血管紧张素转化酶2对Ang Ⅱ诱导的牛乳腺上皮细胞炎症损伤的缓解作用
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摘要
以不同浓度血管紧张素Ⅱ(AngⅡ)诱导牛乳腺上皮细胞(MAC-T)炎症损伤,探讨血管紧张素转化酶2(ACE 2)的变化及与AngⅡ的相互作用。研究包括:ELISA检测细胞上清中细胞炎性因子的分泌或释放;Western blot检测细胞ACE 2和ACE的蛋白表达变化,并进行相关性分析;添加ACE 2活性蛋白对AngⅡ诱导损伤的保护作用。结果显示:1)AngⅡ处理MAC-T,细胞上清中促炎因子TNF-α、IL-6、IL-8含量显著(P<0.05)或极显著(P<0.01)增加,抗炎因子IL-10含量显著降低(P<0.05);2)不同浓度AngⅡ处理细胞后,ACE 2蛋白表达均有降低,10~(-6) mol·L~(-1) AngⅡ处理后显著降低(P<0.05);ACE蛋白表达结果相反;ACE 2与AngⅡ浓度之间存在显著负相关;3)添加外源性ACE 2活性蛋白,细胞上清中TNF-α、IL-6和IL-8浓度均有一定程度的下降,IL-10浓度升高。本研究表明ACE 2/ACE轴的失衡是AngⅡ诱导细胞炎性损伤的主要原因。高水平AngⅡ可激活炎症因子促进炎症反应,是促进炎症反应的主要介质,ACE 2可以通过降解AngⅡ,抑制其对炎症反应的上调效应。
Inflammatory damage of bovine mammary epithelial cells(MAC-T) was induced by different concentrations of angiotensin Ⅱ(Ang Ⅱ), and the changes of angiotensin-converting enzyme 2(ACE 2) and the interaction with Ang Ⅱ were explored. The research includes: Detecting the secretion or release of inflammatory factors in cell supernatant by ELISA; Western blot analysis of protein expression changes of ACE 2 and ACE, and correlation analysis; The protective effect of ACE 2 active protein on Ang Ⅱ induced injury. Results were as follows: 1) The levels of pro-inflammatory factors TNF-α, IL-6 and IL-8 in the supernatant of bovine MAC-T cells in the Ang Ⅱ treatment were significantly(P<0.05) or very significantly increased(P<0.01), the anti-inflammatory factor IL-10 content was significantly reduced(P<0.05); 2) Treatment with different concentrations of Ang Ⅱ, the expression of ACE 2 protein was decreased, and the 10~(-6) mol·L~(-1) Ang Ⅱ treatment group was significantly decreased(P<0.05); the expression of ACE protein was reversed. There was a significant negative correlation between ACE 2 and Ang Ⅱ concentrations; 3) After adding exogenous ACE 2 active protein, the results showed that the concentration of TNF-α, IL-6 and IL-8 in the supernatant of the cells decreased to some extent, and the concentration of IL-10 increased. The imbalance of the ACE 2/ACE axis is the main cause of inflammatory injury induced by Ang Ⅱ. The conclusion showed that high level of Ang Ⅱ can activate inflammatory factors to promote inflammatory response and is the main mediator of inflammatory response. ACE 2 can inhibit the up-regulation of inflammatory response by degrading Ang Ⅱ.
Inflammatory damage of bovine mammary epithelial cells(MAC-T) was induced by different concentrations of angiotensin Ⅱ(Ang Ⅱ), and the changes of angiotensin-converting enzyme 2(ACE 2) and the interaction with Ang Ⅱ were explored. The research includes: Detecting the secretion or release of inflammatory factors in cell supernatant by ELISA; Western blot analysis of protein expression changes of ACE 2 and ACE, and correlation analysis; The protective effect of ACE 2 active protein on Ang Ⅱ induced injury. Results were as follows: 1) The levels of pro-inflammatory factors TNF-α, IL-6 and IL-8 in the supernatant of bovine MAC-T cells in the Ang Ⅱ treatment were significantly(P<0.05) or very significantly increased(P<0.01), the anti-inflammatory factor IL-10 content was significantly reduced(P<0.05); 2) Treatment with different concentrations of Ang Ⅱ, the expression of ACE 2 protein was decreased, and the 10~(-6) mol·L~(-1) Ang Ⅱ treatment group was significantly decreased(P<0.05); the expression of ACE protein was reversed. There was a significant negative correlation between ACE 2 and Ang Ⅱ concentrations; 3) After adding exogenous ACE 2 active protein, the results showed that the concentration of TNF-α, IL-6 and IL-8 in the supernatant of the cells decreased to some extent, and the concentration of IL-10 increased. The imbalance of the ACE 2/ACE axis is the main cause of inflammatory injury induced by Ang Ⅱ. The conclusion showed that high level of Ang Ⅱ can activate inflammatory factors to promote inflammatory response and is the main mediator of inflammatory response. ACE 2 can inhibit the up-regulation of inflammatory response by degrading Ang Ⅱ.
