高精料日粮诱导奶牛乳腺应激和凋亡中血管紧张素转化酶2的作用
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摘要
[目的]本试验在已有研究血管紧张素系统(RAS)在乳腺中保护作用的基础上,对该家族的主要成员血管紧张素转换酶2(ACE2)在对抗高精料所致奶牛乳腺氧化应激、炎性损伤及细胞凋亡中的作用及机制进行了探讨。[方法]6头健康的泌乳期荷斯坦奶牛,随机分为对照组[m(精)∶m(粗)=4∶6]和高精料组[m(精)∶m(粗)=6∶4],采用单栏、定时、定量饲喂。饲喂20周后,活体取乳腺组织,进行如下试验:1)测定乳腺组织中氧化应激指标·OH水平及TNOS、SOD活性;2)ELISA法测定炎性指标TNFα、IL-1β和凋亡指标Caspase-3、Bax、Bcl-2;3)Western blot分析乳腺组织中ACE2和ACE蛋白的表达;ELISA法测定AngⅡ和Ang1-7的含量。[结果]高精料长期饲喂,奶牛乳腺组织中·OH自由基水平显著升高(P<0.05),SOD活性降低,TNOS活性降低显著(P<0.05);炎症因子TNF-α和IL-1β水平均显著升高(P<0.05);促凋亡蛋白Caspase-3和Bax水平显著升高(P<0.05),抑制凋亡蛋白Bcl-2水平显著降低(P<0.05);乳腺组织中AngⅡ浓度(P<0.05)和ACE蛋白表达水平极显著升高(P<0.01),ACE2蛋白表达水平降低(P>0.05),但Ang1-7浓度显著降低(P<0.05)。[结论]长期饲喂高精料可导致奶牛乳腺自由基激活、炎症因子释放、细胞凋亡,乳腺局部损伤。乳腺局部组织中RAS处于激活状态,2条轴互相拮抗,表现为AngⅡ水平升高,ACE2水平降低。
[Objectives]This paper is based on the previous study on the breast-protecting function of the renin-angiotensin system(RAS).Here we further studied one of the main members in this family,angiotensin converting enzyme2(ACE2),on its function and mechanism in AngⅡ induced oxidative stress,inflammation injury and apoptosis. [Methods]The Holstein cows with 6 healthy lactation periods were randomly divided into the control group[m(cencontrate)∶m(forage)=4∶6]and the high grain diet group[m(cencontrate)∶m(forage)=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-α,IL-1β inflammatory index and Caspase-3,Bax,Bcl-2 apoptosis index;3)Western blot analyzed the expression of ACE2 and ACE protein in breast tissue. [Results]Comparing with control group,the level of ·OH in the mammary gland tissues of dairy cows fed the high grain diet 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-α and IL-1β 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 significantly decreased(P<0.05);the concentration of AngⅡ and the expression of ACE protein in breast tissue increased significantly(P<0.05),and the concentration of Ang1-7 and the expression of ACE2 protein decreased significantly(P<0.05). [Conclusions]Long-term feeding of high grain 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-AngⅡ-AT1 R axis,ACE and AngⅡ,were significantly increased,and the key members of the ACE2-Ang1-7-MasR axis ACE2 and Ang1-7 were significantly reduced. The results suggest that the imbalance of the two axis of the RAS system in the local mammary gland,with lower level of ACE2 and the high level of AngⅡ,may be the main mechanism of breast injury.
[Objectives]This paper is based on the previous study on the breast-protecting function of the renin-angiotensin system(RAS).Here we further studied one of the main members in this family,angiotensin converting enzyme2(ACE2),on its function and mechanism in AngⅡ induced oxidative stress,inflammation injury and apoptosis. [Methods]The Holstein cows with 6 healthy lactation periods were randomly divided into the control group[m(cencontrate)∶m(forage)=4∶6]and the high grain diet group[m(cencontrate)∶m(forage)=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-α,IL-1β inflammatory index and Caspase-3,Bax,Bcl-2 apoptosis index;3)Western blot analyzed the expression of ACE2 and ACE protein in breast tissue. [Results]Comparing with control group,the level of ·OH in the mammary gland tissues of dairy cows fed the high grain diet 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-α and IL-1β 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 significantly decreased(P<0.05);the concentration of AngⅡ and the expression of ACE protein in breast tissue increased significantly(P<0.05),and the concentration of Ang1-7 and the expression of ACE2 protein decreased significantly(P<0.05). [Conclusions]Long-term feeding of high grain 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-AngⅡ-AT1 R axis,ACE and AngⅡ,were significantly increased,and the key members of the ACE2-Ang1-7-MasR axis ACE2 and Ang1-7 were significantly reduced. The results suggest that the imbalance of the two axis of the RAS system in the local mammary gland,with lower level of ACE2 and the high level of AngⅡ,may be the main mechanism of breast injury.
