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. 2024 Aug 5;103(11):104161. doi: 10.1016/j.psj.2024.104161

Proteomics analysis for key molecules in adrenal glands of Wenchang chickens for their resistance to heat stress

Yiduo Lin 1, Zeping Ji 1, Chengyun Li 1, Qijun Liang 1, Jiachen Shi 1, Zhiqing Su 1, Xu Yao 1, Xiaohui Zhang 1,1
PMCID: PMC11396071  PMID: 39190996

Abstract

Rising temperatures and intensified agricultural practices have heightened heat stress (HS)-related challenges in poultry farming, notably heat-induced sudden death in chickens. Wenchang chickens, recognized for their heat resistance, have emerged as the potential candidates for improving the economic efficiency of poultry farming. The adrenal gland plays a crucial role in preventing HS-induced heart failure sudden death by secreting hormones. However, little is known about the damage to and resilience of Wenchang chicken adrenal glands during HS. In this study, 34 healthy Wenchang chickens with similar weights were selected for formal experimentation, with 10 as the control group (Con). Following a single exposure to acute HS of 42 ± 1°C and 65% relative humidity for 5 h, 15 deceased individuals formed the HS death (HSD) group, and 9 survived comprised the HS survival (HSS) group. ELISA revealed significant higher (P < 0.05) levels of COR and NE in the HSS and the lowest levels of CORT and EPI in the HSD. Histopathological analysis indicated major degeneration in HSS cortical and chromaffin cells and extensive cell necrosis (nuclear pyknosis) in HSD. Proteomic analysis identified 572 DEPs in HSD vs. Con and 191 DEPs in HSS vs. Con. Bioinformatics highlighted ER protein processing, especially ERAD as a key pathway for heat stress resistance (HSR) in the adrenal gland, with HSPH1, DNAJA1, HSP90AA1, HSPA8 and HERPUD1 identified as regulating key molecules. Western blotting validated significantly higher (P < 0.01) protein levels in both HSS and HSD compared to the Con. Immunohistochemical staining showed increased cytoplasmic HSPH1-positive signal intensity under HS and enhanced HSP90AA1 nuclear signals, strongest in HSS. In summary, HS induces pathological damage in Wenchang chicken adrenal glands, affecting hormone secretion, and various heat shock proteins play crucial roles in cellular resistance. These results elucidate the biological basis of HSR in Wenchang chickens from the perspective of the adrenal gland and provide necessary research foundations for enhancing economic performance of various broilers in high-heat environments and screening drugs for HS treatment.

Key words: key molecule, proteomics, heat-stress, adrenal gland, Wenchang chicken

INTRODUCTION

Poultry plays a significant role in the global food supply chain of meat and egg, providing a substantial contribution to the agricultural industry and human society worldwide. Heat stress (HS) arises from an imbalance between the energy released by the external environment and the internal dissipation ability. As avian lack sweat glands, they exhibit the limited tolerance to high temperatures, especially when combined with high humidity (Fathi et al., 2022). Consequently, heat stress can instigate multiple biochemical and structural changes as well as pathological damage in different organs (Khan et al., 2011; Kilic and Simsek, 2013; Yu et al., 2008), with a significant proportion of broilers experiencing sudden death as the most severe outcome (Tang et al., 2016). In the face of escalating global temperatures as well as intensification of agriculture, heat stress poses an increasingly serious challenge to poultry production (Kumar et al., 2021). Sudden death in broilers is closely related to heat stress-induced heart failure stemming from pathological damage to myocardial cells, with hormonal secretion from the adrenal glands playing a pivotal role in influencing cardiac function.

Based on the duration of exposure, heat stress can be classified into acute heat stress (AHS) and chronic heat stress (CHS). Acute heat stress triggers the rapid release of catecholamines (CA) and glucocorticoids (GC) from the adrenal cortex and medulla (Wilckens and De Rijk, 1997). On the other hand, the long-term stress response, mediated through the hypothalamic-pituitary-adrenal (HPA) axis, stimulates the secretion of cortisol (corticosterone in birds) from the adrenal cortex to exert an anti-heat stress injury effect (Bureau et al., 2009). Failure to recover from intense stress, whether acute or chronic, can lead to cardiac exhaustion, ultimately resulting in mortality. Therefore, ensuring normal adrenal function is crucial for the survival of chickens under heat stress conditions.

Throughout domestication, chicken have globally migrated and adaptively evolved into thousands of breeds with distinct phenotypic characteristics (Chen et al., 2022). Tropical regions, known for their high heat and humidity, pose specific challenges for poultry (Oladokun and Adewole, 2022). Nonetheless, the Wenchang chicken, a local breed flourishing in Hainan Province, China for over 400 yr, has gained recognition for its remarkable resistance and low mortality rates in the face of heat stress conditions (Fu et al., 2022), making it a potential candidate for improving the economic viability of poultry farming. Previous study conducted by our group indicated that Wenchang chickens exhibited improved cardiac function and a lower death rate when subjected to heat stress, hinting at a potential superior adjustment mechanism, likely originating from the adrenal gland. However, the changes in the structure and function of the adrenal glands in Wenchang chickens during heat stress, as well as their self-regulation mechanism within the constituent cells, have not been clearly reported. The widespread adoption of proteomic methods allows for large-scale screening of differentially expressed proteins, enabling direct measurements of protein expression levels and providing valuable insights into the functional activity of target proteins (Rozanova et al., 2021). In this study, we conducted label-free quantitative proteomics (LFQP) on the adrenal tissue of heat-stressed Wenchang chickens to identify key molecules associated with heat stress resistance (HSR). The aim was to uncover the mechanisms behind the heat resistance observed in tropical poultry, and ultimately guiding molecular breeding and screening for drugs to treat heat stress in chickens (Radwan, 2020).

MATERIALS AND METHODS

Experimental Animals Management

A total of forty 70-day-old Wenchang chickens were obtained from Longquan Wenchang Chicken Industrial Co., Ltd (Wenchang, China). Following a week of pre-feeding, 34 healthy individuals with similar weights were selected for the formal test. The entire animal care and experimental procedures were conducted following the guidelines of the Animal Ethics Committee of Hainan Province, China, and approved by the Ethical Committee on Animal Research at Hainan University. The chickens were raised under controlled conditions (22±2°C, 50 ± 10% humidity, 12h dark/12h light) in the animal facility of Hainan University, with free access to feed and water ad libitum.

Heat Stress Treatment and Sample Collection

Twenty-four chickens were randomly selected and exposed one-time to acute heat-stress at 42 ± 1°C and 65% relative humidity for 5 h, while the remaining 10 served as the control group (Con) without heat stress treatment. All maintained normal diet and water intake. Subsequently, the heat-stressed individuals were further categorized based on survival status into the heat stress death group (HSD) and the heat stress survivor group (HSS). Clinical performances were continuously observed and recorded during the heat stress period. Blood samples were withdrawn from the wing veins, centrifuged at 4,500 rpm. The supernatant was collected, and stored at -80°C for later use. Following blood withdrawal, chickens were promptly euthanized by cervical dislocation. Both sides of the adrenal glands were collected, with one divided into 3 portions and stored at -80°C for proteomic analysis, Western blotting, and immunohistochemistry experiments. The other side was fixed in 10% neutral formalin solution for histopathological examination. In total, 10, 15, and 9 original samples were collected from the Con, HSD, and HSS respectively for label-free quantitative proteomics (LFQP) analysis. Within each group, 3 samples (each 200 mg) were randomly selected from 3 chickens and combined into one centrifuge tube, resulting in 3 sample tubes, and snap-frozen with liquid nitrogen.

Detection of Typical Adrenal Hormones by Enzyme-Linked Immunosorbent Assay

The plasma hormone levels of cortisol (COR), corticosterone (CORT), epinephrine (EPI), and norepinephrine (NE) were quantitatively detected by ELISA kits (FineTest, Wuhan, China) following the manufacturer's instructions. Measurements were conducted in triplicate for each sample.

