氨气浓度对仔猪气管和肺组织及免疫功能的影响
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摘要
为了研究不同浓度氨气刺激下断奶仔猪气管和肺组织结构和免疫功能的变化,选取体重相近、同日龄的12头杜×长×大三元断奶仔猪,随机分为4组,对照组不提供氨气,试验组的氨气浓度分别为22.77、45.54和68.30 mg·m~(-3),每天持续刺激8 h。适应期3 d,试验期11 d,于试验第15天早空腹采血、屠宰取样,通过显微镜观察气管和肺的形态结构变化,并测定相关基因表达量以及血清中细胞因子的含量。结果显示:与对照组相比,处理组仔猪气管粘膜纤毛脱落,鳞状上皮细胞增生,肺泡破裂成空泡,肺泡隔增厚;各处理组气管和肺中Claudin-1 mRNA的表达量显著降低(P<0.05);45.54 mg·m~(-3)组肺组织Caspase-3 mRNA的表达量显著上调(P<0.05),而68.30 mg·m~(-3)组肺与气管Caspase-3 mRNA的表达量都显著上调(P<0.05);45.54和68.30 mg·m~(-3)组气管Muc5ac mRNA的表达量均显著升高(P<0.05)。与对照组相比,3个处理组血清中的IL-2含量均显著降低(P<0.05),但45.54和68.30 mg·m~(-3)组间无显著差异(P>0.05),而IL-6、IL-1β、INF-γ和IL-10的含量显著升高(P<0.05);45.54 mg·m~(-3)组IL-6和IL-10含量与22.77和68.30 mg·m~(-3)组无显著差异(P>0.05),但68.30 mg·m~(-3)组IL-6和IL-10含量均显著高于22.77 mg·m~(-3)组(P<0.05);3个处理组间IL-1β含量均无显著差异(P>0.05);各处理组血清INF-γ含量均显著高于对照组,但45.54和68.30 mg·m~(-3)组间INF-γ含量无显著差异(P>0.05)。结果表明,22.77 mg·m~(-3)以上浓度氨气通过影响断奶仔猪基因的表达量,造成气管和肺粘膜被破坏、细胞凋亡和细胞间紧密连接损伤,发生了炎症反应,并且随着氨气浓度升高而加剧。
The study was carried out to determine the effect of different ammonia gas concentrations on the structural and immunal changes in trachea and lung of weaned piglets. A total of 12 weaned pigs(Landrace×Large White×Duroc) were randomly assigned to four groups randomly. The experimental groups were exposed to 22.77, 45.54 or 68.30 mg·m~(-3) of NH_3 for 11 days after 3 days adaption, respectively. The control group remained untreated. Blood samples were collected on the morning of the 15 th day of the experiment with empty stomach. The morphological structure changes of trachea and lung were observed by microscopes, and the expression levels of related genes and the content of cytokines in serum were determined. The result showed that tracheal mucosal cilia and squamous epithelial cell were changed in treatment groups. The cilia of tracheal mucosa fell off and the squamous epithelial cells proliferated. The alveoli was destroyed to vacuoles and the alveolar septum became thicker in the treatment groups compared with the control group. Compared with the control group, the mRNA expression levels of Claudin-1 in trachea and lung of each treatment groups were significantly decreased(P<0.05).The mRNA expression level of Caspase-3 in lung of 45.54 mg·m~(-3) group was significantly increased compared with the control group(P<0.05), while the expression of Caspase-3 in lung and trachea of 68.30 mg·m~(-3) group was significantly increased(P<0.05). The Muc5 ac mRNA expression levels in trachea of 45.54 and 68.30 mg·m~(-3) group were both significantly increased(P<0.05). The IL-2 levels in serum of the three treatment groups were significantly decreased compared with the control(P<0.05), but there was no significant difference between 45.54 and 68.30 mg·m~(-3) groups(P>0.05). The levels of IL-6, IL-1β, INF-γ and IL-10 were significantly increased in the three treatment groups(P<0.05). The levels of IL-6 and IL-10 in 45.54 mg·m~(-3) group were not significantly different from those in 22.77 and 68.30 mg·m~(-3) groups(P>0.05). But the IL-6 and IL-10 levels in 68.30 mg·m~(-3) group were significantly higher than those in 22.77 mg·m~(-3) group(P<0.05). There was no significant difference in IL-1β content among the three treatment groups(P>0.05). The serum INF-γ content in each treatment group was significantly higher than that in the control, but there was no significant difference between 45.54 and 68.30 mg·m~(-3) groups(P>0.05). The findings indicate that ammonia exceeding 22.77 mg·m~(-3) can cause mucosa destruction,apoptosis and damage in tight junctions by affecting gene expression in the trachea and lung of weaned piglets. And the higher ammonia concentrations were, the more damage the trachea and lung were observed. Ammonia exposure above 22.77 mg·m~(-3) can result in inflammation.
