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Hesperidin Alleviated Intestinal Barrier Injury, Mitochondrial Dysfunction, and Disorder of Endoplasmic Reticulum Mitochondria Contact Sites under Oxidative Stress
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Hesperidin Alleviated Intestinal Barrier Injury, Mitochondrial Dysfunction, and Disorder of Endoplasmic Reticulum Mitochondria Contact Sites under Oxidative Stress
橙皮苷减轻氧化应激屏障损伤线粒体功能障碍内质线粒体接触紊乱
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  • Feiyang Gou  飞扬沟
    Feiyang Gou
    Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
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  • Qian Lin  钱琳
    Qian Lin
    Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
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  • Xiaodian Tu  涂小典
    Xiaodian Tu  涂小典
    Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
    浙江大学动物科学学院, 分子动物营养教育部重点实验室(浙江大学), 杭州 310058
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    更多涂晓电的作品
  • Jiang Zhu  江珠
    Jiang Zhu  江珠
    Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
    浙江大学动物科学学院, 分子动物营养教育部重点实验室(浙江大学), 杭州 310058
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    更多朱江的作品
  • Xin Li  李欣
    Xin Li
    Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
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  • Shaokui Chen*  陈少奎*
    Shaokui Chen  陈少奎
    Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
    浙江大学动物科学学院, 分子动物营养教育部重点实验室(浙江大学), 杭州 310058
    School of Animal Science and Nutritional Engineering, Wuhan Polytechnic University, Wuhan 430023, China
    武汉工业大学动物科学与营养工程学院, 武汉 430023
    *Email: loveskchen@163.com
    *邮箱: loveskchen@163.com
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  • Caihong Hu*  胡彩虹*
    Caihong Hu
    Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
    *Email: chhu@zju.edu.cn
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Journal of Agricultural and Food Chemistry

Cite this: J. Agric. Food Chem. 2024, 72, 29, 16276–16286
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https://doi.org/10.1021/acs.jafc.4c02265 IF: 5.7 Q1
Published July 9, 2024
Copyright © 2024 American Chemical Society

Abstract  抽象的

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As primary flavonoids extracted from citrus fruits, hesperidin has been attracting attention widely for its capacity to act as antioxidants that are able to scavenge free radicals and reactive oxygen species (ROS). Many factors have made oxidative stress a risk factor for the occurrence of intestinal barrier injury, which is a serious health threat to human beings. However, little data are available regarding the underlying mechanism of hesperidin alleviating intestinal injury under oxidative stress. Recently, endoplasmic reticulum (ER) mitochondria contact sites (ERMCSs) have aroused increasing concerns among scholars, which participate in mitochondrial dynamics and Ca2+ transport. In our experiment, 24 piglets were randomly divided into 4 groups. Piglets in the diquat group and hesperidin + diquat group received an intraperitoneal injection of diquat (10 mg/kg), while piglets in the hesperidin group and hesperidin + diquat group received hesperidin (300 mg/kg) with feed. The results indicated that hesperidin alleviated growth restriction and intestinal barrier injury in piglets compared with the diquat group. Hesperidin ameliorated oxidative stress and restored antioxidant capacity under diquat exposure. The mitochondrial dysfunction was markedly alleviated via hesperidin versus diquat group. Meanwhile, hesperidin alleviated ER stress and downregulated the PERK pathway. Furthermore, hesperidin prevented the disorder of ERMCSs by downregulating the level of ERMCS proteins, decreasing the percentage of mitochondria with ERMCSs/total mitochondria and the ratio of ERMCSs length/mitochondrial perimeter. These results suggested hesperidin could alleviate ERMCS disorder and prevent mitochondrial dysfunction, which subsequently decreased ROS production and alleviated intestinal barrier injury of piglets under oxidative stress.
作为从柑橘类水果中提取的主要黄酮类化合物,橙皮苷因其作为抗氧化剂的能力而受到广泛关注,能够清除自由基和活性氧(ROS)。多种因素使得氧化应激成为肠道屏障损伤发生的危险因素,严重威胁人类的健康。然而,关于橙皮苷减轻氧化应激肠道损伤的潜在机制的数据很少。近年来,内质(ER)线粒体接触位点(ERMCSs)引起了学者们越来越多的关注,其参与线粒体动力学和Ca 2+转运。在我们的实验中,24 头仔猪被随机分为 4 组。敌草快组和橙皮苷+敌草快组仔猪腹腔注射敌草快(10 mg/kg),橙皮苷组和橙皮苷+敌草快组仔猪腹腔注射橙皮苷(300 mg/kg)随饲料。结果表明,与敌草快组相比,橙皮苷减轻了仔猪的生长受限和肠道屏障损伤橙皮苷可改善敌草快暴露下的氧化应激并恢复抗氧化能力。 与敌草快组相比,橙皮苷显着减轻了线粒体功能障碍。同时,橙皮苷缓解ER应激并下调 PERK 通路。此外,橙皮苷通过下调ERMCS蛋白水平、降低具有ERMCS的线粒体/总线粒体的百分比以及ERMCS长度/线粒体周长的比率来预防ERMCS的紊乱。这些结果表明橙皮苷可以缓解ERMCS紊乱并预防线粒体功能障碍,从而减少ROS的产生并减轻氧化应激下仔猪的肠道屏障损伤

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1. Introduction  一、简介

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It has been well-reported that hesperidin (3,5,7-trihydroxy flavanone-7-rhamnoglucoside, PubChem CID: 10621), an abundant flavanone glycoside present in plants of the rutaceous family, possesses multiple biological activities, of which biological effects have gained wide attention recently. (1) Popularly, hesperidin in citrus fruits varied from 0 to 20 mg/g dry weight (DW) in peels, from 2 to 4 mg/g DW in pulps, and from 0 to 0.199 mg/mL fresh weight in juice. (2,3) As reported by a previous study, the pharmacological properties of hesperidin, including anti-inflammatory, antioxidant, antitumor, and neuroprotective properties, were associated with its capacity to act as antioxidants that are able to scavenge free radicals and reactive oxygen species (ROS). (4) Study in mice demonstrated that hesperidin showed advantages in ameliorating colitis and restoring intestinal barrier function. (5) According to a previous study, hesperidin showed the potential for alleviating amyloid-β (Aβ)-induced oxidative stress and mitochondrial dysfunction in mice. (6) To our knowledge, despite some studies reporting the effects of hesperidin on oxidative stress and intestinal barrier integrity damage, the specific underlying mechanism is not fully understood.
据报道,橙皮苷(3,5,7-三羟基黄烷酮-7-鼠李糖苷,PubChem CID:10621)是芸香科植物中含量丰富的黄烷酮苷,具有多种生物活性,其中生物效应有近期受到广泛关注。 (1)一般来说,柑橘类水果中橙皮苷的含量在果皮中为 0 至 20 mg/g 干重 (DW),果肉中为 2 至 4 mg/g DW,果汁中为 0 至 0.199 mg/mL 鲜重。 (2,3)正如之前的研究报道,橙皮苷的药理特性,包括抗炎、抗氧化、抗肿瘤和神经保护特性,与其作为抗氧化剂清除自由基和活性氧的能力相关。物种(ROS)。 (4)小鼠研究表明橙皮苷在改善结肠炎和恢复肠道屏障功能方面具有优势。 (5)根据之前的一项研究,橙皮苷显示出缓解淀粉样蛋白-β (Aβ) 诱导的小鼠氧化应激线粒体功能障碍的潜力。 (6)据我们所知,尽管有一些研究报告了橙皮苷氧化应激肠道屏障完整性损伤的影响,但具体的潜在机制尚不完全清楚。
The intestinal epithelial barrier is well accepted as the significant barrier to protect against antigens and pathogens, which also mainly attack targets of oxidative stress. (7) It has been well-reported that many factors could induce oxidative stress and intestinal barrier injury in daily life, including pathogens, pollutants, radiation, diet, and lifestyle. (8) As a nonselective quick-acting herbicide, diquat chronically released after entering the body, which subsequently induced excessive production of free radicals and release of a large number of ROS and other oxidizing substances. (9) After that, excess ROS damaged lipids, proteins, and DNA, ultimately inducing intestinal barrier damage. Therefore, diquat was widely used to establish oxidative stress model in piglets. Hence, safe and effective therapeutic measures are urgently required to ameliorate intestinal oxidative stress.
上皮屏障被广泛认为是抵御抗原和病原体的重要屏障,这些抗原和病原体也主要攻击氧化应激的目标。 (7)已有大量报道表明,日常生活中许多因素可诱发氧化应激肠道屏障损伤,包括病原体、污染物、辐射、饮食和生活方式。 (8)敌草快作为一种非选择性速效除草剂,进入体内后会长期释放,导致自由基过量产生,释放大量ROS等氧化性物质。 (9)此后,过量的ROS会损害脂质、蛋白质和DNA,最终导致肠道屏障受损。因此,敌草快被广泛用于建立仔猪氧化应激模型。因此,迫切需要安全有效的治疗措施来改善肠道氧化应激
It is widely accepted that the mitochondria are the main energy factory in various cells, in which the oxidative respiratory chain produces a large body of energy for maintaining intestinal barrier function, while endoplasmic reticulum (ER) as the largest membranous organelle in eukaryotes plays essential roles in the synthesis and metabolism of carbohydrates, lipids, and glycogen and synthesis of protein. (10,11) There was evidence suggesting that the ER stress (ERS) and mitochondrial dysfunction were closely associated with the crosstalk between mitochondria and ER. (12) Recently, physical contacts among different organelles, such as membrane contact sites (MCSs) between the mitochondria, ER, and peroxisomes, were found to be responsible for the transport of information and substances in restricted regions, which have gained wide attention among scholars. (13) Regions of close association between mitochondria and ER which are defined as ER mitochondria contact sites (ERMCSs) were subsequently reported to be abundant in a series of proteins responsible for lipid synthesis and transfer between the mitochondria and ER. (14,15) It has been well-reported that ERMCSs are shown to exist as specialized structures that facilitate coordinated energy metabolism, Ca2+ transfer, and mitochondrial dynamics. (16) More recently, ERMCSs have been reported to be closely related with a number of pathologies, for example, increase of metabolic activities in ERMCSs is closely related to Alzheimer’s disease. (17,18) Although there have been many studies on ERMCSs in the field of life science and medicine, no data have been reported about the effects of hesperidin on ERMCSs, mitochondrial function, and ERS under oxidative stress.
人们普遍认为线粒体是各种细胞的主要能量工厂,其中氧化呼吸链产生大量能量维持肠道屏障功能,而内质(ER)作为真核生物最大的膜细胞器发挥着至关重要的作用参与碳水化合物、脂质、糖原的合成和代谢以及蛋白质的合成。 (10,11)有证据表明内质网应激(ERS) 和线粒体功能障碍线粒体和内质网之间的串扰密切相关。 (12)最近,不同细胞器之间的物理接触,如线粒体、内质网和过氧化物酶体之间的膜接触位点(MCS)被发现负责限制区域内的信息和物质的运输,引起了人们的广泛关注。学者。 (13)随后报道称,线粒体和内质网之间密切相关的区域(被定义为内质网线粒体接触位点(ERMCS))富含一系列负责脂质合成以及线粒体和内质网之间转移的蛋白质。 (14,15)据充分报道,ERMCS 作为特殊结构存在,可促进协调能量代谢、Ca 2+转移和线粒体动力学。 (16)最近,ERMCSs被报道与许多病理学密切相关,例如,ERMCSs代谢活动的增加与阿尔茨海默病密切相关。 (17,18)虽然生命科学和医学领域对ERMCSs有很多研究,但目前尚未有关于橙皮苷氧化应激下ERMCSs、线粒体功能和ERS影响的数据报道。
Therefore, we employed a widely used oxidative stress model by diquat in piglets, which is an ideal model for studying intestinal oxidative stress. (19) We hypothesized that hesperidin could alleviate intestinal barrier injury under oxidative stress via restoring the formation and function of ERMCSs and ameliorating ERS and mitochondrial dysfunction. Hence, we investigated the effects of hesperidin on the formation of ERMCSs, intestinal barrier, ERS, and mitochondria under oxidative stress. Collectively, this investigation provided a promising strategy to use hesperidin to prevent intestinal barrier injury under oxidative stress.
因此,我们采用广泛使用的敌草快仔猪氧化应激模型,这是研究肠道氧化应激的理想模型。 (19)我们假设橙皮苷可以通过恢复ERMCS的形成和功能、改善ERS和线粒体功能障碍来减轻氧化应激下的肠道屏障损伤。因此,我们研究了橙皮苷氧化应激下 EMCS、屏障、ERS 和线粒体形成的影响。总的来说,这项研究为使用橙皮苷预防氧化应激下的肠道屏障损伤提供了一种有前途的策略。

