Abstract 【Objective】Cow mastitis has been one of the biggest challenges in dairy farming and dairy products industry, which restricts the healthy development of dairy industry. Effective prevention and treatment of cow mastitis can provide a good guarantee for the health of cows and the production of high-quality dairy products. This experiment explored the effects of tea tree oil on LPS-induced mastitis in dairy cows, and explored the feasibility of using tea tree oil instead of antibiotics to treat mastitis in dairy cows. This experiment provides a reference for the treatment of dairy cow mastitis with tea tree oil. 【Methods】The cells in good condition, were selected, which were added 50, 100, 200, 500, and 1 000 μg·mL-1 LPS respectively in the culture of these cells to detect the relevant indicators. The tea tree oil and LPS were added to the model for co-culture. The model of dairy cow mastitis induced LPS was established by the CCK-8 method, flow cytometry, real-time fluorescence quantification and ELISA assay. Antagonistic effect of tea tree oil on LPS in dairy cow mastitis cell model: 0.0002%, 0.0004%, 0.0006%, 0.0008%, 0.001%, 0.002%, 0.004%, 0.006%, 0.008% and 0.01% tea tree oil were added to the dairy cow mastitis cell model induced by 200 μg·mL-1 LPS for 12 hours to detect the related indexes. 【Result】CCK-8 method was used to detect the cell proliferation activity. The results showed that under the condition of 100 μg·mL-1 LPS poisoning, the activity of the cells began to decline in varying degrees. There was not a large number of apoptosis in 100 μg·mL-1 LPS after 12 hours of induction, while about 46% of the cells showed early and late apoptosis in 200 μg·mL-1 LPS. 200 μg·mL-1 LPS induced for 12 hours was the best condition for the establishment of mastitis model. The results also showed that when tea tree oil concentration was 0.0004%, 0.0006% and 0.0008%, the apoptosis rate of the cells decreased. Among them, when tea tree oil concentration was 0.0006%, the protection effect was the most obvious. The proportion of living cells was 71.95%, the proportion of early apoptotic cells was 22.15%, and the proportion of late apoptotic cells was 5.11%; compared with the living cells, the proportion of the mastitis model group of tree oil increased by about 22%. After that, the expression of cytokines and apoptotic factors were detected by qPCR in the three groups with protective effect. With the increase of the concentration of tea tree oil, the expression of TNF-α was down regulated more, the expression of IL-6 was down regulated less (P< 0.01), and the expression of STAT1 was up regulated slightly when 0.0004% tea tree oil was added, while down regulated slightly when 0.0006% and 0.0008% tea tree oil were added, and the expression of tea tree oil with 0.0006% concentration was the lowest (P< 0.05). The expression of NF-κB, MAPK and caspase-3 was significantly reduced in the three groups of tea tree oil adding concentration. Among them, the expression of inflammatory response protein in 0.0006% tea tree oil group was the lowest, about 50% of that in the blank control group. The protein expression was almost the same, about 55% of the blank control group (P< 0.05).【Conclusion】Tea tree oil had a certain antagonistic effect on LPS within the appropriate concentration range, which could reduce the proportion of apoptosis, improve the survival proportion of normal cells, and down regulate the expression of inflammatory factors, apoptosis factors and corresponding proteins. Keywords:tea tree oil;cow;LPS;mastitis;effect mechanism