引文
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[14] 刘小倩,刘颖,荣超,等.ACE 2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)
[15] AOUN E,CHEN J,REIGHARD D,et al.Diagnostic accuracy of interleukin-6 and interleukin-8 in predicting severe acute pancreatitis:a meta-analysis[J].Pancreatology,2009,9(6):777-785.
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[17] BOUDJELLAB N,CHAN-TANG H S,LI X,et al.Interleukin 8 response by bovine mammary epithelial cells to lipopolysaccharide stimulation[J].Am J Vet Res,1998,59(12):1563-1567.
[18] LEE J,ZHAO X.Recombinant human interleukin-8,but not human interleukin-1β,induces bovine neutrophil migration in an in vitro co-culture system[J].Cell Biol Int,2000,24(12):889-895.
[19] GONZáLEZ G E,RHALEB N E,D’AMBROSIO M A,et al.Cardiac-deleterious role of galectin-3 in chronic angiotensin Ⅱ-induced hypertension[J].Am J Physiol Heart Circ Physiol,2016,311(5):H1287-H1296.
[20] 郑书国,任尤楠,赵梦秋,等.芝麻素含药血清对血管紧张素Ⅱ诱导大鼠心肌细胞凋亡的影响[J].中药材,2015,38(5):1013-1017.ZHENG S G,REN Y N,ZHAO M Q,et al.Effect of serum containing sesamin on angiotensin Ⅱ-induced apoptosis in rat cardiomyocytes[J].Journal of Chinese Medicinal Materials,2015,38(5):1013-1017.(in Chinese)
[21] PATEL V B,BASU R,OUDIT G Y.ACE2/Ang 1-7 axis:A critical regulator of epicardial adipose tissue inflammation and cardiac dysfunction in obesity[J].Adipocyte,2016,5(3):306-311.
[22] NIAZI Z R,SILVA G C,RIBEIRO T P,et al.EPA:DHA 6:1 prevents angiotensin Ⅱ-induced hypertension and endothelial dysfunction in rats:role of NADPH oxidase- and COX-derived oxidative stress[J].Hypertens Res,2017,40(12):966-975.
[23] IMAI Y,KUBA K,PENNINGER J M.The discovery of angiotensin-converting enzyme 2 and its role in acute lung injury in mice[J].Exp Physiol,2008,93(5):543-548.
[24] HAN J B,YE S J,ZOU C P,et al.Angiotensin Ⅱ causes biphasic STAT3 activation through TLR4 to initiate cardiac remodeling[J].Hypertension,2018,72(6):1301-1311.
[25] YU X Y,XIA Y F,ZENG L Y,et al.A blockade of PI3Kγ signaling effectively mitigates angiotensin Ⅱ-induced renal injury and fibrosis in a mouse model[J].Sci Rep,2018,8:10988.
[26] SODHI C P,WOHLFORD-LENANE C,YAMAGUCHI Y,et al.Attenuation of pulmonary ACE2 activity impairs inactivation of des-Arg9 bradykinin/BKB1R axis and facilitates LPS-induced neutrophil infiltration[J].Am J Physiol-Lung Cell Mol Physiol,2018,314(1):L17-L31.
[27] SIM?ES ESILVA A C,TEIXEIRA M M.ACE inhibition,ACE2 and angiotensin-(1-7) axis in kidney and cardiac inflammation and fibrosis[J].Pharmacol Res,2016,107:154-162.
[28] HAO Y W,LIU Y.Osthole alleviates bleomycin-induced pulmonary fibrosis via modulating angiotensin-converting enzyme 2/angiotensin-(1-7) axis and decreasing inflammation responses in rats[J].Biol Pharm Bull,2016,39(4):457-465.