2. Materials and Methods  2. 材料与方法

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2.1. Ethics Statement  2.1.道德声明

The experiment design and procedures were approved by the Committee of Animal Care and Use of Zhejiang University. Animal procedures in this experiment were approved by the animal care and use committee of Zhejiang University (approval number: ZJU20230247).
实验设计和程序经浙江大学动物保护与使用委员会批准。本实验的动物程序经浙江大学动物保护与使用委员会批准(批准号:ZJU20230247)。

2.2. Experimental Design and Diets
2.2.实验设计和饮食

Twenty-four piglets were used in this experiment. The piglets (35 days old, weaned at 21 d, Duroc × Landrace × Yoshire, initial body weight of 9.70 kg) were assigned into four groups using a randomized complete block design. With six piglets (half male and half female) in each group, there were four groups: control group (basal diet + 0.9% saline injection), hesperidin group (basal diet + 300 mg/kg hesperidin + 0.9% saline injection), diquat group (basal diet + diquat injection), and hesperidin + diquat group (basal diet + diquat injection + 300 mg/kg hesperidin). (20) At the beginning, piglets received an intraperitoneal injection of diquat or sterilized saline at 10 mg/kg of body weight. The dosage of diquat (10 mg/kg) was chosen to induce oxidative stress according to a previous study. (21) The diquat (Sigma-Aldrich, St. louis, MO, USA) and saline were filter-sterized before intraperitoneal injection. The basal diet was formulated according to the NRC (2012) (Table S1). The experiment lasted for 14 days, during which water and feed were freely available. For the duration of the experiments, we calculated the average daily gain (ADG), the average daily feed intake (ADFI), and the ratio of feed to gain (F:G) of piglets.
本实验使用了二十四头仔猪。将仔猪(35日龄,21日断奶,杜洛克×长白×约夏,初始体重9.70 kg)采用随机完全区组设计分为4组。每组6头仔猪,雌雄各半,分为4组:对照组(基础日粮+0.9%生理盐水注射)、橙皮苷组(基础日粮+300 mg/kg橙皮苷+0.9%生理盐水注射)、敌草快组。组(基础饮食+敌草快注射液)、橙皮苷+敌草快组(基础饮食+敌草快注射液+300mg/kg橙皮苷) )。 (20)开始时,仔猪按10mg/kg体重腹腔注射敌草快或灭菌盐水。根据先前的研究,选择敌草快的剂量(10 毫克/千克)来诱导氧化应激(21)敌草快 (Sigma-Aldrich, St. louis, MO, USA) 和生理盐水在腹腔注射前过滤灭菌。基础饮食根据NRC(2012)制定(表S1 )。实验持续14天,期间免费提供水和饲料。在实验期间,我们计算了仔猪的平均日增重 (ADG)、平均日采食量 (ADFI) 和饲料增重比 (F:G)。

2.3. Sample Collection  2.3.样品采集

At the end of the experiment, the piglets were euthanized by intramuscular injection of xylazine-hydrochloride. Before the abdominal cavity was cut open carefully, the skin was wiped with 75% ethanol. After that, the intestines were harvested, and a section of the intestinal tissues were cut and washed with cold normal saline. After the tissues were obtained, the intestinal tract samples were preserved in formaldehyde and glutaraldehyde, respectively, for the immunohistochemical analysis and transmission electron microscopy (TEM) experiment. In addition, intestinal mucosa was frozen in liquid nitrogen and stored at −80 °C until further study.
实验结束时,肌肉注射盐酸赛拉嗪对仔猪实施安乐死。小心切开腹腔前,用75%乙醇擦拭皮肤。之后,收获肠,切下一段组织并用​​冷生理盐水清洗。获得组织后,将肠道样本分别保存在甲醛和戊二醛中,用于免疫组化分析和透射电子显微镜(TEM)实验。此外,将肠粘膜冷冻在液氮中并保存在-80°C直至进一步研究。

2.4. Using Chamber Experiment
2.4.使用室实验

Transepithelial electrical resistance (TER, Ω·cm–2) and fluorescein isothiocyanate-dextran 4 kDa (FD4, ng·cm–2·h–1) were used to assess the jejunum permeability. The Ussing chamber experiment was performed as per the report of Hu et al. (22) Basically, we used the EasyMount Ussing chamber system to detect the TER of jejunum mucosa. FD4 was placed in the mucosal side of the Ussing chamber. The flux of FD4 was recorded through a fluorescence microplate reader (Flx800, Bio-Tek).
跨上皮电阻(TER,Ω·cm –2 )和异硫氰酸荧光素-葡聚糖4 kDa(FD4,ng·cm –2 ·h –1 )用于评估空肠通透性。 Ussing 室实验是根据 Hu 等人的报告进行的。 (22)基本上,我们使用 EasyMount Ussing 室系统来检测空肠粘膜的 TER。 FD4被放置在Ussing室的粘膜侧。通过荧光酶标仪(Flx800,Bio-Tek)记录FD4的通量。

2.5. Intestinal Morphology
2.5.肠道形态学

Hematoxylin-eosin (H&E) staining was used to measure the morphology of jejunal according to previous studies. (23,24) In short, 4% paraformaldehyde was used to fix to the fresh isolated segment of jejunum. After being embedded in paraffin, sectioned, and stained, we used a microscope (Mshot, MF52, China) to capture the images of the samples. After that, we calculated the crypt depth, the villus height, and the ratio of villus height to crypt depth using Case viewer (V2.4) to measure the jejunal morphology.
根据之前的研究,使用苏木精-伊红(H&E)染色来测量空肠的形态。 (23,24)简而言之,使用 4% 多聚甲醛固定新鲜分离的空肠段。石蜡包埋、切片、染色后,我们使用显微镜(Mshot,MF52,中国)捕获样品的图像。之后,我们使用 Caseviewer (V2.4) 计算隐窝深度、绒毛高度以及绒毛高度与隐窝深度的比率,以测量空肠形态。

2.6. Assay of Activity of Serum Transaminase, Antioxidative Enzyme, and Level of MDA
2.6。血清转氨酶、抗氧化酶活性及MDA水平测定

ELISA kits were used (Beyotime Institute of Biotechnology, Shanghai, China) to detect the activity of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), superoxide dismutase (SOD), and catalase (CAT) and level of malondialdehyde (MDA).
采用ELISA试剂盒(上海碧云天生物技术研究所)检测血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性以及丙二醛(MDA)水平。 。

2.7. TEM Experiment  2.7.透射电镜实验

The TEM experiment was performed according to Cao et al. (25) We used 2.5% glutaraldehyde to fix the isolated segments of jejunum at 4 °C overnight. After that, the samples were post fixed with 1% OsO4. After a series of processing of the samples, such as dehydration, infiltration, embedding, ultrathin sectioning, and staining, we used TEM (Japan, Hitachi Model H-7650) to observe the samples.
TEM实验按照Cao等人的方法进行。 (25)我们使用 2.5% 戊二醛将分离的空肠片段在 4°C 下固定过夜。之后,用1%OsO 4后固定样品。样品经过脱水、浸润、包埋、超薄切片、染色等一系列处理后,使用TEM(日本,日立型号H-7650)对样品进行观察。

2.8. Measurement of Mitochondrial Membrane Potential, ATP Content, Mitochondrial Calcium, and Activity of Mitochondria Respiratory Chain Complex I–III
2.8.线粒体膜电位、ATP 含量、线粒体钙和线粒体呼吸链复合物 I-III 活性的测量

