两种动物胆汁酸对热应激小鼠肝脏和空肠保护效果的比较研究

doi:10.16431/j.cnki.1671-7236.2023.07.014

摘要/Abstract

摘要： 【目的】比较猪胆汁酸与羊胆汁酸对热应激小鼠肝脏以及空肠的保护效果。【方法】试验选取40只7周龄BALB/c健康雄性小鼠，随机分为空白对照组（MOCK组）、热应激组（HS组）、热应激-猪胆汁酸组（PBAs组）、热应激-羊胆汁酸组（SBAs组）。试验前7 d，MOCK、HS组小鼠饲喂基础饲粮，PBAs、SBAs组分别在基础饲粮中添加400 mg/kg猪、羊胆汁酸。于试验第8天时，MOCK组小鼠室温放置，其余3组小鼠置于36~38 ℃、相对湿度60%培养箱中2 h，采集小鼠血液、肝脏和空肠组织，HE染色观察空肠组织病理变化。利用ELISA法检测血清中尿素氮（BUN）含量、碱性磷酸酶（AKP）、谷丙转氨酶（ALT）、谷草转氨酶（AST）活性及肝脏和空肠组织中丙二醛（MDA）、还原型谷胱甘肽（GSH）含量以及超氧化物歧化酶（SOD）活性；采用实时荧光定量PCR检测肝脏和空肠中热休克蛋白60（HSP60）、HSP70及炎症因子白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）、IL-10基因mRNA表达量。【结果】与MOCK组相比，HS组空肠绒毛高度极显著下降（P<0.01），隐窝深度显著增加（P<0.05）；血清ALT活性、肝脏和空肠组织SOD活性均显著降低（P<0.05）；肝脏和空肠组织HSP60、HSP70基因相对表达量极显著升高（P<0.01）。与HS组相比，PBAs、SBAs组空肠绒毛高度、绒毛高度与隐窝深度比值（V/C）均极显著升高（P<0.01）；SBAs组血清ALT活性、空肠组织SOD活性显著或极显著升高（P<0.05；P<0.01）；PBAs、SBAs组肝脏组织MDA和GSH含量、HSP60和HSP70基因相对表达量均极显著降低（P<0.01），IL-1β、TNF-α基因相对表达量均极显著升高（P<0.01）。另外，SBAs组肝脏、空肠组织HSP60基因的相对表达量极显著或显著低于PBAs组（P<0.01；P<0.05），肝脏组织IL-1β、TNF-α基因相对表达量显著高于PBAs组（P<0.05）。【结论】在热应激条件下，饲粮中添加羊胆汁酸对于提高小鼠血清ALT活性，提高肝脏和空肠组织抗氧化能力、降低热休克蛋白表达以及修复空肠结构损伤方面优于猪胆汁酸的作用效果，但猪胆汁酸在抗炎效果方面优于羊胆汁酸。

关键词: 热应激; 胆汁酸; 损伤; 保护效果

Abstract: 【Objective】 The purpose of this study was to compare the protective effects of porcine bile acid and sheep bile acid on liver and jejunum of heat stressed mice.【Method】 Forty BALB/c healthy male mice aged 7 weeks were randomly divided into blank control group (MOCK group), heat stress group (HS group), heat stress-pig-bile acid group (PBAs group) and heat stress-sheep-bile acid group (SBAs group).Seven days before heat stress, mice in MOCK and HS groups were fed a basal diet, and those in PBAs and SBAs groups were fed the basal diet supplemented with 400 mg/kg pig bile acid and sheep bile acid, respectively.On day 8, mice in MOCK group were placed at room temperature, and mice in other 3 groups were placed in incubators at 36-38 ℃ and 60% relative humidity for 2 h.The blood, liver and jejunum tissues of mice were collected, and the pathological changes of jejunum tissues were observed by HE staining.The content of urea nitrogen (BUN), the activities of alkaline phosphatase (AKP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, and the contents of malondialdehyde (MDA), reduced glutathione (GSH) and the activities of superoxide dismutase (SOD) in liver and jejunum tissue were determined by ELISA.The mRNA expression of heat shock protein 60 (HSP60), HSP70 and inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and IL-10 genes in liver and jejunum were detected by Real-time quantitative PCR.【Result】 Compared with MOCK group, the villus height of jejunum in HS group was extremely significantly decreased (P<0.01), and the crypt depth was significantly increased (P<0.05).The activity of ALT in serum and SOD activity in liver and jejunum were significantly decreased (P<0.05).The relative expressions of HSP60 and HSP70 genes in liver and jejunum were extremely significantly increased (P<0.01).Compared with HS group, villus height, and the ratio of villus height and crypt depth (V/C) in jejunum of PBAs and SBAs groups were extremely significantly increased (P<0.01).ALT activity of serum and SOD activity of jejunal in SBAs group were significantly or extremely significantly increased (P<0.05 or P<0.01). The contents of MDA and GSH, and the relative expressions of HSP60 and HSP70 genes in liver of PBAs and SBAs groups were extremely significantly decreased (P<0.01), while the relative expressions of IL-1β and TNF-α genes were extremely significantly increased (P<0.01).In addition, the relative expression of HSP60 gene in liver and jejunum of SBAs group was extremely significantly or significantly lower than that of PBAs group (P<0.01 or P<0.05), the relative expressions of IL-1β and TNF-α genes in liver were significantly higher than those in PBAs group (P<0.05).【Conclusion】 Under the condition of heat stress, dietary supplementation of sheep bile acid had better effects than that of porcine bile acid on increasing serum ALT activity, improving antioxidant capacity of liver and jejunum, reducing heat shock protein expression and repairing jejunum structural damage, but porcine bile acid had better anti-inflammatory effect than sheep bile acid.

