect areas after operation, fibroblasts will poduce extracellular matrix components and collagen. When their productions are out of control, they may lead to the formation of scar tissue and finally induce the EF. It is known that hydroxyproline accounts for about 12.5% of the amino acid content in collagen fiber, so the content of hydroxyproline could be regarded as the reflection of collagen(Edwards and O'Brien 1980). In the laminectomy model, multiple parameters such as macroscopic scoring system, hydroxyproline content analysis, histological evaluation and the number of fibroblasts revealed that the MTX application could indeed suppress the EF by reducing the number of fibroblasts and preventing EF after laminectomy. ER is a multifunctional organelle which plays a vital role in calcium homeostasis and synthesis of proteins. The UPR can prevent the accumulation of misfolded/unfolded proteins in the ER lumen, then restore the regular functions of the cells. However, chronic or prolonged ER stress will lead to apoptosis mediated through CHOP pathway. Previous studies have shown that PERK is critical regulator of CHOP expression, activation of PERK will phosphorylate eIF2α and ATF4 synthesis, together with C/EBP-b then binds to the composite site and transactivates the CHOP promoter(Pan et al. 2012). Results of Western blot showed that GRP78 and CHOP, which are known as markers of ER stress, were expressed, indicating that MTX could induce ER stress. Next, we investigated PERK, IRE1α and ATF6, which are three sensors of the ER stress response. Their expression increased with the concentration of MTX; this result further demonstrated that MTX could induce apoptosis through ER stress(Fig. 2A and B). Furthermore, eIF2α, which is the downstream protein of PERK, was increased in a
dose-dependent manner, indicates that PERK plays a important role in this apoptotic signalling pathways. Immunohistochemical analysis demonstrated that, in the epidural fibrotic tissues, the expression of GRP78 and CHOP were upregulated. Collectively, these results support those studies about the anti-proliferative and anti-fibrotic properties of MTX, and these effects may work through ER stress. Based on previous reports and our present study, we propose a possible mechanism of the topical application of MTX on preventing EF. After treatment with MTX, chronic stress or a failed adaptive response in the ER may trigger the accumulation of GRP78 and activate PERK, IRE1α and ATF6. Then PERK phosphorylates eukaryotic initiation factor-2 (eIF2α), regulating expression of ER stress target genes, including CHOP, and increasing the Bax/Bcl-2 ratio; this eventually induces the apoptosis of fibroblasts in epidural fibrotic tissues and prevents EF. In conclusion, this study provides a novel agent to treat FBSS. We further investigated the possible mechanism of the anti-apoptotic properties of MTX. However, the mechanism of MTX on preventing epidural scar adhesion was complicated; our result only shows one possible mechanism. As an anti-cancer agent, unlike other agents such as MMC, although both of them could prevents epidural fibrosis(Lee et al. 2004b), however, topical application of MTX to laminectomy sites may be beneficial to neurons and glia(Bakar et al. 2013, WestlandPollard and Sumner 1990). It may also inhibit the proliferation of various cells and has a negative effect on wound healing. Therefore, further studies should be performed before clinical application.
Acknowledgments Funding was provided by the National Natural Science Foundation of China (Grants#81271994, 81301550 and 81371971); Jiangsu Province Health Department Foundation (H201250); and Nature Science Foundation (BK2011433). We thank all the workers of the pathology laboratory of Yangzhou University.
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403-15. Westland, K., J. D. Pollard & A. J. Sumner (1990) Mitomycin C induces a delayed and prolonged demyelination and conduction block due to Schwann cell destruction. Clin Exp Neurol, 27, 65-78. WOESSNER, J. J. (1961) The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys, 93, 440-7. Xu, J., Y. Chen, Y. Yue, J. Sun & L. Cui (2012) Reconstruction of epidural fat with engineered adipose tissue from adipose derived stem cells and PLGA in the rabbit dorsal laminectomy model. Biomaterials, 33, 6965-73. Yan, L., X. Li, J. Wang, Y. Sun, D. Wang, J. Gu, J. He, H. Hu, G. Chen, Q. Wang & X. Feng (2013) Immunomodulatory effectiveness of tacrolimus in preventing epidural scar adhesion after laminectomy in rat model. Eur J Pharmacol, 699, 194-9. Yang, J., B. Ni, J. Liu, L. Zhu & W. Zhou (2011) Application of liposomeencapsulated hydroxycamptothecin in the prevention of epidural scar formation in New Zealand white rabbits. Spine J, 11, 218-23. Yildiz, K. H., F. Gezen, M. Is, S. Cukur & M. Dosoglu (2007) Mitomycin C, 5fluorouracil, and cyclosporin A prevent epidural fibrosis in an experimental laminectomy model. Eur Spine J, 16, 1525-30.