Histopathological Analysis of Adrenal Glands in Experimental Chickens

Fixed adrenal tissues underwent a process of gradient dehydration, paraffin embedding, and serial cutting (4 μm). The cellular nuclei and cytoplasm were characterized by a hematoxylin and eosin (H&E) staining kit (Lab-test Biotechnology, Beijing, China) according to the manufacturer's instructions. Slides were mounted with coverslips using PermountTM mounting medium. Twenty-seven representative fields at 40× magnification, 400× magnification (near the tissue capsule), and 400× magnification (near the histological center of adrenal glands) were selected for histopathological evaluation using an optical microscope (Mingmei Optoelectronics Technology, Guangzhou, China). Three fields were selected for each magnification, presenting the most representative results. For histopathological assessment, 2 pathologists blinded to treatments independently evaluated all slices. Scores were assigned individually based on the severity and scope of pathological changes: no obvious pathological changes (0 points), cell swelling and mild hydropic and/or fatty degeneration (1 points), moderate hydropic and/or fatty degeneration (2 points), severe hydropic and/or fatty degeneration (3 points), degeneration accompanied by cell necrosis (4 points) and extensive cell necrosis (5 points).

Label-Free Quantitative Proteomics Analysis

The experiments were completed at Biotree (Shanghai, China) Co. Ltd., and methods are described as follows:

Total proteins were extracted and quantified using BCA kits (Beyotime, Shanghai, China). After acetone precipitation, the protein was dissolved in 100 μL protein resolubilization solution and reduced with dithiothreitol (DTT, 5 μL, 1 mol/L) at 55°C for 20 min, then cooled to room temperature. Iodoacetamide (IAA, 20 μL, 1 mol/L) was added to a final concentration and allowed to react for 30 min in the dark. The samples were then trypsin-digested for 14 h at 37°C. Following SDC removal, peptides were desalted on a Strata X C18 column and vacuum-dried overnight at 4°C for LFQP analysis.

After acetone precipitation, total peptides (2 μg) from each sample were separated and analyzed using a nano-UPLC liquid chromatography system (EASY-nLC1200, Thermo Scientific, Waltham, MA) coupled with a mass spectrometer (QExactive HFX, Thermo Scientific) equipped with a nanoelectrospray ion source. The mobile phase consisted of solvent A (0.1% formic acid, 2% acetonitrile/water) and solvent B (0.1% formic acid, 80% acetonitrile/water). Separation was performed using a reversed­phase column (100 μm ID ×15 cm, Reprosil-Pur 120 C18­AQ, 1.9 μm, Dr. Maisch, Ammerbuch, Germany). The column was equilibrated with 100% phase A, and the sample was then introduced for peptide separation at a constant flow rate of 300 nL/min over a 120-min gradient. Peptide separation proceeded over a 2-h gradient at 300 nL/min flow rate, with a gradual increase in solvent B to 100%. The separated peptides underwent a 2-h mass spectrometry analysis in positive ion detection mode. The top 20 ions with the highest intensity from the full scan (range: 350–1600 m/z, resolution: 120k @ 200 m/z) were selected through quadrupole filtering (1.2 m/z for isolation window, 27% for NCE, 1E5 for AGC, and 110 ms for max IT). Subsequently, high-energy collision-induced dissociation (HCD) was employed for fragmentation, followed by fragmentation ion scanning (15k resolution). A dynamic exclusion time of 45 s was implemented based on the peak width, with exclusion criteria for singly charged and >6-valent ions in the secondary scan.

Vendor's raw MS files were processed by Proteome Discoverer (PD) software (Version 2.4.0.305) with the Sequest HT search engine. Peptide identification was conducted by searching tandem MS spectra against the UniProt FASTA database (uniprot-Gallus gallus_9031-2021-09.fasta). The criteria were set as follows: initial precursor mass tolerance ≤10 ppm; fragment mass tolerance: 0.02 Da; false discovery rate (FDR) <1%; fixed modification: carbamidomethylation; variable modifications: oxidation and acetylation (protein N-terminus); enzyme: trypsin; maximum allowed missed cleavages: 2. Proteins were quantified by unique peptides and razor peptides with normalization based on total peptide amounts. Missing values were imputed using the minimum value bisection method.

Bioinformatics Analysis of the Quantitative Proteomics

Differentially expressed proteins (DEPs) were defined by t-tests with a significance level of P < 0.05 and a fold change (FC) of ≤ 0.83 or ≥ 1.2 as criteria. As for the subcellular localization, WolfPsort (https://wolfpsort.hgc.jp/) was used to predict the sites of the DEPs. Gene Ontology (GO) analyses specific to Gallus gallus (chicken) were conducted with DEPs subjected to the Database for Annotation Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/) (Dennis et al., 2003; Huang et al., 2009). Protein expression profiles were further analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (www.kegg.jp/kegg/pathway.html) (Kanehisa et al., 2016). Spearman's correlation coefficient was employed for the correlation analysis, and significant differences were assessed using t-test. Correlation analysis network mapping, hierarchical cluster analysis, and volcano plot visualization were visualized by the corrplot, gplots, and ggplot2 packages in R (version 4.3.1).

Western Blotting

Twenty μg total proteins extracted from the tested adrenal glands in each group were loaded onto SDS-PAGE for separation. Beta (β)-actin from Cell Signaling Technology (Danvers, MA) was simultaneously loaded with equal amounts as a control. Following electrophoresis and transfer to a PVDF membrane, the proteins were incubated overnight at 4°C with the specified primary antibody. Subsequently, the membrane was incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (Abcam, Cambridge, UK) for 2 h at room temperature. After 5 final washes by Tris-buffered saline with Tween-20, the membranes were visualized by a gel documentation system (Bio-Rad Laboratories, Hercules, CA). Gray values of the bands were then analyzed using Image J software (v2.9.0). The primary antibodies purchased include anti- heat shock protein HSP 90-alpha (HSP90AA1) (ab2928, 1:1000) from Abcam (Cambridge, UK) and anti-heat shock 110 kDa protein (HSPH1) (Q92598, 1:2000) from Bio-techne (Minneapolis, MN).

Immunohistochemistry

Fixed adrenal tissue was processed through gradient dehydration, paraffin embedding, and sectioning (4 μm). Sections were then deparaffinized, hydrated, and subjected to antigen retrieval. After sequential blocking with 0.1% Triton X-100 and goat serum at 37°C, primary antibodies against HSPH1 (ab04064; Abcam, Cambridge, UK) and HSP90AA1 (ab2928; Abcam, Cambridge, UK), each diluted to 1:100, were applied to the slides and incubated for 1 h at 37°C. Following PBST washing, the slides were incubated with HRP-conjugated secondary antibodies in the dark at 37°C for 30 min. Finally, the slides were washed with PBST, counterstained with hematoxylin, and imaged under the optical microscope. A most representative field from each group was respectively chosen for presentation. Two pathologists, blinded to the treatment, independently scored 3 representative fields (400× magnification) from each group. The scoring criteria were as follows: staining intensity: negative (0 points), light yellow (1 point), light brown (2 points), dark brown (3 points); positive range: 0% to 25% (1 point), 26% to 50% (2 points), 51% to 75% (3 points), 76% to 100% (4 points). Scores from both criteria were combined to generate a comprehensive positive signal score.

Statistical Analysis

The variables are presented as means ± standard deviation from 3 replicates. Statistical analysis of differentially expressed proteins was conducted using PD software (Version 2.4.0.305). The SPSS (version 26, IBM Corp., Armonk, NY) with t-test was employed to analyze the ELISA, pathological scoring, Western blotting and immunohistochemistry scoring results between groups. Significance levels of P value were indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

RESULTS

Clinical Performance

Upon initiation of the heat stress treatment, both of the broilers in HS groups (HSD and HSS) exhibited increased respiratory rate and water consumption, as well as mouth breathing and uneasiness. As the heat stress treatment progressed (3–5 h), a large proportion in the HS groups displayed symptoms including depression and watery stools, extensively leading to a 62.5% mortality.

