Aflatoxin B1 (AFB1), an important fungal toxin, exists mainly in plant feed ingredients and animals consuming feed contaminated with AFB1 will have reduced growth and impaired health condition mainly due to oxidative stress and reduced immunity. Our previous study found that AFB1 caused oxidative damage and inhibited muscle development of zebrafish. 4-Methylesculetin (4-ME), a coumarin derivative, is now used in biochemistry and medicine widely because of its antioxidant function. Whether 4-ME could alleviate the inhibition of muscle development in grass carp induced by AFB1 has not been reported. In this experiment, 720 healthy grass carp (11.40 ± 0.01 g) were randomly divided into 4 groups with 3 replicates of 60 fish each, including control group, AFB1 group (60 μg/kg diet AFB1), 4-ME group (10 mg/kg diet 4-ME), and AFB1+4-ME group (60 μg/kg diet AFB1 + 10 mg/kg 4-ME diet), for a 60-d growth experiment. In vitro, we also set up 4 treatment groups for grass carp primary myoblast, including control group, AFB1 group (15 μmol/L AFB1), 4-ME group (0.5 μmol/L 4-ME) and AFB1+4-ME group (15 μmol/L AFB1+0.5 μmol/L 4-ME). The results showed that dietary AFB1 decreased growth performance of grass carp, damaged the ultrastructure and induced oxidative damage in grass carp muscle, and significantly decreased the mRNA and protein expression levels of myogenin (MyoG), myogenic differentiation (MyoD), myosin heavy chain (MYHC), as well as the protein expression levels of laminin β1, fibronectin and collagen Ⅰ (P < 0.05), significantly activated the protein expression levels of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and phosphorylate-38 mitogen-activated protein kinase (p38 MAPK) both in grass carp muscle and grass carp primary myoblast (P < 0.05). Supplementation of AFB1 with 4-ME significantly improved the growth performance inhibition and alleviated the muscle fiber development inhibition and extracellular matrix (ECM) degradation in grass carp induced by AFB1 (P < 0.05). The present results revealed that supplementation of AFB1 contaminated feed with 4-ME reduced the inhibition of growth and muscle development by alleviating AFB1-induced ECM degradation in grass carp, which might be related to the p38 MAPK/uPA/MMP/ECM pathway. The results implied that 4-ME could be used as a valuable mycotoxin scavenger in animal feed.

中文翻译：

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减轻黄曲霉毒素 B1 对草鱼 （Ctenopharyngodon idella） 肌肉发育的抑制作用的新见解：4-甲基七叶苷在体内和体外的影响


黄曲霉毒素 B1 （AFB1） 是一种重要的真菌毒素，主要存在于植物饲料成分中，食用受 AFB1 污染的饲料的动物会因氧化应激和免疫力下降而生长发育不良和健康状况受损。我们之前的研究发现，AFB1 引起氧化损伤并抑制斑马鱼的肌肉发育。4-甲基乙二醇素 （4-ME） 是一种香豆素衍生物，由于其抗氧化功能，现在广泛用于生物化学和医学。4-ME 是否能减轻 AFB1 诱导的草鱼肌肉发育的抑制尚未见报道。将 720 条健康草鱼 （11.40 ± 0.01 g） 随机分为 4 组，每组 60 条鱼，包括对照组、AFB1 组（60 μg/kg 饮食 AFB1）、4-ME 组（10 mg/kg 饮食 4-ME）和 AFB1+4-ME 组（60 μg/kg 饮食 AFB1 + 10 mg/kg 4-ME 饮食）， 进行 60 天生长实验。在体外，我们还建立了 4 个草鱼原代成肌细胞处理组，包括对照组、AFB1 组 （15 μmol/L AFB1）、4-ME 组 （0.5 μmol/L 4-ME） 和 AFB1+4-ME 组 （15 μmol/L AFB1+0.5 μmol/L 4-ME）。结果表明，日粮AFB1降低了草鱼的生长性能，破坏了草鱼肌肉的超微结构并诱导了氧化损伤，显著降低了肌瘤素（myogenin，MyoG）、肌原分化（myogeniation，MyoD）、肌球蛋白重链（myosin， heavy chain，MYHC）的mRNA和蛋白表达水平，以及层粘连蛋白β1、纤连蛋白和I.型胶原的蛋白表达水平（P < 0.05） 显著激活草鱼肌肉和草鱼初级成肌细胞中尿激酶型纤溶酶原激活物 （uPA） 、基质金属蛋白酶-2 （MMP-2） 、基质金属蛋白酶-9 （MMP-9） 和磷酸化-38 丝裂原活化蛋白激酶 （p38 MAPK） 的蛋白表达水平 （P < 0.05）。添加 4-ME 可显著提高草鱼生长性能抑制，减轻 AFB1 诱导的草鱼肌纤维发育抑制和细胞外基质 （ECM） 降解 （P < 0.05）。目前的结果表明，在 AFB1 污染的饲料中添加 4-ME 通过减轻 AFB1 诱导的草鱼 ECM 降解来减少对生长和肌肉发育的抑制，这可能与 p38 MAPK/uPA/MMP/ECM 通路有关。结果表明，4-ME 可用作动物饲料中有价值的霉菌毒素清除剂。