肾素血管紧张素系统(RAS)两条轴相互负向调节与大鼠非酒精性脂肪肝病(NAFLD)肝损伤的关系研究
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摘要
探讨了肾素血管紧张素系统(renin angiotensin system,RAS)两条轴对大鼠非酒精性脂肪肝病(non-alcoholic fatty liver disease,NAFLD)肝脏损伤的相互负向调节作用。30只雄性SD大鼠随机分为正常对照组、模型组和用药组。除正常对照组外,其余2组饲喂高脂饲料,用药组另外给伐他汀50 mg/kg·d。3周后,宰杀大鼠,测定血清中甘油三酯(TG)、谷丙转氨酶(ALT)、谷草转氨酶(AST)的含量;测定肝组织匀浆羟自由基(·OH)、一氧化氮合酶(NOS)、超氧化物歧化酶(SOD)、总抗氧化能力(T-AOC)酶活性;ELISA法测定组织匀浆中血管紧张素Ⅱ(AngⅡ)、血管紧张素1-7(Ang1-7)及白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)含量;Western blot分析肝组织血管紧张素转化酶(ACE)、血管紧张素转化酶2(ACE2)、血管紧张素Ⅱ受体1亚型(AT1R)、Mas受体(MasR)蛋白水平。结果:与正常对照组相比,模型组大鼠血清TG、AST和ALT含量以及·OH和NOS活性均显著升高(P<0.05), T-AOC、SOD活性显著降低(P<0.05);IL-1β和TNF-α含量极显著升高(P<0.01);ACE、ACE2的蛋白表达水平与ACE/ACE2的比值均显著升高(P<0.05),AngⅡ、Ang1-7含量与AT1R表达升高(P<0.05),MasR表达有升高趋势(P>0.05)。结论:高脂饲料连续饲喂3周可诱导大鼠发生NAFLD,肝脏局部RAS两条轴均处于激活状态,ACE介导AngⅡ-AT1R经典轴促进肝损伤,而ACE2介导Ang1-7-MasR轴抵抗肝损伤;ACE2负性调节RAS作用,对大鼠NAFLD时肝脏氧化应激及炎性损伤有一定保护作用。
This paper explores the negative regulation effect of the two axes of the renin angiotensin system(RAS) on liver injury in rats with non-alcoholic fatty liver disease(NAFLD). Thirty male SD rats were randomly divided into a normal control group, a model group and a medication group. Except the normal control group, the other two groups were fed with a high-fat diet. The medication group was additionally given Lovastatin 50 mg/kg·d as a positive control every day. Three weeks later, blood samples were drawn and livers were taken from the rats. The contents of TG, ALT and AST in their serum were determined. The liver homogenate was prepared and the activities of ·OH, NOS, SOD and T-AOC were measured. The contents of angiotensinⅡ(AngⅡ), angiotensin 1-7(Ang1-7) and IL-1β and TNF-α in the rat liver tissues were determined using ELISA. The levels of angiotensin converting enzyme(ACE), angiotensin converting enzyme Ⅱ(ACE2), AngⅡ type 1 receptor(AT1R) and MasR in the liver tissues were analyzed by Western blot. The results were as follows: Compared with the normal control group, the levels of TG, ALT and AST in the serum of the model group significantly increased(P<0.05); the oxidative stress index, the activities of ·OH and NOS in rat livers increased significantly(P<0.05); the activities of anti-oxidative stress T-AOC and SOD significantly decreased( P < 0. 05); the levels of inflammatory factors IL-1β and TNF-αsignificantly increased( P <0. 01); the protein expression levels of ACE,ACE2 and the ratio of ACE/ACE2 in liver tissues of the model group were significantly higher than those in the normal control group and the medication group( P<0. 05); the expression of AT1R increased( P<0. 05); MasR expression increased although the difference was not significant( P>0. 05); the level of Ang1-7 and AngⅡ in the liver tissues of the model group increased significantly( P<0. 05). In conclusion,continuous feeding of a high-fat diet for 3 weeks induced NAFLD in rats. The two axes of liver local RAS were activated. ACE mediated the classical axis AngⅡ-AT1R which aggravated liver injury. However,ACE2 mediated the Ang1-7-MasR axis against liver damage. ACE2 negatively regulated RAS and had a protective effect on hepatic oxidative stress and inflammatory injury in rats with NAFLD.