Alterations in Plasma Adrenal-Related Hormone Levels in Heat-Stressed Chickens

To validate the impact of heat stress on adrenal function, we assessed the secretion levels of 4 typical adrenal hormones. Compared to Con, plasma levels of COR were significantly higher in HSS, but not significantly higher in HSD (Figure 1A). CORT levels in HSS were not significantly higher than Con, but significantly higher than HSD, while levels in HSD were significantly lower than Con (Figure 1B). EPI levels in HSS were not significantly higher than Con, but significantly higher than HSD, whereas EPI levels in HSD were significantly lower than Con (Figure 1C). NE levels in HSS were significantly higher than Con, but not significantly higher than HSD. NE levels in HSD were not significantly higher than Con (Figure 1D).

Figure 1.

Figure 1

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Plasma adrenal hormone levels in heat-stressed Wenchang chickens. (A–D) ELISA detection results of COR, CORT, EPI, and NE levels in the plasma of Wenchang chickens.

Histological Changes of Chicken Adrenal Tissues

We further assessed the morphological effects of heat stress on adrenal tissue through histopathological examination and scoring. As shown in Figure 2, the adrenal tissue of the Con exhibited intact capsules, with medullary cells (chromaffin cells) arranged in cord-like structures intermingled with cortical cells. The proportion of cortical cells was slightly larger than that of chromaffin cells, and vacuolization could be observed in a small amount of cortical cells. Hematoxylin and eosin staining revealed that extensive cell necrosis (nuclear pyknosis) of cortical and chromaffin cells was found in HSD, while swelling of cortical cells and fatty degeneration of chromaffin cells was also obvious, accompanied by a reduction of the ratio of medulla to cortex. As for HSS, it was observed that the boundaries between cortex and medulla became blurred, with thinner capsules of the adrenal glands. Cortical cells displayed significant hydropic degeneration, whereas chromaffin cells showed obvious fatty degeneration. Both cortical cells and chromaffin cells also exhibited slight cell necrosis. From the viewpoint of pathological scores, the pathological damage scores in HSD and HSS were significantly higher than that in Con, with HSD higher than HSS.

Figure 2.

Figure 2

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Histopathological assessment and scoring. (A) H&E staining of Wenchang chicken adrenal tissue to observe the adverse effects of heat stress. Magnification for each group (top to bottom): 40x, 400x, and 400x. (B) Pathological damage scores of the adrenal glands. Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Screening and Annotation of Differentially Expressed Proteins

In the comparisons of HSD vs. Con and HSS vs. Con, we respectively identified 94 and 54 upregulated proteins (12 shared proteins), and 478, 137 downregulated proteins (47 shared proteins) as the differentially expressed proteins (DEPs), as depicted in the Venn diagram (Figures 3A and 3B). Hierarchical cluster analysis of the identified proteins revealed clear clustering patterns and distinct abundance profiles (Figures 3C and 3D). Volcano plots showed discernible fold changes between upregulated and downregulated proteins, collectively underscoring the significant impact of heat stress on adrenal protein expression (Figures 3E and 3F). Subcellular localization analysis revealed predominant subcellular sites in the nucleus and cytoplasm of the DEPs (Figures 3G and 3H).

Figure 3.

Figure 3

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LFQP screening of DEPs in the adrenal tissue of heat-stressed Wenchang chickens. (A–B) Quantitative analysis of upregulated and downregulated proteins in two pairwise comparisons (HSD vs. Con and HSS vs. Con) of the adrenal proteins, visualized by Venn diagrams to show commonly identified DEPs. (C–D) Hierarchical cluster analysis of the identified DEPs. (E–F) Volcano plots of HSD vs. Con and HSS vs. Con. (G–H) Results of subcellular localization analysis for HSD vs. Con and HSS vs. Con.

Functional Annotation and Enrichment Analysis

GO annotation enrichment analysis was conducted to gain deeper insights into functions of proteins. As shown in Figure 4A, metabolic process, cell, and binding were the most dominant biological process (BP), cellular component (CC), and molecular function (MF) in the HSD vs. Con comparison. For HSS vs. Con, cellular component organization or biogenesis, cytoplasm, and protein binding were identified to be the most enriched terms (Figure 4B).

Figure 4.

Figure 4

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GO enrichment analysis of DEPs. (A) GO enrichment analysis of DEPs in HSD vs. Con, listing the top 10 terms for biological processes, cellular components, and molecular functions. (B) Top 10 GO enrichment terms identified from DEPs in HSS vs. Con, including 3 categories: biological processes, cellular components, and molecular functions.

Analysis of Differentially Expressed Proteins in Biological Process

Through comparative analysis between HSD vs. Con and HSS vs. Con, we observed a widespread enrichment of binding terms in the molecular function of both comparisons (Figures 5A and 5B). In order to gain further insights into the functionality of these DEPs, we performed KEGG enrichment annotation. A standard of P < 0.05 and a rich factor > 0 was employed to identify significant pathways. The results revealed that the top 3 enriched pathways with the highest rich factors in the HSD vs. Con comparison were Oxidative phosphorylation, Herpes simplex virus 1 infection, and Protein processing in endoplasmic reticulum (ER) (Figure 5A). In the comparison of HSS with Con, the top 3 enriched pathways were Focal adhesion, Protein processing in ER, and Extracellular matrix (ECM)-receptor interaction (Figure 5B). For further refinement, we focused on the top 8 pathways in each comparison and sorted the top and bottom 15 DEPs based on logFC values. Two chord diagram were generated to display the corresponding pathway relationships (Figures 5C and 5D). Notably, protein processing in endoplasmic reticulum was found as the common enriched pathway in both comparisons. HSPH1, DnaJ homolog subfamily A member 1 (DNAJA1), heat shock 70 kDa protein 8 (HSPA8), and homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 protein (HERPUD1) were identified as 4 shared proteins, all of which belong to protein processing in ER.

Figure 5.

Figure 5

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KEGG enrichment pathway analysis of DEPs. (A) KEGG pathway analysis of DEPs in HSD vs. Con. (B) KEGG pathway analysis of DEPs in HSS vs. Con. (C) Chord diagram of the KEGG enrichment analysis results for HSD vs. Con, featuring the top 8 enriched pathways as well as the the top and bottom 15 DEPs selected by logFC ranking. (D) Chord diagram of the KEGG enrichment analysis results for HSS vs. Con, featuring the top 8 enriched pathways and a total of 26 involved DEPs.

Analysis of the Shared DEPs and Correlation Network Construction

Based on LFQP detection results, both of the relative quantification of HSPH1, DNAJA1, HSPA8, and HERPUD1 revealed significant higher levels in HSS compared to Con, with a trend of HSD higher than Con and HSS higher than HSD. Incorporating the literature reports, we also investigated the typical heat stress indicator HSP90AA1, which exhibited a similar expression trend to the 4 proteins aforementioned in LFQP results (Figures 6A–6E). Additionally, based on the pathological findings, we investigated the LFQP quantitative results of the main protein CHGA in chromaffin cells, revealing a significantly lower expression level of in the HSD compared to the Con (Figure 6F). To investigate the potential HSR mechanisms in the adrenal glands of Wenchang chickens, we further conducted correlation analysis among these proteins mentioned above. The results showed a significant positive correlation among the proteins, with each pair of the 5 proteins exhibiting a positive correlation with each other (R2 > 0.7, P < 0.05) (Figure 6G). Specifically, HSPH1 and HERPUD1 exhibited the highest pairwise correlation (R2 = 0.98, P < 0.001). Based on these findings, we proceeded to construct a correlation network for these 5 DEPs (Figure 6H), which consists of 9 edges (correlations), with each node connected to 4 others within the network. Consequently, we ultimately identify these five proteins as key molecules in the HSR mechanism of the adrenal gland in Wenchang chickens.

Figure 6.