Key words: heat stress; bile acids; injure; protective effect

中图分类号:

S852.2

李宁, 蒋昀轩, 刘德凤, 闫国宁, 申明月, 张树宇, 黄博涵, 贾宝玉, 徐力, 魏凯. 两种动物胆汁酸对热应激小鼠肝脏和空肠保护效果的比较研究[J]. 中国畜牧兽医, 2023, 50(7): 2729-2739.

LI Ning, JIANG Yunxuan, LIU Defeng, YAN Guoning, SHEN Mingyue, ZAHNG Shuyu, HUANG Bohan, JIA Baoyu, XU Li, WEI Kai. Comparative Study on the Protective Effects of Two Animal Bile Acids on Liver and Jejunum in Heat-stressed Mice[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2729-2739.

参考文献

[1] SCANES C G.Biology of stress in poultry with emphasis on glucocorticoids and the heterophil to lymphocyte ratio[J]. Poultry Science, 2016, 95(9):2208-2215.
[2] AZAD M A K, KIKUSATO M, MAEKAWA T, et al.Metabolic characteristics and oxidative damage to skeletal muscle in broiler chickens exposed to chronic heat stress[J].Comparative Biochemistry and Physiology Part A:Molecular & Integrative Physiology, 2010, 155(3):401-406.
[3] ALHENAKY A, ABDELQADER A, ABUAJAMIEH M, et al.The effect of heat stress on intestinal integrity and salmonella invasion in broiler birds[J]. Journal of Thermal Biology, 2017, 70:9-14.
[4] YANG L, TAN G Y, FU Y Q, et al.Effects of acute heat stress and subsequent stress removal on function of hepatic mitochondrial respiration, ROS production and lipid peroxidation in broiler chickens[J].Comparative Biochemistry and Physiology Part C:Toxicology & Pharmacology, 2010, 151(2):204-208.
[5] BAGATH M, KRISHNAN G, DEVARAJ C, et al.The impact of heat stress on the immune system in dairy cattle:A review[J]. Research in Veterinary Science, 2019, 126:94-102.
[6] CALIK A, EMAMI N K, SCHYNS G, et al.Influence of dietary vitamin E and selenium supplementation on broilers subjected to heat stress, part Ⅱ:Oxidative stress, immune response, gut integrity, and intestinal microbiota[J].Poultry Science, 2022, 101(6):101858.
[7] 周旭香, 刘丹, 叶晓川.羊胆汁化学成分及药理作用研究进展[J].中国草食动物科学, 2021, 41(4):42-45. ZHOU X X, LIU D, YE X C.Research progress on chemical constituents and pharmacological action of sheep bile[J].Chinese Herbivore Science, 2021, 41(4):42-45.(in Chinese)
[8] 陈昊杰.胆汁酸对草鱼生长、脂质代谢及肠道微生物区系的影响[D].杨凌:西北农林科技大学, 2016. CHEN H J.Effects of bile acids on growth, lipid metabolism and intestinal microflora of grass carp[D].Yangling:Northwest A&F University, 2016.(in Chinese)
[9] LAI W, HUANG W, DONG B, et al.Effects of dietary supplemental bile acids on performance, carcass characteristics, serum lipid metabolites and intestinal enzyme activities of broiler chickens[J].Poultry Science, 2018, 97(1):196-202.
[10] YIN C, XIA B, TANG S, et al.The effect of exogenous bile acids on antioxidant status and gut microbiota in heat-stressed broiler chickens[J].Frontiers in Nutrition, 2021, 8:747136.
[11] 王一博, 王春雨, 曲范娜.高效液相色谱-电雾式检测器同时测定猪、牛、羊、熊胆粉中5种胆汁酸的含量[J].分析化学, 2014, 42(1):109-112. WANG Y B, WANG C Y, QU F N.Simultaneous determination of five bile acids in pig, bovine, sheep and bear bile powder by high performance liquid chromatography with electrospray detector[J].Analytical Chemistry, 2014, 42(1):109-112.(in Chinese)
[12] 姜姣姣, 杨鹏, 时新刚.动物胆汁的成分及药理作用研究进展[J].食品与药品, 2017, 19(3):227-230. JIANG J J, YANG P, SHI X G.Research progress on the composition and pharmacological action of animal bile[J]. Food and Medicine, 2017, 19(3):227-230.(in Chinese)
