A apoptose é uma forma de morte celular intimamente envolvida com condições normais e patológicas e está relacionada com desenvolvimento das doenças neurodegenerativas, tal como a Esclerose Múltipla. Os Inibidores de Fosfodiesterase-5, tais como o Sildenafil e o Tadalafil, tem tido efeitos neuroprotetores em diversos modelos experimentais de neurodegeneração/neuroinflamação, porém ainda não se sabe se o Sildenafil é capaz de modular a apoptose no modelo de EAE. Portanto, este artigo explora os mecanismos de ação do Sildenafil nas vias extrínseca e intrínseca da apoptose na medula espinhal de camundongos C57BL/6 induzidos à EAE.
Sildenafil ameliorates EAE by decreasing apoptosis in the spinal cord of C57BL/6 mice
Eduardo Duarte-Silva1,3, Shyrlene Meiry da Rocha Araújo1,Wilma Helena Oliveira1, Deniele Bezerra de Lós4, Maria Eduarda Rocha de França2, Amanda Pires Bonfanti5, Gabriela Peron5,
Livia de Lima Thomaz5, Liana Verinaud5, Ana Karolina de Santana Nunes2, Christina Alves Peixoto1,6*
1
Laboratory of Ultraestructure, Aggeu Magalhães Institute (IAM), PE, Brazil. 2
Federal University of Pernambuco (UFPE), Recife, PE, Brazil 3
Postgraduate Program in Biosciences and Biotechnology for Health (PPGBBS), Oswaldo Cruz Foundation (FIOCRUZ-PE)/ Aggeu Magalhães Institute (IAM), Recife, PE, Brazil. 4
Laboratory of Neuromuscular Plasticity, Federal University of Pernambuco (UFPE), PE, Brazil.
5
Departament of Structural and Functional Biology, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil.
6
Institute of Science and Technology on Neuroimmunomodulation (INCT-NIM)
Corresponding author:
*Christina Alves Peixoto
ABSTRACT
Apoptosis is one form of cell death that is intimately related to health and pathological conditions. In most neuroinflammatory and/or neurodegenerative diseases, apoptosis is associated with disease development and pathology and inhibition of this process leads to considerable amelioration. It is becoming evident that apoptosis also participates in the pathogenesis of Multiple Sclerosis (MS) and its animal model, Experimental Autoimmune Encephalomyelitis (EAE). Drugs such as Sildenafil, a Phosphodiesterase type 5 Inhibitors (PDE5Is), has proven to be neuroprotective in MS models, however, it is not known whether Sildenafil is able to modulate cell death, specifically apoptosis, in EAE mice. Therefore, the aim of this study was to determine the effects of Sildenafil on extrinsic and intrinsic apoptosis pathway in the spinal cord of C56BL/6 with EAE. TUNEL analysis showed that EAE mice had elevated number of TUNEL+ cells and that treatment with Sildenafil led to reduced number of dying cells, indicating that Sildenafil was able to inhibit cell death. We observed that both extrinsic and intrinsic pathways of apoptosis were governing the dynamics of EAE progression. We showed that in mice with EAE there were increased levels of extrinsic (Caspase-8, -3, TNF-α, FADD) and intrinsic (Caspase-9, Bax and Cytochrome C) apoptosis markers. Bcl-2, an anti- apoptotic protein, was downregulated in EAE animals. We also demonstrated that EAE mice had increased levels of non-caspase mediators of cell survival/cell death (p-IκBα and p-MAPK p38). Besides, EAE mice presented augmented demyelination. Nevertheless, this is the first research to demonstrate that Sildenafil, when administered concomitant to disease induction, modulated the expression of pro- and anti-apoptotic proteins from the extrinsic and intrinsic pathways, as well as diminished the expression of non-caspase mediators and promoted remyelination in the spinal cord, indicating neuroprotective effects. Thus, the present study demonstrated that Sildenafil inhibits apoptosis by two distinct, although interconnected, mechanisms: directly by modulating caspase expression (through extrinsic and intrinsic pathways) and indirectly by modulating the expression of molecules involved with cell death and/or cell survival.
1. INTRODUCTION
Multiple Sclerosis (MS) is a complex chronic disease of the central nervous system (CNS) that has a autoimmune and inflammatory nature (Broux et al., 2013). In spite of all efforts and research done so far, there is not a single animal model that can capture the whole complexity of this pathology in human beings (Denic et al., 2011). Nevertheless, there are some widely used animal models of MS, such as Experimental Autoimmune Encephalomyelitis (EAE), which shares a lot of similarities with MS, like inflammation and axonal degeneration (Steinman and Zamvil, 2005). Besides, Copaxone, Mitoxantrone and Natalizumab, drugs approved for MS treatment, were first tested in this model. Thus, EAE is a relevant model for studying the effects of drugs in animals with MS-like symptoms (Steinman and Zamvil, 2006).
