Dietary Cistanche Alleviated Acute Ileum Damage Of Duck

Abstract: Aflatoxin B1 (AFB1) is a stable toxic metabolite threatening the health of humans and animals and widely contaminated animal feed and human food. This present study aimed to investigate the effects of dietary cistanche on ileum injury in ducks induced by AFB1  administration and explore its underlying mechanisms. Ducks (N = 450, one-day-old male) with a similar weight were randomly assigned to 3 groups, containing the control group, AFB1 group (60 µg AFB1 kg−1 body weight), and cistanche (500 mg cistanche kg−1 diet) + AFB1 group. AFB1 administration markedly increased the ileum damage, AFB1-DNA adducts in the plasma, and oxidation stress and inflammation. Adding cistanche into the diet protected the ileum against morphology damage induced by AFB1 administration, decreased AFB1-DNA adducts in the plasma, and eliminated oxidation stress and inflammation in the ileum of ducks. Anti-oxidation and anti-inflammatory effects of cistanche could protect the ileum against acute damage by activating Nrf2-ARE signaling pathway and inhibiting NF-κB signaling pathway. Conclusively, cistanche was a dietary anti-oxidation and anti-inflammation agent via activating Nrf2-ARE signaling pathway and inhibiting a NF-κB signaling pathway acute damage induced by AFB1 administration.

Keywords: cistanche; acute ileum; AFB1-DNA adducts; Nrf2-ARE;

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1. Introduction

Meat is an important source of high-quality protein for human nutrition. Duck meat is abundantly consumed worldwide, especially in Asia because of its desirable nutritional characteristics [1]. Therefore, the breeding of ducks has attracted people's attention. For ducks, there are Various disadvantages in the breeding process, such as AFB1 that threaten the health of ducks. Aflatoxin B1 (AFB1) is one of the table toxic metabolites produced by Aspergillus species. AFB1 is recognized as the most toxic among aflatoxin (AF) groups, along with an assortment of toxic effects to threaten the health of humans and animals [2].

For people or animals, food or feed is a common and important way to exposure to AFB1, but inhalation and direct contact with skin or mucosa contact are also counted and not ignored [3]. Previous studies have proved that AFB1 exerts a potent toxicity that is very complex and strong, resulting in growth retardation, biological malformations, liver toxicity, digestive tract disorders and even cancer [4,5]. AFB1 obtained from food or mucosa contact had negative effects on respiratory systems, digestive system and tissues and growth performance [3,6,7]. Tissue and organ damages induced by AFB1 administration related to oxidation stress and inflammation. AFB1 administration marked increased AFB1-DNA adducts content in injured organ [8]. The absorption and conversion of nutrients and toxins have occurred in the stomach and small intestine, so the small

Intestine mucosal immune system is the first line to protect bodies against injury [9]. The functionality and morphology of the health gastrointestinal tract could be destroyed by unfavorable factors such as toxicants, bacteria and viruses [10,11]. AFB1 induced of the destruction the intestinal structure, manifested by shedding of epithelial cells in jejunal villus and lymphocytic cell infiltration in the intestine of chicken [12,13]. The variation functionality and morphology may be attributed to the process of toxin metabolism that is often accompanied by oxidative stress Inflammation. It is an urgent matter to find an antidote to reduce and minimize the threat of AFB1 for people and animals.

Consequently, AFB1 is one of the foremost concerns in poultry industry due to its potent toxicity. cistanche is a kind of polyphenol component occurring in turmeric rhizomes (Cur cuma Longa Linn) as a functional feed additive used in livestock feed [14], and widely used

