壳寡糖对仔猪脑海马氧化应激的抑制作用及机制研究

doi:10.11843/j.issn.0366-6964.2023.02.031

摘要/Abstract

摘要： 氧化应激与动物的生长发育迟缓、疾病的发生和发展密切相关。壳寡糖（chito-oligosaccharide，COSs）具有抗氧化应激的作用效果，但其对仔猪脑海马氧化应激的抑制作用与分子机制尚不清楚。本研究对仔猪进行肌注葡聚糖酐铁（FeDex）以构建氧化应激模型，并在此基础上考察COSs对仔猪平均日增重、血清和部分组织氧化和抗氧化相关指标、细胞凋亡相关蛋白基因及神经肽可卡因和苯丙胺调节转录物（cocaine-and amphetamine-regulated transcript，CART）基因表达水平的影响，且进一步将CART基因的表达水平与凋亡相关蛋白基因的表达水平进行相关性分析。结果表明，与对照组相比，氧化应激仔猪的日增重、血清及肝、肺、脑海马和大脑皮质谷胱甘肽（glutathione，GSH）和总抗氧化能力（total antioxidant capacity，T-AOC）的水平均显著降低（P<0.01或P<0.05），而过氧化氢（hydrogen peroxide，H₂O₂）和丙二醛（malondialdehyde，MDA）含量显著增加（P< 0.01或P< 0.05）。根据体重灌胃50 mg·kg^-1剂量的COSs可显著增加氧化应激仔猪的日增重、血清及肝、肺、脑海马和大脑皮质GSH和T-AOC的水平（P<0.01或P<0.05），而显著降低H₂O₂和MDA的含量（P<0.01或P<0.05）。与对照组相比，氧化应激仔猪脑海马Cleaved Caspase-3和Bax基因的表达水平显著上调（P<0.05），而Bcl-2基因的表达水平显著下调（P<0.05）。COSs显著降低了氧化应激仔猪脑海马Bax和Cleaved Caspase-3基因的表达水平（P<0.05），而显著增加了Bcl-2的表达水平（P<0.05）。进一步研究发现，与对照组相比，氧化应激仔猪脑海马神经肽CART基因的表达水平显著降低（P<0.01），而COSs添加可显著增加CART基因的表达水平（P<0.01）。相关性分析结果显示，CART与凋亡相关蛋白基因的表达水平显著相关。COSs可通过下调凋亡信号通路抑制仔猪脑海马的氧化应激，神经肽CART可能介导了该过程。

关键词: 壳寡糖, 仔猪, 海马, 氧化应激, 细胞凋亡, 神经肽-CART

Abstract: Oxidative stress is closely related to the growth retardation and the occurrence and development of diseases in animals. Chito-oligosaccharide (COSs) has the effect of anti-oxidative stress, but the effect and molecular mechanism of COSs in inhibiting oxidative stress in hippocampus of brain in piglets are not clear. In this study, the oxidative stress model of piglets was established by intramuscular injection of dextran iron (FeDex), and on this basis, the effects of COSs on the average daily gain, oxidation and anti-oxidation related indexes of serum and some tissue, expression levels of apoptosis related protein gene and neuropeptide cocaine and amphetamine regulated transcript (CART) genes of hippocampus in piglets were investigated. Furthermore, the correlation between the expression levels of CART gene and apoptosis-related protein gene was analyzed. The results showed that compared with the control group, the daily gain, the levels of glutathione (GSH) and total anti-oxidant capacity (T-AOC) in serum, liver, lung, hippocampus and cerebral cortex of piglets with oxidative stress were significantly decreased (P<0.01 or P<0.05), while the contents of hydrogenperoxide (H₂O₂) and malondialdehyde (MDA) were significantly increased (P<0.01 or P<0.05). Intragastrical administration of 50 mg·kg^-1 COSs significantly increased the daily gain, the levels of GSH and T-AOC in serum, liver, lung, hippocampus and cerebral cortex (P<0.01 or P<0.05), while decreased the contents of H₂O₂ and MDA in serum and liver, lung, hippocampus and cerebral cortex of piglets with oxidative stress (P<0.01 or P<0.05). Compared with the control group, the expression levels of Cleaved Caspase-3 and Bax genes were significantly up-regulated in hippocampus of piglets with oxidative stress (P<0.05), while the expression level of Bcl-2 gene was significantly down-regulated (P<0.05). COSs significantly decreased the expression levels of Bax and Cleaved Caspase-3 genes in the hippocampus of piglets with oxidative stress (P<0.05), while significantly increased the expression level of Bcl-2 (P<0.05). Further study showed that compared with the control group, the expression levels of CART gene were significantly decreased in piglets with oxidative stress (P<0.01), while COSs significantly increased the expression levels of CART gene (P<0.01). The results of correlation analysis showed that CART was significantly correlated with the expression level of apoptosis-related protein genes. COSs can inhibit oxidative stress of hippocampus of piglets by weakening apoptosis signal pathway, which may be mediated by neuropeptide CART.

