Cilostazol disrupts TLR-4, Akt/GSK-3β/CREB, and IL-6/JAK-2/STAT-3/SOCS-3 crosstalk in a rat model of Huntington's disease

Countless neurodegenerative diseases are associated with perverse multiple targets of cyclic nucleotide signalling, hastening neuronal death. Cilostazol, a phosphodiesterase-III inhibitor, exerts neuroprotective effects against sundry models of neurotoxicity, however, its role against Huntington’s disease (HD) has not yet been tackled. Hence, its modulatory effect on several signalling pathways using the 3-nitropropionic acid (3-NP) model was conducted. Animals were injected with 3-NP (10 mg/kg/day, i.p) for two successive weeks with or without the administration of cilostazol (100 mg/kg/day, p.o.). Contrary to the 3-NP effects, cilostazol largely preserved striatal dopaminergic neurons, improved motor coordination, and enhanced the immunohistochemical reaction of tyrosine hydroxylase enzyme. The anti-inflammatory effect of cilostazol was documented by the pronounced reduction of the toll like receptor-4 (TLR-4) protein expression and the inflammatory cytokine IL-6, but with a marked elevation in IL-10 striatal contents. As a consequence, cilostazol reduced IL-6 downstream signal, where it promoted the level of suppressor of cytokine signalling 3 (SOCS3), while abated the phosphorylation of Janus Kinase 2 (JAK-2) and Signal transducers and activators of transcription 3 (STAT-3). Phosphorylation of the protein kinase B/glycogen synthase kinase-3β/cAMP response element binding protein (Akt/GSK-3β/CREB) cue is another signalling pathway that was modulated by cilostazol to further signify its anti-inflammatory and antiapoptotic capacities. The latter was associated with a reduction in the caspase-3 expression assessed by immunohistochemical assay. In conclusion the present study provided a new insight into the possible mechanisms by which cilostazol possesses neuroprotective properties. These intersecting mechanisms involve the interference between TLR-4, IL-6-IL-10/JAK-2/STAT-3/SOCS-3, and Akt/GSK-3β/CREB signalling pathways.


Introduction
One of the progressive neurodegenerative disorders is the Huntington's disease (HD), which is an autosomal inherited disease that targets mainly the striatum. [1]. Psychiatric disturbances, cognitive debility, as well as waning of motor function are the triad that signifies the clinical a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 pathological changes in HD [2]. The 3-nitropropionic acid  is an irreversible inhibitor of succinate dehydrogenase (SDH), a mitochondrial complex II enzyme, responsible for the oxidation of succinate to fumarate in the Kreb's cycle. In turn, it hinders the electron transport in the oxidative phosphorylation flow and impairs mitochondrial function, hence, resulting in a decrease in ATP levels and oxidative burst, events that integrate to cause neuronal injury [3,4]. 3-NP closely simulates many of the neuropathological and behavioral features of HD; hence, 3-NP treated rodents provided a relevant model to examine possible treatments for HD [5]. More interestingly, repeated administration of 3-NP at low doses provokes motor alteration from hyperactivity to hypoactivity depicting the progression from chorea to a parkinsonian-like syndrome in human [6].
The level of cyclic adenosine monophosphate (cAMP) decreases in various neuropathological conditions [7][8][9] and aggravation of neuroinflammatory processes in neurodegenerative diseases was robustly correlated with aberrant cAMP signalling, possibly originating from abnormal PDEs expression [10]. The study of cyclic nucleotide signalling in the last decades has revealed a stunning complexity and may exploit several cellular pathways [11].
Cilostazol, a type III phosphodiesterase inhibitor, increases intracellular cAMP levels; the drug was approved by the Food and Drug Administration for the treatment of intermittent claudication, besides, its principal actions including inhibition of platelet aggregation, antithrombotic action in cerebral ischemia, and vasodilation mediated by the increased cAMP levels [12]. Cilostazol has been shown in a multicenter, randomized, and double-blind clinical trial to provide a considerable risk reduction in patients with recurrent cerebral infarction [13]. Several in vivo studies revealed that cilostazol may be a powerful candidate to protect against brain lesions and cognitive impairment associated with chronic cerebral hypoperfusion and focal cerebral ischemia [14][15][16][17][18][19].
Based on the previous background, the present study was conducted to investigate the potential of cilostazol on the striatal neuropathological as well as behavioural derangements induced by 3-NP in rats. It also extended to explore some of the possibly integrating signalling cues that could offer neuroprotection against 3-NP model.

Ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH publication No.85-23, 1996). The protocol was reviewed and approved by the Ethics Research Committee of Faculty of Pharmacy, Cairo University (PT: 2052). All efforts were exerted to minimize animal suffering during the experimental period. Where, the duration of the experiment was as short as possible, the number of animals was kept to a minimum and all animals were sacrificed by decapitation under light anaesthesia.

Animals
Adult male Wistar rats (150 ± 20 g) were obtained from National Research Centre (NRC, Giza, Egypt). Animals were allowed to acclimatize in the animal facility of Faculty of Pharmacy (Cairo University) for one week prior to starting any experimental procedure. Rats were allowed free access to standard chow pellet and water ad libitum and were kept under controlled environmental conditions (constant temperature of 23±2˚C, humidity of 60±10%, and light/dark (12/12 h) cycle with lights on at 6:00 am). All behavioural tests were carried out in a sound isolated laboratory.

Experimental design
Rats were divided randomly into 4 groups (n = 12); group 1 represents the normal control group and animals received saline, while group 2 depicts cilostazol (Otsuka pharmaceutical Co. S.A.E, Cairo, Egypt) normal treated group and rats were gavaged cilostazol orally (100 mg/ kg/day [20]). In groups 3 and 4, rats were injected daily with 3-NP (Sigma-Aldrich, MO, US; 10 mg/kg, i.p; [21]) without and with cilostazol for 14 days to serve as the HD model and the cilostazol treated groups, respectively. On the last day of the experiment, rats were evaluated for behavioural tests, then sacrificed and the striata were collected for biochemical, western blot, and immunohistochemical analysis.

Behavioural tests
Twenty-four hours after the last dose of treatments, rats were screened for motor performance using manual gait analysis, pole, and cylinder tests.
Manual gait analysis. This experiment was adopted from previous 6-hydroxydopaminelesioned rat model [22,23] to assess the 3-NP-induced sensorimotor dysfunction. The experiment assessed both stride length (distance cut in centimetres by one foot through the gait cycle using 3 hind paw strides when the animal moves at constant pace; [23]) and stride width (mean of side-to-side distance between the two hind paws in three consecutive strides; [24]). At the beginning of experiment and before injecting 3-NP, rats were trained to cross directly into their home via a horizontal path; afterwards the hind limbs were immersed in a non-toxic paint (Crayola LLC, NY, USA), and the animals were allowed to travel the corridor to their home cage on a piece of paper.
Pole test. Muscle rigidity and postural instability were assessed by this test, by placing each rat, head-up, on top of a wooden pole (69 cm long, 8-10 cm in diameter) that was placed in the home cage. The time required for each animal to descend down the pole to the ground was determined and the mean time of 5 trials for each rat was calculated [25].
Cylinder test. Rats were placed in a clear plexiglass cylinder (height/diameter = 30/20 cm) and video recorded for 5 min. When the animal raises forelimbs above shoulder level and touches the cylinder wall with either one or both forelimbs, this is considered as a rear. Additionally, rat must remove both forelimbs from the cylinder wall and touch the cylinder base before another rear was scored. Hence, total rearing time, in addition to rearing frequency in 5 minutes were determined [26,27].

