Heat-Induced Oxidative Stress and Use of Astaxanthin on the NF-kB, NFE2L2 Transcription Factors and Cytoprotective Capacity in the Thymus of Broilers

1. Introduction

The thymus is a crucial central lymphoid organ in animals, playing an integral role in the immune response by serving as the site for T cell development, differentiation, and maturation [1,2,3]. These mature T cells subsequently colonize secondary lymphoid organs to combat invading pathogens [4]. Beyond its role in adaptive immunity, the thymus is also a critical mediator of innate immune responses, providing protection against tumors, pathogens, and tissue damage [5]. The integrity of the thymus is essential for maintaining tissue homeostasis and a fully functional immune system. Thymic injury can lead to immune impairment, resulting in significant consequences due to the development of an immature immune system, which can leave the organism immunocompromised [6,7,8].

Stress represents a physiological and biochemical defense mechanism through which the body responds to adverse environmental effects. This response helps the organism adapt to its environment and maintain internal equilibrium [9]. While moderate stress can enhance immunity, excessive stress can negatively impact growth, development, and production performance in animals. Importantly, it can also lead to immune suppression, increasing susceptibility to diseases and potentially resulting in death [10]. Persistent oxidative stress (OS), in particular, impairs immune function through mechanisms such as cellular DNA damage and biomolecule fragmentation [11,12,13]. Normally, the body's oxidation-antioxidant system maintains a dynamic balance, but OS occurs when the antioxidant (AOX) defenses are overwhelmed [14,15]. This condition can lead to slower growth rates, decreased feed conversion efficiency, reduced production performance, and in severe cases, significant economic losses in industries like poultry farming [16,17]. OS initiates various signaling pathways and inflammatory responses, further compounding its impact on health [18].

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling pathway is a key mediator of OS-induced inflammation [19,20,21,22]. Activation of NF-kB leads to the production of inflammatory cytokines, which are part of the body's response to harmful stimuli. Inflammation, closely linked to the immune system, is a pathological response to such stimuli [23]. Additionally, the transcription factor nuclear factor, erythroid 2-like-2 (NFE2L2/NRF2) plays a significant role in cytoprotection by stimulating the expression of AOX and detoxifying enzymes, including NAD(P)H:quinone oxidoreductase-1 (NQO-1), glutathione S-transferase (GST), and heme-oxygenase-1 (HO-1) [24]. The AOX defense system comprises both enzymatic AOXs (such as SOD, peroxidase, CAT, and GPx1) and non-enzymatic AOXs (including vitamin E, carotenoids, and vitamin C). An imbalance between oxidative and AOX systems can lead to excessive reactive oxygen species (ROS) and reactive nitrogen species (RNS) accumulation, resulting in the destruction of lipids, proteins, and nucleic acids, ultimately causing cellular damage and cell death. OS is thus implicated in a variety of pathological conditions, including inflammatory reactions and tumor development [25,26,27]. Peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear receptor that regulates the genes involved in lipid metabolism, fatty acid oxidation and inflammation, and plays a crucial role in maintaining metabolic homeostasis. PPARα has been shown to produce significant anti-inflammatory effects [28,29] and inhibit the activation of NF-kB [30].

Heat-induced OS poses a particular challenge in animals, affecting their ability to metabolize carotenoids, which necessitates dietary supplementation [31]. Astaxanthin (AST), a potent lipid-soluble AOX from the carotenoid family, has demonstrated exceptional free radical scavenging and anti-inflammatory properties [32,33,34,35]. Its unique structure, featuring hydroxyl and keto groups on each ionone ring, enhances its AOX activity, thereby protecting cellular membranes from oxidation [36]. Despite its presence in aquatic organisms and birds, AST cannot be synthesized by animals and must be obtained from dietary sources such as green algae, red yeast, and crustacean byproducts, with Haematococcus pluvialis being a particularly rich source [37,38]. Supplementation with AST has shown numerous health benefits, including anti-inflammatory, immunomodulatory, cardiovascular, neuroprotective, and anticancer effects [39]. In poultry farming, particularly under heat-stress conditions, AST holds promise as a natural AOX feed supplement, potentially mitigating the adverse effects of heat stress and improving overall health and productivity.

