1. Introduction
Amino acids play fundamental roles in the optimum functionality of the intestine with increasing evidence of regulation of intestinal barrier function by concentration of amino acids in the diets [
1]. There is evidence for the role of amino acids such as glutamine (Gln), arginine (Arg), and threonine (Thr) having positive effects on controlling permeability, promoting cell proliferation, and stimulating several metabolic pathways such as mTOR [
1,
2].
As an essential amino acid for chickens, mounting evidence is emerging on various fundamental roles that Arg plays in different metabolic pathways, regulation of intestinal function, and ultimately protein synthesis and performance. For example, the synthesis of nitric oxide and polyamines is dependent on Arg [
3]. Production of nitric oxide along with enterocyte migration is crucial for restoration of epithelial continuity [
4]. Therefore, Arg can modulate immune response, inflammation, and process of recovery from injury or stress. Recent data with poultry suggest that there is a potential for increasing feed specification for Arg beyond current industry practices to support optimum growth and intestinal functions [
5]. Supplementation of Arg can therefore improve the gut barrier function as shown by reducing the ileal permeability measured in Ussing chambers by Zhang, et al. [
6]. The specific role of Arg in low protein diets for intestinal barrier function has recently been investigated [
7] and showed supplementing additional 5 g/kg Arg positively impacted feed efficiency and some aspects of intestinal permeability and tight junction expression. However, such results need to be confirmed. More importantly, additional Arg may not be economically feasible at such high supplementation level. Therefore, the combination of amino acids or specifically tailored products may be suitable alternatives. Gln and Thr also serve as functional amino acids with specific effects on intestinal permeability and immune function of broiler chickens [
8,
9]. The combination of these amino acids is hypothesized to influence intestinal barrier function by promoting growth performance, anti-inflammatory effects, and underlying mechanisms associated with such response.
Polyphenolic compounds including flavonoids are commonly plant-derived compounds known for having antioxidant capacity and protecting different cells from oxidative damage [
10]. With growing evidence for some phenolic compounds to improve gut health and performance of broiler chickens, it is hypothesized that inclusion of grape extract containing flavonoids alongside amino acids (as part of an amino acid-based supplement) may further enhance the intestinal barrier function and reduce stress-induced intestinal inflammation. To our knowledge, there is no previous research in which such approach has been tested to investigate the mode of action of different combinations of amino acids or polyphenols in counteracting intestinal inflammation, enteric and stress-related issues in broiler chickens.
Intestinal barrier dysfunction can be induced by various physiological or nutritional factors [
11]. One such model is through the use of dexamethasone (DEX), a synthetic glucocorticoid (GC) that causes increased intestinal permeability, differential expression of tight junction proteins, and increased inflammation [
12]. As the use of GC can increase gluconeogenesis and therefore accelerate catabolism of amino acids [
12], this model may be useful to investigate possible counteracting and mechanistic roles of amino acids at least at the intestinal level.
Thus, the current project was conducted to investigate the effects of additional Arg; a combination of Arg and Gln; or an amino acid-based solution containing Arg, Gln, Thr, and grape extract on performance, intestinal barrier function, and mechanistic genes involved in gut barrier function, inflammation, protein synthesis, and apoptosis using DEX as a gut barrier dysfunction model.
4. Discussion
The growth performance results of the current project exceeded the Ross 308 standards for body weight on day 35 [
23]. In line with previous research [
7], additional Arg improved FCR at any stage of the study except for the finisher phase. It appears that for optimal feed efficiency of broilers fed reduced protein diets, a higher concentration of Arg than the current industry recommendations may be required. Noteworthy, a combination of Arg and Gln was equally effective in improving the FCR. This result may be related to the presence of corn in the diets compared with previous studies [
7,
24] conducted with wheat-based diets in which Gln failed to improve growth performance of the birds. Broilers fed corn-based diets are more conducive to Gln supplementation due to lower concentration of Gln in corn relative to wheat particularly when reduced protein diets are used [
25]. The positive growth performance by feeding a combination of Gln and Arg may also be explained by their stimulatory effects on gut development (e.g., provision of energy source for enterocytes, synthesis of polyamines), immune response and function, as well as epithelial cells regeneration [
2,
26].