This paper explores the negative regulation effect of the two axes of the renin angiotensin system(RAS) on liver injury in rats with non-alcoholic fatty liver disease(NAFLD). Thirty male SD rats were randomly divided into a normal control group, a model group and a medication group. Except the normal control group, the other two groups were fed with a high-fat diet. The medication group was additionally given Lovastatin 50 mg/kg·d as a positive control every day. Three weeks later, blood samples were drawn and livers were taken from the rats. The contents of TG, ALT and AST in their serum were determined. The liver homogenate was prepared and the activities of ·OH, NOS, SOD and T-AOC were measured. The contents of angiotensinⅡ(AngⅡ), angiotensin 1-7(Ang1-7) and IL-1β and TNF-α in the rat liver tissues were determined using ELISA. The levels of angiotensin converting enzyme(ACE), angiotensin converting enzyme Ⅱ(ACE2), AngⅡ type 1 receptor(AT1R) and MasR in the liver tissues were analyzed by Western blot. The results were as follows: Compared with the normal control group, the levels of TG, ALT and AST in the serum of the model group significantly increased(P<0.05); the oxidative stress index, the activities of ·OH and NOS in rat livers increased significantly(P<0.05); the activities of anti-oxidative stress T-AOC and SOD significantly decreased( P < 0. 05); the levels of inflammatory factors IL-1β and TNF-αsignificantly increased( P <0. 01); the protein expression levels of ACE,ACE2 and the ratio of ACE/ACE2 in liver tissues of the model group were significantly higher than those in the normal control group and the medication group( P<0. 05); the expression of AT1R increased( P<0. 05); MasR expression increased although the difference was not significant( P>0. 05); the level of Ang1-7 and AngⅡ in the liver tissues of the model group increased significantly( P<0. 05). In conclusion,continuous feeding of a high-fat diet for 3 weeks induced NAFLD in rats. The two axes of liver local RAS were activated. ACE mediated the classical axis AngⅡ-AT1R which aggravated liver injury. However,ACE2 mediated the Ang1-7-MasR axis against liver damage. ACE2 negatively regulated RAS and had a protective effect on hepatic oxidative stress and inflammatory injury in rats with NAFLD.
引文
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[12] 张伟.β-酪啡肽-7对STZ诱导的糖尿病肾病大鼠肾损伤的保护作用及其机制研究[D].南京农业大学,2013.
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[14] 姚瑶,李龙龙,姜志浩,等.棕榈酸对BRL-3A细胞胰岛素抵抗和脂代谢的影响[J].南京农业大学学报,2019,42(1):130-136.
[15] Mantena S K,King A L,Andringa K K,et al.Mitochondrial dysfunction and oxidative stress in the pathogenesis of alcohol- and obesity-induced fatty liver diseases[J].Free Radical Bio Med,2008,44(7):1259-1272.
[16] Rolo A P,Teodoro J S,Palmeira C M.Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis[J].Free Radical Bio Med,2012,52(1):59-69.
[17] Tiniakos D G,Vos M B,Brunt E M.Nonalcoholic fatty liver disease:pathology and pathogenesis[J].Annu Rev Pathol,2010,5(5):145-171.
[18] Santos R A S,Sampaio W O,Alzamora A C,et al.The ACE2/angiotensin-(1-7)/Mas axis of the renin-angiotensin system:focus on angiotensin-(1-7)[J].Physiol Rev,2018,98(1):505-553.
[19] de Macedo S M,Guimaraes T A,Feltenberger J D,et al.The role of renin-angiotensin system modulation on treatment and prevention of liver diseases[J].Peptides,2014,62:189-196.
[20] Vergniol J,Barbu V,Lemoine M,et al.Progression of NAFLD in humans is associated with the activation of the renin-angiotensin system[J].J Hepatol,2011,54(11):S348-S348.
[21] Cao X,Yang F Y,Shi T T,et al.Angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis activates Akt signaling to ameliorate hepatic steatosis[J].Sci Rep,2016,6:21592.
[22] Cengiz M,Ozenirler S,Yilmaz G,et al.Impact of hepatic immunoreactivity of angiotensin-converting enzyme 2 on liver fibrosis due to nonalcoholic steatohepatitis[J].Clin Res Hepatol Gastroenterol,2015,39(6):692-698.
[23] 刘波,吴小翎,张霞,等.福辛普利对非酒精性脂肪性肝炎大鼠肝组织ACE和ACE2基因mRNA转录水平的影响[J].中国生物制品学杂志,2012,25(12):1658-1662.
[2] Goh G B,Pagadala M R,Dasarathy J,et al.Renin-angiotensin system and fibrosis in non-alcoholic fatty liver disease[J].Liver Int,2015,35(3):979-985.
[3] Frantz E D,Penna-de-Carvalho A,Batista T D,et al.Compar-ative effects of the renin-angiotensin system blockers on nonalcoholic fatty liver disease and insulin resistance in C57Bl/6 Mice[J].Metab Syndr Relat D,2014,12(4):191-201.
