Pi303

Brain tissue samples were obtained at corresponding time points after SAH. Moreover, rhwnt1, siwnt1 RNA, and anti-Frizzled1 interventions were applied once SAH models were established. Levels of IL-1β, IL-6, and TNF-α in brain tissue were measured using specific enzyme linked immunosorbent assay kits for rats (PI303, PI328, PT516; Beyotime), in accordance with the manufacturer’s instructions. Briefly, equal quantities of brain tissue from different groups were cut and ground to generate protein samples. Standard samples were diluted to different concentrations and target samples added into corresponding wells of 100 μL. Reaction wells were sealed with transparent film, and incubated at room temperature for 120 minutes. Next, prepared biotinylated antibody was added into the reaction well, and incubated for 1 hour at room temperature. Horseradish peroxidase was added to detect streptavidin, and incubated for 20 minutes at room temperature. Finally, 3,3′,5,5′-tetramethylbenzidine solution was added and incubated for 20 minutes at room temperature. After stop solution was added, absorbance values were measured at 450 nm immediately using a spectrophotometer (FilterMax F5, Molecular Devices, San Francisco, CA, USA). Values were expressed as pg/mL.

The levels of interleukin (IL)-1β, IL-6, IL-18, and tumor necrosis factor alpha (TNF-α) in H9C2 cells were tested as per the protocols of commercially available ELISA kits (PI303, PI328, PI555, and PT516; Beyotime).

The serum levels of insulin (kt30476, MSKbio, Wuhan, China), TC (kt30156, MSKbio, Wuhan, China), TGs (kt50023, MSKbio, Wuhan, China), LDL-C (kt25323, MSKbio, Wuhan, China) and LPS (E-EL-R0589c, Elabscience, Wuhan, China) as well as tumour necrosis factor-alpha (TNF-α, ml002859, mlbio, Shanghai, China), interleukin 6 (IL-6, ml064292, mlbio, Shanghai, China) and IL-1β (PI303, Beyotime, Beijing, China) levels in liver tissue were detected by specific ELISA kits. Briefly, rat serum was added into the enzyme-labelled plates and incubated at 37 °C for 90 min. Then, the fluid in the plates was discarded, and 100 μL biotinylated antibody was added into the plates. After 60 min incubation at 37 °C, the plates were washed by the buffer solution thrice, added with the enzyme-conjugated working solution and incubated at 37 °C for 30 min. Subsequently, the plates were supplemented with the substrate Reagent and incubated at 37 °C for 15 min. Following these, H₂SO₄ (2 M, 50 μL) was added to each well to terminate the reaction. Ultimately, the optical density at 450 nm was recorded under a microplate reader (ELx808, BioTek, Winooski, VT).
Later, homeostasis model assessment-insulin resistance (HOMA-IR) was calculated according to this formula (Jia N et al. 2018 ):
$$\text{HOMA}-\text{IR}=\left[\text{fasting}\mathrm{ }\text{blood}\mathrm{ }\text{glucose}\mathrm{ }\left(\text{FBG},\mathrm{ }\text{mmol}/\mathrm{L}\right)\mathrm{ }\times \mathrm{ }\text{fasting}\mathrm{ }\text{insulin}\mathrm{ }\text{level}\mathrm{ }\left(\text{FINS},\mathrm{ }\text{mUI}/\mathrm{L}\right)/22.5\right].$$