Effects of compound probiotics and aflatoxin-degradation enzyme on alleviating aflatoxin-induced cytotoxicity in chicken embryo primary intestinal epithelium, liver and kidney cells

Aflatoxin B1 (AFB1) is one of the most dangerous mycotoxins for humans and animals. This study aimed to investigate the effects of compound probiotics (CP), CP supernatant (CPS), AFB1-degradation enzyme (ADE) on chicken embryo primary intestinal epithelium, liver and kidney cell viabilities, and to determine the functions of CP + ADE (CPADE) or CPS + ADE (CPSADE) for alleviating cytotoxicity induced by AFB1. The results showed that AFB1 decreased cell viabilities in dose-dependent and time-dependent manners. The optimal AFB1 concentrations and reactive time for establishing cell damage models were 200 µg/L AFB1 and 12 h for intestinal epithelium cells, 40 µg/L and 12 h for liver and kidney cells. Cell viabilities reached 231.58% (p < 0.05) for intestinal epithelium cells with CP addition, 105.29% and 115.84% (p < 0.05) for kidney and liver cells with CPS additions. The further results showed that intestinal epithelium, liver and kidney cell viabilities were significantly decreased to 87.12%, 88.7% and 84.19% (p < 0.05) when the cells were exposed to AFB1; however, they were increased to 93.49% by CPADE addition, 102.33% and 94.71% by CPSADE additions (p < 0.05). The relative mRNA abundances of IL-6, IL-8, TNF-α, iNOS, NF-κB, NOD1 (except liver cell) and TLR2 in three kinds of primary cells were significantly down-regulated by CPADE or CPSADE addition, compared with single AFB1 group (p < 0.05), indicating that CPADE or CPSADE addition could alleviate cell cytotoxicity and inflammation induced by AFB1 exposure through suppressing the activations of NF-κB, iNOS, NOD1 and TLR2 pathways.


Introduction
Mycotoxins are toxigenic fungal secondary metabolites that mainly produced by Aspergillus, Penicillium and Fusarium to have great threat to human and animal health globally. The Food and Agriculture Organization (FAO) showed that approximately 25% of worldwide agricultural raw materials were contaminated with mycotoxins, leading to health problems and enormous economic losses (FAO 2013). So far, at least 400 kinds of mycotoxins such as aflatoxins, zearalenone, deoxynivalenol, fumonisin, patulin, T-2 toxin and ochratoxins have been identified (Cimbalo et al. 2020). There are more than 20 types of aflatoxins including aflatoxin B 1 (AFB 1 ), B 2 , G 1 , G 2 and M 1 , among them AFB 1 is the most toxic mycotoxin with high frequency of contamination in various cereals such as nuts, corn and rice (Negash 2018). AFB 1 is able to cause poor feed efficacy, hepatotoxic, carcinogenic, teratogenic, immunosuppressive and other devastating effects on humans and animals (Meissonnier et al. 2008;Trebak et al. 2015;Zhang et al. 2016). Therefore, it is classified as the category one carcinogen by the International Agency for Research on Cancer (IARC 2012).
Poultry is more sensitive to AFB 1 than the other kinds of animals. AFB 1 residues in poultry body will cause potential health hazard for humans and itself . It is known that moldy food contains large amounts of AFB 1 , especially in moldy peanuts and cereals. In poultry farming, AFB 1 can severely affect the immune system to cause immunosuppression . AFB 1 can also cause apoptosis, gross and histopathological lesions in different organs, especially in liver, kidney, muscles and bursa of Fabricius Peng et al. 2014). It was reported that AFB 1 intoxication could increase mortality, liver and kidney pathology, and decrease bodyweight and feed intake for broilers (Saleemi et al. 2019). Therefore, it is necessary to develop effective detoxification strategies to increase AFB 1 degradation and alleviate AFB 1 -induced inflammatory and immunosuppression in chickens.
