Sildenafil Alleviates Murine Experimental Autoimmune Encephalomyelitis by Triggering Autophagy in the Spinal Cord

Abstract

Multiple Sclerosis (MS) is a neuroinflammatory and chronic Central Nervous System (CNS) disease that affects millions of people worldwide. The search for more promising drugs for the treatment of MS has led to studies on Sildenafil, a phosphodiesterase type 5 Inhibitor (PDE5I) that has been shown to possess neuroprotective effects in the Experimental Autoimmune Encephalomyelitis (EAE), an animal model of MS. We have previously shown that Sildenafil improves the clinical score of EAE mice via modulation of apoptotic pathways, but other signaling pathways were not previously covered. Therefore, the aim of the present study was to further investigate the effects of Sildenafil treatment on autophagy and nitrosative stress signaling pathways in EAE. 24 female C57BL/6 mice were divided into the following groups: (A) Control - received only water; (B) EAE - EAE untreated mice; (C) SILD - EAE mice treated with 25mg/kg of Sildenafil s.c. The results showed that EAE mice presented a pro-nitrosative profile characterized by high tissue nitrite levels, lowered levels of p-eNOS and high levels of iNOS. Furthermore, decreased levels of LC3, beclin-1 and ATG5, suggests impaired autophagy, and decreased levels of AMPK in the spinal cord were also detected in EAE mice. Surprisingly, treatment with Sildenafil inhibited nitrosative stress and augmented the levels of LC3, beclin-1, ATG5, p-CREB and BDNF and decreased mTOR levels, as well as augmented p-AMPK. In conclusion, we propose that Sildenafil alleviates EAE by activating autophagy via the eNOS-NO-AMPK-mTOR-LC3-beclin1-ATG5 and eNOS-NO-AMPK-mTOR-CREB-BDNF pathways in the spinal cord.

Keywords: EAE (Experimental Autoimmune Encephalomyelitis); Sildenafil citrate (Viagra); autophagy; neuroinflammation; nitrosative stress.

Figure 1

Mice clinical score. Control mice (green line) did not develop motor dysfunction, while mice with EAE (purple line) progressively developed motor dysfunction. However, the prophylactic treatment with Sildenafil (blue line) prevented the development of more severe motor symptoms. The evaluation of clinical signs of the disease was performed daily throughout the experiment and scored on a scale of 0 to 5: 0 = no sign; 1 = loss of caudal tone; 2 = weakness in hind limbs; 3 = paralysis of hind limbs; 4 = paralysis of hind limbs and weakness in anterior limbs; 5 = complete paralysis or death. The values are presented as mean ± SEM (n = 8 mice/group). Two-way ANOVA followed by Tukey’s post-test statistical analysis showed statistically significant differences between all groups over time. ****p< 0.0001. This experiment was repeated twice.

Figure 2

Immunohistochemistry for p-eNOS in the spinal cord (A). Statistical differences were analyzed by one-way ANOVA [F (2, 15) = 6.826, P = 0.0078] followed by Tukey’s post-test. Control group presented basal expression of p-eNOS, while EAE mice had decreased labeling of this protein in immunohistochemistry). Treatment with Sildenafil increased p-eNOS immunoreactivity ^#p < 0.05 when CONTROL vs. EAE; *p < 0.05 when EAE vs. SILD. Immunohistochemistry for iNOS in the spinal cord (B). Statistical differences were analyzed by one-way ANOVA [F (2, 23) = 7.101, P = 0.0040] followed by Tukey’s post-test. Control group presented basal expression of iNOS, while EAE mice had increased labeling of this protein in immunohistochemistry. Treatment with Sildenafil decreased iNOS expression. Quantification of pixels (left panels). Values are presented as mean ± SD. ^#p < 0.01 when CONTROL vs. EAE; *p < 0.05 when EAE vs. SILD. n= 6-8 images/group. These experiments were repeated twice.

