Fermented black radish (Raphanus sativus L. var. niger) attenuates methionine and choline deficient diet-induced nonalcoholic fatty liver disease in mice

Abstract

As one of the wide-ranging form of chronic liver disease, there are only limited therapeutic options for nonalcoholic fatty liver disease (NAFLD). We evaluated whether fermented black radish (Raphanus sativus L. var. niger; FBR) ameliorates lipid accumulation, inflammation, and hepatic fibrosis, which are characteristics of the pathogenesis of NAFLD. Fermented black radish treatment reduced lipid accumulation in 3T3-L1 adipocytes, which appeared to be associated with the downregulation of adipogenic transcription factors, including sterol regulatory element-binding protein 1c, CCAAT/enhancer-binding protein α, peroxisome proliferator-activated receptor γ, and lipid accumulation-related genes including adipocyte protein-2 and fatty acid synthase. Administration of FBR to C57BL/6J mice challenged with methionine and choline deficient (MCD) diet significantly attenuated the increased serum levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and triglyceride. In addition, treatment with FBR interestingly repressed the hepatic inflammation induced with MCD diet, by lowering the expression of inducible nitric oxide synthase and suppressing the inactivation of macrophages and Kupffer cells in the liver. Fermented black radish was also shown to mitigate liver fibrosis through the inhibition of alpha-smooth muscle actin, transforming growth factor beta-1, and collagen type I alpha 1 chain. Our results indicate that FBR ameliorates NAFLD and its related metabolic disease by regulating multiple pathways, suggesting that FBR may be an effective dietary supplement for ameliorating NAFLD.

Keywords: MCD diet; fermented black radish; fibrosis; inflammation; steatosis.

Figure 1

Schematic drawing of the in vivo experimental schedule

Figure 2

Intracellular lipid droplets were stained with Oil Red O and visualized by microscopy in 3T3‐L1 cells (a) and liver tissues (b). Bar graphs show the semiquantitative analysis of lipid droplets (c). Triglyceride contents were quantified using individual kits (d). Data are presented as the mean ± standard error (SE). ^#p < .05, ^##p < .01 versus control cells; *p < .05, ***p < .001 versus vehicle‐treated cells

Figure 3

Representative immunoblots of ap2, C/EBPα, C/EBPβ, PPARγ, SREBP1c, FAS, and β‐actin expression (a), and expression of ap2, C/EBPα, PPARγ, SREBP1c, and FAS relative to β‐actin level in FBR‐treated 3T3‐L1 cells (b and c). Data are presented as the mean ± SE. n = 3 per group. ^#p < .05, ^##p < .01, ^###p < .001 versus control cells; *p < .05 versus vehicle‐treated cells

Figure 4

Each panel shows a photograph from each group, representatively (a‐f). The histological characteristics of NAFLD were assessed in each group by three blind observers using hematoxylin and eosin staining (g). Data are presented as the mean ± SE. ^## p < .01, ^### p < .001 versus normal controls; *p < .05, **p < .01 versus vehicle‐treated group. Scale bars in a–f represent 20 μm

Figure 5

Immunohistochemical staining of Iba‐1 in liver sections (a‐f). Bar graph shows the semiquantitative analysis of Iba‐1‐positive area (g) and real‐time PCR analysis of mRNA levels of hepatic iNOS (n = 5 per group) (h). Scale bars in (a–f) = 50 μm. Values in (g and h) are means ± SE. ^# p < .05, ^## p < .01 versus normal controls; *p < .05, **p < .01 versus vehicle‐treated group

Figure 6

Sirius red staining was applied to evaluate hepatic collagen deposition (a–d). Bar graph shows the semiquantitative analysis of Sirius red‐positive area (e) and Ishak staining system (f) in each group. Data are presented as the mean ± SE (n = 5 per group). #p < .05, ##p < .01, ###p < .001 versus normal controls; *p < .05, **p < .01 versus vehicle‐treated group. Scale bars in a–d represent 100 μm

Figure 7

Liver mRNA expression of αSMA (a), TGFβ1 (b), and Col1A1 (c). Data are presented as the mean ± SE. n = 5 per group. ###p < .001 versus normal controls; *p < .05, **p < .01 versus vehicle‐treated group

Figure 8

Schematic diagram of the proposed molecular effects of FBR in in vitro and in vivo