[13] 姚令文, 石岩, 魏锋.羊胆中胆汁酸类成分的考察、测定与探讨[J].药物分析杂志, 2021, 41(10):1718-1723. YAO L W, SHI Y, WEI F.Investigation, determination and discussion of bile acids in sheep bile[J].Journal of Pharmaceutical Analysis, 2021, 41(10):1718-1723.(in Chinese)
[14] 李慧峰, 李培锋, 赵珍.羊胆汁中甘氨胆酸提取工艺研究[J].安徽农业科学, 2008, 36(20):8415. LI H F, LI P F, ZHAO Z.Study on extraction technology of glycine cholic acid from sheep bile[J].Anhui Agricultural Science, 2008, 36(20):8415.(in Chinese)
[15] 李培锋, 郝文利.牛磺胆酸的解热作用及其机制的研究[J].中国兽药杂志, 2006, 40(6):11-14. LI P F, HAO W L.Study on antipyretic effect of taurocholic acid and its mechanism[J].Chinese Journal of Veterinary Medicine, 2006, 40(6):11-14.(in Chinese)
[16] ISLAM K B, FUKIYA S, HAGIO M, et al.Bile acid is a host factor that regulates the composition of the cecal microbiota in rats[J].Gastroenterology, 2011, 141(5):1773-1781.
[17] 沙裴隆有限公司.包含牛磺脱氧胆酸或其药学上可接受的盐作为有效成分的用于预防或治疗炎症性肠病的组合物:CN202080005732.3[P].2021-06-11. SHAPERONG Co., Ltd., A composition containing taurodeoxycholic acid or its pharmaceutically acceptable salt as an active ingredient for the prevention or treatment of inflammatory bowel disease:CN202080005732.3[P].2021-06-11.(in Chinese)
[18] 姜雪薇.羊胆汁中提取牛磺胆酸工艺的优化及牛磺胆酸HPLC的方法学研究[D].呼和浩特:内蒙古农业大学, 2016. JIANG X W.Optimization of extraction process of taurocholic acid from sheep bile and methodological study of taurocholic acid HPLC[D].Hohhot:Inner Mongolia Agricultural University, 2016.(in Chinese)
[19] YANG B, HUANG S, LI S, et al.Safety evaluation of porcine bile acids in laying hens:Effects on laying performance, egg quality, blood parameters, organ indexes, and intestinal development[J].Frontiers in Veterinary Science, 2022, 9:895831.
[20] LAI W, CAO A, LI J, et al.Effect of high dose of bile acids supplementation in broiler feed on growth performance, clinical blood metabolites, and organ development[J].Journal of Applied Poultry Research, 2018, 27(4):532-539.
[21] KATSUKI K, FUJIMOTO M, ZHANG X Y, et al.Feeding induces expression of heat shock proteins that reduce oxidative stress[J].FEBS Letters, 2004, 571(1-3):187-191.
[22] SUN J, YIN B, TANG S, et al.Vitamin C mitigates heat damage by reducing oxidative stress, inducing HSP expression in TM4 sertoli cells[J]. Molecular Reproduction and Development, 2019, 86(6):673-685.
[23] HEADS R J, YELLON D M, LATCHMAN D S.Differential cytoprotection against heat stress or hypoxia following expression of specific stress protein genes in myogenic cells[J].Journal of Molecular Cellular Cardiology, 1995, 27:1669-1678.
[24] HUO C, XIAO C, SHE R, et al.Chronic heat stress negatively affects the immune functions of both spleens and intestinal mucosal system in pigs through the inhibition of apoptosis[J]. Microbial Pathogenesis, 2019, 136:103672.
[25] 王丹, 仲庆振, 孙泽威.热应激对肉鸡的影响及热休克蛋白70的研究进展[J].中国畜牧兽医, 2011, 38(1):51-54. WANG D, ZHONG Q Z, SUN Z W.Effects of heat stress on broilers and research progress of heat shock protein 70[J].China Animal Husbandry & Veterinary Medicine, 2011, 38(1):51-54.(in Chinese)
