四氢生物蝶呤经氧化应激相关途径对心肌损伤及心脏功能的影响机制研究
发布时间：2019-05-22 01:44
【摘要】：目的:四氢生物蝶呤(BH_4)是一氧化氮合酶(NOS)合成一氧化氮(NO)必须的辅助因子。基础及临床证据表明,BH_4治疗能够改善由于BH_4缺乏所致的病理生理障碍。交感神经兴奋与氧化应激相关,β-受体阻滞剂可抑制交感兴奋带来的心脏损害,二者共用是否具有协同作用尚未可知。本研究旨在探讨奈必洛尔联合BH_4治疗对心肌氧化应激水平以及对一氧化氮/环磷酸鸟苷/蛋白激酶G(NO/cGMP/PKG)途径信号分子表达水平的影响,从而证实其是否对自发性高血压大鼠(SHRs)心脏舒张功能不全具有保护作用,二者联合治疗是否可产生协同效应,并初步阐明其相关机制。方法:12周龄SHRs 40只,随机分为5组(n=8):基线组、安慰剂对照组、BH_4组、奈必洛尔组、BH_4+奈必洛尔组及同品系的雄性正常血压对照魏-凯二氏大鼠(WKYs)组(n=10)。应用小动物心脏彩超及右颈动脉插管导管法测定心脏功能;荧光探针法测定心肌活性氧类物质(ROS);ELISA法测定心肌3-硝基酪氨酸(3-NT)含量、NO的生成量和环磷酸鸟苷(cGMP)在心肌的浓度;应用~(14)C标记L-精氨酸转化为~(14)C-L瓜氨酸的转化率,测定心肌内皮源性一氧化氮合酶(e NOS)活性;应用RT-PCR以及Western-blot测定eNOS、受磷蛋白(PLN)、肌质网钙泵2α亚型(SERCA2α)mRNA及相应蛋白表达水平,同时测定心脏β_3肾上腺素能受体(β_3-AR)、蛋白激酶G(PKG)在心肌表达水平。结果:1.与WKYs大鼠组比较,SHRs存在相对性舒张功能相关指标减低,舒张早期血流峰值速度(E)与舒张早期二尖瓣环最大运动速度(E′)的比值(E/E′)增高(P0.01),而其与舒张晚期二尖瓣环运动速度(A′)比值(E′/A′)降低(P0.01)。2.与对照组相比,BH_4干预对SHRs血压和心率的影响无明显差异(P0.05),而奈必洛尔干预可显著降低血压和心率(P0.01);与安慰剂对照组和BH_4单药治疗组相比较,二者联合治疗可使心率和血压明显降低(P0.01),然而与奈必洛尔单药治疗组相比血压和心率没有变化(P0.05);与对照组相比,BH_4和(或)奈必洛尔单药干预有效减低左室舒张末压力(LVEDP)和E/E′,而增加E′/A′(P均0.01),并缩短左室松弛时间时间常数(tau),使左心室压力下降最大速率(-dP/dt_(max))与左室收缩压力(LVSP)的比值(-dP/dt_(max)/LVSP)更低(P均0.01);二者联合治疗与单药治疗(BH_4或奈必洛尔)比较具有协同作用(P0.05)。3.与对照组比较,BH_4和(或)奈必洛尔单药干预后,心肌eNOS m RNA的表达水平、eNOS二聚体的表达、eNOS活性、NO的生成量、p-PLN/SERCA2a、β_3-AR、cGMP和PKG的表达水平明显增加(P0.05或P0.01),而3-NT和ROS表达量明显降低(P0.05或P0.01),并且联合治疗组具有协同效应(P0.05或P0.01)。结论:1.BH_4或奈必洛尔单独治疗均可明显改善心脏舒张功能,其可能的机制为二者均促使心肌eNOS再耦联、增加eNOS活性,提高心肌NO生成;2.奈必洛尔阻断β_1-AR,降低心率和血压,同时兴奋β_3-AR,再耦联eNOS,改善心脏舒张功能;3.BH_4与奈必洛尔相应剂量的联合治疗对降低心肌氧化应激、提高BH_4/BH2的比值、增加eNOS活性、增加NO的生成,具有协同效应,从而对心脏舒张功能不全产生更明显的保护效应,其主效应经由再耦联eNOS,并通过NO/c GMP/PKG途径而发挥心脏保护作用。目的:肿瘤放射治疗诱发的心肌损害在临床较为常见,但其发生机制有待进一步探讨,治疗手段有限。本研究旨在探讨BH_4干预是否对X线辐射诱导抑制心肌细胞增殖、促进凋亡、氧化应激等损伤产生保护效应及其相关的作用机制。方法:应用克隆形成法实验检测X射线和X射线+BH_4干预对H9c2心肌细胞增殖活力的影响;测定X射线和X射线+BH_4干预对心肌细胞乳酸脱氢酶(LDH)含量、超氧化物歧化酶(SOD)和丙二醛(MDA)水平,以明确辐射对H9c2心肌细胞氧化应激水平的变化情况;Hoechst 33342染色检测细胞凋亡变化情况;碘化丙锭染色,流式细胞术检测辐射对H9c2心肌细胞周期分布的影响;采用Western blot法检测PI3K/Akt/P53信号通路蛋白的表达水平以及内皮型一氧化氮合酶(e NOS)蛋白表达水平,同时也明确诱导型一氧化氮合酶(iNOS)表达变化情况。结果:X线辐射剂量依赖性地抑制H9c2心肌细胞的增殖、促进细胞凋亡和细胞周期G_0/G_1期阻滞(P0.01);增加LDH释放、MDA浓度和NO的产生,降低SOD在心肌细胞中浓度(P0.05或P0.01,而BH_4治疗则显著逆转X射线辐射造成的损伤(P0.05或P0.01);X线辐射可显著上调Bax、p53和caspase-3的表达水平,同时下调PI3K、Akt、p-Akt和Bcl-2的表达水平;BH_4治疗显著下调Bax、p53和caspase-3的表达,而上调PI3K,Akt、p-Akt和Bcl-2的表达(P0.05或P0.01);BH_4上调2、4 Gy辐射组eNOS表达,同时下调iNOS的表达(P0.05或P0.01),最终的净效应是增加eNOS来源NO的生成量。结论:BH_4干预可以改善X射线诱导的H9c2心肌细胞的损伤,其机制可能是通过增加eNOS的再耦联而不是iNOS再耦联,同时降低由X线诱导心肌细胞氧化应激水平及抑制细胞凋亡而发挥对放疗心肌损伤的保护效应。
[Abstract]:Objective: To study the effect of tetrahydrobiopterin (BH _ 4) on the synthesis of nitric oxide (NO) by nitric oxide synthase (NOS). The basis and clinical evidence show that BH _ 4 treatment can improve the pathophysiological disorder due to the lack of BH _ 4. The sympathetic nerve stimulation is related to oxidative stress, and the co-receptor blocker can inhibit the cardiac damage caused by sympathetic excitation. The aim of this study was to investigate the effect of nebivolol combined with BH _ 4 on the level of myocardial stress and the expression level of NO/ cGMP/ PKG pathway in the NO/ cGMP/ PKG pathway. So as to confirm whether it has a protective effect on the cardiac diastolic function of the spontaneously hypertensive rats (SHRs), and whether the