乳房炎奶牛血浆源外泌体对LPS诱导的MAC-T细胞炎症的影响

Effect of Plasma-derived Exosomes on LPS-induced MAC-T Cell Inflammation in Dairy Cows with Mastitis

【目的】外泌体是细胞通讯和组织微环境建立的关键因素,其对炎症过程具有一定的调节作用。本研究旨在探究奶牛血浆源外泌体对脂多糖(LPS)诱导的奶牛乳腺上皮细胞(MAC-T细胞)炎症的调控作用。【方法】采集健康及患有乳房炎的奶牛静脉血,离心后获得血浆,并对血浆进行超速离心,分离得到血浆源外泌体。通过实时荧光定量PCR检测孵育奶牛血浆源外泌体后MAC-T细胞中促炎因子的表达。Western blotting验证孵育奶牛血浆源外泌体后NF-κB通路相关蛋白Toll样受体4(TLR4)、髓样分化因子88(MyD88)和肿瘤坏死因子α(TNF-α)的表达。通过CCK8和Hoechst染色验证奶牛血浆源外泌体对MAC-T细胞的增殖及凋亡效果的影响。【结果】实时荧光定量PCR结果显示,与LPS组相比,奶牛乳房炎血浆源外泌体可显著下调促炎因子白细胞介素-6(IL-6)、IL-23、IL-1α、NFRK、MyD 88、TNF-α、IL-1β基因mRNA的表达(P<0.05);健康奶牛血浆源外泌体仅对IL-1β基因mRNA表达有显著抑制作用(P<0.05)。Western blotting结果显示,与LPS组相比,奶牛乳房炎血浆源外泌体显著降低NF-κB通路蛋白TLR4、MyD88和TNF-α的表达(P<0.05);健康奶牛血浆源外泌体NF-κB通路蛋白表达与LPS组无显著差异(P>0.05)。CCK8与Hoechst染色结果显示,与LPS组相比,奶牛乳房炎血浆源外泌体可促进细胞增殖,缓解细胞凋亡;健康奶牛血浆源外泌体可恢复一定的细胞活力。【结论】奶牛乳房炎血浆源外泌体可通过NF-κB通路缓解LPS诱导的MAC-T细胞炎症。本研究结果将为血浆源外泌体在奶牛乳房炎中发挥的抗炎作用提供理论依据,同时为血浆外泌体的临床应用奠定基础。

【Objective】Exosomes are key factors in cellular communication and establishment of tissue microenvironment,and they have a regulatory role in inflammatory processes.This study was aimed to investigate the role of plasma-derived exosomes from dairy cows in the regulation of lipopolysaccharide(LPS)-induced inflammation in mammary epithelial cells(MAC-T)of dairy cows.【Method】In this study,venous blood was collected from healthy and mastitis-affected dairy cows,and plasma was obtained after centrifugation,and plasma-derived exosomes were isolated by ultracentrifugation.The expression of pro-inflammatory factors after incubation of plasma-derived exosomes from dairy cows was detected by Real-time quantitative PCR.Western blotting was used to verify the expression of NF-κB pathway proteins TLR4,MyD88 and TNF-αafter incubation of plasma-derived exosomes from dairy cows.The effects of plasma-derived exosomes from dairy cows on the proliferation as well as apoptosis of MAC-T cells were verified by CCK8 and Hoechst staining.【Result】Real-time quantitative PCR results showed that plasma-derived exosomes from dairy cows with mastitis significantly down-regulated the expression of pro-inflammatory factors IL-6,IL-23,IL-1α,NFRK,MyD 88,TNF-αand IL-1βgenes mRNA compared with LPS group(P<0.05).Plasma-derived exosomes from healthy cows significantly inhibited mRNA expression of the IL-1βgene(P<0.05).Western blotting results showed that the plasma-derived exosomes from dairy cows with mastitis significantly decreased the expression of the NF-κB pathway proteins TLR4,MyD88 and TNF-α(P<0.05).NF-κB pathway protein expression in plasma-derived exosomes from healthy cows was not significantly different from that of the LPS group(P>0.05).CCK8 and Hoechst staining results showed that plasma-derived exosomes from dairy cows with mastitis could promote cell proliferation and alleviate apoptosis compared with LPS group.Plasma-derived exosomes from healthy cows restored some cell viability.【Conclusion】The results indicated that plasma-derived exosomes from dairy cows with mastitis could alleviate LPS-induced inflammation of MAC-T cells through NF-κB pathway.This study would provide a theoretical basis for the anti-inflammatory role played by plasma-derived exosomes in dairy cow mastitis,and at the same time laid the foundation for the clinical application of plasma-derived exosomes.