Clinical changes caused by heat stress
We measured the body weight, rectal temperature, and forehead temperature of pigs on days 1 to 7 of implementing the heat stress treatment. The body weight of pigs was significantly reduced on day 7 of heat stress (Fig. 1A). Under heat stress conditions, both forehead temperature and rectal temperature of pigs were higher than control, the most difference between heat stressed and control pigs appearing on day 7 (Fig. 1B, C). These results indicated that heat stress significantly affected the growth performance (in terms of the abovementioned traits) of pigs.
iTRAQ-based DEP identification and quantitative analysis of swine hypothalamic tissue
All protein and peptide identifications were obtained by database searching and stringent data filtering. The LC–MS/MS analysis produced 7072 spectra, corresponding to 2882 unique peptides; 360 proteins were identified at a false discovery rate (FDR) of ≤0.01 (Figure 2A). According to the level of protein abundance, proteins were regarded as different proteins when the difference multiple reached 1.5 or more, and the statistical test showed significant difference (P < 0.05). Thus, there were 295 DEPs identified in the hypothalamus, of which 148 were up-regulated and 147 were down-regulated. Table 1 provides information about 40 key DEPs, 20 up-regulated and 20 down-regulated. A list of all identified DEPs has been provided in Supplementary Table 2.
The molecular weights of most DEPs were in the range of 20–60 kD (157 DEPs) (Fig. 2B). In addition, the identified DEPs had high peptide coverage, of which 80% and 54% showed more than 10% and 20% sequence coverage, respectively (Fig. 2C). About 74% of the identified DEPs had three or more peptides (Fig. 2D).
Subcellular localization and canonical pathways of identified DEPs
To elucidate the functional characteristics of DEPs in the hypothalamus under HS, the DEPs of the hypothalamic tissues were analyzed based on the basic biological functions, clustering of molecular functions, and cell locations of proteins in the UniProtKB/Swiss-Prot, TrEMBL, and Gene Ontology databases. The DEPs identified from hypothalamus tissue altered by heat stress localized in various subcellular regions: cytoplasmic vesicles (14.9%), microtubule cytoskeleton (11.6%), cytoskeleton (19.3%), high-density lipoprotein particles (1.1%), triglyceride-rich lipoprotein particles (1.5%), endoplasmic reticulum part (5.5%), SNARE protein (1.5%), primary lysosomes (0.7%), pigment granules (5.1%), mitochondrial matrix (2.0%), mitochondria lumen (2.9%), clathrin-coated vesicles (5.1%), synaptic vesicles (2.2%), clathrin-coated vesicles membrane (2.2%), and non-membrane border organelles (24.4%) (Fig. 3A).
To better understand these DEPs, further analysis was undertaken using the Ingenuity Pathway Analysis (IPA) tool. Canonical pathways were examined first; the top 30 pathways are shown in Fig. 3B, including pathways related to inflammation and immunity, such as Huntington’s disease signaling, clathrin-mediated endocytosis signaling, and Liver X receptor/Retinoic X receptor activation (Fig. 3B).
Functional characteristics and bioinformatics analysis of DEPs
Compared with the human genome database, annotation in the pig genome database is relatively scarce, and many protein features are not identified or classified. The differential proteins identified in our study were converted to the human protein gene bank identification (gi) number. The gi numbers and regulatory levels of these proteins were entered into the IPA software, and based on the database, protein–protein interaction signal pathways were constructed. The proteins identified by iTRAQ in the hypothalamus were clustered according to different functions; four statistically significant functional groups were found, namely, diseases and disorders, molecular and cellular functions, physiological and phylogenetic functions, and toxicological functions (Fig. 4).
The DEPs identified in the hypothalamus of pigs under HS were consistent and corresponded to 23 diseases and disorders including proteins that are related to neurological diseases, psychological disorders, skeletal and muscular disorders, hereditary disorders, metabolic diseases, dermatological diseases and conditions, hematological diseases, immunological diseases, and inflammatory diseases, among others (Fig. 4A). The DEPs could also be assigned to 27 molecular and cellular functions groups, including cell death and survival, cell assembly and organization, cell function and maintenance, molecular transport, free radical scavenging, cell morphology, small molecule biochemistry, nucleic acid metabolism, lipid metabolism, and cellular development (Fig. 4B); 16 physiological system development and functions groups, including nervous system development and function, tissue morphology, organ morphology, organismal development, tissue development, behavior, organismal survival, embryonic development, organismal function, hematological system development and function, immune cell trafficking, and organ development (Fig. 4C); and 12 toxicity functions groups, namely, liver hyperplasia/hyper-proliferation, renal damage, renal tubule injury, cardiac necrosis/cell death, kidney failure, cardiac arrhythmia, cardiac damage, cardiac inflammation, renal dilation, cardiac dysfunction, liver fibrosis (Fig. 4D).
Among the DEPs identified in the hypothalamus, 13 functional networks were constructed (Fig. 5). The four networks of interest correspond to (1) Cell assembly and tissue, neural development and function, intercellular signal and interaction (Fig. 5A); (2) Nucleic acid metabolism, small molecular biochemistry, and cell morphology (Fig. 5B); (3) Cell assembly and tissue, cell function and maintenance, and neuropathic disease (Fig. 5C); (4) Free radical scavenging, small molecule biochemistry, and cancer (Fig. 5D). Proteins that are present in these pathways and identified as up-regulated DEPs in our analysis are depicted in shades of red and those identified as down-regulated are shown in green. Proteins in the network, but not identified as DEPs in our study, are depicted in white.
We also predicted the upstream regulators of hypothalamic DEPs by IPA and found that cytokines, kinases, chemical agents, chemical kinase activators, mature microRNAs, and growth factor were activators of these DEPs, while auxins, transcription regulators, and chemicals were inhibitors of these DEPs. These predicted upstream regulators of DEPs responsive to heat stress may have an important role in regulating hormone secretion and signal transduction in pigs.
Validation of Protein Identification and Quantification
Histone H2A is a type of innate immune molecule discovered in recent years that plays a key role in the phagocytosis of neutrophils and in the clearance of pathogenic microorganisms. To verify the reliability of DEPs identified by iTRAQ, the hypothalamus of pigs on day 7 of HS was used to detect the expression of Histone H2A by western blotting. As shown in Figure 6, compared with the control group, the expression level of Histone H2A in the HS group was significantly up-regulated (p < 0.01), which was in agreement with the results of the iTRAQ analysis.