Figure 6

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Analysis of common DEPs and their correlation network construction (A–E) LFQP quantification results of HSPH1, DNAJA1, HSP90AA1, HSPA8, and HERPUD1. (F) LFQP quantification results of CHGA. (G) Correlation analysis result of HSPH1, DNAJA1, HSP90AA1, HSPA8 and HERPUD1. (H) Correlation network of HSPH1, DNAJA1, HSP90AA1, HSPA8 and HERPUD1. Data represented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Validation of ER Protein Processing-Related DEPs

To improve the reliability of the results, we selected HSPH1 and HSP90AA1 for further validation (Figure 7A). According to the Western blotting results, HSPH1 exhibits significantly higher relative gray values in both HSD vs. Con and HSS vs. Con, with HSS significantly higher than HSD. Similarly, the relative gray value of HSP90AA1 is significantly higher in HSD and HSS compared to Con, and HSS exhibits a significant higher value compared to HSD. These observations align with the LFQP results (Figures 7B and 7C). Immunohistochemical analysis revealed a significantly higher intensity of HSPH1-positive signal in HSD and HSS compared to Con. The positive signals in HSS were stronger than in HSD, and both groups of the HSPH1 primarily distributed in the cytoplasm. Regarding HSP90AA1, a certain level of positive signals for HSP90AA1 was observed in the cytoplasm of Con. However, in the cytoplasm of cortical cells in HSD, the HSP90AA1-positive signal intensity was slightly lower than that of the Con, although nuclei positive signals of cortical and chromaffin cells were stronger. In the HSS, both of the positive signals of HSP90AA1 in the nuclei and cytoplasm of cortical and chromaffin cells were significantly higher compared to the Con and HSD. The scores of positive signals of HSPH1 and HSP90AA1 in both HSD and HSS were significantly higher compared to Con, with HSPH1 scoring significantly higher in HSS than in HSD.

Figure 7.

Figure 7

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Validation of ER Protein Processing-Related DEPs. (A) Western blot validation of HSPH1 and HSP90AA1 in adrenal tissue of heat-stressed Wenchang chickens, with β-actin as the control. (B–C) Relative gray values of HSPH1 and HSP90AA1 from Western blotting results. (D) Positive signals of HSPH1 and HSP90AA1 in control and heat-stressed adrenal tissue of Wenchang chickens observed through immunohistochemical staining. (E–F) Scores of positive signals of HSPH1 and HSP90AA1. Data represented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

DISCUSSION

In this study, on the basis of assessing heat stress-induced damage to Wenchang chicken adrenal tissue, we conducted LFQP analysis, focusing on DEPs and particularly comparing HSD vs. Con and HSS vs. Con. The ELISA results showed significantly higher (P < 0.05) COR and NE plasma hormone levels in HSS and non-significantly higher levels in HSD compared to the Con. CORT and EPI levels were significantly lower in HSD compared to both Con and HSS (P < 0.05), with the highest levels observed in the HSS. Histopathological analysis revealed main degeneration in HSS cortical and chromaffin cells, severe cell necrosis in HSD, and significantly higher pathological scores (P < 0.05) in HSD and HSS compared to Con, with HSD scoring the highest (P < 0.01). These results collectively underscore the detrimental effects of fatal acute heat stress on the adrenal gland tissues of Wenchang chickens. For HSD vs. Con and HSS vs. Con, 94, 54 upregulated DEPs and 478, 137 downregulated DEPs were respectively identified based on the criteria of FC ≥ 1.2 or FC ≤ 0.83 and P < 0.05. Through GO and KEGG analysis, we identified a shared significantly enriched pathway "Protein processing in endoplasmic reticulum." Subsequent logFC sorting revealed the top and bottom 15 DEPs (30 in total) in HSD vs. Con and 26 DEPs in the HSS vs. Con comparison. Spearman's correlation analysis ultimately unveiled a correlation network comprising 5 DEPs: HSPH1, DNAJA1, HSP90AA1, HSPA8, and HERPUD1. Validation of HSPH1 and HSP90AA1 expression through Western blotting confirmed the consistent expression trends with the LFQP results. Immunohistochemistry further supported these findings, confirming the analysis results of LFQP and subcellular localization. These results collectively suggest that HSPH1, DNAJA1, HSP90AA1, HSPA8, and HERPUD1 may play dynamic network regulatory roles in the HSR process of adrenal gland cells in Wenchang chicken.

Under acute heat stress, catecholamines (EPI and NE, hormones of the Sympathetic-Adrenal-Medullary axis) and glucocorticoids (COR and CORT, hormones of the HPA axis) are primarily synthesized (Padgett and Glaser, 2003; Marketon and Glaser, 2008; Nawaz et al., 2021), enhance hepatic glycogenolysis and glucose synthesis (Barth et al., 2007), inhibit the production of cytokine and pro-inflammatory mediator (Wilckens and De Rijk, 1997), and promote the release of anti-inflammatory mediators, exerting protective effects against heat stress-induced damage. Chronic heat stress is mainly mediated by adrenal corticosteroids, triggering self-protective responses and serving as a major response mode in avians (Lin et al., 2006). These adrenal-related hormone levels are commonly used to assess the physiological status of animals under stress conditions (Armario et al., 2020), among which COR and CORT are considered to be the key biomarkers for evaluating avian heat stress status (Post et al., 2003; Kinlein et al., 2019; Li et al., 2020), with CORT being the predominant (Carsia, 2022). However, it is worth noting that corticosteroid hormone secretion follows diurnal and seasonal cycles, and is influenced by various factors including negative feedback regulation of the HPA axis, food intake, and other stressors (Mormède et al., 2007). Studies by Mack et al. (2013) and Calefi et al. (2019) found no significant difference in serum CORT levels between heat-stressed and non-stressed avian species. Therefore, the combined use of SAM axis markers can provide us a more comprehensive understanding of animal stress responses (Martínez-Miró et al., 2016; Lensen et al., 2019). In our study, we observed that the plasma NE levels in 2 heat stress groups were higher than those in the Con, and there was a significant difference in HSS vs. Con, indicating a direct impact of heat stress on the avian endocrine system, consistent with previous research by Lyte et al. (2023). Contrary to previous reports (Moudgal et al., 1990), the secretion levels of EPI in the HSD were significantly lower than those in the Con; however, these results were similar to the findings by Edens and Siegel (1975). A noteworthy observation is that the HSD exhibited significantly lower plasma CORT levels compared to the Con, which is a finding that will be further discussed in subsequent sections.

Pathological structural damage is a crucial factor affecting adrenal hormone secretion. Different from mammals, in typical avian adrenal medulla, adrenal cortical cells are intermingling with chromaffin cells (Carsia, 2022). Recognized by their distinct granular structure, chromaffin cells have emerged as a pivotal cell type in avian adrenal pathology research. Chromogranin A (CHGA) is a primary soluble protein in chromaffin granules of adrenal medulla (Blaschko et al., 1967), regarded as a potential stress marker for high heat (Yoshida et al., 2011). In this study, we observed the most extensive degeneration of adrenal cells in the HSS and the most severe necrosis in HSD, along with more significantly elevated pathological scores in HSD than HSS. This observation aligns with reduced plasma levels of EPI. LFQP results revealed significantly lower CHGA expression in the HSD compared to the Con, consistent with findings reported by Zheng et al. (2021). Therefore, we infer that the death of chickens under heat stress may result from the necrosis of chromaffin cells, thereby impairing catecholamine secretion and subsequently impacting cardiac function. Similarly, the significantly lower CORT levels in the HSD compared to the Con likely stem from heat-induced damage to cortical cells. GO and KEGG functional analysis of DEPs revealed a significant downregulation of metabolic processes and oxidative phosphorylation (OXPHOS) pathways in HSD. The cellular energy demands increase during the initial stages of heat stress pathophysiology. Research by Yang et al. (2010) found that acute heat treatment could promptly double the broiler cell energy consumption, reaching twice the normal physiological levels.