The main objectives of the present study were to investigate the protective properties of AST against heat-induced oxidative stress and apoptosis in the chicken thymus. Our research question is, “How does heat-induced oxidative stress affect the NF-kB, NFE2L2, PPARα, cytoprotective capacity genes and apoptotic pathways in the broiler thymus, and can astaxanthin help mitigate the stress?” We hypothesize that heat-induced oxidative stress in the broiler thymus correlates with poor growth performance, disrupting the physiological and biochemical defense mechanisms; and H. pluvialis-derived astaxanthin dietary supplementation mitigates the effects through modulation of transcription pathways.

2. Materials and Methods

2.1. Ethics Statement

Animal protocol (Protocol No. 17-2605) used in this study was approved by the University of Hawaii Institutional Animal Care and Use Committee (IACUC). Animals were raised under animal welfare guidelines and euthanized in accordance with humane protocols in preparation for necropsy.

2.2. Experimental Animal Design

Cob 500 unsexed broiler chicks were obtained from Asagi Hatchery (Honolulu, Hawaii, USA). Several mitigation strategies were simultaneously tested in parallel and the findings reported separately based on treatment. In this trial the feed additive treatment is AST. The animals were raised from Day 0 to 6 weeks in deep litter pens with pine shavings on concrete flooring, four birds in each pen. The animals were reared under two temperature conditions: TN (n=24) at 21-22°C and 50% RH, and HS (n=36) at 32-35°C and 42-50% RH. Animals were provided a normal starter feed from 0 to 21 days, and a normal finisher feeds on 22-42 days with free access to feed and water. Nutritional compositions of the supplemented diets are listed in Table 1. After 14 days, the HS group was further divided into two dietary regimens, i.e., basal diet HS (n=18, treatment 1), and basal diet with 1.33 mg/kg AX supplement HSAX (n=18, treatment 2). The light cycle was set at 1:23 dark:light cycles throughout the trial. The broilers were euthanized on day 42, and ileum tissue samples from randomly selected 6 birds of each group were collected at necropsy.

2.3. Astaxanthin-rich Dietary Supplement

The diet was supplemented with P25HB provided by AstaReal®, Inc. (Burlington, New Jersey, USA). PH25B contains 2.5% (w/w) dried Haematococcus pluvialis-algae, and other components including modified starch, gum Arabic, mixed tocopherols, L-ascorbyl palmitate, silicon dioxide, xanthan gum, γ –cyclodextrin, polysorbate 80, rosemary extract and ferulic acid. A comparable nutritional composition of H. pluvialis-algae is listed in Appendix A (Table A1). The natural forms of astaxanthin comprise of mainly mono-esterified, followed by di-esterified and free forms, 3,3’ –dihydroxy-β, β-carotene-4, 4’ –dione (C₄₀H₅₂0₄ free form).

2.4. Growth Performance

Weekly feed intake was recorded, and the average daily feed intake (ADFI), average daily gain ratio (ADGR), and feed conversion ratio (FCR) were calculated. The body weight (BW) of each bird was recorded using a Mettler Toledo scale before heat stress treatment and at the end of the heat treatment.

2.5. Tissue Sample Collection

Immediately after euthanizing, ileum tissues were collected from randomly selected 6 birds of each group, snapped-frozen in liquid nitrogen, and stored at −80°C.

2.6. Total RNA extraction and cDNA preparation

Total RNA was isolated from frozen tissues (50–100mg) using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The concentration of total RNA was determined using NanoPhotometer® P330 (IMPLEN, Los Angeles, CA). Complementary DNA (cDNA) was synthesized from 1 µg of total RNA (20 µl reaction of RT mixture) using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA), and further diluted with nuclease free–water (1:25) for the qPCR reaction below.

