| This study focused on the expression,localization and anti-injury effects of ACE2 in mammary tissue.The research consists of three parts:1.The expression and location of ACE2 in mammary gland epithelial cells and the relationship between RAS and inflammatory injury induced by LPSWe made sure the expression and location of RAS in mammary gland epithelial cells via RT-PCR,Western-Blot and immunofluorescence.We used LPS to establish an inflammation injury in mammary gland epithelial cells and discuss the relationship between the two axes of RAS and inflammatory injury in cells.The mouse mammary gland epithelial cells were selected to do all experiments as follows:1)we detected the mRNA and protein expression of ACE2 in mammary gland epithelial cells;2)we performed immunofluorescence to verify the ACE2 location in mammary gland epithelial cells;3)We used different concentration of LPS stimulate mammary gland epithelial cells at different time points to induce inflammation in mammary gland epithelial cells;4)we analyzed the expression levels of ACE2,MasR,ACE and AT1R in cells exposed to different concentration of LPS via Western Blot and detected the content of Angll and Angl-7 in cell supernatant by RIA and ELISA;5)we analyzed the correlation among LPS,TNF-?,IL-6,IL-8,ACE2,MasR,ACE,AT1R,Ang? and Angl-7 via spearman correlation analysis.All the results were list as follows:1)ACE2 mRNA and protein expressed in mammary gland epithelial cells and located on the membrane of cells;2)the inflammatory injury was successfully established in mammary gland epithelial cells after cells were exposed to LPS for 9 h according to the cells relative viability,inflammatory factors and mitochondrial membrane potential in cells;3)after cells were exposed to LPS for 9 h,the levels of ACE2,MasR and the content of Angl-7 had a declining expression with the increase of LPS concentration,and the levels of ACE,AT1R and the content of Ang? had an increasing expression with the increase of LPS concentration.These results suggest that there are all members of RAS expressing in mammary gland epithelial cells,and were involved to the process of inflammatory injury induced by LPS in mammary gland epithelial cells.Meanwhile,the ACE/Ang?/ATIR axis was activated and the ACE2/Angl-7/MasR axis was depressed with the increase of LPS concentration.ACE2 was negatively correlated with the inflammatory injury induced by LPS.2.The anti-inflammatory injury role of ACE2 in mammary gland and its mechanism In former chapter of this study,it has been found that ACE2 has a significant negative correlation with the degree of inflammation induced by LPS,which suggests that ACE2 may have a role of anti-inflammatory injury.In order to furtherly make sure the role of ACE2 against inflammatory injury in mammary gland epithelial cells,we planned to establish ACE2 gene knock out mammary gland epithelial cells and give a ACE2 active protein to normal cells,to clarify the function of ACE2 on inflammatory injury of mammary gland epithelial cells,and reveal its signaling mechanism according to the mitochondrial membrane potential in cells,the content of inflammatory factors in cell supernatant.All the results are listed as follows:1)the ACE2 gene knock out cells was successfully established in this chapter;2)the inflammatory injury of ACE2-knock out cells induced by LPS was worse than that of normal cells,with a decrease of mitochondrial membrane potential in cells and a rise of inflammatory factors release including TNF-?,IL-6 and IL-8;however,the inflammatory injury of normal cells was attenuated by ACE2 active protein with a recovery of mitochondrial membrane potential and a downregulation of inflammatory factors,which would be offset with an administration of A779,a blocker of MasR;3)the phosphorylation levels of p65 and ERK were upregulated by LPS in NF-?B and MAPK signaling pathways of cells,which would be reversed by ACE2 active protein.It was been proved that ACE2 had a role of anti-inflammatory injury induced by LPS in mammary gland epithelial cells.ACE2 attenuated the inflammatory injury induced by LPS in mammary gland epithelial cell via degrading Ang? and activating ACE2/Angl-7/MasR axis,then inhibiting the activation of NF-?B/ERK signaling pathway,even reducing the expression and release of inflammatory factors.3.ACE2 protects mouse mammary gland from inflammatory injury and its anti-inflammatory mechanismIn former two chapter,we have proved that ACE2 could protect mammary gland epithelial cells from an inflammatory injury,and discussed its signaling pathway mechanism.We will make sure the anti-inflammatory role of ACE2 in vivo in this chapter.Mouse is the research object in this chapter.LPS was used to induce a mastitis in mouse with an ACE2 pretreatment group.All the experiments are listed as follows:1)the observation of inflammatory lesions in local mammary gland tissue;2)the detection of inflammatory factors,myeloperoxidase(MPO)and n-acetyl-?-glucosaminidase(NAG)in mammary gland tissue;3)the observation of ACE2 expression change in mammary gland tissue among all groups;4)the detection of Ang?,Angl-7 and inflammatory factors levels in mammary gland tissue by ELISA,the analysis of ACE2 level,MasR level and the key protein phosphorylation level in NF-?B/ERK pathway of cells by western blot.All the experiment results were listed as follows:1)ACE2 expressed in mammary gland and locates in mammary gland epithelial cell;2)LPS administration induced an increase of TNF-?,IL-6,IL-8 content and the MPO,NAG activity(P<0.05),with an obvious inflammatory lesions in mammary gland tissue;a ACE2 pretreatment could reduce the above markers and attenuate the local inflammatory lesions;3)LPS administration led to a rise of Ang? content and a decline of Ang1-7,also upregulated the phosphorylation of p65 and ERK,which was reversed by ACE2 active protein.All these results suggested that ACE2 attenuate mastitis induced by LPS and protect mammary gland via degrading Ang?,generating Ang1-7,inhibiting NF-?B and EPK and reducing the release of inflammatory factors. |
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