The study was carried out to determine the effect of different ammonia gas concentrations on the structural and immunal changes in trachea and lung of weaned piglets. A total of 12 weaned pigs(Landrace×Large White×Duroc) were randomly assigned to four groups randomly. The experimental groups were exposed to 22.77, 45.54 or 68.30 mg·m~(-3) of NH_3 for 11 days after 3 days adaption, respectively. The control group remained untreated. Blood samples were collected on the morning of the 15 th day of the experiment with empty stomach. The morphological structure changes of trachea and lung were observed by microscopes, and the expression levels of related genes and the content of cytokines in serum were determined. The result showed that tracheal mucosal cilia and squamous epithelial cell were changed in treatment groups. The cilia of tracheal mucosa fell off and the squamous epithelial cells proliferated. The alveoli was destroyed to vacuoles and the alveolar septum became thicker in the treatment groups compared with the control group. Compared with the control group, the mRNA expression levels of Claudin-1 in trachea and lung of each treatment groups were significantly decreased(P<0.05).The mRNA expression level of Caspase-3 in lung of 45.54 mg·m~(-3) group was significantly increased compared with the control group(P<0.05), while the expression of Caspase-3 in lung and trachea of 68.30 mg·m~(-3) group was significantly increased(P<0.05). The Muc5 ac mRNA expression levels in trachea of 45.54 and 68.30 mg·m~(-3) group were both significantly increased(P<0.05). The IL-2 levels in serum of the three treatment groups were significantly decreased compared with the control(P<0.05), but there was no significant difference between 45.54 and 68.30 mg·m~(-3) groups(P>0.05). The levels of IL-6, IL-1β, INF-γ and IL-10 were significantly increased in the three treatment groups(P<0.05). The levels of IL-6 and IL-10 in 45.54 mg·m~(-3) group were not significantly different from those in 22.77 and 68.30 mg·m~(-3) groups(P>0.05). But the IL-6 and IL-10 levels in 68.30 mg·m~(-3) group were significantly higher than those in 22.77 mg·m~(-3) group(P<0.05). There was no significant difference in IL-1β content among the three treatment groups(P>0.05). The serum INF-γ content in each treatment group was significantly higher than that in the control, but there was no significant difference between 45.54 and 68.30 mg·m~(-3) groups(P>0.05). The findings indicate that ammonia exceeding 22.77 mg·m~(-3) can cause mucosa destruction,apoptosis and damage in tight junctions by affecting gene expression in the trachea and lung of weaned piglets. And the higher ammonia concentrations were, the more damage the trachea and lung were observed. Ammonia exposure above 22.77 mg·m~(-3) can result in inflammation.
引文
[1] 李雪,陈凤鸣,熊霞,等.饲养密度对猪群健康和猪舍环境的影响[J].动物营养学报,2017,29(7):2 245-2 251.
[2] NI J Q,LIU S,DIEHL C A,et al.Emission factors and characteristics of ammonia,hydrogen sulfide,carbon dioxide,and particulate matter at two high-rise layer hen houses[J].Atmospheric Environment,2017,154:260-273.
[3] 赵天,王国军,彭孝坤,等.氨气和硫化氢应激对肉羊免疫及抗氧化功能的影响[J].畜牧兽医学报,2018,49(10):2 191-2 204.