Mitochondria, as the cellular energy factories, primarily generate cellular energy through OXPHOS facilitated by the electron transport chain (ETC) and ATP synthase. However, prolonged exposure to adverse heat stress conditions may hinder ETC-related functions, including complex I inactivation, protein thiol oxidation, glycolytic enzyme carbonylation, and reductions in NAD+ and ATP levels. This results in a decrease in electron flow along the respiratory chain, impairing mitochondrial ATP synthesis, and can ultimately lead to cell death (Belhadj-Slimen et al., 2016). Our LFQP results of Con vs. HSD revealed significant downregulation of several NADH-ubiquinone oxidoreductase (NDUF) family members, including NDUFS2, NDUFS4, NDUFC2, and NDUFB7, under heat stress conditions. This suggests that heat stress may potentially disrupt the mitochondrial oxidative phosphorylation function of adrenal cells by affecting ETC complex I, ultimately resulting in metabolite damage.

A significant discovery in our study was the identification of a shared KEGG enrichment pathway: endoplasmic reticulum protein processing, significantly enriched in both comparisons of HSD vs. Con and HSS vs. Con. Through analysis of shared DEPs and subsequent correlation analysis, we discovered a correlation network composed of 5 DEPs: HSPH1, DNAJA1, HSP90AA1, HSPA8, and HERPUD1, serving as the key molecules in Wenchang chicken resistance to heat stress-induced damage. ER is a crucial membrane-bound organelle responsible for regulating protein production, including folding, assembly, modification, quality control, and recycling (Braakman and Bulleid, 2011). In adrenal cells, numerous ribosomes are attached to the surface of the ER, imparting strong protein secretion capabilities (Dara et al., 2011). However, elevated temperatures can impede protein synthesis, causing protein denaturation, thereby reducing yields of the protein and subsequently affecting protein metabolism (Baumgard and Rhoads, 2013). Currently, protein denaturation is also considered the signal that activate the biological response to heat stress (Huang et al., 2015).

Heat shock proteins (HSPs) are typical proteins involved in the cellular response to heat stress and are commonly regarded as important biochemical indicators (Tomanek, 2010; Varasteh et al., 2015). Their primary function is to act as molecular chaperones, maintaining proper protein folding and facilitating the maturation of functional conformations, preventing misfolding, aggregation, and accumulation of the proteins (Balchin et al., 2016). The HSP70 family constitutes a major component of the cellular chaperone network and stress response. It is typically induced by various stress and pathological conditions and is widely expressed in organs including muscles, liver, heart, kidneys, and blood vessels (Sun et al., 2007; Zhang et al., 2014). HSPA8 (Hsc70), as the constitutive protein in the HSP70 family (Hightower et al., 1994), engages in various housekeeping chaperoning functions, including nascent polypeptides folding, translocation of membrane proteins, facilitating chaperone-mediated autophagy, preventing protein aggregation under stress, and orchestrating ubiquitination and degradation of intracellular proteins (Daugaard et al., 2007), which is crucial in cell survival. Despite the stress-inducible members being commonly recognized as major proteins in the heat stress response, HSPA8 is also believed to function in HSR (di Iorio et al., 1996). Research by Leung et al. (1996) observed conformational changes in bovine Hsc70 under heat conditions, suggesting a potential role in acquired thermotolerance. Zaprjanova et al. (2013) demonstrated that HSPA8 is mobilized to prevent apoptosis in the testes and epididymis as well as aiding HSP72 in repairing stress-induced protein conformational changes in the testes and epididymis. Moreover, Cheng et al. (2018) observed a significant upregulation of HSPA8 expression in B strain Taiwan Country hens following acute heat stress treatment without recovery. HSP40, also known as DNAJ due to the presence of J domains, is classified into 3 subfamilies (Craig and Marszalek, 2017). It functions as a co-chaperone with HSPA8, regulating ATPase activity to assist in facilitating protein folding, assembly, and degradation, thereby playing a vital role in the chaperone network (Wentink et al., 2020). DNAJA1 belongs to the DNAJ/HSP40 family, and is one of the most abundant DNAJ co-chaperones of HSPA8 (Baaklini et al., 2012). Li et al. (2020) found that the expression of DNAJA1, DNAJB12 and DNAJC8 was induced at different levels under acute and chronic heat stress. Among them, DNAJA1 exhibited a potentially stronger resistance role to heat stress compared to DNAJB12 and DNAJC8, suggesting its potential as a key factor in thermotolerance in honeybees. HSPH1 belongs to the HSP70 family and is a crucial member of the major heat shock proteins, widely distributed in various animal tissues and cells (Subjeck et al., 1982; Lee-Yoon et al., 1995). In ER, HSPH1 primarily participates in ER-associated degradation (ERAD) and acting as a nucleotide exchange factor (NEF) for HSPA8, assisting in the conformational cycling of HSPA8 through DNAJ/HSP40 co-chaperones to facilitate the degradation of misfolded proteins (Smith et al., 2011; Rosenzweig et al., 2019). HSPH1 also plays an indispensable role in the animal HSR mechanism. Research by Hightower and Guidon, 1989 revealed a rapid increase in the expression levels of HSPH1 in rat embryonic cells under heat stress conditions. Furthermore, studies by Luo et al. (2014) on comparative population genomics of broiler chickens using gene microarray analysis and by Asadollahpour et al. (2022) on indigenous Iranian chickens, suggest that HSPH1 is also an important protein molecule in the HSR mechanism. In our LFQP results, we observed a coordinated increase in HSPA8, DNAJA1, and HSPH1 in the adrenal tissue of heat-stressed Wenchang chickens, with significant differences between the HSD and HSS compared to the Con. These DEPs are collectively enriched in the endoplasmic reticulum protein processing pathway and exhibit a high degree of correlation with each other (R2 > 0.7, P < 0.05), indicating a strong synergistic regulatory effect. Notably, HSPH1 exhibited the largest fold change and significantly higher gray value compared to the Con in Western blotting validation.

HERPUD1 (HERP) is an ER-resident protein upregulated under various stress conditions (Ma and Hendershot, 2004). It is considered to be an important component of ERAD, functioning as a scaffold protein on the ER membrane to facilitate retrotranslocation of misfolded protein substrates in the absence of enzymatic activity and interacts with proteins involved in ubiquitination and degradation, thereby regulating ERAD, increasing cellular autophagy while suppressing apoptosis (Schulze et al., 2005; Ge et al., 2017; Chen et al., 2019). However, as suggested by Hendriksen et al. (2006), its role in cells may be dual: it inhibits apoptosis in the short term but induces apoptosis in the long term (Hendriksen et al., 2006). Our LFQP results demonstrated a coordinated upregulation of HERPUD1, which is significantly correlated with HSPA8, DNAJA1, and HSPH1, highlighting the importance of protein quality control in the ER. Based on these findings, we hypothesize that the ubiquitin-proteasome-mediated ERAD process may be the key mechanism for broiler adrenal tissue cells to resist heat stress-induced damage. HSP90 is primarily a cytoplasmic protein, responsible for maintaining protein homeostasis and exerting cellular protective functions through interactions with various client proteins (Prodromou, 2016). HSP90AA1 is the stress-inducible isoform of HSP90, playing a crucial role in cell stress response (Hu et al., 2023), and is regarded as the classic biomarker of heat stress. Previous work from our research group demonstrated the significant protective role of HSP90AA1 in primary chicken cardiomyocytes under heat stress (Yao et al., 2023). Additionally, studies by Chen et al. (2020) have suggested that enhanced cellular resistance to heat stress-induced damage may be associated with the nuclear translocation of HSP90AA1. In this study, LFQP analysis revealed a significant increase in HSP90AA1 expression in the HSS, consistent with Western blotting results. Immunohistochemical staining and scoring results also demonstrated enhanced positive signals of HSP90AA1 in the nuclei and cytoplasm of adrenal cortical cells and chromaffin cells in the HSS, with a trend of higher nuclear positive signal intensity in the HSD and HSS compared to the Con as the heat stress progressed. Therefore, we propose that during heat stress, the adrenal glands of Wenchang chicken may exert HSR by upregulating the expression levels of HSP90AA1, potentially involving nuclear translocation of the HSP90AA1. Based on the above results, we have obtained a possible explanation for the previous ELISA results: the significant reduction in CORT levels in HSD is highly likely to be associated with impaired ERAD and related HSR functions, as well as dysfunction of cortical and chromaffin cells, especially cell necrosis, thereby resulting in compromised hormone secretion. Overall, these dynamically altered proteins in adrenal glands are expected to be the key molecules for regulating the HSR mechanism in Wenchang chicken.

