Griess assay kit

To measure the NO levels, present in the RAW264.7 cells, a Griess Assay Kit (Beyotime) was employed. For this purpose, the RAW264.7 cells were cultured in a 12‐well plate (10⁵ cells per well), wherein they were grouped and treated as described in Section 2.3, then the supernatants of the different groups were collected for subjected to centrifugation (300× g for 5 min) to remove all cellular debris. The obtained supernatants were subsequently transferred to a 96‐well plate (50 μL per well), and Griess Assay Kit solutions 1 and 2 (50 μL each) were added as per the manufacturer's instructions. The levels of NO were calculated by measuring the absorbance at 540 nm.

Nitrites are oxidized metabolites of NO, which was evaluated by the Griess assay kit (Beyotime Biotechnology Co., Ltd., Jiangsu, China) to reflect the content of NO in cell supernatant. EA.hy 926 cells (n = 6 donors) were seeded in a 96-well culture plate and analyzed separately. Upon confluency, the medium was changed to FBS-free DMEM and incubated overnight for synchronization prior to treatment. Cells were separately incubated with 1 μM sildenafil (Sigma, USA) and SFI at three concentrations (10, 20, or 40 μl/ml) for 24 h. The amount of accumulated nitrite derived from NO metabolism was determined according to the kit operating instructions. After incubation at room temperature for 10 min, absorbance was measured at 540 nm as the reference wavelength by a microcontent multifunction microplate reader (Infinite M200, NanoQuant, Switzerland). Nitrite concentrations were calculated using a NaNO₂ standard curve (0-100 μM in cell culture medium). A nitrite standard reference curve was established for each assay.

The seeds of A. villosum were purchased from Wenshan Autonomous Prefecture, Yunnan Province, China: 2019 harvest. DEAE-52 Cellulose and Sephadex G-100 were purchased from Solarbio (Beijing, China). Monosaccharide standards and LPS were purchased from Sigma-Aldrich Chemical Co. (St., Louis, MO, USA). Deuterium oxide and trifluoroacetate acid (TFA) were purchased from Sigma-Aldrich Co. (Beijing, China). Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco Life Technologies (Grand Island, NY, USA). Chromatographic grade acetonitrile was purchased from Oceanpak (Goteborg, Sweden). The CCK-8 was acquired from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The Griess assay kit was obtained from Beyotime Biotechnology (Shanghai, China). Mouse ELISA kits to detect TNF-α, IL-6 and IL-10 were purchased from Neobioscience Technology Co., Ltd. (Shenzhen, China). All other reagents were of analytical grade.

Plasma concentrations of NO were measured with Griess assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The expressions of Bax, Bcl-2, p-Akt, Akt, p-endothelial nitric oxide synthase (eNOS), eNOS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were assessed using Western blot as described recently [15 (link)]. Proteins were measured with Pierce BCA Protein Assay Kit (Thermo). Hippocampal protein lysates (50 mg/well) were separated using (SDS/PAGE) under reducing conditions. Following electrophoresis, the separated proteins were transferred to a PVDF membrane (Millipore). Subsequently, non-specific proteins were blocked using blocking buffer (5% skim milk or 5% BSA in T-TBS containing 0.05% Tween 20), followed by overnight incubation with primary rabbit anti-rat antibodies specific for target proteins as mentioned before (Cell Signaling Technology) at 4°C. Blots were rinsed three times (5 min each) with T-TBS and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000, Proteintech) for 2 h at room temperature. The blots were visualized by using enhanced chemiluminescence (ECL) method (Thermo). GAPDH was applied to be the internal control protein. Intensity of the tested protein bands was quantified by densitometry.

The release of nitric oxide was detected by Griess assay kit (Beyotime, China) as the instructions indicated. The absorbance at 540 nm was measured on an ELX Ultra microplate reader (BioTek, USA).

NO release was detected using the Griess assay kit (S0021S; Beyotime Biotechnology, Haimen, China). An immunoassay kit (KGE004B, R&D Systems, Minneapolis, USA) was used to quantify PGE2 release. HDAC6 activity was measured using a commercial HDAC6 fluorometric assay kit (ab284549; Abcam, Cambridge, UK).