Il 1β

Western blot was performed to examine the expression of proangiogenic and proinflammatory proteins in control or treated cells according to a standard protocol (29 (link)). In brief, cells were harvested and lysed using RIPA lysis buffer. Protein concentration of cell lysates was determined using a Bradford assay. Equal amounts of total protein (30 µg) were resolved by SDS-PAGE (10 or 12.5% gels). The resolved proteins were then transferred onto a PVDF membrane (Bio-Rad Laboratories, Inc.) and processed for analysis. The membrane was incubated with antibodies to RANTES (Santa Cruz Biotechnology, Inc., cat. no. sc-1410, 1:1,000 dilution), VEGF (Santa Cruz Biotechnology, Inc., cat. no. sc-7269, 1:1,000 dilution), IL-1β (Elabscience Biotechnology Inc; cat. no. E-AB-52153, 1:1,000 dilution) overnight at 4°C, followed by respective horseradish peroxidase (HRP) antibodies (anti-goat HRP, cat. no. sc-2020, anti-rabbit HRP, cat. no. sc-2005, anti-mouse HRP, cat. no. sc-2004, Santa Cruz Biotechnology, Inc., 1:2,000 dilution) for 1 h at room temperature. β-actin (Santa Cruz Biotechnology, Inc., cat. no. sc-1615, 1:2,000 dilution) was used as a loading control. All the blots were visualized using Clarity Western ECL (Bio-Rad Laboratories, Inc.) reagent.

All herbs were purchased from Beijing Tongrentang Co., Ltd. (Beijing, China) and were authenticated by Xiaozhong Chen, associate professor of Heilongjiang University of Chinese medicine. D-galactose was purchased from Shanghai Macklin Biochemical Co., Ltd. Piracetam was purchased from Tianjin Jinshi Pharmaceutical Co., Ltd; The primary antibodies for Bcl-2, Caspase-1, Bax, PSD95, SYP and β-actin were purchased from Wuhan Boster Biological Technology Co., Ltd. IL-1β, IL-18 and TNF-α ELISA kits were obtained from Elabscience Biotechnology Co., Ltd. DAPI purchased from Beijing Solarbio Science & Technology Co., Ltd.

After fasting (> 8 hours), venous blood (5 mL) was aspirated from patients and controls between 8:00 and 10:00 a.m. utilizing disposable needles and plastic syringes, and samples were transferred into a clean plain tube. Blood was left at room temperature for 15 min for clotting and centrifuged at 3,000 rpm for 10 minutes. Serum was then separated and stored in two Eppendorf at -80 °C for later analysis. Commercial ELISA sandwich kits were used to measure serum CCL11, IL-1β, and TNF-α (Elabscience®, Inc., Houston, USA). The sensitivities of the kits are 9.38 pg/mL for CCL11 kit, 4.69pg/mL for IL-1β kit, and 4.69 pg/mL for TNF-α kit. All the measured concentrations were greater than the sensitivity of the assays. We did not apply left-censoring and used the measured concentration in the statistical analyses. The procedures were followed according to the manufacturer’s instructions without modifications. The intra-assay coefficient of variation (CV) (precision within an assay) was < 6.22%. Serum CRP was measured using a kit supplied by Spinreact® (Barcelona, Spain). The test is based on the principle of latex agglutination.