**3. Results**

As shown in Figure 1A, the cell viability significantly decreased when the concentrations of H2O2 were more than 0.8 mM (*P* < 0.05). The cell viability was reduced at 35.15% when 1 mM H2O2 was added in the medium compared with the control (0 mM H2O2). Therefore, 1 mM H2O2 was selected to induce oxidative damage in this study. As shown in Figure 1B, the cell viability was not changed when cells were treated with the RDE at the concentrations of 0–200 μg/mL (*P* > 0.05). As shown in Figure 1C, when cells were pre-treated with 1 mM H2O2 for 3 h and then incubated with different concentrations of RDE for 24 h, RDE showed a dose-dependent recovery of the cell viability that was compromised by 1 mM H2O2 and the cells treated with 100 μg/mL of RDE had similar viability with the control cells (*P* > 0.05). Therefore, 100 μg/mL of RDE was used in the subsequent experiments.

**Figure 1.** Effect of H2O2 and red-osier dogwood extracts (RDE) on the cell viability of Caco-2 cells. (**A**) Cells were treated with different concentrations of H2O2 for 24 h, and then cell viability was detected. (**B**) Cells were treated with RDE for 24 h, and then cell viability was detected. (**C**) Cells were treated with 1.0 mM H2O2 for 3 h, and then treated with a medium containing different RDE concentrations for 24 h. Cells were seeded in a 96 well plate at a density of 2 × 10<sup>4</sup>/well and cultured for 5 d. Cell viability was expressed as a percentage of control (without H2O2 and RDE). The data were presented as mean ± SD, *n* = 5. Different lower case letters indicate a significant difference at *P* < 0.05.

H2O2 treatment induced a significant amount of IL-8 secretion and they were increased almost two-fold when compared with the control cells (NC) (*P* < 0.05) (Figure 2A). The 100 μg/mL of RDE reduced IL-8 secretion in the cells when compared with the treatment control cells (TC) (*P* < 0.05). However, IL-8 secretion was still higher in the cells treated with RDE (T) than in the control cells (NC) (*P* < 0.05). As shown in Figure 2B, the intracellular fluorescent intensity of cells treated with H2O2 (TC) was higher than those of the control cells (NC) and the incubation of H2O2-pretreated cells with RDE for 24 h reduced the ROS to the level of control. The H2O2 treatment increased total GSH content (*P* < 0.05) while the reduced GSH/ GSSS ratio remained the same (*P* > 0.05) when compared with the control cells (NC). The RDE treatment reduced both total GSH and GSSG (*P* < 0.05, Figure 2C) and had a higher reduced GSH/GSSG ratio when compared with both the NC and TC groups (*P* < 0.05, Figure 2D).

**Figure 2.** The effect of red-osier dogwood extracts (RDE) on interleukin (IL)-8 secretion (**A**), reactive oxygen species (ROS) level (**B**), glutathione (GSH) content (**C**), and reduced GSH/oxidized GSH (GSSG) (**D**) of Caco-2 cells after H2O2 treatment. NC indicates negative control: cells were cultured with a medium without H2O2 and RDE for 27 h (3 h + 24 h). TC indicates treatment control: cells were cultured with a medium containing 1 mM of H2O2 for 3 h, and then treated with a medium for 24 h. T indicates RDE treatment: cells were cultured with a medium containing 1 mM of H2O2 for 3 h, and then treated with a medium containing 100 μg/mL of RDE for 24 h. The data were presented as mean ± SD, *n* = 3. Different lower case letters and Greek letters indicate a significant difference at *P* < 0.05.

As shown in Figure 3A, the Nrf-2 protein abundance was lower (*P* < 0.05) in the cells treated with H2O2 (TC) than in the control cells (NC). The Nrf-2 protein abundance was higher (*P* < 0.05) in the cells treated with RDE (T) than in the cells treated with H2O2 (TC) but still lower (*P* < 0.05) than in the control cells (NC). HO-1 mRNA abundance was increased (*P* < 0.05) by 5.29 folds under H2O2 treatment compared with that of the control cells (NC) and HO-1 mRNA abundance was the highest (*P* < 0.05) in the cells treated with RDE (Figure 3B). H2O2treatment significantly decreased the mRNA

levels of SOD and GSH-Px, and the mRNA levels of the two enzymes were increased by followed RDE treatment (*P* < 0.05). However, there was no significant difference (*P* > 0.05) observed in the mRNA level of CAT among the three treatment groups (Figure 3C).

