Nebivolol elicits a neuroprotective effect in the cuprizone model of multiple sclerosis in mice: emphasis on M1/M2 polarization and inhibition of NLRP3 inflammasome activation

Abstract

Background and aim: Multiple sclerosis (MS) is a demyelinating neurodegenerative inflammatory disease affecting mainly young adults. Microgliosis-derived neuroinflammation represents a key hallmark in MS pathology and progression. Nebivolol (Neb) demonstrated antioxidant, anti-inflammatory and neuroprotective properties in several brain pathologies. This study was conducted to investigate the potential neuroprotective effect of Neb in the cuprizone (Cup) model of MS.

Methods: C57Bl/6 mice were fed 0.2% Cup mixed into rodent chow for 5 weeks. Neb (5 and 10 mg/kg/day) was administered by oral gavage during the last 2 weeks.

Results: Neb prevented Cup-induced weight loss and motor deficits as evidenced by increased latency to fall in the rotarod test and enhanced locomotor activity as compared to Cup-intoxicated mice. Neb reversed Cup-induced demyelination as confirmed by Luxol fast blue staining and myelin basic protein western blotting. Administration of Neb modulated microglial activation status by suppressing M1 markers (Iba-1, CD86, iNOS, NO and TNF-α) and increasing M2 markers (Arg-1 and IL-10) as compared to Cup-fed mice. Furthermore, Neb hindered NLRP3/caspase-1/IL-18 inflammatory cascade and alleviated oxidative stress by reducing lipid peroxidation, as well as increasing catalase and superoxide dismutase activities.

Conclusion: These findings suggest the potential neuroprotective effect of Neb in the Cup-induced model of MS in mice, at least partially by virtue of shifting microglia towards M2 phenotype, mitigation of NLRP3 inflammasome activation and alleviation of oxidative stress.

Keywords: Cuprizone; M1/M2 polarization; Multiple sclerosis; NLRP3; Nebivolol.

Fig. 1

Study timeline showing Cup and Neb dosing regimens

Fig. 2

The effect of Neb on Cup-induced motor abnormalities. a Locomotor activity test. b Rotarod test. Data are presented as means ± SD (n = 6). a, b, c: Statistically significant from the control, Cup and Cup + Neb (5 mg/kg)-treated groups, respectively, at P < 0.05. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test for multiple comparisons between groups

Fig. 3

The effect of Neb on Cup-induced body weight changes. a, b:Statistically significant from the control and Cup-treated groups, respectively, at P < 0.05. Statistical analysis was performed using two-way ANOVA followed by Bonferroni test for multiple comparisons between groups

Fig. 4

a Representative photomicrograph of LFB stained CC sections (Control group) showing normal positive staining intensity (arrow). b Cup group showing a significant decrease in staining intensity (star). c, d Cup + Neb (5 and 10 mg/kg), respectively, both showing elevated staining intensity as compared to Cup-intoxicated mice (arrow). e Neb (10 mg/kg) showing positive staining intensity (arrow). Magnification 400× . f Percentage of myelinated neurofibers resulting from LFB expressed as a percentage of control. Data are presented as means ± SD (n = 6). a, b, c: Statistically significant from the control, Cup and Cup + Neb (5 mg/kg)-treated groups, respectively, at P < 0.05. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test for multiple comparisons between groups

Fig. 5

a Western blot analysis of brain MBP. b Densitometric quantitation of MBP protein expression. Data are presented as means ± S.D. (n = 3). a, b, c: Statistically significant from the control, Cup and Cup + Neb (5 mg/kg)-treated groups, respectively, at P < 0.05. Statistical analysis was performed using one-way ANOVA followed by Tukey's test for multiple comparisons between groups

Fig. 6

a Western blot analysis of brain Iba-1, CD86 and iNOS. b, c and d Densitometric quantitation of Iba-1, CD86 and iNOS protein expression, respectively. Data are presented as means ± SD. (n = 3). a, b, c: Statistically significant from the control, Cup and Cup + Neb (5 mg/kg)-treated groups, respectively, at P < 0.05. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test for multiple comparisons between groups

Fig. 7

The effect of Neb on TNF-α (a) and nitrite (b) levels in the brain tissues of Cup-treated mice. Data are presented as means ± SD (n = 6). a, b, c: Statistically significant from the control, Cup and Cup + Neb (5 mg/kg)-treated groups, respectively, at P < 0.05. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test for multiple comparisons between groups

Fig. 8

a Western blot analysis of brain Arg-1. b Densitometric quantitation of Arg-1 protein expression. Data are presented as means ± S.D. (n = 3). a, b, c: Statistically significant from the control, Cup and Cup + Neb (5 mg/kg)-treated groups, respectively, at P < 0.05. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test for multiple comparisons between groups

Fig. 9

The effect of Neb on IL-10 levels in the brain tissues of Cup-treated mice. Data are presented as means ± SD (n = 6). a, b, c: Statistically significant from the control, Cup and Cup + Neb (5 mg/kg)-treated groups, respectively, at P < 0.05. Statistical analysis was performed using one-way ANOVA followed by Tukey's test for multiple comparisons between groups

Fig. 10

a Western blot analysis of brain NLRP3 and cleaved caspase-1. b Densitometric quantitation of NLRP3 protein expression. c Densitometric quantitation of cleaved caspase-1 protein expression. Data are presented as means ± SD. (n = 3). a, b, c: Statistically significant from the control, Cup and Cup + Neb (5 mg/kg)-treated groups, respectively, at P < 0.05. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test for multiple comparisons between groups

Fig. 11

The effect of Neb on IL-18 levels in the brain tissues of Cup-treated mice. Data are presented as means ± SD (n = 6). a, b, c: Statistically significant from the control, Cup and Cup + Neb (5 mg/kg)-treated groups, respectively, at P < 0.05. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test for multiple comparisons between groups

Fig. 12

The effect of Neb on oxidative stress markers: MDA levels a, catalase b and SOD c activities in the brain tissues of Cup-intoxicated mice. Data are presented as means ± SD (n = 6). a, b, c: Statistically significant from the control, Cup and Cup + Neb (5 mg/kg)-treated groups, respectively, at P < 0.05. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test for multiple comparisons between groups