Figure 3

(A) Immunohistochemistry for beclin-1 in the spinal cord. Statistical differences were analyzed by one-way ANOVA [F (2, 9) = 11.05, P = 0.0038] followed by Tukey’s post-test. Control group presented basal expression of beclin-1, while EAE mice had decreased labeling of this protein in immunohistochemistry. Treatment with Sildenafil increased beclin-1 immunoreactivity. Immunohistochemistry for ATG5 in the spinal cord. Statistical differences were analyzed by one-way ANOVA [F (2, 14) = 7.343, P = 0.0066] followed by Tukey’s post-test. Control group presented basal expression of ATG5, while EAE mice had decreased labeling of this protein in immunohistochemistry. Treatment with Sildenafil increased ATG5 immunoreactivity. Immunohistochemistry for mTOR in the spinal cord. Statistical differences were analyzed by one-way ANOVA [F (2, 30) = 4.817, P = 0.0153] followed by Tukey’s post-test. Control group presented basal expression of mTOR while EAE mice had increased labeling of this protein in immunohistochemistry. Treatment with Sildenafil decreased immunoreactivity mTOR immunoreactivity. (C–E) Quantification of pixels. Values are presented as mean ± SD. ^#p < 0.05 when CONTROL vs. EAE; *p < 0.01 when EAE vs. SILD. n= 6-8 images/group. These experiments were repeated twice. (B) Immunohistochemistry for LC3 in the spinal cord. Statistical differences were analyzed by one-way ANOVA [F (2, 31) = 8.718, P = 0.0010] followed by Tukey’s post-test. Control group presented basal expression of LC3, while EAE mice had decreased labeling of this protein in immunohistochemistry. Treatment with Sildenafil increased LC3 immunoreactivity. ^#p < 0.05 when CONTROL vs. EAE; *p < 0.01 when EAE vs. SILD. Immunohistochemistry for p-CREB in the spinal cord. Statistical differences were analyzed by one-way ANOVA [F (2, 10) = 6.987, P = 0.0126] followed by Tukey’s post-test. Control group presented basal expression of p-CREB, while EAE mice had decreased labeling of this protein. Treatment with Sildenafil increased p-CREB immunoreactivity. ^#p < 0.05 when CONTROL vs. EAE; *p < 0.01 when EAE vs. SILD. Immunohistochemistry for BDNF in the spinal cord. Statistical differences were analyzed by one-way ANOVA [F (2, 15) = 14.26, P = 0.0003] followed by Tukey’s post-test. Control group presented basal expression of BDNF, while EAE mice had decreased labeling of this protein. Treatment with Sildenafil increased BDNF immunoreactivity. ^#p < 0.05 when CONTROL vs. EAE; *p < 0.001 when EAE vs. SILD (F–H) Quantification of pixels. Values are presented as mean ± SD. n= 6-8 images/group. These experiments were repeated twice.

Figure 4

Western blot for p-AMPK (A) Statistical differences were analyzed by one-way ANOVA [F (2, 3) = 34.57, P = 0.0085] followed by Tukey’s post-test. Control group presented basal expression of p-AMPK, while EAE mice displayed decreased p-AMPK levels. Treatment with Sildenafil increased p-AMPK levels. (B) Densitometric analysis of p-AMPK. Values are presented as mean ± SD. ^#p < 0.01 when CONTROL vs. EAE; *p <0.05 when EAE vs. SILD n= 5 mice/group. This experiment was repeated twice. (C) Levels of nitrite in the spinal cord showed difference among groups by one-way ANOVA [F (2, 6) = 13.72, P = 0.0058]. Control group presented basal expression of NO, while EAE mice had increased levels of nitrite compared with control group. Conversely, treatment with Sildenafil reduced nitrite levels compared to EAE group. Values are presented as mean ± SD. ^#p < 0.01 when CONTROL vs. EAE; *p < 0.05 when EAE vs. SILD. n= 5 mice/group. This experiment was repeated twice.

Figure 5

Schematic summarizing the signaling pathways modulated by Sildenafil to improve EAE pathology. Sildenafil increases the levels of p-AMPK and reduces the levels of mTOR, which leads to an increase in the levels of LC3, promoting autophagy. Furthermore, Sildenafil enhances the expression of p-eNOS, leading to the production of NO, which further activates AMPK. Moreover, NO triggers the activation of the NO-sGC-cGMP-PKG pathway, which leads to increased levels of p-CREB and BDNF, further promoting autophagy. Furthermore, Sildenafil inhibits iNOS, which when activated produces NO in excess, thus inhibiting nitrosative stress. Altogether, the activation of the aforementioned signaling pathways improves EAE pathology. Red bars: inhibition; Green arrows: activation.