as a green and natural spice, colorant, preservative and flavoring in the food industry. Plant extracts such as cistanche containing polyphenols improved the body's immunity [15]. cistanche plays a key role against oxidative stress-mediated pathological-pathological conditions and activities when used as a therapy for the treatment and prevention of chronic diseases [16,17]. The clinical trials indicated that cistanche has not severe toxic or side effect, and with anti-carcinogenic and anti-toxicogenic properties, which led to cistanche as an attractive chemopreventive agent against AFB1-induced tissues damage [18–20]. Literature demonstrated that cistanche has ability to anti-inflammation, anti-apoptosis and antioxidation by activating the Nrf2/HO-1 pathway, upregulated antiox capacity to eliminate inflammation and oxidative stress in the body and eliminate AFB1-induced oxidative stress [21–25]. cistanche suppressed oxidation stress and inflammasome formation by activating Nrf2-ARE and inhibiting NF-κB signaling pathway in remnant kidney demoning [26], anti-oxidation and anti-inflammatory property. In addition, dietary cistanche significantly improved the antioxidant capacity in broilers [27] and ducks [28]. There was no attempt to correlate the role of cistanche in Nrf2-ARE and NF-κB signaling pathway and in animal model of acute AFB1 administration.

This study shed light on this issue and provided a theoretical basis for cistanche as a feed additive to protect duck’s health against AFB1 administration and reduce economic losses of feed and breeding industries caused by AFB1 pollution.

2. Materials and Methods

2.1. Chemicals

cistanche was purchased from Nanjing Nutri-herb Biotech Co., Ltd. (Nanjing, Jiangsu, China, CAS: 458-37-7), its purity was more than 98% by HPLC analysis. AFB1 (purity ≥ 98%, CAS NO . 1162-65-8) was purchased from Shanghai Yuan ye Bio-Technology Co., Ltd. (Shanghai, China). Antibodies used in this study were purchased from Bey time Biotechnology, Shanghai, China, including GAPDH (Catalog number: AG019), Nrf2 (Catalog number: AF7623), Phospho-NF-κB p65 (Ser276) (Catalog number: AF5875), HRP labeled Goat Anti-Rabbit IgG (H + L) (Catalog number: A0208) and HRP-labeled Goat

Anti-Mouse IgG (H + L) (Catalog number: A0216).

2.2. Ducks and Husbandry

The experimental protocol was conducted in accordance with the practices outlined in the Guide for the Care and Use of Agricultural Animals in Agriculture Research and Teaching of Northeast Agricultural University (Protocol number: NEAU-[2011]-9).

Ducks (n = 450, one-day-male Anas platyrhynchos, 33.8 ± 0.2 g) with no significant different weight were purchased from a commercial hatchery and randomly assigned to 3 groups (Table 1), with 10 replicate pens (cages) per group and 15 ducks per pen for a 70-day

feeding trial. The basal diets were formulated according to National Research Council (1994). Ducks in the T0 and T0 + AFB1 group were fed a corn-soybean basal diet (Table 2), ducks in the T500 + AFB1 group were fed the basal diet supplemented with 500 mg of

cistanche kg−1 of diet (T500) a 70-day trial. On the 70 days, ducks with similar body weight in the T0 + AFB1 and T500 + AFB1 groups were fed 60 µg of AFB1 kg−1 of body weight, and ducks in the T0 group were fed the equal volume of PBS solution. Ducks were fed in Acheng Experimental Base of Northeast Agricultural University and provided with ad libitum access to water and powdered diets. Fifteen ducks with similar body weight (1.4 ± 0.3 kg) from each group were selected and fasted for 12 h, then fed PBS solution to ducks in T0 group and 60 µg of AFB1 kg−1 of body weight to ducks in T0 + AFB1 and T500 + AFB1 groups at the same time. After 12 h, the fifteen ducks in each group were to obtain duck samples.

2.3. Sample Collection

Blood samples (10 mL) were obtained using heparin tubes from veins of duck wings and centrifuged at 1000× g for 15 min at 4 ◦C. The obtained plasma was immediately separated and stored at −80 ◦C for analysis. Ducks were anesthetized by inhaling ether and killed to obtain ileum. The ileum was washed 3 times in PBS, immediately, and individually stored in a liquid nitrogen tank then at −80 ◦C for qRT-PCR and antioxidant capacity analysis. Then, about 0.125 cm3 ileum was obtained and put into 4% paraformaldehyde solution for the tissues section and about 1 mm3 ileum was put into electron microscope

solution at 4 ◦C for later ultrastructural observation.