Apoptosis is one form of programmed cell death that plays a major role in normal and pathological conditions. For instance, during normal development, specifically in the brain development, there is elimination of unwanted extra cells, such as neurons, in order to have their number precisely regulated (Bibel and Barde, 2000; Singh, 2007). On the other hand, deregulated apoptosis is related to initiation and progression of infectious, autoimmune and neurodegenerative diseases, and also cancer (Singh, 2007). Apoptosis is triggered either by intrinsic or extrinsic stimuli. The extrinsic pathway is activated when a specific ligand, such as TNF or Fas ligand, binds to the death receptor in the cell surface, such as TNFR and FasR. This binding induces the activation of the receptors, which in turn rearrange in order to form a homotrimeric complex, which then participates in the recruitment of TRADD and FADD (Kiechle and Zhang, 2002). The intrinsic pathway is related to the mitochondria and happens when this organelle responds to stress signals releasing pro-apoptotic factors, such as AIF, SMAC/DIABLO, Endonuclease G and Cytochrome C (Kiechle and Zhang, 2002).
Many studies demonstrates that apoptosis is one of the pathological mechanism in MS and EAE (Das et al., 2008; Dasgupta et al., 2013, 2012; Meyer et al., 2001). For that reason, studies addressing the apoptotic pathway are essential, once new molecular targets can be discovered and new drugs can be used to treat neuroinflammatory diseases, such as MS.
Phosphodiesterase-5 (PDE-5) is an enzyme responsible for the regulation of intracellular levels of the second messenger 3,5-cyclic guanosine monophosphate (cGMP) (Corbin, 2004; Corbin and Francis, 1999), which is involved in a plethora of cellular processes, such as cell proliferation and differentiation (Hertz and Beavo, 2011),
neuroplasticity (W. Reierson et al., 2011) and memory (Domek-Łopacińska and Strosznajder, 2005). Interestingly, a study suggests that increased levels of cGMP is associated with anti-inflammatory effects (Rapôso et al., 2014). Sildenafil (Viagra ®) is PDE-5 selective inhibitor and it has classically been used as a treatment for erectile dysfunction (ED) (GOLDSTEIN et al., 1998) and pulmonary hypertension (Shekerdemian et al., 2002). Besides, some studies showed that Sildenafil’s use is correlated with angiogenesis and axonal remodeling (Ding et al., 2011, 2008), oligodendrogenesis (Zhang et al., 2012), improvement in memory, cognition and learning (Cuadrado- Tejedor et al., 2011; Shahidi et al., 2014), decreased microglial activation and reduction in pro-inflammatory cytokines secretion and oxidative stress (Nunes et al., 2012; Raposo et al., 2013). Some studies show that the PDE5 inhibitors (PDE5Is) can interfere with apoptosis, either by inducing or by preventing it from happening. One study about idiopathic pulmonary arterial hypertension (IPAH) showed that administration of Tadalafil lead to apoptosis of pulmonary arterial smooth muscle cells (PASMCs) with subsequent amelioration of IPAH (Yamamura et al., 2017). Mei and coworkers showed that Sildenafil induced apoptosis of human colorectal cancer cells (Mei et al., 2015). On the other hand, Bolnick et al. demonstrated that Sildenafil was able to prevent the apoptosis of the human first-trimester trophoblast cells when they were exposed to oxidative stress (Bolnick et al., 2015). Another study revealed that Sildenafil prevented apoptosis of cardiomyocytes in an animal model of cardiotoxicity (Fisher et al., 2005). Nevertheless, more research is yet to be performed in order to elucidate its therapeutic potential, mechanisms of action and safety regarding its use in central nervous system (CNS) diseases. Therefore, the aim of this study was to evaluate the effects of Sildenafil on apoptosis in C57BL/6 mice spinal cord.
2. MATERIAL AND METHODS
2.1 Animals
A total of 37 C57BL/6 female mice aged 8-12 weeks and weighing 25-30 g from biotery from Aggeu Magalhães Institute were used and distributed in the following experimental groups: a) Control (n=9) - they received only water; b) EAE (n=13) – they were EAE induced and received only vehicle (water); c) SILD (n=14) – they were EAE induced and received 25 mg/kg of Sildenafil subcutaneously (s.c). Mice were kept under a controlled temperature (22°C) and photoperiod environment (12h/12h light/dark) and received water and
standard chow ad libitum throughout the entire experiment. The experiment was approved by and performed in accordance with the guidelines of the Aggeu Magalhães Institute Ethics Committee/Oswaldo Cruz Foundation (87/2015 CEUA/FIOCRUZ).