[4] 李聪.不同浓度氨气对肉鸡生长性能及呼吸道黏膜屏障的影响[D].北京:中国农业科学院,2014.
[5] 蒋载阳,江书忠,曹霞,等.论猪舍中氨气的危害[J].中国猪业,2018,13(8):58-60,62.
[6] 叶俏.吸入刺激性气体所致肺损伤的临床诊治[C]// 中国毒理学会.中国毒理学会灾害与应急毒理学专业委员会第一次全国学术交流会论文集.北京:《国际药学研究杂志》编辑部,2016:790-790.
[7] DAVID B,MEJDELL C,MICHEL V,et al.Air quality in alternative housing systems may have an impact on laying hen welfare:Part II Ammonia[J].Animals,2015,5(3):886-896.
[8] 徐彬,魏凤仙,李绍钰.氨气的产生及其对肉鸡健康与生产性能的影响[J].中国家禽,2018,40(7):1-5.
[9] 魏凤仙.湿度和氨暴露诱导的慢性应激对肉仔鸡生长性能、肉品质、生理机能的影响及其调控机制[D].杨凌:西北农林科技大学,2012.
[10] 熊嫣.温热环境因子及氨气对肉鸡健康的影响机制研究[D].北京:中国农业科学院,2017.
[11] 黄龙辉.氨气刺激对仔猪呼吸道粘液分泌的影响[D].武汉:华中农业大学,2017.
[12] 中华人民共和国农业部质量标准办公室.畜禽场环境质量标准:NY/T 388-1999[J].养猪,2005(1):42-43.
[13] 江楠,刘美辰,周泠,等.猪带绦虫重组Bb-TSO45W-4B疫苗诱导仔猪保护力及其免疫机制研究[J].中国病原生物学杂志,2015(10):900-904.
[14] VERHOEVEN D,GEORGE M D,HU W,et al.Enhanced innate anti-viral gene expression,interferon-α,and cytolytic responses are predictive of mucosal immune recovery during SIV infection[J].Journal of Immunology,2014,192(7):3 308-3 318.
[15] QUINN S P,ZAHID M J,DURKIN J R,et al.Automated identification of abnormal respiratory ciliary motion in nasal biopsies[J].Science Translational Medicine,2015,7(299):299ra124.
[16] GARCIA M A,NELSON W J,CHAVEZ N.Cell-cell junctions organize structural and signaling networks[J].Cold Spring Harbor Perspectives in Biology,2018,10(4):a029181.
[17] JOHN L J,FROMM M,SCHULZKE J D.Epithelial barriers in intestinal inflammation[J].Antioxidants & Redox Signaling,2011,15(5):1 255-1 270.
[18] 杨慧,王晓楠,丁磊,等.Claudin家族基因剔除小鼠表型的研究进展[J].医学综述,2015(18):3 265-3 268.
[19] COPIN M C,DEVISME L,BUISINE M P,et al.From normal respiratory mucosa to epidermoid carcinoma:Expression of human mucin genes[J].International journal of cancer.Journal international du cancer,2000,86(2):162-168.
[20] PU X,STORR S J,ZHANG Y,et al.Caspase-3 and casp ase-8 expression in breast cancer:caspase-3 is associated with survival[J].Apoptosis,2017,22(3):357-368.
[21] 龙杰,王羽,刘晓翠,等.氧化应激对原代培养乳鼠心房肌细胞中内质网应激及凋亡因子的影响[J].中国应用生理学杂志,2017,33(2):185-188.
[22] GU Z T,WANG H,LI L,et al.Heat stress induces apoptosis through transcription-independent p53-mediated mitochondrial pathways in human umbilical vein endothelial cell[J].Scientific Reports,2014,4:4 496.
[23] 张千,李发弟,李飞.反刍动物应激反应中糖皮质激素对免疫系统的调节机理[J].畜牧兽医学报,2017,48(05):785-792.
[24] ROSENBERG S A.IL-2:The first effective immunotherapy for human cancer[J].The Journal of Immunology,2014,192(12):5 451-5 458.