Up to date, several strategies have been reported to alleviate AFB 1 toxicity including physical, chemical and biological methods. The physical detoxification methods (absorption, heating and irradiation) and chemical detoxification methods (ammonization, solvent extraction and oxidation) have many defects such as nutritional losses, expensive equipment requirement and low efficiency (Gregorio et al. 2014;Arzandeh and Jinap 2015;Zhu et al. 2016). It was found that the biological method was more effective to degrade mycotoxins than other ones (Das et al. 2014;Melvin et al. 2014;Fernández et al. 2015). Many species of microbes such as bacteria, molds and yeasts have demonstrated the capability to alleviate AFB 1 toxicity, due to their metabolic transformation or adsorption ability for AFB 1 . It was reported that addition of lactic acid bacteria and S. cerevisiae to AFB 1 -contaminated diet could reduce AFB 1 residues and prevent degenerative changes in the liver and kidney of broilers (Śliżewska et al. 2019). Aspergillus oryzae has been reported to be able to degrade AFB 1 (Alberts et al. 2009). The other reports showed that the cooperation of compound probiotics (CP) and AFB 1 -degradation enzyme (ADE) could degrade AFB 1 effectively (Zuo et al. 2013;Huang et al. 2019).
It was reported that liver and kidney were the primary target organs attacked by AFB 1 (Gholami-Ahangaran et al. 2016;Pérez-Acosta et al. 2016). In addition, the small intestine is the physical barrier which usually first contacts with and absorbs AFB 1 , as a result intestinal heath is seriously influenced by AFB 1 (Pinton and Oswald 2014). However, the optimal strategies for alleviating the negative effects of AFB 1 on intestine, liver and kidney cells of chickens have not been reported. Therefore, small intestine, liver and kidney cells of chickens were selected in this study to investigate the toxic effects of AFB 1 on chicken embryo primary cells, and explore the efficacy of CPADE or CPSADE for alleviating AFB 1 -induced cytotoxicity and inflammatory of chickens.
The AFB 1 -degradating enzyme was extracted from solid-state fermentation of Aspergillus oryzae (A. oryzae, CGMCC3.4437) according to the previous protocol (Huang et al. 2019). The crude enzyme solution of 10% AFB 1 -degrading enzyme was diluted with cell medium and stored at 4 °C for further use. The AFB 1 -degrading enzyme activity in 10% crude enzyme solution was determined to be 51 U/mL according to the previous protocol (Gao et al. 2011).

Primary chicken embryo intestinal epithelium, liver and kidney cell preparation
The 14-day-old fertilized chicken eggs were purchased from Kaifeng Breeding Chicken Co., Ltd. Kaifeng, China, which were cleaned by 75% alcohol, placed in a verticalflow clean bench ultra-clean, and handled with ultraviolet irradiation for 20 min. The air chamber of embryo was carefully broken with the tweezers, the chicken embryo was taken out and quickly decapitated, followed by taking out small intestine, liver and kidney tissues, and rinsed in PBS containing 1% penicillin (10,000 U/mL)-streptomycin (10 mg/mL) (Beijing Solarbio Biotechnology Co., Ltd. Beijing, China).
The mesentery of small intestine was carefully exfoliated in PBS solution, cut into 1 mm size, put into 5 mL centrifuge tube, and washed with PBS until the supernatant was clear. After removing the washing solution, 1 mL 0.25% pancreatin was added to digest the tissues at 37 °C for 10 min with shaking once every 2 min. The tissues were centrifuged at 1000 r/min for 5 min to remove supernatant, and then 2 mL DMEM/F12 medium supplemented with 10% FBS and 1% penicillin-streptomycin were added. The filtrate was collected using 200-mesh sieve, and the cells were cultured in a 5% CO 2 incubator at 37 °C for 2 h. The supernatant was removed after centrifuged with 1000 r/min for 10 min, the cells were adjusted to 5.0 × 10 5 cells/mL with DMEM/F12 supplemented with 2.5% FBS and 1% penicillin-streptomycin. 0.2 mL or 2 mL cells were put in a 96-well or 12-well culture plate, and cultured at 37 °C in a 5% CO 2 incubator. The incubating cell medium was replaced every 2 days.
