急性冷应激对绵羊免疫功能和不同组织热休克蛋白70家族基因表达的影响

doi:10.11843/j.issn.0366-6964.2019.08.011

摘要/Abstract

摘要： 旨在研究急性冷应激对绵羊免疫功能和不同组织热休克蛋白70（HSP70）家族基因表达的影响。本研究选取8只健康、体况接近的（12±0.5）月龄小尾寒羊×湖羊杂交F₁母羊，单笼饲养于保温舍内（风寒温度（-7.14±2.53）℃），适应7 d。第8天将母羊移置舍外（风寒温度（-27.40±3.12）℃）急性冷应激12 h，采集冷应激前后试验个体血样和组织样。利用ELISA法测定血清免疫指标（细胞因子和免疫球蛋白G含量），通过实时荧光定量PCR法测定不同组织（肝、肾、脾、心、背最长肌和十二指肠）HSP70家族基因（HSPA1A、HSPA6和HSPA8）和热休克转录因子1基因（HSF1）的表达量。结果显示：1）与冷应激前相比，冷应激后试验羊血清肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-2（IL-2）和白细胞介素-6（IL-6）浓度显著升高（P<0.05）；血清白细胞介素-4（IL-4）和免疫球蛋白G（IgG）浓度显著下降（P<0.05）。2）冷应激后，试验羊HSPA1A mRNA表达量在肝、肾、心和背最长肌中显著升高（P<0.05）；HSPA6 mRNA表达量在心和背最长肌中显著升高（P<0.05）；HSPA8 mRNA表达量在背最长肌中显著升高（P<0.05），在脾和十二指肠中显著下降（P<0.05）；HSF1 mRNA表达量在肝、心和背最长肌中显著升高（P<0.05），在脾和十二指肠中显著下降（P<0.05）。在本试验条件下，急性冷应激能抑制绵羊的免疫功能； HSP70家族基因（HSPA1A、HSPA6和HSPA8）在各组织中的表达水平不同，其中HSPA1A对温度更敏感，宜作为绵羊冷应激的生物标记物；肝、心和背最长肌等组织中HSP70家族基因表达量升高可能与其保护组织细胞维持正常产热有关。

关键词: 急性冷应激, 绵羊, 风寒温度, 免疫功能, 热休克蛋白70家族基因

Abstract: This experiment was conducted to explore the effect of acute cold stress on immune function and expression of HSP70 family genes in different tissues in sheep. Eight (12±0.5)-month-old healthy female sheep (F₁ of Small Tail Han sheep×Hu sheep) were selected and raised separately in cage in heat-preserved sheep house (wind chill temperature:(-7.14±2.53)℃). The adaptation period lasted for 7 d. At the 8th day, female sheep were treated with acute cold stress for 12 h in outdoor (wind chill temperature:(-27.40±3.12)℃), and then blood and tissue samples were collected before and after cold stress. Serum immune indexes (cytokine and immunoglobulin G content), the expression of HSP70 family genes (HSPA1A, HSPA6 and HSPA8) and heat shock transcription factor 1 (HSF1) in different tissues (liver, kidney, spleen, heart, longissimus dorsi muscle and duodenum) were measured by ELISA and RT-PCR method, respectively. The results showed as the follows:1) Compared with before acute cold stress, the serum levels of TNF-α, IL-1β, IL-2 and IL-6 increased significantly after acute cold stress(P<0.05), while the serum levels of IL-4 and IgG decreased significantly (P<0.05). 2) The mRNA expression of HSPA1A increased significantly in liver, kidney, heart and longissimus dorsi muscle after acute cold stress(P<0.05). HSPA6 expression level increased significantly in the longissimus dorsi muscle and heart (P<0.05). HSPA8 expression level increased significantly in longissimus dorsi muscle(P<0.05), and decreased significantly in spleen and duodenum(P<0.05). HSF1 expression level increased significantly in liver, heart and longissimus dorsi muscle (P<0.05), and decreased significantly in spleen and duodenum(P<0.05). In this experiment, acute cold stress could inhibit the immune function, and the expression levels of HSP70 family genes (HSPA1A, HSPA6 and HSPA8) were different in different tissues. The HSPA1A was more sensitive to temperature and could be suitable to as a biomarker for cold stress. The increased expression of HSP70 family genes in liver, heart and longissimus dorsi muscle might be related to keeping the normal heat production for tissue cells.

