野百合碱肺动脉高压大鼠SIRT3和Warburg效应关键酶的变化
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摘要
目的:研究野百合碱(MCT)肺动脉高压大鼠沉默信息调节因子3(SIRT3)和Warburg效应关键酶的表达变化。方法:16只SD大鼠随机分为对照组和MCT组,每组8只,MCT组大鼠腹腔注射MCT1次,对照组腹腔注射等体积生理盐水,常规饲养28 d,超声检测肺动脉血流动力学、右心功能相关指标,右心导管测定右心室收缩压,称重并计算右心室/(左心室+室间隔)的比值,苏木素伊红染色检测肺动脉重构,Western blot和免疫组织化学法检测SIRT3和Warburg效应关键酶的表达。结果:与对照组相比,MCT组肺动脉血流加速时间缩短[(22.33±1.53)ms对(33.67±5.51)ms,P<0.05],右心室内径增大[(3.33±0.22)mm对(2.29±0.21)mm,P<0.05],右心室收缩压明显升高[(30.90±4.28)mmHg对(7.83±0.67)mmHg,P<0.05],三尖瓣收缩期位移缩短[(2.01±0.09)mm对(2.59±0.19)mm,P<0.05],右心室/(左心室+室间隔)的比值增加(0.63±0.10对0.29±0.02,P<0.05)。苏木素伊红染色显示MCT组大鼠肺动脉中膜较对照组明显增厚[(378.47±129.97)μm对(105.16±61.17)μm,P<0.05]。Western blot结果显示,MCT组大鼠肺组织中的葡萄糖转运体1(Glut1,1.61±0.96对1.13±0.65,P<0.05)、葡萄糖转运体4(Glut4,0.98±0.63对0.69±0.47,P<0.05)、乳酸脱氢酶(LDH,1.14±0.12对0.66±0.12,P<0.05)、单羧酸转运蛋白4(MCT4,1.01±0.23对0.62±0.11,P<0.05)的蛋白表达水平明显高于对照组,丙酮酸脱氢酶(PDH,0.77±0.30对0.92±0.36,P<0.05)和SIRT3(0.91±0.11对1.44±0.11,P<0.05)的蛋白表达水平明显低于对照组。免疫组织化学染色结果显示,MCT组大鼠肺小动脉中Glut1 (0.24±0.07对0.20±0.04,P<0.05)、Glut4(0.26±0.02对0.23±0.02,P<0.05)、LDH (0.50±0.07对0.24±0.06,P<0.05)、MCT4(0.22±0.02对0.16±0.02,P<0.05)的蛋白表达水平明显高于对照组,PDH(0.13±0.01对0.22±0.01,P<0.05)和SIRT3(0.13±0.01对0.21±0.02,P<0.05)的蛋白表达水平明显低于对照组。结论:MCT肺动脉高压大鼠模型中存在肺动脉重构,重构肺动脉中存在Warburg效应增强及SIRT3表达下调。
Objective:To study the expression of key enzymes of Warburg effect and SIRT3 in rats with pulmonary arterial hypertension induced by monocrotaline(MCT). Methods:Sixteen SD rats were randomly divided into the control and MCT groups,each group of eight.MCT group rats were intraperitoneal injected with 1% MCT solution,while control group rats were intraperitoneal injected with saline solution.MCT and control group rats were conventional bred for 28 d and performed with hematoxylin eosin(HE)stain for detecting the pathological changes of pulmonary artery remodeling,echocardiography for detection of pulmonary artery hemodynamics and right ventricular function,right cardiac catheter for measurement of right ventricular systolic pressure and calculation of the weight ratio of right ventricle/(left ventricle+interventricular septum).The key enzymes of Warburg effect and SIRT3 were assessed by western blot and immunohistochemistry,respectively. Results:MCT group rats significantly shortened pulmonary artery acceleration time [(22.33±1.53)ms vs.(33.67±5.51)ms,P<0.05],increased right ventricular internal dimension [(3.33±0.22)mm vs.(2.29±0.21)mm,P<0.05],increased right ventricular systolic pressure[(30.90±4.28)mmHg vs.(7.83±0.67)mmHg,P<0.05],decreased tricuspid annular plane systolic excursion[(2.01±0.09)mm vs.(2.59±0.19)mm,P<0.05],and increased right ventricle/left ventricle+interventricular septum(0.63±0.10 vs.0.29±0.02,P<0.05),as compared with control group rats.HE stain showed that the wall thickness of the pulmonary artery significantly increased in MCT group compared with control group[(378.47±129.97)μm vs.