Anti ho 1

After cell lysates were obtained from control and agent-treated cells, approximately 40 μg proteins were subjected to 10% SDS–page and transferred to nitrocellulose membrane. Membranes were blocked at room temperature for 1 h in blocking buffer containing 5% nonfat dry milk to prevent nonspecific binding and then incubated with primary antibodies overnight at 4°C. The primary antibodies used in the present study was anti-Nrf2 (Biorbyt, Cambridge, 1 : 200), anti-NLRP3 (MyBioSource, LLC, San Diego, California, 1 : 1000, anti-IL-1β (ab-9722, Abcam, 1 : 1000), anti-HO-1 (Bioss Antibodies, Beijing, 1 : 1,000), and Anti-NQO1 (Bioss Antibodies, 1 : 1,000). The membranes were washed in TBST (50 mmol/L Tris-HCl, pH 7.6; 150 mmol/L NaCl; 0.1% Tween 20) for 30 min and incubated with an appropriate secondary antibody (Sigma, 1 : 2000 dilution). Bound antibody was visualized with a commercial enhanced chemiluminescence kit (Amersham Pharmacia Biotech) and Kodak film. The level of each protein was expressed relative to the amount of actin.

Expressions of Nrf2, HO-1, CAT, MnSOD, Capase3, Bax, and Bcl-2 proteins in the kidneys of chickens were determined by western blot. The Protein Extraction Kit (Beyotime, Wuhan, China) was used to extract total proteins from the tissues. The BCA Protein Assay Kit (Solarbio, Beijing, China) was used to measure total protein content. The following antibodies were used to detect their respective proteins: anti-Caspase3 (1 : 1000, Abcam, Tokyo, Japan), anti-Bax (Immunoway, Suzhou, China), anti-HO-1, anti-Bcl-2, anti-Nrf2 (1 : 1000, Bioss, Beijing, China), anti-MnSOD (1 : 6000, Enzo Clinical Labs, Farmingdale, NY, USA), anti-CAT (1 : 700, Biorbyt, Cambridge, UK), anti-Actin (1 : 10000, Abcam, Tokyo, Japan). An HRP-labeled goat anti-rabbit IgG (1 : 10000, Jackson Immuno Research Labs, West Grove, PA, USA) was used as the secondary antibody. Relative band intensities were detected on a DNR Bio Imaging system by using the NcmECL Ultra method (Ncmbio, Suzhou, China). Relative intensities of these bands were normalized according to the β-actin.

The dry powder of noni puree was provided by Morinda Inc. The Noni powder was suspended in water and prepared to a concentration of 0.5 mg/10 mL. Hydrocortisone injection was provided by TianJin KingYork Ltd. (CAT H12020887). Acetylcholine (ACh), 5-Hydroxytryptamine (5-HT), dopamine (DA), and noradrenaline (NA) ELISA kits were purchased from Jiangsu Meimian Industrial Co., Ltd. (CAT MB3256B, MB3179B, MB3092B, and MB3269B). Total superoxide dismutase (T-SOD), catalase (CAT), lipid peroxidation (LPO), and malondialdehyde (MDA) kits were purchased from the Nanjing Jiancheng Bioengineering Institute (CAT A001-1-1, A007-1-1, A106-1-2, and A003-1-1). Anti-Nrf2, anti-KEAP1, anti-HO-1, and anti-β-actin were purchased from Bioss (Beijing, China) (CAT BJ07138310, BJ07012178, BJ02265489, and AH11286487).