2.7. Bioinformatics: Genome Assembly and Gene Primer Design

The National Center for Biotechnology Information (NCBI) genome browser was used to search and compile genes for Gallus gallus domesticus related to heat stress, oxidative stress, cytoprotective, epithelial integrity, transcription factors and housekeeping genes. The NCBI-Basic Local Alignment Search Tool (BLAST) was used to design primers for polymerase chain reaction (PCR) from the accession numbers obtained from the list of genes (Table 2). The primer parameters were set for a PCR product size between a minimum of 100 and maximum of 250 for 5 primers to return. The primer melting temperatures were set for a minimum of 55°C, optimum of 57°C, and maximum of 60°C with a maximum Tm difference of 3°C. The exon junction span was set that the primers must span an exon-exon junction. The organism specified was Gallus gallus (taxid 9031). The forward and reverse primer sequences (5’->3’) were then submitted to Integrated DNA Technologies (Coralville, IA, USA) for synthesis.

2.8. Quantitative Real-time RT-PCR (qPCR)

The qPCR was performed using PerfeCTa SYBR Green FastMix (Quantabio, Beverly, MA, USA) on a Q - qPCR instrument (Quantabio, Beverly, MA, USA). The qPCR reaction mixture consisted of 2 µl of cDNA, 10 µl PerfeCTa SYBR Green FastMix, 1 µl of each forward and reverse primers (5 µmol concentration), and 6 µl of sterile deionized water to make a final reaction mixture of 20 µl. Specific primer pairs for the detection of each gene were designed using the NCBI Primer-Blast tool. The qPCR reaction was carried out following standard cycling mode. The amplification conditions were 50°C for 2 min (hold), 95°C for 2 min (hold), followed by 40 repeat cycles of 95°C for 15 s (denaturation), 60°C for 15 s (annealing), and 72°C for 1 min (extension). A melting curve was also generated to confirm SYBR Green-based objective amplicon, and further qPCR products were confirmed using 2% agarose gel electrophoresis. Three house-keeping genes Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Beta-actin (β-actin), and TATA-Box Binding Protein (TBP) were analyzed in triplicates in each bird to determine the most stable house-keeping gene. Based on the uniformity of expression level across samples, β-actin was chosen as the housekeeping gene. Gene expression level was determined using cycle threshold (Ct) values following the standard curve method after normalization with housekeeping genes. Fold change for each gene was calculated using the 2^−ΔΔCt method and presented as mean ± standard error [40].

2.9. Gene Ontology

Significantly differentially expressed genes identified from the qPCR procedure were searched in the Ensembl genome database for chicken (GRCg6a) species to obtain Gene ontologies (GO) information (https://uswest.ensembl.org/Gallus_gallus/Info/Index) [41]. The GO included the cellular component, molecular function and biological process of these genes identified with ENSGAL Transcript IDs.

2.10. Statistical Analysis

The statistical analysis was performed using the Kruskal-Wallis rank sum test with statistical significance set at p<0.05, followed by the Dunn post hoc test for comparison between three groups: TN, HS, and HSAX, and the p-value adjusted using the Bonferroni method. Growth performance measurements were calculated based on the data collected at the end of the 42-day trial period. Analysis was conducted using the R open source program, libraries ‘FSA’, ‘dunn.test’, and ‘gplots’, R Core Team (2023)(https://www.R-project.org/) [42].

3. Results

3.1. Growth Performance

At the end of the 42-day poultry trial, the TN group growth performance indicators were found to be significantly higher for BW, ADFI, ADGR, compared to the HS and HSAX groups by conducting a Kruskal-Wallis test to evaluate differences among the three groups (p<0.01). Although the results clearly showed negative impacts of heat on the HS and HSAX groups, when performing the post hoc pairwise comparisons using Dunn’s test and applying the Bonferroni correction to control increased risk of error due to multiple comparisons, the negative impacts were more statistically significant comparing HS vs TN (p<0.01), rather than HSAX vs TN (p<0.05). In addition, the FCR showed the TN group was significantly lower in feed conversion, requiring less feed to maintain body weight compared to the HS vs TN, and HSAX vs TN (p<0.05) (Table 3, Figure 1).

