MS14 down-regulates lipocalin2 expression in spinal cord tissue in an animal model of multiple sclerosis in female C57BL/6

Abstract

Background: Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis, which is a demyelinating and an inflammatory disease of central nervous system. Recent studies have established that some molecules such as Lipocaline2 (LCN2), which expresses during inflammatory conditions, play an important role in EAE pathogenesis and might involve in its treatment process. Recently, it has been proved that MS14, an herbal-marine drug, has anti-inflammatory properties through reduction of TNF-α and IL-1β. Thus, the present study investigated the effects of MS14 on the course of EAE and its relation to LCN2 expression in both protein and gene levels. Methods: EAE was induced in female C57BL/6 mice using Hooke kits. Animals were scored for clinical signs of the disease according to a 10-point EAE scoring system. On 21^st and 35^th days after immunization, mice (n = 4/group) were deeply anesthetized, and the spinal cords were removed. Inflammatory cell infiltration and LCN2 expression in spinal cord were assessed by hematoxylin and eosin staining, immuno-histochemistry, and real-time PCR methods. Results: MS14 significantly ameliorated EAE symptoms and decreased lymphocyte infiltration into the spinal cord (P<0.05). Our data also revealed that LCN2 expression was significantly down-regulated in acute and chronic phases of EAE both at protein and gene levels after MS14 treatment (P<0.05). Conclusion: The results demonstrated that MS14 regulatory effect on EAE is accompanied by LCN2 down-regulation after treatment with the herb; however, more studies are required for clarifying the other involved mechanisms.

Keywords: Lipocalin2; MS14; Experimental autoimmune; encephalomyelitis; Multiple sclerosis.

Fig. 1

Effects of MS14 on daily clinical score of EAE during 35 days after immunization. Daily clinical score of MS14-treated group was compared to control group using Mann Whitney test. Values are shown as means ± SEM. MS14 suppressed significantly the signs of the disease from 22 days after immunization to the end (*P<0.05). Open (○) and black circles (●) represent mean daily clinical score for MS14-treated and control groups, respectively. Asterisk shows significant difference

Fig.2

Effects of MS14 on inflammatory cell infiltration into spinal cord. Lymphocyte infiltrations in spinal cord have been evaluated by hematoxylin and eosin staining and arrows have been demonstrated lymphocyte infiltration. Infiltrated cells were measured at axial section of spinal cord in control and MS14-treated groups on the 35^th day after immunization. Values are shown as mean ± SEM., the infiltrated surface of the spinal cord was significantly smaller in MS14-treated (A = 7.08 ± 1.50) than control group (B = 26.70 ± 6.50) group (*P<0.05). Asterisk shows significant difference. Magnification (100 × and 400 ×).

Fig. 3

Effects of MS14 on Lipocalin-2 (LCN2) protein induction. LCN2/ neutrophil gelatinase-associated lipocalin was detected in paraformaldehyde-fixed paraffin-embedded sections of spinal cord using monoclonal anti-mouse LCN2 antibody. MS14 treatment attenuates the LCN2 expression (A) in comparison with the control group (B).

Fig. 4

Effects of MS14 on LCN2 gene expression. (A) LCN2 gene expression determined by RT-PCR in control and MS14 groups at acute and chronic phases. (A) Lane 1 (normal), lane 2 (control at acute phase), lane 3 (MS14 at acute phase), lane 4 (control at chronic phase), and lane 5 (MS14 at chronic phase). Up-regulation of LCN2 is shown in acute phase of EAE. (B) β-actin expression is shown in all groups. (C) Fold changes of LCN2 expression in both control and MS14 groups at acute and chronic phases. The level of LCN2 expression is higher at acute phase and strongly is affected by MS14 at chronic phase (*P<0.05). Asterisks show significant differences. Our results showed a significant decrease in LCN2 expression in MS14-treated group during acute phase (2.65 ± 0.34) compared to control group (5.32 ± 0.37, P<0.05). Similar results were shown in control and MS14 groups (4.34 ± 0.29 vs. 1.123 ± 0.17) during chronic phase. M, marker