Phlorotannin Alleviates Liver Injury by Regulating Redox Balance, Apoptosis, and Ferroptosis of Broilers under Heat Stress

Abstract

Heat stress (HS) poses a great challenge to the poultry industry by inducing oxidative damage to the liver, endangering the health and production of broilers. As an important type of seaweed polyphenols, phlorotannin has been shown to have antioxidant properties. The present study evaluated the protective effects of dietary phlorotannin on HS-induced liver injury in broilers based on oxidative damage parameters. A total of 108 twenty-one days old male Arbor Acres plus (AA+) broilers were randomly divided into three groups: TN group (thermoneutral, 24 ± 1 °C, fed with basal diet), HS group (HS, 33 ± 1 °C for 8 h/day, fed with basal diet), and HS + phlorotannin group (HS + 600 mg/kg phlorotannin). Each group has six replicate cages with six birds per cage. The feeding experiment lasted 21 days. At the termination of the feeding experiment (42 days old), samples were collected for analysis of morphological and biochemical features. The results showed that HS decreased the liver index, serum albumin (ALB) content, hepatic antioxidant enzymes activities of catalase (CAT), total superoxide dismutase (T-SOD), glutathione S-transferase (GST), and glutathione peroxidase (GSH-Px) (p < 0.05), while increasing the hepatic histopathology score, apoptosis rate, and malondialdehyde (MDA) content (p < 0.05) in 42-day-old broilers. Compared with the HS group, dietary phlorotannin improved the activities of antioxidant enzymes (GST and GSH-Px) but decreased the histopathology score and apoptosis rate in the liver (p < 0.05). Moreover, HS down-regulated hepatic mRNA expression of CAT1, NQO1, HO-1, and SLC7A11 (p < 0.05), while up-regulated hepatic mRNA expression of Keap1, MafG, IκBα, NF-κB P65, IFN-γ, TFR1, ACSL4, Bax, and Caspase-9 (p < 0.05). Compared with HS group, dietary phlorotannin up-regulated hepatic mRNA expression of Nrf2, CAT1, MafF, GSTT1, NQO1, HO-1, GCLC, GPX1, TNF-α, Fpn1, and SLC7A11 (p < 0.05), while down-regulated hepatic mRNA expression of IκBα, Bax, Caspase-9, and TFR1 (p < 0.05). In conclusion, dietary supplementation of 600 mg/kg phlorotannin could alleviate HS-induced liver injury via regulating oxidative status, apoptosis, and ferroptosis in broilers; these roles of phlorotannin might be associated with the regulation of the Nrf2 signaling pathway.

Keywords: broilers; heat stress; liver; oxidative stress; seaweed polyphenols.

Figure 1

Effect of Phlorotannin on liver changes in heat-stressed broilers at 42 days old (n = 6). (A) Observation of the liver by the naked eye. Histopathological changes in the liver were stained by hematoxylin and eosin ((B) ×200, (C) ×400). (D) Pathology scores of broiler liver. (E) Effects of phlorotannin on the liver index of heat-stressed broilers. TN: thermoneutral group, the broilers reared at 24 ± 1 °C throughout the experimental period, fed with basic diet; HS: heat stress group, the broilers reared at 33 ± 1 °C for 8 h/day (9:00 am to 17:00 pm), fed with basic diet; HS + PT: HS + phlorotannin group, the broilers reared at 33 ± 1 °C for 8 h/day (9:00 am to 17:00 pm), fed with 600 mg/kg phlorotannin in the basic diet. CV: central vein; H: hepatocyte; S: hepatic sinusoid; P: portal area. Inflammatory cell infiltration (), vacuolization (), dilated hepatic sinusoids (). ^ab Indicates that the same columns carrying various superscripts are significantly different at p < 0.05.

Figure 2

Effects of phlorotannin on apoptosis in the liver of heat-stressed broilers (TUNEL assay). (A) TUNEL staining results of broiler liver tissues (200×), blue fluorescence is a normal cell nucleus, and apoptotic cells were dyed red. (B) Percentage of TUNEL-positive cells. The results are represented as mean ± SEM (n = 6). TN: thermoneutral group, the broilers reared at 24 ± 1 °C throughout the experimental period, fed with basic diet; HS: heat stress group, the broilers reared at 33 ± 1 °C for 8 h/day (9:00 am to 17:00 pm), fed with basic diet; HS + PT: HS + phlorotannin group, the broilers reared at 33 ± 1 °C for 8 h/day (9:00 am to 17:00 pm), fed with 600 mg/kg phlorotannin in the basic diet. DAPI: 4’,6-diamidino-2-phenylindole, a fluorescent dye that can bind strongly to DNA; TUNEL: terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling; Merged: a double staining technique; ^ab Indicates that the same columns carrying various superscripts are significantly different at p < 0.05.

