基于iTRAQ技术挖掘猪肾组织高原低氧适应性关键蛋白

doi:10.11843/j.issn.0366-6964.2021.05.010

摘要/Abstract

摘要： 旨在探究高原低氧环境下藏猪与大约克猪肾组织中低氧适应的差异表达蛋白（differentially expressed protein，DEPs）。本试验选择藏猪与大约克猪两个品种无亲缘关系的去势公猪，饲养于海拔3 000 m的高原，按自由采食方式饲养至180日龄，每个品种随机选择生长情况相近的 9头猪采集肾组织。利用同位素标记相对和绝对定量（isobaric tags for relative and absolute quantification，iTRAQ）技术对藏猪和大约克猪肾组织中蛋白差异表达情况进行分析，并对差异表达蛋白进行GO（Gene Ontology）功能注释及KEGG通路分析。结果发现，在两猪种肾脏组织中共获得4 370个蛋白质，以FC>1.2或<0.833，P<0.05为筛选条件共筛选出181个DEPs，其中在藏猪中上调的蛋白质有138个，下调的有43个。这些DEPs主要功能富集在HIF-1信号通路、PPAR信号通路、补体和凝血级联信号通路、氧化还原酶活性、碳代谢、血压调节、线粒体等与低氧适应相关的条目和通路上，进一步综合分析后共筛选出5个与低氧适应相关的重要DEPs（CD59、A2M、eNOS、CDKN1B和PGK1）。这些DEPs分别在维持血液动力学、调节能量代谢与生理环境稳态等方面的调节中起到重要作用，为进一步研究猪的高原低氧适应性提供一定理论依据。

关键词: iTRAQ, 猪, 肾, 低氧适应性

Abstract: The aim of this study was to explore the differentially expressed proteins(DEPs) of hypoxic adaptation in kidney tissues of Tibetan pigs and Yorkshire pigs under high altitude hypoxic environment. Unrelated castrated boars from Tibetan pig and Yorkshire pig were selected as experimental animals, and fed ad libitum on the plateau with altitude of 3 000 m, up to 180-day-old, 9 pigs with similar growth and body condition were randomly selected from each breed to collect kidney tissue. In this study, isobaric tags for relative and absolute quantification(iTARQ) technique was used to analyze the difference of protein expression in kidney tissues between Tibetan pigs and Yorkshire pigs, the differentially expressed proteins were analyzed by GO functional annotation and KEGG pathway. The results showed that, a total of 4 370 proteins were obtained from kidney tissues of two pig breeds, FC>1.2 or <0.833 and P<0.05 as the criterion, a total of 181 DEPs were screened out, among them, 138 proteins were up-regulated and 43 proteins were down-regulated in the Tibetan pigs. In the functional analysis, DEPs were mainly enriched in items and pathways related to hypoxic adaptation, such as HIF-1 signaling pathway, PPAR signaling pathway, complement and coagulation cascades signaling pathway, oxidoreductase activity, carbon metabolism, regulation of blood pressure, mitochondria, etc., after further comprehensive analysis, 5 important DEPs related to hypoxic adaptation were screened out(CD59, A2M, eNOS, CDKN1B and PGK1). These DEPs play an important role in maintaining hemodynamics, regulating energy metabolism and physiological environment homeostasis, the result of this study provides a theoretical basis for further study on the adaptability of pigs to hypoxic at high altitude.

Key words: iTRAQ, pig, kidney, hypoxic adaptation

中图分类号:

S828.2

李梦柔, 韦明邦, 张健, 邢璐, 徐士军, 肖青青, 叶幼荣, 董世雄, 段梦琪, 商鹏. 基于iTRAQ技术挖掘猪肾组织高原低氧适应性关键蛋白[J]. 畜牧兽医学报, 2021, 52(5): 1238-1246.

LI Mengrou, WEI Mingbang, ZHANG Jian, XING Lu, XU Shijun, XIAO Qingqing, YE Yourong, DONG Shixiong, DUAN Mengqi, SHANG Peng. Mining Key Proteins of High Altitude Hypoxic Adaptability in Porcine Kidney Tissue Based on iTRAQ Technology[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1238-1246.