Fig. 1. MTX caused fibroblasts apoptosis. (A) Dose-dependent effects on fibroblasts after treatment with various concentrations of MTX overnight. (B) Timedependent effects on fibroblasts at 1 μM for various durations. The viability was determined by CCK-8 assay. (C, D) Apoptotic cells were stained by annexin V/PI dual-staining with 1 μM MTX for 24 hours. (E) Western blots revealed that MTX induces cleaved PARP. β-actin was included as a control. Gels were run in triplicate. The data are presented as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01).
Fig. 2. MTX induced ER stress in fibroblasts. (A) The expression of CHOP, Bcl-2 and Bax were determined by Western blot analysis in cells after treatment with 0.001,
0.01, 0.1, 1 and 10 μM MTX overnight. β-actin was used as a control. (B) The dosedependent effects of MTX on ER stress-associated proteins, including GRP78, pPERK, PERK, ATF6 p90, p-IRE1 and T-IRE1, were determined by Western blot analysis in cells after treatment with 0.001, 0.01, 0.1, 1 and 10 μM MTX overnight. βactin was used as a control. (C) The dose-dependent effects of MTX on eIF2α and peIF2α were determined by Western blot analysis in cells after treatment with 0.001, 0.01, 0.1, 1 and 10 μM MTX overnight. β-actin was used as a control.
Fig. 3. Histological analysis of fibroblasts in epidural scar tissue of the laminectomy operation areas treated with saline, 0. 5 mg/ml, 1 mg/ml and 2mg/ml MTX. (A) Dense scar tissues (*) adhered to dura maters were found in control group. Moderate scar tissues (*) were found in 0.5 mg/ml MTX group. Loose scar tissues (*) without adherence to dura mater were found in 1 and 2 mg/ml MTX groups. ‘‘S’’ represents spinal cord, and ‘‘L’’ represents laminectomy defect. The sections were stained with H&E, and the magnification was ×40. (B, C) The number of fibroblasts was decreased as the concentration of MTX increased, which shows that the effect of MTX on epidural adhesion tissues was in a dose-dependent manner. The sections were stained with H&E, and the magnification was ×200. Fig. 4. Effect of MTX on epidural collagen tissue in rats. (A) The collagen density of epidural adhesion tissues in each group. The collagen tissues are blue in the section with Masson’s trichrome staining under the light microscope (×40). MTX reduced collagen synthesis and fibrosis. (B) HPC was expressed as μg/mg. The amount of hydroxyproline was decreased when the concentration of MTX increased. *P < 0.05 and **P < 0.01 compared with the HPC in control group. The density of collagen tissue and hydroxyproline analysis shows that the decreased effect was in a dosedependent manner.
Fig. 5. Immunohistochemical analysis of GRP78 and CHOP in epidural scar tissue applied with MTX (0.5, 1 or 2 mg/ml) or saline. As the dosage of MTX increased, the expression of GRP78 (A) and CHOP (B) increased.
Table.1. The degree of epidural adhesion according to Rydell’s classification. Grade Group 0 1 2 Saline 0 0 0 0.5mg/ml 0 1 2 1.0mg/ml** 2 3 1 2.0mg/ml** 4 2 0 Compares with the score of adhesion in control group. * < 0.05, ** < 0.01.
Figure Fig.1
3 6 3 0 0
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PII: DOI: Reference:
S0014-2999(16)30815-9 http://dx.doi.org/10.1016/j.ejphar.2016.12.032 EJP70994
To appear in: European Journal of Pharmacology Received date: 14 November 2016 Revised date: 12 December 2016 Accepted date: 20 December 2016 Cite this article as: Hui Chen, Lianqi Yan, Jingcheng Wang, Yu Sun, Xiaolei Li, Shuai Zhao, Daxin Wang, Gengyao Zhu and Yuan Liang, Methotrexate prevents epidural fibrosis through endoplasmic reticulum stress signalling pathway, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2016.12.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Methotrexate prevents epidural fibrosis through endoplasmic reticulum stress signalling pathway Hui Chen1, Lianqi Yan1, Jingcheng Wang*, Yu Sun*, Xiaolei Li, Shuai Zhao, Daxin Wang, Gengyao Zhu, Yuan Liang Department of Orthopedics, Clinical medical college of Yangzhou University, Subei People's Hospital of Jiangsu Province, Yangzhou, 225001, China [email protected] (JC Wang) [email protected] (Y Sun) *
Corresponding authors: Jingcheng Wang and, Address: Tel: +86 13301456789 (Pro.
