RAS系统在LPS诱导bMECs炎性损伤过程中的调控作用  

The Regulatory Role of RAS in LPS-induced Inflammatory of bMECs

作　　者：张想军 周学章 ZHANG Xiang-Jun;ZHOU Xue-Zhang(College of Life Science,Ningxia University/Key Laboratory of the Ministry of Education for the Conservation and Utilization of Special Biological Resources of Western China,Yinchuan 750021,China)

机构地区：[1]宁夏大学生命科学学院/西部特色生物资源保护与利用教育部重点实验室,银川750021

出　　处：《农业生物技术学报》2021年第10期1958-1967,共10页Journal of Agricultural Biotechnology

基　　金：宁夏自然科学基金项目(2020AAC03074)。

摘　　要：本研究旨在探索肾素-血管紧张素系统(renin-angiotensin system, RAS)在奶牛乳腺上皮细胞(bovine mammary epithelial cells, bMECs)中的表达定位及RAS对损伤的调节作用。用不同浓度脂多糖(lipopolysaccharide, LPS)处理bMECs后,CCK-8(Cell Counting Kit-8)法检测细胞活性,qPCR检测IL-1β、IL-6、IL-8、TNF-α、ACE2、ACE、MasR和AT1R基因表达水平,酶联免疫吸附剂测定(enzyme linked immunosorbent assay, ELISA)检测上清中AngⅡ和Ang 1-7含量;采用Western blot和细胞免疫荧光测定ACE2蛋白在bMECs中的表达和位置。实验结果表明,ACE2在bMECs中有表达,且定位于胞膜和胞浆;LPS处理bMECs 24 h后产生明显炎性损伤,IL-1β、IL-6、IL-8、TNF-α、ACE、AT1R基因和AngⅡ蛋白的表达随LPS浓度升高而增加,ACE2和MasR基因的表达随LPS浓度增加而先升高后降低,Ang 1-7蛋白的表达随LPS浓度增加而下降。本研究表明bMECs中存在ACE2、ACE、MasR、AT1R、AngⅡ和Ang1-7等RAS成员,且ACE2/MasR/Ang 1-7和ACE/AngⅡ/AT1R通路参与了LPS诱导的b MECs炎性损伤调控。本实验为研究RAS在乳腺炎中的调控作用及发掘抗炎药物作用新靶点奠定了基础。The aim of this study is to explore the renin-angiotensin system’s(RAS) expression and location in bovine mammary epithelial cells(bMECs), and its regulatory role in LPS-induced inflammatory injury. After treating bMECs with different concentrations of LPS, CCK-8 method was used to examine cell viability, and qPCR was used to detect IL-1β, IL-6, IL-8, TNF-α, ACE2, ACE, MasR 和 AT1 R mRNA expression. Enzyme linked immunosorbent assay(ELISA) was used to test the content of Ang Ⅱ and Ang 1-7 protein in the supernatant. Western blot and cellular immunofluorescence were used to verify the expression and location of ACE2 protein in bMECs. The results showed that ACE2 was expressed and localized in the cell membrane and cytoplasm of b MECs. Producing obvious inflammatory damage by LPS treatment of bMECs for 24 h, the expression of IL-1β, IL-6, IL-8, TNF-α, ACE, AT1 R gene, and Ang Ⅱ proein increased with the LPS concentration. The expression of ACE2 and MasR m RNA firstly increased and then decreased, while the expression of Ang 1-7 protein decreased along with the LPS concentration. This study indicates the presence of RAS members such as ACE2, ACE, MasR, AT1 R, Ang Ⅱ and Ang 1-7 in bMECs, and the ACE2/MasR/Ang 1-7 and ACE/Ang Ⅱ/AT1 R pathways involved in the regulation of bMECs inflammatory injury induced by LPS. This experiment lays the foundation for studying the regulatory role of RAS in mastitis and discovering new targets for the action of anti-inflammatory drug.

关 键 词：奶牛乳腺上皮细胞 炎性反应 肾素血管紧张素系统(RAS) 血管紧张素转换酶2(ACE2) 

分 类 号：S8[农业科学—畜牧兽医]