A087 1 2

Triglyceride (TG), glucose, and nonesterified fatty acid (NEFA) in serum were determined using the commercial kits (A110-1-1, F006-1-1, and A042-2-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. Insulin in serum was determined with an ELISA kit (CSB-E06829p; Cusabio, Wuhan, China) according to the manufacturer's instructions. Homeostasis model assessment‐insulin resistance (HOMA‐IR) = [(fasting insulin, mIU/L) × (fasting glucose, mmol/L)]/22.5; homeostasis model assessment‐insulin sensitivity (HOMA‐IS) = 1/[(fasting insulin, mIU/L) × (fasting glucose, mmol/L)] [21 (link)]. TG, NEFA, malondialdehyde (MDA), protein carbonyl, 8-hydroxy-2′-deoxyguanosine urine (8-OHdG), and glutathione (GSH) in the placenta were determined using the respective commercial kits (A110-1-1, A042-2-1, A003-1-2, A087-1-2, H165, and A006-2-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Placental reactive oxygen species (ROS) production was measured using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) according to the manufacturer's protocol (E004; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) as described previously [22 (link)].

The liver and spleen tissues were examined microscopically after being fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin [18 (link)]. The total antioxidant capacity (T-AOC) and the activities of glutathione peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT), along with concentrations of malondialdehyde (MDA) were measured by specific assay kits (A003, A005, A001, A007–1, and A087–1–2) that were purchased from the Nanjing Jiancheng Bioengineering Institute of China. The protein concentrations were measured by the bicinchoninic acid assay.

The duodenum, jejunum, ileum tissues were microscopically examined after fixing in 10% neutral-buffered formalin and processing for paraffin embedding, sectioning at 5 μm, and then staining with hematoxylin and eosin [17 (link)]. The concentrations of lipopolysaccharide (LPS), malondialdehyde (MDA), protein carbonyl (PC) and reduced glutathione (GSH) and activity of diamine oxidase (DAO), superoxide dismutase (SOD), total antioxidant capacity (T-AOC) were measured by a colorimetric method with the use of specific assay kits (H255, A003-1-2, A087-1-2, A006-1-1, A088-1-1, A001-1-2 and A015-1-2) from the Nanjing Jiancheng Bioengineering Institute of China. Protein concentration was measured by the bicinchoninic acid assay (Beyotime Institute of Biotechnology, Jiangsu, China).