Key words: chitooligosaccharides, piglets, oxidative stress, apoptosis, neuropeptide-CART

中图分类号:

S857.3

杨成迎, 汪锴, 黄子晴, 林海烂, 王乃秀, 李雨航, 刘炎青, 刘煜萱, 朱燕, 何道领, 陈红跃, 甘玲. 壳寡糖对仔猪脑海马氧化应激的抑制作用及机制研究[J]. 畜牧兽医学报, 2023, 54(2): 744-756.

YANG Chengying, WANG Kai, HUANG Ziqing, LIN Hailan, WANG Naixiu, LI Yuhang, LIU Yanqing, LIU Yuxuan, ZHU Yan, HE Daoling, CHEN Hongyue, GAN Ling. Study on the Inhibitory Effect of Chito-oligosaccharide on Oxidative Stress inHippocampus of Piglets and Its Mechanism[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(2): 744-756.

参考文献 50

[1]	REUTER S, GUPTA S C, CHATURVEDI M M, et al. Oxidative stress, inflammation, and cancer:how are they linked?[J]. Free Radic Biol Med, 2010, 49(11):1603-1616.
[2]	CROSS C E, HALLIWELL B, BORISH E T, et al. Oxygen radicals and human disease[J]. Ann Int Med, 1987, 107(4):526-545.
[3]	邢帅兵. 高温环境对断奶仔猪生长性能、肠道健康和抗氧化状态的影响及上限温度临界值的探究[D]. 雅安:四川农业大学, 2020.XING S B. Effects of high temperature on growth performance, intestinal health and antioxidant status in weaned piglets and exploration of the upper critical temperature[D]. Ya'an:Sichuan Agricultural University, 2020. (in Chinese)
[4]	PLUSKE J R, WILLIAMS I H. Reducing stress in piglets as a means of increasing production after weaning:administration of amperozide or co-mingling of piglets during lactation?[J]. Anim Sci, 1996, 62(1):121-130.
[5]	BEN-SHAUL V, LOMNITSKI L, NYSKA A, et al. The effect of natural antioxidants, NAO and apocynin, on oxidative stress in the rat heart following LPS challenge[J]. Toxicol Lett, 2001, 123(1):1-10.
[6]	DIPLOCK A T, CHARULEUX J L, CROZIER-WILLI G, et al. Functional food science and defence against reactive oxidative species[J]. Br J Nutr, 1998, 80(S1):S77-S112.
[7]	MENDIS E, KIM M M, RAJAPAKSE N, et al. An in vitro cellular analysis of the radical scavenging efficacy of chitooligosaccharides[J]. Life Sci, 2007, 80(23):2118-2127.
[8]	LI X J, PIAO X S, KIM S W, et al. Effects of chito-oligosaccharide supplementation on performance, nutrient digestibility, and serum composition in broiler chickens[J]. Poult Sci, 2007, 86(6):1107-1114.
[9]	LUO Z G, DONG X X, KE Q, et al. Chitooligosaccharides inhibit ethanol-induced oxidative stress via activation of Nrf2 and reduction of MAPK phosphorylation[J]. Oncol Rep, 2014, 32(5):2215-2222.
[10]	XU Y Y, ZHANG Q, YU S, et al. The protective effects of chitooligosaccharides against glucose deprivation-induced cell apoptosis in cultured cortical neurons through activation of PI3K/Akt and MEK/ERK1/2 pathways[J]. Brain Res, 2011, 1375:49-58.
[11]	ZHOU S L, YANG Y M, GU X S, et al. Chitooligosaccharides protect cultured hippocampal neurons against glutamate-induced neurotoxicity[J]. Neurosci Lett, 2008, 444(3):270-274.
[12]	XU W, HUANG H C, LIN C J, et al. Chitooligosaccharides protect rat cortical neurons against copper induced damage by attenuating intracellular level of reactive oxygen species[J]. Bioorg Med Chem Lett, 2010, 20(10):3084-3088.
[13]	杨成迎, 林海烂, 黄子晴, 等. 外源性神经肽CART对大鼠海马神经元氧化应激的抵抗作用及机制研究[J]. 畜牧兽医学报, 2022, 53(4):1231-1240.YANG C Y, LIN H L, HUANG Z Q, et al. Effects and mechanisms of exogenous neuropeptide CART on oxidative stress in hippocampal neurons[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4):1231-1240. (in Chinese)