Wang), Fax: +86 514 87937406 *
Corresponding author. Yu Sun. Tel: +86 18051060619 (Yu Sun M.D); Fax: +86 514
87937406 ABSTRACT Lumbar laminectomy is one of the most common treatments for lumbar disc herniation and other lumbar disorders with serious complications, such as failed back surgery syndrome, mainly caused by epidural fibrosis (EF). The developing fibrosis causes radicular pain after the laminectomy or discectomy. Methotrexate (MTX) is a folic acid antagonist that has shown anti-proliferative effects in previous studies. The aim of our experiment is to study whether MTX has positive effects on the outcome of the laminectomy in rats. Our finding first demonstrated the beneficial effect of topical application of MTX in laminectomy models. As the results of a macroscopic scoring system, hydroxyproline content analysis, histological evaluation, the number of fibroblasts and immunohistochemistry showed that MTX suppressed the EF compared with the control group, and the inhibiting effect was in a dose-dependent manner. Furthermore, we hypothesized that the endoplasmic reticulum (ER) stress mediated the suppression effect of the EF. To verify this point of view, fibroblast cells cultured from epidural scar tissues of rats were used. CCK-8 assay, Western blot (for apoptotic genes, such as cleaved PARP) and annexin V-FITC/PI double-labelling 1
Hui Chen and Lianqi Yan equally contributed to paper.
showed that MTX could induce cell apoptosis. The expression of CHOP and GRP78 and the activation of ER stress-associated genes strongly suggested that ER stress mediated the apoptotic signalling pathway; immunohistochemistry of GRP78 and CHOP further verified this. Our findings indicate that topical application of MTX could indeed reduce EF, and the application of MTX could induce apoptosis through ER stress in rats. Keywords: Methotrexate, endoplasmic reticulum stress, fibroblast apoptosis, epidural fibrosis
1. Introduction Lumbar laminectomy is one of the most common treatments for lumbar disc herniation and other lumbar disorders; however, it may cause a series of symptoms, including “failed back surgery syndrome” (FBSS)(GuyerPatterson and Ohnmeiss 2006, Emmez et al. 2008), which could result in poor clinical outcomes. These clinical outcomes include recurrent, persistent low back pain and disability(Xu et al. 2012). There are many etiologic reasons for FBSS, but epidural fibrosis (EF) after laminectomy is implicated as the main contributing factor(SiqueiraKranzler and Dharkar 1983, Ross et al. 1996). The developing fibrosis causes radicular pain by leading compression or tethering the nerve roots and impeding their normal motion, then these constrained nerve roots were stretched by the movement of the vertebral column(Andrychowski et al. 2013, Yang et al. 2011). Thus, preventing EF formation is believed to be the best approach to manage this problem. A number of methods have been studied to prevent EF, including surgical methods and material agents(Abitbol et al. 1994); however, effects of these treatments are not satisfactory, so their clinical applications are still limited. Therefore, preventing the occurrence of EF is still a great challenge to surgeons. Many drugs, including some anti-cancer agents, such as mitomycin C (MMC), 5fluorouracil (5-FU) and hydroxycamptothecin (HCPT)(Lee et al. 2004, Yildiz et al. 2007, Sun et al. 2008), and some immunosuppressive agents, such as tacrolimus
(FK506) and pimecrolimus(Cemil et al. 2009, Yan et al. 2013), have been studied to prevent this condition, but experiments showed that they still have quite a lot of limitations before clinical trials. Methotrexate (MTX) is a folic acid antagonist(Belinsky et al. 2007), which is used in the treatment of cancer chemotherapy, rheumatoid arthritis and psoriasis(Arena et al. 2012a). Recently, it was reported that MTX induces apoptosis in CCRF-CEM and Nalm6 cells through the activation of endoplasmic reticulum (ER) stress(Kuznetsov et al. 2011). The ER is the place where protein synthesis, folding, assembly and transport occurs(Marciniak and Ron 2006). When the unfolded/misfolded proteins assemble under a certain degree, the unfolded protein response (UPR) provides a protective effect to the cells from the injury and then restore regular functions(Lenna and Trojanowska 2012). However, chronic stress or a failed adaptive response in the ER causes apoptosis by triggering the accumulation of glucose-regulated protein 78 (GRP78/BiP) and activation of the dsRNA-activated protein kinase (PKR)-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and transcription factor 6 (ATF6). Then the PERK phosphorylates eukaryotic initiation factor-2 (eIF2α), regulates the expression of ER stress target genes, including CCAAT/enhancer binding protein homologous protein (CHOP)(Gotoh et al. 2004, Tajiri et al. 2004). The Bcl-2 family is the downstream of CHOP, which plays a vital role in the ER stress-mediated cell death pathways(Hetz 2007). Therefore, we are interested in whether MTX induces fibroblast apoptosis by ER stress and whether it could reduce and prevent EF, in the hopes of treating EF in the future.
