茶树油对脂多糖诱导的奶牛瘤胃上皮细胞炎症因子表达的影响

Effects of Tea Tree Oil on Expression of Inflammatory Factors in Bovine Rumen Epithelial Cells Induced by Lipopolysaccharide

本试验旨在研究茶树油(TTO)对脂多糖(LPS)诱导的奶牛瘤胃上皮细胞(BRECs)炎症因子表达的影响。利用CCK-8法测定浓度为0(对照)、0.00625%、0.0125%、0.025%、0.05%、0.1%的TTO对BRECs活力的影响,每组6个重复,根据试验结果筛选出TTO的适宜浓度为0.05%,以此浓度做后续试验。再利用实时荧光定量PCR、蛋白质印迹法,分别探究BRECs中炎症因子在基因水平以及蛋白水平表达量的变化。试验分为4组:对照组不添加TTO和LPS;TTO组仅添加0.05%的TTO;LPS组仅添加1μg/mL LPS;LPS+TTO组添加1μg/mL LPS和0.05%TTO,每组6个重复。结果表明:1)与对照组相比,添加0.05%TTO显著提高BRECs的活力(P<0.05);添加0.1%TTO显著抑制BRECs的活力(P<0.05)。2)与对照组相比,TTO组白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和Toll样受体-4(TLR-4)的基因表达量均显著降低(P<0.05),LPS组白细胞介素-1β(IL-1β)、IL-6、白细胞介素-8(IL-8)、TNF-α、TLR-4基因表达量显著升高(P<0.05);与LPS组相比,在LPS中添加TTO显著降低IL-1β、IL-6、IL-8、TNF-α、TLR-4的基因表达量(P<0.05)。3)与对照组相比,LPS可以提高核因子-κB(NF-κB)通路中p65、磷酸化-p65(p-p65)蛋白表达量,添加LPS+TTO与仅添加LPS相比可以降低p65、p-p65蛋白表达量。综上所述,低浓度的TTO可以提高BRECs的活力,抑制LPS诱导的BRECs炎症信号通路NF-κB相关蛋白表达,进而抑制促炎因子产生。

The purpose of this study was to investigate the effects of tea tree oil(TTO)on the expression of inflammatory factors in bovine rumen epithelial cells(BRECs)induced by lipopolysaccharide(LPS).The cell counting kit-8(CCK-8)was used to determine the effects of different TTO concentrations[0(control),0.00625%,0.0125%,0.025%,0.05%and 0.1%TTO]on the activity of BRECs and each concentration was performed six replicates.The concentration of 0.05%TTO was selected according to the above tests for subsequent experiments.Then,qRT-PCR and Western blot were used to investigate the changes of expression levels of inflammatory factors at gene and protein levels in BRECs.The experiment was divided into four groups:control group(CON group,without adding TTO and LPS),TTO group(only adding 0.05%TTO),LPS group(only adding 1μg/mL LPS),LPS+TTO group(both adding 1μg/mL LPS and 0.05%TTO).Each group was performed six replicates.The results showed as follows:1)compared with the CON group,the cell viability of BRECs was significantly improved by adding 0.05%TTO(P<0.05)and was significantly inhibited by adding 0.1%TTO(P<0.05).2)Compared with the CON group,the expression levels of interleukin(IL)-1β,IL-6,IL-8,tumor necrosis factor-α(TNF-α)and Toll like receptor-4(TLR-4)genes were significantly increased when LPS was added(P<0.05).Compared with LPS group,the expression levels of IL-1β,IL-6,IL-8,TNF-αand TLR-4 genes were significantly decreased by adding TTO to LPS(P<0.05).3)Compared with the CON group,LPS could increase the protein expression levels of p65 and p-p65 in nuclear factor-κB(NF-κB)pathway but adding both LPS and TTO could decrease the protein expression levels of p65 and p-p65.In summary,low concentration of TTO can enhance the activity of BRECs and inhibit the expression of proteins associated with inflammatory signaling pathway NF-κB induced by inflammation of BRECs which caused by LPS,and then can inhibit the production of proinflammatory factor.