The correlation between inflammatory injury induced by LPS and RAS in EpH4-Ev cells
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 9 h. 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 9 h. Meanwhile, the ACE/AngII/AT1R axis was activated and the ACE2/Ang1-7/MasR axis was depressed.