combination therapy can generate a synergistic effect and preliminarily clarify the relevant mechanism thereof. Methods: Forty-four-week-old SHRs were randomly divided into 5 groups (n = 8): the baseline group, the placebo control group, the BH _ 4 group, the nebivolol group, the BH _ 4 + nebivolol group and the male normal blood pressure control of the same line (WKYs) group (n = 10). The heart function was determined by using a small animal's heart color ultrasound and the right carotid artery catheterization method. The reactive oxygen species (ROS) in the myocardium were determined by the fluorescence probe. The content of 3-nitrotyrosine (3-NT), the amount of NO and the concentration of the cyclophosphonic acid (cGMP) in the myocardium were determined by ELISA. The activity of endogenic nitric oxide synthase (e-NOS) was determined by the conversion of ~ (14) C-labeled L-arginine to ~ (14) C-L citrulline, and the expression levels of eNOS, phosphoprotein (PLN),2-(SERCA2-1) mRNA and corresponding protein were determined by RT-PCR and Western-blot. At the same time, the expression level of the adrenergic receptor (HCO3 _ 3-AR) and protein kinase G (PKG) in the heart of the heart was determined. Results:1. Compared with the WKYs group, the correlation index of the relative diastolic function of the SHRs was reduced, and the ratio of the peak velocity (E) of the early diastolic blood flow to the maximum velocity (E) of the early diastolic mitral annulus (E/ E) was increased (P0.01). The ratio of the velocity of the mitral annulus to the end-diastolic mitral annulus (A-A) was decreased (P 0.01). Compared with the control group, the effect of BH _ 4 intervention on the blood pressure and heart rate of SHRs was not significantly different (P0.05). However, there was no change in blood pressure and heart rate compared with the control group (P0.05). Compared with the control group, the combination of BH _ 4 and (or) nebivolol was effective in reducing the left ventricular end-diastolic pressure (LVEDP) and E/ E ratio (P <0.01). The left ventricular relaxation time constant (tau) was shortened, and the ratio of left ventricular pressure (-dP/ dt _ (max)) to left ventricular systolic pressure (LVSP) (-dP/ dt _ (max)/ LVSP) was lower (P <0.01). Compared with the control group, the expression level of eNOS mRNA, the expression of eNOS dimer, eNOS activity, NO production, p-PLN/ SERCA2a, HCO3 _ 3-AR, cGMP and PKG were significantly increased (P0.05 or P0.01). The expression of 3-NT and ROS decreased significantly (P0.05 or P0.01), and the combined treatment group had a synergistic effect (P0.05 or P0.01). Conclusion: 1.BH _ 4 or nebivolol can obviously improve the diastolic function of the heart, and the possible mechanism is that the myocardial eNOS is recoupled, the eNOS activity is increased, and the NO production of the myocardium is improved. nebivolol is used to block the blood pressure, reduce the heart rate and blood pressure, and is also excited to be 3-ar, and then the eNOS is coupled to improve the diastolic function of the heart;3. the combined treatment of the corresponding dose of BH _ 4 and nebivolol can reduce the oxidative stress of the