3.2. Quantitative Real-time RT-PCR (qPCR) Gene Expression

For gene expression studies, three genes were considered for housekeeping genes, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), ACTB (β-actin), and TBP (TATA-box binding protein). β-actin was selected for its high and relatively stable expression under experimental conditions performed, making it a reliable reference for normalizing the gene expression data collected.

3.2.1. Statistically Significant Genes Identified in Differential Expression Analysis

Table 4. Genes exhibiting significant differential expression for the thymus tissue of Cob-500 broilers with β-actin as the housekeeping gene for normalization. Three groups were compared: TN, HS, and HSAX. Statistical analysis based on the Kruskal-Wallis rank sum test, followed with the Dunn post hoc test for comparisons, and p-value adjusted with the Bonferroni method, and where “ns” is considered “not significant”. * 0.05>p>0.01, ** 0.01>p>0.001.

	Kruskal-Wallis	Dunn/Bonferroni p-value
Gene	p-value	TN vs HS	TN vs HSAX	HS vs HSAX
NFKB1	0.0112	0.0283	ns	0.0283
RELA	0.0119	0.0088	ns	ns
IKBKB	0.0090	0.0105	0.0694	ns
NFE2L2	0.0394	0.0331	ns	ns
KEAP1	0.0112	0.0283	0.0283	ns
MAF	0.0298	0.0241	ns	ns
PPARA	0.0644	0.0601	ns	ns
RXRA	0.0041	0.0029	ns	ns
PPARG	0.0034	0.0105	0.0105	ns
CD36	0.0417	0.0448	ns	ns
SOD1	0.1284	ns	ns	ns
SOD2	0.0489	ns	ns	0.0694
SOD3	0.0446	0.0386	ns	ns
CAT	0.2911	ns	ns	ns
GPX1	0.2086	ns	ns	ns
GPX2	0.0050	0.0061	ns	0.0448
GPX3	0.0852	ns	ns	ns
GPX4	0.2359	ns	ns	ns
PRDX1	0.1169	ns	ns	ns
PRDX3	0.1345	ns	ns	ns
PRDX4	0.0260	0.0283	ns	ns
PRDX6	0.0060	0.0061	ns	0.0694
BCL2	0.3123	ns	ns	ns
CASP3	0.0005	ns	ns	0.0002
TP53	0.5671	ns	ns	ns

Table 4. Genes exhibiting significant differential expression for the thymus tissue of Cob-500 broilers with β-actin as the housekeeping gene for normalization. Three groups were compared: TN, HS, and HSAX. Statistical analysis based on the Kruskal-Wallis rank sum test, followed with the Dunn post hoc test for comparisons, and p-value adjusted with the Bonferroni method, and where “ns” is considered “not significant”. * 0.05>p>0.01, ** 0.01>p>0.001.

	Kruskal-Wallis	Dunn/Bonferroni p-value
Gene	p-value	TN vs HS	TN vs HSAX	HS vs HSAX
NFKB1	0.0112	0.0283	ns	0.0283
RELA	0.0119	0.0088	ns	ns
IKBKB	0.0090	0.0105	0.0694	ns
NFE2L2	0.0394	0.0331	ns	ns
KEAP1	0.0112	0.0283	0.0283	ns
MAF	0.0298	0.0241	ns	ns
PPARA	0.0644	0.0601	ns	ns
RXRA	0.0041	0.0029	ns	ns
PPARG	0.0034	0.0105	0.0105	ns
CD36	0.0417	0.0448	ns	ns
SOD1	0.1284	ns	ns	ns
SOD2	0.0489	ns	ns	0.0694
SOD3	0.0446	0.0386	ns	ns
CAT	0.2911	ns	ns	ns
GPX1	0.2086	ns	ns	ns
GPX2	0.0050	0.0061	ns	0.0448
GPX3	0.0852	ns	ns	ns
GPX4	0.2359	ns	ns	ns
PRDX1	0.1169	ns	ns	ns
PRDX3	0.1345	ns	ns	ns
PRDX4	0.0260	0.0283	ns	ns
PRDX6	0.0060	0.0061	ns	0.0694
BCL2	0.3123	ns	ns	ns
CASP3	0.0005	ns	ns	0.0002
TP53	0.5671	ns	ns	ns

Figure 2. Gene expression fold change heatmap for thymus tissue of Cob-500 broilers. Three groups were compared: TN, HS, and HSAX. The HS group expression values show shades of red with significant higher expression and the HSAX to a lesser extent, whereas, blue indicates a lower level of expression in the TN group.