JC-1 assay kit was used (Beyotime Institute of Biotechnology, Shanghai, China) to measure the change of mitochondrial membrane potential (MMP). After the samples were processed, we used the microplate reader to measure the fluorescence. ATP detection kit was used (Beyotime Institute of Biotechnology, Shanghai) to determine the ATP content of samples. According to a previous study, with the calcium test kit (Sigma-Aldrich), the concentration of mitochondrial Ca2+ was calculated after mitochondrial membrane permeabilization and the ensuing release of Ca2+. (26) The activity of mitochondrial respiratory chain complexes I–III was measured by the quantitative determination kits (Genmed Scientifics Shanghai, China). The activity of mitochondrial respiratory chain complexes is determined by measuring the OD values of their substrates at their respective excitation wavelengths.
使用JC-1测定试剂盒(碧云天生物技术研究所,上海,中国)测量线粒体膜电位(MMP)的变化。样品处理后,我们使用酶标仪测量荧光。采用ATP检测试剂盒(上海碧云天生物技术研究所)测定样品的ATP含量。根据先前的研究,使用钙测试试剂盒(Sigma-Aldrich),在线粒体膜透化和随后释放的Ca 2+后计算线粒体Ca 2+的浓度。 (26)通过定量测定试剂盒(上海吉迈科技有限公司)测定线粒体呼吸链复合物I-III的活性。线粒体呼吸链复合物的活性是通过测量其底物在各自激发波长下的 OD 值来确定的。

2.9. Real-Time Quantitative PCR Analysis
2.9.实时定量 PCR 分析

According to Shi et al., (27) relative mRNA expression level was assessed. Real-time quantitative PCR (RT-qPCR) was performed according to the manufacturer’s instructions (qPCR, master mix, Vazyme, Nanjing, China). The information on primers used for RT-qPCR is listed in Table S2.
根据 Shi 等人的说法, (27)评估了相对 mRNA 表达水平。根据制造商的说明(qPCR,master mix,Vazyme,南京,中国)进行实时定量PCR(RT-qPCR)。用于RT-qPCR的引物信息列于表S2中。

2.10. Western Blotting Analysis
2.10.蛋白质印迹分析

According to He et al., the relative expression level of protein was assessed by the Western blot assay. (28) After the samples from the jejunum mucosa were extracted, purified, denatured, etc., we obtained the pure total proteins. The total proteins were run and isolated through SDS-PAGE. Then, the proteins were incubated with specific primary antibodies overnight and then with secondary antibodies. The primary antibodies used in the experiment are listed in Table S3.
根据He等人的说法,蛋白质的相对表达水平是通过Western blot测定来评估的。 (28)空肠粘膜样品经提取、纯化、变性等处理后,得到纯的总蛋白。通过 SDS-PAGE 运行并分离总蛋白。然后,将蛋白质与特定一抗孵育过夜,然后与二抗孵育。实验中使用的一抗列于表S3中。

2.11. Determination of Mitochondrial ROS Production
2.11.线粒体ROS 产生的测定

According to the study by Yang et al., the DCFH-DA kit was used (Beyotime Biotechnology, Shanghai, China) to detect the level of mitochondrial ROS via a fluorometric method. (29) After measuring, the results of each group were calculated as fold changes versus control.
根据Yang等人的研究,使用DCFH-DA试剂盒(Beyotime Biotechnology,上海,中国)通过荧光法检测线粒体ROS水平。 (29)测量后,将每组的结果计算为相对于对照组的倍数变化。

2.12. Immunofluorescence of Jejunal Sections and Colocalization Analysis of Correlation Scatter Plots
2.12.空肠切片的免疫荧光和相关散点图的共定位分析

According to the description of Yu et al., (30) the paraffin sections were subjected to a series of procedures, including dewaxing to water, antigen retrieval, serum blocking, and incubation with the corresponding primary and secondary antibodies. Finally, the samples were observed and captured under a fluorescence microscope (Mshot, MF52, China) to visualize the labeling of the target antigens. Image-J was used to analyze the colocalization of the correlation scatterplot between the mitochondria marker and the ER marker, which demonstrated the interaction between mitochondria and ER.
根据Yu等人的描述, (30)石蜡切片经过一系列程序,包括脱蜡至水、抗原修复、血清封闭以及与相应的一抗和二抗孵育。最后,在荧光显微镜(Mshot,MF52,中国)下观察和捕获样品,以可视化目标抗原的标记。使用Image-J分析线粒体标记和ER标记之间的相关散点图的共定位,证明线粒体和ER之间的相互作用。

2.13. Statistical Analysis
2.13.统计分析

Data were subject to one-way analysis of variance with Duncan’s multiple range tests through SPSS 20.0 (SPSS Inc., Chicago, IL). Difference was considered to be significant when P < 0.05. * < 0.05, ** < 0.01, and *** < 0.001 vs control group and # < 0.05, ## < 0.01, and ### < 0.001 vs diquat group, respectively. Each group had six replicates, and “ns” means not significant.
通过 SPSS 20.0(SPSS Inc.,芝加哥,伊利诺斯州)对数据进行单向方差分析,采用 Duncan 的多范围检验。当P < 0.05 时,认为差异显着。与对照组相比,* < 0.05、** < 0.01 和 *** < 0.001,与敌草快组相比,# < 0.05、## < 0.01 和 ### < 0.001。每组有 6 个重复,“ns”表示不显着。

3. Results  3. 结果

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3.1. Dietary Hesperidin Alleviated Growth Restriction and Intestinal Barrier Injury in Piglets under Oxidative Stress
3.1.日粮橙皮苷减轻氧化应激下仔猪的生长受限和屏障损伤

Figure1a–c shows that diquat-induced oxidative stress markedly decreased the final body weight (FBW) compared with the control (P < 0.001), while dietary hesperidin significantly alleviated oxidative stress-induced decrease of FBW (P < 0.01), which indicated that hesperidin alleviated the growth restriction under oxidative stress. Meanwhile, dietary hesperidin group revealed substantial elevation on ADG (P < 0.01) and ADFI (P < 0.001) compared with the diquat group, which were strikingly decreased by diquat treatment (P < 0.001). The ratio of feed to gain (F:G) was not influenced among four groups (P > 0.05). According to Figure 1d,e, hesperidin supplementation significantly alleviated oxidative stress-induced decrease of TER (P < 0.01) and increase of FD4 flux (P < 0.01) compared with diquat treatment. The diquat treatment induced abnormalities in the crypt-villus structure of jejunum according to the H&E staining, which was alleviated by hesperidin supplementation (Figure 1f). As can be seen in Figure 1g,h, the villus height and the ratio of villus height to crypt depth were markedly decreased (P < 0.001 and P < 0.01) by diquat challenge and were markedly increased by dietary hesperidin (P < 0.01 and P < 0.05). Meanwhile, the difference of crypt depth among four groups was not significant (P > 0.05). Figure 1i,j shows that the diquat challenge strikingly decreased the level of tight junction proteins (ZO-1, Occludin and Claudin-1) (P < 0.01, P < 0.05, and P < 0.05, respectively) compared with the control group, which was strikingly increased by hesperidin (P < 0.001, P < 0.001, and P < 0.01, respectively). Considered together, these data indicated that dietary hesperidin alleviated intestinal barrier injury and growth retardation of piglets under oxidative stress.
图1 a-c显示,与对照相比,敌草快诱导的氧化应激显着降低了最终体重(FBW)( P < 0.001),而膳食橙皮苷显着缓解了氧化应激诱导的FBW降低( P < 0.01),这表明表明橙皮苷缓解了氧化应激下的生长限制。同时,与敌草快组相比,膳食橙皮苷组的 ADG ( P < 0.01) 和 ADFI ( P < 0.001) 显着升高,而敌草快处理则显着降低 ( P < 0.001)。四组间料重比(F:G)均不受影响( P > 0.05)。根据图1d 、e,与敌草快处理相比,补充橙皮苷显着减轻了氧化应激引起的TER减少( P <0.01)和FD4通量增加( P <0.01)。根据 H&E 染色,敌草快治疗引起空肠隐窝绒毛结构异常,补充橙皮苷可缓解这种异常(图 1 f)。从图 1 g,h 中可以看出,敌草快攻击后绒毛高度以及绒毛高度与隐窝深度之比显着降低( P < 0.001 和P < 0.01),而膳食橙皮苷则显着增加( P < 0.01 和 P < 0.01)。 P <0.05)。 同时,四组间隐窝深度差异无显着性( P >0.05)。图 1 i,j 显示与对照组相比,敌草快挑战显着降低了紧密连接蛋白(ZO-1、Occludin 和 Claudin-1)的水平(分别为P < 0.01、 P < 0.05 和P < 0.05) ,橙皮苷显着增加(分别为P < 0.001、 P < 0.001 和P < 0.01)。综合考虑,这些数据表明日粮橙皮苷可减轻氧化应激下仔猪的肠道屏障损伤和生长迟缓。

Figure 1  图1

Figure 1. Dietary hesperidin alleviated oxidative stress-induced growth restriction and intestinal barrier injury. (a–c) Initial and final body weight (IBW, FBW) (a), ADG and ADFI (b), and ratio of feed to gain (F/G) (c) of piglets. (d, e) TER (d) and FD4 flux (e) of jejunum in piglets. (f–h) Images of piglets’ jejunum morphology (scale bars = 100 μm) (f), Quantification of the villus height and crypt depth (g) and the ratio of villus height to crypt depth (h) in the jejunum of piglets. (i, j) Tight junction protein (ZO-1, Occludin, Claudin-1) expression (i) by Western blot and the quantification of expression level (j).
图 1. 膳食橙皮苷可减轻氧化应激引起的生长受限和肠道屏障损伤。 (a-c) 仔猪的初始体重和最终体重(IBW、FBW)(a)、ADG 和 ADFI(b)以及饲料增重比(F/G)(c)。 (d, e) 仔猪空肠的 TER (d) 和 FD4 通量 (e)。 (f–h) 仔猪空肠形态图像(比例尺 = 100 μm) (f),仔猪空肠绒毛高度和隐窝深度 (g) 的量化以及绒毛高度与隐窝深度 (h) 的比率。 (i, j) 紧密连接蛋白 (ZO-1、Occludin、Claudin-1) 表达 (i) 通过蛋白质印迹和表达水平定量 (j)。