[14]	BELUJON P, GRACE A A. Hippocampus, amygdala, and stress:interacting systems that affect susceptibility to addiction[J]. Ann N Y Acad Sci, 2011, 1216(1):114-121.
[15]	LATHE R. Hormones and the hippocampus[J]. J Endocrinol, 2001, 169(2):205-231.
[16]	MAO P Z. Oxidative stress and its clinical applications in dementia[J]. J Neurodegener Dis, 2013, 2013:319898.
[17]	FANSELOW M S, DONG H W. Are the dorsal and ventral hippocampus functionally distinct structures[J]. Neuron, 2010, 65(1):7-19.
[18]	KOYLU E O, COUCEYRO P R, LAMBERT P D, et al. Cocaine- and amphetamine-regulated transcript peptide immunohistochemical localization in the rat brain[J]. J Comp Neurol, 1998, 391(1):115-132.
[19]	HSIEH Y S, CHEN P N, YU C H, et al. Involvement of oxidative stress in the regulation of NPY/CART-mediated appetite control in amphetamine-treated rats[J]. Neurotoxicology, 2015, 48:131-141.
[20]	KROEMER G, DALLAPORTA B, RESCHE-RIGON M. The mitochondrial death/life regulator in apoptosis and necrosis[J]. Annu Rev Physiol, 1998, 60:619-642.
[21]	SHA D J, LI L L, YE L, et al. Icariin inhibits neurotoxicity of β-amyloid by upregulating cocaine-regulated and amphetamine-regulated transcripts[J]. NeuroRep, 2009, 20(17):1564-1567.
[22]	YANG B Y, MEI H Y, ZUO F Y, et al. Expression of microRNAs associated with oxidative stress in the hippocampus of piglets[J]. Genes Genom, 2017, 39(7):701-712.
[23]	LANGIE S A S, KOWALCZYK P, TUDEK B, et al. The effect of oxidative stress on nucleotide-excision repair in colon tissue of newborn piglets[J]. Mutat Res, 2010, 695(1-2):75-80.
[24]	LIPIŃSKI P, STARZYŃSKI R R, CANONNE-HERGAUX F, et al. Benefits and risks of iron supplementation in anemic neonatal pigs[J]. Am J Pathol, 2010, 177(3):1233-1243.
[25]	GHATE N B, CHAUDHURI D, PANJA S, et al. Nerium indicum leaf alleviates iron-induced oxidative stress and hepatic injury in mice[J]. Pharm Biol, 2015, 53(7):1066-1074.
[26]	GAN L, YANG B Y, MEI H Y. The effect of iron dextran on the transcriptome of pig hippocampus[J]. Genes Genom, 2017, 39(1):1-14.
[27]	田刚, 黄琳惠, 宋晓华, 等. 壳寡糖对氧化应激仔猪生长性能、抗氧化能力及空肠养分消化和转运能力的影响[J]. 动物营养学报, 2018, 30(7):2652-2661.TIAN G, HUANG L H, SONG X H, et al. Effects of chitooligosaccharides on growth performance, antioxidant capacity and nutrient digestion and transport capacity of jejunum of oxidative stress piglets[J]. Chinese Journal of Animal Nutrition, 2018, 30(7):2652-2661. (in Chinese)
[28]	宋晓华. 壳寡糖对断奶仔猪肠道氧化应激损伤的保护研究[D]. 雅安:四川农业大学, 2016.SONG X H. Protection of chitooligosaccharide on intestinal oxidative stress damage in weaning piglets[D]. Ya'an:Sichuan Agricultural University, 2016. (in Chinese)
[29]	FRIEL J K, FRIESEN R W, HARDING S V, et al. Evidence of oxidative stress in full-term healthy infants[J]. Pediatr Res, 2004, 56(6):878-882.
[30]	JOMOVA K, BAROS S, VALKO M. Redox active metal-induced oxidative stress in biological systems[J]. Transition Met Chem, 2012, 37(2):127-134.
[31]	YIN J, REN W K, DUAN J L, et al. Dietary arginine supplementation enhances intestinal expression of SLC7A7 and SLC7A1 and ameliorates growth depression in mycotoxin-challenged pigs[J]. Amino Acids, 2014, 46(4):883-892.