2. Materials and Methods 2.1. Fibroblast culture and treatment Fibroblast cells were obtained from epidural scar tissue isolated from rats that underwent laminectomies. Fibroblasts were cultured at 37°C under 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY),
supplemented with 20% foetal bovine serum (FBS; Gibco), 100 U/ml penicillin and 100 mg/ml streptomycin (PS; Thermo, Rockford, IL). Cells in exponential growth phase between passages 3 and 6 were used for the experiments. Fibroblast cell monolayers were seeded into 96-well plates, 6-well plates, or 10-cm dishes overnight until reaching approximately 50–80% density and then were washed with phosphatebuffered saline (PBS; pH 7.4) and treated with MTX purchased from Sigma (St. Louis, MO, USA) in various concentrations of reagent and in various times. The controls were treated with 5-min applications of PBS only. After treatment, the cells were immediately washed three times with PBS and were maintained in the growth medium for subsequent experiments. 2.2. Cell Viability Cell viability was measured using a Cell Counting Kit-8 (CCK-8;Dojindo, Tokyo, Japan). The cells were cultured in triplicate in 96-well plates and were treated with 105
to 10-9 M MTX. In another group, the cells were maintained in DMEM for 0, 12, 24,
36, 48 or 72 h, as described previously, and the cells were further incubated with 10 μl WST-8 (2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfonyl]-2Htetrazolium; Dojindo Laboratories, Kumamoto, Japan) for 1 h at 37°C. Cells that stained positively with WST-8 were considered viable cells and were expressed as a percentage compared with the control cells. 2.3. Western Blot Analysis Treated cells were lysed on ice in lysis buffer (Beyotime, Hangzhou, China), according to the manufacturer’s instructions. The protein concentration was determined by the BCA Protein Assay Kit (Beyotime, Hangzhou, China). Equal amounts (60 μg/lane) of total proteins were subjected to electrophoresis on a 6%, 10% or 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 5% skim milk in tris-buffered saline and Tween 20 (TBST) for 2 h at room temperature and then incubated with the primary antibodies. Anti-78-kDa glucose-regulated protein (GRP78), anti-CHOP, anti-phosphoIRE1α (S724) antibodies, anti-activating transcription factor 6 (ATF6), anti- phospho-eIF2α,
anti- eIF2α, anti-Bax and anti-Bcl-2 antibodies were obtained from Abcam (Abcam, Hong Kong, China). Anti-phospho-PERK and anti-inositol-requiring enzyme 1α (IRE1α) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antipoly ADP-ribose polymerase (PARP), anti-PERK and anti-β-actin antibody were purchased from Cell Signalling Technology (Cell Signaling Technology, Beverly, MA, USA). The membranes were next washed three times in TBST and incubated with horseradish peroxidase-conjugated goat antimouse or anti-rabbit IgG (Santa Cruz Biotechnology) (diluted 1:5,000) for 2 h and then washed with TBST three times. The immune complexes were visualised via fluorography enhanced by the electrochemiluminescence system (Millipore, Bedford, USA). 2.4. Flow cytometry analysis of cell apoptosis The cells were plated in 6-well plates and incubated overnight at 37°C. After treatment with 1 μM MTX for 24 h, the detached and adherent cells were collected and washed three times with ice-cold PBS buffer. Cells were then resuspended in binding buffer at 1 × 106 cells/well and incubated with annexin V-FITC and PI (BD Biosciences, Singapore) for double-staining, according to the manufacturer’s protocol. Before the analysis, the mixture was incubated in the dark for 15 min at room temperature. 2.5. Animals A total of 48 Sprague-Dawley, young-adult, male rats (purchased from the experimental animal centre of Yangzhou university, China) with weights of 250 ± 20 g were used for this study. All animals received care in compliance with the principles of Laboratory Animal Care of international recommendations, and the experimental protocol was approved by the Animal Care and Research Committee of the Yangzhou University, China. The rats were randomly divided into four groups (12 rats per group): MTX (0.5 mg/ml), MTX (1 mg/ml), MTX (2 mg/ml) or control (saline). The rats were acclimated to the environment for 1 week before the experiment. 2.6. Animal mode Laminectomy models were performed on rats according to the procedure in the previous study(Sun et al. 2007). After anaesthesia by intraperitoneal injection of