3.2. Dietary Hesperidin Decreased the Activity of Serum ALT and AST and Increased the Antioxidant Capacity in the Jejunum of Piglets under Oxidative Stress
3.2.日粮橙皮苷降低氧化应激下仔猪血清 ALT 和 AST 活性并增加空肠抗氧化能力

The activity of serum ALT and AST was increased under diquat exposure (P < 0.01), while diet supplemented with hesperidin significantly decreased serum ALT and AST levels in comparison to the diquat group (P < 0.01) (Figure 2a,b). As can be seen in Figure 2c–e, diquat treatment resulted in a notable decrease in the CAT and SOD activity and increase in the MDA level (P < 0.05, P < 0.01, and P < 0.01, respectively), while hesperidin supplementation markedly increased the activity of CAT and SOD (P < 0.01, P < 0.05) and decreased the MDA level (P < 0.05) versus diquat treatment. According to the result of RT-qPCR, the mRNA level of antioxidant enzymes (SOD1, GPX-1, and GPX-4) markedly decreased (P < 0.05, P < 0.01, and P < 0.01, respectively) under diquat exposure, which were strikingly increased with hesperidin supplementation (P < 0.05, P < 0.05, and P < 0.01, respectively) (Figure 2f).
敌草快暴露下血清 ALT 和 AST 活性升高( P < 0.01),而与敌草快组相比,添加橙皮苷的饮食显着降低血清 ALT 和 AST 水平( P < 0.01)(图 2 a、b)。如图2c -e 所示,敌草快处理导致 CAT 和 SOD 活性显着降低,MDA 水平升高(分别为P < 0.05、 P < 0.01 和P < 0.01),而补充橙皮苷与对照组相比,CAT和SOD活性显着升高( P < 0.01, P < 0.05),MDA水平显着降低( P < 0.05)。敌草快治疗。 RT-qPCR结果显示,敌草快暴露下抗氧化酶( SOD1GPX-1GPX-4 )的mRNA水平显着降低(分别为P < 0.05、 P < 0.01和P < 0.01)。补充橙皮苷后显着增加(分别为P < 0.05、 P < 0.05 和P < 0.01)(图2f )。

Figure 2  图2

Figure 2. Dietary hesperidin alleviated oxidative stress-induced decrease of activity of serum ALT, AST, and antioxidant capacity. (a, b) Activity of serum ALT (a) and AST (b) in piglets. (c–e) Activity of CAT (c), SOD (d), and MDA level (e) in piglets. (f) Heat map of mRNA level of antioxidant enzyme (SOD1, GPX-1, and GPX-4).
图 2. 膳食橙皮苷可缓解氧化应激引起的血清 ALT、AST 活性和抗氧化能力下降。 (a, b) 仔猪血清 ALT (a) 和 AST (b) 的活性。 (c-e) 仔猪中 CAT (c)、SOD (d) 和 MDA 水平 (e) 的活性。 (f) 抗氧化酶(SOD1、GPX-1 和 GPX-4)的 mRNA 水平热图。

3.3. Dietary Hesperidin Alleviated Mitochondrial Injury and Dysfunction in the Jejunum of Piglets under Oxidative Stress
3.3.日粮橙皮苷可减轻氧化应激下仔猪空肠的线粒体损伤功能障碍

According to Figure 3a,b, the diquat treatment induced a noteworthy elevation (P < 0.001) in mitochondria with ultrastructural abnormities such as mitochondrial pyknosis and mitochondria with broken cristate, which was alleviated via hesperidin supplementation (P < 0.001). According to Figure 3c–f, in comparison to the control group, the diquat treatment induced an escalation (P < 0.01) in the mitochondrial ROS level and marked reduction in MMP and ATP contents (P < 0.05, P < 0.01), while hesperidin supplementation significantly alleviated the increase of ROS level (P < 0.01) and decrease of MMP and ATP contents (P < 0.01, P < 0.05) induced by diquat. Meanwhile, the dietary hesperidin exhibited statistically significant elevation on the activity of mitochondrial respiratory chain complex I–III of jejunum (P < 0.05, P < 0.01, and P < 0.05, respectively), which were markedly decreased under diquat exposing (P < 0.01, P < 0.01, and P < 0.05, respectively). According to the result of RT-qPCR, the mRNA level of mitochondrial biogenesis proteins (PGC-1α and NRF-1) were significantly decreased under diquat exposure (P < 0.001, P < 0.05), which were strikingly augmented with hesperidin supplementation (P < 0.05, P < 0.01) (Figure 3g). As shown in Figure 3h,i, compared with the control, the diquat treatment resulted in a noteworthy elevation on Bax (P < 0.001), while Bcl-2 was significantly decreased under diquat exposure (P < 0.01). Meanwhile, the hesperidin supplementation alleviated the increase of Bax (P < 0.05) and decrease of Bcl-2 versus diquat group (P < 0.001). The level of Cyto Cyt-c was increased (P < 0.01) and that of Mtio Cyt-c was decreased (P < 0.001) under oxidative stress, while hesperidin alleviated the increase in Cyto Cyt-c (P < 0.05) and decrease in Mtio Cyt-c (P < 0.001) as shown in Figure 3h–k. As can be seen in Figure 3l–n, hesperidin supplementation alleviated (P < 0.01) increase in the concentration of jejunal mitochondrial Ca2+ compared with the diquat group. Additionally, the diquat challenge induced a notable increase in the level of mitochondrial calpain-1 (P < 0.01), while hesperidin supplementation markedly diminished the mitochondrial calpain-1 level versus diquat group (P < 0.05). The result suggested that dietary hesperidin could ameliorate mitochondrial injury and dysfunction in jejunum of piglets under oxidative stress.
根据图 3 a、b,敌草快治疗引起线粒体显着升高( P < 0.001),并伴有超微结构异常,例如线粒体固缩和线粒体嵴破裂,通过补充橙皮苷可以缓解这种异常( P < 0.001)。根据图 3 c-f,与对照组相比,敌草快处理引起线粒体ROS 水平升高( P < 0.01),MMP 和 ATP 含量显着降低( P < 0.05, P < 0.01),而补充橙皮苷可显着缓解ROS水平的升高( P < 0.01)以及MMP和ATP含量的降低( P < 0.01, P < 0.05)由敌草快诱导。同时,膳食橙皮苷对空肠线粒体呼吸链复合物 I-III 的活性具有统计学意义的显着升高(分别为P < 0.05、 P < 0.01 和P < 0.05),而敌草快暴露则显着降低( P < 0.01)。 、 P < 0.01 和P < 0.05 分别)。根据RT-qPCR结果,敌草快暴露下线粒体生物发生蛋白( PGC- 1α和NRF-1 )的mRNA水平显着降低( P <0.001,P<0.05),而补充橙皮苷则显着增加(P<0.001, P <0.05)。 P < 0.05, P < 0.01)(图 3 g)。 如图 3 h,i 所示,与对照相比,敌草快处理导致 Bax 显着升高( P < 0.001),而 Bcl-2 在敌草快暴露下显着降低( P < 0.01)。同时,与敌草快组相比,补充橙皮苷减轻了Bax的升高( P <0.05)和Bcl-2的降低( P <0.001)。氧化应激下Cyto Cyt-c水平升高( P <0.01),Mtio Cyt-c水平降低( P <0.001),而橙皮苷则缓解了Cyto Cyt-c的升高( P <0.05)和Mtio Cyt-c的降低(P<0.05)。 Mtio Cyt-c( P < 0.001)如图 3 h-k 所示。如图 3 l-n 所示,与敌草快组相比,补充橙皮苷减轻了空肠线粒体Ca 2+浓度的增加( P < 0.01)。此外,与敌草快组相比,敌草快挑战诱导线粒体calpain-1 水平显着增加( P < 0.01),而橙皮苷补充显着降低了线粒体calpain-1 水平( P < 0.05)。结果表明日粮橙皮苷可以改善氧化应激下仔猪空肠的线粒体损伤功能障碍

Figure 3  图3

Figure 3. Dietary hesperidin alleviated mitochondria injury and dysfunction under oxidative stress. (a, b) Mitochondria ultrastructure in the jejunum (a) and quantitative statistics (b) (scale bars represent 1 μm, red arrows indicate damaged mitochondria). (c–f) MMP (c), quantification of ROS (d), ATP content (e), and activity of mitochondria respiratory chain complex I–III (f). (g) mRNA level of mitochondrial biogenesis. (h–k) Representative bands (h, j) and the quantification (i, k). (l–n) Mitochondrial calcium content (l), Western blot bands (m), and protein quantification (n) of mitochondrial calpain-1.
图 3. 膳食橙皮苷可减轻氧化应激下的线粒体损伤功能障碍。 (a,b)空肠线粒体超微结构(a)和定量统计(b)(比例尺代表1μm,红色箭头表示受损的线粒体)。 (c-f) MMP (c)、ROS 定量 (d)、ATP 含量 (e) 和线粒体呼吸链复合物 I-III 的活性 (f)。 (g)线粒体生物发生的 mRNA 水平。 (h–k) 代表性波段 (h, j) 和量化 (i, k)。 (l–n)线粒体钙含量 (l)、蛋白质印迹条带 (m) 和线粒体calpain-1 的蛋白质定量 (n)。

3.4. Dietary Hesperidin Alleviated ERS and Downregulated Related Pathway in the Jejunum of Piglets under Oxidative Stress
3.4.日粮橙皮苷减轻氧化应激下仔猪空肠中的 ERS ​​并下调相关通路

