Alleviation of Carbon-Tetrachloride-Induced Liver Injury and Fibrosis by Betaine Supplementation in Chickens

Abstract

Betaine is a food component with well-reported hepatoprotection effects. However, the effects and mechanisms of betaine on liver fibrosis development are still insufficient. Because metabolic functions of chicken and human liver is similar, we established a chicken model with carbon Tetrachloride- (CCl4-) induced fibrosis for studying antifibrotic effect of betaine in vivo and in vitro. Two-week-old male chicks were supplemented with betaine (1%, w/v) in drinking water for 2 weeks prior to the initiation of CCl4 treatment (i.p.) until sacrifice. Primary chicken hepatocytes were treated with CCl4 and betaine to mimic the in vivo supplementation. The supplementation of betaine significantly alleviated liver fibrosis development along with the inhibition of lipid peroxidation, hepatic inflammation cytokine, and transforming growth factor-β1 expression levels. These inhibitive effects were also accompanied with the attenuation of hepatic stellate cell activation. Furthermore, our in vitro studies confirmed that betaine provides antioxidant capacity for attenuating the hepatocyte necrosis by CCl4. Altogether, our results highlight the antioxidant ability of betaine, which alleviates CCl4-induced fibrogenesis process along with the suppression of hepatic stellate cells activation. Since betaine is a natural compound without toxicity, we suggest betaine can be used as a potent nutritional or therapeutic factor for reducing liver fibrosis.

Figure 1

Strategy of the experimental design used to evaluate the effects of betaine on CCl₄-induced hepatic injury in chickens. P.O.: peanut oil.

Figure 2

Betaine supplementation suppressed CCl₄-induced liver fibrosis development. (a) Liver sections with Masson's staining. (b) Quantification of collagen area in Masson's staining by pixel calculation. (c) Concentration of total betaine in chicken liver. (d) Oxidative stress in chicken liver. (e) Chicken liver MDA concentrations after CCl₄ challenge. Data were analyzed by two-way ANOVA (n = 6). Means with the same letter were not significantly different at P ≤ 0.05. 8-OHdG: 8-hydroxy-2-deoxyguanosine, MDA: malondialdehyde analysis.

Figure 3

Effects of betaine on liver mRNA expressions of fibrogenesis related genes with CCl₄-induced liver fibrosis (n = 6). Values were presented as the mean ± SEM. Data were analyzed by two-way ANOVA. Means with the same letter were not significantly different at P ≤ 0.05. Alpha smooth muscle actin (ACTA2), collagen type1-α1 (COL1A1), collagen type3-α1 (COL3A1), interleukin-6 (IL-6), and transforming growth factor-beta 1 (TGF-β1).

Figure 4

Effects of betaine on liver mRNA expressions of selected liver injury biomarkers and plasma DPP4 and GST concentrations with CCl₄-induced liver fibrosis. (a) mRNA expressions of selected liver injury biomarkers. (b) Plasma DPP4 concentrations. (c) Plasma GST concentrations. Values were presented as the mean ± SEM. Data were analyzed by two-way ANOVA (n = 6). Means with the same letter were not significantly different at P ≤ 0.05. Acetoacetyl-CoA synthetase (AACS), D-dopachrome tautomerase (DDT), dipeptidyl-peptidase 4 (DPP4), glutamate synthase (GLUL), and glutathione S-transferase (GST).

Figure 5

Effects of betaine on cell survival, lipid peroxidation, and proinflammation gene expression in chicken hepatocytes. (a) Cell survival after betaine and CCl₄ treatments of hepatocytes for 20 hours (n = 4). (b) MDA levels of cell culture medium when cells were treated with CCl₄ or betaine for 20 hours (n = 4). (c) IL-6 mRNA levels and (d) TGF-β1 mRNA levels in hepatocytes treated with CCl₄ or betaine for 20 hours (n = 4). Values were presented as the mean ± SEM. Data were analyzed by two-way ANOVA. Means with the same letter were not significantly different at P ≤ 0.05. Malondialdehyde (MDA), interleukin-6 (IL-6), and transforming growth factor-beta 1 (TGF-β1).

Figure 6

Antioxidant capacity of betaine and its role in CCl₄-induced liver fibrosis. Under CCl₄ stimulation, cytochrome P450 2E1 (CYP2E1) transforms CCl₄ to CCl₃ ⁻. CCl₃ ⁻ is a free radical, which induces lipid peroxidation (LPO) and causes oxidative stress. The cellular elevated oxidative stress induces secretion of inflammatory cytokines, including interleukin-6 (IL-6). Inflammation cytokines upregulate glutathione S-transferase (GST) and transforming growth factor-beta 1 (TGF-β1). GST as an antioxidant provides the self-protective mechanism to eliminate free radicals and LPO. Hepatocyte secreted TGF-β1 activates hepatic stellate cells (Ac-HSCs) through binding with TGF-β receptors. Further, the activated HSCs process fibrogenesis and synthesize collagens, finally leading to liver fibrosis. Moreover, Ac-HSCs generate and secret the dipeptidyl-peptidase 4 (DPP4), which leads us to detect the increasing plasma DPP4 concentrations in liver fibrosis chicken. Betaine provides the antioxidant capacity to alleviate the oxidative stress, which suppresses the effects of CCl₄ in the very beginning.