myocardium, increase the ratio of BH _ 4/ BH2, increase the activity of the eNOS, increase the generation of NO, It has a synergistic effect, which results in a more significant protective effect on the diastolic function of the heart, and its main effect is through recoupling the eNOS and the protective effect of the heart through the NO/ c GMP/ PKG pathway. Objective: The treatment of myocardial damage induced by radiotherapy is more common in clinic, but its mechanism is still to be discussed further, and the treatment is limited. The purpose of this study is to investigate whether BH _ 4 intervention can inhibit the proliferation of myocardial cells induced by X-ray radiation, promote apoptosis, oxidative stress, and so on. Methods: The effects of X-ray and X-ray + BH _ 4 on the activity of H9c2 myocardial cells were measured by the method of clone formation. The levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by X-ray and X-ray + BH _ 4 intervention. The changes of oxidative stress level of H9c2 myocardial cells were determined by explicit radiation. Hoechst 33342 staining and flow cytometry were used to detect the effect of radiation on the cycle distribution of H9c2 myocardial cells. The expression level of PI3K/ Akt/ P53 signaling pathway protein and the expression level of endothelial nitric oxide synthase (e-NOS) were detected by Western blot, and the expression of inducible nitric oxide synthase (iNOS) was also determined. Results: X-ray radiation dose-dependently inhibited the proliferation of H9c2 myocardial cells, and promoted cell apoptosis and cell cycle G _ 0/ G _ 1 arrest (P0.01), increased LDH release, MDA concentration and NO production, and decreased the concentration of SOD in myocardial cells (P0.05 or P0.01). The expression level of Bax, p53 and caspase-3 was significantly increased by X-ray radiation, and the expression level of PI3K, Akt, p-Akt and Bcl-2 was downregulated, and the expression of Bax, p53 and caspase-3 was down-regulated by BH _ 4, and the expression of PI3K and Akt was up-regulated. The expression of p-Akt and Bcl-2 (P0.05 or P0.01), the increase of eNOS expression in the radiation group of 2 and 4 Gy in BH _ 4, and the down-regulation of iNOS expression (P0.05 or P0.01), and the final net effect was to increase the generation of NO in eNOS. Conclusion: BH _ 4 intervention can improve the damage of H9c2 myocardial cells induced by X-ray, and the mechanism may be to increase the recoupling of eNOS instead of iNOS recoupling, while reducing the oxidative stress level induced by X-ray and the inhibition of cell apoptosis.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96
本文编号：2482570
[Abstract]:Objective: To study the effect of tetrahydrobiopterin (BH _ 4) on the synthesis of nitric oxide (NO) by nitric oxide synthase (NOS). The basis and clinical evidence show that BH _ 4 treatment can improve the pathophysiological disorder due to the lack of BH _ 4. The sympathetic nerve stimulation is related to oxidative stress, and the co-receptor blocker can inhibit the cardiac damage caused by sympathetic excitation. The aim of this study was to investigate the effect of nebivolol combined with BH _ 4 on