Figure 2. Gene expression fold change heatmap for thymus tissue of Cob-500 broilers. Three groups were compared: TN, HS, and HSAX. The HS group expression values show shades of red with significant higher expression and the HSAX to a lesser extent, whereas, blue indicates a lower level of expression in the TN group.

3.2.2. NF-kB Transcription Signaling Pathway Genes

The studies showed the impact of HS and AOX treatment expressed through the NF-kB transcription factor signaling pathway and pairwise comparisons. Gene expression results where HS was significantly upregulated higher than TN were found in NF-kB, TN vs HS (p=0.0283), proto-oncogene, NF-kB subunit (RELA), TN vs HS (p=0.0088) and inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), TN vs HS (p=0.0105). HS was also significantly higher as compared to HSAX in the expression of NF-kB, HS vs HSAX (p=0.0283). There was a marginal difference between HSAX elevated in expression over TN of IKBKB, TN vs HSAX (p=0.0694).

Figure 3. Effects of heat stress and astaxanthin treatment in the NF-kB transcription factor signaling pathway in the thymus tissue of Cob-500 broilers. Three groups were compared: TN, HS, and HSAX. Kruskal-Wallis test and Dunn post hoc test for statistical significance represented: (A) NFKB1, KW p=0.0112, TN vs HS (p=0.0283), HS vs HSAX (p=0.0283); (B) RELA, KW p=0.0119, TN vs HS (p=0.0088); and (C) IKBKB, KW p=0.0090, TN vs HS (p=0.0105), TN vs HSAX (p=0.0694).

Figure 3. Effects of heat stress and astaxanthin treatment in the NF-kB transcription factor signaling pathway in the thymus tissue of Cob-500 broilers. Three groups were compared: TN, HS, and HSAX. Kruskal-Wallis test and Dunn post hoc test for statistical significance represented: (A) NFKB1, KW p=0.0112, TN vs HS (p=0.0283), HS vs HSAX (p=0.0283); (B) RELA, KW p=0.0119, TN vs HS (p=0.0088); and (C) IKBKB, KW p=0.0090, TN vs HS (p=0.0105), TN vs HSAX (p=0.0694).

3.2.3. NFE2L2-Mediated Signaling Pathway Genes

The results of the impact of HS and AOX treatment on the expression of the NFE2L2-mediated signaling pathway showed the HS treatment group upregulated as compared to the TN group, NFE2L2, TN vs HS (p=0.0331), kelch like ECH associated protein 1 (KEAP1), TN vs HS (p=0.0283), and musculoaponeurotic fibrosarcoma (MAF), TN vs HS (p=0.0241). Additionally, the AST treatment was shown to be significantly upregulated over the TN group, KEAP1, TN vs HSAX (p=0.0283).

Figure 4. Effects of heat stress and astaxanthin treatment in the NFE2L2-mediated signaling pathway in the thymus tissue of Cob-500 broilers. Three groups were compared: TN, HS, and HSAX. Kruskal-Wallis test and Dunn post hoc test for statistical significance represented: (A) NFE2L2, KW p=0.0394, TN vs HS (p=0.0331); (B) KEAP1, KW p=0.0112, TN vs HS (p=0.0283), TN vs HSAX (p=0.0283); and (C) MAF, KW p=0.0298, TN vs HS (p=0.0241).