Liver cells were prepared as above and modified as following: 1 mL collagen protease and 1 mL neutral protease were added to digest the tissues at 37 °C for 30 min with shaking once every 3 min. Then 2 mL M199 medium supplemented with 10% FBS and 1% penicillin-streptomycin were added. After shaking up and down, the filtrates were collected with a 200-mesh sieve, and then centrifuged with 1000 r/min for 10 min to remove the supernatant. 1.5 mL M199 medium supplemented with 10% FBS and 1% penicillin-streptomycin were added to the centrifuge tube, and then 3 mL 50% percoll separation solution were added and mixed well, centrifuged for 15 min at 3000 r/ min. After centrifugation, the upper layer was removed, and the middle layer was taken out and put into a new centrifuge tube, then equivalent volume M199 medium was added to the new centrifuge tube, centrifuged for 10 min at 1000 r/min. At last the liver cells were resuspended with M199 medium supplemented with 10% FBS and 1% penicillin-streptomycin, adjusted and cultured as above. Kidney cells were prepared with the same protocol as liver cells, modified by using DMEM/F12 medium to replace M199 medium.

Cell viability assay and experimental design
Three kinds of primary cells were seeded into 96-well plates. Cell viability was measured by MTT assay every 2 days (Fotakis and Timbrell 2005). The growth curves of three kinds of cells were plotted with time as the abscissa and absorbance value as the ordinate. The following experiments were carried out in the logarithmic phase of cells. The experimental designs were as follows: 1. Effect of different AFB 1 concentrations on cell damage: three kinds of cells were seeded into 96-well plates with a density of 5.0 × 10 5 cells/mL, cultured to their logarithmic phases, followed by removing the culture medium and washing twice with PBS, and subsequently incubated with different concentrations of AFB 1 for 6, 12, 24 and 48 h, respectively. AFB 1 concentrations were 0, 40, 80, 120, 160 and 200 µg/L for intestinal epithelium cells; 0, 10, 20, 40 and 80 µg/L for the liver and kidney cells. AFB 1 was diluted with the corresponding cell media without serum and antibiotics. 2. Effect of CP or CPS on cell viability: the cells were prepared as above. CP and CPS were diluted with the corresponding cell media without serum and antibiotics. The cells were incubated with the different concentrations of CP or CPS for 12, 24 and 48 h, respectively. 3. Effect of ADE on cell viability: ADE was diluted with the cell medium without serum and antibiotics to make the final concentrations at 0, 0.0001%, 0.001%, 0.01%, 0.1% and 1%, which was incubated with cells for 6, 12, 24 and 48 h, respectively. 4. The functions of CPADE and CPSADE for alleviating cytotoxicity: The cell culture was 12 h. The detail design was listed in Table 1. The previous report in our laboratory showed that CPADE and CPSADE were more effective than CP, CPS and ADE for degrading AFB 1 (Huang et al. 2018); therefore, CP, CPS and ADE were not considered for alleviating cytotoxicity induced by AFB 1 in this study.
At the end of above cell incubations, each well was added with 10 µL 5 mg/mL MTT and incubated for 4 h. Then the cell supernatants were removed and 150 µL DMSO was added to each well. Thereafter, the plates were shaken for 10 min at room temperature. The absorbances (A) were determined at 490 nm wavelength with a reference wavelength of 630 nm by an ELx 800 microplate reader (BIO-TEK Instruments Inc., Winooski, VT, USA). The cell viability (%) = (A 490nm − A 630nm value in the experimental groups)/(A 490nm − A 630nm in the control groups) × 100%.