[29] LIU C X,XIAO L P,LI F D,et al.Generation of outbred Ace2 knockout mice by RNA transfection of TALENs displaying colitis reminiscent pathophysiology and inflammation[J].Transgenic Res,2015,24(3):433-446.
[30] WANG J,LIU R X,QI H Y,et al.The ACE2-angiotensin-(1-7)-Mas axis protects against pancreatic cell damage in cell culture[J].Pancreas,2015,44(2):266-272.
[31] HE H L,LIU L,CHEN Q H,et al.MSCs modified with ACE2 restore endothelial function following LPS challenge by inhibiting the activation of RAS[J].J Cell Physiol,2015,230(3):691-701.
[2] RODRIGUES P T R,ROCHA N P,MIRANDA A S,et al.The anti-inflammatory potential of ACE2/angiotensin-(1-7)/mas receptor axis:evidence from basic and clinical research[J].Curr Drug Targets,2017,18(11):1301-1313.
[3] HAN J B,ZOU C P,MEI L Q,et al.MD2 mediates angiotensin Ⅱ-induced cardiac inflammation and remodeling via directly binding to Ang Ⅱ and activating TLR4/NF-κB signaling pathway[J].Basic Res Cardiol,2017,112(1):9.
[4] 丁文飞,李雪,吴蔚桦,等.阿利吉仑抑制糖尿病肾病大鼠血管紧张素Ⅱ/血管紧张素1-7(Ang Ⅱ/Ang1-7)信号途径[J].细胞与分子免疫学杂志,2018,34(10):891-895.DING W F,LI X,WU W H,et al.Aliskiren inhibits angiotensin Ⅱ/angiotensin 1-7(Ang Ⅱ/Ang1-7) signal pathway in rats with diabetic nephropathy[J].Chinese Journal of Cellular and Molecular Immunology,2018,34(10):891-895.(in Chinese)
[5] ASGHAR W,AGHAZADEH-HABASHI A,JAMALI F.Cardiovascular effect of inflammation and nonsteroidal anti-inflammatory drugs on renin-angiotensin system in experimental arthritis[J].Inflammopharmacology,2017,25(5):543-553.
[6] STANKE-LABESQUE F,DEVILLIER P,BEDOUCH P,et al.Angiotensin Ⅱ-induced contractions in human internal mammary artery:effects of cyclooxygenase and lipoxygenase inhibition[J].Cardiovasc Res,2000,47(2):376-383.
[7] 黄丽,应涛,何文明.不同肾素血管紧张素系统阻滞剂对ACE2-Ang-(1-7)及炎症因子的影响[J].心电与循环,2017,36(2):77-80.HUANG L,YING T,HE W M.The effects of different renin-angiotensin system inhibitors on ACE2-Angiotensin(1-7) and inflammatory factors[J].Journal of Electrocardiology and Circulation,2017,36(2):77-80.(in Chinese)
[8] ZHANG M L,GAO Y H,ZHAO W G,et al.ACE-2/ANG1-7 ameliorates ER stress-induced apoptosis in seawater aspiration-induced acute lung injury[J].Am J Physiol-Lung Cell Mol Physiol,2018,315(6):L1015-L1027.
[9] WANG K,LIU X Q,HANG X,et al.The correlation between inflammatory injury induced by LPS and RAS in EpH4-Ev cells[J].Int Immunopharmacol,2017,46:23-30.
[10] MEHAFFEY E,MAJID D S A.Tumor necrosis factor-α,kidney function,and hypertension[J].Am J Physiol Renal Physiol,2017,313(4):F1005-F1008.
[11] PETERSEN A M,PEDERSEN B K.The role of IL-6 in mediating the anti-inflammatory effects of exercise[J].J Physiol Pharmacol,2006,57 Suppl 10:43-51.
[12] REDONDO E,GáZQUEZ A,VADILLO S,et al.Induction of interleukin-8 and interleukin-12 in neonatal ovine lung following experimental inoculation of bovine respiratory syncytial virus[J].J Comp Pathol,2014,150(4):434-448.
[13] QIN S Y,JIANG H X,LU D H,et al.Association of interleukin-10 polymorphisms with risk of irritable bowel syndrome:a meta-analysis[J].World J Gastroenterol,2013,19(48):9472-9480.
[14] 刘小倩,刘颖,荣超,等.ACE 2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)
[15] AOUN E,CHEN J,REIGHARD D,et al.Diagnostic accuracy of interleukin-6 and interleukin-8 in predicting severe acute pancreatitis:a meta-analysis[J].Pancreatology,2009,9(6):777-785.