the level of myocardial stress and the expression level of NO/ cGMP/ PKG pathway in the NO/ cGMP/ PKG pathway. So as to confirm whether it has a protective effect on the cardiac diastolic function of the spontaneously hypertensive rats (SHRs), and whether the combination therapy can generate a synergistic effect and preliminarily clarify the relevant mechanism thereof. Methods: Forty-four-week-old SHRs were randomly divided into 5 groups (n = 8): the baseline group, the placebo control group, the BH _ 4 group, the nebivolol group, the BH _ 4 + nebivolol group and the male normal blood pressure control of the same line (WKYs) group (n = 10). The heart function was determined by using a small animal's heart color ultrasound and the right carotid artery catheterization method. The reactive oxygen species (ROS) in the myocardium were determined by the fluorescence probe. The content of 3-nitrotyrosine (3-NT), the amount of NO and the concentration of the cyclophosphonic acid (cGMP) in the myocardium were determined by ELISA. The activity of endogenic nitric oxide synthase (e-NOS) was determined by the conversion of ~ (14) C-labeled L-arginine to ~ (14) C-L citrulline, and the expression levels of eNOS, phosphoprotein (PLN),2-(SERCA2-1) mRNA and corresponding protein were determined by RT-PCR and Western-blot. At the same time, the expression level of the adrenergic receptor (HCO3 _ 3-AR) and protein kinase G (PKG) in the heart of the heart was determined. Results:1. Compared with the WKYs group, the correlation index of the relative diastolic function of the SHRs was reduced, and the ratio of the peak velocity (E) of the early diastolic blood flow to the maximum velocity (E) of the early diastolic mitral annulus (E/ E) was increased (P0.01). The ratio of the velocity of the mitral annulus to the end-diastolic mitral annulus (A-A) was decreased (P 0.01). Compared with the control group, the effect of BH _ 4 intervention on the blood pressure and heart rate of SHRs was not significantly different (P0.05). However, there was no change in blood pressure and heart rate compared with the control group (P0.05). Compared with the control group, the combination of BH _ 4 and (or) nebivolol was effective in reducing the left ventricular end-diastolic pressure (LVEDP) and E/ E ratio (P <0.01). The left ventricular relaxation time constant (tau) was shortened, and the ratio of left ventricular pressure (-dP/ dt _ (max)) to left ventricular systolic pressure (LVSP) (-dP/ dt _ (max)/ LVSP) was lower (P <0.01). Compared with the control group, the expression level of eNOS mRNA, the expression of eNOS dimer, eNOS activity, NO production, p-PLN/ SERCA2a, HCO3 _ 3-AR, cGMP and PKG were significantly increased (P0.05 or P0.01). The expression of 3-NT and ROS decreased significantly (P0.05 or P0.01), and the combined treatment group had a synergistic effect (P0.05 or P0.01). Conclusion: 1.BH _ 4 or nebivolol can obviously improve the diastolic function of the heart, and the possible mechanism is that the myocardial eNOS is recoupled, the eNOS activity is increased, and the NO production of the myocardium is improved. nebivolol is used to block the blood pressure, reduce