Hesperetin, a Citrus Flavonoid, Ameliorates Inflammatory Cytokine-Mediated Inhibition of Oligodendroglial Cell Morphological Differentiation

Abstract

Oligodendrocytes (oligodendroglial cells) are glial cells that wrap neuronal axons with their differentiated plasma membranes called myelin membranes. In the pathogenesis of inflammatory cytokine-related oligodendroglial cell and myelin diseases such as multiple sclerosis (MS), typical inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) are thought to contribute to the degeneration and/or progression of the degeneration of oligodendroglial cells and, in turn, the degeneration of naked neuronal cells in the central nervous system (CNS) tissues. Despite the known involvement of these inflammatory cytokines in disease progression, it has remained unclear whether and how TNFα or IL-6 affects the oligodendroglial cells themselves or indirectly. Here we show that TNFα or IL-6 directly inhibits morphological differentiation in FBD-102b cells, which are differentiation models of oligodendroglial cells. Their phenotype changes were supported by the decreased expression levels of oligodendroglial cell differentiation and myelin marker proteins. In addition, TNFα or IL-6 decreased phosphorylation levels of Akt kinase, whose upregulation has been associated with promoting oligodendroglial cell differentiation. Hesperetin, a flavonoid mainly contained in citrus fruit, is known to have neuroprotective effects. Hesperetin might also be able to resolve pre-illness conditions, including the irregulated secretion of cytokines, through diet. Notably, the addition of hesperetin into cells recovered TNFα- or IL-6-induced inhibition of differentiation, as supported by increased levels of marker protein expression and phosphorylation of Akt kinase. These results suggest that TNFα or IL-6 itself contributes to the inhibitory effects on the morphological differentiation of oligodendroglial cells, possibly providing information not only on their underlying pathological effects but also on flavonoids with potential therapeutic effects at the molecular and cellular levels.

Keywords: Akt kinase; IL-6; TNFα; differentiation; hesperetin; oligodendrocyte.

Figure 1

Treatment of cells with TNFα inhibits oligodendroglial cell morphological differentiation. (A,B) FBD-102b cells were treated with TNFα or control vehicles and were allowed to be differentiated for 0 or 3 days. Lower panels a and b are magnified dotted squares a and b of middle panels. Differentiation efficiencies were divided into 3 categories (cells with primary processes were classified as category 1; cells with secondary processes branched from primary processes were classified as category 2; and cells with third processes branched from secondary processes or with widespread membranes were classified as category 3) and depicted in graphs (**, p < 0.01; n = 10 [taking one picture each from 10 independent experiments]).

Figure 2

Effects of TNFα on oligodendroglial cell marker protein expression. (A,B) Cells were treated with TNFα or control vehicles. The PLP1, Sox10, or control actin blot is shown. Immunoreactive band intensities were also compared to be depicted in graphs (**, p < 0.01; n = 3 blots [obtaining one sample each from 3 independent experiments]).

Figure 3

Treatment of cells with IL-6 inhibits oligodendroglial cell morphological differentiation. (A,B) FBD-102b cells were treated with IL-6 or control vehicles and were allowed to be differentiated. Lower panels a and b are magnified dotted squares a and b of middle panels. Differentiation efficiencies were divided into 3 categories and depicted in graphs (**, p < 0.01; *, p < 0.05; n = 10 [taking one picture each from 10 independent experiments]).

Figure 4

Effects of IL-6 on oligodendroglial cell marker protein expression. (A,B) Cells were treated with IL-6 or control vehicles. The PLP1, Sox10, or control actin blot is shown. Immunoreactive band intensities were also compared to be depicted in graphs (*, p < 0.05; n = 3 blots [obtaining one sample each from 3 independent experiments]).

Figure 5

TNFα or IL-6 inhibits phosphorylation of Akt kinase. (A,B) Cells were treated with control vehicles (-), interleukin-1α (IL-1α as a positive control)/interleukin-1α (IL-1β as a negative control) or tumor necrosis factor α (TNFα). The (pS473)Akt1 or Akt1 blot is shown. Immunoreactive band intensities for (pS473)Akt1 were also compared to be depicted in graphs (**, p < 0.01; n = 3 blots [obtaining one sample each from 3 independent experiments]). (C,D) Cells were treated with control vehicles (-) or interleukin-6 (IL-6). The (pS473)Akt1 or Akt1 blot is shown. Immunoreactive band intensities for (pS473)Akt1 were also compared to be depicted in graphs (**, p < 0.01; n = 3 blots [obtaining one sample each from 3 independent experiments]). The vertical values in graphs were evaluated as (pS473)Akt1/total Akt1.

Figure 6

Hesperetin recovers TNFα inhibition of oligodendroglial cell morphological differentiation. (A,B) Differentiation of FBD-102b cells treated with TNFα was induced in the presence of hesperetin (Hes) or control vehicles. Lower panels a and b are magnified dotted squares a and b of middle panels. Differentiation efficiencies were divided into 3 categories and depicted in graphs (**, p < 0.01; n = 10 [taking one picture each from 10 independent experiments]).

Figure 7

Effects of hesperetin on expression levels of oligodendroglial cell marker proteins in the presence of TNFα. (A,B) Cells were treated with TNFα in the presence of hesperetin (Hes) or control vehicles. The PLP1, Sox10, or control actin blot is shown. Immunoreactive band intensities were also compared to be depicted in graphs (**, p < 0.01; n = 3 blots [obtaining one sample each from 3 independent experiments]).

Figure 8

Hesperetin recovers IL-6 inhibition of oligodendroglial cell morphological differentiation. (A,B) Differentiation of FBD-102b cells treated with IL-6 was induced in the presence of hesperetin (Hes) or control vehicles. Lower panels a and b are magnified dotted squares a and b of middle panels. Differentiation efficiencies were divided into 3 categories and depicted in graphs (**, p < 0.01; n = 10 [taking one picture each from 10 independent experiments]).

Figure 9

Effects of hesperetin on expression levels of oligodendroglial cell marker proteins in the presence of IL-6. (A,B) Cells treated with IL-6 in the presence of hesperetin (Hes) or control vehicles. The PLP1, Sox10, or control actin blot is shown. Immunoreactive band intensities were also compared to be depicted in graphs (*, p < 0.05; n = 3 blots [obtaining one sample each from 3 independent experiments]).

Figure 10

Hesperetin recovers inhibition of Akt kinase phosphorylation by TNFα or IL-6. (A,B) Cells were treated with TNFα or IL-1α in the presence of control vehicles or hesperetin (Hes). The (pS473)Akt1 or Akt1 blot is shown. Immunoreactive band intensities for (pS473)Akt1 were also compared to be depicted in graphs (**, p < 0.01; n = 3 blots [obtaining one sample each from 3 independent experiments]). (C,D) Cells were treated with IL-6 in the presence of control vehicles or hesperetin. The (pS473)Akt1 or Akt1 blot is shown. Immunoreactive band intensities for (pS473)Akt1 were also compared to be depicted in graphs (**, p < 0.01; n = 3 blots [obtaining one sample each from 3 independent experiments]). The vertical values in graphs were evaluated as (pS473)Akt1/total Akt1.