C010 2

The ALT and AST levels in the plasma were measured with assay kits purchased from Nanjing Jiancheng (C009-2, C010-2). The livers were soaked in 4% formaldehyde for 1 day, and subsequently embedded in paraffin for histologic assessment. The prepared slices were further deparaffinized and stained with hematoxylin-eosin staining or Sirius red staining.

The serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in the rat blood were measured with assays purchased from Nanjing Jiancheng (C010-2, C009-2). For the histologic assessment, the livers were fixed in 4% formaldehyde for 24 hours and embedded in paraffin. Liver sections (4μm) were deparaffinized and fixed. The sections were stained with H&E.

The AST and ALT levels were measured by assay kits (C010-2, C009-2) purchased from Nanjing Jiancheng (Nanjing, China). For the histologic assessment, the livers were fixed in 4% formaldehyde for 24 h and embedded in paraffin. Liver sections (5 μm) were deparaffinized and fixed. In all experimental groups, the 5-μm-thick sections of the formalin-fixed and paraffin-embedded livers were processed for hematoxylin and eosin staining (H&E) to estimate the degree of hepatic lesions.

The hearts, liver, and kidney were fixed by 4% PFA for overnight, embedded in paraffin or OCT, and sectioned at 7 µm. Slides were stained with Masson's trichrome (ab150686, Abcam) and HE (C0105S, Beyotime) to identify areas of fibrosis and pathology. The levels of aspartate aminotransferase (AST, C010‐2, Nanjing jiancheng) and Alanine aminotranferease (ALT, C009‐2, Nanjing jiancheng) in serum were measured according to the manufacturer's instructions.

The blood collected from right ventricle was stored at room temperature for 4 h and centrifuged at a speed of 3000 r.p.m. for 10 min. Then, the supernatant was separated. The content of serum alanine aminotransferase (ALT) and aspartate transaminase (AST) were detected following the manufacturer’s protocol of respective assay kits (C009-2 and C010-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The OD value at 510 nm of each well was detected by ELIASA (Infinite 200 PRO, Tecan), and the absolute OD values were obtained from the difference between test well and its paired control well. The unit of ALT or AST expressed as U/L was transformed from the absolute optical density (OD) of each sample according to the standard curve.

Abdominal aortic blood was centrifuged at 3,000 rpm for 20 min to obtain the serum for further analysis. Liver tissue homogenate was obtained by adding homogenate medium (900 µL) to 100 mg of liver tissue, followed by centrifugation of the mixture at 3,000 rpm for 10 min, and collection of the supernatant for further analysis. The serum levels of total cholesterol (TC), serum triglyceride (TG), serum low-density lipoprotein cholesterol (LDL-C), serum aspartate aminotransferase (AST) (C010-2), serum alanine aminotransferase (ALT) (C009-2) (Jiancheng, Nanjing, China) and liver TG (A0-10017; Dongou, Zhejiang, China) were evaluated according to the manufacturer’s instructions. Hepatic FFA (A042-2-1) and ATP (A095-1-1) (Jiancheng, Nanjing, China) levels were examined using an assay kit according to the manufacturer’s instructions. The content of serum insulin was examined via using a commercial kit (90080; Crystal Chem, Elk Grove Village, IL, USA). HOMA-IR = (fasting blood glucose × fasting insulin content)/22.5 (Haffner et al. 1997 (link)).