2.2 Experimental Autoimmune Encephalomyelitis (EAE) induction
The EAE was induced as described by Verinaud et al. (2015), with a few modifications. Briefly, each animal as immunized with 200 ng of MOG-35-55 (Genscript, USA) emulsified in Freund`s Complete Adjuvant (CFA, Sigma, USA) supplemented with dried out tuberculosis (H37RA) through an injection via s.c. in the upper back (the region immediately after the head) in the tail base (100 µl of emulsion in each region). 240 ng of Pertussis toxin (Ptx, Sigma) was administered via i.p. at 0h and 48h after the immunization. Clinical signs of the disease were followed and graded daily according to a 0-5 score scale, in which: 0, no sign; 1, limp tail; 2, hind limbs weakness; 3, hind limb paralysis; 4, hind limb paralysis and fore limb weakness; 5, full paralysis/death. In the last day of experiment, mice were anesthetized via i.p with 80 mg/kg of ketamine and 10mg/kg of xylazine. The spinal cord was removed by ejection. After dissection, a 10mL syringe with PBS (1X) was inserted in the spinal canal lumen of the thoracic or sacral region and pressure was applied until it was removed completely. The spinal cord was washed in PBS (1X) and used for analysis of the inflammatory and apoptotic pathways.
2.3 Sildenafil treatment
Sildenafil treatment (Viagra®, Pfizer) was initiated in the first day after the immunization. The drug was diluted in a salt solution (NaCl 0.2%) in a concentration of 25 mg/kg and was administered subcutaneously (s.c) twice a day during 21 days.
2.4 Immunohistochemistry
The immunohistochemical analysis was performed as described by Oliveira et al. (2016). Briefly, after anesthesia, the animals were transcardially perfused with 20 mL of physiological saline followed by 40 mL of 4% paraformaldehyde (PFA, Sigma) in 0.1 M phosphate sodium phosphate monobasic and dibasic heptahydrate – Sigma-Aldrich) buffered
saline (PBS), pH 7.2. After euthanasia, mice spinal cord was removed and overnight (O/N) post-fixed in the same fixative. The samples were processed for paraffin, as routine. The endogenous peroxidase activity was blocked with hydrogen peroxide (3%) diluted in methanol for 20 min and unspecific sites with blocking solution of bovine serum albumin (1%) (Miles, Naperville, IL, USA), TWEEN 20 e PBS (1X) for 1h room temperature (RT). Next, the samples were incubated O/N at 4 ºC with the following primary antibodies: Caspase-8 (Santa Cruz, sc5263, 1:100), Caspase-3 (ABCAM, ab4051, 1:100) and TNF-α (Prepotech, #500-P64, 1:50). After being washed with PBS (1X), the slides were incubated for 1h AT with a biotin-conjugated secondary antibody (DakoCytomation, USA, Biotinylated Link Universal HRP; #K0690) and visualized with 3'-3- diaminobenzidine (DAB) as chromogen. All slides were mounted in Entellan (Merck, #1079610100).
2.5 Immunofluorescence of paraffin-embedded tissue
Mice spinal cord was processed as described in section 2.4. The slides were heated to 58ºC for 20 min and then immersed in two baths of xylene, alcohol 100%, 95% and 70% followed by distilled water. Then, the slides were submitted to antigen retrieval in Citrate buffer (pH 6.0, 60ºC in the steamer) for 35 min. After cooling for 20 min AT, the slides were washed three times with PBS (1X), immersed in Tritox- X-100 0,5% for 15min and again washed in the same solution. Unspecific sites were blocked by blocking solution (BSA 1%+TWEEN 20+PBS 1X) for one hour. Next, samples were incubated O/N at 4ºC with the following primary antibodies: anti-caspase-9 (Santa Cruz, #sc56076, 1:100) and anti-MBP (Abcam, #ab7349, 1:100). In the next day, the slides were washed five times with PBS (1X) and incubate with the following fluorophore conjugated-secondary antibody for one hour AT: Alexa Fluor 488 goat anti- rabbit (Life Technologies, #A11008, 1:500) and Alexa Fluor 488 goat anti-mouse (Invitrogen, #A11001, 1:500). The slides were then washed again in PBS and DAPI (Vector laboratories, Burlingame, CA, USA, 1:500) was applied for 5 min. Finally, samples were washed in distilled water, mounted in gelatin medium and analyzed using a fluorescence microscope (Leica DM2500) coupled with digital camera (Leica DFC345 FX).
