Proteome and Transcriptome Analysis of the Antioxidant Mechanism in Chicken Regulated by Eucalyptus Leaf Polyphenols Extract

Eucalyptus leaf polyphenols extract (EPE) has been proved to have various bioactivities, but few reports focus on its antioxidant mechanism in vivo. The purpose of this study was to elucidate the effect and mechanism of EPE dietary supplements on antioxidant capacity in chicken. A total of 216 chickens were randomly selected for a 40-day experiment. Four treatment groups received diets including the control diet only, the control diet + low EPE (0.6 g/kg), the control diet + moderate EPE (0.9 g/kg), and the control diet + high EPE (1.2 g/kg). Compared with control group, the glutathione peroxidase (GSH-Px) activity and glutathione (GSH) content in the breast muscle of the moderate EPE treatment group was significantly higher (p < 0.05), while the malonaldehyde (MDA) content in the moderate EPE group was reduced (p < 0.05). Moreover, proteomic and transcriptomic analyses of the breast muscle revealed that glutathione metabolism and the peroxisome were the two crucial metabolic pathways responsible for increased antioxidant capacity of the muscle. Accordingly, nine candidate genes and two candidate proteins were identified related to improved antioxidant status induced by EPE supplements. This research provides new insights into the molecular mechanism of antioxidant capacity in chickens treated with EPE dietary supplements.


Introduction
The genus Eucalyptus comprises over 900 species of economically valuable plants endemic to Australia [1,2]. In China, the eucalyptus plantation area reached 4.5 million hm 2 by the end of 2016 [3]. There is a growing need to develop alternative uses for eucalyptus products, and eucalyptus plants have attracted broad interest among researchers to investigate their functionality and potential applications [4][5][6]. Eucalyptus leaves are known to contain numerous bioactive substances including, but not limited to, terpenoids, tannins, flavonoids, and phloroglucinol derivatives, and eucalyptus has also been shown to function as antioxidants [7,8]. Accordingly, bioactive components derived from eucalyptus leaves and their functions for use in the food industry are widely researched, with many studies focused on their antioxidant capacity [9][10][11].
significantly improves antioxidant status [17,18]. Eucalyptus leaves extracts with immense biomass and great antioxidant potential benefited to improve growth performance and health status of broilers, which could be useful for the poultry industry [19,20]. However, few studies have investigated the antioxidant mechanism of EPE in chicken. Therefore, this study was performed to reveal EPE dietary supplements on the antioxidant mechanism of chicken using proteomic and transcriptomic analysis.

Materials and Methods
2.1. Experimental Material. All the Huiyang beard chickens used in this study were provided by Xingtai Modern Agricultural Limited Company of Huizhou in Guangdong, China. EPE: Eucalyptus leaves (Eucalyptus grandis × Eucalyptus urophylla GL9) were picked in October in Zhanjiang and airdried naturally (7~9 d, 28 ± 2°C, moisture content 15:53 ± 2:11%). The eucalyptus leaves were extracted by 70% ethanol solvent (v/v) at 75°C. And then the extraction solution was vacuum-concentrated and spray-dried (the yield of EPE was 25:78 ± 2:03%), the EPE was stored at 4°C for the subsequent experiment.
The total polyphenol content in EPE was determined using the Folin-phenol method [21]. Additionally, the EPE was analyzed using high-performance liquid chromatography (HPLC) with a Diamonsil C18 column (250 × 4:6 mm, 5 μm, Diamonsil, China). EPE was reconstituted in buffer A (H 2 O) and loaded onto the column. It was then eluted using gradient buffer B (Methanol) in the following order: 10% at 0 min and 10~90% at 60 min. The flow rate was 1 mL/min and the injection volume was 10 μL. The column temperature was 37°C and a detection wavelength of 270 nm.

Animal Experimental Design.
A total of 216 chickens (90 days old) were randomly assigned to one of four treatments (6 pens/treatment and 6 chicken/pen), and the duration of the trial was 40 days (from 90 to 130 days). Four treatment groups received diets including control diet only (Table 1), low EPE (control diet+0.6 g/kg EPE), moderate EPE (control diet+0.9 g/kg EPE), and high EPE (control diet+1.2 g/kg EPE). The experimental design and procedures were approved by following the requirements of the Regulations for the Administration of Affairs Concerning Experimental Animals of China.