引文
[1] Muňoz-Durango N,Fuentes C,Castillo A,et al.Role of the renin-angiotensin-aldosterone system beyond blood pressure regulation:molecular and cellular mechanisms involved in end-organ damage during arterial hypertension[J].International Journal of Molecular Sciences,2016,17(7):797.
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[3] Santos R A S,Ferreira A J,Verano-Braga T,et al.Angiotensin-converting enzyme 2,angiotensin-(1-7)and Mas:new players of the renin-angiotensin system[J].Journal of Endocrinology,2013,216(2):1-17.
[4] Xiao H L,Zhao L X,Yang J,et al.Association between ACE2/ACE balance and pneumocyte apoptosis in a porcine model of acute pulmonary thromboembolism with cardiac arrest[J].Molecular Medicine Reports,2018,17(3):4221-4228.
[5] Li Y,Cao Y,Zeng Z,et al.Angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis prevents lipopolysaccharide-induced apoptosis of pulmonary microvascular endothelial cells by inhibiting JNK/NF-κB pathways[J].Scientific Reports,2015,5:8209.
[6] 刘小倩,刘颖,荣超,等.ACE2在奶牛乳腺中的表达定位及其抗炎性损伤作用的研究[J].畜牧兽医学报,2017,48(3):552-560.Liu X Q,Liu Y,Rong C,et al.Expression Localization of angiotensin converting enzyme 2(ACE 2)in dairy cow mammary gland and its anti-inflammation effect[J].Acta Veterinaria et Zootechnica Sinica,2017,48(3):552-560(in Chinese with English abstract).
[7] Wang K,Liu X,Hang X,et al.The correlation between inflammatory injury induced by LPS and RAS in EpH4-Ev cells[J].International Immunopharmacology,2017,46:23-30.
[8] 曹洋,叶平生,荣超,等.高精料日粮对干奶期奶山羊肝脏中游离氨基酸重分配的影响[J].南京农业大学学报,2016,39(2):289-296.DOI:10.7685/jnau.201507017.Cao Y,Ye P S,Rong C,et al.The effects of high concentrate diets on the redistribution offree amino acids in the liver of goats in dry period[J].Journal of Nanjing Agricultural University,2016,39(2):289-296(in Chinese with English abstract).
[9] Ribeiro-Oliveira A,Jr,Nogueira A I,Pereira R M,et al.The renin-angiotensin system and diabetes:an update[J].Vascular Health & Risk Management,2008,4(4):787-803.
[10] Sadik N A,Metwally N S,Shaker O G,et al.Local renin-angiotensin system regulates the differentiation of mesenchymal stem cells into insulin-producing cells through angiotensin type 2 receptor[J].Biochimie,2017,137:132-138.
[11] 荣超.血管紧张素转换酶2(ACE2)在奶牛乳腺中的表达分布及其抗损伤作用[D].南京:南京农业大学,2016.Rong C.Study on angiotensin converting enzyme2(ACE2)in bovine mammary:its expression localization and function of anti-injury[D].Nanjing:Nanjing Agricultural University,2018(in Chinese with English abstract).
[12] Agarwal D,Welsch M A,Keller J N,et al.Chronic exercise modulates RAS components and improves balance between pro- and anti-inflammatory cytokines in the brain of SHR[J].Basic Research in Cardiology,2011,106(6):1069-1085.
[13] Diep Q N,Amiri F,Touyz R M,et al.PPAR alpha activator effects on AngⅡ-induced vascular oxidative stress and inflammation[J].Hypertension,2002,40(6):866-871.
[14] Maneesai P,Bunbupha S,Kukongviriyapan U,et al.Effect of asiatic acid on the AngⅡ-AT1R-NADPH oxidase-NF-κB pathway in renovascular hypertensive rats[J].Naunyn-Schmiedeberg’s Archives of Pharmacology,2017,390(10):1073-1083.
[15] de Morais S D B,Shanks J,Zucker I H.Integrative physiological aspects of brain ras in hypertension[J].Current Hypertension Reports,2018,20(2):10.
[16] Chen Q F,Kuang X D,Yuan Q F,et al.Lipoxin A4 attenuates LPS-induced acute lung injury via activation of the ACE2-Ang-(1-7)-Mas axis[J].Innate Immun,2018,5(24):285-296.
[17] 刘小倩.血管紧张素转化酶2(ACE2)在奶牛乳腺细胞应激和凋亡损伤中的保护作用[D].南京:南京农业大学,2018.Liu X Q.Protective effect of ACE2 on cellular stress and apoptosis injuries in mammary gland epithelial cells of cows[D].Nanjing:Nanjing Agricultural University,2018(in Chinese with English abstract).
