Biochemical analyzer

The levels of AST, ALT, and ALP, which are liver enzymes, and that of albumin, which is a protein, were measured using a biochemical analyzer (Hitachi, Tokyo, Japan). All of these are affected by liver damage. The AST, ALT, and ALP levels are elevated when the liver is injured, while lower than normal levels of albumin might indicate liver damage^{34 (link)}. Moreover, the AST/ALT ratio is an important risk factor for predicting liver damage because the levels of AST increase more than those of ALT when hepatocellular death increases^{19 (link)}.

Blood samples at each time point were collected in heparin-containing tubes and centrifuged at 5000×g for 10 min. Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured with a biochemical analyzer (Hitachi Co. Ltd., Tokyo, Japan). Formalin-fixed liver sections (5 µm) were stained with hematoxylin and eosin (HE) and Masson's trichrome (MT). The fibrotic area by MT staining was quantified by image analysis (Image-Pro Plus 6.0; Media Cybernetics, Bethesda, MD) and the histological features were independently assessed by two pathologists who were blinded to other details of the experiments.

Blood was gathered in EDTA-coated tubes, and the serum was collected by centrifuging blood sample at 3000 rpm for 20 min at 4 °C. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), and glucose levels in serum were analyzed by a biochemical analyzer (Hitachi, Tokyo, Japan). Low-density lipoprotein cholesterol (LDL) was evaluated using LDL-Cholestest Kit (SEKISUI MEDICAL Co., Ltd., Tokyo, Japan). Glucagon-like peptide-1 (GLP-1) was determined in the serum using a GLP-1 Multispecies ELISA Kit (Invitrogen, Waltham, MA, USA) and absorbance reader (800TS, BioTek, Winooski, VT, USA).

The rats were sacrificed 6 h after the induction of AP. Venous blood, liver, lung, and pancreas were collected. Tissues were immersed in neutral formalin for 24 h and then dehydrated with ethanol (70%, 80%, 90%, 95%, and 100%) followed by xylene. Tissues were embedded in paraffin and cut into sections, which were used for HE staining (LEICA AUTO STAINER XL). The venous blood was collected in disposable blood collection tubes (separation gel & coagulation accelerator) under sterile conditions and centrifuged at 860 g for 15 min to yield serum. Serum was obtained and used to measure triglycerides, cholesterol, amylase, and lipase using an automatic HITACHI biochemical analyzer.

Mice serum was obtained and analyzed for ALT, AST and LDH levels by a biochemical analyzer (Hitachi, Tokyo, Japan). All ELISA procedures were performed referring to the manufacture descriptions (mouse IL2, IL6, IL-12, IFN-γ and TNF-α ΕLISA detection kits from Ebioscience, USA, neutrophil elastase ELISA kits from Cusabio, China and myeloperoxidase (MPO) mouse ELISA kit from Invitrogen, USA). The concentrations of different cytokines in samples were calculated according to their respective standard curves.