Striatal processing
After behavioural testing, all animals were sacrificed by decapitation under thiopental sodium (5 mg/kg) anaesthesia [28]. Brains were rapidly dissected out and the striata were isolated and stored at -80˚C. The right striata of 8 animals were homogenized in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris HCl, pH 8, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% SDS) provided with protease inhibitor cocktail and used for the estimation of phosphorylated cAMP response element binding protein (p-S133 CREB), p-S473 Akt, and toll like receptor (TLR)-4. The left striata of the previous 8 rats were homogenized in ice cold phosphate buffer saline (PBS; pH = 7.4) and used for the ELISA determination of interleukin (IL)-6, IL-10, nuclear factor kappa B (NF-κB) p65, phosphorylated glycogen synthase kinase-3 β (p-S9 GSK-3β), Janus Kinase 2 (p-Y1007/1008 JAK-2), signal transducers and activators of transcription (p-Y705 STAT-3) and suppressor of cytokine signalling 3 (SOCS3) in addition to, caspase-3 activity. These parameters were normalized to protein content, measured according to Bradford [29]. The striata of the remaining 4 rats were used to determine the immunohistochemical protein expression of tyrosine hydroxylase (TH) and caspase-3, besides the histopathological assessment.
Immunohistochemical assay of tyrosine hydroxylase (TH) and caspase-3. For immunohistochemical examination, striatal tissues were fixed in 10% neutral buffered formalin overnight and then embedded in paraffin, to be sectioned at 4-μm thickness. The paraffin embedded sections were deparaffinized in xylene, and hydrated by ethyl alcohol in decreasing concentrations (100%, 95% and 70%). Antigen retrieval was achieved by heating slides (in a 200 ml Coplin jar filled with 10 mM citrate buffer) in a commercial microwave oven operating at a frequency of 2.45 GHz and 600 W power setting. After two heating cycles of 5 minutes each, slides were allowed to cool at room temperature and thoroughly washed in PBS (pH 7.4). Striatal sections were stained by applying the labelled streptavidin-biotin-peroxidase method according to the manufacturer staining protocol (Vectastain Elit ABC peroxidase kit, Vector Laboratories, Burlingame, CA, USA). Brifly, endogenous peroxidase activity was quenched by incubating the specimen for 5 min with 3% hydrogen peroxide. Sections were incubated with corresponding primary antibody for TH (Biorbyt Co., Cambridge, UK; cat#:orb43362) and caspase-3 (Abcam Co., Cambridge, UK;cat#:ab184787) at 4˚C overnight. After conjugation with streptavidin-biotin-peroxidase complex, colouring was performed with 3, 30-diaminobenzidine substrate-chromogen and hematoxylin was used for counter staining the reacted caspase-3 (1:100) and TH (1:100) antibodies. Microscopical examination was performed and five serial fields were captured at 400× magnification and the intensity of reaction into striatal area for each antibody among different groups was determined using Leica Application Suite imaging software (Leica Microsystems, Germany).
Determination of caspase-3 activity. Caspase-3 activity was estimated using a colorimetric assay kit (R&D Systems, Inc., USA; cat#: BF3100). The results were expressed as fold increase in optical density relative to the normal group.

Histopathological analysis
The paraffin embedded slides were stained using H&E stain for histopathological evaluation. Neuronal cells stained with H&E were viewed and the striatal neurons were outlined, then the pathological changes in striatum were examined at high power (×400 magnification) in each group. In H & E staining, each section was assigned a damage score between 0 and 3 for each of five parameters, namely, necrosis of neurons, neurophagia, cellular edema, congestion of blood vessels and focal gliosis. The scores for the five parameters measured for each rat were summed to obtain the "total histology score", being maximally 15 (three being the maximum for the five parameters examined). An experienced pathologist who was blinded to the experiment groups performed all histopathological examinations.

Statistical analysis
Data are expressed as means ± SEM. GraphPad Prism 1 software package, version 6 (Graph-Pad Software Inc., CA, USA) was used to carry out all statistical tests. For parametric analysis, multiple comparisons were performed using one way analysis of variance (ANOVA) test followed by Tukey's Multiple Comparison test. For non-parametric data, one-way analysis of variance test (Kruskel-Wallis Test) followed by Dunn's multiple comparisons test were used. P<0.05 was set as the significance limit for all comparison.

Results
Groups treated with cilostazol alone without 3-NP injections showed no significant difference as compared to the normal control group, thus, all comparisons were carried against the normal control group. Additionally, using the cylindrical test 3-NP-lesioned animals exhibited a progressive decrease in the (C) rearing time and (D) rearing frequency (rearing/5 min) in comparison to normal control group. Treatment with cilostazol, on the other hand, increased both the number and time of rearing significantly compared to 3-NP group. Finally, results of the pole test revealed that insulted rats showed a significant decrease in (E) T−total compared to normal group, whereas cilostazol reverted the 3-NP effect.