Experimental Basic Fodder and Management.
Ingredients and nutrient compositions of the diets are shown in Table 1, and the diets were used throughout the whole experimental period. The chickens were raised in cages in a controlled environment and had free access to food and drinking water. The temperature in the house was 24~29°C, and the relative humidity was 60~78%. Pens were routinely disinfected to maintain appropriate standards of cleanliness. All animal procedures were conducted under the protocol (SCAU-AEC-2010-0416) approved by the Animal Ethics Committee of South China Agricultural University.
2.4. Sample Collection. After 40 days, 12 chickens were randomly selected from each treatment group (2 broiler chickens from each replication), and blood samples were drawn from the neck vein with a sterile syringe. The blood was centrifuged at 3000 × g for 15 min to obtain serum samples, and they were stored at -80°C until analysis. Chickens were then euthanized, and the whole breast muscle tissues were preserved in liquid nitrogen.

Statistical
Analysis. Data were analyzed by one-way ANOVA (SPSS, 2010). Differences among treatment groups were evaluated using Duncan's multiple range tests, with a significance threshold of p < 0:05.

Quality of EPE.
According to the Folin-reagent method described by Wang et al. (Gallic acid as the equivalent polyphenol) [21], the total polyphenols content in EPE was 317:08 ± 16:49 mg/g. Further, HPLC of EPE ( Figure 1) indicated that the main active substance was of comparable qual-ity to the EPE reported in our previous research, based on the standard substance retention time [23]. Table 2, the concentrations of GSH-Px, T-SOD, T-AOC, and MDA in serum were not significantly different among the treatment groups (p > 0:05). Compared with the control group, the T-SOD activity and T-AOC increased in the EPE treatment groups, and the GSH-Px activity in the moderate EPE group increased (0.9 g/kg) by 13.8% (p > 0:05). Table 3, the T-SOD activity and T-AOC of the breast muscle in the moderate EPE (0.9 g/kg) and high EPE (1.2 g/kg) groups showed an increasing trend compared to

4
Oxidative Medicine and Cellular Longevity the control group (p > 0:05). The GSH-Px activity of the breast muscle tissue in the moderate (0.9 g/kg) EPE group was 44.5%, higher than that of the control group (p < 0:05), and there was an upward trend in the low (0.6 g/kg) and high (1.2 g/kg) EPE groups (p > 0:05). The GSH content of breast muscle in the moderate and high EPE groups increased by 23.3% and 20.7% compared with the control group (p < 0:05). Additionally, MDA content in the breast muscle of the moderate (0.9 g/kg) EPE group was reduced by 25.4% (p < 0:05), though the other two groups did not show a significant change (p > 0:05).

Screening and Annotation of DEGs in Muscle Induced by EPE Dietary Supplements in Chicken Based on RNA-Seq
Techniques.
To ensure high quality data, raw sequencing reads were filtered based on coverage, sequence saturation, the redundant distribution frequency map, and the distribution of sequencing reads across the chromosomes. A total of 14621 genes were identified as differentially regulated, of which 289 were significantly different between the EPE treatment group and the control group. The overall distribution of gene expression differences was visualized by a volcanic map ( Figure 2).

Gene Ontology Enrichment
Analysis of the DEGs. Gene Ontology (GO) enrichment analysis was used to annotate these DEGs by biological process, cellular component, and molecular function ( Figure 3). Overall, 47 different biological processes were enriched, including oxygen transport, oxidation-reduction, aminophospholipid transport, phospholipid translocation, and lipid translocation. Cellular component analysis revealed that the apical plasma membrane, membrane region, membrane part, cellular component, integral component of membrane, plasma membrane region, brush border, hemoglobin complex, and substrate-specific transporter activity were significantly enriched. Additionally, oxygen binding, oxygen transporter activity, phospholipidtranslocating ATPase activity, and other 22 molecular function items were identified by the molecular function analysis. Based on the above GO enrichment analysis, glutathione metabolic process (biological process), glutathione transferase activity (molecular function), and peroxisome (cellular component) were the enriched GO terms most clearly associated with antioxidant capacity (Table 4). Overall, 4 antioxidant genes (gamma-glutamyltransferase 1, GGT1; microsomal glutathione S-transferase 1, MGST1; glutathione S-transferase alpha 4, GSTA4L; and glutathione S-transferase class-alpha, GSTAL1) in the EPE group were substantially enhanced in glutathione metabolic process relative to the control group. Similarly, three of these genes (MGST1, GSTA4L, and GSTAL1) were also associated with glutathione metabolic process and glutathione transferase activity, which play an