参考文献 42

[1]	COLOMBO J S,JIA S H,D’SOUZA R N.Modeling hypoxia induced factors to treat pulpal inflammation and drive regeneration[J].J Endod,2020,46(S9):S19-S25.
[2]	JIN J,CHEN L,LIU G Q,et al.Proteomic analysis of anti-angiogenic effects by conbercept in the mice with oxygen induced retinopathy[J].Int J Ophthalmol,2020,13(12):1844-1853.
[3]	HU M M,ZHANG T M,GU M,et al.Pretreatment to posttreatment hypoxia inducible factor-1α ratios as a potentially predictive marker for first-line treatment in nonsmall cell lung cancer patients without known driver mutations[J].Genet Test Mol Bioma,2020,24(12):798-803.
[4]	SHI Y,LIU Z,ZHANG Q,et al.Phosphorylation of seryl-tRNA synthetase by ATM/ATR is essential for hypoxia-induced angiogenesis[J].PLoS Biol,2020,18(12):e3000991.
[5]	BARCLAY H,MUKERJI S,KAYSER B,et al.Respiratory alkalinization and posterior cerebral artery dilatation predict acute mountain sickness severity during 10 h normobaric hypoxia[J].Exp Physiol,2021,106(1):175-190.
[6]	NORTON C E,JEMIGAN N L,WALKER B R,et al.Membrane depolarization is required for pressure-dependent pulmonary arterial tone but not enhanced vasoconstriction to endothelin-1 following chronic hypoxia[J].Pulm Circ,2020,10(4):1-10.
[7]	GAMBOA M.Hemocyanin and hexamerins expression in response to hypoxia in stoneflies (Plecoptera, Insecta)[J]. Arch Insect Biochem Physiol,2020,105(3):e21743.
[8]	WILKES M,LONG G,MASSEY H,et al.Cognitive function in simulated paragliding flight[J].Aerosp Med Hum Perform,2019,90(10):851-859.
[9]	VARIS N,PARKKOLA K I,LEINO T K.Hypoxia hangover and flight performance after normobaric hypoxia exposure in a hawk simulator[J].Aerosp Med Hum Perform,2019,90(8):720-724.
[10]	STORZ J F,SCOTT G R.Phenotypic plasticity,genetic assimilation,and genetic compensation in hypoxia adaptation of high-altitude vertebrates[J].Comp Biochem Physiol A Mol Integr Physiol,2020,253:110865.
[11]	ZHANG B,CHAMBA Y,SHANG P,et al.Comparative transcriptomic and proteomic analyses provide insights into the key genes involved in high-altitude adaptation in the Tibetan pig[J].Sci Rep,2017,71(1):3654.
[12]	BARRETT H L,DONAGHUE K C,FORBES J M.Going in early:hypoxia as a target for kidney disease prevention in diabetes?[J].Diabetes,2020,69(12):2578-2580.
[13]	KIM D J K,DREW R C,SICA C T,et al.Renal medullary oxygenation decreases with lower body negative pressure in healthy young adults[J].J Appl Physiol,2021,130(1):48-56.
[14]	YAP D Y H,MCMAHON L P,HAO C M,et al.Recommendations by the Asian Pacific society of nephrology (APSN) on the appropriate use of HIF-PH inhibitors[J].Nephrology (Carlton),2021,26(2):105-118.
[15]	LI X L,CHEN W H,FENG J F,et al.The effects of HIF-1α overexpression on renal injury,immune disorders and mitochondrial apoptotic pathways in renal ischemia/reperfusion rats[J].Transl Androl Urol,2020,9(5): 2157-2165.
[16]	HONG S E,AN J H,YU S L,et al.Ceria-zirconia antioxidant nanoparticles attenuate hypoxia-induced acute kidney injury by restoring autophagy flux and alleviating mitochondrial damage[J].J Biomed Nanotechnol,2020, 16(7): 1144-1159.
[17]	MAZZALI M,JEFFERSON J A,NI Z,et al.Microvascular and tubulointerstitial injury associated with chronic hypoxia-induced hypertension[J].Kidney Int,2003,636(6):2088-2093.
[18]	PALUBISKI L M,O’HALLORAN K D,O’NEILL J.Renal physiological adaptation to high altitude:a systematic review[J].Front Physiol,2020,11:756.
[19]	YU W J,ZENG H H,CHEN J Z,et al.miR-20a-5p is enriched in hypoxia-derived tubular exosomes and protects against acute tubular injury[J].Clin Sci (Lond),2020,134(16):2223-2234.
[20]	YAN Z P,XU G S.A novel choice to correct inflammation-induced anemia in CKD:oral hypoxia-inducible factor prolyl hydroxylase inhibitor roxadustat[J].Front Med (Lausanne),2020,7:393.
[21]	NAVARRO-YEPES J,BURNS M,ANANDHAN A,et al.Oxidative stress,redox signaling,and autophagy:cell death versus survival[J].Antioxid Redox Signal,2014,21(1):66-85.
[22]	GOURIOU Y,ALAM M R,HARHOUS Z,et al.ANT2-mediated ATP import into mitochondria protects against hypoxia lethal injury[J].Cells,2020,9(12):2542.