(105.16±61.17)μm,P<0.05)].Western blot results showed that the lung tissues of MCT group rats significantly upregulated the expression of Glut1(1.61±0.96 vs.1.13±0.65,P<0.05),Glut4(0.98±0.63 vs.0.69±0.47,P<0.05),LDH(1.14±0.12 vs.0.66±0.12,P<0.05)and MCT4(1.01±0.23 vs.0.62±0.11,P<0.05),while downregulated the expression of PDH(0.77±0.30 vs.0.92±0.36,P<0.05)and SIRT3(0.91±0.11 vs.1.44±0.11,P<0.05),as compared with those of control group rats.Immunohistochemistry results suggested that the Glut1(0.24±0.07 vs.0.20±0.04,P<0.05),Glut4(0.26±0.02 vs.0.23±0.02,P<0.05),LDH(0.50±0.07 vs.0.24±0.06,P<0.05)and MCT4(0.22±0.02 vs.0.16±0.02,P<0.05)in pulmonary arterioles of MCT group rats were significantly higher,while the PDH(0.13±0.01 vs.0.22±0.01,P<0.05)and SIRT3(0.13±0.01 vs.0.21±0.02,P<0.05)were significantly lower than those of control group. Conclusions:Remodeled pulmonary arterial exists in pulmonary arterial hypertension rats induced by MCT, which is accompanied by enhanced Warburg effect and downregulated expression of SIRT3.
Objective:To study the expression of key enzymes of Warburg effect and SIRT3 in rats with pulmonary arterial hypertension induced by monocrotaline(MCT). Methods:Sixteen SD rats were randomly divided into the control and MCT groups,each group of eight.MCT group rats were intraperitoneal injected with 1% MCT solution,while control group rats were intraperitoneal injected with saline solution.MCT and control group rats were conventional bred for 28 d and performed with hematoxylin eosin(HE)stain for detecting the pathological changes of pulmonary artery remodeling,echocardiography for detection of pulmonary artery hemodynamics and right ventricular function,right cardiac catheter for measurement of right ventricular systolic pressure and calculation of the weight ratio of right ventricle/(left ventricle+interventricular septum).The key enzymes of Warburg effect and SIRT3 were assessed by western blot and immunohistochemistry,respectively. Results:MCT group rats significantly shortened pulmonary artery acceleration time [(22.33±1.53)ms vs.(33.67±5.51)ms,P<0.05],increased right ventricular internal dimension [(3.33±0.22)mm vs.(2.29±0.21)mm,P<0.05],increased right ventricular systolic pressure[(30.90±4.28)mmHg vs.(7.83±0.67)mmHg,P<0.05],decreased tricuspid annular plane systolic excursion[(2.01±0.09)mm vs.(2.59±0.19)mm,P<0.05],and increased right ventricle/left ventricle+interventricular septum(0.63±0.10 vs.0.29±0.02,P<0.05),as compared with control group rats.HE stain showed that the wall thickness of the pulmonary artery significantly increased in MCT group compared with control group[(378.47±129.97)μm vs.(105.16±61.17)μm,P<0.05)].Western blot results showed that the lung tissues of MCT group rats significantly upregulated the expression of Glut1(1.61±0.96 vs.1.13±0.65,P<0.05),Glut4(0.98±0.63 vs.0.69±0.47,P<0.05),LDH(1.14±0.12 vs.0.66±0.12,P<0.05)and MCT4(1.01±0.23 vs.0.62±0.11,P<0.05),while downregulated the expression of PDH(0.77±0.30 vs.0.92±0.36,P<0.05)and SIRT3(0.91±0.11 vs.1.44±0.11,P<0.05),as compared with those of control group rats.Immunohistochemistry results suggested that the Glut1(0.24±0.07 vs.0.20±0.04,P<0.05),Glut4(0.26±0.02 vs.0.23±0.02,P<0.05),LDH(0.50±0.07 vs.0.24±0.06,P<0.05)and MCT4(0.22±0.02 vs.0.16±0.02,P<0.05)in pulmonary arterioles of MCT group rats were significantly higher,while the PDH(0.13±0.01 vs.0.22±0.01,P<0.05)and SIRT3(0.13±0.01 vs.0.21±0.02,P<0.05)were significantly lower than those of control group. Conclusions:Remodeled pulmonary arterial exists in pulmonary arterial hypertension rats induced by MCT, which is accompanied by enhanced Warburg effect and downregulated expression of SIRT3.