Four-to-ten pictures of the spinal cord were selected and their pixels were quantified by GIMP2.6.11 software (GNU Image Manipulation Program software, CNET Networks, Inc. Australia).
2.7 Western Blotting
The Western Blotting analysis was performed as described by Oliveira et al. (2016). Briefly, the animals were anesthetized and euthanized by cervical dislocation. The spinal cord was removed and stored in liquid nitrogen until use. Spinal cords were homogenized in extraction cocktail in order to obtain a pool (n=5). Proteins (30 mg) were separated by 12% and/or 14% polyacrilamide gel electrophoresis and transferred to nitrocellulose membranes (BioRad, CA, USA). Unspecific sites were blocked with 5% skim milk for 1h RT and the membranes were incubated O/N at 4ºC with primary antibody against BAX (Abcam, #ab7977, 1:1000), BCL-2 (Abcam, #ab7973, 1:1000), Cytochrome C (Santa Cruz, #sc13156, 1:1000), FADD (Santa Cruz, #sc6036, 1:1000), p-P38-MAPK (Cell Signaling, #9211S, 1:1000) and p-IκBα (Cell Signaling, #9246L, 1:1000). Next, the membranes were washed and incubated with anti-rabbit HRP-conjugated secondary antibody (ABCAM, #ab6721, 1:3000) for 1.5 h. The bands were detected by a chemiluminescent reagent (Super Signal, Pierce, catalog number 34080) and visualized in C-DiGit Blot Scanner (LI-COR Biosciences, USA) by the Image Studio Digits (LI-COR Biosciences, USA, v. 5.0) software. Pixels were quantified by using Image J 1.38 (NIH, Bethesda, MD, USA) software. The analyses were done in duplicate and β-actin (Sigma-Aldrich, catalog number A2228, 1:3000) was used as control.
2.8 TUNEL assay
TUNEL assay was performed using TACS® 2 TdT-Fluor In Situ Apoptosis Detection Kit (Trevigen, cat#:4812-30-K) according to the manufacturer’s instructions. Slides were warmed to 57ºC for 5 minutes and then immersed in 2 changes of xylene and 100%, 95%, and 70% ethanol for 5 minutes each. Slides were washed in 1X PBS for 10 minutes. In a humidity chamber, the slides were covered with Cytonin and incubated for 20 minutes AT. Next, they were washed twice with ddH2O for 5 minutes. Slices were immersed in 1X TdT Labeling Buffer for 5 minutes and covered with 50 µL of Labeling Reaction Mix and incubated at 37°C
for 1h in the humidity chamber. Next, slices were immersed in 1X TdT Stop Buffer RT to interrupt the labeling reaction. Washing with 1X PBS twice took place in order to remove the unbound conjugates. In the humidity chamber, slices were covered with 50 µL of Strep-Flour and incubated RT for 20 minutes. The slices were washed twice with 1X PBS for 2 minutes and mounted in a fluorescent appropriate medium for visualization. For control, samples were not treated with TdT enzyme and treated with TACS-Nuclease. The analysis was done in duplicate. The sections were analyzed in a fluorescence microscope (Leica DM2500) coupled with digital camera (Leica DFC345 FX).
2.9 Statistical analysis
The mice clinical score was analyzed by the Kruskal-wallis test, followed by Dunn’s post-test. The statistical differences for Immunohistochemistry and Immunofluorescence were analyzed by one-way ANOVA followed by Turkey’s post- test. Mice weight was analyzed by two-way ANOVA followed by Tukey’s post-test. Student’s t test was used for two group comparisons (Western Blot). The results, except the clinical score, are presented as mean ± standard deviation. The chi-squared test was used to compare the number of apoptotic cells between groups in TUNEL assay. All analysis was done by using Graphpad Prism (version 6.0, GraphPad Software Inc., USA) software.
3. RESULTS
3.1 Sildenafil prevents weight loss due to EAE
In order to investigate the effect of Sildenafil administered concomitant with EAE induction we analyzed mice daily by observing whether they presented weight loss or not, a pattern commonly seen in EAE mice. The control group displayed a stable body weight throughout the entire experiment (Figure 1). The differences in body weight were observed in the EAE and the SILD group. The EAE group displayed reduced body weight starting from the 13th day of experiment. On the other hand, the EAE mice treated with Sildenafil were not affected by weight loss, but remained similar to the control group until the end of the experiment, showing that Sildenafil was able to prevent weight loss in diseased animals (Figure 1).
