In the early stage of raising caged laying ducks, the pressure level changed substantially, which not only caused a certain degree of liver tissue damage, but also increased the expression of inflammatory injury factors . Our previous study found that during the peak of stress response in laying ducks, the content of VA/RA in serum changed. At the same time, the caged laying ducks showed intestinal barrier dysfunction, and the related genes affecting RA biosynthesis in the intestine changed. Therefore, we believe that VA/RA is related to intestinal barrier function. We designed the experiment to investigate the effect of VA on intestinal barrier function. VA is a multifunctional vitamin involved in a variety of biological processes. Its control of the immune system and function may not only be the most effective for development but also for protective or regulatory adaptive immunity . This effect is especially important at the intestinal border where dietary vitamin A is first absorbed. Most of the effects of vitamin A are produced by its metabolite retinoic acid (RA). Li et al found that vitamin A increased Toll-like receptor 4 (TLR4) expression through its receptor, thereby enhancing intestinal barrier function in colonic mucosal cells, suggesting the connection between inflammation and VA supplementation .
Changes in organisms during stress are postulated to be linked to accumulating lipid peroxidation products, such as free radicals, peroxy radicals, hydroperoxides, aldehydes and ketones . Lipid peroxidation is defined as a parameter of oxidative stress . T-SOD acts as a catalyst in the removal of toxic superoxide radicals . An elevated level of T-SOD activity is a defense mechanism against an increased level of free radicals under stressful conditions . After H2O2 treatment, significant differences in T-SOD, T-AOC and MDA levels were observed between the CG1 and those in the CG2. In this experiment, the activity of T-AOC and T-SOD was increased and the content of MDA was decreased in cells treated with appropriate concentration of VA/RA, which proved that VA/RA improve the level of antioxidant in cells. It has been consistently reported that TEER is one of the most commonly used indicators for the permeability of intestinal epithelial cells [17, 18]. The enhancement of barrier function was manifested by the decrease of paracellular permeability and the increase of TEER. In the present study, we determined that the addition of VA/RA increased cell viability after oxidative stress stimulation. The TEER test showed that VA metabolism affected intestinal permeability. Primary IECs using duck embryos have basic structure, metabolism and functions comparable to intestinal epithelial cells in vivo and can be used as a good model for studying poultry gastrointestinal response mechanisms under stress conditions . Oxidative stress generates a large amount of reactive oxygen species (ROS). Studies have shown that ROS can cause endoplasmic reticulum stress leading to apoptosis . Destruction of the intestinal barrier function is related to intestinal epithelial cell apoptosis . Studies have shown that vitamin A reverses LPS-induced intestinal barrier damage by increasing the expression of intestinal barrier proteins . However, research on the specific regulatory mechanism is still very limited, and comprehensive, in-depth and continuous research is still needed. We used RNA-seq to screen differentially expressed genes, and study the protective mechanism of VA/RA on H2O2-induced oxidative stress in primary duck IECs.
Antioxidants and lipid peroxidation are key factors in the response to stress. Lipid peroxidation is defined as a parameter of oxidative stress. Peroxisome proliferator-activated receptors (PPARs) can regulate the function and expression of complex gene networks, such as energy homeostasis and inflammation [22, 23]. This family includes three known members: PPARα, PPARβ and PPARγ, also known as NR1C1, NR1C2 and NR1C3. PPARγ is related to oxidative stress, and oxidative stress can activate a variety of transcription factors, including NF-κB, P53, PPAR γ and Nrf2 .
The role of PPARγ in oxidative stress is mainly to inhibit damage and promote cell survival, in this defense system, PPARγ does not act alone, but forms a positive feedback loop with Nrf2 to exert its anti-inflammatory effect and inhibit NF-κB pathway . Nrf2 is a key transcription factor in oxidative stress that responds to cell damage induced by high levels of ROS. It binds to the antioxidant response element (ARE) and mediates the expression of heme oxygenase-1 (HO-1), which plays an important role in inhibiting cell oxidative damage and is the first line of defense against oxidative stress damage [27, 28]. HO-1 reduces oxidative stress, inflammation, and apoptosis by reducing the production of ROS . PPARγ upregulates HO-1 expression and protects cells from endothelial cell toxicity induced by TNF-α . Studies have shown that retinoic acid can reduce H2O2-induced cell oxidative stress damage by upregulating Nrf2/HO-1 expression, which is an important cytoprotective mechanism against oxidative damage [31, 32]. In this research, the relative gene expression levels of PPAR, Nrf2 and HO-1 in the TGs were significantly higher than those in the CG2. The results showed that treatment with VA/RA = 1 × 10− 8 M:3 × 10− 8 M can improve the antioxidant function of IECs under oxidative stress, which may be achieved by regulating the PPAR-mediated Nrf2/HO-1 signaling pathway.
HO-1 is involved in endothelial barrier dysfunction induced by oxidative stress, such as intestinal epithelial dysfunction [33, 34]. Previous studies have found that HO-1 can protect intestinal epithelial cells from oxidative damage by upregulating tight junction protein (TJP) levels [35, 36]. The integrity of epithelial structure and function depends on the normal function and regulation of tight junction proteins, including claudins and ZOs. Studies have shown that changes in ZO-1 and claudin-1 are related to intestinal epithelial dysfunction [21, 37]. KEGG pathway analysis revealed that the signaling pathways were mainly enriched in the categories of cell adhesion molecules, immune response and cell regulation. Cell adhesion molecules are involved in focal adhesion and tight junctions. Similarly, qRT-PCR results showed that H2O2 stimulation can cause downregulation of ZO-1, claudin-1, and Occludin expression, which indicates that these TJP are essential in abnormal intestinal barrier function, and the addition of VA/RA leads to upregulation of TJP expression.