Kyoto Encyclopedia of Genes and Genomes (KEGG)
Analysis of the DEGs. DEGs were enriched in 148 pathways in the KEGG enrichment analysis, and 38 KEGG pathways were identified to have significant changes. Based on the previous analysis of physiological indicators, the peroxisomes and the glutathione metabolism pathway were selected for further evaluation of the antioxidant capacity of the muscle by KEGG enrichment metabolic pathway analysis. Significant DEGs that were enriched in the peroxisomes and glutathione metabolism pathway showed some overlap with the genes related to antioxidant status identified by the GO enrichment analysis, including HAO1, HAO2, and GGT1 (Table 5).

Screening and Annotation of DEPs in Muscle Induced by EPE Dietary Supplements in Chicken Based on iTRAQ Techniques
3.5.1. Identification and GO Analysis of DEPs Induced by EPE. The total number of tested proteins was 1430, and there were 149 protein differences between the EPE group and the control group. According to the standard of differential protein screening, there were 14 significant differences in protein concentration (>1.2-fold change, p < 0:05), 10 of which were upregulated and 4 were downregulated (Table 6). These proteins were annotated (cellular components, molecular functions, and biological processes) by GO enrichment analysis (Figure 4). Biological process analysis revealed that most proteins were associated with 31 different GO terms, including p53 class mediator, threonyl-tRNA aminoacylation, and   Figure 3: GO enrichment histogram for DEGs. Note: differentially expressed genes (DEGs). Each column in the figure is a GO term, and the abscissa text indicates the name and classification of the GO term. The height of the column, that is, the ordinate, indicates the enrichment rate. The color indicates the significance of enrichment, that is, FDR. The darker the color, the more significant the enrichment of the GO term, wherein the mark with FDR < 0:001 is * * * , the mark with FDR < 0:01 is * * , and the mark with FDR < 0:05 is * , the right color gradient indicates the FDR size. 6 Oxidative Medicine and Cellular Longevity regulation of DNA damage checkpoint. When DEPs were annotated by the cell components, significantly enriched terms included chylomicron, plasma lipoprotein particle, very-lower-density lipoprotein particle, triglyceride-rich lipoprotein particle, protein-lipid complex, and five other cellular components terms. A total of five DEPS were annotated by molecular function, and there was significant enrichment of nineteen GO terms, including threonine-tRNA ligase activity, nutrient reservoir activity, p53 binding, and glutathione transferase activity.

KEGG Pathway Analysis of the DEPs.
DEPs were analyses by KEGG pathway analysis and the results indicated that cytochrome b-c1 complex subunit Rieske (Q5ZLR5), A0A0A0MQ61, and F1P372 were main proteins associated with oxidative phosphorylation, glutathione metabolism, aminoacyl-tRNA biosynthesis, metabolism of xenobiotics by cytochrome P450, etc. (Table 7).

Combined Analysis of Transcriptomics and Proteomics.
All proteins and their associated transcripts in both the transcriptomic and proteomic analyses were classified into nine categories ( Figure 5). Of the 61 proteins and genes that were upregulated in the EPE group, the A0A0A0MQ61 protein and the glutathione S-transferase alpha-like 2 gene (GSTAL2) played a crucial role in enhancing antioxidant status. The glutathione metabolism in the transcriptomic analysis indicated that GGT1, MGST1, and GSTA4L were downstream genes regulated by glutathione, which correspond to the observed increase in GSH-Px activity in muscle tissue induced by EPE supplements. Accordingly, A0A0A0MQ61, the protein product of the glutathione S-transferase gene, was substantially upregulated in glutathione metabolism, which was in line with the expression of GGT1, MGST1, and GSTA4L ( Figure 6). Therefore, EPE may enhance the antioxidant status of chicken by improving the activity of specific antioxidant proteins.

