1. Introduction
Changes in dietary components, the use of medications and vaccines, and mycotoxin contamination of feeds, among other things, may result in the excessive formation of reactive oxide species (ROS), which causes oxidative stress in pigs in the current intensive swine production [
1]. In particular, intestinal damage can result from severe oxidative stress [
2]. The primary sources of ROS generation are found in abundance in intestinal epithelial cells’ mitochondria [
3]. In addition to inducing apoptosis and preventing cell proliferation, ROS also interferes with intestinal function and retards intestinal development [
4,
5]. Therefore, dietary regulation is crucial to reducing intestinal damage brought on by oxidative stress.
The most recent type of cell death to be discovered is ferroptosis, which has recently been linked to oxidative stress [
6,
7]. The major features of ferroptosis are the buildup of iron ions in cells, the decreased ability of glutathione peroxidase 4 (GPX4) to repair lipid peroxidation injury, and the oxidation of polyunsaturated fatty acids including phospholipids [
8]. Ferroptosis cells exhibit morphological traits such as ruptured mitochondrial outer membranes, reduced or absent mitochondrial cristae, and damaged cell membrane integrity [
6]. In terms of biochemistry, ferroptosis may cause glutathione to be depleted and GPX4 activity to decline [
4].
Glycine is typically viewed as a nutritionally unnecessary amino acid because the body can produce it [
9]. However, a wealth of data has demonstrated that synthesized de novo glycine was unable to support piglets’ neonatal growth and development [
10,
11,
12]. More and more studies have recently claimed that glycine is vital for relieving oxidative stress and liver damage [
13,
14,
15]. Glycine is an important part of the antioxidant glutathione peroxidase (GSH-PX), which is a crucial regulator of ferroptosis [
16]. Glycine may, therefore, have the ability to alleviate ferroptosis.
In order to induce intestinal oxidative stress and damage in the weanling piglets, the pigs received an intraperitoneal injection of diquat. Intraperitoneal injection of diquat is a common and mature model to establish oxidative stress [
17]. Some studies showed that an injection of diquat induced a reduction in productive performance, organ injury and increased production of ROS and so on [
18,
19]. The goal was to determine if glycine might enhance intestinal health by modulating the anti-oxidative capability and ferroptosis signaling pathway in piglets’ intestinal mucosa.
4. Discussion
This experiment aimed to investigate whether intestinal cells would undergo ferroptosis after the establishment of diquat-induced oxidative stress model in piglets, and to explore the protective effect and mechanism of glycine supplementation diet on intestinal injury in weaned piglets.
The morphological changes in intestinal tissue may be a direct reflection of how well the intestinal barrier is functioning. Intestinal VH, CD, and VH/CD are crucial indicators of intestinal mucosal morphology [
25,
26]. Disaccharides need to be further broken down into monosaccharides by intestinal mucosal cells (such as sucrase, lactase, and maltase) in order to be absorbed, because intestinal epithelial cells cannot do this directly. As a result, the activity of intestinal mucosal disaccharides is one of the key markers indicating intestinal digestive capacity [
27,
28]. Protein, RNA, and DNA are indicators of intestinal growth and development and damage repair. Protein/DNA and RNA/DNA can indicate the ability of protein synthesis [
29,
30]. In this study, oxidative stress induced by diquat stimulation significantly changed the morphological structure of intestinal villi, and decreased the activity of disaccharidase, protein content, RNA/DNA, and protein/DNA in piglets. Consistent with our research, Xiao et al. showed that oxidative stress can lead to impaired intestinal barrier function in piglets [
31]. Xu et al. showed that oxidative stress can cause intestinal injury in piglets and significantly reduce the activity of intestinal digestive enzymes [
32]. The above results were consistent with the results of this study. Interestingly, dietary glycine ameliorated intestinal morphological and structural damage, and increased disaccharidase activity, protein content, RNA/DNA, and protein/DNA, which demonstrated that glycine may be able to positively regulate intestinal structural and functional damage caused by oxidative stress in this study. Similar to our results, a variety of studies have shown that dietary glycine can improve the intestinal barrier function, and promote the villus growth of jejunum and ileum, which is beneficial to the development of small intestinal mucosa of piglets [
30,
33].
