Total Antioxidant Capacity (TAC)

The TAC of FRB was determined according to the manufacturer's instruction (Cayman Chemical, Ann Arbor, MI, USA). The optical density (OD) was read at 750 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

### Di(Phenyl)-(2,4,6-trinitrophenyl) Iminoazanium (DPPH) Antioxidant Assay

Radical-scavenging activity was quantified by a DPPH antioxidant assay using a commercial kit (D678-01, Dojindo, Rockville, MD, USA) and measuring the absorbance at 517 nm.

### *4.4. Measurements and Analytical Procedures*

4.4.1. Determination of Liver Damage

### Liver Function Index

Activities of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were detected with the ADVIA® Chemistry XPT System (Siemens Healthcare Diagnostics, Eschborn, Germany).

### Liver Histopathological Examination

The caudate lobe of liver tissues was fixed in a 10% formaldehyde solution. Sections of liver tissues were then stained with hematoxylin A- and eosin (H&E) and evaluated by a veterinarian. Images of the tissues were captured with a digital camera at 200× magnification. Histopathological evaluations of macrovesicular steatosis, microvesicular steatosis, hypertrophy, and the number of inflammatory foci were separately scored, and the severity was graded as described by Liang et al. [57] with minor modifications. Adding the scores of the above four parameters was used to calculate the NAFLD score.

### Liver Cytokine Levels

The extraction method of liver tissues was described by Chen et al. [58]: 0.5 g of liver tissues was homogenized in 1.5 mL of ice-cold buffer containing 50 mM Tris (pH 7.2), 150 mM NaCl, 1% Triton X-100, and 0.1% protease inhibitor (PI) (HYK0010, MedChem-Express, Monmouth Junction, NJ, USA). The homogenized solution was then centrifuged at 3000 rpm for 15 min at 4 ◦C, and the supernatant was collected. Concentrations of hepatic TNF-α, IL-1β, IL-6, and IL-10 were determined by corresponding enzyme-linked immunosorbent assay (ELISA) kits, including rat TNF-α (BioLegend Systems, San Diego, CA, USA), IL-1β (Rat IL-1, R&D Systems, Minneapolis, MN, USA), IL-6 (Rat IL-6, R&D Systems) and IL-10 (Rat IL-10, R&D Systems, Minneapolis, MN, USA). The OD was read at 450 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA) for all cytokines.

### Plasma and Liver Lipid Peroxidation

Liver tissues (0.1 g) were homogenized as described for the method of liver cytokines. Plasma and the supernatants of liver homogenates were used for the lipid peroxidation analysis. Lipid peroxidation in plasma and the liver was measured by TBARS levels with a TBARS kit (TBARS 10009055 (TCA method) Assay Kit, Cayman Chemical, MI, USA) according to the assay kit instructions.

### 4.4.2. Determination of Lipid Metabolism

### Hepatic TC and TG Concentrations

For the liver TC determination, 0.01 g of liver tissue was homogenized in 200 µL of solvent (chloroform: isopropanol: nonyl phenoxypolyethoxylethanol (NP-40) of 7: 11: 0.1) and centrifuged at 8876 rpm for 10 min at 4 ◦C. Avoiding the pellet, the liquid (organic phase) was transferred to a new tube, air dried at 50 ◦C to remove the chloroform, and samples were placed under a vacuum for 30 min to remove trace amounts of the organic solvent. Dried lipids were then dissolved in 200 µL of 1× assay diluent with vortexing until the solution turned homogenous and then was stored at −80 ◦C. To measure the concentrations of liver TGs, 0.1 g of liver tissue was homogenized in 500 µL of diluted NP-40 reagent containing protease inhibitors. The mixture was centrifuged at 7247 rpm for 10 min at 4 ◦C, and the supernatant (including a layer of insoluble fat) was collected and stored at −80 ◦C. TC and TG contents were further analyzed with a cholesterol colorimetric assay kit (Cell Biolabs, San Diego, CA, USA) and colorimetric TG assay kit (Cayman Chemical), and results were expressed as milligrams per gram (mg/g) of liver tissue.

### Hepatic Lipid Metabolism-Related Protein Expressions

A Western blot analysis was performed to determine expressions of SIRT1, AMPKα, p-AMPKα, leptin receptor and AdipoR2. The method of crude extraction preparation of

liver tissues was assessed according to previously described protocols [59]. Liver tissue (0.1 g) was homogenized in 0.5 mL of RIPA buffer containing 1% of a PI (HYK0010, Med-ChemExpress) and phosphatase inhibitor (PPI) (HYK0022, MedChemExpress). After being placed in an ice bath for 30 min, the homogenate was centrifuged at 7939 rpm for 15 min at 4 ◦C, followed by collection of the supernatant. Liver proteins (50 µg) were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then electroblotted onto polyvinylidene difluoride (PVDF) transfer membranes (Pall, Port Washington, NY, USA) and incubated with 5% bovine serum albumin (BSA). These blots were incubated with primary antibodies listed in Table 9, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The blots were finally treated with goat anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP; Croyez Bioscience, Taipei, Taiwan), and specific bindings of anti-bodies were assayed by the UVP Chemidoc it 515 Imaging System (UVP, Upland, CA, USA) using a T Western Lightning kit (PerkinElmer Lifesciences, Boston, MA, USA). Bands were quantified using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA).

**Table 9.** Antibodies used for Western blotting.


SIRT1, NAD-dependent sirtuin-1; AMPKα, adenosine monophosphate-activated protein kinase-α; p-AMPKα, phosphorylated-AMPKα; AdipoR2, adiponectin receptor 2.