Discussion
Phytogenic feed additives have been gaining attention in improving the health status of the flocks [31,32]. Our previous study revealed that diet with polyphenols obtained from eucalyptus leaves exhibited a positive effect on growth performance in laying hens [23]. In the present study, different concentrations of EPE in chicken diet did not exhibit significant positive or negative effect on chicken performance, including average daily gain, average daily feed take, and feed conversion rate  Gamma-glutamyltranspeptidase 1, microsomal glutathione S-transferase 1, glutathione S-transferase class-alpha, glutathione S-transferase alpha 4 GGT1, MGST1, GSTAL1, GSTA4L Note: differentially expressed genes (DEGs). The screening criteria for significantly KEGG enrichment pathways were p < 0:05. Hydroxyacid oxidase 1 (HAO1); hydroxyacid oxidase 2 (HAO2); bile acid-CoA: amino acid N-acyltransferase (BAAT); microsomal glutathione S-transferase 1 (MGST1); glutathione Stransferase class-alpha (GSTAL1); glutathione S-transferase alpha 4 (GSTA4L).  The color indicates the significance of enrichment, that is, FDR. The darker the color, the more significant the enrichment of the GO term, wherein the mark with FDR < 0:001 is * * * , the mark with FDR < 0:01 is * * , and the mark with FDR < 0:05 is * , the right color gradient indicates the FDR size. 8 Oxidative Medicine and Cellular Longevity (data not shown), which is in agreement with the report of Sedaghat and Torshizi [33], and might be due to that the chickens were in the adulthood.