Cilostazol alleviated 3-NP-induced alternations in tyrosine hydroxylase (TH) and caspase-3
The deficit motor performance observed after 3-NP treatment was associated with a reduction in the optical density of striatal TH antibody immuno-reactivity as compared to normal group (Fig 2). Simultaneously, 3-NP-insulted rats exhibited an upsurge in caspase-3 expression as shown by immunohistochemical assay as well as caspase-3 activity to reach 4 fold the normal group (Fig 3).
Nevertheless, co-administration of cilostazol protected striatal neuronal cells from degenerative apoptotic death as it suppressed both the expression and the activity of pro-apoptotic protein caspase-3 and consequently preserved dopaminergic neurons as shown by increased immunostaining density of striatal TH as compared to 3-NP control group.

Cilostazol enhanced the striatal p-(S473) Akt, p-(S9) GSK-3β, and p-(S133) CREB
As depicted in Fig 5, 3-NP abated the striatal (A) p-GSK-3β, as well as the protein expression of (B) p-Akt and (C) p-CREB, as compared to normal control group. On the other hand, cilostazol increased the phosphorylation of the three signalling molecules, as compared to 3-NPlesioned rats.

Cilostazol-preserved neurons in striata against 3-NP-induced degeneration
Histopathological photomicrographs (Fig 7) show that (C) 3-NP section caused a significant neuronal damage compared to sections of the normal (A) saline and (B) cilostazol groups.  3-NP resulted in (C) severe neuronal necrosis and neuronophagia, (D) focal gliosis, and (E) cellular edema with congested blood capillaries. The total histology score was markedly increased in rats with 3-NP insults (G). On the other hand, co-administration of cilostazol

Fig 7. Effect of cilostazol (100 mg/kg, p.o) on striatal histopathological photomicrographs in 3-NP-treated rats.
Sections A and B show normal neuronal structure of rats receiving saline and cilostazol, respectively. Section of (C) 3-NP treated group show a significant neuronal damage with severe neuronal necrosis and neuronophagia, (D) focal gliosis, and (E) cellular edema with congested blood capillaries. However, section of (F) cilostazol treated group show an improvement in histopathological changes induced by 3-NP, except for mild cellular edema. (G) Total histology score, data are expressed as box plots of the median of 6 animals. Statistical analysis was done using Kruskal-Wallis test followed by Dunn's test. a Significantly different from normal group at P<0.05, b Significantly different from 3-NPtreated group at P<0.05.
https://doi.org/10.1371/journal.pone.0203837.g007 resulted in a prominent decrease in total histology score (G) as well as improvement in histopathological changes induced by 3-NP, except for mild cellular edema (F).