According to Figure 4a,b, the expression level of ERS-related proteins such as caspase-12, Grp78, CHOP, and ATF6 was significantly increased under diquat exposure (P < 0.05, P < 0.01, P < 0.01, and P < 0.05, respectively), while hesperidin supplementation induced a decrease on Caspase-12, Grp78, and CHOP (P < 0.01, P < 0.05, and P < 0.05, respectively). ATF6 was decreased by hesperidin, which was not statistically significant (P > 0.05). Based on Figure 4c–e, under diquat exposure, p-PERK and p-PERK/PERK were markedly upregulated (P < 0.01, P < 0.05), which indicated that diquat treatment induced ERS pathway in piglets. The hesperidin supplementation strikingly downregulated the level of p-PERK and p-PERK/PERK (P < 0.001, P < 0.001). Moreover, the level of p-eIF2α and p-eIF2α/eIF2α was strikingly increased with diquat injection versus control (P < 0.05, P < 0.05). The dietary hesperidin resulted in a noteworthy reduction in the level of p-eIF2α and p-eIF2α/eIF2α versus diquat treatment (P < 0.01, P < 0.05).
根据图 4a 、b,敌草快暴露下 ERS ​​相关蛋白如 caspase-12、Grp78、CHOP 和 ATF6 的表达水平显着增加( P < 0.05、 P < 0.01、 P < 0.01 和P <分别为 0.05),而补充橙皮苷可导致 Caspase-12、Grp78 和 CHOP 的减少( P <分别为 0.01、 P < 0.05 和P < 0.05)。橙皮苷使ATF6降低,但差异无统计学意义( P >0.05)。根据4c-e,在敌草快暴露下,p-PERK和p-PERK/PERK显着上调( P <0.01, P <0.05),这表明敌草快处理诱导仔猪的ERS途径。补充橙皮苷显着下调 p-PERK 和 p-PERK/PERK 水平( P < 0.001, P < 0.001)。此外,与对照相比,敌草快注射后 p-eIF2α 和 p-eIF2α/eIF2α 的水平显着增加( P < 0.05, P < 0.05)。与敌草快治疗相比,膳食橙皮苷导致 p-eIF2α 和 p-eIF2α/eIF2α 水平显着降低( P < 0.01, P < 0.05)。

Figure 4  图4

Figure 4. Dietary hesperidin alleviated ERS-induced unfolded protein response in the jejunum of piglets. (a, b) Representative bands of Western blot analysis of ERS (a) and quantification of relative proteins (b). (c–e) Representative bands of Western blot analysis of PERK pathway proteins (e) and quantification of relative proteins (d, e).
图 4. 日粮橙皮苷减轻仔猪空肠中 ERS ​​诱导的未折叠蛋白反应。 (a, b) ERS ​​的蛋白质印迹分析的代表性条带 (a) 和相关蛋白的定量 (b)。 (c-e) PERK 通路蛋白的蛋白质印迹分析的代表性条带 (e) 和相关蛋白的定量 (d, e)。

3.5. Dietary Hesperidin Influenced the Formation of ERMCSs and ERMCSs-Related Proteins in the Jejunum of Piglets under Oxidative Stress
3.5.日粮橙皮苷影响氧化应激下仔猪空肠中ERMCS和ERMCS相关蛋白的形成

According to Figure 5a–c, the diquat challenge led to a substantial elevation in the percentage of mitochondria with ERMCSs/total mitochondria (P < 0.01) and the ratio of length of ERMCSs to mitochondria perimeter (P < 0.01), suggesting that the formation of ERMCSs was disturbed in the jejunum of piglets under oxidative stress. Meanwhile, with hesperidin supplementation, the formation of ERMCSs was markedly decreased compared with diquat treatment (P < 0.05). According to relative IF of mitochondrial marker and ER marker (Figure 5d,e), the diquat challenge markedly increased the Pearson’s correlation coefficient of the mitochondrial marker and ER marker (P < 0.01) of jejunum in piglets. Meanwhile, hesperidin supplementation led a noteworthy reduction (P < 0.01) in the Pearson’s correlation coefficient of the mitochondrial marker and ER marker under diquat exposure, which indicated that dietary hesperidin alleviated the interaction between mitochondria and ER. According to Figure 5f–h, the diquat injection led to a positive effect on the formation of ERMCSs by which the mRNA level of ERMCSs-relative proteins such as mfn2, VAPB, Grp75, IP3R, and PACS2 was strikingly increased (P < 0.01, P < 0.05, P < 0.001, P < 0.01, and P < 0.05, respectively). Meanwhile, the dietary hesperidin strikingly alleviated the increase in the mRNA level of these proteins in comparison with the diquat group (P < 0.05, P < 0.01, P < 0.05, P < 0.05, and P < 0.01, respectively). Moreover, there was no significant difference (P > 0.05) in the mRNA level of Mfn1, VDAC1, and PTPIP51 between four groups. As the ERMCS marker proteins, the expression levels of Mfn2 and Grp75 were markedly increased (P < 0.01 and P < 0.05) under diquat exposure, while Mfn1 and VDAC1 were not influenced among four groups (P > 0.05). Compared with the diquat group, the expression levels of Mfn2 and Grp75 were strikingly decreased (P < 0.05 and P < 0.01) with the supplementation of hesperidin. In accordance with Figure 5i,j, the expression of AMPKα between four groups was not influenced (P > 0.05). The expression of p-AMPKα and the ratio of p-AMPKα/AMPKα were significantly upregulated via oxidative stress (P < 0.05), while dietary hesperidin markedly intensified the expression of p-AMPKα and the ratio of p-AMPKα/AMPKα (P < 0.01 and P < 0.05) compared with the diquat group. Herein, the present study suggested that dietary hesperidin alleviated the disorder of ERMCSs under oxidative stress.
根据图5a -c,敌草快挑战导致带有ERMCS的线粒体/总线粒体的百分比显着升高( P <0.01)以及ERMCS的长度与线粒体周长的比率( P <0.01),这表明氧化应激下仔猪空肠中 EMCCS 的形成受到干扰。同时,与敌草快处理相比,补充橙皮苷后,ERMCSs的形成明显减少( P < 0.05)。根据线粒体标记和ER标记的相对IF(图5d ,e),敌草快挑战显着增加了仔猪空肠线粒体标记和ER标记的Pearson相关系数( P <0.01)。同时,补充橙皮苷导致敌草快暴露下线粒体标记物和ER标记物的皮尔逊相关系数显着降低( P < 0.01),这表明膳食橙皮苷减轻了线粒体和ER之间的相互作用。根据图5f -h,敌草快注射对ERMCSs的形成产生积极影响,ERMCSs相关蛋白如mfn2VAPBGrp75IP3RPACS2的mRNA水平显着增加( P <0.01) , P < 0.05, P < 0.001, P < 0.01, P < 0.05, 分别)。 同时,与敌草快组相比,膳食橙皮苷显着减轻了这些蛋白质 mRNA 水平的增加(分别为P < 0.05、 P < 0.01、 P < 0.05、 P < 0.05 和P < 0.01)。此外,四组间Mfn1VDAC1PTPIP51 mRNA水平差异均无统计学意义( P >0.05)。作为 EMCS 标志蛋白,Mfn2 和 Grp75 在敌草快暴露下表达水平显着升高( P < 0.01 和P < 0.05),而 Mfn1 和 VDAC1 在四组中未受影响( P > 0.05)。与敌草快组相比,添加橙皮苷后,Mfn2和Grp75的表达水平显着降低( P <0.05和P <0.01)。由图 5 i、j 可见,四组间 AMPKα 表达均未受影响( P > 0.05)。氧化应激显着上调 p-AMPKα 表达及 p-AMPKα/AMPKα 比值( P < 0.05),膳食橙皮苷显着增强 p-AMPKα 表达及 p-AMPKα/AMPKα 比值( P < 0.05)。 0.01 和P < 0.05)与敌草快组相比。在此,本研究表明膳食橙皮苷可减轻氧化应激下 EMCS 的紊乱

Figure 5  图5

Figure 5. Dietary hesperidin influenced the formation of ERMCSs under oxidative stress. (a–c) Ultrastructure of ERMCSs in the jejunum (a) (green arrows indicate mitochondria; red arrows indicate ER; blue arrows indicate ERMCSs), quantitative statistics of the percentage of mitochondria with ERMCSs (b), and quantitative statistics of the length of ERMCSs/mitochondrial perimeter (c). (d, e) Representative images (d) of mitochondria marker (Tomme 20) and ER marker (PDI) staining and colocalization analysis of correlational scatterplot and quantification (e). (f–h) Relative mRNA level of ERMCSs-related proteins (f), representative bands of Western blot analysis of ERMCSs marker proteins (g), and quantification (h). (i and j) Representative bands of Western blot analysis of p-AMPKα and AMPKα (i) and quantification of p-AMPKα and AMPKα and the ratio of p-AMPKα/AMPKα (j).
图 5. 膳食橙皮苷影响氧化应激下 EMCS 的形成。 (a~c)空肠中ERMCS的超微结构(a)(绿色箭头为线粒体;红色箭头为ER;蓝色箭头为ERMCS),含ERMCS的线粒体比例定量统计(b),长度定量统计EMCS/线粒体周长 (c)。 (d, e)线粒体标记 (Tomme 20) 和 ER 标记 (PDI) 染色的代表性图像 (d) 以及相关散点图和量化 (e) 的共定位分析。 (f–h) EMCSs 相关蛋白的相对 mRNA 水平 (f)、ERMCSs 标记蛋白的蛋白质印迹分析的代表性条带 (g) 和定量 (h)。 (i 和 j) p-AMPKα 和 AMPKα (i) 的蛋白质印迹分析的代表性条带以及 p-AMPKα 和 AMPKα 的定量以及 p-AMPKα/AMPKα 的比率 (j)。