[4] Wu Y,Ma K L,Zhang Y,et al.Lipid disorder and intrahepatic renin-angiotensin system activation synergistically contribute to non-alcoholic fatty liverdisease[J].Liver Int,2016,36(10):1525-1534.
[5] Xue H,Zhou L,Yuan P,et al.Counteraction between angiotensin Ⅱ and angiotensin-(1-7) via activating angiotensin type I and Mas receptor on rat renal mesangial cells[J].Regul Peptides,2012,177(1-3):12-20.
[6] Santos R A S,Ferreira A J,Silva A C E.Recent advances in the angiotensin-converting enzyme 2-angiotensin(1-7)-Mas axis[J].Exp Physiol,2008,93(5):519-527.
[7] Simoes e Silva A C,Silveira K D,Ferreira A J,et al.ACE2,angiotensin-(1-7) and Mas receptor axis in inflammation and fibrosis[J].Br J Pharmacol,2013,169(3):477-492.
[8] Cao X,Yang F Y,Xin Z,et al.The ACE2/Ang-(1-7)/Mas axis can inhibit hepatic insulin resistance[J].Mol Cell Endocrinol,2014,393(1-2):30-38.
[9] Sousa Santos S H,Oliveira Andrade J M,Fernandes L R,et al.Oral Angiotensin-(1-7) prevented obesity and hepatic inflammation by inhibition of resistin/TLR4/MAPK/NF-kappa B in rats fed with high-fat diet[J].Peptides,2013,46:47-52.
[10] Zhang W,Miao J,Li P,et al.Up-regulation of components of the renin-angiotensin system in liver fibrosis in the rat induced by CCl4[J].Res Vet Sci,2013,95(1):54-58.
[11] 中华医学会肝病学分会脂肪肝和酒精性肝病学组,中国医师学会脂肪性肝病专家委员会.非酒精性脂肪性肝病防治指南(2018年更新版)[J].临床肝胆病杂志,2018,34(05):947-957.
[12] 张伟.β-酪啡肽-7对STZ诱导的糖尿病肾病大鼠肾损伤的保护作用及其机制研究[D].南京农业大学,2013.
[13] Spahis S,Delvin E,Borys J M,et al.Oxidative stress as a critical factor in nonalcoholic fatty liver disease pathogenesis[J].Antioxid Redox Signal,2017,26(10):519.
[14] 姚瑶,李龙龙,姜志浩,等.棕榈酸对BRL-3A细胞胰岛素抵抗和脂代谢的影响[J].南京农业大学学报,2019,42(1):130-136.
[15] Mantena S K,King A L,Andringa K K,et al.Mitochondrial dysfunction and oxidative stress in the pathogenesis of alcohol- and obesity-induced fatty liver diseases[J].Free Radical Bio Med,2008,44(7):1259-1272.
[16] Rolo A P,Teodoro J S,Palmeira C M.Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis[J].Free Radical Bio Med,2012,52(1):59-69.
[17] Tiniakos D G,Vos M B,Brunt E M.Nonalcoholic fatty liver disease:pathology and pathogenesis[J].Annu Rev Pathol,2010,5(5):145-171.
[18] Santos R A S,Sampaio W O,Alzamora A C,et al.The ACE2/angiotensin-(1-7)/Mas axis of the renin-angiotensin system:focus on angiotensin-(1-7)[J].Physiol Rev,2018,98(1):505-553.
[19] de Macedo S M,Guimaraes T A,Feltenberger J D,et al.The role of renin-angiotensin system modulation on treatment and prevention of liver diseases[J].Peptides,2014,62:189-196.
[20] Vergniol J,Barbu V,Lemoine M,et al.Progression of NAFLD in humans is associated with the activation of the renin-angiotensin system[J].J Hepatol,2011,54(11):S348-S348.
[21] Cao X,Yang F Y,Shi T T,et al.Angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis activates Akt signaling to ameliorate hepatic steatosis[J].Sci Rep,2016,6:21592.
[22] Cengiz M,Ozenirler S,Yilmaz G,et al.Impact of hepatic immunoreactivity of angiotensin-converting enzyme 2 on liver fibrosis due to nonalcoholic steatohepatitis[J].Clin Res Hepatol Gastroenterol,2015,39(6):692-698.
[23] 刘波,吴小翎,张霞,等.福辛普利对非酒精性脂肪性肝炎大鼠肝组织ACE和ACE2基因mRNA转录水平的影响[J].中国生物制品学杂志,2012,25(12):1658-1662.