Csb e04640r

The blood was taken by heart puncture, and serum was immediately separated through centrifugation (3000 rpm for 10 min) and kept under −80 °C until analysis. The concentrations of Tumor Necrosis Factor α (TNF-α), Interluekin-6 (IL-6), Free fatty acids (FFA), insulin, and leptin in serum were measured using rat ELISA kits (CSB-E11987r, CSB-E04640r, CSB-E08770r, CSB-E05070r and CSB-E07433r, Cusabio Biotech Co., Ltd, China). Glucose, Aspartate Aminotransferase (AST), Alanine aminotransferase (ALT), Triglyceride and Cholesterol were measured using automatic measuring analyzer (Roche Diagnostic).

The serum IL-6 level of dams were used to validate the establishment of MIA model (Parker-Athill and Tan, 2010 (link)). The blood of dams in part Ⅱ was collected via caudal vein 3 h and 6 h after poly(I:C) injection. Samples were placed at room temperature for 20 min and centrifuged at 1,600 g for 15 min to separate serum. The concentration of serum IL-6 was assessed following the kit instruction (CUSABIO, CSB-E04640r, Wuhan, China).

The inflammatory factors IL-6, TNFα, and IL-1β in serum and pancreatic tissue were estimated by using assay kits (CSB-E04640r, CSB-E11987r, CSB-E08055r, CusaBio, Wuhan, China) according to the manufacturer’s protocols.

Blood was centrifuged at 1000 g for 15 min at 2–8 °C. The supernatant was collected. After that, the detection of inflammatory cytokines TNF‐α, IL1β and IL‐6 in each group of rats was performed using ELISA kits (CSB‐E11987r, CSB‐E08055r, CSB‐E04640r; CusaBio, Wuhan, China), respectively. The experimental instructions were followed strictly. A microplate reader was used to monitor the absorbance (A) values of each group at 450 nm wavelength (A_450 nm). The content of blood urea nitrogen (BUN), creatinine (CRE), GSH, MDA, SOD and albumin (ALB) was detected by the kits (#C013‐2‐1, #C011‐2‐1, #A028‐2‐1, #A006‐2‐1, #A003‐1 and #A001‐3; Nanjing Jiancheng Bioengineering Institute, Nanjing, China), respectively. The operation was performed according to the manufacturer's instructions. The microplate reader (MB‐530; HEALES, Shenzhen, China) was used to measure the A values of each group at the wavelengths of 640 nm (BUN), 546 nm (CRE), 628 nm/630 nm (GSH), 405 nm (MDA), 532 nm (SOD) and 450 nm (ALB). The urinary ALB to CRE ratios (UACRs) were calculated.

Blood was collected from the abdominal vena cava after inhalational anesthesia, and divided into EDTA tubes and conical tubes for analysis. For hematological examination, blood was put into an EDTA tube (DB Caribe, Ltd., Washington, DC, USA), it was rotated on a roll mixer for about 30 min, and, then, a blood analyzer (Hemavet 950Fs, Drew Scientific Inc., Dallas, TX, USA) was used to measure white blood cells, lymphocytes, granulocytes, and mid-size cells.
On the other hand, the blood collected in the conical tube was coagulated at room temperature for 30 min for cytokine analysis, and the serum separated in a centrifuge at 3000 rpm for 10 min was used with an ELISA kit to detect the inflammatory cytokine TNF-α (CSB-E11987r, Cusabio, TX, USA), IL-6 (CSB-E04640r, Cusabio, TX, USA), PGE2 (CSB-E07967r, Cusabio, TX, USA), MMP-2 (ab213910, Abcam, Cambridge, UK), and nitric oxide (ab65328, Abcam, Cambridge, UK) contents were measured.

For inflammatory cytokine measurement, Raw 264.7 cells were dispensed in a 24-well plate to be 8 × 10⁴ cells/well and cultured for 24 h. After that, the samples were treated and reacted by concentration, and each well was treated with LPS and cultured for 24 h. After incubation, the supernatant of the cell lysate was centrifuged. The inflammatory cytokines TNF-α (CSB-E11987r, Cusabio, TX, USA), IL-6 (CSB-E04640r, Cusabio, TX, USA), PGE2 (CSB-E07967r, Cusabio, TX, USA), MMP-2 (ab213910, Abcam, Cambridge, UK), and MMP-9 (RMP900, R&D Systems, Minneapolis, MN, USA) were measured using ELISA kits, following the manufacturers’ instructions. The experimental results of inflammatory cytokines are shown in Figure S1.