Nano2

The cells inoculated in 24-well plates were divided into the of groups of blank, LPS, and SL+LPS and incubated for 24 h. Then, the supernatant was carefully pipetted off. For the blank group, 500 µL of the DMEM solution was added. For the LPS group, 500 µL of the DMEM solution containing LPS (1 µg/mL) was added. For the SL+LPS group, the DMEM solution with different concentrations of SL (6, 12.5, 20 µg/mL) was pretreated for 2 h, and then the final concentration was kept to 1 µg/mL by adding LPS and incubated in the incubator for another 24 h. After that, the supernatants used for the assays of NO and cytokine were collected. For the measurement of NO, the above supernatant was mixed with an equal volume of Griess reagent (S0021S, Beyotime Inc., Shanghai, China) and then incubated for 10 min at room temperature. The concentration of nitrogen oxide was measured by oxygen demand (OD) analysis at 540 nm. NaNO₂ (S0021S, Beyotime Inc., China) was used as a standard regent, and the enzyme-linked immunosorbent assay kits (Absin Bioscience Inc., Shanghai, China) were used to determine the concentration of cytokine for IL-6 and TNF-α.

Nitrogen oxide were used as markers of oxidative stress and to detect the extent of brain damage. The NO generation was analyzed as previously described with minor modification (Yu, Lin, Zhang, & Guo, 2004). The levels of NO in primary cortical neurons were measured following the relevant quantitative kit instructions (Jiancheng, Nanjing, China). The neurons were pulverized in liquid nitrogen and suspended in homogenization buffer (NaCl 0.1 M, Tris‐HCl 0.01 M, pH 7.6, egtazic acid 1 mM, aprotinin 1 mg/L, and PMSF 100 mg/L). After centrifugation, 50 µl Griess reagent (equal volume of 1% sulfanilamide in HCl 0.1 M and 0.1% N‐ [−1‐naphthyl‐ethylenediamine dihydrochloride]) was added to 50 µl of suspending media. Nitrite concentration was determined by spectrophotometry (560 nm) from a standard curve (0–100 mM) derived from NaNO₂ (Beyotime Biotechnology). The data were expressed as mean ± SD (nitrite) in µM.