ketamine (100 mg/kg body weight), the fur around the location of L1 and L2 were shaved, then antisepsis of the exposed skin was performed with iodophor. The L1 vertebral plate was exposed by a midline skin incision and separation of the paraspinal muscles. The dura mater was exposed after removing the spinous process and vertebral plate of L1 by rongeur forceps. A total laminectomy of L1 was performed. 2.7. Topical application of drugs After depilation, disinfection and hemostasis of the lumbar region, MTX in various concentrations of 0.5, 1 and 2 mg/ml or saline were administered to the laminectomy areas with cotton pads (4 × 4 mm) for 5 min. The surrounding tissues were covered by wet gauzes to avoid touching the agent. After the cotton pads were removed, the decorticated areas of laminectomy were irrigated with saline to get rid of the remaining MTX immediately. The wounds were then sutured to close. After the operations, an intramuscular injection of Cefazolin sodium was administered for 3 days to the rats to prevent infection after the operation. 2.8. Macroscopic assessment of EF Six rats were randomly selected from each group after 4 weeks for macroscopic evaluation. The surgical sites were reopened, and epidural scar adhesion was evaluated under double-blind trials to prevent bias, and the amount of adhesion was judged based on the Rydell classification(Rydell 1970): grade 0, epidural scar tissue was not adherent to the dura mater; grade 1, epidural scar tissue was adherent to the dura mater but easily dissected; grade 2, epidural scar tissue was adherent to the dura mater and dissected with difficulty without disrupting the dura matter; grade 3, epidural scar tissue was firmly adherent to the dura mater and could not be dissected. 2.9. Determination of hydroxyproline content in epidural scar tissue After macroscopic evaluation, approximately 5 mg of wet-weight scar tissue was obtained from the decorticated areas for hydroxyproline content (HPC) analysis, according to a previous study(WOESSNER 1961). Briefly, the samples were lyophilised, ground and hydrolysed with 6 mol/l HCl at 110°C for 24 h. Then 1 ml hydroxyproline developer (β–dimethylaminobenzaldehyde solution) was added to the
samples and standards. The absorbances were evaluated at 558 nm using a spectrophotometer. In the end, the HPC/mg scar tissue was calculated according to the standard curve constructed with serial concentrations of commercial hydroxyproline. 2.10. Histological analysis Six remaining rats were picked, and the histological analysis was performed after 4 weeks. The rats were anaesthetised and underwent intracardial perfusion with 4% paraformaldehyde. The whole L1 spinal column, including the paraspinal muscles and epidural fibrotic tissue, was removed and immersed in 10% buffered formalin. The specimens were decalcified and then embedded in paraffin. Successive 4-μm transverse sections were made through the L1 vertebra from the top to bottom. The sections were stained with hematoxylin–eosin (H&E) and Masson’s trichrome stains. The EF and epidural collagen tissues were evaluated by light microscopy using ×40 and ×200 magnifications. 2.11. Immunohistochemistry Immunohistochemistry for GRP78 and CHOP proteins was performed using the avidin-biotin-peroxidase complex method. For antigen retrieval, the sections for GRP78 were treated with 0.5% trypsin at 37°C for 10 min. After blocking with 1% skim milk, tissue sections were incubated with anti-grp78/BiP polyclonal antibody and anti-CHOP antibody overnight at 4°C and then washed in PBS solution. Biotinylated anti-rabbit IgG was used as the secondary antibody, and the sections were visualised as previously described. 2.12. Statistical analysis The statistical analysis was performed by SPSS 19.0 statistical software. Significant differences between the treatment groups were analyzed via one-way ANOVA. All of our data were expressed as mean ± standard deviation (S.D.) values. Statistical significance was defined as a P value < 0.05. 3. Results 3.1 MTX induces apoptotic cell death in fibroblasts To study whether MTX could induce cell death in fibroblasts, we used different concentrations of MTX to treat the cells for 24 h, then cell viability was determined