[25] LIAO W,LIN J X,WANG L,et al.Modulation of cytokine receptors by IL-2 broadly regulates differentiation into helper T cell lineages[J].Nature Immunology,2011,12(6):551-559.
[26] YANG Z Z,GROTE D M,ZIESMER S C,et al.Soluble IL-2Rα facilitates IL-2-mediated immune responses and predicts reduced survival in follicular B-cell non-Hodgkin lymphoma[J].Blood,2011,118(10):2 809-2 820.
[27] GRINBERG B Y,BAEYENS A,YOU S,et al.IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells[J].Journal of Experimental Medicine,2010,207(9):1 871-1 878.
[28] 涂浩,赵星灿,杨占娜,等.猪重组IL-2、IL-4和IFN-γ对口蹄疫合成肽疫苗的免疫增强作用研究[J].畜牧兽医学报,2015,46(8):1 390-1 399.
[29] MCGEOUGH M D,PENA C A,MUELLER J L,et al.Cutting edge:IL-6 is a marker of inflammation with no direct role in inflammasome-mediated mouse models[J].The Journal of Immunology,2012,189(6):2 707-2 711.
[30] ROPELLE E R,FLORES M B,CINTRA D E,et al.IL-6 and IL-10 anti-inflammatory activity links exercise to hypothalamic insulin and leptin sensitivity through IKKbeta and ER stress inhibition[J].Plos Biology,2010,8(8):697-704.
[31] 樊延霞.缺血性脑卒中与血清白介素-1β、白介素-2、肿瘤坏死因子-α的相关性[D].泰安:泰山医学院,2015.
[32] CHAUDHRY H,ZHOU J,ZHONG Y,et al.Role of cytokines as a double-edged sword in sepsis[J].Vivo,2013,27(6):669-684.
[33] 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):2 246-2 253.
[2] NI J Q,LIU S,DIEHL C A,et al.Emission factors and characteristics of ammonia,hydrogen sulfide,carbon dioxide,and particulate matter at two high-rise layer hen houses[J].Atmospheric Environment,2017,154:260-273.
[3] 赵天,王国军,彭孝坤,等.氨气和硫化氢应激对肉羊免疫及抗氧化功能的影响[J].畜牧兽医学报,2018,49(10):2 191-2 204.
[4] 李聪.不同浓度氨气对肉鸡生长性能及呼吸道黏膜屏障的影响[D].北京:中国农业科学院,2014.
[5] 蒋载阳,江书忠,曹霞,等.论猪舍中氨气的危害[J].中国猪业,2018,13(8):58-60,62.
[6] 叶俏.吸入刺激性气体所致肺损伤的临床诊治[C]// 中国毒理学会.中国毒理学会灾害与应急毒理学专业委员会第一次全国学术交流会论文集.北京:《国际药学研究杂志》编辑部,2016:790-790.
[7] DAVID B,MEJDELL C,MICHEL V,et al.Air quality in alternative housing systems may have an impact on laying hen welfare:Part II Ammonia[J].Animals,2015,5(3):886-896.
[8] 徐彬,魏凤仙,李绍钰.氨气的产生及其对肉鸡健康与生产性能的影响[J].中国家禽,2018,40(7):1-5.
[9] 魏凤仙.湿度和氨暴露诱导的慢性应激对肉仔鸡生长性能、肉品质、生理机能的影响及其调控机制[D].杨凌:西北农林科技大学,2012.
[10] 熊嫣.温热环境因子及氨气对肉鸡健康的影响机制研究[D].北京:中国农业科学院,2017.
[11] 黄龙辉.氨气刺激对仔猪呼吸道粘液分泌的影响[D].武汉:华中农业大学,2017.
[12] 中华人民共和国农业部质量标准办公室.畜禽场环境质量标准:NY/T 388-1999[J].养猪,2005(1):42-43.
[13] 江楠,刘美辰,周泠,等.猪带绦虫重组Bb-TSO45W-4B疫苗诱导仔猪保护力及其免疫机制研究[J].中国病原生物学杂志,2015(10):900-904.