Key words: acute cold stress, sheep, wind chill temperature, immune function, heat shock protein 70 family genes

中图分类号:

S826.4

彭孝坤, 张宇, 黄晓瑜, 周广琛, 邢晓南, 张恩平. 急性冷应激对绵羊免疫功能和不同组织热休克蛋白70家族基因表达的影响[J]. 畜牧兽医学报, 2019, 50(8): 1625-1634.

PENG Xiaokun, ZHANG Yu, HUANG Xiaoyu, ZHOU Guangchen, XING Xiaonan, ZHANG Enping. Effects of Acute Cold Stress on Immune Function and Expression of Heat Shock Protein 70 Family Genes in Different Tissues in Sheep[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(8): 1625-1634.

参考文献 58

[1]	赵有璋.羊生产学[M].3版.北京:中国农业出版社,2011.
	ZHAO Y Z.Sheep production[M].3rd ed.Beijing:China Agriculture Press,2011.(in Chinese)
[2]	杨莉,王凤丽,刘海燕,等.寒冷应激对阿勒泰羊硬脂酰辅酶A去饱和酶表达的影响以及序列分析[J].中国兽医学报,2015,35(6):989-994.
	YANG L,WANG F L,LIU H Y,et al.Effects of cold stress on the expression of stearoyl-coenzyme A desaturase of Altay sheep and its sequence analysis[J].Chinese Journal of Veterinary Science,2015,35(6):989-994.(in Chinese)
[3]	刘璐.冷应激对饥饿断奶羔羊生理生化指标及内分泌的影响[D].乌鲁木齐:新疆农业大学,2014.
	LIU L.Effect of cold stress on physiological,biochemical and endocrinological values in fasting weaned lambs[D]. Urumqi:Xinjiang Agricultural University,2014.(in Chinese)
[4]	PHILLIPS C.The welfare risks and impacts of heat stress on sheep shipped from Australia to the Middle East[J].Vet J,2016, 218:78-85.
[5]	KAUFMAN J D,SAXTON A M,RÍUS A G,et al.Short communication:relationships among temperature-humidity index with rectal,udder surface,and vaginal temperatures in lactating dairy cows experiencing heat stress[J].J Dairy Sci,2018,101(7):6424-6429.
[6]	BERMAN A,HOROVITZ T,KAIM M,et al.A comparison of THI indices leads to a sensible heat-based heat stress index for shaded cattle that aligns temperature and humidity stress[J].Int J Biometeorol,2016,60(10):1453-1462.
[7]	高玉红,李宏双,郭建军,等.坝上寒区不同建筑类型奶牛舍冬季温热环境评价[J].东北农业大学学报,2015,46(12):52-57,93.
	GAO Y H,LI H S,GUO J J,et al.Assessment of thermal environment of different styles of dairy houses in winter in chill region of Bashang[J].Journal of Northeast Agricultural University,2015,46(12):52-57,93.(in Chinese)
[8]	单春花,陈伟,李宏双,等.舍饲散栏有窗奶牛舍温热环境的年动态研究[J].中国畜牧兽医,2017,44(10):3106-3112.
	SHAN C H,CHEN W,LI H S,et al.Annual dynamic of thermal environment of dairy cowshed in free stall-feeding with window[J].China Animal Husbandry & Veterinary Medicine,2017,44(10):3106-3112.(in Chinese)
[9]	HAQUE N,SINGH M,HOSSAIN S A.Improved milk production through PG-PL system by provision of in-house shelter management in lactating Murrah buffaloes during winter season[J].J Anim Physiol Anim Nutr (Berl),2018,102(1):166-174.
[10]	刘晓丹.肉鸡冷应激相关基因的mRNA表达谱分析及差异表达基因的研究[D].哈尔滨:东北农业大学,2012.
	LIU X D.Analysis of mRNA expression profile of cold stress gene in broilers and study of differential expression genes[D].Harbin:Northeast Agricultural University,2012.(in Chinese)
[11]	SU Y Y,WEI H D,BI Y J,et al.Pre-cold acclimation improves the immune function of trachea and resistance to cold stress in broilers[J]. J Cell Physiol,2019,234(5):7198-7212.
[12]	ZHAO F Q,ZHANG Z W,QU J P,et al.Cold stress induces antioxidants and Hsps in chicken immune organs[J].Cell Stress Chaperones, 2014,19(5):635-648.