by CCK-8 assay. According to the results of the CCK-8 assay, the cell viability was reduced sharply as the concentration increased. In addition, cells were treated with MTX (1 μM) for various durations (0–60 h) and then examined by CCK-8 assay. These data showed that MTX reduced cell viability in a dose- and time-dependent manner in fibroblast (Fig. 1A and B). Similar results were observed using the annexin V-FITC/PI double labelling (Fig. 1C, and D), which demonstrated that the apoptosis rate was significantly increased. Furthermore, we examined cleaved-PARP after different durations of incubation with 1 μM MTX because it is regarded as a marker of apoptosis(Oliver et al. 1998). The result showed that the cleaved-PARP was increased which confirmed our hypothesis (Fig. 1E). Collectively, these results suggest that MTX induces apoptotic cell death in fibroblasts. 3.2. MTX induces ER stress-mediated apoptosis We determined whether ER stress is induced by MTX. According to previous studies, GRP78 and CHOP are markers of ER stress. When treated with MTX for various concentrations, the anti-apoptogenic Bcl-2 expression decreased significantly, whereas the apoptogenic Bax and CHOP expressions increased. The ratio of Bcl2/Bax was decreased markedly (Fig. 2A). After treatment with different concentrations of MTX overnight, cells were collected; as shown in Fig. 2B and 2C the GRP78 expression was markedly increased. MTX also induced the activation of IRE-1, PERK and ATF-6, which are the three sensors of the ER stress response, the eIF2α, which is the downstream protein of PERK was also being phosphorylated. These data demonstrated that MTX induced ER stress, which eventually decreased the Bcl-2/Bax ratio in the apoptosis signalling pathways of fibroblasts. 3.3. Macroscopic evaluation of epidural scar adhesion To study the effect of MTX on EF, laminectomy models were established. The results of macroscopic observation, classified according to Rydell’s classification, suggested that grade 3 epidural adhesions existed in all of the control group rats. Grade 0, 1 and 2 were found in the MTX group (Table 1). 3.4. Histological evaluation In Fig. 3A, the H&E images show that topical application of MTX could reduce
EF, and the loose fibrotic tissues did not adhere to the dura mater in the laminectomy areas. Fibroblast counting showed that the number of fibroblasts was decreased as the concentration of MTX rose (Fig. 3B and C); this means the degree of EF in the MTX group was in a dose-independent manner. These results revealed that MTX could reduce EF in rats. 3.5. Effect of MTX on epidural collagen tissue in rats In Fig. 4A, Masson’s trichrome-stained images show that MTX treatment could reduce the density of epidural collagen tissue, and the results were coincidence with H&E staining. In the control group, the collagen density of epidural tissue was much more than the MTX groups. In addition, we observed that the degree of epidural collagen in the MTX group was in a dose-independent manner. The results of the HPC analysis were in accordance with the Masson’s trichrome staining. The HPCs of the epidural fibrotic tissue from the different groups are shown in Fig. 4B. Compared to the control group, topical application of MTX could markedly decrease HPC, and the tendency of the HPC of the MTX group was also coincidence with the concentrations. The HPC in the 1 mg/ml group was less than that in the control and 0.5 mg/ml group; the 2 mg/ml group remained the least. These results demonstrate that topical application of MTX could reduce collagen density and HPC in epidural fibrotic tissue of rats. 3.6. MTX suppresses the EF through ER stress To determine whether ER stress is involved in MTX-mediated EF suppression, we further assessed the GRP78 and CHOP expression through immunohistochemical analysis. As discussed previously, GRP78 and CHOP are markers of ER stress, when the ER stress response is activated, GRP78 and CHOP are located exclusively in the cytoplasm. Epidural fibrotic tissues were isolated and were 4% paraformaldehyde fixed and paraffin embedded. The results of the immunohistochemical analysis are shown in Fig. 5A and B. In contrast to the control group, the immunostained granules of cytoplasmic GRP78 and CHOP were significantly increased in the MTX-treated groups. The laboratory findings are strongly suggestive that ER stress is activated in MTX-mediated EF suppression.