Oxidative Medicine and Cellular Longevity
Recently, natural polyphenols used in animal diets have been reported to build an integrated antioxidant system to prevent from damage led by free radicals [34][35][36][37]. This antioxidant capacity is assumed to result from the free radicalscavenging properties of phenolic compounds [38][39][40]. Our previous study reported that EPE effectively scavenged DPPH• and ABTS• free radicals in vitro [21]. In the present study, the antioxidant effects in serum were not obviously affected by the EPE supplement. However, an increasing trend in GSH-Px was observed with a higher concentration of the EPE diet, whereas the MDA content showed the opposite effects. This result was supported by the previous report that diet with 0.8 g/kg polyphenols from eucalyptus leaves increased the GSH-Px activity in serum of laying hen and an insignificant decrease in MDA was observed in quails supplement with eucalyptus leaves [23,31]. Interestingly, our study revealed that EPE dietary supplements significantly improved GSH-Px activity and GSH content and decreased MDA content in breast muscle tissues. Higher concentration of the EPE diet led to higher GSH-Px activity, which is consistent with the report by Fathi et al. [31]. Similarly, MDA content can reflect the degree of lipid peroxidation; thereby, indirectly reflect cell damage and freshness of meat [41]. In agreement with our results, a diet with green tea extract led to a remarkably decreasing MDA content in meat tissue [42]. Polyphenols might be accumulated to exhibit antioxidant effects in meat tissues [35]. To explain the antioxidant capacity of polyphenols, it is crucial to understand how they are absorbed, metabolized, and eliminated from the body. It is stated that the intestinal utilization of polyphenols depended on their degree of polymerization and galloylation [43]. Monomeric and some oligomeric polyphenols tended to be easily absorbed at small intestine compared with the polymeric forms of polyphenols [44]. This suggested that monomeric and some oligomeric polyphenols in EPE, such as gallic acid, pedunculagin, hyperoside, and other com-pounds, could be absorbed and functioned as an antioxidant in chickens. This hypothesis was supported by Chamorro et al. that monomeric (catechin, epicatechin, gallic acid, and epicatechin-O-gallate) and dimeric (procyanidin B1 and procyanidin B2) catechins in grape pomace were easily digested and absorbed in chickens [45]. Overall, EPE dietary supplements are able to improve the antioxidant capacity of chicken.
The results of the transcriptomic analysis indicated that glutathione transferase activity, glutathione metabolic process, and the peroxisome were the three GO enrichment terms related to antioxidant activity. That is, several significantly upregulated antioxidant genes, such as GGT1, MGST1, GSTA4L, HAO1, and HAO2, are all believed to improve antioxidant status. Moreover, previous research indicated that GGT1 and PGDS play an important role in the synthesis of glutathione, and its upregulation exerts a prooxidant action [46]. Shinno et al. [47] reported that microsomal glutathione S-transferase 1 (MGST1) could be activated by gallic acid to protect the membrane against damage caused by oxidative stress. These results suggested that these antioxidant genes were vital to improve antioxidant status in chicken. Likewise, the KEGG pathway enrichment analysis also indicated that the peroxisomes and the glutathione metabolism pathway were the two crucial antioxidant pathways. Peroxisomes are rich in enzymes, mainly including oxidases, catalase, and peroxidase. Catalase, in particular, is known to protect cells by hydrolyzing the hydrogen peroxide generated in redox reactions [48,49]. Additionally, several differentially expressed antioxidant-related genes were significantly enriched in the glutathione metabolism pathway, and the content of glutathione may be altered in cells as a result of EPE supplements. Glutathione is known to effectively scavenge free radicals and other reactive oxygen species, and it can be oxidized to form GSSG [50,51]. In this study, significantly improved GSH-Px converted GSSG into GSH to enhance antioxidant status. In addition, SOD and APOA4 genes were notably identified to be upregulated in this study, though the protein products Cardiac muscle contraction 1 ko04932 Nonalcoholic fatty liver disease (NAFLD) 1 ko05010 Alzheimer's disease 1 ko05012 Parkinson's disease 1 ko05016 Huntington's disease 1 ko00480 Glutathione metabolism 1 A0A0A0MQ61 (uncharacterized protein) ko05204 Chemical carcinogenesis 1 ko00980 Metabolism of xenobiotics by cytochrome P450 1 ko00982 Drug metabolism-cytochrome P450 1 ko00970 Aminoacyl-tRNA biosynthesis 1 F1P372 (uncharacterized protein) Note: differentially expressed proteins (DEPs). Q5ZLR5 (cytochrome b-c1 complex subunit Rieske, mitochondrial), A0A0A0MQ61 (uncharacterized protein), F1P372 (uncharacterized protein).    Figure 5: A scatter plots of the expression levels of all proteins and their associated transcripts in both groups. Note: the abscissa in the figure indicates the difference in the expression of the protein in the EPE treatment group and the control group, and the ordinate indicates the difference in the FPKM value of the corresponding transcript in the experimental group and the control group. Each point represents a protein and its associated transcript; in the upper left corner, rho represents the Pearson's correlation coefficient between the two omics, p represents the correlation test p value; when rho > 0, it is called the negative correlation; When rho < 0, it is called positive correlation; when rho = 0, it is called zero correlation, that is, there is no correlation; the larger the|rho | , the greater the correlation between the two omics.
remained unchanged. They may also contribute to the antioxidation of muscle, given the known functions of SOD and the known APOA4 [52]. In the present study, the gene expression variation of superoxide dismutase was consistent with the increasing trend of SOD enzyme activity in the breast muscle in the moderate (0.9 g/kg) EPE treatment groups compared to the control group. This result suggests that EPE improved the activity of SOD, but supplementation with EPE over a long period of time or an increased dose of EPE in chicken feed also substantially impacted the animal's antioxidant capacity. Previous research indicated that the expressions of GST, Prdx6, PPIA, alfatoxin aldehyde reductase, and SOD regulated by albusin B were enhanced to activate the systemic antioxidant defense [16], and SOD, catalase (CAT), GST, and GSH-Px were considered as AOE to protect against oxidative stress [53]. Overall, the functions of these upregulated genes were highly correlated with antioxidant status and could be linked to the improvement of antioxidant capacity induced by EPE supplements. Many previous studies reported that glutathione is associated with glutathione metabolism pathways and that it likely functions as a reducing milieu to improve antioxidant function in cells [54]. As such, the results of the proteomic analysis indicated that antioxidant-related glutathione transferase activity was significantly increased in response to EPE supplements. The corresponding protein was a kind of glutathione S-transferase (A0A0A0MQ61, EC2.5.1.18) that is known to function in glutathione metabolism in Gallus gallus. Additionally, A0A0A0MQ61, an antioxidant protein, was significantly increased as a result of EPE dietary supple-ments. Further, the upregulated peroxiredoxin-1 protein in the peroxisome metabolic pathway is a known redoxregulating protein and is considered to be an antioxidant enzyme to eliminate various ROS [55]. Based on the above analyses, the antioxidant mechanism of muscle tissue in chicken treated with EPE dietary supplements is inferred and given in Figure 7. As shown, EPE treatments significantly improved GSH-Px activity and decreased MDA content of the breast muscle. Transcriptomic and proteomic analyses revealed that nine candidate genes and two candidate proteins were identified, which are responsible for improving antioxidant status induced by EPE supplements. Overall, this study promotes the understanding of the antioxidant mechanism in chicken regulated by EPE treatment.

Conclusion
Overall, EPE dietary supplements appeared to improve the antioxidant status of chicken by enhancing GSH-Px activity and reducing MDA content in muscle tissues. Glutathione metabolism and the peroxisome were considered the key metabolic pathways underlying the upregulation of AOE. Furthermore, all of the changes induced by EPE supplements may contribute to the systemic antioxidant defense of chicken, and these altered proteins and genes should be further explored in the future.