An amino acid-based solution with the combination of Arg, Gln, Thr, and grape extract with only 1 g/kg dietary inclusion was able to improve performance similar to other treatments. Noteworthy, in the experiment, the objective was not to separate the effect of all individual amino acids or grape extract but rather investigate their combined effects on promoting growth performance and mechanisms surrounding intestinal barrier function of broiler chickens. Therefore, the main part of the study utilized DEX injections as a gut barrier dysfunction model. There was no interaction between DEX and diet for performance-related parameters and permeability, which was expected given the response of birds is markedly higher to DEX than diets. The performance data of experiment 2 were only presented as indicative of the birds’ status. The lack of diet effect on performance parameters of these individual birds is explained by the relatively short stay in cages (one week) as opposed to the group-housed birds on raised floor pens in experiment 1.
Repeated injections of DEX as a synthetic GC cause strong and wide-ranging effects on intestinal barrier function and inflammation by induing stress-like response when preferentially binding to type II GC receptors [
12]. At the systemic level, suppression of immune response was expected as evidenced by atrophy of bursa and spleen observed in the current study, while there were also substantial metabolic consequences as shown by hepatic enlargement in this study that may be the result of metabolic changes in processes such as lipogenesis and steatosis [
27]. On the other hand, dietary treatments reduced the weight of liver independently, which may be related to a possible reduction in inflammation or changes in fat accumulation and need further investigation.
Intestinal epithelial cells play crucial roles in maintaining the intestinal barrier to avoid entry of harmful and unwanted bacterial toxins and pathogens while allowing passage of essential nutrients, water, and ions. Intestinal permeability, as the key function of the intestinal barrier, is controlled by tight junction proteins.
Claudins 1 and
3 are among barrier-forming tight-junction proteins and alongside other tight junctions such as zonula occludens from strong crosslinks with F-actin and myosin from the membrane cytoskeleton [
28]. Changes in claudin levels and
ZOs are expected to influence gut barrier function and permeability. In the current study, the ileal tissue was selected to study the gene expression including that of tight junction proteins as it was found in other studies that gut barrier-related genes were comparatively more responsive in the ileum than jejunum in broiler chickens [
7,
12]. Transcriptional expression of
claudin 1 and
3 as well as
ZO1 and
ZO2 remained unaffected by dietary treatments supplemented with tested amino acids. However, overexpression of
Claudin 3 and
ZO2 in DEX-injected birds was consistent with a previous study [
12]. Although increased expression of these tight junctions is not in line with increased permeability observed in DEX-injected birds, it may be related to a compensatory response to restore the intestinal barrier in the challenged birds. This is possible as the tissue sampling was conducted almost 24 h after the last DEX injection in the current experiment. The intestinal epithelial cells and the tight junction proteins are highly dynamic and rapidly replaced [
28], therefore, it is possible that within that timeframe these proteins were highly expressed to restore the dysfunctional intestinal barrier.
In the present study, the FITC-d was given proportionate to the BW of the birds while in two other studies in our laboratory the same dosage of FITC-d was given to the birds regardless of their BW [
7,
12]. Consistently, there was still a significant increase in FITC-d concentration in serum of birds, which may indicate that the damage to the intestinal barrier and tight junction integrity is more critical than BW or dosage of FITC-d for detection of differences in intestinal permeability in broiler chickens. There was a trend for change in intestinal permeability measured by FITC-d associated with diets in which Arg and then MIX tended to decrease the intestinal permeability in a higher proportion than other treatments. This observation concurred with a previous study [
7] in the case of Arg. As a functional amino acid, Arg can impact epithelial integrity through its secondary functions including production of nitric oxide and polyamines [
29]. However, the response to Arg observed in this study cannot be explained by gene expression of selected tight junction proteins and there may be other mechanisms involved.
Amongst various cytokines,
IL1β, a proinflammatory cytokine, is involved in the development of intestinal inflammation and depending on its concentration can increase intestinal permeability [
30]. This increase in permeability at least in part is reported to be associated with upregulation of myosin light chain kinase [
30]. At a therapeutic dosage, DEX is expected to suppress the
IL1β, but the results obtained in this study are consistent with a previous study highlighting that repeated injections of DEX indeed upregulated the expression of selected cytokines including
IL1β at the transcriptional level in ileal tissues [
31]. In the current experiment, birds injected with DEX had a higher intestinal permeability (elevated FITC-d in serum) in all dietary treatments but showed a complex interaction for the gene expression of
IL1β in birds fed different diets. All amino acid supplements were able to restrain the overexpression of
IL1β compared to control birds, but the extent of response was more pronounced for Arg and Arg-Gln fed birds.