4. Discussion According to previous studies, EF often results in negative effects on outcome after lumbar laminectomy. To solve the problem, numerous methods have been studied to prevent EF through surgical methods and new materials, including hyaluronic acid and fat grafts(Robertson et al. 1993, Sandoval and HernandezVaquero 2008); however, clinical application of these methods have been limited because the effects were not as satisfactory as expected. Recently, it was reported that many anti-cancer and some immunosuppressive agents have been studied in light of this problem. Though experimental results of these agents demonstrated that they indeed have positive effects on FBSS, toxic and side effects limit their clinical applications. MTX is a folic acid antagonist and it is used in the treatment of cancer chemotherapy, rheumatoid arthritis, and psoriasis. Numerous studies have investigated its properties. The medication time, dosage and route of administration will significantly affect its function. Long-term therapy of MTX orally induces pulmonary toxicity and liver stiffness(Arena et al. 2012b, Ohbayashi et al. 2014a); when taken intraperitoneally for a week, it will induce oxidative tissue damage on spinal tissue(Ayromlou et al. 2011). Recent studies have found that, low-dose MTX has a beneficial effect by reducing early neutrophil infiltration and decreasing lipid peroxidation levels, and has significantly protective effects on injured spinal cord tissue in the first 24 h after spinal cord injury, showing it has a beneficial effect on neurons and glia(Hashizume et al. 2000, Bakar et al. 2013, Sanli et al. 2012). In previous studies, MTX induced apoptosis in CCRF-CEM and Nalm6 cells through the activation of ER stress. It is known that apoptosis is an important way of cell death; it generally occurs via extrinsic and intrinsic pathways, also known as receptor-mediated or mitochondrial-mediated. Recently, it was reported that ER stress also plays an important role in cell apoptosis(Oyadomari and Mori 2004). Thus, it is reasonable to doubt whether topical application of MTX could induce the EF suppression with little side effects.
In this present study, topical application of MTX could effectively prevent EF. The concentration of MTX chosen was based on previous studies(Heenen et al. 1998, Hashizume et al. 2000, Ohbayashi et al. 2014b, Jiang et al. 2011). Our experimental data from CCK-8 assays show that MTX treatment could reduce the cell viability in a dose- and time-dependent manner. Further studies included annexin V-FITC/PI double labelling and Western blotting for cleaved-PARP, demonstrating that the reduction of cell viability was occurring through apoptosis. In bone defect areas after operation, fibroblasts will poduce extracellular matrix components and collagen. When their productions are out of control, they may lead to the formation of scar tissue and finally induce the EF. It is known that hydroxyproline accounts for about 12.5% of the amino acid content in collagen fiber, so the content of hydroxyproline could be regarded as the reflection of collagen(Edwards and O'Brien 1980). In the laminectomy model, multiple parameters such as macroscopic scoring system, hydroxyproline content analysis, histological evaluation and the number of fibroblasts revealed that the MTX application could indeed suppress the EF by reducing the number of fibroblasts and preventing EF after laminectomy. ER is a multifunctional organelle which plays a vital role in calcium homeostasis and synthesis of proteins. The UPR can prevent the accumulation of misfolded/unfolded proteins in the ER lumen, then restore the regular functions of the cells. However, chronic or prolonged ER stress will lead to apoptosis mediated through CHOP pathway. Previous studies have shown that PERK is critical regulator of CHOP expression, activation of PERK will phosphorylate eIF2α and ATF4 synthesis, together with C/EBP-b then binds to the composite site and transactivates the CHOP promoter(Pan et al. 2012). Results of Western blot showed that GRP78 and CHOP, which are known as markers of ER stress, were expressed, indicating that MTX could induce ER stress. Next, we investigated PERK, IRE1α and ATF6, which are three sensors of the ER stress response. Their expression increased with the concentration of MTX; this result further demonstrated that MTX could induce apoptosis through ER stress(Fig. 2A and B). Furthermore, eIF2α, which is the downstream protein of PERK, was increased in a
dose-dependent manner, indicates that PERK plays a important role in this apoptotic signalling pathways. Immunohistochemical analysis demonstrated that, in the epidural fibrotic tissues, the expression of GRP78 and CHOP were upregulated. Collectively, these results support those studies about the anti-proliferative and anti-fibrotic properties of MTX, and these effects may work through ER stress. Based on previous reports and our present study, we propose a possible mechanism of the topical application of MTX on preventing EF. After treatment with MTX, chronic stress or a failed adaptive response in the ER may trigger the accumulation of GRP78 and activate PERK, IRE1α and ATF6. Then PERK phosphorylates eukaryotic initiation factor-2 (eIF2α), regulating expression of ER stress target genes, including CHOP, and increasing the Bax/Bcl-2 ratio; this eventually induces the apoptosis of fibroblasts in epidural fibrotic tissues and prevents EF. In conclusion, this study provides a novel agent to treat FBSS. We further investigated the possible mechanism of the anti-apoptotic properties of MTX. However, the mechanism of MTX on preventing epidural scar adhesion was complicated; our result only shows one possible mechanism. As an anti-cancer agent, unlike other agents such as MMC, although both of them could prevents epidural fibrosis(Lee et al. 2004b), however, topical application of MTX to laminectomy sites may be beneficial to neurons and glia(Bakar et al. 2013, WestlandPollard and Sumner 1990). It may also inhibit the proliferation of various cells and has a negative effect on wound healing. Therefore, further studies should be performed before clinical application.