4. Discussion  4. 讨论

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It is widely accepted that intestinal epithelial barrier, as the primary line of defense against a hostile environment within the intestinal lumen, is crucial for protecting the body against toxin, antigens, and pathogens. (31) As one of the most common flavonoids in citrus fruits, hesperidin is known to have antioxidant and anti-inflammatory activities (4) and has been widely used. In the present study, a well-established oxidative stress model via diquat intraperitoneal injection in piglets was used in our investigation to explore the effects of hesperidin on growth performance, intestinal barrier function, and antioxidant capacity in piglets under oxidative stress. Based on the result, the dietary hesperidin alleviated oxidative stress-induced decrease in growth performance. At the same time, we also found that with hesperidin supplementation, the jejunal villus height and the ratio of villus height to crypt depth were markedly increased in piglets versus diquat group, which implied that hesperidin alleviated intestinal barrier injury. According to Western blot analysis, the level of tight junction proteins was increased by hesperidin compared with the diquat group. Similarly, Chen et al. found hesperetin (an aglycone of hesperidin) increased the expression of tight junction proteins. (32) Furthermore, hesperidin supplementation increased the activity of CAT and SOD, decreased the level of MDA, and upregulated the mRNA level of related antioxidant enzymes, which indicated that the decrease in antioxidant capacity induced by oxidative stress was attenuated with hesperidin in the jejunal of piglets. Similarly, Chen et al. found that hesperidin enhanced the activity of SOD and GPX and glutathione reductase, which may indicate the improvement in the total antioxidant function of H2O2-injuried chondrocytes. (33) Herein, the present study suggested that hesperidin alleviated the intestinal barrier injury and recovered the antioxidant capacity in the piglets’ jejunum, which subsequently increased the growth performance of piglets under oxidative stress. In addition, hesperidin alleviated the increase in serum ALT and AST under oxidative stress, which indicated the protective effects of hesperidin on hepatic injury in piglets. (34)
人们普遍认为,上皮屏障是抵御肠腔内恶劣环境的主要防线,对于保护身体免受毒素、抗原和病原体的侵害至关重要。 (31)作为柑橘类水果中最常见的黄酮类化合物之一,橙皮苷具有抗氧化和抗炎活性(4) ,并已被广泛应用。本研究采用成熟的仔猪腹腔注射敌草快氧化应激模型,探讨橙皮苷氧化应激条件下仔猪生长性能、肠道屏障功能和抗氧化能力的影响。根据结果​​,膳食橙皮苷减轻了氧化应激引起的生长性能下降。同时,我们还发现,与敌草快组相比,补充橙皮苷后,仔猪的空肠绒毛高度以及绒毛高度与隐窝深度之比显着增加,这表明橙皮苷减轻了肠道屏障损伤。根据蛋白质印迹分析,与敌草快组相比,橙皮苷增加了紧密连接蛋白的水平。同样,陈等人。发现橙皮素(橙皮苷的一种苷元)增加了紧密连接蛋白的表达。 (32)此外,补充橙皮苷可增加 CAT 和 SOD 的活性,降低 MDA 水平,并上调相关抗氧化酶的 mRNA 水平,这表明空肠中橙皮苷可减轻氧化应激引起的抗氧化能力下降。仔猪。同样,陈等人。发现橙皮苷增强SOD和GPX以及谷胱甘肽还原酶的活性,这可能表明H 2 O 2 -损伤的软骨细胞的总抗氧化功能得到改善。 (33)在此,本研究表明橙皮苷减轻了仔猪肠道屏障损伤并恢复了仔猪空肠的抗氧化能力,从而提高了仔猪在氧化应激下的生长性能。此外,橙皮苷还能缓解氧化应激下血清ALT和AST的升高,说明橙皮苷对仔猪肝损伤具有保护作用。(34)
Studies have shown that mitochondria are the primary site of energy production, which play an indispensable role in intestinal barrier function. (11) Under situations of stress, a large body of endogenous ROS was produced via mitochondrial oxidative respiratory chain, which subsequently induced mitochondria injury and decreased ATP production. (35) However, almost no investigation has reported the effect of hesperidin on the mitochondrial dysfunction under oxidative stress in piglets currently. Therefore, we proceeded to assess the effects of hesperidin on the mitochondrial function of jejunum in piglets under oxidative stress. According to the result of TEM, we found the ultrastructure of mitochondria with a complete bilayer membrane and a dense matrix in the hesperidin group, which was essential to mitochondrial function, (36) while the ultrastructure of mitochondria was disturbed in the diquat group. As one of the most important indicators of mitochondrial function, MMP was the important part of the energy-storage process. ROS was a byproduct of mitochondrial oxidative phosphorylation to produce ATP, while excess ROS was also one of the factors leading to mitochondrial dysfunction. ATP production was largely determined by the activity of the mitochondrial respiratory chain complex, which is a significant indicator of mitochondrial function. In our experiment, the hesperidin supplementation ameliorated mitochondrial dysfunction via increasing MMP and ATP contents, decreasing ROS production, and upregulated activity of respiratory chain complex I–III under oxidative stress. Tsai et al. found that hesperidin supplements restored mitochondrial activity exposed to H2O2 in human articular chondrocytes, which supported our study. (37) Similarly, Wang et al. found that hesperidin markedly improved the activity of mitochondrial respiratory chain complex I–IV in APPswe/PS 1dE9 mice, which supported our findings. (38) As can be seen in Figure 3g, the mRNA level of PGC-1α and NRF-1 was increased with dietary hesperidin, which indicated that hesperidin restored mitochondrial biogenesis via activating the PGC-1α pathway. According to the result of Western blotting of mitochondrial apoptosis-related proteins, hesperidin downregulated the level of Bax and Cyto Cyt-C and upregulated the Bcl-2 and Mito Cyt-C, which suggested a potential mechanism by which hesperidin protected mitochondria and inhibited apoptosis. (39) One previous study has suggested that calcium overload in mitochondria could lead to the opening of the mitochondrial permeability transition pore and, subsequently, lead to mitochondrial dysfunction and cell death. (40) In the present study, the result showed that with hesperidin supplementation, the mitochondrial calcium content and the level of calpain-1 were decreased versus the diquat group, suggesting that hesperidin may alleviate calcium overload in mitochondria.
研究表明,线粒体是能量产生的主要场所,在肠道屏障功能中发挥着不可或缺的作用。 (11)应激情况下,线粒体氧化呼吸链产生大量内源性ROS,导致线粒体损伤,ATP产生减少。 (35)然而,目前几乎没有研究报道橙皮苷氧化应激下仔猪线粒体功能障碍的影响。因此,我们着手评估橙皮苷氧化应激下仔猪空肠线粒体功能的影响。根据TEM结果,我们发现橙皮苷组的线粒体超微结构具有完整的双层膜和致密的基质,这对线粒体功能至关重要(36) ,而敌草快组的线粒体超微结构受到干扰。 MMP作为线粒体功能最重要的指标之一,是能量储存过程的重要组成部分。 ROS是线粒体氧化磷酸化产生ATP的副产物,过量的ROS也是导致线粒体功能障碍的因素之一。 ATP 的产生很大程度上取决于线粒体呼吸链复合体的活性,这是线粒体功能的重要指标。在我们的实验中,橙皮苷补充剂通过增加 MMP 和 ATP 含量、减少 ROS 产生以及上调氧化应激下呼吸链复合物 I-III 的活性来改善线粒体功能障碍。蔡等人。发现橙皮苷补充剂恢复了人类关节软骨细胞中暴露于 H 2 O 2 的线粒体活性,这支持了我们的研究。 (37)同样,Wang 等人。发现橙皮苷显着提高了 APPswe/PS 1dE9 小鼠线粒体呼吸链复合物 I-IV 的活性,这支持了我们的发现。 (38)如图3g所示,膳食橙皮苷使PGC-1α和NRF-1的mRNA水平增加,这表明橙皮苷通过激活PGC-1α途径恢复线粒体生物发生。线粒体凋亡相关蛋白Western blotting结果显示,橙皮苷下调Bax和Cyto Cyt-C水平,上调Bcl-2和Mito Cyt-C水平,提示橙皮苷保护线粒体、抑制细胞凋亡的潜在机制。 。 (39)先前的一项研究表明,线粒体中的钙超载可能导致线粒体通透性转换孔打开,随后导致线粒体功能障碍和细胞死亡。 (40)在本研究中,结果表明,补充橙皮苷后,与敌草快组相比,线粒体钙含量和 calpain-1 水平降低,表明橙皮苷可以缓解线粒体钙超载。
As the largest membranous organelle in eukaryotes, ER plays essential roles in the synthesis and metabolism of carbohydrates, lipids, glycogen, and synthesis of protein. (10) Under oxidative stress, the homeostasis of ER was disturbed, and the dysfunction of ER could activate the unfold protein reaction (UPR). (41) ER-resident molecular chaperons GRP78 could bind to the misfolded proteins to be dissociated from PERK, ATF6, and IRE1 to further activate downstream ERS signaling and restore the ER to homeostasis. (41) In the present study, the data demonstrated that the level of ERS-related proteins (Caspase-12, Grp78, CHOP, and ATF6) was upregulated under oxidative stress, which was alleviated by dietary hesperidin. It is well-known that the PERK–eIF2α–CHOP–caspase-12 pathway is closely associated with UPR and ERS. (42) The result showed that with hesperidin supplementation, the level of phosphorylation of PERK and eIF-2α was downregulated, which reflected that hesperidin ameliorated ERS. Take together, these results showed that hesperidin may improve the folding capacity of proteins and alleviate ERS-induced apoptosis via the PERK–eIF2α–CHOP–caspase-12 pathway, which eventually restored ER to homeostasis.