CONCLUSIONS

In summary, our study investigated the adverse effect of heat stress on adrenal gland tissue of Wenchang chickens. LFQP and bioinformatics analysis revealed the shared enriched pathway of endoplasmic reticulum protein processing and unveiled a correlation network comprising HSPH1, DNAJA1, HSP90AA1, HSPA8, and HERPUD1, highlighting the significant role of ERAD process in mitigating heat stress-induced injury in adrenal tissue cells of Wenchang chicken. These findings offer valuable insights into understanding the mechanisms underlying adrenal resistance to heat stress-induced damage in Wenchang chicken and identifying key molecules for their heat resistance, thereby providing theoretical basis for screening the treatment drugs of heat stress and heat-resistant poultry breeding.

DISCLOSURES

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

This research was funded by the National Natural Science Foundation Regional Funding Project of China (grant number 32260868), Youth Fund Project of Hainan Provincial Natural Science Foundation of China (grant number 322QN246), Key Research and Development project in Hainan Province (grant number ZDYF2024XDNY218), Research Project of Collaborative Innovation Center of Hainan University (grant number XTCX2022NYC05), and the Initial Scientific Research Foundation in Hainan University (grant number KYQD(ZR)-2200C6).

REFERENCES

  1. Armario A., Labad J., Nadal R. Focusing attention on biological markers of acute stressor intensity: Empirical evidence and limitations. Neurosci. Biobehav. Rev. 2020;111:95–103. doi: 10.1016/j.neubiorev.2020.01.013. [DOI] [PubMed] [Google Scholar]
  2. Asadollahpour Nanaei H., Kharrati-Koopaee H., Esmailizadeh A. Genetic diversity and signatures of selection for heat tolerance and immune response in Iranian native chickens. BMC Genom. 2022;231:224. doi: 10.1186/s12864-022-08434-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baaklini I., Wong M.J., Hantouche C., Patel Y., Shrier A., Young J.C. The DNAJA2 substrate release mechanism is essential for chaperone-mediated folding. J. Biol. Chem. 2012;287:41939–41954. doi: 10.1074/jbc.M112.413278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Balchin D., Hayer-Hartl M., Hartl F.U. In vivo aspects of protein folding and quality control. Science. 2016;353:aac4354. doi: 10.1126/science.aac4354. [DOI] [PubMed] [Google Scholar]
  5. Barth E., Albuszies G., Baumgart K., Matejovic M., Wachter U., Vogt J., Calzia E. Glucose metabolism and catecholamines. Crit. Care Med. 2007;35:508–518. doi: 10.1097/01.CCM.0000278047.06965.20. [DOI] [PubMed] [Google Scholar]
  6. Baumgard L.H., Rhoads R.P., Jr. Effects of heat stress on postabsorptive metabolism and energetics. Annu. Rev. Anim. Biosci. 2013;1:311–337. doi: 10.1146/annurev-animal-031412-103644. [DOI] [PubMed] [Google Scholar]
  7. Belhadj-Slimen I., Najar T., Ghram A., Abdrrabba M. Heat stress effects on livestock: molecular, cellular and metabolic aspects, a review. J. Anim. Physiol. Anim. Nutr. Berl. 2016;100:401–412. doi: 10.1111/jpn.12379. [DOI] [PubMed] [Google Scholar]
  8. Blaschko H., Comline R.S., Schneider F.H., Silver M., Smith A.D. Secretion of a chromaffin granule protein, chromogranin, from the adrenal gland after splanchnic stimulation. Nature. 1967;215:58–59. doi: 10.1038/215058a0. [DOI] [PubMed] [Google Scholar]
  9. Braakman I., Bulleid N.J. Protein folding and modification in the mammalian endoplasmic reticulum. Annu. Rev. Biochem. 2011;80:71–99. doi: 10.1146/annurev-biochem-062209-093836. [DOI] [PubMed] [Google Scholar]
  10. Bureau C., Hennequet-Antier C., Couty M., Guémené D. Gene array analysis of adrenal glands in broiler chickens following ACTH treatment. BMC Genom. 2009;10:1–8. doi: 10.1186/1471-2164-10-430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Calefi A.S., Quinteiro-Filho W.M., de Siqueira A., Lima A.P.N., Cruz D.S.G., Hazarbassanov N.Q., Palermo-Neto J. Heat stress, Eimeria spp. and C. perfringens infections alone or in combination modify gut Th1/Th2 cytokine balance and avian necrotic enteritis pathogenesis. Vet. Immunol. Immunopathol. 2019;210:28–37. doi: 10.1016/j.vetimm.2019.03.001. [DOI] [PubMed] [Google Scholar]
  12. Carsia, R. V. 2022. Chapter 33 - Adrenals. Pages 881-914 in Sturkie's Avian Physiology (7th edition), C. G. Scanes and S. Dridi, eds. Academic Press, San Diego, California.
  13. Chen B., Yang B., Zhu J., Wu J., Sha J., Sun J., Bao E., Zhang X. Hsp90 relieves heat stress-induced damage in mouse kidneys: Involvement of antiapoptotic PKM2-AKT and autophagic HIF-1α signaling. Int. J. Mol. Sci. 2020;21:1646. doi: 10.3390/ijms21051646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chen F., Wen X., Lin P., Chen H., Wang A., Jin Y. HERP depletion inhibits zearalenone-induced apoptosis through autophagy activation in mouse ovarian granulosa cells. Toxicol. Lett. 2019;301:1–10. doi: 10.1016/j.toxlet.2018.10.026. [DOI] [PubMed] [Google Scholar]
  15. Chen X., Bai X., Liu H., Zhao B., Yan Z., Hou Y., Chu Q. Population genomic sequencing delineates global landscape of copy number variations that drive domestication and breed formation of in chicken. Front. Genet. 2022;13 doi: 10.3389/fgene.2022.830393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cheng C.Y., Tu W.L., Chen C.J., et al. Proteomic analysis of thermal regulation of small yellow follicles in broiler-type Taiwan country chickens. J. Poult. Sci. 2018;55:120–136. doi: 10.2141/jpsa.0170069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Craig E.A., Marszalek J. How do J-proteins get Hsp70 to do so many different things? Trends Biochem. Sci. 2017;42:355–368. doi: 10.1016/j.tibs.2017.02.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dara L., Ji C., Kaplowitz N. The contribution of endoplasmic reticulum stress to liver diseases. Hepatology. 2011;53:1752–1763. doi: 10.1002/hep.24279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Daugaard M., Rohde M., Jäättelä M. The heat shock protein 70 family: highly homologous proteins with overlapping and distinct functions. FEBS Lett. 2007;581:3702–3710. doi: 10.1016/j.febslet.2007.05.039. [DOI] [PubMed] [Google Scholar]
  20. Dennis G., Jr, Sherman B.T., Hosack D.A., Yang J., Gao W., Lane C.H., Lempicki R.A. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 2003;4:3. [PubMed] [Google Scholar]
  21. di Iorio P.J., Holsinger K., Schultz R.J., Hightower L.E. Quantitative evidence that both Hsc70 and Hsp70 contribute to thermal adaptation in hybrids of the livebearing fishes Poeciliopsis. Cell Stress Chaperones. 1996;1:139–147. doi: 10.1379/1466-1268(1996)001<0139:qetbha>2.3.co;2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Edens F.W., Siegel H.T. Adrenal responses in high and low ACTH response lines of chickens during acute heat stress. Gen Comp Endocrinol. 1975;25:64–73. doi: 10.1016/0016-6480(75)90040-4. [DOI] [PubMed] [Google Scholar]