引文
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[14] Cimen H,Han MJ,Yang YJ,et al.Regulation of succinate dehydrogenase activity by SIRT3in mammalian mitochondria[J].Biochemistry,2010,49(2):304-311.
[15] Ozden O,Park SH,Wagner BA,et al.SIRT3deacetylates and increases pyruvate dehydrogenase activity in cancer cells[J].Free Radic Biol Med,2014,76:163-172.
[16] Zhang XK,Ji RP,Liao XH,et al.MicroRNA-195regulates metabolism in failing myocardium via alterations in sirtuin 3expression and mitochondrial protein acetylation[J].Circulation,2018,137(19):2052-2067.
[2] Paulin R,Michelakis ED.The metabolic theory of pulmonary arterial hypertension[J].Circ Res,2014,115(1):148-164.
[3] Zhang R,Jing ZC.Energetic metabolic roles in pulmonary arterial hypertension and right ventricular remodeling[J].Curr Pharm Des,2016,22(31):4780-4795.
[4] Xiong YL,Wang MX,Zhao JB,et al.Sirtuin 3:a Janus face in cancer[J].Int J Oncol,2016,49(6):2227-2235.
[5] Koppenol WH,Bounds PL,Dang CV.Otto warburg′s contributions to current concepts of cancer metabolism[J].Nat Rev Cancer,2011,11(5):325-337.
[6] Archer SL,Gomberg-Maitland M, Maitland ML,et al.Mitochondrial metabolism,redox signaling,and fusion:a mitochondria-ROS-HIF-1alpha-Kv1.5O2-sensing pathway at the intersection of pulmonary hypertension and cancer[J].Am J Physiol Heart Circ Physiol, 2008, 294(2):H570-H578.
[7] Anderson KA,Green MF,Huynh FK,et al.Snapshot:mammalian sirtuins[J].Cell,2014,159(4):956.
[8] Li R,Quan Y,Xia W.SIRT3inhibits prostate cancer metastasis through regulation of FOXO3A by suppressing Wnt/β-catenin pathway[J].Exp Cell Res,2018,364(2):143-151.
[9] Paulin R, Dromparis P,Sutendra G,et al.Sirtuin 3deficiency is associated with inhibited mitochondrial function and pulmonary arterial hypertension in rodents and humans[J].Cell Metab,2014,20(5):827-839.
[10] Wei T, Huang G, Gao J,et al.Sirtuin 3 deficiency accelerates hypertensive cardiac remodeling by impairing angiogenesis[J].J Am Heart Assoc,2017,6(8):e006114.
[11] Ansari A,Rahman MS,Saha SK,et al.Function of the SIRT3 mitochondrial deacetylase in cellular physiology,cancer,and neurodegenerative disease[J].Aging Cell,2017,16(1):4-16.
[12] Fan J,Shan CL,Kang HB,et al.Tyr phosphorylation of PDP1toggles recruitment between ACAT1and SIRT3to regulate the pyruvate dehydrogenase complex[J].Mol Cell,2014,53(4):534-548.
[13] Zou XH, Zhu YE, Park SH,et al. SIRT3-mediated dimerization of IDH2 directs cancer cell metabolism and tumor growth[J].Cancer Res,2017,77(15):3990-3999.
[14] Cimen H,Han MJ,Yang YJ,et al.Regulation of succinate dehydrogenase activity by SIRT3in mammalian mitochondria[J].Biochemistry,2010,49(2):304-311.
[15] Ozden O,Park SH,Wagner BA,et al.SIRT3deacetylates and increases pyruvate dehydrogenase activity in cancer cells[J].Free Radic Biol Med,2014,76:163-172.
[16] Zhang XK,Ji RP,Liao XH,et al.MicroRNA-195regulates metabolism in failing myocardium via alterations in sirtuin 3expression and mitochondrial protein acetylation[J].Circulation,2018,137(19):2052-2067.