[14] VERHOEVEN D,GEORGE M D,HU W,et al.Enhanced innate anti-viral gene expression,interferon-α,and cytolytic responses are predictive of mucosal immune recovery during SIV infection[J].Journal of Immunology,2014,192(7):3 308-3 318.
[15] QUINN S P,ZAHID M J,DURKIN J R,et al.Automated identification of abnormal respiratory ciliary motion in nasal biopsies[J].Science Translational Medicine,2015,7(299):299ra124.
[16] GARCIA M A,NELSON W J,CHAVEZ N.Cell-cell junctions organize structural and signaling networks[J].Cold Spring Harbor Perspectives in Biology,2018,10(4):a029181.
[17] JOHN L J,FROMM M,SCHULZKE J D.Epithelial barriers in intestinal inflammation[J].Antioxidants & Redox Signaling,2011,15(5):1 255-1 270.
[18] 杨慧,王晓楠,丁磊,等.Claudin家族基因剔除小鼠表型的研究进展[J].医学综述,2015(18):3 265-3 268.
[19] COPIN M C,DEVISME L,BUISINE M P,et al.From normal respiratory mucosa to epidermoid carcinoma:Expression of human mucin genes[J].International journal of cancer.Journal international du cancer,2000,86(2):162-168.
[20] PU X,STORR S J,ZHANG Y,et al.Caspase-3 and casp ase-8 expression in breast cancer:caspase-3 is associated with survival[J].Apoptosis,2017,22(3):357-368.
[21] 龙杰,王羽,刘晓翠,等.氧化应激对原代培养乳鼠心房肌细胞中内质网应激及凋亡因子的影响[J].中国应用生理学杂志,2017,33(2):185-188.
[22] GU Z T,WANG H,LI L,et al.Heat stress induces apoptosis through transcription-independent p53-mediated mitochondrial pathways in human umbilical vein endothelial cell[J].Scientific Reports,2014,4:4 496.
[23] 张千,李发弟,李飞.反刍动物应激反应中糖皮质激素对免疫系统的调节机理[J].畜牧兽医学报,2017,48(05):785-792.
[24] ROSENBERG S A.IL-2:The first effective immunotherapy for human cancer[J].The Journal of Immunology,2014,192(12):5 451-5 458.
[25] LIAO W,LIN J X,WANG L,et al.Modulation of cytokine receptors by IL-2 broadly regulates differentiation into helper T cell lineages[J].Nature Immunology,2011,12(6):551-559.
[26] YANG Z Z,GROTE D M,ZIESMER S C,et al.Soluble IL-2Rα facilitates IL-2-mediated immune responses and predicts reduced survival in follicular B-cell non-Hodgkin lymphoma[J].Blood,2011,118(10):2 809-2 820.
[27] GRINBERG B Y,BAEYENS A,YOU S,et al.IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells[J].Journal of Experimental Medicine,2010,207(9):1 871-1 878.
[28] 涂浩,赵星灿,杨占娜,等.猪重组IL-2、IL-4和IFN-γ对口蹄疫合成肽疫苗的免疫增强作用研究[J].畜牧兽医学报,2015,46(8):1 390-1 399.
[29] MCGEOUGH M D,PENA C A,MUELLER J L,et al.Cutting edge:IL-6 is a marker of inflammation with no direct role in inflammasome-mediated mouse models[J].The Journal of Immunology,2012,189(6):2 707-2 711.
[30] ROPELLE E R,FLORES M B,CINTRA D E,et al.IL-6 and IL-10 anti-inflammatory activity links exercise to hypothalamic insulin and leptin sensitivity through IKKbeta and ER stress inhibition[J].Plos Biology,2010,8(8):697-704.
[31] 樊延霞.缺血性脑卒中与血清白介素-1β、白介素-2、肿瘤坏死因子-α的相关性[D].泰安:泰山医学院,2015.
[32] CHAUDHRY H,ZHOU J,ZHONG Y,et al.Role of cytokines as a double-edged sword in sepsis[J].Vivo,2013,27(6):669-684.
[33] 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):2 246-2 253.