Wang Kun, Liu Xiaoqian, Xiao Hang, Wang Huanhuan, Zhang Yuanshu
College of Veterinary Medicine, Nanjing Agricultural University, Weigang No.1, Nanjing 210095, China.
College of Veterinary Medicine, Nanjing Agricultural University, Weigang No.1, Nanjing 210095, China.
Int Immunopharmacol. 2017 May;46:23-30. doi: 10.1016/j.intimp.2017.02.016. Epub 2017 Feb 27.
Renin-angiotensin system (RAS) plays an important role of regulating inflammatory injury. However, it is not clear about the correlation between renin-angiotensin system (RAS) and inflammation induced by LPS in mammary gland cells. So immunofluorescence was performed to verify the ACE2 expression in mammary gland cells. MTT assay was performed to detect cell viability. ELISA was performed to detect cytokines in cell supernatant. Western Blot was performed to analyze RAS levels and ACE2 level change was observed by immunofluorescence. The TLR4 level and p65 phosphorylation were detected by Western Blot. The ACE2 protein intensively located on the cell membrane. According to the results of MTT assay and TNF-α level, the injury was evidently induced by high concentration LPS after 9h. The TNF-α, IL-6, IL-8, ACE, AT1R and AngII had an increasing expression with the rise of cell injury. In contrast, the MasR, Ang1-7 and ACE2 had a declining expression with the increase of cell injury degree. The TLR4 level and p65 phosphorylation in high concentration LPS group was significantly higher than that of control group. These results suggest that a valid inflammatory injury was induced after the cells were treated by high concentration of LPS for 9h. Meanwhile, the ACE/AngII/AT1R axis was activated and the ACE2/Ang1-7/MasR axis was depressed.
肾素-血管紧张素系统（RAS）在调节炎症损伤中起重要作用。然而，肾素-血管紧张素系统（RAS）与脂多糖（LPS）诱导的乳腺细胞炎症之间的相关性尚不清楚。因此，进行免疫荧光以验证乳腺细胞中血管紧张素转换酶2（ACE2）的表达。进行MTT法检测细胞活力。进行酶联免疫吸附测定（ELISA）以检测细胞上清液中的细胞因子。进行蛋白质免疫印迹法（Western Blot）分析RAS水平，并通过免疫荧光观察ACE2水平变化。通过蛋白质免疫印迹法（Western Blot）检测Toll样受体4（TLR4）水平和p65磷酸化。ACE2蛋白主要位于细胞膜上。根据MTT法检测结果和肿瘤坏死因子-α（TNF-α）水平，高浓度LPS作用9小时后明显诱导了细胞损伤。随着细胞损伤的增加，TNF-α、白细胞介素-6（IL-6）、白细胞介素-8（IL-8）、血管紧张素转换酶（ACE）、血管紧张素Ⅱ1型受体（AT1R）和血管紧张素Ⅱ（AngII）表达增加。相反，随着细胞损伤程度的增加，Mas受体（MasR）、血管紧张素1-7（Ang1-7）和ACE2表达下降。高浓度LPS组的TLR4水平和p65磷酸化明显高于对照组。这些结果表明，高浓度LPS处理细胞9小时后诱导了有效的炎症损伤。同时，ACE/AngII/AT1R轴被激活，而ACE2/Ang1-7/MasR轴被抑制。