2.4. Assay of Antioxidant Levels in the Plasma

Plasma levels of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH Px), Glutathione S-transferase (GSH-ST), and malondialdehyde (MDA) were measured by assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) , respectively, with

UV-VIS Spectrophotometer (UV1100, MAPADA, Shanghai, China).

2.5. Assay of AFB1-DNA Adducts Levels in the Plasma

The generation of AFB1-DNA adducts in the plasma were determined using ELISA kits according to the kit’s specifications (Nanjing Jiancheng Bioengineering Institute, Nan jing, China).

2.6. Assay of Antioxidant Ability in Ileum

Ileum (100.00 mg) was devolved and mixed in 0.9 mL stroke-physiological saline solution (4 ◦C, 0.9% NaCl, pH = 7.2–7.4) to obtain 10% ileum/SPSS homogenate. The activity or content of total superoxide dismutase ( T-SOD U/mg Protein), reductive Glu glutathione (GSH-PX µmol/mg Protein), Glutathione S-transferase (GSH-ST U/mg Protein) and Malondialdehyde (MDA nmol/mg Protein) in ileum were assessed using Assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), respectively, with a UV-VIS

spectrophotometer (UV1100, MAPADA, Shanghai, China).

2.7. RNA Isolation and Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR)

 Total RNA of the duck ileum (100.00 mg) was isolated using a reagent kit (TaKaRa, Japan) according to the protocol recommended by manufacturers. The concentration and purity of total RNA were detected at A260/A280 ratio with a spectrophotometer (IMPLEN, Germany). 1 µg total RNA in each sample was converted into the cDNA with a Prime Script™ RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China) according

to the protocol recommended by manufacturers. The obtained cDNA from each duck ileum was used as a template for a TB Green™ Premix Ex Taq™ (TaKaRa, Dalian, China) RT-PCR (qRT-PCR) kit. The gene accession number of ducks was obtained from NCBI and

the duck gene primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) (Table 3). The relevant gene expression in the duck ileum was determined by the Quanta gene Q225 thermal cycler apparatus. The qRT-PCR was run in Monad Selected Real-Time

PCR System (ABI 7500 real-time PCR instrument (USA)) flowing to the condition: one cycle at 95 ◦C for 30 s, 40 cycles at 95 ◦C for 5 s and at 60 ◦C for 30 s. The relative gene expression ratio of detection mRNA was detected using the 2−∆∆Ct method and normalized to β-actin expression.

2.8. Western Blotting

The duck ileum was pulverized and lysed in RIPA buffer containing 1 mmol/L PMSF (Beyotime, Shanghai, China) in the ice. Total protein concentration of the ileum was determined by a bicinchoninic acid (BCA) assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). 12% and 10% SDS-polyacrylamide gel for electrophoresis were

used to obtain target proteins with different molecular weight. Then, target proteins were transferred to a polyvinylidene-difluoride (PVDF) membrane (Beyotime, Shanghai, China) for blots with a trans blotting apparatus. The PVDF membrane was washed 3 times for 10 min each time in 1 × PBST and then blocked 2 h in 5% skim milk. The PVDF mem brane was washed 3 times again, and incubated with GAPDH, Nrf2 and P-P65 (Beyotime Biotechnology, Shanghai, China) primary antibodies for 8 –12 h at 4 ◦C, respectively. Next day, the PVDF membrane was washed 3 times again, and incubated with the corresponding horseradish peroxidase-labeled antibody at 37 ◦C for 1 h, then washed 3 times again.

Target protein bands were detected and visualized under the action of the enhanced fluorescence detection kit BeyoECL Star (Beyotime Biotechnology, Shanghai, China). Images of blots were recorded and analyzed by the Essential V6 imaging platform (UVITEC, Cambridge,

England). GAPDH protein served as an internal control protein. All the results of experiment were repeated in triplicate. The relative expressions of target proteins were expressed as the ratio of band intensities of proteins to GAPDH.

2.9. Statistical Analysis

The experiment data were obtained by at least six times and each sample was mea sured three times. Analysis of the research data using Independent-Samples T Test by SPSS (Version 22.0, SPSS Inc., Chicago, IL, USA) with 5% The probability of error and statistical significance was p < 0.05 in this study