*2.4. Cytoprotective Activity*

#### 2.4.1. Cell Viability Assay

In this study, a Cell Counting Kit-8 (CCK8) kit was conducted to assess the viability of the cells. IEC-6 cells were seeded into a 96-well plate, 104 cells/well. After incubation in 5% CO2 and 37 ◦C for 24 h, CYP (200,400, and 800 μg/mL) was inserted into the well for co-culturing 24 h, CCK-8 solution was added and incubated with IEC-6 cells for 30 min, followed by measuring the absorbance at 450 nm using a microplate reader.

Different concentrations (100–500 μM) of H2O2 were used to stimulate IEC-6 cells for 4 h in 96-well plates, respectively. After adding CCK-8 solution for 4 h, the absorbance was measured at 450 nm to screen the damage concentration of H2O2.

After incubating IEC-6 cells in 5% CO2 at 37 ◦C for 24 h in 96-well plates, CYP at 200, 400, and 800 μg/mL was incubated in the wells together for 24 h, respectively. IEC-6 cells were stimulated with H2O2 for 4 h, and then CCK-8 assay was performed.

#### 2.4.2. Measurement of SOD and MDA Levels

The cells (2 × 105 cells/well) were seeded onto a six-well plate and incubated for 24 h to adhere to the bottom of the well plates. After CYP (200, 400, and 800 μg/mL, respectively) were added for co-culturing 24 h, cells were exposed to a final concentration of 300 μM of H2O2 for 4 h. Finally, the content of MDA and SOD contents were measured according to the manufacturer's instructions of the assay kits.

#### 2.4.3. Measurement of Intracellular ROS Level

Intracellular ROS was tested by using 2,7-dichlorofuorescin diacetate (DCFH-DA). IEC-6 cells (2 × 105 cells/well, 2 mL/well) were seeded onto a six-well plate. After incubation for 24 h, cells were exposed to H2O2 (300 μM) for 4 h before different concentrations of CYP pre-treated cells for 4 h. Fluorescent dye was added to bind to the cells for 30 min, then washed with PBS and 1640 medium, respectively. Finally, the fluorescence intensity was detected by fluorescence spectrophotometry.

#### 2.4.4. Western Blot Analysis

Aiming to further evaluate the expression of proteins in the mitogen-activated protein kinase (MAPK) pathway associated with cellular oxidative stress, pre-treat IEC-6 cells with different concentrations of CYP, after adding H2O2 to stimulate for 4 h, the cells were washed twice with PBS and then lysed the cells with RIPA buffer. After centrifugation, the samples buffer was added to the supernatant and heated at 95 ◦C for 5 min to obtain the total protein. Immediately prior to electrophoresis with 10% SDS-PAGE, protein samples were transferred onto PVDF membranes (Millipore Co., Belford, MA, US). After blocking with 5% bovine serum albumin (BSA), the membranes were incubated with primary antibody overnight at 4 ◦C and secondary antibody at room temperature for 1 h. Following incubation with developer, the fluorescent signal was detected using a Molecular Imager ChemiDoc™ XRS Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).
