*2.5. IL-8 Determination*

IL-8 was determined using IL-8 Human Uncoated ELISA Kit (cat. #88-8086, Invitrogen, Carlsbad, CA, USA). Cells were cultured in a 12-well plate with the inoculum density of 2 × 10<sup>5</sup> cells/well and treated with H2O2 and RDE as described above. After, 100 μL of the medium was used to determine IL-8 content according to the kit instructions.

#### *2.6. Reactive Oxygen Species (ROS) Assay*

Cellular ROS was measured using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) [26]. Cells were cultured in a T-25 flask with a inoculum density of 1 × 10<sup>6</sup> cells/well until 90% confluence reached. They were treated with H2O2 and RDE as described above. After treatments, cells were washed with PBS two times and treated with 1 mL DCFH-DA solution (10 μM in PBS) for 30 min at 37 ◦C. Then, cells were washed with PBS two times and fixed with 4% PFA for 15 min at room temperature. After that, they were digested with 0.25% trypsin (at 37 ◦C) to monoplast, washed

with PBS, and then centrifuged to collect cells for flow cytometry analysis. A total of 10,000 cells was calculated, and fluorescence intensity was measured.

## *2.7. Glutathione Determination*

Total glutathione (GSH) and oxidized glutathione (GSSG) were determined using a Glutathione Colorimetric Detection Kit (cat. #EIAGSHC, Invitrogen). Cells were cultured in 12-well plate with a inoculum density of 2 × 10<sup>5</sup> cells/well and treated with H2O2 and RDE as described above. Afterward, cells were washed with ice-cold PBS and resuspended in 200 μL of ice-cold 5% aqueous 5-sulfo-salicylic acid dehydrate (SSA) and followed by the freeze–thaw cycling to lyse cells. After, lysed cells were centrifuged at 14,800× *g* at 4 ◦C for 15 min. The dilution and assay were conducted by following the kit instructions. The reduced GSH was calculated by an equation: reduced GSH = total GSH − GSSG.

#### *2.8. Transepethial Electrical Resistant (TEER) Measurement*

The TEER of cell monolayers was measured using a Millicell Electrical Resistance System (ESR-2) (Millipore-Sigma) according to our previously published procedure [10]. Caco-2 cells were seeded onto Transwells (Polyester membrane, 12 wells, 12 mm diameter inserts, Corning Costar, Fisher Scientific) at a density of 1 × 10<sup>5</sup> cells/insert. The TEER value was monitored every other day. When a monolayer of cells was completely di fferentiated (about 10 days), cells were treated with H2O2 and RDE, and TEER values were measured after H2O2 treatment for 6 h and after RED treatment for 24 h, respectively. The data were presented as a percentage of initial value before treatments.

#### *2.9. Measurement of Cell Permeability*

To quantify the paracellular permeability of cell monolayers, 1 mg/mL of FD4 (46944-500MG-F, Sigma-Aldrich) was added to the apical side of the inserts (Polyester membrane, 12 wells, 12 mm diameter inserts, Corning Costar, Fisher Scientific, Ottawa, ON, Canada). The cell culture and the treatments were the same as that of TEER measurement. The basolateral medium aliquots were taken after 2 h of incubation at 37 ◦C in an atmosphere of 5% CO2. Then, 100 μL of the medium was transferred into 96-well plates and the di ffused fluorescent tracer was then measured by fluorometry (excitation, 485 nm; emission, 528 nm) using a Synergy ™ H4 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA) [10].

#### *2.10. RNA Extraction and Real-Time PCR*

Total RNA isolation and real-time PCR were conducted according to Omonijo et al. [10]. The reference gene was GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The primers for real-time PCR analysis were designed with the Primer-Blast based on the published cDNA sequence in the Gene Bank (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The information of genes detected and the primers are shown in Table 1.
