The ameliorative role of phlorotannin on aflatoxin B1-induced liver oxidative stress and mitochondrial injury is related to the activation of Nrf2 and Nrf1 signaling pathways in broilers

Abstract

Background: Aflatoxin B₁ (AFB₁) risks animal and human health, and the liver is considered the most crucial detoxification organ. Phlorotannin (PT) is a polyhydroxy phenol that has a wide range of biological activities, including anti-oxidation and hepatoprotection, which can promote the ability of liver detoxification. This study aimed to elucidate the protective effect of PT on AFB₁-induced liver damage in broilers.

Results: In vivo experiment showed that the PT reduced AFB₁ content and AFB₁-exo-8,9-epoxide DNA (AFBO-DNA) concentration in serum and liver (P < 0.05), improved the histomorphology of liver and hepatic mitochondria, and activated nuclear factor erythroid 2-related factor 2 (Nrf2)-related antioxidant and detoxification pathway by upregulating the activities of antioxidant enzymes (catalase [CAT], glutathione S-transferase [GST]) and total antioxidant capacity (T-AOC) level (P < 0.05), and inhibited the mRNA expression of CYP1A1 (cytochrome P450 family 1 subfamily A member 1) and phase II detoxification enzyme related genes (GPX1, GSTT1, and NQO1) of broilers exposed to AFB₁ (P < 0.05). Meanwhile, PT upregulated the Nrf1 pathway-related mitochondrial biosynthetic genes (Nrf1, mitochondrial transcription factor A [TFAM], mitofusin 1 [MFN1]) in broilers fed AFB₁ contaminated diet (P < 0.05). In vitro verification study suggested that the use of Nrf2/Nrf1 inhibitors suppressed the ameliorative role of PT on AFB₁-induced liver injury of broilers, which was manifested in the mRNA expression of Nrf2, NQO1, GSTT3, Nrf1, TFAM, and other genes decreasing (P < 0.05), and down-regulation of the protein expression of Nrf2, total and nucleus p-Nrf2, and total and nucleus p-Nrf1 (P < 0.05).

Conclusion: The PT ameliorates oxidative stress and hepatotoxicity by activating the Nrf2-mediated phase II detoxification enzymes pathway and maintains mitochondrial homeostasis by activating the Nrf1 signaling pathway in broilers exposed to AFB₁.

Keywords: Aflatoxin B1; Biological detoxification; Broiler chickens; Liver injury; Phlorotannin.

Fig. 1

Effect of phlorotannin on AFB₁-induced the growth performance and hepatic function in broilers. A Outline of experimental procedures to examine the role of PT in AFB₁-induced broilers. B Growth performance and liver index. ADFI, Average daily feed intake; ADG, Average daily gain; F/G, Average daily feed intake/Average daily gain. C Serum TP and ALB contents and serum ALT and AST activity in the indicated broilers. TP, Total protein; ALB, Albumin; ALT, Alanine aminotransferase; AST, Aspartate transaminase. AFB₁, Aflatoxin B₁; PT, Phlorotannin; Control, basal diet; AFB₁, basal diet + 0.1 mg/kg AFB₁; AFB₁ + PT₂₀₀, basal diet + 0.1 mg/kg AFB₁ + 200 mg/kg PT; AFB₁ + PT₄₀₀, basal diet + 0.1 mg/kg AFB₁ + 400 mg/kg PT; AFB₁ + PT₆₀₀, basal diet + 0.1 mg/kg AFB₁ + 600 mg/kg PT; AFB₁ + PT₈₀₀, basal diet + 0.1 mg/kg AFB₁ + 800 mg/kg PT. Data are shown as mean ± SEM (n = 6). ^a–dDifferent superscript letters indicate significant differences (P < 0.05)

Fig. 2

Effect of phlorotannin on AFB₁-induced liver structure and apoptosis of broilers. A H&E staining images of liver sections in indicated broilers. CV: Central vein; H: Hepatocytes; S: Hepatic sinusoid; P: Portal area. Black triangle represents steatosis. Black ellipse represents inflammatory infiltration. The scar bar is 50 μm of 200 × and 25 μm of 400 ×. B Inflammation score by H&E staining images. C Representative image of immunofluorescence staining for TUNEL in broiler’s liver. The scar bar is 200 ×. D Apoptosis rate by TUNEL staining images. AFB₁, Aflatoxin B₁; PT, Phlorotannin; Control, basal diet; AFB₁, basal diet + 0.1 mg/kg AFB₁; AFB₁ + PT₆₀₀, basal diet + 0.1 mg/kg AFB₁ + 600 mg/kg PT. Data are shown as mean ± SEM (n = 6). ^a,bDifferent superscript letters indicate significant differences (P < 0.05)

