GPR120的激活与抑制对LTA诱导奶牛乳腺上皮细胞相关促炎基因表达及细胞活力的影响

Effect of Activation and Inhibition of GPR120 on LTA-induced Expression of Pro-inflammatory Genes and Cell Viability in Mammary Epithelial Cells of Dairy Cows

为探讨GPR120基因对LTA介导的奶牛乳腺上皮细胞的炎症反应的缓解作用,试验使用GPR120激活剂TUG891与抑制剂AH7614处理Mac-T细胞,选用20μg·m L^(-1)的LTA刺激Mac-T细胞构,建奶牛乳腺炎症细胞模型;通过q RT-PCR、Western blot与CCK-8法检测各组GPR120的表达水平及激活与抑制GPR120后在LTA诱导的Mac-T细胞炎症应答过程中,细胞内相关促炎因子的m RNA表达变化及对细胞活力的影响。结果表明:使用GPR120激活剂与抑制剂处理细胞3 h,GPR120的m RNA表达水平均达到极显著上调与抑制(P<0.01);使用20μg·m L^(-1)LTA刺激Mac-T细胞24 h后,检测促炎细胞因子TNF-α、IL-1β、IL-6的m RNA表达水平均明显上调(P<0.05),表明基于使用LTA构建的奶牛乳房炎的Mac-T细胞模型成功,且LTA刺激显著促进GPR120的表达水平(P<0.05);激活GPR120可显著缓解由LTA诱导的炎性细胞因子TNF-α、IL-6和IL-1β的释放(P<0.05);而抑制GPR120可进一步显著加剧由LTA诱导的Mac-T细胞产生的促炎细胞因子TNF-α、IL-1β、IL-6的高表达(P<0.01),蛋白表达水平结果与上述一致。用LTA刺激Mac-T细胞后48 h内,细胞活力显著下降(P<0.01);而使用激活剂处理后可显著缓解由LTA诱导引发细胞活力的降低(P<0.01),使用抑制剂处理后进一步引发细胞活力降低。由此可见,GPR120参与了LTA诱导的牛乳腺上皮细胞的炎症反应,并且GPR120的激活减缓了炎症反应,这一研究结果可为筛选奶牛乳房炎抗性基因提供重要依据。

To investigate the effect of the GPR120 gene on the mitigation of LTA-mediated inflammation in mammary epithelial cells of dairy cows,Mac-T cells were treated with GPR120 activator TUG891 and inhibitor AH7614,and Mac-T cells were stimulated with 20μg·mL^(-1) of LTA to construct a mammary inflammatory cell model.The mRNA expression of the relevant intracellular pro-inflammatory factors and their effects on cell viability were examined by qRT-PCR and CCK-8 assay in the Mac-T cell inflammatory response.The results showed that the mRNA expression levels of GPR120 were significantly up-regulated and inhibited by the treatment of GPR120 activator and inhibitor for 3 h.The mRNA expression levels of pro-inflammatory cytokines TNF-α,IL-1βand IL-6 were significantly up-regulated by the stimulation of Mac-T cells with 20μg·mL^(-1) LTA for 24 h(P<0.05).The expression levels of the pro-inflammatory cytokines TNF-α,IL-1βand IL-6 were significantly up-regulated(P<0.05),indicating that the Mac-T cell model of mastitis in cows constructed using LTA was successful,and that LTA stimulation significantly promoted the expression level of GPR120(P<0.05);activation of GPR120 significantly alleviated the release of the inflammatory cytokines TNF-α,IL-6 and IL-1βinduced by LTA(P<0.05);and inhibition of GPR120 significantly increased the expression level of GPR120(P<0.05).further significantly exacerbated the high expression of pro-inflammatory cytokines TNF-α,IL-1βand IL-6 produced by Mac-T cells induced by LTA(P<0.01).Within 48 h of stimulation of Mac-T cells with LTA,cell viability was significantly reduced(P<0.01),while treatment with activator significantly alleviated the reduction in cell viability induced by LTA(P<0.01),which was further triggered by treatment with inhibitor.It suggested that GPR120 was involved in the LTA-induced inflammatory response in bovine mammary epithelial cells and that activation of GPR120 slowed down the inflammatory response,which provided an important basis for screening for mastitis resistance genes in cows.