[2] Suzuki Y,Ruiz-Ortega M,Lorenzo O,et al.Inflammation and angiotensin Ⅱ[J].The International Journal of Biochemistry & Cell Biology,2003,35(6):881-900.
[3] Santos R A S,Ferreira A J,Verano-Braga T,et al.Angiotensin-converting enzyme 2,angiotensin-(1-7)and Mas:new players of the renin-angiotensin system[J].Journal of Endocrinology,2013,216(2):1-17.
[4] Xiao H L,Zhao L X,Yang J,et al.Association between ACE2/ACE balance and pneumocyte apoptosis in a porcine model of acute pulmonary thromboembolism with cardiac arrest[J].Molecular Medicine Reports,2018,17(3):4221-4228.
[5] Li Y,Cao Y,Zeng Z,et al.Angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis prevents lipopolysaccharide-induced apoptosis of pulmonary microvascular endothelial cells by inhibiting JNK/NF-κB pathways[J].Scientific Reports,2015,5:8209.
[6] 刘小倩,刘颖,荣超,等.ACE2在奶牛乳腺中的表达定位及其抗炎性损伤作用的研究[J].畜牧兽医学报,2017,48(3):552-560.Liu X Q,Liu Y,Rong C,et al.Expression Localization of angiotensin converting enzyme 2(ACE 2)in dairy cow mammary gland and its anti-inflammation effect[J].Acta Veterinaria et Zootechnica Sinica,2017,48(3):552-560(in Chinese with English abstract).
[7] Wang K,Liu X,Hang X,et al.The correlation between inflammatory injury induced by LPS and RAS in EpH4-Ev cells[J].International Immunopharmacology,2017,46:23-30.
[8] 曹洋,叶平生,荣超,等.高精料日粮对干奶期奶山羊肝脏中游离氨基酸重分配的影响[J].南京农业大学学报,2016,39(2):289-296.DOI:10.7685/jnau.201507017.Cao Y,Ye P S,Rong C,et al.The effects of high concentrate diets on the redistribution offree amino acids in the liver of goats in dry period[J].Journal of Nanjing Agricultural University,2016,39(2):289-296(in Chinese with English abstract).
[9] Ribeiro-Oliveira A,Jr,Nogueira A I,Pereira R M,et al.The renin-angiotensin system and diabetes:an update[J].Vascular Health & Risk Management,2008,4(4):787-803.
[10] Sadik N A,Metwally N S,Shaker O G,et al.Local renin-angiotensin system regulates the differentiation of mesenchymal stem cells into insulin-producing cells through angiotensin type 2 receptor[J].Biochimie,2017,137:132-138.
[11] 荣超.血管紧张素转换酶2(ACE2)在奶牛乳腺中的表达分布及其抗损伤作用[D].南京:南京农业大学,2016.Rong C.Study on angiotensin converting enzyme2(ACE2)in bovine mammary:its expression localization and function of anti-injury[D].Nanjing:Nanjing Agricultural University,2018(in Chinese with English abstract).
[12] Agarwal D,Welsch M A,Keller J N,et al.Chronic exercise modulates RAS components and improves balance between pro- and anti-inflammatory cytokines in the brain of SHR[J].Basic Research in Cardiology,2011,106(6):1069-1085.
[13] Diep Q N,Amiri F,Touyz R M,et al.PPAR alpha activator effects on AngⅡ-induced vascular oxidative stress and inflammation[J].Hypertension,2002,40(6):866-871.
[14] Maneesai P,Bunbupha S,Kukongviriyapan U,et al.Effect of asiatic acid on the AngⅡ-AT1R-NADPH oxidase-NF-κB pathway in renovascular hypertensive rats[J].Naunyn-Schmiedeberg’s Archives of Pharmacology,2017,390(10):1073-1083.
[15] de Morais S D B,Shanks J,Zucker I H.Integrative physiological aspects of brain ras in hypertension[J].Current Hypertension Reports,2018,20(2):10.
[16] Chen Q F,Kuang X D,Yuan Q F,et al.Lipoxin A4 attenuates LPS-induced acute lung injury via activation of the ACE2-Ang-(1-7)-Mas axis[J].Innate Immun,2018,5(24):285-296.
[17] 刘小倩.血管紧张素转化酶2(ACE2)在奶牛乳腺细胞应激和凋亡损伤中的保护作用[D].南京:南京农业大学,2018.Liu X Q.Protective effect of ACE2 on cellular stress and apoptosis injuries in mammary gland epithelial cells of cows[D].Nanjing:Nanjing Agricultural University,2018(in Chinese with English abstract).