Reverse transcription PCR and quantitative real-time PCR
The primary intestinal epithelium, liver and kidney cells were seeded with a density of 5.0 × 10 5 cells/mL in 12-well culture plates and allowed to adhere for 24 h, respectively. After four treatments (Control, AFB 1 , CPADE or CPSADE, CPADE or CPASDE + AFB 1 ) for three kinds of primary cells for 12 h respectively, total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) according to the standard manufacturer's instructions, and then dissolved in 50 µL RNase-free water, stored at − 80 °C. The quality and concentration of RNA samples were measured by NanoDrop ND-1000 Spectrophotometer (Nano-Drop Technologies, Wilmington, DE, U.S.). Approximately 1 µg total RNA from each sample was reversely transcribed into cDNA by TB GREEN kit (TaKaRa, Dalian, China). Quantitative RT-PCR was performed with CFX Connect ™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). All the primers used in this study were listed in Table 2. The β-actin was used as a house-keeping gene, and the relative gene abundances in chicken embryo primary intestinal epithelium, liver and kidney cells were

Statistical analysis
All experimental data were presented as means ± standard deviations. The data were analyzed using one-way analysis of variance (ANOVA) by the Duncan method with SPSS 20.0 software (Sishu Software, Shanghai Co., Ltd. Shanghai, China). All graphs were generated using GraphPad Prism 8. Differences were considered as statistically significance at p < 0.05.

The growth curves of primary intestinal epithelium, liver and kidney cells of chicken embryo
Figure 1 demonstrated that the logarithmic growth phases of intestinal epithelium, liver and kidney cells appeared during the incubation periods of 8-12, 6-12 and 6-12 days, and reached the logarithmic peak on the 10th, 12th and 6th day, respectively (p < 0.05).
Effects of AFB 1 on the viabilities of primary intestinal epithelium, liver and kidney cells Table 3 showed that AFB 1 decreased cell viability in dosedependent and time-dependent manners. The higher  AFB 1 concentrations and longer incubation time caused more serious damages for three kinds of cells. AFB 1 had insignificant effect on intestinal epithelium cell viability when its concentration was below 80 μg/L within 48 h incubation (p > 0.05); however, it was significantly influenced when AFB 1 concentration were more than 80 μg/L (p < 0.05), especially under the condition that the incubation time was 48 h. Liver and kidney cells of chicken embryo were more sensitive to AFB 1 than intestinal epithelium cells. They were significantly influenced by 80 μg/L AFB 1 within 6 h incubation, 40 μg/L AFB 1 within 12 h incubation, 20 μg/L AFB 1 within 24 h incubation, 10 μg/L AFB 1 within 48 h incubation (p < 0.05), compared with the control group. After considering the above results, AFB 1 concentrations and reaction time were confirmed as 200 μg/L and 12 h for intestinal epithelium cells, 40 μg/L AFB 1 and 12 h for liver and kidney cells in the subsequent experiments. (p < 0.05) at CPS3 levels after 12 h incubation for kidney cells. According to the above results, the optimal incubation time was selected as 12 h in the subsequent experiment. In general, the liver and kidney cells can't directly contact with microbes; therefore, CPS was selected in the subsequent experiments for liver and kidney cell incubations. Figure 2 showed that the relative viabilities of three kinds of cells were significantly decreased (p < 0.05) when ADE concentrations were between 0.01 and 1%; however, the cell viabilities were significantly increased when ADE concentrations were between 0.001 and 0.0001% (p < 0.05). Therefore, the optimal ADE content was selected as 0.001% in the subsequent experiment.

Discussions
Aflatoxins are the ubiquitous dietary contaminants all over the world, which lead to low feed intake, low efficiency and substantial economic losses (Tedesco et al. 2004). Aflatoxin B 1 is frequently detected in cereals,  Guo et al. AMB Expr (2021) 11:35 feedstuffs and diets to cause liver damage and immune inhibition of domestic animals (Kraieski et al. 2016;Yuan et al. 2016). AFB 1 residues in domestic animal products will be harmful to human and public health. Liver is the main target organ of AFB 1 , but AFB 1 is also detected in kidney and intestinal tract of animals. Therefore, it is necessary to find an effective and safe method to alleviate AFB 1 for animal and human. Nowadays, probiotics have been widely used to degrade mycotoxins. It was reported that Bacillus subtilis could germinate in intestinal tract, and reduce AFB 1 absorption and residues in the internal organs of broilers (Salem et al. 2018). The compound probiotics of B. subtilis, L. casei and C. utilis were reported to increase production performance, alleviate histological lesions, degrade mycotoxins and decrease mycotoxin residues in broilers (Chang et al. 2020). In order to increase the efficiency of alleviating AFB 1 -induced cell damage, the compound probiotics was combined with AFB 1 -degrading enzyme in this study.