ER作为真核生物中最大的膜细胞器,在碳水化合物、脂质、糖原的合成和代谢以及蛋白质的合成中发挥着重要作用。 (10)氧化应激下,ER稳态被扰乱,ER功能失调可激活未折叠蛋白反应(UPR)。 (41) ER 驻留分子伴侣 GRP78 可以与错误折叠的蛋白质结合,从 PERK、ATF6 和 IRE1 解离,进一步激活下游 ERS ​​信号传导并使 ER 恢复稳态。 (41)在本研究中,数据表明,ERS 相关蛋白(Caspase-12、Grp78、CHOP 和 ATF6)的水平在氧化应激下上调,而膳食橙皮苷可缓解这种情况。众所周知,PERK-eIF2α-CHOP-caspase-12 通路与 UPR 和 ERS ​​密切相关。 (42)结果表明,补充橙皮苷后,PERK 和 eIF-2α 的磷酸化水平下调,这反映出橙皮苷改善了 ERS。综上所述,这些结果表明橙皮苷可以通过 PERK-eIF2α-CHOP-caspase-12 途径提高蛋白质的折叠能力并减轻 ERS ​​诱导的细胞凋亡,最终使 ER 恢复稳态。
Recently, as a physical contact composed with a series of proteins on the outer mitochondrial membrane and the ER membrane, the ERMCSs have garnered growing attention from researchers in life sciences and have been demonstrated as a signaling hub controlling cell physiology. (13,14,16) Since the discovery of ERMCSs by J.E. Vance, scholars defined ERMCSs as dynamic lipid rafts spanning 10–30 nm between the mitochondrial membrane and the ER membrane and studied the function of proteins in ERMCSs, such as Mfn1, Mfn2, VDAC1, Grp75, IP3R, VAPB, PTPTIP51, and PACS2. (15,43) However, the effect of hesperidin on the formation and function of ERMCSs in piglets is rarely reported. Therefore, we employed a wide-used oxidative stress model in piglets to investigate the effects of hesperidin on the formation of ERMCSs in jejunum. With the TEM experiment, the morphological structure of ERMCSs was observed, and the result indicated that hesperidin strikingly inhibited the formation of ERMCSs compared with the diquat group. Moreover, in order to observe the interaction between mitochondria and ER, Tomm20 and PDI were used as markers for mitochondria and ER as they are well characterized mitochondria and ER, respectively. (44) Under fluorescent microscopy, the result suggested that the degree of coupling between ER and mitochondria was reduced with hesperidin supplementation through Pearson’s coefficient. On the basis of previous research, activation of AMPKα is beneficial for cellular homeostasis and senescence prevention, which is closely related to the oxidative stress. (45) In our experiment, oxidative stress upregulated the ratio of p-AMPKα/AMPKα, which was consistent with the previous reports. (46) Hesperidin strikingly augmented AMPK phosphorylation after diquat treatment, which was similar to the study by Li et al. (47) Therefore, the present study suggested that AMPK might participate in hesperidin in alleviating ERMCS disorder and mitochondrial dysfunction through interaction with mfn2, and further study is needed. As the basis for the formation of ERMCSs, a serial of tethering proteins in ERMCSs play essential roles in forming this functional platform, such as Mfn1 (Mfn2)-Mfn2, VDAC1-Grp75 -IP3R, and VAPB-PTPTIP51. Thus, we used RT-qPCR to detect the mRNA level of relative proteins. We found that with hesperidin supplementation, the formation of ERMCSs was decreased by downregulating the level of Mfn2, VDAC1, Grp75, IP3R, VAPB, and PACS2. Among these tethering proteins, Mfn2, Grp75, and VDAC1 are widely accepted as the ERMCS markers in a previous study: Mfn2 mainly maintains the formation of ERMCSs between ER and mitochondria; VDAC1 has been proved to be a vital target for mediating a variety of ions and metabolites into and out of mitochondria; Grp75 is cytosolic chaperone that promotes ERMCS formation through the IP3R-GRP75-VDAC1 complex. (48) According to the result of Western blot, dietary hesperidin attenuated the increase on the expression level of Mfn2 and Grp75 compared with diquat group, which indicated that hesperidin alleviated the disorder of ERMCSs under oxidative stress.
近年来,ERMCS作为线粒体外膜和内质网膜上一系列蛋白质组成的物理接触点,越来越受到生命科学研究人员的关注,并被证明是控制细胞生理学的信号中枢。 (13,14,16)自JE Vance发现ERMCS以来,学者们将ERMCS定义为线粒体膜和内质网膜之间跨度10~30 nm的动态脂筏,并研究了ERMCS中蛋白质的功能,如Mfn1、Mfn2 、VDAC1、Grp75、IP3R、VAPB、PTPTIP51 和 PACS2。 (15,43)然而,橙皮苷对仔猪 EMCS 形成和功能的影响却鲜有报道。因此,我们采用了广泛使用的仔猪氧化应激模型来研究橙皮苷对空肠中ERMCS形成的影响。通过TEM实验观察ERMCSs的形态结构,结果表明,与敌草快组相比,橙皮苷显着抑制ERMCSs的形成。此外,为了观察线粒体和 ER 之间的相互作用,Tomm20 和 PDI 被用作线粒体和 ER 的标记,因为它们分别很好地表征了线粒体和 ER。 (44)在荧光显微镜下,结果表明通过皮尔逊系数补充橙皮苷可以降低内质网和线粒体之间的耦合程度。 根据前期研究,AMPKα的激活有利于细胞稳态和预防衰老,与氧化应激密切相关。 (45)在我们的实验中,氧化应激上调了p-AMPKα/AMPKα的比率,这与之前的报道一致。 (46)敌草快处理后,橙皮苷显着增强 AMPK 磷酸化,这与 Li 等人的研究相似。 (47)因此,本研究表明AMPK可能通过与mfn2相互作用参与橙皮苷缓解ERMCS紊乱线粒体功能障碍,还需要进一步研究。作为ERMCS形成的基础,ERMCS中的一系列束缚蛋白在该功能平台的形成中发挥着重要作用,例如Mfn1(Mfn2)-Mfn2、VDAC1-Grp75-IP3R和VAPB-PTPTIP51。因此,我们使用RT-qPCR来检测相关蛋白的mRNA水平。我们发现,补充橙皮苷后,ERMCS 的形成通过下调Mfn2、VDAC1、Grp75、IP3R、VAPBPACS2的水平而减少。在这些束缚蛋白中,Mfn2、Grp75和VDAC1在之前的研究中被广泛认为是ERMCS标记:Mfn2主要维持ER和线粒体之间ERMCS的形成; VDAC1已被证明是介导多种离子和代谢物进出线粒体的重要靶标; Grp75 是胞质伴侣,通过 IP3R-GRP75-VDAC1 复合物促进 EMCS 形成。 (48) Western blot结果显示,与敌草快组相比,膳食橙皮苷减弱了Mfn2和Grp75表达水平的增加,这表明橙皮苷减轻了氧化应激下ERMCS的紊乱
Actually, these tethering proteins not only serve as the basis for the formation of ERMCSs, but many functions of ERMCSs such as ER-mitochondria calcium transfer also depend on them. To our knowledge, the VDAC1-Grp75-IP3R complex is closely related to the Ca2+ transport between mitochondria and ER, which may be responsible for mitochondria calcium overload and dysfunction. (49) Li et al. have demonstrated that deoxynivalenol induced the increased interaction strength of IP3R and VDAC1 and mitochondrial calcium overload in the jejunum of piglets, which subsequently induced mitochondrial dysfunction and intestinal barrier injury. (50) In accordance with the study we have done, the decrease in the mitochondria calcium by hesperidin may be related to the negative effect of hesperidin on the formation of ERMCSs and the calcium channel between ER and mitochondria. Therefore, based on the results obtained, we hypothesized that hesperidin alleviated mitochondrial calcium overload and dysfunction via downregulating the calcium channel such as VDAC1-Grp75-IP3R complexes and inhibiting the Ca2+ transport from the ER to the mitochondria. To further understand the potential mechanisms of the effect of hesperidin on ERMCS disorder in the jejunum of piglets under oxidative stress, more work is required in the follow-up study. For example, flow cytometry is used to detect the level of mitochondria Ca2+, and immunofluorescence and immunoprecipitation are used to determine the colocalization and interaction of the calcium channels such as VDAC1-Grp75-IP3R complex, by which we could further investigate the role of ERMCSs and calcium channels in the alleviation of mitochondrial calcium overload and dysfunction by hesperidin under oxidative stress.
实际上,这些束缚蛋白不仅是ERMCS形成的基础,ERMCS的许多功能如ER-线粒体钙转移也依赖于它们。据我们所知,VDAC1-Grp75-IP3R复合物与线粒体和内质网之间的Ca 2+转运密切相关,这可能是线粒体钙超载和功能障碍的原因。 (49)李等人。已经证明,脱氧雪腐镰刀菌烯醇诱导仔猪空肠中 IP3R 和 VDAC1 相互作用强度的增加和线粒体钙超载,从而导致线粒体功能障碍屏障损伤(50)根据我们所做的研究,橙皮苷减少线粒体钙可能与橙皮苷对ERMCSs的形成以及ER和线粒体之间的钙通道的负面影响有关。因此,基于所获得的结果,我们假设橙皮苷通过下调VDAC1-Grp75-IP3R复合物等钙通道并抑制Ca 2+从内质网到线粒体的转运来减轻线粒体钙超载和功能障碍。 为了进一步了解橙皮苷氧化应激下仔猪空肠ERMCS紊乱影响的潜在机制,还需要开展更多的后续研究。例如,利用流式细胞术检测线粒体Ca 2+水平,利用免疫荧光和免疫沉淀测定VDAC1-Grp75-IP3R复合物等钙通道的共定位和相互作用,从而进一步研究其作用。橙皮苷氧化应激下缓解线粒体钙超载和功能障碍中的ERMCS和钙通道的作用。
Collectively, the current study demonstrated that hesperidin alleviated intestinal barrier injury, preserved antioxidative capacity in piglets’ intestine under oxidative stress. Importantly, our work indicated that hesperidin could alleviate the disorder of ERMCSs and attenuated mitochondrial dysfunction and ERS, which in turn decreases ROS production and prevents intestinal barrier injury under oxidative stress. The results supported possible applications of nutrition therapy such as hesperidin targeting the restoration of the formation and function of ERMCSs, which may be a useful approach for addressing intestinal barrier injury and mitochondrial dysfunction in piglets under oxidative stress.
总的来说,目前的研究表明,橙皮苷可以减轻肠道屏障损伤,在氧化应激下保留仔猪肠道的抗氧化能力。重要的是,我们的工作表明,橙皮苷可以缓解 EMCS紊乱,减轻线粒体功能障碍和 ERS,从而减少 ROS 产生并防止氧化应激下的肠道屏障损伤。这些结果支持了营养疗法的可能应用,例如橙皮苷,其目标是恢复 EMCS 的形成和功能,这可能是解决氧化应激下仔猪肠道屏障损伤线粒体功能障碍的有效方法。

