iTRAQ-based quantitative proteomic analyses the cycle chronic heat stress affecting liver proteome in yellow-feather chickens

Abstract

Heat stress (HS) is one of the main environmental factors affecting the efficiency of poultry production. The yellow-feather chickens (YFC) as an indigenous strain of chicken is a popular poultry breed in China. Our previous study used the RNA-seq to analyze the gene expression profiles of male YFC under HS and showed that the lipid and energy metabolism pathways are activated in livers of YFC exposed to acute HS (38°C, 4 h and 25°C recovery 2 h). In this study, we used quantitative proteome analysis based on iTRAQ to study the liver response of YFC to cycle chronic HS (38 ± 1°C, 8 h/d, 7 d, CyCHS). The male YFCs treatment used the CyCHS from 22 to 28 days of age. The liver tissue samples were collected at 28 d old. A total of 39,327 unique peptides matches were detected using iTRAQ analysis and 4,571 proteins exhibited a false discovery rate of 1% or less. Forty-six significant differentially expressed proteins (DEPs) were detected in the CyCHS group compared with the control group for the liver samples, including up- and down-regulated DEPs were 18 and 28, respectively. We found that the enriched biological process terms of the DEPs expressed in the liver were related to DNA metabolic process, oxidation-reduction process, oxidative stress and gluconeogenesis. In KEGG pathway analysis. Most of the hepatic DEPs were annotated to glutathione metabolism and TCA cycle in response to CyCHS. The up-regulation of 5 DEPs (GPX1, GSTT1, GSTT1L, RRM2, and LOC100859645) in the glutathione metabolism pathway likely reflects an attempt to deal with oxidative damage by CyCHS. The down-regulation of 3 DEPs (Isocitrate dehydrogenase [IDH3A], IDH3B, and phosphoenolpyruvate carboxykinase 1) in the TCA cycle pathway contributes to the regulation mechanism of energy metabolism and probably to cope with the balance of heat production and dissipation during CyCHS in order to adapt to high temperature environments. Our results provide insights into the potential molecular mechanism in heat-induced oxidative stress and energy in YFCs and future studies will investigate the functional genes associated with the response to HS.

Keywords: cycle chronic heat stress; liver; proteome; yellow-feather chicken.

Figure 1

Analysis of differentially abundant proteins. Red, green, and black dots on the graph represent the significantly up-regulated, down-regulated, and unchanged, respectively, between the cycle chronic heat stress and control group. The DEPs, including GPX1, GSTT1, GSTT1L, LOC100859645 and RRM2, enriched in glutathione metabolism KEGG pathway. The DEPs, including IDH3A, IDH3B and PCK1, enriched in citrate cycle KEGG pathway.

Figure 2

Verified the selected eight DEPs by Parallel reaction monitoring (PRM). PRM was used to determine the eight candidate proteins, and the results were consistent with those of LC-MS/MS analysis. Data are presented as mean ± SE (n = 9 per group). Control: control group (temperature constant maintained at 25 ± 1°C). CyCHS: cycle chronic heat stress group (38±1°C 8h/d, 7d). ** indicates significant difference (P < 0.01). A, B, C, D, E, F, G and H represent GPX1, LOC100859645, GSTT1, IDH3A, HAO2, ST13P5, HSPH1, and TIMD4, respectively.

Figure 3

Verified the selected eight DEPs by real-time PCR. qRT-PCR was used to determine the eight candidate proteins, and the results were consistent with those of LC-MS / MS analysis. Data are presented as mean ± S.E (n = 9 per group). Control: control group (temperature constant maintained at 25 ± 1°C). CyCHS: cycle chronic heat stress group (38 ± 1°C 8 h/d, 7 d). * or ** indicate significant difference (P < 0.05 or 0.01, respectively). A, B, C, D, E, F, G and H represent GPX1, LOC100859645, GSTT1, IDH3A, HAO2, ST13P5, HSPH1, and TIMD4, respectively.