3.2 Sildenafil prevents EAE progression
In order to determine whether Sildenafil could interfere with EAE progression or not, we decided to observe mice daily starting from the onset (day 13-14th). The control group did not present any signs of neurological disability (Figure 2), which was seen only in the EAE and SILD group starting from the 14th day of experiment. EAE mice displayed a progressive increased score throughout the experiment, whereas EAE mice treated with Sildenafil had delay in this progression, showing that Sildenafil was able to prevent EAE development in the concomitant treatment regimen (Figure 2).
3.3 Sildenafil decreases the number of TUNEL+ cells in the EAE mice spinal cord
This experiment was conducted in order to demonstrate the effects of Sildenafil on apoptosis in the mice spinal cord. For that reason, we used the TUNEL assay to detect whether cells were dying or not after EAE induction. However, we were not able to determine precisely what types of cells were dying or what pathways were governing apoptosis. Preliminary data show that control group presented a few TUNEL+ cells (Figure 3A). EAE mice had increased number of cells undergoing apoptosis, as seen by the fluorescence intensity (Figure 3B). On the other hand, EAE mice treated with Sildenafil had fewer positive cells then the EAE group (Figure 3C) (Chi-sq=60.93; p<0.0001), showing that Sildenafil was able to interfere with the apoptotic pathway, either direct or indirectly, and to diminish the number of TUNEL+ cells in the spinal cord.
3.4 Sildenafil restores demyelination by increasing the expression of MBP in the spinal cord of mice with EAE
The next experiment was based on the fact that EAE is a severe demyelinating disease of the CNS and other reports by our laboratory suggested that Sildenafil could have a remyelinating or protective effect on the myelin sheath in the Cuprizone-Induced Demyelination model of MS (de Santana Nunes et al., 2016; Nunes et al., 2012). Furthermore, apoptosis observed in TUNEL assay could be related to the death of oligodendrocytes and thus to demyelination. Therefore, we aimed at investigating if Sildenafil could promote remyelination and/or prevent demyelination in the EAE mouse model of MS. For that reason,
we performed immunofluorescence against spinal cord MBP and observed that control group showed intense labelling of MBP (Figure 4A). Mice with EAE showed clear signs of demyelination, seen by decreased expression of MBP in the white matter (Figure 4B). However, treatment with Sildenafil concomitant with disease induction was able to augment MBP labelling and prevent and or/restore demyelination seen in sick mice (Figure 4C), showing that Sildenafil was able to modulate the expression of this myelin-related protein probably by affecting oligodendrocytes, myelin-forming cells.
3.5 Sildenafil inhibits the extrinsic pathway of apoptosis by diminishing the expression of Caspase-8, -3, TNF-α and FADD
In order to determine whether the extrinsic pathway of apoptosis was active during EAE progression in mice, we used antibodies against extrinsic pathway components, such as Caspase-8, -3, TNF-α and FADD. The EAE group presented a significant labelling for Caspase-8, -3 in both Gray and White matter, and increased levels of TNF-α and FADD expression, when compared with the control group (Figure 5A, 6A, 7A and 9B). On the other hand, EAE mice treated with Sildenafil had reduced expression of caspases in both Gray and White matter when compared to the EAE group, as well as, less TNF-α and FADD labeling (5C, 6C, 7C and 9B), showing that Sildenafil was able to modulate, either directly or indirectly, the expression of the molecules involved in the extrinsic pathway of apoptosis.
3.6 Sildenafil modulates the intrinsic pathway of apoptosis by diminishing the expression of pro-apoptotic proteins Bax, Cytochrome C and Caspase-9 and increasing the expression of anti-apoptotic protein Bcl-2
Here, our goal was to determine whether the intrinsic pathway of apoptosis also played a role in disease progression in mice and if it occurred at the same time as the extrinsic pathway. To accomplish that, we used antibodies against classical proteins of the intrinsic pathway, such as Bax, Bcl-2, Cytochrome C and Caspase-9. EAE mice had increased expression of Bax, Cytochrome C and Caspase-9 (Figure 9C, 9E and 8B) and decreased the expression of Bcl-2 (Figure 9D). However, mice with EAE treated with Sildenafil presented decreased expression of pro-apoptotic proteins (Figure 8C and 9C and 9E) and increased expression of anti-apoptotic protein Bcl-2 (Figure 9D), showing that Sildenafil could
modulate, either directly or indirectly, the expression of proteins involved in the intrinsic pathway of apoptosis.
3.6 Sildenafil modulates cell death by decreasing the expression of pMAPK-p38 and p-IκBα
In this final experiment, our goal was to determine the whether the expression of other molecules involved in cell survival and/or death, such as p-MAPK-p38 and p- IKBα was up or downregulated after treatment with Sildenafil. EAE group had increased expression of