Trichloroacetic acid

Methanol, acetonitrile, and acetic acid, chromatographically pure, were purchased from Thermo Fisher Technology China Co., LTD. (Shanghai, China). Sodium hydroxide was provided by the Tianjin Tianshun Lye Co., LTD. (Tianjin, China), edible salt was purchased from the Jiangxi Jiangyan Huakang Industrial Co., LTD. (Jiangxi China), and copper sulfate (food grade) was provided by the Xilong Technology Co., LTD. (Guangdong, China). Casein, trichloroacetic acid, and folinol were purchased from the Beijing Solarbio Technology Co., LTD. (Beijing, China). Sulfosalicylic acid was purchased from the Tianjin Damao Chemical Reagent Factory (Tianjin, China). Standard products: 5′-cytidine monophosphate (CMP), 5′-uridine monophosphate (UMP), succinic acid, 5′-adenosine monophosphate (AMP), 5′-guanosine monophosphate (GMP), 5′-inosine monophosphate (IMP), and 5′-xanthosidic monophosphate (XMP) were purchased from the Beijing Solarbio Technology Co., LTD. (Beijing, China).

BBB permeability was assessed by Evans blue (EB) staining. Rats were anesthetized with a mixture of 2%–2.5% isoflurane and 97.5%–98% air and EB (2%, 4 ml/kg, normal saline, Solarbio) was injected into the tail vein of the stroke rat. After the EB cycle for 1 h, the left ventricle was slowly perfused with normal saline to wash out the EB. The brain tissue was cut into five pieces and photographed quickly. Left hemispheres were isolated and homogenized with 50% trichloroacetic acid (1.5 ml/g, Solarbio) for 1 min to take immune fluorescence. The tissue was kept at 4 °C overnight, centrifuged at 14 000 rpm for 15 min, and the supernatant was collected. The fluorescence intensity was detected by a full-wavelength microplate reader (excitation at 620 nm and emission at 680 nm). Data are represented as concentration of EB dye (μg)/tissue weight (g).

After the treatments, MDA production was determined by thiobarbituric acid colorimetry. The cells were rinsed in phosphate-buffered saline (PBS), scraped, and lysed in precooled lysis solution. Then, 500 μL of the lysis supernatant, 15% trichloroacetic acid, and 0.67% thiobarbituric acid (400 μL, Solarbio Life Science) were added to a 5 mL tube. After mixing and incubating for 20 min at 95°C, the tubes were cooled, and 3 mL of isopropyl alcohol was added to extract the pigments. The OD_532nm value was measured, and total protein content in the cells was determined with a kit. MDA production was calculated according to the formula: MDA production (ng/mg Pro) = MDA content (ng/mL) × 1.5 mL/total protein weight (mg).