Intestinal antioxidant capacity is closely related to intestinal health; however, diquat can induce oxidative stress and decrease intestinal antioxidant capacity [
34]. T-AOC displays the overall amount of total antioxidants in the body or organs, and MDA reflects the degree of peroxidation in the body, which is an essential marker of oxidative stress [
35]. GSH is a cofactor of GSH-PX, which is a natural free radical scavenger in animals. GSH-PX plays a significant function in antioxidant damage and can accelerate the reduction of lipid peroxides [
36]. In this study, it was found that under normal physiological conditions of piglets, dietary glycine significantly enhanced the activities of T-AOC in jejunum and GSH-PX in ileum. This may be because glycine is a vital precursor for the synthesis of GSH, and with the increase in dietary glycine, intestinal antioxidant capacity was enhanced. When piglets were exposed to oxidative stress induced by diquat, the activities of T-AOC and GSH-PX and GSH content in jejunum were significantly decreased, MDA content was significantly increased, and the activities of T-AOC and GSH-PX in ileum were significantly decreased. However, GSH-PX activity and GSH content in jejunum of piglets in the glycine supplementation group were significantly increased, MDA content was significantly decreased, and GSH content in ileum was significantly enhanced. These results suggested that glycine can enhance the antioxidant capacity and mitigate the damage of oxidative stress by increasing the activity of antioxidant enzymes in jejunum of piglets. Some studies have shown that supplementation of glycine in the diet can increase intestinal GSH content, enhance the intestinal antioxidant level and effectively relieve oxidative stress in mice [
36,
37]. Moreover, Hua et al. found that dietary glycine could increase the activities of T-AOC, GSH-PX, and GSH in the liver, reduce the content of MDA, and alleviate the oxidative stress induced by diquat injection in piglets [
38]. This further illustrated that glycine may improve intestinal antioxidant capacity by increasing the GSH content [
30,
33].
Intestinal injury is inevitably accompanied by cell death; however, it is possible that intestinal injury brought on by oxidative stress has a distinct cell death mechanism rather than the usual classical cell death mode [
39]. Ferroptosis is a non-apoptotic, iron-dependent cell death form that is intimately associated with the oxidative stress process. [
6]. In this study, the mRNA and protein expression levels of intestinal ferroptosis-related signaling pathway were detected to explore its influencing mechanism. System Xc
−/GPX4 signaling pathway is closely related to ferroptosis [
40]. TFR1 acts as a carrier to transfer iron ions (Fe
3+) into the cell’s inner membrane when cells undergo ferroptosis. HSPB1 is a molecular chaperone of several small heat shock proteins, which can reduce the concentration of Fe
3+ by inhibiting the expression of TFR1, thereby alleviating the intensity of ferroptosis [
41]. System Xc
− is composed of SLC7A11 and SLC3A2 subunits through disulfide bonds, which can transfer glutamate out of the cell through the cell membrane and cystine into the cell membrane at the same time. Finally, cystine is converted into cysteine and GSH is synthesized. GSH can reduce ROS under the action of the antioxidant enzyme GPX4 and alleviate oxidative damage to cells [
42]. In this study, under diquat stimulation, the mRNA expressions of TFR1 and GPX4 in jejunum and TFR1 and HSPB1 in ileum were significantly increased, while the mRNA expression of SLC7A11 in ileum was significantly decreased, indicating that diquat stimulation caused a large amount of Fe
3+ to enter cells, leading to oxidative stress in the intestine. At this time, the body alleviated oxidative stress injury by enhancing the activities of GPX4 and HSPB1. Meanwhile, dietary glycine significantly increased the mRNA expressions of SLC7A11 and GPX4 in jejunum and ileum, and significantly decreased the mRNA expression of TFR1 in jejunum and ileum after diquat stimulation. These results indicated that glycine can enhance the intestinal antioxidant system to inhibit the occurrence of ferroptosis, which is consistent with the results of intestinal antioxidant capacity. According to the results of mRNA expression and protein expression, glycine significantly reduced the expression of TFR1 protein in jejunum and ileum after diquat stimulation, and significantly increased the expression of GPX4 protein in jejunum. Consistent with the results of many studies, it was found that the expression of the GPX4 gene could inhibit iron death and alleviate cell damage [
43,
44,
45]. Xu et al. have shown that dietary glycine can effectively inhibit the expression of TFR1, a key gene of ferroptosis, and promote the expression of GPX4, thereby alleviating liver injury induced by diquat [
38]. These results suggested that dietary glycine can enhance the synthesis of GPX4, inhibit the occurrence of ferroptosis, and then protect the gut from the damage caused by ferroptosis.