茶树油对LPS诱导的奶牛乳腺炎的作用及其机制

Effect and Mechanism of Tea Tree Oil on LPS Induced Mastitis in Dairy Cows

【目的】奶牛乳腺炎一直是奶牛养殖业和奶制品行业最大的挑战之一,制约着奶业的健康发展。有效进行奶牛乳腺炎的防治,可以为奶牛的健康、生产优质乳制品提供良好保障。探究茶树油对LPS诱导的奶牛乳腺炎作用效果,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。【方法】在牛乳腺上皮细胞的培养中分别添加50、100、200、500、1 000μg·mL^(-1)的LPS进行相关指标的检测。通过CCK-8法检测细胞增殖活性、流式细胞仪检测细胞凋亡、实时荧光定量检测细胞因子表达量以及ELISA检测相关蛋白的表达量等方法检测乳腺上皮细胞的相关指标。研究构建了LPS诱导的奶牛乳腺炎模型。在200μg·mL^(-1) LPS诱导12h的奶牛乳腺炎细胞模型中分别添加0.0002%、0.0004%、0.0006%、0.0008%、0.001%、0.002%、0.004%、0.006%、0.008%、0.01%的茶树油进行相关指标的检测。【结果】CCK-8法检测细胞增殖活性,结果显示100μg·mL^(-1)的LPS攻毒情况下,细胞已开始出现不同程度的活性下降情况。进一步使用流式细胞术检测细胞凋亡情况,结果显示在诱导12h的情况下,100μg·mL-1的LPS并未出现大量的细胞凋亡,而200μg·mL^(-1)的LPS诱导情况下约有46%左右的细胞出现了早期凋亡和晚期凋亡。以上结果表明:试验中,200μg·mL^(-1) LPS诱导12h为构建奶牛乳腺炎模型的最佳条件。添加茶树油浓度为0.0004%、0.0006%、0.0008%时细胞的凋亡比例会有所下降,其中添加茶树油浓度为0.0006%的这一组保护效果最为明显,其活细胞比例71.95%,早期凋亡细胞比例22.15%,晚期凋亡细胞比例5.11%,与未添加茶树油的乳腺炎模型组活细胞相比提高了约22%。之后对有保护效果的3组进行qPCR检测细胞因子与凋亡因子表达量,结果显示随着加入茶树油的浓度上升,TNF-α表达量下调的较多,IL-6的表达量下调的较少(P<0.01),STAT1的表达量在加入0.0004%浓度茶树油时有轻微的上调,在0.0006%和0.0008%浓度时表达量略微下调,其中添加0.0006%浓度茶树油表达量最低(P<0.05)。进一步使用ELISA法检测炎症蛋白和凋亡蛋白表达量,结果显示3组浓度的茶树油添加组均极显著降低了NF-κB、MAPK和Caspase-3的表达量,其中,0.0006%浓度茶树油组较其余浓度组的炎症反应蛋白的表达量最低,约为空白对照组的50%(P<0.05),而这3组的凋亡反应相关蛋白表达量则几乎持平,约为空白对照组的55%(P<0.05)。【结论】茶树油对于奶牛乳腺炎有一定的拮抗作用,可以降低细胞凋亡比例,提高正常细胞的存活比例,并下调炎症因子与凋亡因子以及相应蛋白的表达量。

【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 1000μg·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 200μg·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 100μg·mL^(-1) LPS poisoning,the activity of the cells began to decline in varying degrees.There was not a large number of apoptosis in 100μg·mL^(-1) LPS after 12 hours of induction,while about 46%of the cells showed early and late apoptosis in 200μg·mL^(-1) LPS.200μg·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-αwas 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.