[26] 张霞, 屈平平, 丛霞.热应激对小鼠胚胎细胞HSP70表达和超微结构的影响[J].畜牧兽医学报, 2011, 42(10):1463-1469. ZHANG X, QU P P, CONG X.Effects of heat stress on HSP70 expression and ultrastructure of mouse embryonic cells[J].Acta Veterinaria et Zootechnica Sinica, 2011, 42(10):1463-1469.(in Chinese)
[27] 黄帆, 吕秋凤.热应激诱导的氧化应激对动物肠道组织的损伤[J].动物营养学报, 2017, 29(6):1856-1860. HUANG F, LYU Q F.Effects of oxidative stress induced by heat stress on intestinal tissue of animals[J].Chinese Journal of Animal Nutrition, 2017, 29(6):1856-1860.(in Chinese)
[28] LIU L, FU C, YAN M, et al.Resveratrol modulates intestinal morphology and HSP70/90, NF-κB and EGF expression in the jejunal mucosa of Black-boned chickens on exposure to circular heat stress[J]. Food and Function, 2016, 7(3):1329-1338.
[29] 郭月, 刘小宝, 李健.马齿苋水提物对热应激小鼠空肠结构及吸收功能的影响[J].畜牧兽医学报, 2020, 51(4):873-880. GUO Y, LIU X B, LI J.Effects of portulaca oleracea aqueous extract on jejunal structure and absorption function in heat stressed mice[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(4):873-880.(in Chinese)
[30] MUJAHID A, YOSHIKI Y, AKIBA Y, et al.Superoxide radical production in chicken skeletal muscle induced by acute heat stress[J]. Poultry Science, 2005, 84(2):307-314.
[31] HYDER M A, HASAN M, MOHIELDEIN A H.Com-parative levels of ALT, AST, ALP and GGT in liver as-sociated diseases[J].European Journal of Experimental Biology, 2013, 3(2):280.
[32] HUANG S C, FU Y F, REHMAN M U, et al.Effect of the acute heat stress on serum endotoxin concentration and the expression of TLR4 mRNA in liver of arbor acres broiler chickens[J]. Animal Production Science, 2018, 58(12):2200.
[33] MUJAHID A, PUMFORF N R, BOTTJE W, et al.Mitochondrial oxidative damage in chicken skeletal muscle induced by acute heat stress[J].Journal of Poultry Science, 2007, 44(4):439-445.
[34] SCHIENER M, CHANDEL N S.ROS function in redox signaling and oxidative stress[J].Current Biology, 2014, 24(10):453-462.
[35] TSIKAS D.Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples:Analytical and biological challenges[J]. Analyical Biochemistry, 2017, 524:13-30.
[36] LIU L L, HE J H, XIE H B, et al.Resveratrol induces antioxidant and heat shock protein mRNA expression in response to heat stress in black-boned chickens[J]. Poultry Science, 2014, 93(1):54-62.
[37] 李培锋.牛羊胆汁中有效成分的提取纯化试验研究[D].呼和浩特:内蒙古农业大学, 2007. LI P H.Experimental study on extraction and purification of effective components from bile of cattle and sheep[D].Hohhot:Inner Mongolia Agricultural University, 2007.(in Chinese)
[38] 乌云夫.甘氨胆酸对小鼠的抗氧化作用研究[D].呼和浩特:内蒙古农业大学, 2013. WU Y F.Study on antioxidation of glycocholic acid in mice[D].Hohhot:Inner Mongolia Agricultural University, 2013.(in Chinese)
[39] CAO L, TANG J, LI Q, et al.Expression of selenoprotein genes is affected by heat stress in IPEC-J2 cells[J].Biological Trace Element Research, 2016, 172(2):354-360.
[40] YANG H H, HOU C C, LIN M T, et al.Attenuating heat-induced acute lung inflammation and injury by dextromethorphan in rats[J]. American Journal Respiratory Cell Molecular Biology, 2012, 46(3):407-413.
[41] GUO C, XIE S, CHI Z, et al.Bile acids control inflammation and metabolic disorder through inhibition of NLRP3 inflammasome[J]. Immunity, 2016, 45(4):802-816.