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

Abstract

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.

Fig 1

Effect of cilostazol (100 mg/kg, p.o) on (A) stride width, (B) stride length, (C) rearing time, (D) rearing frequency, and (E) T−total in 3-NP-treated rats. Non-parametric data are presented as median (max-min) using Kruskel–Wallis test followed by Dunn’s as a post-hoc test. Parametric data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's Multiple Comparison. ^a Significantly different from normal group at P<0.05, ^b Significantly different from 3-NP- treated group at P<0.05.

Fig 2. Effect of cilostazol (100 mg/kg, p.o) on protein expression of TH in 3-NP-treated rats.

Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's Multiple Comparison. ^a Significantly different from normal group at P<0.05, ^b Significantly different from 3-NP- treated group at P<0.05, ^ab Significantly different from both normal and 3-NP- treated groups at P<0.05.

Fig 3

Effect of cilostazol (100 mg/kg, p.o) on (A) protein expression and (B) activity of caspase-3 in 3-NP-treated rats. Caspase-3 protein expression is presented as mean ± SEM and caspase-3 activity is presented as fold increase relative to normal group. Statistical analysis was performed using one-way ANOVA followed by Tukey's Multiple Comparison. ^a Significantly different from normal group at P<0.05, ^b Significantly different from 3-NP- treated group at P<0.05, ^ab Significantly different from both normal and 3-NP- treated groups at P<0.05.

Fig 4

Effect of cilostazol (100 mg/kg, p.o) on striatal protein expression/contents of (A) TLR-4, (B) NF-κB p65, (C) IL-6, and (D) IL-10 in 3-NP-treated rats. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's Multiple Comparison. ^a Significantly different from normal group at P<0.05, ^b Significantly different from 3-NP- treated group at P<0.05, ^ab Significantly different from both normal and 3-NP- treated groups at P<0.05.

Fig 5

Effect of cilostazol (100 mg/kg, p.o) on striatal protein expression/contents of (A) p-GSK-3β, (B) p-Akt, and (C) p-CREB in 3-NP-treated rats. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's Multiple Comparison. ^a Significantly different from normal group at P<0.05, ^b Significantly different from 3-NP- treated group at P<0.05, ^ab Significantly different from both normal and 3-NP- treated groups at P<0.05.

Fig 6

Effect of cilostazol (100 mg/kg, p.o) on striatal contents of (A) p-JAK-2, (B) p-STAT-3, and (C) SOCS3 in 3-NP treated rats. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's Multiple Comparison. ^a Significantly different from normal group at P<0.05, ^b Significantly different from 3-NP- treated group at P<0.05, ^ab Significantly different from both normal and 3-NP- treated groups at P<0.05.

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-NP- treated group at P<0.05.