  23. Fathi M.M., Galal A., Radwan L.M., Abou-Emera O.K., Al-Homidan I.H. Using major genes to mitigate the deleterious effects of heat stress in poultry: an updated review. Poult. Sci. 2022;101:102–157. doi: 10.1016/j.psj.2022.102157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Fu A., Gu L., Chen Y. Overview of Wenchang chicken and analysis of the whole industry chain. Chin. Livestock Poult. Breeding. 2022;18:30–33. [Google Scholar]
  25. Ge M., Luo Z., Qiao Z., Zhou Y., Cheng X., Geng Q., Cai Y., Wan P., Xiong Y., Liu F., Wu K., Liu Y., Wu J. HERP binds TBK1 to activate innate immunity and repress virus replication in response to endoplasmic reticulum stress. J. Immunol. 2017;199:3280–3292. doi: 10.4049/jimmunol.1700376. [DOI] [PubMed] [Google Scholar]
  26. Hendriksen P.J., Dits N.F., Kokame K., Veldhoven A., van Weerden W.M., Bangma C.H., Trapman J., Jenster G. Evolution of the androgen receptor pathway during progression of prostate cancer. Cancer Res. 2006;66:5012–5020. doi: 10.1158/0008-5472.CAN-05-3082. [DOI] [PubMed] [Google Scholar]
  27. Hightower L.E., Guidon P.T.Jr. Selective release from cultured mammalian cells of heat-shock (stress) proteins that resemble glia-axon transfer proteins. J. Cell. Physiol. 1989;138:257–266. doi: 10.1002/jcp.1041380206. [DOI] [PubMed] [Google Scholar]
  28. Hightower, L. E., S. E. Sadis, and I. M. Takenaka. 1994. Interactions of vertebrate hsc70 and hsp70 with unfolded proteins and peptides. Pages 179-207 in The Biology of Heat Shock Proteins and Molecular Chaperones. R.I. Morimoto, A. Tissieres, and C. Georgopoulos, eds. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY.
  29. Hu L., Fang H., Abbas Z., et al. The HSP90AA1 gene is involved in heat stress responses and its functional genetic polymorphism are associated with heat tolerance in Holstein cows. J. Dairy Sci. 2024;107:5132–5149. doi: 10.3168/jds.2023-24007. [DOI] [PubMed] [Google Scholar]
  30. Huang C., Jiao H., Song Z., Zhao J., Wang X., Lin H. Heat stress impairs mitochondria functions and induces oxidative injury in broiler chickens. J. Anim. Sci. 2015;93:2144–2153. doi: 10.2527/jas.2014-8739. [DOI] [PubMed] [Google Scholar]
  31. Huang D.W., Lempicki R.A., Sherman B.T. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols. 2009;4:44–57. doi: 10.1038/nprot.2008.211. [DOI] [PubMed] [Google Scholar]
  32. Kanehisa M., Sato Y., Kawashima M., Furumichi M., Tanabe M. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 2016;44:457–462. doi: 10.1093/nar/gkv1070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Khan R.U., Naz S., Nikousefat Z., Tufarelli V., Javdani M., Rana N., Laudadio V. Effect of vitamin E in heat-stressed poultry. Worlds Poult. Sci. J. 2011;67:469–478. [Google Scholar]
  34. Kilic I., Simsek E. The effects of heat stress on egg production and quality of laying hens. J Anim Vet Adv. 2013;12:42–47. [Google Scholar]
  35. Kinlein S.A., Phillips D.J., Keller C.R., Karatsoreos I.N. Role of corticosterone in altered neurobehavioral responses to acute stress in a model of compromised hypothalamic-pituitary-adrenal axis function. Psychoneuroendocrinology. 2019;102:248–255. doi: 10.1016/j.psyneuen.2018.12.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kumar M., Ratwan P., Dahiya S.P., Nehra A.K. Climate change and heat stress: impact on production, reproduction and growth performance of poultry and its mitigation using genetic strategies. J. Therm. Biol. 2021;97 doi: 10.1016/j.jtherbio.2021.102867. [DOI] [PubMed] [Google Scholar]
  37. Lee-Yoon D., Easton D., Murawski M., Burd R., Subjeck J.R. Identification of a major subfamily of large hsp70-like proteins through the cloning of the mammalian 110-kDa heat shock protein. J. Biol. Chem. 1995;270:15725–15733. doi: 10.1074/jbc.270.26.15725. [DOI] [PubMed] [Google Scholar]
  38. Lensen R.C., Moons C.P., Diederich C. Physiological stress reactivity and recovery related to behavioral traits in dogs (Canis familiaris) PLoS One. 2019;14 doi: 10.1371/journal.pone.0222581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Leung S.M., Senisterra G., Ritchie K.P., Sadis S.E., Lepock J.R., Hightower L.E. Thermal activation of the bovine Hsc70 molecular chaperone at physiological temperatures: physical evidence of a molecular thermometer. Cell Stress Chaperones. 1996;1:78–89. doi: 10.1379/1466-1268(1996)001<0078:taotbh>2.3.co;2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Li D., Tong Q., Shi Z., Li H., Wang Y., Li B., Yan G., Chen H., Zheng W. Effects of chronic heat stress and ammonia concentration on blood parameters of laying hens. Poult Sci. 2020;99:3784–3792. doi: 10.1016/j.psj.2020.03.060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Li G., Zhao H., Guo H., Wang Y., Cui X., Li H., Xu B., Guo X. Analyses of the function of DnaJ family proteins reveal an underlying regulatory mechanism of heat tolerance in honeybee. Sci. Total Environ. 2020;716 doi: 10.1016/j.scitotenv.2020.137036. [DOI] [PubMed] [Google Scholar]
  42. Lin H., Decuypere E., Buyse J. Acute heat stress induces oxidative stress in broiler chickens. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2006;144:11–17. doi: 10.1016/j.cbpa.2006.01.032. [DOI] [PubMed] [Google Scholar]
  43. Luo Q.B., Song X.Y., Ji C.L., Zhang X.Q., Zhang D.X. Exploring the molecular mechanism of acute heat stress exposure in broiler chickens using gene expression profiling. Gene. 2014;546:200–205. doi: 10.1016/j.gene.2014.06.017. [DOI] [PubMed] [Google Scholar]
  44. Lyte J.M., Lyte M., Daniels K.M., Oluwagbenga E.M., Fraley G.S. Catecholamine concentrations in duck eggs are impacted by hen exposure to heat stress. Front Physiol. 2023;14 doi: 10.3389/fphys.2023.1122414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ma Y., Hendershot L.M. Herp is dually regulated by both the endoplasmic reticulum stress-specific branch of the unfolded protein response and a branch that is shared with other cellular stress pathways. J. Biol. Chem. 2004;279:13792–13799. doi: 10.1074/jbc.M313724200. [DOI] [PubMed] [Google Scholar]
  46. Mack L.A., Felver-Gant J.N., Dennis R.L., Cheng H.W. Genetic variations alter production and behavioral responses following heat stress in 2 strains of laying hens. Poult. Sci. 2013;92:285–294. doi: 10.3382/ps.2012-02589. [DOI] [PubMed] [Google Scholar]
  47. Marketon J.I.W., Glaser R. Stress hormones and immune function. Cell. Immunol. 2008;252:16–26. doi: 10.1016/j.cellimm.2007.09.006. [DOI] [PubMed] [Google Scholar]
  48. Martínez-Miró S., Tecles F., Ramón M., Escribano D., Hernández F., Madrid J., Orengo J., Martínez-Subiela S., Manteca X., Cerón J.J. Causes, consequences and biomarkers of stress in swine: an update. BMC Vet. Res. 2016;12:1–9. doi: 10.1186/s12917-016-0791-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Mormède P., Andanson S., Aupérin B., Beerda B., Guémené D., Malmkvist J., Manteuffel G., Prunet P., van Reenen C.G., Richard S., Veissier I. Exploration of the hypothalamic–pituitary–adrenal function as a tool to evaluate animal welfare. Physiol. Behav. 2007;92:317–339. doi: 10.1016/j.physbeh.2006.12.003. [DOI] [PubMed] [Google Scholar]
