Abstract Glycinin is a major anti-nutritional factor of soybean. Glycinin inhibits growth and impairs intestinal health in grass carp and Jian carp, but the underlying mechanisms are largely unknown. This study used grass carp to examine whether the negative influences of dietary glycinin are related to intestinal apoptosis signalling and tight junctions (TJs). The results showed that 8% dietary glycinin caused higher diamine oxidase activities and D-lactate content in the serum than did the control, indicating impairment of intestinal structural integrity. For the apoptosis indices, in the midgut and hindgut, glycinin aggravated the DNA fragmentation phenomenon, promoted caspase-3, −8, and − 9 activities, and increased cytochrome c protein abundance in the cytoplasm (apart from mitochondria) compared to those the control. In the midgut, glycinin increased the mRNA levels of pro-apoptotic molecules, including caspase-3, tumour necrosis factor-α (TNF-α), B-cell lymphoma 2 (Bcl-2)-associated X (Bax), and apoptotic protease activating factor 1 (Apaf-1), and decreased the mRNA levels of the anti-apoptotic molecule Bcl-2 compared to those in the control. In the hindgut, glycinin increased caspase-3, −8, and − 9, TNF-α, Fas-ligand (FasL), Bax, and Apaf-1 mRNA levels and decreased Bcl-2, myeloid cell leukaemia 1 (Mcl-1), and inhibitors of apoptosis proteins (IAP) mRNA levels compared to those in the control. For TJs, compared to that in the control, glycinin reduced the mRNA abundance of barrier-forming TJs—including claudin-c in the foregut; ZO-1, ZO-2b, occludin, and claudin-7a in the midgut; and ZO-1, ZO-2b, occludin, claudin-3c, claudin-7a, claudin-7b, and claudin-11 in the hindgut. In contrast, glycinin increased the mRNA abundance of pore-forming TJs, including claudin-15a and claudin-15b, in the midgut and hindgut. As an important nuclear factor of TJ transcription, hepatic nuclear factor 4 alpha (HNF-4α) protein abundance was reduced by glycinin in the midgut and hindgut. Glutamine alleviated apoptosis in the midgut. Glycinin aggravated apoptosis associated with both extrinsic and intrinsic signalling and disrupted TJ related to HNF-4α signalling in the midgut and hindgut, ultimately disrupting the intestinal structural integrity of grass carp.

中文翻译：

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大豆甘氨酸蛋白破坏了与细胞凋亡加重相关的肠道结构完整性，并下调了草鱼幼鱼肠道中紧密连接蛋白的转录（Ctenopharyngodon idella）

摘要 大豆球蛋白是大豆的主要抗营养因子。甘氨酸抑制草鱼和剑鱼的生长并损害肠道健康，但其潜在机制尚不清楚。本研究使用草鱼来检查膳食大豆球蛋白的负面影响是否与肠道凋亡信号传导和紧密连接 (TJ) 相关。结果表明，8% 的膳食大豆球蛋白导致血清中二胺氧化酶活性和 D-乳酸含量高于对照，表明肠道结构完整性受损。对于细胞凋亡指数，在中肠和后肠中，与对照组相比，大豆球蛋白加剧了 DNA 断裂现象，促进了 caspase-3、-8 和 -9 的活性，并增加了细胞质（除线粒体外）中细胞色素 c 蛋白的丰度. 在中肠，大豆球蛋白增加促凋亡分子的 mRNA 水平，包括 caspase-3、肿瘤坏死因子-α (TNF-α)、B 细胞淋巴瘤 2 (Bcl-2) 相关 X (Bax) 和凋亡蛋白酶激活因子 1 (Apaf-1)，并且与对照相比降低了抗凋亡分子 Bcl-2 的 mRNA 水平。在后肠中，大豆球蛋白增加 caspase-3、−8 和 − 9、TNF-α、Fas-配体 (FasL)、Bax 和 Apaf-1 mRNA 水平并降低 Bcl-2、髓细胞白血病 1 (Mcl-1 )，以及与对照组相比的凋亡蛋白抑制剂 (IAP) mRNA 水平。对于 TJs，与对照组相比，大豆球蛋白降低了形成屏障的 TJs 的 mRNA 丰度——包括前肠中的密蛋白-c；中肠中的 ZO-1、ZO-2b、occludin 和 claudin-7a；和后肠中的 ZO-1、ZO-2b、occludin、claudin-3c、claudin-7a、claudin-7b 和 claudin-11。相比之下，大豆球蛋白增加了中肠和后肠中成孔 TJ 的 mRNA 丰度，包括 claudin-15a 和 claudin-15b。作为 TJ 转录的重要核因子，肝核因子 4 α (HNF-4α) 蛋白丰度被中肠和后肠中的大豆球蛋白降低。谷氨酰胺减轻了中肠的细胞凋亡。Glycinin 加剧了与外在和内在信号相关的细胞凋亡，并破坏了中肠和后肠中与 HNF-4α 信号相关的 TJ，最终破坏了草鱼的肠道结构完整性。中肠和后肠中的大豆球蛋白降低了肝核因子 4 α (HNF-4α) 蛋白丰度。谷氨酰胺减轻了中肠的细胞凋亡。Glycinin 加剧了与外在和内在信号相关的细胞凋亡，并破坏了中肠和后肠中与 HNF-4α 信号相关的 TJ，最终破坏了草鱼的肠道结构完整性。中肠和后肠中的大豆球蛋白降低了肝核因子 4 α (HNF-4α) 蛋白丰度。谷氨酰胺减轻了中肠的细胞凋亡。Glycinin 加剧了与外在和内在信号相关的细胞凋亡，并破坏了中肠和后肠中与 HNF-4α 信号相关的 TJ，最终破坏了草鱼的肠道结构完整性。