C010 2 1

The concentrations of estradiol (E2), total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST) in serum, and fasting plasm glucose (FPG) were measured according to the manufacturer’s protocols for each kit. The determination kit for E2 (CSB-E05109m, Wuhan, China) was purchased from CUSABIO BIOTECH CO. Ltd, and determination kits for TC (A111-1-1, Nanjing, China), TG (A110-1-1, Nanjing, China), HDL-C (A112-1-1, Nanjing, China), LDL-C (A113-1-1, Nanjing, China), ALT (C009-2-1, Nanjing, China), AST (C010-2-1, Nanjing, China), and FPG (F006-1-1, Nanjing, China) were purchased from Nanjing Jiancheng Bioengineering Institute.

The blood samples were collected from ND and DIO mice after intrasplenic injection. The spinning condition for blood specimen tube was 4000 rpm for 15 min. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST)were measured using a commercial kit (Nanjing Jian Cheng Bioengineering Institute, C009-2-1, C010-2-1).

Age-matched eight- to ten-week-old male mice were treated with either an isocaloric ethanol or pair-fed control diet following the protocol of NIAAA model 21 (link). Briefly, all mice were first fed ad libitum with the control Lieber-DeCarli liquid diet (Bio-Serv, product No. F1259SP) for 5 days, Ethanol groups were then fed with a liquid diet (Bio-Serv, product No. F1258SP) containing 5% (vol/vol) ethanol for 10 days and control groups were fed with isocaloric control diet for 10 days. At day 11, mice were administrated by oral gavage in the morning with a single bolus of ethanol (5 g/kg body weight) or isocaloric maltose dextrin solution (9 g/kg body weight), respectively. The mice were sacrificed and blood and tissue samples were collected 9 h post-gavage. Liquid diets were freshly prepared from powder daily. Food intake was recorded daily. Depilation, refusal to eat, intolerable pain, and dramatic weight loss were used as humane endpoints in this experiment.
Serum and liver triglyceride (TG), total cholesterol (T-CHO) and serum alanine transaminase (ALT), aspartate transaminase (AST) levels were determined using biochemical assay kits (Nanjing Jiancheng Bioengineering Institute, A110-1-1, A111-1-1, C009-2-1 and C010-2-1) according to the manufacturer's specifications.

The mouse serum was collected by centrifugation of clotted blood at 3000 × g for 15 min in a refrigerated centrifuge and stored at −80 °C. Commercial kits were used to measure serum levels of ALT (C009-2-1, Jiancheng, Nanjing, China) and AST (C010-2-1, Jiancheng, Nanjing, China) according to the manufacturer’s instructions.

Plasma samples were taken from the turbots at 24 h after PBS/LPS injection described at 2.8 and 2.9. Serum levels of GPT (C009-2-1; Jiancheng, China), GOT (C010-2-1; Jiancheng, China) as markers of hepatic damage were measured performed according to the manufacturer’s instructions.