In general,
IL-10 is produced by activated macrophages and is an anti-inflammatory cytokine with a higher expression that enhances the intestinal barrier function [
32]. Although
IL-10 can be expected to be inhibited by therapeutic doses of GC, excessive or repeated injections of DEX can be detrimental, leading to upregulation of
IL-10 [
33], which concurs with the results of our study. Besides,
IL-10 may have immunostimulatory properties on the production of
IFN-γ, which can counteract its anti-inflammatory properties [
34]. The overexpression of
IL-10,
IFN-γ, and
IL-1β in birds injected with DEX in the current study may therefore suggest that their transcriptional expression was affected by the concentration of one or another despite their different functions. Both Arg and Gln were shown to reduce proinflammatory cytokines. For Arg, the signaling pathways include
NF-κB,
iNOS, and MAPK, while for Gln,
MAPK/ERK,
mTOR, and
NF-κB are involved in reducing the intestinal inflammation [
35]. Similarly, Thr can reduce intestinal inflammation by increasing MUC-2, IgA, and barrier function via
NF-κB,
mTOR, and
MAPK [
35]. We studied
NF-κB,
mTOR, and
MUC-2, which all remained unaffected by the experimental treatments. This suggests that other mechanisms may have been involved in the response to the tested amino acid combinations for affecting expression of
IL1β as well as changes in the intestinal permeability. Whole RNA sequencing may provide insights into possible mechanistic explanations in future studies.
Apoptosis is the programmed process of death in various cells including intestinal epithelial cells. When apoptotic pathways are disrupted, unwanted, damaged or infected cells cannot be fully eliminated. In this study, two apoptotic regulating proteins of
BCL-2 and
Caspase-3 were studied by mRNA expression. These two genes were not influenced in this study by either DEX or the tested supplements. Glucocorticoids are expected to promote apoptosis and reduce cell proliferation as they increase oxidative stress in various cells [
36]. The lack of effect in this study may be related to sampling time or involvement of other pathways not specifically investigated here. Various amino acids [
37] and grape extracts are also shown to affect pathways of apoptosis but none of the treatments affected the selected apoptotic-related genes studied here.
mTOR Complex 1 pathways are known to be affected by dietary amino acids that in turn affect protein syntheses. In this study, three genes related to this pathway including
mTOR,
Raptor, and
RPS6KB1 were studied. Only
Raptor was upregulated in birds fed MIX, which had a combination of Arg, Thr, Glu, and grape extract only compared with birds fed a combination of Arg-Glu. The upregulation of
Raptor is shown to promote protein synthesis and it interacts with
mTOR to form a nutrient sensitive complex [
38]. Therefore, it is possible that the addition of Thr and grape extract may have had some positive stimulatory impacts on
mTORC-1 through activation of
Raptor. Given the nature of the product tested in this study, it is not possible to pinpoint the effect to a particular amino acid, but this positive impact is consistent with improved feed efficiency observed in this group of birds even though a lower concentration of amino acids were fed compared with other treatments. The positive effect of Arg on gene expression of
Raptor and
RPS6KB1 is shown by other researchers [
5].
Glucose absorption through the intestinal epithelium is facilitated by
SGLT1 that are apically located and play an important role in making the glucose available for maintaining cellular and organic functions. While an increase in
SGLT-1 can be perceived positive for enhancing growth performance and gut development [
39], its overexpression under stress conditions may increase paracellular permeability [
40] and promote fat deposition [
39]. As there was no significant change in intestinal permeability in birds fed a diet supplemented with MIX, the increased expression of
SGLT-1 may have been a possible result of a synergistic stimulatory effect of grape extract and a combination of tested amino acids, which deserves further investigation. Apart from transferring peptides,
PepT-1 can be upregulated in a cell survival mechanism under stress conditions [
41]. The lack of changes in expression of
PepT-1 in the current experiment was somewhat unexpected as both lower feed intake [
42] and the stress stimulated by DEX [
12] are shown to upregulate expression of
PepT-1.
Under various stress conditions including physiological stress, heat shock proteins such as
HSP70 are expected to be highly expressed as an endogenous protection mechanism to maintain cellular hemostasis and prevent apoptotic processes [
43]. Noteworthy, the measurement of this gene was unaltered in the current study by either diet or DEX challenge. However, as an enzyme involved in synthesis of glutathione,
GSS was affected independently by both diet and DEX. The response to DEX by upregulation of
GSS may indicate an oxidative/antioxidative balance response under physiological stress induced by GC. In future studies, measurement glutathione content may provide more insight into the potential interactive effect of treatments under stress stimulated with DEX. Among tested treatments, MIX containing grape extract caused the highest expression of
GSS, which may be related to flavonoids present in that treatment as dietary grape proanthocyanidins are shown to improve antioxidant indices such as glutathione content [
44].