This result showed that the viabilities of three kinds of primary cells were decreased with increasing AFB 1 concentrations and incubation time, suggesting that both of them are the main factors for determining the extent of AFB 1 toxicity. In general, liver and kidney cells are more sensitive to AFB 1 than intestinal cells, which may be related to the different responses from the different cell types and organs (Zain 2011). AFB 1 can be metabolized to high reactive metabolites by cytochrome P450 enzyme system in liver cells, resulting in formation of AFB 1 -DNA adducts to cause carcinogenesis and mutations (Valeria et al. 2020;Owumi et al. 2020). The kidney cells can be directly damaged by AFB 1 through increasing cell apoptosis and death . For the intestinal epithelium cells, AFB 1 damage was mainly presented from barrier function loss and inflammatory response (Hernández-Ramírez et al. 2019). Because intestinal epithelium cells usually contact with AFB 1 directly, the long-term adaptation makes them be insensitive to AFB 1 than liver and kidney cells. The addition of compound probiotics and mycotoxin-degrading enzyme could contribute to cell proliferations and alleviate the toxicity induced by AFB 1 , which might be from mycotoxin biodegradation (Huang et al. 2018). It was found that the different concentrations of CP or CPS at different reaction time had different effects on the viabilities of three kinds of cells; therefore, the optimal CP or CPS concentrations and reaction time were selected for improving viabilities of different cell types. It was also indicated that CP was more effective than CPS for increasing cell viabilities, maybe due to the interaction between primary cells and microbes.
The previous researches have indicated that lactic acid bacteria can synthesize a wide variety of polysaccharides during their growth process (Round et al. 2011;Poole et al. 2018). These polysaccharides can be classified into two kinds, one kind can be tightly linked to the cell surface forming the capsular polysaccharides, which are loosely attached to the extracellular surface, or secreted to the environment as exopolysaccharides (Castro-Bravo et al. 2018). Capsular polysaccharide adhesion to intestinal epithelial cells is believed to help probiotic bacteria to transiently colonize and persist on epithelial cells for decreasing the colonization of intestinal pathogens (Castro-Bravo et al. 2018). Another kind is called extracellular polysaccharides, which can modulate intestinal immunity and reduce the secretion of proinflammatory cytokines (Laiño et al. 2016). Enterococcus faecalis can directly produce extracellular polysaccharide (Rossi et al. 2015), which may be the reason why CP is able to improve cell vitality more than CPS in this study. However, the long-term incubation of CP or CPS was harmful to cells, the reason may be due to the secondary metabolites produced by probiotics to influence cell growth.
Aspergillus oryzae can produce many kinds of enzymes such as protease and amylase except for AFB 1 -degradation enzyme, which may affect cell paste and growth. The reason why high ADE concentrations could influence cell viability might be due to the high levels of enzymes existing in ADE to damage cells, so low ADE concentration was selected in this study. It was reported that supplementation of L. bulgaricus or L. rhamnosus could produce significant protective effect against AFB 1 -induced liver damage and inflammatory response . Moreover, the addition of compound probiotics and mycotoxin-degradation enzyme could prevent broilers from damages induced by AFB 1 (Zuo et al. 2013). In this study, four kinds of compound probiotics plus AFB 1 -degradation enzyme additions significantly increased the cell viability induced by AFB 1 , inferring that CPDE or CPSADE could alleviate the toxicology induced by AFB 1 in three kinds of primary cells.