Acknowledgments Funding was provided by the National Natural Science Foundation of China (Grants#81271994, 81301550 and 81371971); Jiangsu Province Health Department Foundation (H201250); and Nature Science Foundation (BK2011433). We thank all the workers of the pathology laboratory of Yangzhou University.
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Fig. 1. MTX caused fibroblasts apoptosis. (A) Dose-dependent effects on fibroblasts after treatment with various concentrations of MTX overnight. (B) Timedependent effects on fibroblasts at 1 μM for various durations. The viability was determined by CCK-8 assay. (C, D) Apoptotic cells were stained by annexin V/PI dual-staining with 1 μM MTX for 24 hours. (E) Western blots revealed that MTX induces cleaved PARP. β-actin was included as a control. Gels were run in triplicate. The data are presented as the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01).
Fig. 2. MTX induced ER stress in fibroblasts. (A) The expression of CHOP, Bcl-2 and Bax were determined by Western blot analysis in cells after treatment with 0.001,
0.01, 0.1, 1 and 10 μM MTX overnight. β-actin was used as a control. (B) The dosedependent effects of MTX on ER stress-associated proteins, including GRP78, pPERK, PERK, ATF6 p90, p-IRE1 and T-IRE1, were determined by Western blot analysis in cells after treatment with 0.001, 0.01, 0.1, 1 and 10 μM MTX overnight. βactin was used as a control. (C) The dose-dependent effects of MTX on eIF2α and peIF2α were determined by Western blot analysis in cells after treatment with 0.001, 0.01, 0.1, 1 and 10 μM MTX overnight. β-actin was used as a control.
Fig. 3. Histological analysis of fibroblasts in epidural scar tissue of the laminectomy operation areas treated with saline, 0. 5 mg/ml, 1 mg/ml and 2mg/ml MTX. (A) Dense scar tissues (*) adhered to dura maters were found in control group. Moderate scar tissues (*) were found in 0.5 mg/ml MTX group. Loose scar tissues (*) without adherence to dura mater were found in 1 and 2 mg/ml MTX groups. ‘‘S’’ represents spinal cord, and ‘‘L’’ represents laminectomy defect. The sections were stained with H&E, and the magnification was ×40. (B, C) The number of fibroblasts was decreased as the concentration of MTX increased, which shows that the effect of MTX on epidural adhesion tissues was in a dose-dependent manner. The sections were stained with H&E, and the magnification was ×200. Fig. 4. Effect of MTX on epidural collagen tissue in rats. (A) The collagen density of epidural adhesion tissues in each group. The collagen tissues are blue in the section with Masson’s trichrome staining under the light microscope (×40). MTX reduced collagen synthesis and fibrosis. (B) HPC was expressed as μg/mg. The amount of hydroxyproline was decreased when the concentration of MTX increased. *P < 0.05 and **P < 0.01 compared with the HPC in control group. The density of collagen tissue and hydroxyproline analysis shows that the decreased effect was in a dosedependent manner.
Fig. 5. Immunohistochemical analysis of GRP78 and CHOP in epidural scar tissue applied with MTX (0.5, 1 or 2 mg/ml) or saline. As the dosage of MTX increased, the expression of GRP78 (A) and CHOP (B) increased.
Table.1. The degree of epidural adhesion according to Rydell’s classification. Grade Group 0 1 2 Saline 0 0 0 0.5mg/ml 0 1 2 1.0mg/ml** 2 3 1 2.0mg/ml** 4 2 0 Compares with the score of adhesion in control group. * < 0.05, ** < 0.01.
Figure Fig.1
3 6 3 0 0
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Fig.5