Figure 4. Effects of heat stress and astaxanthin treatment in the NFE2L2-mediated signaling pathway in the thymus tissue of Cob-500 broilers. Three groups were compared: TN, HS, and HSAX. Kruskal-Wallis test and Dunn post hoc test for statistical significance represented: (A) NFE2L2, KW p=0.0394, TN vs HS (p=0.0331); (B) KEAP1, KW p=0.0112, TN vs HS (p=0.0283), TN vs HSAX (p=0.0283); and (C) MAF, KW p=0.0298, TN vs HS (p=0.0241).

3.2.4. PPARα Signaling Pathway Genes

Consistent with the findings for the NF-kB and NFE2L2 pathways, the HS treatments resulted in upregulation over the TN groups in the PPARα pathway genes. The findings showed retinoid X receptor alpha (RXRA), TN vs HS (p=0.0029), peroxisome proliferator-activated receptor gamma (PPARγ), TN vs HS (p=0.0105), cluster of differentiation 36 (CD36), TN vs HS (p=0.0448), and a marginal difference in PPARα, TN vs HS (p=0.0601). Significant upregulation was also found in PPARγ, TN vs HSAX (p=0.0105).

Figure 5. Effects of heat stress and astaxanthin treatment in the PPARα signaling pathway in the thymus tissue of Cob-500 broilers. Three groups were compared: TN, HS, and HSAX. Kruskal-Wallis test and Dunn post hoc test for statistical significance represented: (A) PPARα, KW p=0.0644, TN vs HS (p=0.0601)(not significant); (B) RXRA, KW p=0.0041, TN vs HS (p=0.0029); (C) PPARγ, KW p=0.0034, TN vs HS (p=0.0105), TN vs HSAX (p=0.0105); and (D) CD36, KW p=0.0417, TN vs HS (p=0.0448).

Figure 5. Effects of heat stress and astaxanthin treatment in the PPARα signaling pathway in the thymus tissue of Cob-500 broilers. Three groups were compared: TN, HS, and HSAX. Kruskal-Wallis test and Dunn post hoc test for statistical significance represented: (A) PPARα, KW p=0.0644, TN vs HS (p=0.0601)(not significant); (B) RXRA, KW p=0.0041, TN vs HS (p=0.0029); (C) PPARγ, KW p=0.0034, TN vs HS (p=0.0105), TN vs HSAX (p=0.0105); and (D) CD36, KW p=0.0417, TN vs HS (p=0.0448).

3.2.5. Cytoprotective Capacity Genes

Results of the cytoprotective capacity genes provided a variation of expression. Upregulation of the HS over the TN was significant for superoxide dismutase 3 (SOD3), TN vs HS (p=0.0386), glutathione peroxidase 2 (GPX2), TN vs HS, (p=0.0061), peroxiredoxin 4 (PRDX4), (p=0.0283) and peroxiredoxin 6 (PRDX6), (p=0.0061). Comparing the HS and HSAX groups, GPX2 was significantly upregulated, HS vs HSAX (p=0.0448). Marginal differences were found in SOD2, HS vs HSAX (p=0.0694) and PRDX6, HS vs HSAX (p=0.0694), but with opposing outcomes.

Figure 6. Effects of heat stress and astaxanthin treatment in the cytoprotective capacity gene expression in the thymus tissue of Cob-500 broilers. Three groups were compared: TN, HS, and HSAX. Kruskal-Wallis test and Dunn post hoc test for statistical significance represented: (A) SOD2, KW p=0.0489, HS vs HSAX (p=0.0694); (B) SOD3, KW p=0.0446, TN vs HS (p=0.0386); (C) GPX2, KW p=0.0050, TN vs HS (p=0.0061), HS vs HSAX (p=0.0448); (D) PRDX4, KW p=0.0260, TN vs HS (p=0.0283); and (E) PRDX6, KW p=0.0060, TN vs HS (p=0.0061), HS vs HSAX (p=0.0694).