Discussion
The present study highlighted the potential neuroprotective effect of cilostazol, the phosphodiesterase III inhibitor, in a 3-NP-induced HD model. Cilostazol strongly ameliorated 3-NPinduced neuro-inflammation and striatal degeneration. It amended the 3-NP-induced lesions in the striatal dopaminergic neurons as evidenced by the increased protein expression of TH along with the marked improvement in histopathological changes. These restorations were reflected on the functional tests, where cilostazol enhanced neurobehavioral performance (locomotor activity, motor coordination, muscle rigidity, and postural stability) as well. The present study shed light on some of the multiple downstream signalling targets of cyclic nucleotide that may account for the cilostazol neuroprotective effects.
Cilostazol signified its anti-inflammatory effect by suppressing the inflammatory event resulting from the 3-NP-induced elevation in NF-κB p65, IL-6, and TLR-4 protein expression. Indeed, the inflammatory cascades provoked as a response to many types of brain insults are pivotal to control and counteract detrimental effects on neurons. However, severe or chronic inflammation, can itself damage neurons due to the excessive activation of astrocytes and microglia via the utilization of TLRs [30,31]. These receptors recognize damage-associated molecular patterns and mediate host-inflammatory responses to injury through the activation of transcription factors, such as NF-κB p65, as shown herein, with the subsequent production of pro-inflammatory cytokines, as IL-6, TNF-α and IL-1β [32,33].
The upregulation of TLR-4 expression has been reported in Parkinson's disease and multiple systems atrophy post-mortem brain tissue, suggesting clinical relevance of TLR-4 in the progression of many widespread neurodegenerative diseases [34,35]. Moreover, chronic or excessive activation of glia by IL-6 is a common denominator in neuronal loss in several neurodegenerative diseases [36]. In the present study, injection of 3-NP markedly increased the striatal IL-6, which in turn increases TLR-4, these results concur with that of Tamandl et al. [37] who showed that IL-6 upregulated TLR-4 resulting in hyper-responsiveness to lipopolysaccharides (LPS). The anti-inflammatory effect of cilostazol can be attributed partly to the inhibition of the key inflammatory transcription factor NF-κB p65 that entails its downstream cytokines as reported herein and previously [38]. In support to the current findings, Park et al. [39] reported that cilostazol was able to suppress the expression of TLR-4 along with cytokine production in macrophages from patients with rheumatoid arthritis.
Apart from abating the inflammatory mediators, cilostazol spared the anti-inflammatory cytokine IL-10 possibly by the phosphorylation/inactivation of GSK-3β. In fact, GSK3β, a crucial modulator of innate inflammatory processes, potently suppresses the production of the cytokine IL-10, while concurrently augments the production of pro-inflammatory cytokines in response to TLR-4 signalling pathway [40,41]. Additionally, cilostazol has activated/ phosphorylated protein kinase B (PKB or Akt) and the downstream molecule CREB besides GSK-3 β to pin down its anti-inflammatory character and to concur with previous studies in different models [42,43]. Ample evidence have emphasized the neuroprotective effect of p-Akt/p-GSK-3β in different models of neurotoxicity in rats [44][45][46]. Therefore, the ability of 3-NP to reduce the phosphorylated forms of the Akt/GSK-3β/CREB cue certified the occurrence of inflammatory milieu to highlight the role of neuro-inflammation in the progression of 3-NP-induced striatal degeneration, as confirmed herein and reported earlier [6,47]. Cilostazol-mediated activation of Akt is responsible for the current inactivation/ phosphorylation of GSK-3β at the serine site to match previous work [48] and to endorse the activation of many downstream transcription factors, such as CREB [49]. This can provide a molecular understanding of how GSK-3β inactivation promotes an anti-inflammatory immune response after TLR activation. Inactive/phosphorylated GSK-3β enhances CREB association with CREB binding protein (CBP), while reducing the interaction between NF-κB p65 with CBP required for its optimal transcriptional activity [40]. A previous in vitro study [50] harmonizes with the present findings, where cilostazol significantly increased the expression of p-CREB and decreased nuclear NF-κB p65 (level and DNA binding activity). Moreover, p-CREB plays an essential role in the production of IL-10 via binding to its promotor region to induce its transcription [51] to be one explanation for the boosted IL-10 content.
Besides its anti-inflammatory role, p-CREB plays a role in the improved motor activity noticed herein via increasing the gene transcription of TH as stated previously by Park et al. [50] and as reported in our findings. In the present work, cilostazol-mediated activation of CREB was associated also by an increase in the protein expression of TH to increase the production of dopamine and to support the intact functional dopaminergic neurons [52].