Fig. 3

Effect of phlorotannin on AFB₁-induced liver antioxidant capacity and Nrf2-mediated phase II etoxification enzyme ability of broilers. A Liver and serum AFB₁ and AFBO contents in broilers. B Liver antioxidant levels and detoxification ability of broilers. C Relative mRNA levels of Phase I detoxification enzyme in broilers liver. D Relative mRNA levels of Phase II detoxification enzyme in broilers liver. E Relative mRNA levels of Nrf2 pathway-related gene in broilers liver. AFB₁, Aflatoxin B₁; PT, Phlorotannin; Control, basal diet; AFB₁, basal diet + 0.1 mg/kg AFB₁; AFB₁ + PT₆₀₀, basal diet + 0.1 mg/kg AFB₁ + 600 mg/kg PT. Data are shown as mean ± SEM (n = 6). ^a–cDifferent superscript letters indicate significant differences (P < 0.05)

Fig. 4

Effect of phlorotannin on AFB₁-induced liver mitochondrial damage of broilers. A Transmission electron microscope image of broiler’s liver mitochondria. The scar bar is 6 K and 12 K. Red arrow represents the mitochondrial bilayer membrane structure. Black pentagram represents the blurring or disappearance of the mitochondrial bilayer membrane structure. B The number and area of mitochondria at 6 K. The unit of area is µm². C Relative mRNA levels of Nrf1 pathway-related gene in broilers liver. AFB₁, Aflatoxin B₁; PT, Phlorotannin; Control, basal diet; AFB₁, basal diet + 0.1 mg/kg AFB₁; AFB₁ + PT₆₀₀, basal diet + 0.1 mg/kg AFB₁ + 600 mg/kg PT. Data are shown as mean ± SEM (n = 6). ^a–cDifferent superscript letters indicate significant differences (P < 0.05)

Fig. 5

Effects of phlorotannin with different concentrations on AFB₁-induced LMH cells. A Outline of the experimental grouping of different concentrations of PT on the survival rate of LMH cells. B Survival rate of LMH cells treated with different concentrations of PT for 12 h. C Outline of the experimental grouping of different concentrations of PT on the survival rate of AFB₁-induced LMH cells. D Survival rate of AFB₁-induced LMH cells treated with different concentrations of PT for 12 h. E LMH cell state diagram. The scar bar is 100 ×. AFB₁, Aflatoxin B₁; PT, Phlorotannin; Normal, 10% complete medium; Control, 10% complete medium + 0.025% DMSO; AFB₁, 10% complete medium + 0.1 μg/mL AFB₁; N1, 10% complete medium + 0.1 μg/mL AFB₁ + 1 μg/mL PT; N2, 10% complete medium + 0.1 μg/mL AFB₁ + 2 μg/mL PT; N3, 10% complete medium + 0.1 μg/mL AFB₁ + 3 μg/mL PT; N4, 10% complete medium + 0.1 μg/mL AFB₁ + 4 μg/mL PT; N5, 10% complete medium + 0.1 μg/mL AFB₁ + 5 μg/mL PT. Data are shown as mean ± SEM (n = 6). ^a–dDifferent superscript letters indicate significant differences (P < 0.05)

Fig. 6

Effect of phlorotannin on AFB₁-induced oxidative stress of LMH cells through Nrf2 pathway. A Relative mRNA levels of Nrf2 pathway-related gene in AFB₁-induced LMH cells. B Western blot analysis of Nrf2, total and nuclear p-Nrf2 in AFB₁-induced LMH cells. Lamin B and β-actin served as loading control. C Quantification of protein expression Nrf2, total and nuclear p-Nrf2. D Schematic diagram of AFB₁ metabolism and PT detoxification pathway. AFB₁, Aflatoxin B₁; PT, Phlorotannin; Control, 10% complete medium + 0.025% DMSO; AFB₁, 10% complete medium + 0.1 μg/mL AFB₁; AFB₁ + PT, 10% complete medium + 0.1 μg/mL AFB₁ + 1 μg/mL PT; AFB₁ + PT + Nrf2 inhibitor, 10% complete medium + 0.1 μg/mL AFB₁ + 1 μg/mL PT + 1.9 μmol/L ML385 (Nrf2 inhibitor).– for not adding, + for adding. Data are shown as mean ± SEM (n = 6). ^a–cDifferent superscript letters indicate significant differences (P < 0.05)

Fig. 7

Effect of phlorotannin on AFB₁-induced mitochondrial damage of LMH cells via Nrf1 pathway. A Relative mRNA levels of Nrf1 pathway-related gene in AFB₁-induced LMH cells. B Western blot analysis of total and nuclear Nrf1 in AFB₁-induced LMH cells. Lamin B and β-actin served as loading control. C Quantification of protein expression total and nuclear Nrf1. D Schematic diagram of mitochondrial function mediated by AFB₁ and PT on Nrf1. AFB₁, Aflatoxin B₁; PT, Phlorotannin; Control, 10% complete medium + 0.025% DMSO; AFB₁, 10% complete medium + 0.1 μg/mL AFB₁; AFB₁ + PT, 10% complete medium + 0.1 μg/mL AFB₁ + 1 μg/mL PT; AFB₁ + PT + Nrf1 inhibitor, 10% complete medium + 0.1 μg/mL AFB₁ + 1 μg/mL PT + 10 μmol/L WRR139 (Nrf1 inhibitor). – for not adding, + for adding. Data are shown as mean ± SEM (n = 6). ^a–cDifferent superscript letters indicate significant differences (P < 0.05)