Tnf α

In accordance with the manufacturer’s protocol, the assessment of COX-2 (Cusabio^®, Houston, USA), NF-κB (Elabscience^®, Houston, USA), PGE2 (Elabscience^®, Houston, USA), IL-6 (Immuno-Biological Laboratories^®, Minneapolis, USA), TNF-α (Cloud-Clone Corp.^®, Texas, USA), ADAM-17 or TACE (Elabscience^®, Houston, USA), and TGF-α (LifeSpan Biosciences^®, Houston, USA) in prostatic tissue homogenates was performed using the corresponding rat ELISA kits.

The levels ofIL-6, IL-1β and TNF-α (Cloud-Clone Corp.) in cell culture supernatant were quantified using specific ELISA kits for rats according to the manufacturers’ instructions. Firstly, determined wells for diluted standard, blank and sample, and standard, blank and samples were added to the plates and incubated for 1 h at 37 °C. Next, detection reagent was added to the each well and incubated for 30min, followed by 90ul substrate solution. Then reaction was stopped by adding 50ul of stop solution. Finally, ran the microplate reader and conducted measurement at 450 nm immediately.

A total of 500 μl of blood was collected from the hearts of the mice following anesthesia. Serum samples were obtained by centrifugation of blood at 3,000 rpm for 15 min, and they were stored at −80°C prior to analysis. An ELISA assay was used to measure the plasma concentration of the biomarkers PINP, CTX-1, and TNF-α (Cloud Clone Corp, China) according to the manufacturer’s instructions. The optical absorbance at 450 nm was determined using a microplate absorbance reader (Model 680 Microplate Reader, Bio-Rad).

The hippocampal total JAK2 levels and serum TNF-α levels were determined with commercial ELISA kits (total JAK2, Cloud-Clone Corp., Wuhan, China; TNF-α, Multi Sciences, Hangzhou, China) according to the manufacturer’s instructions. Briefly, 100 µL of each standard or sample were pipetted into 96-well plates coated with primary antibodies and incubated on a plate shaker at room temperature. After several washes, HRP-conjugated streptavidin was added to all wells and incubated again. Then, the wells were repeatedly washed again. Subsequently, the 3,3′,5,5′-tetramethylbenzidine substrate was pipetted into the walls to generate a yellow colour that was stopped by an addition of stop solution. The mean optical density was determined using the Multiskan™ GO (Thermo Fisher Scientific, Waltham, USA) Detector system at a wavelength of 450 nm.

HT-29 (HTB-38) and THP-1 (TIB-202) cells were purchased from the American Type Culture Collection (ATCC). RPMI-1640, D-MEM, fetal bovine serum (FBS), L-glutamine, HEPES, sodium pyruvate, glucose, 2-mercaptoethanol, penicillin/streptomycin mixture, phosphate buffered saline solution (PBS), mouse monoclonal antibody against β-actin, horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary antibodies and other chemicals of analytical grade were from Sigma-Aldrich (Milan, Italy). Lipopolysaccharide (LPS) was from InvivoGen (San Diego, CA, USA). Rabbit monoclonal antibody for nuclear factor erythroid 2-related factor 2 (Nrf2) was from Bioss Antibodies (Aurogene Srl, Rome, Italy). The rabbit antibodies against NOS2 and laminin B1 were purchased from Abcam (Cambridge, UK). Mouse MMP-2 polyclonal antibody was obtained from Chemicon International Inc. (Merck, Milan, Italy). Mouse monoclonal antibodies against SOD2 were from Santa Cruz Biotechnology (D.B.A. Italia S.R.L., Milan, Italy). Pierce ECL Western Blotting Substrate, TRIzol, the high-capacity cDNA archive kit and SYBR Select Master mix were from Life Technologies (Milan, Italy). Specific primers for real-time PCR were from Eurofins Genomics (Ebersberg, Germany). ELISA kits for the quantitative detection of human IL-1β and TNF-α were from Cloud-Clone Corp. (Katy, TX, USA).

Alterations in inflammatory cytokines (IL-1β and TNFα), GSK-3β, and BDNF in the newborn serum were detected using ELISA kits of IL-1β (SEA563Ra) (Cloud-Clone Corp.), TNF-α (BioLegend), GSK-3β (Abbexa LLC, Houston, TX, USA), and BDNF (SEA011Ra) (Cloud-Clone Corp.). All the ELISA kits were used in accordance with the manufacturer's instructions (n = 6 per group); the quantity of these factors was expressed as pg/mL.