Dietary Intervention Accelerates NASH Resolution Depending on Inflammatory Status with Minor Additive Effects on Hepatic Injury by Vitamin E Supplementation

Figures

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  Figure 1. Study overview. Sixty-six guinea pigs were preloaded on either a standard guinea pig chow diet (4% fat, 0% cholesterol, and 0% sucrose) or high-fat diet (20% fat, 0.35% cholesterol, and 15% sucrose) for 25 weeks. Six animals receiving standard guinea pig chow diet and 12 animals receiving high-fat diet were randomly selected for euthanasia prior to intervention start, providing baseline values. The remaining animals were weight stratified into: Control (n = 12), proceeding on the standard guinea pig chow diet (4% fat, 0% cholesterol, and 0% sucrose); Chow (n = 18), receiving the standard guinea pig chow diet and CvitE (n = 18), receiving the standard guinea pig chow diet supplemented with vitamin E (1125 mg/kg feed all-rac vitamin E corresponding to a daily intake of 2000 IU vitE per animal). After eight weeks of intervention, guinea pigs were euthanized. HF, high-fat diet; LF, low-fat diet.

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  Figure 2. Histopathological evaluation of disease severity. (A) Steatosis: Control25 and Control animals showed significantly lower steatosis grade compared with HF. Chow and CvitE showed significantly increased steatosis compared with Control and were not different from HF. (B) Inflammation: Control25 and Control were significantly decreased compared with HF, while Chow and CvitE were significantly increased compared with Control with no difference compared to HF. (C) Ballooning hepatocytes; Control25 and Control were significantly different from HF and Chow significantly different from Control. CvitE was significantly reduced compared with HF and not different from Control. (D) Fibrosis; Control25 and Control had a significantly lower fibrosis score compared with HF and Chow and CvitE had a significantly increased fibrosis score compared with Control with no difference compared to HF. (E) NAFLD activity score (NAS): in the evaluation of disease severity no effect of treatment was observed in Chow and CvitE, which was not different from HF and significantly increased compared with Control. Control25 and Control were significantly reduced compared with HF. Data are presented as individual…

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  Figure 3. Expression of key target genes. (A) Expression of inflammatory target genes (IL-8, TNF-α and MCP-1) showed a significant downregulation in the Control group compared with HF. Furthermore, expression levels in Chow and CvitE were significantly elevated compared with Control, while no difference compared to HF was detected. CYP7A1 expression levels did not show any difference between groups. (B) Expression of the fibrotic target genes TGF-β and Col1a1 showed significant downregulation in the Control group compared with HF, while chow and CvitE showed significant upregulation compared with Control with no difference to HF. In the expression of α-sma, Chow was significantly upregulated compared with Control. Moreover, the Control group appeared downregulated compared with HF and CvitE although not reaching significance. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with Control; ## p < 0.01, #### p < 0.0001, compared with HF. IL-8, interleukin 8; TNF-α, tumor necrosis factor α; MCP-1, monocyte chemotactic protein 1; CYP7A1, cytochrome P450 7A1; TGF-β, transforming growth factor β; Col1a1, collagen 1a1; α-sma, α smooth muscle actin.

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  Figure 4. Subanalyses of histopathological markers of NASH and lipid accumulation relative to inflammatory status. (A,B) For Chow and CvitE, correlation between severity of steatosis and inflammation was detected. Low showed significantly reduced score compared with high in Chow and CvitE. (C,D) A low ballooning hepatocyte score correlated with a low inflammation score, with a significant decrease in low compared with high in Chow and CvitE. (E,F) In Chow, correlation between fibrosis and inflammation, with significant reduction in low compared with intermediate and high was detected. No correlation was observed in CvitE. (G,H) Chow and CvitE showed significant correlation between inflammation and NAS. In Chow, high was significantly increased compared with intermediate and low. In CvitE, high was significantly increased compared with low. (I,J) Correlation was observed between level of hepatic cholesterol and inflammation score in Chow and CvitE. In Chow and CvitE, low had significantly lower cholesterol levels compared with high. (K,L) Chow and CvitE showed correlation between hepatic triglyceride level and inflammation score, with significantly decreased levels in low compared with…

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  Figure 5. Representative histological images of liver sections at study termination. Panels are divided into subgroups: low (inflammatory score 0); intermediate (inflammatory score 1) and high (inflammatory score 2–3). (A–C,G–I) Hematoxylin and eosin stain, scale bar 100 µm. (D–F,J–L) Picro Sirius red stain (fibrosis stained red), scale bar 500 µm. The histological images show the pathological lesions associated with NASH: steatosis (macro and micro vesicular) (blue arrowheads), lobular inflammation (red arrowheads) and bridging fibrosis (purple arrowhead). Ballooning hepatocytes were also assessed, however cannot be identified at this magnification. CV, central vein; PA, portal area.