Automatic biochemical analyzer

ALT and AST levels in serum were detected by an automatic biochemical analyzer (Rayto, China) as standard indexes of hepatocyte injury.

Blood was collected from the postcava and centrifuged at 3500 rpm for 10 min. The perfusate and liver tissues were collected after normothermic reperfusion, and the serum, perfusate, and liver tissues were stored at −80 °C. The cell culture medium was collected after normal culture or modeling. ALT and AST levels in the rat serum and perfusate were measured using automatic analysis in the Zhongnan Hospital of Wuhan University. MDA and SOD activities in the rat liver were measured using commercial kits (Jiancheng, Nanjing, China) according to the manufacturer’s protocol. The activities of the AST, ALT, and LDH in the cell culture medium were measured using an automatic biochemical analyzer (Rayto, Shenzhen, China).

The activity of inflammation factors was evaluated based on the expressed level of TNF-α, interleukin-6 (IL-6), and interleukin 1-β (IL-β). The samples were coronal sectioned around 7 μm for a series of detection. The immunofluorescence antibodies were specially connected to the target protein, and the methods followed the instruction for use. DAPI was used to exhibit the area of the cells. The fluorescence was observed under a fluorescence microscope. The content of malondialdehyde (MAD), GSH-px, and the activities of SOD were investigated for evaluating the anti-oxidative stress effect of Lu. The samples were prepared follow the instructions in the MAD, GSH-px, and SOD kits, and the contents were measured with an automatic biochemical analyzer (Rayto life and analytical sciences Co., Ltd, Shenzhen, China).

Formalin-fixed tissue samples were embedded in paraffin, and 5 μm sections were cut. Sections were stained with hematoxylin and eosin (H&E staining) for the evaluation of necrosis. Neutrophils and γH2AX positive cells were detected using a Ly6G antibody (1:400, Servicebio, Wuhan, China) and a γH2AX antibody (1:500, Huabio, Hangzhou, China) respectively. H&E staining and immunohistochemistry (IHC) analysis were evaluated by microscopy, and five visual fields were randomly selected for observation. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by an automatic biochemical analyzer from Rayto Life and Analytical Sciences Co., Ltd. (Shenzhen, China).