  50. Moudgal R.P., Panda J.N., Jagmohan Catecholamines are present in hen’s egg yolk in fairly stable form: elevated adrenaline indicates stress. Curr. Sci. 1990;19:937–939. [Google Scholar]
  51. Nawaz A.H., Amoah K., Y.Leng Q., Zheng J.H., Zhang W.L., Zhang L. Poultry response to heat stress: its physiological, metabolic, and genetic implications on meat production and quality including strategies to improve broiler production in a warming world. Front. Vet. Sci. 2021;8 doi: 10.3389/fvets.2021.699081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Oladokun S., Adewole D.I. Biomarkers of heat stress and mechanism of heat stress response in Avian species: current insights and future perspectives from poultry science. J. Therm. Biol. 2022;110 doi: 10.1016/j.jtherbio.2022.103332. [DOI] [PubMed] [Google Scholar]
  53. Padgett D.A., Glaser R. How stress influences the immune response. Trends Immunol. 2003;24:444–448. doi: 10.1016/s1471-4906(03)00173-x. [DOI] [PubMed] [Google Scholar]
  54. Post J., Rebel J.M., Ter Huurne A.A. Physiological effects of elevated plasma corticosterone concentrations in broiler chickens. An alternative means by which to assess the physiological effects of stress. Poult. Sci. 2003;82:1313–1318. doi: 10.1093/ps/82.8.1313. [DOI] [PubMed] [Google Scholar]
  55. Prodromou C. Mechanisms of Hsp90 regulation. Biochem. J. 2016;473:2439–2452. doi: 10.1042/BCJ20160005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Radwan L.M. Genetic improvement of egg laying traits in Fayoumi chickens bred under conditions of heat stress through selection and gene expression studies. J. Therm. Biol. 2020;89 doi: 10.1016/j.jtherbio.2020.102546. [DOI] [PubMed] [Google Scholar]
  57. Rosenzweig R., Nillegoda N.B., Mayer M.P., Bukau B. The Hsp70 chaperone network. Nat. Rev. Mol. Cell Biol. 2019;20:665–680. doi: 10.1038/s41580-019-0133-3. [DOI] [PubMed] [Google Scholar]
  58. Rozanova S., Barkovits K., Nikolov M., Schmidt C., Urlaub H., Marcus K. Quantitative mass spectrometry-based proteomics: an overview. quantitative methods in proteomics. Methods Mol. Biol. 2021;2228:85–116. doi: 10.1007/978-1-0716-1024-4_8. [DOI] [PubMed] [Google Scholar]
  59. Schulze A., Standera S., Buerger E., Kikkert M., van Voorden S., Wiertz E., Koning F., Kloetzel P.M., Seeger M. The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway. J. Mol. Biol. 2005;354:1021–1027. doi: 10.1016/j.jmb.2005.10.020. [DOI] [PubMed] [Google Scholar]
  60. Smith M.H., Ploegh H.L., Weissman J.S. Road to ruin: targeting proteins for degradation in the endoplasmic reticulum. Science. 2011;334:1086–1090. doi: 10.1126/science.1209235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Subjeck J.R., Sciandra J.J., Chao C.F., Johnson R.J. Heat shock proteins and biological response to hyperthermia. Br. J. Cancer Suppl. 1982;5:127–131. [PMC free article] [PubMed] [Google Scholar]
  62. Sun P., Liu Y., Wang Q., Wang Z., Bao E. HSP70 and HSP70 mRNA localization in heat-stressed tissues of broilers. Chin. J. Agric. Biotechnol. 2007;4:181–186. [Google Scholar]
  63. Tang S., Yin B., Song E., Chen H., Cheng Y., Zhang X., Bao J., Hartung J. Aspirin upregulates αB-Crystallin to protect the myocardium against heat stress in broiler chickens. Sci. Rep. 2016;6:37273. doi: 10.1038/srep37273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Tomanek L. Variation in the heat shock response and its implication for predicting the effect of global climate change on species' biogeographical distribution ranges and metabolic costs. J. Exp. Biol. 2010;213:971–979. doi: 10.1242/jeb.038034. [DOI] [PubMed] [Google Scholar]
  65. Varasteh S., Braber S., Akbari P., Garssen J., Fink-Gremmels J. Differences in susceptibility to heat stress along the chicken intestine and the protective effects of galacto-oligosaccharides. PLoS One. 2015;10 doi: 10.1371/journal.pone.0138975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wentink A.S., Nillegoda N.B., Feufel J., Ubartaitė G., Schneider C.P., De Los Rios P., Hennig J., Barducci A., Bukau B. Molecular dissection of amyloid disaggregation by human HSP70. Nature. 2020;587:483–488. doi: 10.1038/s41586-020-2904-6. [DOI] [PubMed] [Google Scholar]
  67. Wilckens T., De Rijk R. Glucocorticoids and immune function: unknown dimensions and new frontiers. Immunol. Today. 1997;18:418–424. doi: 10.1016/s0167-5699(97)01111-0. [DOI] [PubMed] [Google Scholar]
  68. Yang L., Tan G.Y., Fu Y.Q., Feng J.H., Zhang M.H. Effects of acute heat stress and subsequent stress removal on function of hepatic mitochondrial respiration, ROS production and lipid peroxidation in broiler chickens. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2010;151:204–208. doi: 10.1016/j.cbpc.2009.10.010. [DOI] [PubMed] [Google Scholar]
  69. Yao X., Zhu J., Li L., Yang B., Chen B., Bao E., Zhang X. Hsp90 protected chicken primary myocardial cells from heat-stress injury by inhibiting oxidative stress and calcium overload in mitochondria. Biochem. Pharmacol. 2023;209 doi: 10.1016/j.bcp.2023.115434. [DOI] [PubMed] [Google Scholar]
  70. Yoshida C., Ishikawa T., Michiue T., Quan L., Maeda H. Postmortem biochemistry and immunohistochemistry of chromogranin A as a stress marker with special regard to fatal hypothermia and hyperthermia. Int J Legal Med. 2011;125:11–20. doi: 10.1007/s00414-009-0374-3. [DOI] [PubMed] [Google Scholar]
  71. Yu J., Bao E., Yan J., Lei L. Expression and localization of Hsps in the heart and blood vessel of heat-stressed broilers. Cell Stress and Chaperones. 2008;13:327–335. doi: 10.1007/s12192-008-0031-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Zaprjanova S., Rashev P., Zasheva D., Martinova Y., Mollova M. Electrophoretic and immunocytochemical analysis of Hsp72 and Hsp73 expression in heat-stressed mouse testis and epididymis. Eur. J. Obstet. Gynecol. Reprod. Biol. 2013;168:54–59. doi: 10.1016/j.ejogrb.2012.12.021. [DOI] [PubMed] [Google Scholar]
  73. Zhang W.W., Kong L.N., Zhang X.Q., Luo Q.B. Alteration of HSF3 and HSP70 mRNA expression in the tissues of two chicken breeds during acute heat stress. Genet. Mol. Res. 2014;13:9787–9794. doi: 10.4238/2014.November.27.6. [DOI] [PubMed] [Google Scholar]
  74. Zheng H.T., Zhuang Z.X., Chen C.J., Liao H.Y., Chen H.L., Hsueh H.C., Chen C.F., Chen S.E., Huang S.Y. Effects of acute heat stress on protein expression and histone modification in the adrenal gland of male layer-type country chickens. Sci. Rep. 2021;111:6499. doi: 10.1038/s41598-021-85868-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

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