[32]	GUÉRAUD F, TACHÉ S, STEGHENS J P, et al. Dietary polyunsaturated fatty acids and heme iron induce oxidative stress biomarkers and a cancer promoting environment in the colon of rats[J]. Free Radic Biol Med, 2015, 83:192-200.
[33]	DINCER Y, ERZIN Y, HIMMETOGLU S, et al. Oxidative DNA damage and antioxidant activity in patients with inflammatory bowel disease[J]. Dig Dis Sci, 2007, 52(7):1636-1641.
[34]	WEI M X, WU Y M, CHEN D Z, et al. Changes of free radicals and digestive enzymes in saliva in cases with deficiency in spleen-yin syndrome[J]. J Biomed Res, 2010, 24(3):250-255.
[35]	TURK R, JURETIĆ D, GEREŠ D, et al. Influence of oxidative stress and metabolic adaptation on PON1 activity and MDA level in transition dairy cows[J]. Anim Reprod Sci, 2008, 108(1-2):98-106.
[36]	HE Y Z, GU X F, LU L H, et al. The oxidative molecular regulation mechanism of NOX in children with phenylketonuria[J]. Int J Dev Neurosci, 2014, 38:178-183.
[37]	RUI D, YANG Y J. Aluminum chloride induced oxidative damage on cells derived from hippocampus and cortex of ICR mice[J]. Brain Res, 2010, 1324:96-102.
[38]	HOU X, ZHANG J F, AHMAD H, et al. Evaluation of antioxidant activities of ampelopsin and its protective effect in lipopolysaccharide-induced oxidative stress piglets[J]. PLoS One, 2014, 9(9):e108314.
[39]	SOUFI F G, MOHAMMAD-NEJAD D, AHMADIEH H. Resveratrol improves diabetic retinopathy possibly through oxidative stress-nuclear factor κB-apoptosis pathway[J]. Pharmacol Rep, 2012, 64(6):1505-1514.
[40]	GUERRINI R. Epilepsy in children[J]. Lancet, 2006, 367(9509):499-524.
[41]	CHOPRA K, KUMAR B, KUHAD A. Pathobiological targets of depression[J]. Expert Opin Ther Targets, 2011, 15(4):379-400.
[42]	NUNOMURA A, MOREIRA P I, LEE H G, et al. Neuronal death and survival under oxidative stress in Alzheimer and Parkinson diseases[J]. CNS Neurol Disord Drug Targets, 2007, 6(6):411-423.
[43]	LI X J, HOU J C, SUN P, et al. Neuroprotective effects of TongLuoJiuNao in neurons exposed to oxygen and glucose deprivation[J]. J Ethnopharmacol, 2012, 141(3):927-933.
[44]	TREVISAN C, MKUPASI E M, NGOWI H A, et al. Severe seizures in pigs naturally infected with Taenia solium in Tanzania[J]. Vet Parasitol, 2016, 220:67-71.
[45]	GREEN D R, REED J C. Mitochondria and apoptosis[J]. Science, 1998, 281(5381):1309-1312.
[46]	ADAMS J M, CORY S. The Bcl-2 protein family:arbiters of cell survival[J]. Science, 1998, 281(5381):1322-1326.
[47]	MYERS K M, FISKUM G, LIU Y B, et al. Bcl-2 protects neural cells from cyanide/aglycemia-induced lipid oxidation, mitochondrial injury, and loss of viability[J]. J Neurochem, 1995, 65(6):2432-2440.
[48]	HAYLEY S, POULTER M O, MERALI Z, et al. The pathogenesis of clinical depression:stressor- and cytokine-induced alterations of neuroplasticity[J]. Neuroscience, 2005, 135(3):659-678.
[49]	PASTORI G M, FOYER C H. Common components, networks, and pathways of cross-tolerance to stress. The central role of "redox" and abscisic acid-mediated controls[J]. Plant Physiol, 2002, 129(2):460-468.
[50]	QIU B, HU S D, LIU L B, et al. CART attenuates endoplasmic reticulum stress response induced by cerebral ischemia and reperfusion through upregulating BDNF synthesis and secretion[J]. Biochem Biophys Res Commun, 2013, 436(4):655-659.