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. 2020 Sep 3:11:556391.
doi: 10.3389/fphar.2020.556391. eCollection 2020.

Oleanolic Acid Acetate Alleviates Symptoms of Experimental Autoimmune Encephalomyelitis in Mice by Regulating Toll-Like Receptor 2 Signaling

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Oleanolic Acid Acetate Alleviates Symptoms of Experimental Autoimmune Encephalomyelitis in Mice by Regulating Toll-Like Receptor 2 Signaling

Minju Kim et al. Front Pharmacol. .

Abstract

Toll-like receptor 2 (TLR2) is expressed by several immune cells in the central nervous system and plays an important role in neuroinflammation. TLR2 upregulation has been reported in multiple sclerosis patients and in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. Therefore, modulating TLR2 signaling can be an effective treatment strategy against MS. Oleanolic acid acetate (OAA) has antiinflammatory and immunomodulatory effects. Hence, this study aimed to examine the effects of OAA on TLR2 signaling and neuroinflammation in EAE. EAE was induced in C57/BL6 mice using synthesized myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, and OAA was administered daily. Hind limb paralysis and inflammatory cell infiltration were observed in the spinal cords of EAE mice. Moreover, T-cell proliferation was significantly stimulated in splenic cells from EAE mice. The expression of proinflammatory cytokines in the spinal cord was upregulated, and their serum protein levels were increased in EAE mice. Furthermore, upregulation of TLR2 and downstream signaling molecules was observed in the spinal cord. These pathological changes were reversed by OAA treatment. Our results suggest that OAA might have promising therapeutic properties and that the TLR signaling pathway is an effective therapeutic target against multiple sclerosis.

Keywords: Toll-like receptor 2; experimental autoimmune encephalomyelitis; inflammation; multiple sclerosis; oleanolic acid acetate.

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Figures

Figure 1
Figure 1
OAA attenuated clinical symptoms of EAE mice. OAA was administered after the onset of clinical symptoms (A). The average scores in the OAA-treated group (black squares) were significantly lower than those of the EAE model group (A). EAE pathogenesis induced weight loss was significantly recovered by OAA treatment (B). Each value is shown as mean ± standard error (SE) (n=8) of the clinical score of the EAE disease, which was scored until day 21 PI. Body weight data represent means standard error (SE) (n=8). *p < 0.05, **p < 0.01 compared to vehicle-treated EAE mice. OAA, oleanolic acid acetate; EAE, experimental autoimmune encephalomyelitis; PI, postimmunization.
Figure 2
Figure 2
OAA inhibited proliferation of T cells and reduced spleen weight. Spleens were collected from EAE mice on day 21 PI, weighed, and cultured as single-cell suspensions. Increase in spleen weight was significantly suppressed in OAA-treated mice (A). OAA treatment significantly suppressed Con A and MOG35-55-induced splenic T cell proliferation (B). Data are represented as means ± standard error (SE) (n=8). *p < 0.05, **p < 0.01 compared to induced splenocytes from vehicle-treated EAE mice. OAA, oleanolic acid acetate; EAE, experimental autoimmune encephalomyelitis; PI, postimmunization; Con A, concanavalin A; MOG35-55, myelin oligodendrocyte glycoprotein peptide.
Figure 3
Figure 3
OAA regulated production of key inflammatory cytokines during EAE. Serum was collected on day 21 PI from mice, including normal, EAE mice, and OAA-treated mice with EAE. ELISA was performed to detect cytokine levels of IL-6, IL-17, IFN-γ, and IL-1β. Cytokine levels were increased in EAE mice compared to those in normal mice (A). However, they were lower in the serum of OAA-treated EAE mice. mRNA levels of proinflammatory cytokines, TNF-α, IL-1β, and IL-6, were significantly suppressed in OAA-treated mice compared to those of normal control mice (B). Data are represented as means ± standard error (SE) of 6 mice. *p < 0.05, **p < 0.01 compared to vehicle-treated EAE mice. OAA, oleanolic acid acetate; IL, interleukin; TNF-α, tumor necrosis factor-alpha.
Figure 4
Figure 4
OAA suppressed infiltration of immune cells and macrophages in the spinal cords of EAE mice. H&E staining showed that OAA administration attenuated inflammatory infiltration in spinal cords compared to the infiltration observed in EAE mice (upper panel). Immunohistochemistry showed that infiltration of CD68+ macrophages was reduced in the spinal cords of OAA-treated mice (lower panel). Expression of TLR2 was elevated in EAE spinal cords and mainly expressed in CD68+ macrophages (lower panel). Scale bars = 100 µm. OAA, oleanolic acid acetate; EAE, experimental autoimmune encephalomyelitis; H&E, hematoxylin and eosin.
Figure 5
Figure 5
OAA downregulated the expression of TLR2 and downstream signaling molecules in the spinal cords of EAE mice. OAA downregulated TLR2 and MyD88 mRNA expression in the spinal cords (A). Western blot analysis of TLR2, MyD88, IRAK4, and TRAF6 (B). The expression level of TLR2, MyD88, IRAK4, and TRAF6 was significantly suppressed in OAA treated group, compared with that of EAE group (B). Data are shown as mean ± standard error (SE), n=6, *p < 0.05 compared to EAE mice. OAA, oleanolic acid acetate; EAE, experimental autoimmune encephalomyelitis; TLR2, Toll-like receptor 2; MyD88, myeloid differentiation primary response protein 88; IRAK4, IL-1 receptor-associated kinase 4: TRAF6, tumor necrosis factor (TNF) receptor-associated factor 6.

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