**4. Discussion**

Numerous studies have shown that the occurrences of the intestinal disease are associated with a defective barrier function that was caused by oxidative damage [27]. Therefore, controlling oxidative damage and repairing the intestinal barrier could be effective ways to prevent many intestinal diseases. Phytochemicals have been reported to reduce the production of cellular ROS [28] and the incidence of intestinal inflammation [10]. The supplementation of ground red-osier dogwood in feeds decreased the serum malondialdehyde content and increased the SOD levels in the *Escherichia coli* F4 K88<sup>+</sup> infected animals, as reported by a previous study [21]. In the present study, an oxidative damage model was established by using H2O2 in Caco-2 cells, aiming to evaluate the therapeutic effects of RDE on intestinal oxidative damage.

To establish an H2O2-induced oxidative injury model with Caco-2 cells, the dosage effects of H2O2 on cell viability was investigated firstly. The results showed that the H2O2 treatment significantly reduced cell viability at more than 0.8 mM, indicating that a high dosage of H2O2 inhibited the growth of the cells or was toxic to the cells. These findings are consistent with previous studies of H2O2 and Caco-2 cells [29]. However, a wide concentration range of RDE (up to 200 μg/mL, equal to 53 μg/mL total phenolic compounds) did not show a negative effect on cell viability, suggesting that higher RDE containing higher phenolic compounds might have better pharmaceutical effectiveness. Moreover, our results demonstrated that 100 μg/mL of RDE completely recovered the cell viability that was compromised by 1 mM H2O2. Therefore, our results sugges<sup>t</sup> that RDE exerts protective effects against H2O2-induced oxidative damage in intestinal epithelial cells in vitro.

H2O2-induced oxidative stress triggered an imbalanced redox state and excessive ROS accumulation in Caco-2 cells, which in turn induced inflammatory responses evidenced by an increased IL-8 level. IL-8, a chemokine that can be produced by epithelial cells, has been recognized as an indicator of the inflammatory response [30]. The mediators can activate signal transduction cascades as well as inducing variations in transcription factors, such as Nrf-2, which mediate immediate cellular stress responses [31]. The oxidative stress and inflammatory responses together exacerbated the damage of both cellular structure and biomacromolecules. After a 3 h treatment with H2O2 and following a 24 h treatment with 100 μg/mL of RDE, the viability of Caco-2 cells was significantly restored, accompanied by decreased levels of IL-8 and ROS, indicating the inflammatory responses and oxidative damage were alleviated. The anti-oxidative components in RDE including rutin and phenolic acids are highly effective at scavenging oxygen radicals [32].

A recent study shows that rutin attenuates inflammatory responses induced by lipopolysaccharide in an in vitro mouse muscle cell (C2C12) model and supplementation of rutin or rutin-containing plant extracts may hold promising potential for attenuating oxidative stress and inflammation [33]. In addition, RDE increased the ratio of reduced GSH/GSSG, and reduced the consumption of reduced GSH. The results indicate that RDE can alleviate the occurrence of inflammation and oxidative damage by scavenging ROS production directly.

To further investigate the therapeutic mechanism of RDE on H2O2-induced oxidative damage, the protein expression of Nrf-2 and gene expression of responsive gene HO-1 and antioxidative enzyme SOD, CAT, and GSH-Px were measured. The protein expression of Nrf-2 was induced (Figure 3A) and the mRNA expression of HO-1 was significantly upregulated after the addition of RDE (Figure 3B). In addition, the mRNA abundance of SOD and GSH-Px also showed the same trend as HO-1 (Figure 3C). This study revealed that RDE activated Nrf-2, mediated the antioxidant gene expression, and enhanced the antioxidant capacity of cells, thereby preventing the inflammation response and oxidative damage.

The intact gu<sup>t</sup> barrier is important to prevent cell damage and restore cellular function caused by intestinal inflammation [10] and oxidative stress [34]. The TEER and permeability, as measured by FD4 di ffusion, were often used to evaluate the integrity and tightness of the barrier formed by epithelial cells. Caco-2 cells were measured until the stable TEER formed by the tight junction. The permeability of the barrier after the 24 h treatment was also investigated by measuring the fluorescence intensity of FITC-Dextran (FD4) in the basolateral side di ffused from the apical side. The H2O2 treatment lowered the TEER value and increased the permeability, suggesting that H2O2 treatment led to the increase in cell permeability [35], and resulted in a defective barrier function of cells. However, TEER values were increased and permeability was decreased by RDE treatment for 24 h, indicating that the barrier function of Caco-2 cells was improved significantly. A recent study showed that intact epithelial barrier functions are necessary in order to maintain the homeostatic state in response to physiological host-gut microbiome cross-talks, and therefore the maintenance of epithelial barrier function is crucial for alleviating gu<sup>t</sup> inflammation [36]. It has been reported that phytochemicals extracted from apples can increase the TEER value of Caco-2 cells [37], and this might be attributed to the antioxidative e ffects of phytochemicals.

The morphology of the cytoskeleton and tight junction was visualized by β-actin and ZO-1 immunofluorescent staining. H2O2 treatment disorganized the β-actin fibrin and di ffused cell tight junction protein ZO-1. This indicates that H2O2 caused oxidative damage to the cell membrane monolayer. The addition of RDE obviously restored cell structure and stabilized morphological characteristics of ZO-1.

In this study, the mRNA and protein abundance of tight junction proteins, including ZO-1, claudin-1, claudin-3, and occluding, were determined. When compared with the sole H2O2 treatment group, RDE supplemented to cells enhanced the mRNA abundance of tight junction protein claudin-1, claudin-3, and occludin, but decreased the ZO-1 mRNA level in the Caco-2 cells. Firstly, this might be attributed to the fact that the sensitivity of gene expression of the tight junction proteins was di fferent when responding to H2O2 [38]. The transcription of ZO-1 was more sensitive to H2O2, and it could rapidly respond to oxidative stress. Secondly, it may also be related to the ordinal expression of di fferent tight junction proteins. Within 24 h after H2O2 treatment, intracellular H2O2 was still playing a role in cells, and the expression of ZO-1, which was sensitive to oxidative stress, was induced. However, when cells were treated with H2O2 for 3 h, followed by RDE incubation of cells for 24 h, ROS was partially removed by RDE due to its strong antioxidant e ffect. Therefore, the expression of ZO-1 was not upregulated.

Interestingly, claudin-3 and occludin could be upregulated by RDE treatment, indicating that there were some unknown components in RDE that could regulate claudin-3 and occludin gene expression. In terms of tight junction proteins expression, H2O2 treatment reduced the contents of ZO-1, claudin-1, claudin-3, and occludin significantly. This was not completely consistent with their mRNA expression results. Firstly, it can be attributed to protein expression lagging behind mRNA expression [24]. In addition, H2O2 treatment significantly disrupted the structure of tight junction proteins, resulting in a decrease in the protein content. After RDE treatment, the protein content of ZO-1 and claudin-3 increased significantly, but the protein content of claudin-1 and occludin did not increase. It was also evident from the staining results that the ZO-1 structure has also been significantly repaired to enhance the cellular barrier function and viability. Based on our observations in this study, there could be two reasons to explain why RDE enhances cellular barrier function. Firstly, RDE is rich in antioxidants [19,32] that have the ability to scavenge ROS directly and repair the structure of tight junction proteins. Moreover, RDE is rich in unknown components that activate the Nrf-2 pathway to enhance the antioxidant capacity of cells and induce the expression of tight junction proteins.