the heart rate and blood pressure, and is also excited to be 3-ar, and then the eNOS is coupled to improve the diastolic function of the heart;3. the combined treatment of the corresponding dose of BH _ 4 and nebivolol can reduce the oxidative stress of the myocardium, increase the ratio of BH _ 4/ BH2, increase the activity of the eNOS, increase the generation of NO, It has a synergistic effect, which results in a more significant protective effect on the diastolic function of the heart, and its main effect is through recoupling the eNOS and the protective effect of the heart through the NO/ c GMP/ PKG pathway. Objective: The treatment of myocardial damage induced by radiotherapy is more common in clinic, but its mechanism is still to be discussed further, and the treatment is limited. The purpose of this study is to investigate whether BH _ 4 intervention can inhibit the proliferation of myocardial cells induced by X-ray radiation, promote apoptosis, oxidative stress, and so on. Methods: The effects of X-ray and X-ray + BH _ 4 on the activity of H9c2 myocardial cells were measured by the method of clone formation. The levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by X-ray and X-ray + BH _ 4 intervention. The changes of oxidative stress level of H9c2 myocardial cells were determined by explicit radiation. Hoechst 33342 staining and flow cytometry were used to detect the effect of radiation on the cycle distribution of H9c2 myocardial cells. The expression level of PI3K/ Akt/ P53 signaling pathway protein and the expression level of endothelial nitric oxide synthase (e-NOS) were detected by Western blot, and the expression of inducible nitric oxide synthase (iNOS) was also determined. Results: X-ray radiation dose-dependently inhibited the proliferation of H9c2 myocardial cells, and promoted cell apoptosis and cell cycle G _ 0/ G _ 1 arrest (P0.01), increased LDH release, MDA concentration and NO production, and decreased the concentration of SOD in myocardial cells (P0.05 or P0.01). The expression level of Bax, p53 and caspase-3 was significantly increased by X-ray radiation, and the expression level of PI3K, Akt, p-Akt and Bcl-2 was downregulated, and the expression of Bax, p53 and caspase-3 was down-regulated by BH _ 4, and the expression of PI3K and Akt was up-regulated. The expression of p-Akt and Bcl-2 (P0.05 or P0.01), the increase of eNOS expression in the radiation group of 2 and 4 Gy in BH _ 4, and the down-regulation of iNOS expression (P0.05 or P0.01), and the final net effect was to increase the generation of NO in eNOS. Conclusion: BH _ 4 intervention can improve the damage of H9c2 myocardial cells induced by X-ray, and the mechanism may be to increase the recoupling of eNOS instead of iNOS recoupling, while reducing the oxidative stress level induced by X-ray and the inhibition of cell apoptosis.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96
【参考文献】
相关期刊论文 前2条
1 ;Diverse Effects of L-arginine on Cardiac Function of Rats Subjected to Myocardial Ischemia and Reperfusion in vivo[J];Acta Biochimica et Biophysica Sinica;2007年03期
2 郭继鸿;;舒张性心衰的新理念[J];中国医刊;2011年02期
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