[23]	左清清,姚娜,董坤哲,等.低氧适应的线粒体调控机制研究进展[J].畜牧兽医学报,2013,44(7):993-999.ZUO Q Q,YAO N,DONG K Z,et al.How mitochondria play a role in hypoxic adaptation[J].Acta Veterinaria et Zootechnica Sinica,2013,44(7):993-999.(in Chinese)
[24]	ZHUANG Z N,GUO S,JI Z P,et al.Hypoxia preconditioning induced HIF-1α promotes glucose metabolism and protects mitochondria in liver I/R injury[J].Clin Res Hepatol Gastroenterol,2015,39(5):610-619.
[25]	MOOG P,SCHAMS R,SCHNEIDINGER A,et al.Effect of hypoxia preconditioned secretomes on lymphangiogenic and angiogenic sprouting:an in vitro analysis[J].Biomedicines,2020,8(9):365.
[26]	DUAN M Q,WANG Z M,GUO X Y,et al.Integrated analysis of transcriptomic and proteomic analyses reveals different metabolic patterns in the livers of Tibetan and Yorkshire pigs[J].Asian-Australas J Anim Sci,2020, doi:10.5713/ajas.20.0342.
[27]	BALOGLU E,NONNENMACHER G,SELENINOVA A,et al.The role of hypoxia-induced modulation of alveolar epithelial Na+- transport in hypoxemia at high altitude[J].Pulm Circ,2020,10(S1):50-58.
[28]	DU X,ZHANG R,YE S L,et al.Alterations of human plasma proteome profile on adaptation to high-altitude hypobaric hypoxia[J].J Proteome Res,2019,18(5):2021-2031.
[29]	ZHANG Y,ZHENG X T,ZHANG Y W,et al.Comparative transcriptomic and proteomic analyses provide insights into functional genes for hypoxic adaptation in embryos of Tibetan chickens[J].Sci Rep,2020,10(1):11213.
[30]	FAN N,LIU C,REN M.Effect of different high altitudes on vascular endothelial function in healthy people[J].Medicine (Baltimore),2020,99(11):e19292.
[31]	KINDERLERER A R,STEINBERG R,JOHNS M,et al.Statin-induced expression of CD59 on vascular endothelium in hypoxia:a potential mechanism for the anti-inflammatory actions of statins in rheumatoid arthritis[J].Arthritis Res Ther,2006,8(4):R130.
[32]	MEVORACH D.Paroxysmal nocturnal hemoglobinuria (PNH) and primary p.Cys89Tyr mutation in CD59: differences and similarities[J].Mol Immunol,2015,67(1):51-55.
[33]	RODRÍGUEZ-CALVO R,FERRÁN B,ALONSO J,et al.NR4A receptors up-regulate the antiproteinase alpha-2 macroglobulin (A2M) and modulate MMP-2 and MMP-9 in vascular smooth muscle cells[J].Thromb Haemost,2015,113(6):1323-1334.
[34]	LEÓN-VELARDE F,MEJÍA O.Gene expression in chronic high altitude diseases[J].High Alt Med Biol,2008, 9(2):130-139.
[35]	张博,强巴央宗,李庆岗,等.藏猪内皮型一氧化氮合酶(eNOS)基因的表达与低氧适应分析[J].中国农业大学学报,2013,18(5):104-108.ZHANG B,CHAMBA Y Z,LI Q G,et al.Endothelial nitric oxide synthase (eNOS) gene expression and hypoxic adaptation analysis in Tibetan pig[J].Journal of China Agricultural University,2013,18(5):104-108.(in Chinese)
[36]	张寿,靳国恩,常兰,等.牦牛和柴达木黄牛低氧通气反应及颈动脉体NO、NOS的比较研究[J].畜牧兽医学报,2018,49(10):2276-2282.ZHANG S,JIN G E,CHANG L,et al.Comparison on hypoxic ventilatory response and NO,NOS of carotid body between yaks and qaidam yellow cattle[J].Acta Veterinaria et Zootechnica Sinica,2018,49(10):2276-2282.(in Chinese)
[37]	王慧,解卫平,左祥荣,等.埃他卡林对长期低氧大鼠肺组织eNOS mRNA和蛋白表达的影响[J].中国药理学通报,2009,25(12):1594-1598.WANG H,XIE W P,ZUO X R,et al.Effects of iptakalim on the mRNA and protein expression of eNOS in pulmonary tissue of chronic hypoxic rats[J].Chinese Pharmacological Bulletin,2009,25(12):1594-1598.(in Chinese)
[38]	GARDNER L B,LI Q,PARK M S,et al.Hypoxia inhibits G1/S transition through regulation of p27 expression[J].J Biol Chem,2001,276(11):7919-7926.
[39]	KRTOLICA A,KRUCHER N A,LUDLOW J W.Hypoxia-induced pRB hypophosphorylation results from downregulation of CDK and upregulation of PP1 activities[J].Oncogene,1998,17(18):2295-2304.
[40]	LI Q,CUI H H,YANG Y J,et al.Quantitative proteomics analysis of ischemia/reperfusion injury-modulated proteins in cardiac microvascular endothelial cells and the protective role of tongxinluo[J].Cell Physiol Biochem, 2017, 41(4):1503-1518.
[41]	BEUTLER E.PGK deficiency[J].Br J Haematol,2007,136(1):3-11.
[42]	SHAO F,YANG X Y,WANG W,et al.Associations of PGK1 promoter hypomethylation and PGK1-mediated PDHK1 phosphorylation with cancer stage and prognosis:a TCGA pan-cancer analysis[J].Cancer Commun (Lond),2019,39(1):54.