Figure 6. Effects of heat stress and astaxanthin treatment in the cytoprotective capacity gene expression in the thymus tissue of Cob-500 broilers. Three groups were compared: TN, HS, and HSAX. Kruskal-Wallis test and Dunn post hoc test for statistical significance represented: (A) SOD2, KW p=0.0489, HS vs HSAX (p=0.0694); (B) SOD3, KW p=0.0446, TN vs HS (p=0.0386); (C) GPX2, KW p=0.0050, TN vs HS (p=0.0061), HS vs HSAX (p=0.0448); (D) PRDX4, KW p=0.0260, TN vs HS (p=0.0283); and (E) PRDX6, KW p=0.0060, TN vs HS (p=0.0061), HS vs HSAX (p=0.0694).

3.2.6. Apoptotic Gene

The results of the apoptotic pathway showed a clear indicator in the upregulation of caspase 3 (CASP3), HS vs HSAX (p=0.0002).

Figure 7. Effects of heat stress and astaxanthin treatment in the apoptotic pathways in the thymus tissue of Cob-500 broilers. Three groups were compared: TN, HS, and HSAX. Kruskal-Wallis test and Dunn post hoc test for statistical significance represented: (A) CASP3, KW p=0.0005, HS vs HSAX (p=0.0002).

Figure 7. Effects of heat stress and astaxanthin treatment in the apoptotic pathways in the thymus tissue of Cob-500 broilers. Three groups were compared: TN, HS, and HSAX. Kruskal-Wallis test and Dunn post hoc test for statistical significance represented: (A) CASP3, KW p=0.0005, HS vs HSAX (p=0.0002).

3.2.7. Gene Ontology (GO) Gene Enrichment

4. Discussion

4.1. Growth Performance

Growth performance is anticipated to suffer under any stress condition, and in this study under HS. Although the growth performance indexes did not show any significant benefit from an AOX supplement, the health of the poultry was further elucidated through the molecular mechanisms of gene expression providing insights affecting the health and wellbeing of the broilers.

4.2. Gene Ontology Enrichment and Expression Analysis

4.2.1. NF-kB Transcription Signaling Pathway Genes

The NF-kB transcription signaling pathway is a critical regulator of immune and inflammatory responses. NF-kB can be found throughout the cell in the nucleus, mitochondrion, chromatin and cytoplasm. When not upregulated, NF-kB is generally sequestered in the cytoplasm by IkB inhibitor proteins such as IKBKB, located in the cytoplasm and cytosol. RELA found in the cytoplasm is a key subunit of the NF-kB transcription factor complex to form heterodimers of NF-kB for translocation into the nucleus upon activation for DNA-binding transcription activity. When activated by stimuli, such as stress, free radicals, pathogens and cytokines, IkBs are phosphorylated by a kinase enzyme for ubiquitination and degradation, freeing the NF-kB to translocate into the nucleus to promote the transcription of target genes involved in inflammation, immune response, cell proliferation, and survival. The NF-kB pathway plays a significant role in responding to cellular stress and homeostasis (Table 5).

The outcome of our studies indicates that the upregulation of the HS group over the TN group is consistent with the function of the NF-kB complex to respond to cellular stress. While there is no significance between the TN and HSAX groups, it would indicate that AST is having some effect in reducing cellular stress, which coincides with the results where there is a significant difference between the upregulation of the HS over the HSAX group.

4.2.2. NFE2L2-Mediated Signaling Pathway Genes

The NFE2L2 (NRF2) plays a crucial role in cellular defense against oxidative stress. NFE2L2 is found throughout the cell in the nucleus, Golgi apparatus, chromatin, plasma membrane and cytoplasm. Under normal conditions, NFE2L2 is bound by KEAP1 in the cytoplasm, and in response to OS or electrophilic stimuli, NFE2L2 is released to translocate to the nucleus where it heterodimerizes with small MAF proteins. This NFE2L2-MAF complex binds to antioxidant response elements (AREs) in the promoters of target genes leading to transcription of a various cytoprotective genes (Table 5).

The results of our study show the HS group upregulated in comparison to the TN group in response to OS throughout the NFE2L2 signaling pathway. Similarly, HSAX is upregulated over the TN group in the KEAP1 gene expression, a critical regulator of the NFE2L2 signaling pathway, acting as a sensor for OS and controlling the activity of NFE2L2, likely a homeostatic feedback mechanism to maintain a balanced redox environment.

