Celastrol Attenuates Multiple Sclerosis and Optic Neuritis in an Experimental Autoimmune Encephalomyelitis Model

Abstract

This study was aimed to evaluate the effects of celastrol, a natural compound with multiple bioactivities, on multiple sclerosis and optic neuritis (ON) in rat experimental autoimmune encephalomyelitis (EAE). EAE was induced in Sprague Dawley rats using myelin basic protein, and the animals received daily intraperitoneal injections of celastrol or vehicle for 13 days. The EAE rats showed abnormal neurobehavior and inflammatory infiltration and demyelination in the spinal cord. Significantly upregulated mRNA expression of pro-inflammatory cytokines interferon-γ and interleukin-17 and downregulated anti-inflammatory cytokines interleukin-4 were found in the spinal cord of EAE rats. In the study of ON, severely inflammatory responses like in the spinal cord were also seen in the optic nerve, as well as obvious microgliosis. Furthermore, activation of nuclear factor kappa-B and upregulated inducible nitric oxide synthase was observed in the optic nerve. In addition, apoptosis of retinal ganglion cells and dysregulation of apoptotic-associated proteins in the optic nerve were found in EAE rats. Treatment of celastrol potently restored these changes. In most of the indexes, the effects of high dose of celastrol were better than the low dose. Our data conclude that administration of celastrol attenuates multiple sclerosis and ON in EAE via anti-inflammatory and anti-apoptotic effects. These findings provide new pre-clinical evidence for the use of celastrol in treatment of multiple sclerosis.

Keywords: apoptosis; celastrol; inflammation; multiple sclerosis; optic neuritis.

FIGURE 1

Chemical structure of celastrol.

FIGURE 2

Treatment of celastrol attenuates neurological severity of experimental autoimmune encephalomyelitis (EAE) rats. Neither low nor high dose of celastrol delayed the onset of EAE, but both of the dosages decreased neurologic severity of EAE. Data were shown as mean ± SD. (n = 5). ^∗∗P < 0.01 versus control group, ^##P < 0.01 versus EAE group.

FIGURE 3

Treatment of celastrol suppressed histopathological alterations and demyelination in spinal cords of EAE rats. H & E (A) and Luxol fast blue (LFB; C) staining showed that celastrol attenuated inflammatory infiltration and demyelination in spinal cord of EAE rats, inflammatory (B) and demyelination (D) scores were significantly lowered in both low and high dosages of celastrol groups. Scale bars: 100 μm. Data were shown as mean ± SD. (n = 5). ^∗∗P < 0.01 versus control group, ^##P < 0.01 versus EAE group.

FIGURE 4

Celastrol regulated production of cytokines and activation of NF-κB in spinal cords of EAE rats. Celastrol dose-dependently downregulated the mRNA expression of INF-γ (A) and IL-17 (B) but upregulated IL-4 (C) in spinal cord of EAE rats. Data were shown as mean ± SD, n = 5. ^∗∗P < 0.01 versus control group, ^##P < 0.01 versus EAE group, ^††P < 0.01 versus low dosage of celastrol group.

FIGURE 5

Celastrol inhibited production of cytokines and microgliosis in optic nerve of EAE rats. Celastrol dose-dependently downregulated the mRNA expression of INF-γ, IL-1β, TNF-α and IL-17 but upregulated IL-4 (A) in optic nerve of EAE rats. The expression of CD11b was decreased after treatment of celastrol (B). Scale bar: 50 μm. Data were shown as mean ± SD, n = 5. ^∗∗P < 0.01 versus control group, ^##P < 0.01 versus EAE group, ^††P < 0.01 versus low dosage of celastrol group.

FIGURE 6

Celastrol inhibited inducible nitric oxide synthase (iNOS) expression and activation of NF-κB in optic nerve in EAE rats. (A) IHC staining of iNOS in optic nerve. (B) Quantification of iNOS-positive areas. (C) Quantitative real-time PCR analysis of iNOS expression. (D) Western blot analysis of iNOS expression. (E–G) Western blot analysis of IκBα, p65 and p-p65 expression, respectively. Treatment of celastrol reduced expression of iNOS and inhibited the activation of NF-κB in optic nerve in EAE rats. Scale bar: 100 μm. Data were shown as mean ± SD, n = 5. ^∗∗P < 0.01 versus control group, ^##P < 0.01 versus EAE group, ^††P < 0.01 versus low dosage of celastrol group.

FIGURE 7

Celastrol attenuates ganglion cells apoptosis in the retina of EAE rats. Treatment of celastrol decreased the number of TUNEL-positive cells (A), upregulated expression of Bcl-2 (B) and downregulated expression of Bax, cleaved-caspase 3 and cleaved-PARP. Scale bar: 100 μm. Data were shown as mean ± SD, n = 5. ^∗∗P < 0.01 versus control group, ^##P < 0.01 versus EAE group, ^††P < 0.01 versus low dosage of celastrol group.