Pt512

Serum insulin (P3376, Beyotime Biotechnology), TNFα (PT512, Beyotime Biotechnology), IL-6 (PI326, Beyotime Biotechnology), and PDGF-AA (SEA523Mu, Cloud-Clone Corp., Wuhan, China) concentrations in the liver tissue and plasma were quantified using mouse ELISA kits according to the manufacturer’s instructions. The levels of secreted PDGF-AA in the cell culture supernatants were determined using a human ELISA kit (SEA523Hu, Cloud-Clone Corp). Active RhoA was detected in a colorimetric RhoA activation assay (G-LISA) (BK124, Cytoskeleton, Denver, CO, USA).

Mice enzyme-linked immunosorbent assay (ELISA) kits were used to measure the contents of TNF-α (#PT512, Beyotime Institute of Biotechnology, Shanghai, China), IL-6 (#PI326, Beyotime Institute of Biotechnology, Shanghai, China), and MCP-1 (#PC125, Beyotime Institute of Biotechnology, Shanghai, China) in the culture supernatant collected above, according to the manufacturer’s instructions. Absorbance was measured at 450 nm.

The ELISA method was used to detect the levels of IL-8 (SEKM-0046, Solarbio, China) and TNF-α (PT512, Beyotime, China) in the whole blood of mice. The blood from the heart of LPS mice in a heparinized tube was collected, 200 μL of heparinized mouse blood was added to a 96-well plate containing 25 μL LPS (final concentration 5 ng/mL), and 25 μL AWRK6 (20 μg/mL), GO (20 μg/mL) and AWRK6/GO (20 μg/mL) were added and incubated at 37°C for 24 hours and centrifuged at 1200 rmp/min for 8 minutes. The upper layer of plasma was analyzed by ELISA for the inflammatory factor IL- 8 levels. The animal study was reviewed and approved by Linyi People’s Hospital.

The concentrations of hs-CRP (E-EL-M0677c, R&D, USA), TNF-α (PT512, Beyotime), and IL-1β (PI301, Beyotime) in each group of cells or mouse left atrium tissues were measured using the ELISA kits, and all operations were implemented in strict accordance with the protocols of the corresponding kits and the literature [32 (link)].

Serum and tissue Tnf-α and Il-1β levels were analyzed using an ELISA kit (PT512 and PI301; Beyotime, Shanghai, China), and D-lactic acid (D-LA) and diamine oxidase (DAO) levels in the serum were also measured (A088-2-1 and H263-1-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Serum was used to detect cytokines TNF-α (Beyotime, Cat. No. PT512) and IL-6 (Beyotime, Cat. No. PI326) levels by ELISA analysis according to the manufacturer’s instructions.

BV2 cell culture supernatant and rmTBI mouse serum were obtained. ELISA detection was performed according to the instructions of tumor necrosis factor-α (TNF-α) (PT512, Beyotime Biotechnology, Co. Ltd., Shanghai, China) and interleukin-10 (IL-10) (PI522, Beyotime) kits. The absorbance value was measured with a microplate reader (BS-1101, DeTie Experimental Equipment, Co. Ltd., Nanjing, China) at 492 nm after zero adjustment with a blank control well. The content of the factor to be tested was determined on the standard curve based on the absorbance value of each sample.