Apart from its role in inflammation, the Akt/GSK-3β/CREB axis plays also a part in cell survival via regulating apoptosis, a cellular death type that has been involved in various neurodegenerative disorders including HD [53,54]. Activation of one or more TLRs in neurons and glial cells increases the vulnerability of neurons to apoptosis [55]. In parallel, downregulation of the defence signal Akt/GSK-3β promotes the neuronal death partially via suppressing CREB activity that correlates with the expression of many survival and proliferation genes [56]. The current immuno-histological examination along with the enzyme activity test revealed that cilostazol markedly curbed caspase-3 activity that was enhanced in 3-NP treated rats, to emphasize its neuroprotective potential. This effect is possibly linked to its modulatory effect on TLR-4 expression and p-Akt/p-GSK-3β/p-CREB signalling pathway. In line with these findings, earlier studies divulged that cilostazol possesses an anti-apoptotic effect via enhancing the phosphorylation/activation of CREB and increasing the anti-apoptotic Bcl-2 in cerebral ischemia model [17,43] and in the white matter region after cerebral hypoperfusion [18].
In our study, the second molecular signalling pathway that was modulated by cilostazol is the p-JAK-2/p-STAT-3/SOCS3 pathway to nail down its anti-inflammatory effect. Activation of p-JAK-2/p-STAT-3 pathway plays a dire role in neuro-inflammatory diseases [57,58], where upon binding to its receptor, IL-6 triggers the phosphorylation of JAK-2 with the subsequent phosphorylation of its downstream molecule STAT-3. Dimerized p-STAT-3 molecules translocate into the nucleus to augment the transcription factor NF-κB [59], a fact that adds to the increased content of this transcription factor. Additionally, nuclear STAT-3 by binding to DNA increases cytokine gene expression, suggesting that by activating STAT-3, IL-6 promotes its own production in a feed-forward loop leading to sustained inflammation [60]; these events support our results. Such inflammatory cascade induces SOCS3 to act as a negative feedback regulator to inhibit inflammation running out of control [61]. Beurel and Jope. [62] previously reported that during neuro-inflammation the glial production of IL-6 prerequisites a strong integration between p-STAT-3 and active GSK-3β, where, GSK-3β was found to be crucial for the activation of STAT3 in primary astrocytes by promoting its tyrosine phosphorylation (Y 705) [63]. Hence, the interference with either the activity of STAT-3 or GSK-3β was associated with a marked suppression in the release of IL-6 to confirm the key role of both molecules in promoting IL-6 production by glial cells [62].
In agreement with the previous in vitro study of Park et al. [50], cilostazol herein impeded the 3-NP-associated elevation in IL-6/p-JAK-2/p-STAT-3 to accentuate the neuroprotective role mediated by suppressing this cue. These data synchronize with that of Qin et al. [64], who also underlined the beneficial effects of inhibiting JAK/STAT pathway in α-synuclein-induced neuroinflammation and dopaminergic neurodegeneration. In what appears to be a vicious cycle, STAT-3 also induces the expression of TLR-4 with proposed binding sites of STAT-3 in the TLR4 promoter region [65,66]. This could partially explain the marked increase in both STAT-3 content and TLR-4 expression after 3-NP administration and their co-inhibition in cilostazol treated rats.
The ability of cilostazol to dampen p-STAT-3 could be attributed to the inactivation of GSK-3β, since GSK-3β profoundly activates STAT-3 [63]. As a compensatory response against the inflammatory events, SOCS-3 was elevated in the 3-NP rats; however, this rise was not enough to oppose the inflammatory upsurge in the 3-NP group. Nevertheless, concomitant administration of cilostazol increased further the production of SOCS-3 to play its role in quelling the inflammatory flare and to support the findings of Gaudy et al. [67], who proved that increased cAMP induces SOCS3 to negatively regulate JAK/STAT signalling pathway. Intriguingly, SOCS3 has a regulatory role in the function of STAT3 induced by IL-6, but not that of IL-10 due to a different affinity of SOCS3 to the two receptors [68]. In a mutual intervention, the cilostazol-induced SOCS-3 expression could indorse the production of IL-10 [69], which in turn, can induce further the release of SOCS3 [70,71]. In addition, the ability of cilostazol to trigger PI3K/Akt/p-GSK-3β trajectory may participate in increasing IL-10 production [72].Thus, the anti-inflammatory effect of IL-10 mediated through STAT-3 is preserved along with that of PI3K/Akt/GSK-3β pathway, which implicated in both the production and responses to IL-10 [72].
In conclusion, the present study highlighted the neuroprotective potential of cilostazol against 3-NP-induced HD model via modulating the crosstalk between TLR-4, Akt/GSK-3β/ CREB and JAK-2/STAT3/SOCS-3 signalling pathways to provide a promising therapeutic tool slowing the progression of HD.