**1. Introduction**

Phytochemicals, also referred to as phytobiotics or phytogenics, are natural bioactive products derived from plants. They include mainly essential oils [1], flavonoids [2], terpenes [3], polysaccharides [4], and phenolic acid [5]. Currently, phytochemicals were studied widely because of their multiple functions in human and animal health [6,7]. Phytochemicals have broad spectrum functions that are antioxidative [8], anti-microbial [9], anti-inflammatory [10], and anticancer in nature [11]. Research has investigated the profile of phenolic compounds and antioxidant activity of finger millet varieties [12,13]. Twenty phenolic compounds were identified including 18 flavonoids with the predominant flavonoids of catechin and epicatechin. Chen et al. [14] revealed that the dominant phytochemicals in wheat were phenolic acids with high antioxidative capacity in vitro. In addition, the beneficial functions of soy isoflavone [15,16] and lutein [8] have drawn extensive attention recently. Burgeoning evidence indicates that phytochemicals extracted from the plants could be a good source of functional foods or potential additives for functional foods and animal feeds.

Red-osier dogwood (*Cornus stolonifera* Michx.) is native to North America and is commonly found in marshes, low meadows, and along river banks from Labrador to Alaska [17]. In Canada, it is a precious plant resource and rich in phenolic components, including anthocyanins with strong antioxidative activity [18]. At present, ground red-osier dogwood has been investigated in animal feeds to improve growth performance, feed intake, disease prevention, and meat quality [19,20]. Researchers [19,20] investigated the e ffects of ground red-osier dogwood supplementation on the digestibility, blood metabolites, and acute phase response in beef heifers. The results showed that ground red-osier dogwood could increase the feed digestibility and plasma concentration of haptoglobin and serum amyloid A in a ground red-osier dogwood dose-dependence. Dietary 4% ground red-osier dogwood supplementation attenuated oxidative stress and improved the growth performance of the *E. coli* infected weaned piglets, as revealed by Koo et al. [21,22] and Jayaraman et al. [21,22]. The beneficial e ffects of ground red-osier dogwood on animal growth and feed digestion might be associated with the oxidative homeostasis of the intestinal cells and the integrity of the intestinal barrier that are maintained by the antioxidative function of phytochemicals in the ground red-osier dogwood.

Cellular oxidative and inflammatory models have been widely used to investigate the potential mechanisms of phytochemicals involved in human and animal health. The intestinal porcine epithelial cell line J2 (IPEC-J2) and human colon adenocarcinoma cell line-2 (Caco-2) provide useful cell models for investigating the antioxidant mechanism of phytochemicals in the intestine [8,21,22]. Omonijo et al. [10] revealed the anti-inflammatory mechanism of thymol in the inflammatory models of lipopolysaccharide (LPS)-induced IPEC-J2 cells. Yang et al. [23] investigated the e ffects of resveratrol on the oxidative model of IPEC-J2 cells, and found that resveratrol protects IPEC-J2 cells from oxidative damage by stimulating the Nrf-2 pathway. Wu et al. [24] revealed that phytochemicals increase the expressions of tight junction proteins including occludin, zona occludens (ZO-1), and induce the upregulation of antioxidant enzymes including superoxidedismutase (SOD), hemeoxygenase-1 (HO-1), catalase (CAT), and glutathione peroxidase (GSH-Px) in the oxidative model of Caco-2 cells. A variety of adverse factors could cause the production of reactive oxygen species (ROS), resulting in oxidative damage of the intestinal barrier. Moreover, persistent oxidative damage could trigger the development of the intestinal diseases, including colon cancer [25]. Therefore, the intake of natural phytochemicals could maintain the health of the gu<sup>t</sup> and be considered an e ffective way to reduce the occurrence of intestinal diseases. Although ground red-osier dogwood has been investigated as potential animal feed additives to improve animal performance and meat quality, the mechanisms of ground red-osier dogwood in maintaining the oxidative homeostasis and the integrity of the intestinal barrier are still not fully understood.

In the present study, it is hypothesized that red-osier dogwood extracts (RDE) act as antioxidant agents capable of exerting protective e ffects against chemical-induced damage in intestinal epithelial cells. Therefore, an oxidative damage model of Caco-2 cells induced by hydrogen peroxide (H2O2) was established, and the mechanisms of RDE in recovering the intestinal cells from oxidative damage were investigated by examining the antioxidant system and the tight junction protein expression.

#### **2. Materials and Methods**

#### *2.1. Materials and Reagents*

RDE powder (moisture 7.3%; total phenolic content 26.51 gallic acid equivalent (GAE)/100 g including gallic acid 1670.09 mg/100 g and rutin 2745.49 mg/100 g) from leaves was provided by Red Dog Enterprises Ltd. (Swan River, MB, Canada). Hydrogen peroxide (H2O2), 2,7dichlorofluorescein diacetate (DCFH-DA), 4 kDa fluorescein isothiocyanate-dextran (FD4) and paraformaldehyde (PFA) were purchased from Sigma-Aldrich (Oakville, ON, Canada). Hanks' balanced salt solution (HBSS), DMEM/F12 medium, trypsin (0.25%), anti-anti (penicillin (100 IU/mL), streptomycin (100 μg/mL), and 0.25 μg/mL of amphotericin B) and fetal bovine serum (FBS) were purchased from Invitrogen (Fisher Scientific, Ottawa, ON, Canada). T-25 or T-75 culture flasks, Transwell permeable supports (0.4 μm pore size), and cell culture plates were purchased from Corning (Fisher Scientific, Ottawa, ON, Canada).