**Figure 3.** The effect of red-osier dogwood extracts (RDE) on the Nrf-2 protein level (**A**), HO-1 mRNA expression (**B**), and antioxidative enzymes mRNA levels (**C**) of Caco-2 cells after H2O2 treatment. NC indicates negative control: cells were cultured with a medium without H2O2 and RDE for 27 h (3 h + 24 h). TC indicates treatment control: cells were cultured with a medium containing 1 mM of H2O2 for 3 h, and then treated with a normal medium for 24 h. T indicates RDE treatment: cells were cultured with medium containing 1 mM of H2O2 for 3 h, and then treated with a medium containing100 μg/mL of RDE for 24 h. The data were presented as mean ± SD, *n* = 4. Different lower case letters and Greek letters indicate a significant difference at *P* < 0.05. Nrf-2: nuclear factor erythroid 2-related factor 2, SOD: superoxide dismutase; HO-1: hemeoxygenase-1; CAT: catalase; GSH-Px: glutathione peroxidase.

In Figure 4A, after a 3 h H2O2 treatment, the TEER values were significantly decreased (set as 0 h from RDE treatment) (*P* < 0.05). There was no significant difference (*P* > 0.05) observed in the TEER values between the TC and NC groups and between the NC and T groups when cells were further cultured for 24 h. However, the TEER value was higher (*P* < 0.05) in the cells treated with RDE (T) than in the treatment control cells (TC). As shown in Figure 4B, the leakage of FD4 was higher (*P* < 0.05) in the TC group than in the NC and T groups. However, there was no significant difference (*P* > 0.05) in the leakage of FD4 observed between the NC and T groups. As shown in Figure 5, the cytoskeletal structure of the β-actin fiber was partially disorganized in the TC group by H2O2 treatment, while incubation with RDE after H2O2 treatment could alleviate the damage induced by H2O2. The morphology of tight junction had similar changes as cytoskeleton after different treatments.

As shown in Figure 6A, ZO-1 mRNA abundance was significantly increased by H2O2 treatment (*P* < 0.05) while there was no difference (*P* > 0.05) observed in the mRNA abundance of claudin-1, claudin-3, and occludin when compared with the control cells (NC). However, the cells treated with RDE had a higher level of claudin-1, claudin-3, and occludin mRNA abundance and a lower level of ZO-1 mRNA abundance than those in the other two treatments (*P* < 0.05). As shown in Figure 6B, H2O2 treatment (TC) significantly decreased the protein abundance of ZO-1, claudin-1, claudin-3, and occludin (*P* < 0.05) when compared with the control cells (NC). RDE treatment (T) significantly increased the protein abundance of ZO-1 and claudin-3 (*P* < 0.05) when compared with the TC group.

**Figure 4.** The effect of red-osier dogwood extracts (RDE) on the transepithelial resistance (TEER) (**A**) and permeability (**B**) of Caco-2 cells after H2O2 treatment. NC indicates negative control: cells were cultured with DMEM solution without H2O2 and RDE for 27 h (3 h + 24 h). TC indicates treatment control: cells were cultured with a medium containing 1 mM of H2O2 for 3 h, and then treated with a medium for 24 h. T indicates RDE treatment: cells were cultured with a medium containing 1 mM of H2O2 for 3 h, and then treated with medium containing 100 μg/mL of RDE for 24 h. TEER was measured after H2O2 treatment for 3 h and after RDE treatment for 24 h, respectively. The data were presented as mean ± SD, *n* = 4. Different lower case letters and Greek letters indicate a significant difference at *P* < 0.05.

**Figure 5.** The effect of red-osier dogwood extracts (RDE) on the morphological changes of β-actin fiber and tight junction in Caco-2 cells after H2O2 treatment. (**A**) The morphological changes of β-actin and (**B**) The morphological changes of ZO-1. NC indicates negative control: cells were cultured with a medium without H2O2 and RDE for 27 h (3 h + 24 h). TC indicates treatment control: cells were cultured with a medium containing 1 mM of H2O2 for 3 h, and then treated with a medium for 24 h. T indicates RDE treatment: cells were cultured with a medium containing 1 mM of H2O2 for 3 h, and then treated with medium containing 100 μg/mL of RDE for 24 h. ZO-1: zonula occludens-1.

**Figure 6.** The effect of red-osier dogwood extracts (RDE) on the tight junction mRNA abundance (**A**) and protein abundance (**B**) of Caco-2 cells after H2O2 treatment. NC indicates negative control: cells were cultured with a medium without H2O2 and RDE for 27 h (3 h + 24 h). TC means treatment control: cells were cultured with a medium containing 1 mM of H2O2 for 3 h, and then treated with a medium for 24 h. T indicates RDE treatment: cells were cultured with a medium containing 1 mM of H2O2 for 3 h, and then treated with a medium containing 100 μg/mL of RDE for 24 h. The data were presented as mean ± SD, *n* = 3. Different lower case letters indicate a significant difference at *P* < 0.05. ZO-1: zonula occludens-1. The black bar, gray bar, and light black bar indicated the treatments NC, TC, and T, respectively.