[13]	ZHANG Z B,CHEN B J,YUAN L,et al.Acute cold stress improved the transcription of pro-inflammatory cytokines of Chinese soft-shelled turtle against Aeromonas hydrophila[J].Dev Comp Immunol,2015,49(1):127-137.
[14]	BANERJEE D,UPADHYAY R C,CHAUDHARY U B,et al.Seasonal variation in expression pattern of genes under HSP70[J]. Cell Stress Chaperones,2014,19(3):401-408.
[15]	DEANE C A S,BROWN I R.Knockdown of heat shock proteins HSPA6(Hsp70B') and HSPA1A (Hsp70-1) sensitizes differentiated human neuronal cells to cellular stress[J].Neurochem Res,2018,43(2):340-350.
[16]	DANGI S S,DANGI S K,CHOUHAN V S,et al.Modulatory effect of betaine on expression dynamics of HSPs during heat stress acclimation in goat (Capra hircus)[J].Gene,2016,575(2):543-550.
[17]	DANGI S S,GUPTA M,DANGI S K,et al.Expression of HSPs:an adaptive mechanism during long-term heat stress in goats (Capra hircus)[J]. Int J Biometeorol,2015,59(8):1095-1106.
[18]	DANGI S S,GUPTA M,NAGAR V,et al.Impact of short-term heat stress on physiological responses and expression profile of HSPs in Barbari goats[J].Int J Biometeorol,2014,58(10):2085-2093.
[19]	NAGAYACH R,GUPTA U D,PRAKASH A,et al.Expression profiling of hsp70 gene during different seasons in goats (Capra hircus) under sub-tropical humid climatic conditions[J].Small Rumin Res,2017,147:41-47.
[20]	TUCKER C B,ROGERS A R,VERKERK G A,et al.Effects of shelter and body condition on the behaviour and physiology of dairy cattle in winter[J].Appl Anim Behav Sci,2007,105(1-3):1-13.
[21]	SHITZER A,DE DEAR R.Inconsistencies in the "New" windchill chart at low wind speeds[J].J Appl Meteor Climatol, 2006, 45(5):787-790.
[22]	MEKIS É,VINCENT L A,SHEPHARD M W,et al.Observed trends in severe weather conditions based on humidex,wind chill, and heavy rainfall events in Canada for 1953-2012[J].Atmos-Ocean,2015,53(4):383-397.
[23]	COSTA W P,FACANHA D A E,LEITE J H G M,et al.Thermoregulatory responses and blood parameters of locally adapted ewes under natural weather conditions of Brazilian semiarid region[J].Sem:Ciênc Agrár,2015,36(6Suppl 2):4589-4600.
[24]	魏凤仙,胡骁飞,张敏红,等.相对湿度和氨气应激对肉仔鸡血氨水平及细胞因子含量的影响[J].动物营养学报,2013,25(10):2246-2253.
	WEI F X,HU X F,ZHANG M H,et al.Effects of relative humidity and ammonia stress on plasma ammonia level and cytokine contents of broilers[J].Chinese Journal of Animal Nutrition,2013,25(10):2246-2253.(in Chinese)
[25]	YANG Z J,LIU C,ZHENG W J,et al.The functions of antioxidants and heat shock proteins are altered in the immune organs of selenium-deficient broiler chickens[J].Biol Trace Elem Res,2016,169(2):341-351.
[26]	SHINI S,HUFF G R,SHINI A,et al.Understanding stress-induced immunosuppression:exploration of cytokine and chemokine gene profiles in chicken peripheral leukocytes[J].Poult Sci,2010,89(4):841-851.
[27]	郭爽,郭景茹,汪志,等.不同时间强度冷刺激对大鼠血清中炎症相关细胞因子的影响[J].中国兽医学报,2013,33(3):352-357,361.
	GUO S,GUO J R,WANG Z,et al.Effects of differentduration of cold stress on production of cytokines in serum of SPF wistar rats[J].Chinese Journal of Veterinary Science,2013,33(3):352-357,361.(in Chinese)
[28]	CHEN J,ZHOU Y X,JIN X D,et al.Expression of interleukin-2 in Candidal balanoposthitis and its clinical significance[J].Chin Med J (Engl),2011,124(17):2776-2778.