4.2.3. PPARα Signaling Pathway Genes

The PPARα signaling pathway plays a vital role in lipid metabolism, energy homeostasis and inflammation. Although PPARα which is predominantly found in the nucleus has only a marginal significance in upregulation of the HS over the TN group, RXRA, PPARγ and CD36 is significantly expressing. PPARγ in the HSAX group is also upregulated in comparison to the TN group. PPARα is generally in an inactive state in the nucleus and is activated by fatty acids or specific ligands to form a heterodimer with RXRA, and further undergo conformational changes for DNA-binding transcription factor activity in the promoter of target genes. Among the target genes is CD36 which facilitates fatty acid uptake and enzymes for lipid metabolism. PPARγ can interact with PPARα to help regulate lipid metabolism, reduce inflammation and maintain energy homeostasis (Table 5).

The results from our study indicate that although there is a subtle response of PPARα to heat stress, PPARγ appears to be involved in the cellular response to heat stress and suggests a role in AOX-mediated response to stress. RXRA forms heterodimers with PPARα and is essential for transcriptional activity. The upregulation of CD36 in the HS group compared to the TN suggests increased lipid metabolism or fatty acid utilization, placing a demand on energy due to heat-induced OS.

4.2.4. Cytoprotective Capacity Genes

Cytoprotective capacity genes safeguard cells from various stressors including oxidative damage by enhancing AOX defense mechanisms to promote cell survival. SOD2 is found in the mitochondrion and responds to OS through the oxidation-reduction process for removal of superoxide radicals. SOD3 is found in the extracellular space and plays a crucial role in scavenging superoxide radicals outside the cell. GPX2 is found in the cytosol, intercellular bridge and mitotic spindle, involved in detoxifying peroxides, such as hydrogen peroxide. PRDX4 is found in the cytoplasm and endoplasmic reticulum, and PRDX6 is also found in the cytoplasm, and also the nucleus. Both peroxiredoxins are involved in scavenging for peroxides, providing peroxide detoxification and protection of cells from OS (Table 5).

The results showed significant upregulation of the HS compared to the TN groups in the expression of SOD3, GPX2, PRDX4 and PRDX6. There was also significant upregulation of the HS group over the HSAX group for GPX2, but only marginally for PRDX6. In addition, when analyzing the expression of SOD3, there was marginal upregulation of HSAX over the HS group. Overall, our findings highlight the dynamic regulation of AOX genes in response to heat-induced OS and the potential modulatory effects of AST-AOX supplementation on cellular antioxidant defense mechanisms.

4.2.5. Apoptotic Pathway Genes

The CASP3 can be found in the cytoplasm and nucleus where it encodes for a cysteine protease involved in the execution phase of apoptosis. BCL2 encodes for an anti-apoptotic protein that inhibits cell death, and TP53 encodes for a tumor suppressor protein involved in cell cycle regulation and apoptosis (Table 5).

Our findings show significant upregulation of CASP3 in the HS group over the HSAX group, which suggests an activation of apoptotic pathways, potentially indicating cellular damage or heat-induced OS. The lack of significant expression of BCL2 and TP53 may suggest that heat stress alone may not induce significant alterations in apoptotic regulators, or that the antioxidant supplementation may be effective in mitigating the response to heat stress.

5. Conclusions

Overall, our experimental findings highlight the dynamic regulation of gene expression in the thymus related to the NF-kB, NFE2L2, PPARα, cytoprotective capacity and apoptotic pathways. Our main objective of the present study was to investigate the protective properties of AST against heat-induced OS and apoptosis in the chicken thymus. Our research provided insights into the molecular regulatory mechanisms that respond to heat-induced OS, and the potential therapeutic implementation of AST-AOX supplementation to mitigate the effects through modulation of transcription pathways. The complexities of such mechanisms, and the varied responses of AOX whether endogenous or applied leaves us with knowledge gaps that require further research to understand the therapeutic potentials of AST-AOX.