The previous studies have demonstrated that AFB 1 exposure can induce inflammation response in different cells and organs Wang et al. 2019;Zhao et al. 2019). Inflammation is a response against infection, illness and injury by the excessive expressions of chemokines and inflammatory cytokines such as TNF-α, IL-6 and IL-8 ( Barutta et al. 2015;Guo et al. 2015). TNF-α is a proinflammatory cytokine, which can stimulate various kinds of cells to produce chemokines to cause tissue damage and inflammation response (Shanmugam et al. 2016). It can be speculated that the degree of AFB 1 -induced damage may be decreased by suppressing the overexpression of inflammatory cytokines. In this study, AFB 1 exposure significantly up-regulated the mRNA abundances of IL-6, IL-8 and TNF-α in the three kinds of primary cells, but CPADE or CPSADE addition significantly down-regulated their mRNA abundances in the intestinal and kidney cells except for TNF-α in liver cells, indicating that probiotic combined with ADE could suppress gene expressions of some pro-inflammatory cytokines such as IL-6 and IL-8 (Weninger and Andrian 2003).
NF-κB is an important nuclear transcription factor and a major regulator for anti-inflammatory. The activated NF-κB plays a vital role in inflammatory response by regulating multiple cytokines (Zhang et al. 2018). In response to the inflammation cytokines, inducible nitric oxide synthase (iNOS) can catalyze the production of NOD which is a potent pro-inflammatory mediator (Surh et al. 2001). NOD1 is an innate immune sensor, which consists of a C-terminal leucine-rich region (LRR), central NOD and N-terminal caspase-activating domain (CARD) (Ma et al. 2020). NOD1 plays an important role in response to pathogen infection to induce activation of intracellular signaling pathway, leading to pro-inflammatory response (Caruso et al. 2014;Robertson et al. 2016). Several studies have showed that TLRs and NODs can participate in production of pro-inflammatory molecules to enhance immune responses ( Van-Heel et al. 2005;Fritz et al. 2005). It was reported that NLRs, NOD1 and NOD2 had the similar domain architectures and functions, but had the different CARD domain numbers (Trindade and Chen 2020). It was confirmed that NOD1 and NOD2 could activate the classical NF-κB and MAPK pathways related to cell inflammation and apoptosis (Seger and Wexler 2016).
TLRs play the vital roles in innate immune system. The effects of different mycotoxins on gene expression of TLR2, TLR4 and TLR7 have been reported (Chen et al. 2013). It was reported that 600 μg/kg AFB 1 in broiler diet could simultaneously down-regulate the expressions of TLR2, TLR4 and TLR7 genes in the intestinal tissues of broilers, and decrease the expressions of cytokines such as IFN-γ and TNF-α to reduce the innate immunity of broilers ). However, another research showed that mixed aflatoxins B and G could up-regulate TLR2 and TLR4 transcripts (Malvandi et al. 2013), corresponding with this study, which may due to the dosedependent effect of aflatoxins .
In this study, AFB 1 exposure could up-regulate NF-κBp65, iNOS, NOD1 and TLR2 mRNA abundances in intestinal, kidney and liver cells to cause to the multiple inflammatory pathway responses, in agreement with the previous report (Yan et al. 2020); however, CPADE or CPADE addition could down-regulate their mRNA abundances except for NOD1 and TNF-α in liver cells, indicating that CPADE or CPADE was able to alleviate cell inflammations and damages induced by AFB 1 through suppressing the pathway activations of NF-κB, iNOS, NOD1 and TLRs.
It can be concluded that CPADE or CPSADE is able to alleviate AFB 1 -induced cytotoxicity and inflammation of chicken embryo primary intestinal epithelium, liver and kidney cells by down-regulating mRNA abundances of inflammation cytokines through suppressing the activations of NF-κB, iNOS, NOD1 and TLRs signal pathways. These findings provide insights into the future development of strategies for CPADE or CPSADE to protect the primary cells from AFB 1 -induced damages.