[29]	ZHAO F Q,ZHANG Z W,YAO H D,et al.Effects of cold stress on mRNA expression of immunoglobulin and cytokine in the small intestine of broilers[J]. Res Vet Sci,2013,95(1):146-155.
[30]	计红,杨焕民,李士泽,等.冷应激对仔猪血浆相关细胞因子和IgG水平的影响[J].应用与环境生物学报,2010,16(5):632-636.
	JI H,YANG H M,LI S Z,et al.Changes in correlated cytokines and IgG of blood plasma of piglets under cold stress[J].Chinese Journal of Applied & Environmental Biology,2010,16(5):632-636.(in Chinese)
[31]	杨莉,张莉,齐亚银,等.冷应激对湖羊血清因子及热休克蛋白70 mRNA表达的影响[J].中国畜牧兽医,2015,42(4):890-895.
	YANG L,ZHANG L,QI Y Y,et al.Effect of cold stress on serum factor and Hsp70 mRNA in Hu sheep[J].China Animal Husbandry & Veterinary Medicine,2015,42(4):890-895.(in Chinese)
[32]	BOD L,DOUGUET L,AUFFRAY C,et al.IL-4-induced gene 1:a negative immune checkpoint controlling B cell differentiation and activation[J].J Immunol,2018,200(3):1027-1038.
[33]	伊亢力.免疫细胞因子基因及相关miRNAs在肉鸡急性冷、热应激中的表达调控研究[D].杨凌:西北农林科技大学,2017.
	YI K I.The expression mechanism of immune cytokine gene and associated miRNAs in acute heat/cold stressed broilers[D]. Yangling:Northwest A&F University,2017.(in Chinese)
[34]	VAN DEBOVENKAMP F S,HAFKENSCHEID L,RISPENS T,et al.The emerging importance of IgG Fab glycosylation in immunity[J]. J Immunol,2016,196(4):1435-1441.
[35]	ARCHANA P R,ALEENA J,PRAGNA P,et al.Role of heat shock proteins in livestock adaptation to heat stress[J].J Dairy,Vet Anim Res, 2017,5(1):00127.
[36]	LIEW P K,ZULKIFLI I,HAIR-BEJO M,et al.Effects of early age feed restriction and heat conditioning on heat shock protein 70 expression,resistance to infectious bursal disease,and growth in male broiler chickens subjected to heat stress[J].Poult Sci, 2003, 82(12):1879-1885.
[37]	刘嘉敏,赵元莙.HSP70的研究进展及其在生物医学中的应用[J].教育教学论坛,2017(50):61-62.
	LIU J M,ZHAO Y J.Research progressfor HSP70 and its application in biomedicine study[J].Education Teaching Forum, 2017(50):61-62.(in Chinese)
[38]	BHARATI J,DANGI S S,CHOUHAN V S,et al.Expression dynamics of HSP70 during chronic heat stress in Tharparkar cattle[J]. Int J Biometeorol,2017,61(6):1017-1027.
[39]	GUPTA M,DANGI S S,MAURYA D,et al.Expression profile of cold shock protein genes in goats (Capra hircus) during different seasons[J]. Iran J Vet Res,2014,15(1):7-12.
[40]	KRAKOWIAK J,ZHENG X,PATEL N,et al.Hsf1 and Hsp70 constitute a two-component feedback loop that regulates the yeast heat shock response[J].eLife,2018,7:e31668.
[41]	高雅君.炎症条件下草鱼HSP70的表达及HSF1对其调控的初步研究[D].成都:电子科技大学,2018.
	GAO Y J.Preliminary study on the expression of HSP70 and its regulation by HSF1 under inflammatory conditions in grass carp[D].Chengdu:University of Electronic Science and Technology of China,2018.(in Chinese)
[42]	ZHANG A Y,GUO Y F,ZHANG S N,et al.Cytokine effects and cellular signaling pathways of grass carp HSP70 in head kidney leukocytes[J]. Fish Shellfish Immunol,2015,46(2):550-556.
[43]	CAO X,YUE L,SONG J Y,et al.Inducible HSP70 antagonizes IL-1β cytocidal effects through inhibiting NF-kB activation via destabilizing TAK1 in HeLa cells[J].PLoS One,2012,7(11):e50059.