PDF (1253KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文 本文引用格式 陈志, 张逸, 路钦越, 郭佳禾, 梁艳, 张明怡星, 杨章平. 茶树油对LPS诱导的奶牛乳腺炎的作用及其机制[J]. 中国农业科学, 2021, 54(14): 3124-3133 doi:10.3864/j.issn.0578-1752.2021.14.017 CHEN Zhi, ZHANG Yi, LU QinYue, GUO JiaHe, LIANG Yan, ZHANG MingYiXing, YANG ZhangPing. Effect and Mechanism of Tea Tree Oil on LPS Induced Mastitis in Dairy Cows[J]. Scientia Acricultura Sinica, 2021, 54(14): 3124-3133 doi:10.3864/j.issn.0578-1752.2021.14.017
开放科学(资源服务)标识码(OSID):
0 引言
【研究意义】乳腺是合成乳汁的主要场所,随着人们营养观念的转变,乳品已成为消费需求的主流[1]。奶牛乳腺炎影响奶牛的健康,制约奶牛业的发展,近期有研究显示,奶牛疾病抗性越强,如奶牛乳房炎和部分繁殖疾病,则长寿性表现越好[2]。因此,奶牛的健康养殖和抗病营养是提高我国奶牛养殖水平、改善奶品质和增强市场竞争力的新型支撑技术[3]。大部分奶牛乳腺炎由病原菌导致,可用抗生素进行治疗。但随着耐药性问题日益突出,如何有效防治奶牛乳腺炎,寻找新的抗菌物质,成为当前奶牛养殖业研究热点。【前人研究进展】奶牛乳腺炎是指由于各种物理、化学、病原菌等因素引起的乳房炎症,导致奶牛乳腺组织发生病理学变化,进而导致所分泌乳汁发生理化性质和细菌学变化[4]。引起奶牛乳腺炎的病因很复杂,但最主要的还是由病原微生物引起,常见的病原菌有20多种[5],其中以金黄色葡萄球菌、无乳链球菌、停乳链球菌和大肠杆菌为主,如李蕊等[6]研究发现金黄色葡萄球菌诱导型乳腺炎影响了奶牛脂肪酸的合成过程,降低了脂肪酸各组分的绝对含量;吴富鑫等[7]也对无乳链球菌在乳腺中的感染和传播的过程进行了讨论。LPS是大肠杆菌等革兰氏阴性菌细胞壁的主要成分之一,是导致乳腺炎发病的主要致病因素。国内外研究已经在多种动物上成功构建LPS诱导的乳腺炎模型[8,9],在大肠杆菌导致的奶牛乳腺炎上,LPS所引起的急性临床指标与乳汁中酶活性和急性期蛋白密切相关[10]。奶牛乳腺炎会导致奶牛产奶量大幅下降,奶品质降低,同时导致奶牛乳腺的伤害。防治奶牛乳腺炎意义非凡,对于奶牛乳腺炎的治疗主要还是使用抗生素,但大部分牛场基本都是在不明确病原菌及抗生素耐药性的情况下滥用抗生素,导致病原菌的耐药及药物残留[11],进而降低了治疗效果。寻找有效的抗生素替代品进行奶牛乳腺炎的防治成为一项具有重要意义的研究项目。茶树油(tea tree oil,TTO)是指取自于桃金娘科(Myrtaceae)白千层(Melaleuca L.)的数种植物的精油,其中最主要的一种为互叶白千层(M.alternifola),故又称为互叶白千层油。茶树油是许多非处方保健品和化妆品的常用成分,随着天然药物的蓬勃发展,越来越多的人使用含有茶树油的产品[12]。茶树油抗菌谱广、抗菌活性强,故被用于治疗由真菌、细菌或病毒引起的相关疾病,如陈昕等[13]研究发现茶树油提取物粉对大鼠免疫功能有较明显的增强作用,其有望开发成为一种可缓解全球“抗菌药物危机”的天然抗菌剂[14]。【本研究切入点】随着抗生素滥用所导致的耐药性问题,寻找新型有效抗菌物质迫在眉睫。本研究旨在探究茶树油对LPS诱导的奶牛乳腺炎的作用及其机制,探索茶树油替代抗生素治疗奶牛乳腺炎的可行性,为茶树油治疗奶牛乳腺炎提供参考。【拟解决的关键问题】构建LPS诱导的奶牛乳腺炎模型以及茶树油对该乳腺炎模型的作用及其机制。
与空白对照组比较,#表示差异显著( P<0.05),##表示差异显著(P<0.05); 与 LPS 组比较,*表示差异显著(P<0.05),** 表示差异极显著(P<0.01)。下同 Fig. 4Effect of TTO on the expression of inflammatory factors and apoptotic factors in mastitis mode
# meansP<0.05 and ## meansP<0.01, vs control group;* meansP<0.05 and ** meansP<0.01, vs LPS group. The same as below
WANG JK, LIU JX. Metabolism of amino acids in the mammary gland of lactation ruminants Scientia Agricultura Sinica, 2005, 38(7):1453-1457. (in Chinese) [本文引用: 1]
ZHANG HL, LIU AX, MI SY, LIX, LUO HP, YAN XY, WANG YC. Longevity traits in dairy cattle breeding Scientia Agricultura Sinica, 2020, 53(19):4070-4082. (in Chinese) [本文引用: 1]
LIU LQ. Dairy cow mastitis and its prevention and treatment Animal Husbandry and Veterinary Science and Technology Information, 2016(9):67-68. (in Chinese) [本文引用: 1]
XU YP. The etiology and prevention of dairy cow mastitis Animal Husbandry and Veterinary Science and Technology Information, 2015(6):57-57. (in Chinese) [本文引用: 1]
LIR, ZHANG CL, GENG XH, LIR, PU JH, MAO YJ, JI DJ, YANG ZP, WU SH, XU XQ, LU CB, REN XL. Effects of Staphylococcus aureus-induced mastitis on fatty acid composition in milk of Chinese Holstein dairy cows Chinese Journal of Animal Nutrition, 2016, 28(2):426-435. (in Chinese) [本文引用: 1]
WU FX, XIONG BH, TONG JJ, JIANG LS. Factors related to streptococcus agalactiae virulence in dairy cows with mastitis Acta Veterinaria et Zootechnica Sinica, 2020, 51(12):2954-2963. (in Chinese) [本文引用: 1]
YANG ZT, YIN RL, CONG YF, YANG ZQ, ZHOU ES, WEI ZK, LIU ZC, CAO YG, ZHANG NS. Oxymatrine lightened the inflammatory response of LPS-induced mastitis in mice through affecting NF-κB and MAPKs signaling pathways Inflammation, 2014, 37(6):2047-2055. DOI:10.1007/s10753-014-9937-7URL [本文引用: 1]
CAO SZ, YANG DY, YU SM, SHEN LH, LIU CS. Establishment of experimental mastitis model by Lipopolysaccharide via teat duct in rabbit Animal Husbandry and Feed Science, 2010, 11:26-27. [本文引用: 1]
