2.6. Experimental Validation

Differentiated rat pheochromocytoma (PC12) cells were obtained from the Shanghai Institute of Life Sciences, Chinese Academy of Sciences. PC12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified incubator at 37°C with 5% CO₂. Caprylic acid CA (T3946), chrysin (T2837), and imperatorin (T2845) were purchased from the Aladdin, China (https://www.aladdin-e.com/). Scutellarein (SCU, T3319), fisetin (T2879), gallic acid (T0877), morin (T2835), and palmatine (T5S0802) were purchased from the Bidepharmatech, China (https://www.bidepharmatech.com/). Their respective purities were > 98.5%, and all of the reagents were dissolved in dimethyl sulfoxide (DMSO) as a stock solution at 0.1 M. The OGD/R model of PC12 cells was established as previously described [26]. Specifically, PC12 cells cultured in glucose-free DMEM were incubated at 37°C and N₂/CO₂ (95%/5%) in a hypoxic incubator chamber (Thermo Scientific, USA). After incubation for 4 h under hypoxic conditions, PC12 cells were transferred back to normal DMEM containing 10% FBS and 4500 mg/L glucose and subsequently incubated under normal culturing conditions to recover [27]. Control cells were not treated with OGD/R.

The cell viability was determined using the MTT assay. Briefly, the cells were incubated with MTT (5 mg/ml) for 4 h after treatment. Formazan, generated by living cells, was dissolved in DMSO. The optical density (OD) was read at 490 nm using a microplate reader (Bio-Rad Model550, CA).

The TUNEL assay was conducted with PC12 cells using the One-step TUNEL Apoptosis Assay Kit (Beyotime). Specifically, PC12 cells were fixed with cold 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 5 min, and subsequently incubated with TUNEL reagents for 1 h at 37°C in the dark. The slides were washed with PBS and counterstained with DAPI staining solution for 5 min. TUNEL-positive cells were imaged using a fluorescence microscopy.

The detection of PC12 cell apoptosis was determined by flow cytometry. Cells from each group were harvested, washed twice with PBS, and suspended in 5 mL binding buffer. Cells were stained with 5 μL Annexin V-fluorescein isothiocyanate (FITC) and PI staining solution for 15 min in the dark at room temperature, and cell apoptosis was detected by flow cytometry (BD Biosciences, USA).

The cell supernatant was obtained from each group and centrifuged at 12,000 × g for 10 min. After collecting the supernatant, ELISA kits (ELISA, Biological Technology, Jiangsu) were used to detect the levels of TNF-α, IL-1β, and IL-6, as described in the ELISA kit instructions. All procedures were repeated at least 3 times.