[44]	HULINA A,RAJKOVIC M G,DESPOT D J,et al.Extracellular Hsp70 induces inflammation and modulates LPS/LTA-stimulated inflammatory response in THP-1 cells[J].Cell Stress Chaperones,2018,23(3):373-384.
[45]	HECK T G,SCOMAZZON S P,NUNES P R,et al.Acute exercise boosts cell proliferation and the heat shock response in lymphocytes:correlation with cytokine production and extracellular-to-intracellular HSP70 ratio[J].Cell Stress Chaperones, 2017, 22(2):271-291.
[46]	REDZOVIC A,GULIC T,LASKARIN G,et al.Heat-shock proteins 70 induce pro-inflammatory maturation program in decidual CD1a+ dendritic cells[J].Am J Reprod Immunol,2015,74(1):38-53.
[47]	VAN EDEN W,BROERE F,VAN DER ZEE R.HSP70 is a major contributor to the MHCⅡ ligandome and inducer of regulatory T cells[M]//ASEA A A A,KAYR P.HSP70 in Human Diseases and Disorders.Cham:Springer,2018:163-171.
[48]	金天明.动物生理学[M].北京:清华大学出版社,2012.
	JIN T M.Animal physiology[M].Beijing:Tsinghua University Press,2012.(in Chinese)
[49]	张冬杰,李忠秋,汪亮,等.低温对猪肌肉和肝脏组织线粒体数量的影响[J].黑龙江畜牧兽医,2018(1):100-102,253.
	ZHANG D J,LI Z Q,WANG L,et al.The effect of low temperature on mitochondrial numbers in pig muscle and liver tissues[J]. Heilongjiang Animal Science and Veterinary Medicine,2018(1):100-102,253.(in Chinese)
[50]	杨焕民.应激肉牛几种组织中HSPs表达的研究[D].长春:中国人民解放军军需大学,2002.
	YANG H M.Studies on the expression of HSPs in tissues of the stressed cattle[D].Changchun:Heping Campus Jilin University, 2002.(in Chinese)
[51]	SHI H Z,YAO R Z,LIAN S,et al.Regulating glycolysis,the TLR4 signal pathway and expression of RBM3 in mouse liver in response to acute cold exposure[J].Stress,2019,22(3):366-376.
[52]	计红,詹雪龙,薛琳琳,等.急性冷刺激对大鼠骨骼肌和7个内脏器官的组织结构影响[J].中国兽医学报,2019,39(1):105-112.
	JI H,ZHAN X L,XUE L L,et al.Effects of cold stimulus on the histology structure of skeletal muscle and seven visceral organs in rats[J].Chinese Journal of Veterinary Science,2019,39(1):105-112.(in Chinese)
[53]	谢永新,史延,黄利华,等.急性冷、热应激对肉鸡肝脏和心脏组织中HSPs表达的影响[J].中国家禽,2018,40(1):7-11.
	XIE Y X,SHI Y,HUANG L H,et al.Effects of acute heat/cold stress on the expression of HSPs in liver and heart of broiler[J].China Poultry,2018,40(1):7-11.(in Chinese)
[54]	刘颖,任文陟,李苏楠,等.冷应激对猪脂肪、肝脏、心脏与肾脏组织中PPARγ2 mRNA及蛋白表达的影响[J].中国兽医学报,2012,32(4):592-597.
	LIU Y,REN W Z,LI S N,et al.PPARγ2 gene expression of swine adipose tissue,liver,heart and kidney in cold stress[J].Chinese Journal of Veterinary Science,2012,32(4):592-597.(in Chinese)
[55]	AFSAL A,BAGATH M,SEJIAN V,et al.Effect of heat stress on HSP70 gene expression pattern in different vital organs of Malabari goats[J]. Biol Rhythm Res,2019,doi:10.1080/09291016.2019.1600270.
[56]	DANG W,XU N,ZHANG W,et al.Differential regulation of Hsp70 expression in six lizard species under normal and high environmental temperatures[J]. Pak J Zool,2018,50(3):1043-1051.
[57]	赵福庆.冷应激对雏鸡组织热休克蛋白和小肠免疫功能的影响[D].哈尔滨:东北农业大学,2013.
	ZHAO F Q.Effect of cold stress on heat shock proteins and immune function of small intestine in chicken[D].Harbin:Northeast Agricultural University,2013.(in Chinese)
[58]	于宪一,李术,陈蕾,等.冷应激对雏鸡肠组织中HSP70 mRNA表达的影响[J].东北农业大学学报,2011,42(3):90-93.
	YU X Y,LI S,CHEN L,et al.Effects of cold stress on HSP70 mRNA expression in intestines of chickens[J].Journal of Northeast Agricultural University,2011,42(3):90-93.(in Chinese)