Figure 3

Effect of dietary phlorotannin on mRNA expression of genes related to antioxidants. TN: thermoneutral group, the broilers reared at 24 ± 1 °C throughout the experimental period, fed with basic diet; HS: heat stress group, the broilers reared at 33 ± 1 °C for 8 h/day (9:00 am to 17:00 pm), fed with basic diet; HS + PT: HS + phlorotannin group, the broilers reared at 33 ± 1 °C for 8 h/day (9:00 am to 17:00 pm), fed with 600mg/kg phlorotannin in the basic diet. Nrf2: nuclear factor-erythroid factor 2-related factor 2; Keap1: kelch-like associated protein 1; CAT1: y+ cationic amino acid transporter 1; MafF: MAF bZIP transcription factor F; MafG: MAF bZIP transcription factor G; MafK: MAF bZIP transcription factor K; GSTT1: glutathione S-transferase theta 1; GSTA3: glutathione S-transferase alpha 3; GSTO1: glutathione S-transferase omega 1; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2; NQO1: NAD(P)H dehydrogenase, quinone 1; HO-1: heme oxygenase 1; GCLC: glutamate-cysteine ligase catalytic subunit; GCLM: glutamate-cysteine ligase modifier subunit; GPX1: glutathione peroxidase 1; GPX3: glutathione peroxidase 3. The results are represented as mean ± SEM (n = 6). ^abc Indicates that the same columns carrying various superscripts are significantly different at p < 0.05.

Figure 4

Effect of dietary phlorotannin on mRNA expression levels of inflammation-related genes. TN: thermoneutral group, the broilers reared at 24 ± 1 °C throughout the experimental period, fed with basic diet; HS: heat stress group, the broilers reared at 33 ± 1 °C for 8 h/day (9:00 am to 17:00 pm), fed with basic diet; HS + PT: HS + phlorotannin group, the broilers reared at 33 ± 1 °C for 8 h/day (9:00 am to 17:00 pm), fed with 600mg/kg phlorotannin in the basic diet. IκBa: NF-κB inhibitor alpha; NF-κB P65: nuclear factor κappa B P65; TNF-α: tumor necrosis factor-α; IFN-γ: interferon gamma; IL-1β: interleukin 1 beta; IL-2: interleukin 2; IL-4: interleukin 4; IL-6: interleukin 6; IL-10: interleukin 10. The results are represented as mean ± SEM (n = 6). ^ab Indicates that the same columns carrying various superscripts are significantly different at p < 0.05.

Figure 5

Effect of dietary phlorotannin on mRNA expression levels of ferroptosis and apoptosis regulatory genes. TN: thermoneutral group, the broilers reared at 24 ± 1 °C throughout the experimental period, fed with basic diet; HS: heat stress group, the broilers reared at 33 ± 1 °C for 8 h/day (9:00 am to 17:00 pm), fed with basic diet; HS + PT: HS + phlorotannin group, the broilers reared at 33 ± 1 °C for 8 h/day (9:00 am to 17:00 pm), fed with 600 mg/kg phlorotannin in the basic diet. TFR1: transferrin receptor; Fpn1: solute carrier family 40 member 1; FTH1: ferritin heavy chain 1; ACSL4: acyl-CoA synthetase long chain family member 4; SLC7A11: solute carrier family 7 member 11; GPX4: glutathione peroxidase 4; PTGS2: prostaglandin-endoperoxide synthase 2; Bcl-2: B cell leukemia/lymphoma 2; Bax: BCL2 associated X; Caspase-3: cysteinyl aspartate specific proteinase 3; Caspase-9: cysteinyl aspartate specific proteinase 9. The results are represented as mean ± SEM (n = 6). ^abc Indicates that the same columns carrying various superscripts are significantly different at p < 0.05.

Figure 6

Spearman correlation analysis. The color legend on the right indicates correlation coefficient values by color. The value corresponding to the intermediate heat map is Spearman correlation coefficient r, which is between −1 and +1. When r < 0, it is a negative correlation, and when r > 0, it is a positive correlation. Histopathology: the pathological score of broiler liver; TUNEL: the apoptosis rate of broiler liver; Nrf2: nuclear factor-erythroid factor 2-related factor 2; CAT1: y+ cationic amino acid transporter 1; MafF: MAF bZIP transcription factor F; GSTT1: glutathione S-transferase theta 1; NQO1: NAD(P)H dehydrogenase, quinone 1; HO-1: heme oxygenase 1; GCLC: glutamate-cysteine ligase catalytic subunit; GPX1: glutathione peroxidase 1; IκBa: NF-κB inhibitor alpha; TNF-α: tumor necrosis factor-α; TFR1: transferrin receptor; Fpn1: solute carrier family 40 member 1; SLC7A11: solute carrier family 7 member 11; Bax: BCL2 associated X; Caspase-9: cysteinyl aspartate specific proteinase 9; GST, glutathione S-transferase; GSH-Px, glutathione peroxidase. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

The proposed mechanism of phlorotannin alleviates heat stress-induced liver injury by regulating redox, apoptotic, and ferroptosis signaling pathways in broilers.