[16] SAHIBZADA H A,KHURSHID Z,KHAN R S,et al.Salivary IL-8,IL-6 and TNF-α as potential diagnostic biomarkers for oral cancer[J].Diagnostics (Basel),2017,7(2):E21.
[17] BOUDJELLAB N,CHAN-TANG H S,LI X,et al.Interleukin 8 response by bovine mammary epithelial cells to lipopolysaccharide stimulation[J].Am J Vet Res,1998,59(12):1563-1567.
[18] LEE J,ZHAO X.Recombinant human interleukin-8,but not human interleukin-1β,induces bovine neutrophil migration in an in vitro co-culture system[J].Cell Biol Int,2000,24(12):889-895.
[19] GONZáLEZ G E,RHALEB N E,D’AMBROSIO M A,et al.Cardiac-deleterious role of galectin-3 in chronic angiotensin Ⅱ-induced hypertension[J].Am J Physiol Heart Circ Physiol,2016,311(5):H1287-H1296.
[20] 郑书国,任尤楠,赵梦秋,等.芝麻素含药血清对血管紧张素Ⅱ诱导大鼠心肌细胞凋亡的影响[J].中药材,2015,38(5):1013-1017.ZHENG S G,REN Y N,ZHAO M Q,et al.Effect of serum containing sesamin on angiotensin Ⅱ-induced apoptosis in rat cardiomyocytes[J].Journal of Chinese Medicinal Materials,2015,38(5):1013-1017.(in Chinese)
[21] PATEL V B,BASU R,OUDIT G Y.ACE2/Ang 1-7 axis:A critical regulator of epicardial adipose tissue inflammation and cardiac dysfunction in obesity[J].Adipocyte,2016,5(3):306-311.
[22] NIAZI Z R,SILVA G C,RIBEIRO T P,et al.EPA:DHA 6:1 prevents angiotensin Ⅱ-induced hypertension and endothelial dysfunction in rats:role of NADPH oxidase- and COX-derived oxidative stress[J].Hypertens Res,2017,40(12):966-975.
[23] IMAI Y,KUBA K,PENNINGER J M.The discovery of angiotensin-converting enzyme 2 and its role in acute lung injury in mice[J].Exp Physiol,2008,93(5):543-548.
[24] HAN J B,YE S J,ZOU C P,et al.Angiotensin Ⅱ causes biphasic STAT3 activation through TLR4 to initiate cardiac remodeling[J].Hypertension,2018,72(6):1301-1311.
[25] YU X Y,XIA Y F,ZENG L Y,et al.A blockade of PI3Kγ signaling effectively mitigates angiotensin Ⅱ-induced renal injury and fibrosis in a mouse model[J].Sci Rep,2018,8:10988.
[26] SODHI C P,WOHLFORD-LENANE C,YAMAGUCHI Y,et al.Attenuation of pulmonary ACE2 activity impairs inactivation of des-Arg9 bradykinin/BKB1R axis and facilitates LPS-induced neutrophil infiltration[J].Am J Physiol-Lung Cell Mol Physiol,2018,314(1):L17-L31.
[27] SIM?ES ESILVA A C,TEIXEIRA M M.ACE inhibition,ACE2 and angiotensin-(1-7) axis in kidney and cardiac inflammation and fibrosis[J].Pharmacol Res,2016,107:154-162.
[28] HAO Y W,LIU Y.Osthole alleviates bleomycin-induced pulmonary fibrosis via modulating angiotensin-converting enzyme 2/angiotensin-(1-7) axis and decreasing inflammation responses in rats[J].Biol Pharm Bull,2016,39(4):457-465.
[29] LIU C X,XIAO L P,LI F D,et al.Generation of outbred Ace2 knockout mice by RNA transfection of TALENs displaying colitis reminiscent pathophysiology and inflammation[J].Transgenic Res,2015,24(3):433-446.
[30] WANG J,LIU R X,QI H Y,et al.The ACE2-angiotensin-(1-7)-Mas axis protects against pancreatic cell damage in cell culture[J].Pancreas,2015,44(2):266-272.
[31] HE H L,LIU L,CHEN Q H,et al.MSCs modified with ACE2 restore endothelial function following LPS challenge by inhibiting the activation of RAS[J].J Cell Physiol,2015,230(3):691-701.