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.4c02265 IF: 5.7 Q1 .
支持信息可免费获取: https://pubs.acs.org/doi/10.1021/acs.jafc.4c02265 如果:5.7 Q1

  • Ingredients and nutrient composition of the control diet, information on primers used for RT-qPCR, and the primary antibody used for Western blot experiment (PDF)
    对照饮食的成分和营养成分、RT-qPCR 所用引物信息以及 Western blot 实验所用一抗 ( PDF )

Hesperidin Alleviated Intestinal Barrier Injury, Mitochondrial Dysfunction, and Disorder of Endoplasmic Reticulum Mitochondria Contact Sites under Oxidative Stress

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Supporting Information  支持信息
Hesperidin alleviated intestinal barrier injury, mitochondrial dysfunction and
橙皮苷可减轻肠道屏障损伤、线粒体功能障碍和
disorder of endoplasmic reticulum mitochondria contact sites under oxidative
氧化作用下内质网线粒体接触部位紊乱
stress  压力
1
Feiyang Gou  飞扬沟
1
, Qian Lin  , 钱琳
1
, Xiaodian Tu  , 涂晓殿
1
, Jiang Zhu  , 江珠
1
, Xin Li  , 李欣
1
, Shaokui Chen  , 陈少奎
1, 2
*,
2
Caihong Hu  胡彩虹
1
*
3
4

3
4
1
Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of
动物分子营养部重点实验室(浙江大学)
5
Education; College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
教育;浙江大学动物科学学院, 杭州 310058
6
2
School of Animal Science and Nutritional Engineering, Wuhan Polytechnic University,
武汉工业大学动物科学与营养工程学院
7
Wuhan 430023, China  中国 武汉 430023
8
9

8
9
*Corresponding author:  *通讯作者:
10
Shaokui Chen; E-mail: loveskchen@163.com
陈少奎;邮箱:loveskchen@163.com
11
Caihong Hu; E-mail: chhu@zju.edu.cn
胡彩虹;邮箱:chhu@zju.edu.cn
13
Table S1. Composition and nutrition levels of the daily diet
Ingredients
g/kg
Analyzed composition
g/kg
Corn
567
Net energy
2
, Kcal/kg
2413.12
Soybean meal
223
crude protein
194.92
Wheat middling
128
lysine
12.7
Soybean oil
14
methionine
4
Fish meal
31
calcium
8.2
Dicalcium phosphate
9
total phosphorus
7.1
Limestone
10
Sodium chloride
4.4
L-lysine HCl
2.9
DL-methionine
0.7
vitamin−mineral
premix
1
10
14
1
Supplied per kilogram of diet: vitamin A, 17,500 IU; vitamin D
3
, 5000 IU; vitamin E,
15
50 IU; vitamin K
3
, 5.0 mg; riboflavin, 12.5 mg; thiamine, 5.0 mg; pyridoxine, 5.0 mg;
16
pantothenic acid, 25 mg; folic acid, 2.5 mg; biotin, 0.2 mg; vitamin B
12
, 0.05 mg; Zn,
17
80 mg; Cu, 6 mg; I, 0.14 mg; Fe, 100 mg; Mn, 4 mg; Se, 0.3 mg.
18
2
Net energy (NE) was calculated from data provide by Feed Database in China (2022).

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Author Information  作者信息

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  • Corresponding Authors  通讯作者
    • Shaokui Chen - Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, ChinaSchool of Animal Science and Nutritional Engineering, Wuhan Polytechnic University, Wuhan 430023, China Email: loveskchen@163.com
      陈少奎-浙江大学动物科学学院, 分子动物营养教育部重点实验室(浙江大学), 杭州 310058 ; 武汉工业大学动物科学与营养工程学院, 武汉 430023 ; 邮箱: loveskchen@163.com
    • Caihong Hu - Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, ChinaOrcidhttps://orcid.org/0000-0001-5445-4532 Email: chhu@zju.edu.cn
      胡彩虹-浙江大学动物科学学院, 分子动物营养教育部重点实验室(浙江大学), 杭州 310058 ; Orcid https://orcid.org/0000-0001-5445-4532邮箱: chhu@zju.edu.cn
  • Authors  作者
    • Feiyang Gou - Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
      苟飞扬-浙江大学动物科学学院, 分子动物营养教育部重点实验室(浙江大学), 杭州 310058
    • Qian Lin - Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
      钱琳-浙江大学动物营养教育部重点实验室, 浙江大学动物科学学院, 杭州 310058
    • Xiaodian Tu - Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
      涂小典-浙江大学动物科学学院, 分子动物营养教育部重点实验室(浙江大学), 杭州 310058
    • Jiang Zhu - Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
      朱江-浙江大学动物科学学院, 分子动物营养教育部重点实验室(浙江大学), 杭州 310058
    • Xin Li - Key Laboratory of Molecular Animal Nutrition (Zhejiang University), Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
      李欣-浙江大学动物科学学院, 分子动物营养教育部重点实验室(浙江大学), 杭州 310058
  • Notes  笔记
    The authors declare no competing financial interest.
    作者声明不存在竞争性经济利益。

Acknowledgments  致谢

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This research was supported by the National Natural Science Foundation of China (32172740 and 32372888), the Zhejiang Provincial Natural Science Foundation of China under grant no. LZ24C170001, and the Key R&D Program of Zhejiang Province (2024C02004).
该研究得到国家自然科学基金(32172740和32372888)、浙江省自然科学基金(批准号:32172740和32372888)的支持。 LZ24C170001,浙江省重点研发计划(2024C02004)。

References  参考

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本文参考 50 种其他出版物。

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Journal of Agricultural and Food Chemistry
农业与食品化学杂志

Cite this: J. Agric. Food Chem. 2024, 72, 29, 16276–16286
引用此:J. Agric。食品化学.2024, 72, 29, 16276–16286
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https://doi.org/10.1021/acs.jafc.4c02265 IF: 5.7 Q1
https://doi.org/10.1021/acs.jafc.4c02265IF:5.7 第一季度
Published July 9, 2024
发布于 2024 年 7 月 9 日
Copyright © 2024 American Chemical Society
版权所有 © 2024 美国化学会

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  • Abstract  抽象的

    Figure 1  图1

    Figure 1. Dietary hesperidin alleviated oxidative stress-induced growth restriction and intestinal barrier injury. (a–c) Initial and final body weight (IBW, FBW) (a), ADG and ADFI (b), and ratio of feed to gain (F/G) (c) of piglets. (d, e) TER (d) and FD4 flux (e) of jejunum in piglets. (f–h) Images of piglets’ jejunum morphology (scale bars = 100 μm) (f), Quantification of the villus height and crypt depth (g) and the ratio of villus height to crypt depth (h) in the jejunum of piglets. (i, j) Tight junction protein (ZO-1, Occludin, Claudin-1) expression (i) by Western blot and the quantification of expression level (j).
    图 1. 膳食橙皮苷可减轻氧化应激引起的生长受限和肠道屏障损伤。 (a-c) 仔猪的初始体重和最终体重(IBW、FBW)(a)、ADG 和 ADFI(b)以及饲料增重比(F/G)(c)。 (d, e) 仔猪空肠的 TER (d) 和 FD4 通量 (e)。 (f–h) 仔猪空肠形态图像(比例尺 = 100 μm) (f),仔猪空肠绒毛高度和隐窝深度 (g) 的量化以及绒毛高度与隐窝深度 (h) 的比率。 (i, j) 紧密连接蛋白 (ZO-1、Occludin、Claudin-1) 表达 (i) 通过蛋白质印迹和表达水平定量 (j)。

    Figure 2

    Figure 2. Dietary hesperidin alleviated oxidative stress-induced decrease of activity of serum ALT, AST, and antioxidant capacity. (a, b) Activity of serum ALT (a) and AST (b) in piglets. (c–e) Activity of CAT (c), SOD (d), and MDA level (e) in piglets. (f) Heat map of mRNA level of antioxidant enzyme (SOD1, GPX-1, and GPX-4).

    Figure 3

    Figure 3. Dietary hesperidin alleviated mitochondria injury and dysfunction under oxidative stress. (a, b) Mitochondria ultrastructure in the jejunum (a) and quantitative statistics (b) (scale bars represent 1 μm, red arrows indicate damaged mitochondria). (c–f) MMP (c), quantification of ROS (d), ATP content (e), and activity of mitochondria respiratory chain complex I–III (f). (g) mRNA level of mitochondrial biogenesis. (h–k) Representative bands (h, j) and the quantification (i, k). (l–n) Mitochondrial calcium content (l), Western blot bands (m), and protein quantification (n) of mitochondrial calpain-1.

    Figure 4

    Figure 4. Dietary hesperidin alleviated ERS-induced unfolded protein response in the jejunum of piglets. (a, b) Representative bands of Western blot analysis of ERS (a) and quantification of relative proteins (b). (c–e) Representative bands of Western blot analysis of PERK pathway proteins (e) and quantification of relative proteins (d, e).

    Figure 5

    Figure 5. Dietary hesperidin influenced the formation of ERMCSs under oxidative stress. (a–c) Ultrastructure of ERMCSs in the jejunum (a) (green arrows indicate mitochondria; red arrows indicate ER; blue arrows indicate ERMCSs), quantitative statistics of the percentage of mitochondria with ERMCSs (b), and quantitative statistics of the length of ERMCSs/mitochondrial perimeter (c). (d, e) Representative images (d) of mitochondria marker (Tomme 20) and ER marker (PDI) staining and colocalization analysis of correlational scatterplot and quantification (e). (f–h) Relative mRNA level of ERMCSs-related proteins (f), representative bands of Western blot analysis of ERMCSs marker proteins (g), and quantification (h). (i and j) Representative bands of Western blot analysis of p-AMPKα and AMPKα (i) and quantification of p-AMPKα and AMPKα and the ratio of p-AMPKα/AMPKα (j).

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  • Supporting Information

    Supporting Information


    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.4c02265 IF: 5.7 Q1 .

    • Ingredients and nutrient composition of the control diet, information on primers used for RT-qPCR, and the primary antibody used for Western blot experiment (PDF)


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