LARSENT, RØNTVED CM, INGVARTSEN KL, VELSL, BJERRINGM. Enzyme activity and acute phase proteins in milk utilized as indicators of acute clinical E. coli LPS-induced mastitis Animal, 2010, 4(10):1672-1679. DOI:10.1017/S1751731110000947URL [本文引用: 1]
CHENX, HAN LL, WEI QH, ZHU XY, CHENX, ZHANG YM. Effects of tea tree oil extract powder on immune function in rats Chinese Journal of Veterinary Medicine, 2020, 56(9):74-77. (in Chinese) [本文引用: 1]
NIUB, LIUY, LIANG JP. Research progress on pharmacological effects and related applications of tea tree oil China Animal Husbandry and Veterinary, 2018. (in Chinese) [本文引用: 1]
LINJ. The protective effect of plant essential oils on LPS-induced injury of bovine mammary epithelial cells [D]. Beijing: Chinese Academy of Agricultural Sciences, 2016. (in Chinese) [本文引用: 1]
LU LM, LI QZ, HUANG JG, GAO XJ. Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis Molecules, 2012, 18(1):263-275. DOI:10.3390/molecules18010263URL [本文引用: 1]
CAO CH, HE YJ, GAO YR, SHI ZD, JI XX. LPS-mediated inflammatory response and its mechanism Journal of Henan University: Medical Edition, 2017(36):76. (in Chinese) [本文引用: 1]
WANG LF, JIA SD, YANG GQ, ZHU HS, LIU RY, YANP, LIM, YANG GY. Effects of lipopolysaccharide on liver metabolomics of dairy goats Scientia Agricultura Sinica, 2015, 48(18):3701-3710. (in Chinese) [本文引用: 1]
LIU LX, LINY, ZHANGL, LI QZ. Effects of lipopolysaccharide on milk cow mammary epithelial cytotoxicity and milk protein synthesis Journal of Northeast Agricultural University, 2015, 46(6):61-66. (in Chinese) [本文引用: 1]
刘珊英. 低剂量LPS激活NF-κB促进胰岛β细胞株NIT-1增殖 中国药理学通报, v. 25(11):1430-1433. [本文引用: 1]
LIU SY. Low dose LPS enhances proliferation of a mouse insulinoma cell line NIT-1 Chinese Pharmacological Bulletin, v. 25(11):1430-1433. (in Chinese) [本文引用: 1]
WANGY, WANG ZW. In vitroexperiment of different sources of tea tree oil inhibiting LPS-induced TNF-α release Journal of the Third Military Medical University, 2007, 29(04):324-327. (in Chinese) [本文引用: 1]
RAINARDP, RIOLLET, CÉLINE. Innate immunity of the bovine mammary gland Veterinary Research, 2015, 37(3):369-400. DOI:10.1051/vetres:2006007URL [本文引用: 1]
WEN YL, ZHANGY, YAO XP, SHEN LH, YU SM, CAO SZ. Protective effect of baicalin on LPS-induced mastitis in rabbits Natural Products Research and Development, 2016(9):1377-1383. (in Chinese) [本文引用: 1]
ZOU XL, YANGX, ZHANG YF, DONG XQ, PENG WX, WANG CY, YU XQ. The relationship between the expression of PPAR-γ, TLR4 and STAT1 signal activation in peritoneal tissues and LPS- induced acute peritonitis in rats Chinese Journal of Microbiology and Immunology, 2009, 29(8):716-722. (in Chinese) [本文引用: 1]
SCHMITZS, PFAFFL MW, MEYER H HD, BRUCKMAIER RM. Short-term changes of mRNA expression of various inflammatory factors and milk proteins in mammary tissue during LPS-induced mastitis Domestic Animal Endocrinology, 2004, 26(2):0-126. [本文引用: 1]
HEINRICHP, BEHRMANNI, HAANS, HERMANNSH, MÜLLER-NEWENG, SCHAPERF. Principles of interleukin (IL)-6-type cytokine signalling and its regulation Biochemical Journal, 2003, 374(1):1. DOI:10.1042/bj20030407URL [本文引用: 1]
LI SH, ZHANG XD. Research progress on the structure and function of STAT1 International Journal of Transplantation and Blood Purification, 2008, 6(1):39-42. (in Chinese) [本文引用: 1]
CHEN JY, WANGC, WANGJ, CAO LR. Research progress of MAPK signaling pathway Chinese Medical Science, 2011, 1(8):32-34. (in Chinese) [本文引用: 1]
XU XX, WENH, HU YZ, YUH, ZHANGY, CHENC, PANX. STAT1-caspase 3 pathway in the apoptotic process associated with steroid-induced necrosis of the femoral head Journal of Molecular Histology, 2014, 45(4):473-485. DOI:10.1007/s10735-014-9571-6URL [本文引用: 1]