| This paper is based on the previous study on the breast-protecting flunetion of the Renin-Angiotensin System(RAS).Here we futher studied one of the main members in this family,ACE2,on its function and mechanism in Ang Ⅱ induced oxidative stress,infammation injury and apoptosis.This study is mainly from the following aspects:1 Preliminary study on the role of local angiotensin converting enzyme2(ACE2)in breast injury induced by high concentrate in dairy cowThe Holstein cows with 6 healthy lactation periods were randomly divided into the control group(4:6)and the high concentrate group(6:4),and the single column fixed time quantitative feeding was used.After feeding for 20 weeks,the breast tissue was taken from the living body.The following experiments were carried out:1)determine the oxidative stress index of OH,TNOS and SOD in mammary tissue;2)ELISA method was used to determine TNF alpha,IL-1 beta inflammatory index and Caspase-3,Bax,Bcl-2 apoptosis index;3)Western Blot analyzed the expression of ACE2 and protein in breast tissue.Results:the concentration of OH in the mammary gland tissues of dairy cows increased significantly(P<0.01),the activity of TNOS and SOD decreased,and the activity of TNOS decreased significantly(P<0.05),the levels of TNF alpha and IL-1 beta of inflammatory factors increased significantly(P<0.05),and Caspase-3 and Bax levels of apoptotic protein increased significantly(P<0.01).The level of inhibition of apoptotic protein Bcl-2 was significantly decreased(P<0.05);the level of Ang Ⅱ and the expression of ACE protein in breast tissue increased significantly(P<0.05),and the level of Ang 1-7 and the expression of ACE2 protein decreased significantly(P<0.05).Conclusion:long-term feeding of high concentrate diet can cause a certain degree of damage to the mammary gland tissue of dairy cows and cause recessive mastitis,which shows a significant increase in the release of OH,the decrease of antioxidant enzyme activity,and the enhancement of the inflammatory response to the apoptosis of the cells.In this process,the local RAS system of the breast was activated,the key members of the ACE-Ang Ⅱ-AT1R axis,ACE and Ang Ⅱ,were significantly increased,and the key members of the ACE2-Ang1-7-MasR axis ACE2 and Ang 1-7 were significantly reduced.The results suggest that the imbalance of the two axes of the RAS system in the local mammary gland and the high level of the ACE2 Ang Ⅱ may be the main mechanism of breast injury.2 The functions of ngiotensin converting enzyme2(ACE2)in Ang Ⅱ induced inflammatory injury and apoptosis in bovine mammary epithelial cells(MAC-T).Angll was used to induced injury in the MAC-T cells to explore the interaction between AngⅡ and ACE2 under cellular injury.This study is as follows:1)the appropriate concentration and treatment time of Angll to induce oxidative stress injury in MAC-T cells was determined by measuring TNOS activity,the concentration of·OH and SOD activity stress indicators;2)the secretion of inflammatory cytokines in cell supernatant was measured by ELISA method;3)the change in the expression of apoptosis regulating proteins in cells were measured using Western blot(4)immunofluorescence was used to determine the presence and position of ACE2 in the cells.Western blot was used to measure the amount of ACE2 and ACE2 proteins in the cells,and then correlation analysis was performed between the two.Results:l)Ang Ⅱ treatment induced oxidative stress in bovine MAC-T cells.The appropriate concentration of Ang Ⅱ was 10-8 and 10-6 mol/L and the action time was 6 h;2)compared with the control group,the release of proinflanmatory factor TNF-α,IL-6,and IL-8 in the supernatant of MAC-T significantly increased(p<0.05)or significantly increased(p<0.01)in the Ang Ⅱ treated,while the release of IL-10 significantly reduced(p<0.05);3)compared with the control group,the expression of apoptotic protein Bax and Caspase-3 in the MAC-T cells was significantly up-regulated(p<0.01),and the expression of Bcl-2 protein was down regulated(p>0.05);4)there existed ACE2 in the bovine MAC-T cells,and ACE2 was located on the cell membrane.After treatment with different concentrations of Angll,the expression of ACE2 protein decreased,and in the 10-6 mol/L Angll treatment group,it decreased significantly(p<0.05);the expression of ACE protein was the just opposite.There was a significant negative correlation between ACE2 and Ang Ⅱ.Conclusion:Ang Ⅱ can induce oxidative stress,inflammatory injury and apoptosis in bovine MAC-T cells.During this process,both ACE2 and ACE enzyme activities were activated,but the effect of ACE2 degradation on Ang Ⅱ was less than that of ACE producing Ang Ⅱ,which means the degradation effect of ACE2 was at a disadvantage.Its specific damage or protection mechanism needs further study.3 Mechanism of ngiotensin converting enzyme2(ACE2)resistance to MAC-T cell damage induced AngⅡThe previous chapter showed that ACE2 was negatively correlated with the degree of cell damage induced by AngⅡ,suggesting that ACE2 may have a certain anti injury effect.In order to further clarify the resistance of ACE2 to MAC-T cell injury induced by Ang Ⅱ,this chapter experiments by giving exogenous ACE2 active protein to the cells,through oxidative stress,To determine the anti injury effect of ACE2 on Ang Ⅱinduced breast cell injury,and to reveal the possible mechanism of the anti injury effect of ACE2 by analyzing the expression or content changes of ACE2,ACE,Ang 1-7,Ang Ⅱ in the cell RAS members.Results:after adding ACE2 active protein 1)the production of ROS,OH and TNOS and SOD increased,and the activity of TNOS and SOD increased;and the content of TNF-alpha,IL-6 and IL-8 decreased,and the release of IL-10 of anti-inflammatory factors increased;3)the expression of apoptotic protein Bax and apoptotic executive protein in the cells decreased and inhibited the loss of apoptosis.The expression level of dead protein Bcl-2 increased;4)the content of Ang Ⅱ in cells decreased significantly,and the content of Ang 1-7 increased significantly,Conclusion:ACE2 has a certain resistance to Ang Ⅱ induced oxidative stress,inflammatory injury and apoptosis in bovine mammary epithelial cells.On the one hand,it can degrade Ang Ⅱ by ACE2,reduce Angll production,and on the other hand,antagonize and inhibit Ang Ⅱ through its downstream product Ang 1-7. |
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