Abstract：
Deoxynivalenol (DON) pollution is prevalent in crops, and can induce oxidative stress and intestinal injury. Hesperidin is one of the major flavonoids in citrus fruits that has various biological activities such as antioxidant and anti-inflammatory activities. However, whether hesperidin could alleviate DON-induced intestinal injury and the mechanism remain unclear. Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) have attracted attention for their crucial signaling points to regulate ER-mitochondria calcium transfer. This study aims to evaluate the effects of hesperidin on the intestinal barrier, mitochondrial function, MAMs, and inositol 1,4,5-triphosphate receptor (IP3R)-mitochondrial calcium uniporter (MCU) calcium axis in the intestine of piglets exposed to DON. Twenty-four piglets were randomly divided into four groups in a 2 × 2 factorial arrangement for a 21-d experiment: Control: basal diet; hesperidin group: basal diet + 300 mg kg-1 hesperidin; DON: basal diet + 1.5 mg kg-1 DON; DON + hesperidin group: basal diet + 1.5 mg kg-1 DON + 300 mg kg-1 hesperidin. The data showed that when compared with the DON group, hesperidin improved growth performance and the intestinal barrier, alleviated intestinal oxidative stress and ER stress, and decreased the serum alanine aminotransferase (ALT) level (P < 0.05). Hesperidin also alleviated mitochondrial dysfunction and ferroptosis in the intestine of piglets exposed to DON (P < 0.05). Importantly, hesperidin prevented excessive MAM formation by downregulating the protein levels of Mitofusin 2 (Mfn2) and glucose-regulated protein 75 (GRP75), decreasing the ratio of the mitochondria with MAMs/total mitochondria and the ratio of MAM length/mitochondrial perimeter and lengthening the mitochondria-ER distance in MAMs (P < 0.05). Furthermore, hesperidin regulated the IP3R-glucose-regulated protein 75 (GRP75)-voltage-dependent anion channel 1 (VDAC1)-MCU calcium axis by decreasing the protein levels of GRP75 and MCU and the calcium level of the mitochondria compared with the DON group (P < 0.05). An in vitro experiment was conducted to further explore whether IP3R-mediated ER-mitochondria calcium transfer was involved in the protective effects of hesperidin on the intestinal epithelium barrier and mitochondria. Data showed that hesperidin may exert protective effects on the intestinal epithelium barrier and mitochondria via inhibiting ER-mitochondrial calcium transfer mediated by IP3Rs. These data suggested that hesperidin could alleviate MAM-mediated mitochondrial calcium overload, thereby improving mitochondrial function and alleviating oxidative stress and intestinal injury in DON-challenged piglets.
摘要：
脱氧雪腐镰刀菌烯醇(DON)污染在农作物中普遍存在,并可引起氧化应激和肠道损伤。橙皮苷是柑橘类水果中主要的黄酮类化合物之一,具有抗氧化、抗炎等多种生物活性。然而,橙皮苷是否能减轻DON引起的肠损伤,其机制尚不清楚。线粒体相关的内质网（ER）膜（MAMs）因其调节ER-线粒体钙转移的关键信号点而受到关注。本研究旨在评价橙皮苷对肠道屏障功能的影响,线粒体功能，MAMs,和暴露于DON的仔猪肠道中的肌醇1,4,5-三磷酸受体（IP3R）-线粒体钙单蛋白（MCU）钙轴。将24只仔猪按2×2因子排列随机分为4组，进行21天实验：对照：基础日粮；橙皮苷组：基础日粮+300mgkg-1橙皮苷；DON：基础日粮+1.5mgkg-1DON；DON橙皮苷组：基础日粮+1.5mgkg-1DON+300mgkg-1橙皮苷。数据显示，与DON组相比，橙皮苷改善生长性能和肠道屏障，减轻肠道氧化应激和ER应激，降低血清丙氨酸转氨酶(ALT)水平(P<0.05)。橙皮苷还能减轻暴露于DON的仔猪肠道线粒体功能障碍和铁凋亡(P<0.05)。重要的是,橙皮苷通过下调Mitofusin2（Mfn2）和葡萄糖调节蛋白75（GRP75）的蛋白水平来防止过多的MAM形成，降低MAMs中线粒体/总线粒体的比例和MAM长度/线粒体周长的比例，延长MAMs中线粒体-ER距离（P<0.05）。此外，橙皮苷调节IP3R-葡萄糖调节蛋白75（GRP75）-电压依赖性阴离子通道1（VDAC1）-MCU钙轴，通过降低GRP75和MCU的蛋白水平和线粒体钙水平，与DON组相比（P<0.05）。进行了体外实验，以进一步探讨IP3R介导的ER-线粒体钙转移是否参与橙皮苷对肠道上皮屏障和线粒体的保护作用。结果表明，橙皮苷可能通过抑制IP3Rs介导的ER-线粒体钙转移对肠上皮屏障和线粒体发挥保护作用。这些数据表明橙皮苷可以缓解MAM介导的线粒体钙超载，从而改善DON攻击仔猪的线粒体功能，减轻氧化应激和肠道损伤。
