四氫生物蝶呤經(jīng)氧化應(yīng)激相關(guān)途徑對心肌損傷及心臟功能的影響機(jī)制研究
發(fā)布時間：2019-05-22 01:44
【摘要】：目的:四氫生物蝶呤(BH_4)是一氧化氮合酶(NOS)合成一氧化氮(NO)必須的輔助因子�；A(chǔ)及臨床證據(jù)表明,BH_4治療能夠改善由于BH_4缺乏所致的病理生理障礙。交感神經(jīng)興奮與氧化應(yīng)激相關(guān),β-受體阻滯劑可抑制交感興奮帶來的心臟損害,二者共用是否具有協(xié)同作用尚未可知。本研究旨在探討奈必洛爾聯(lián)合BH_4治療對心肌氧化應(yīng)激水平以及對一氧化氮/環(huán)磷酸鳥苷/蛋白激酶G(NO/cGMP/PKG)途徑信號分子表達(dá)水平的影響,從而證實其是否對自發(fā)性高血壓大鼠(SHRs)心臟舒張功能不全具有保護(hù)作用,二者聯(lián)合治療是否可產(chǎn)生協(xié)同效應(yīng),并初步闡明其相關(guān)機(jī)制。方法:12周齡SHRs 40只,隨機(jī)分為5組(n=8):基線組、安慰劑對照組、BH_4組、奈必洛爾組、BH_4+奈必洛爾組及同品系的雄性正常血壓對照魏-凱二氏大鼠(WKYs)組(n=10)。應(yīng)用小動物心臟彩超及右頸動脈插管導(dǎo)管法測定心臟功能;熒光探針法測定心肌活性氧類物質(zhì)(ROS);ELISA法測定心肌3-硝基酪氨酸(3-NT)含量、NO的生成量和環(huán)磷酸鳥苷(cGMP)在心肌的濃度;應(yīng)用~(14)C標(biāo)記L-精氨酸轉(zhuǎn)化為~(14)C-L瓜氨酸的轉(zhuǎn)化率,測定心肌內(nèi)皮源性一氧化氮合酶(e NOS)活性;應(yīng)用RT-PCR以及Western-blot測定eNOS、受磷蛋白(PLN)、肌質(zhì)網(wǎng)鈣泵2α亞型(SERCA2α)mRNA及相應(yīng)蛋白表達(dá)水平,同時測定心臟β_3腎上腺素能受體(β_3-AR)、蛋白激酶G(PKG)在心肌表達(dá)水平。結(jié)果:1.與WKYs大鼠組比較,SHRs存在相對性舒張功能相關(guān)指標(biāo)減低,舒張早期血流峰值速度(E)與舒張早期二尖瓣環(huán)最大運(yùn)動速度(E′)的比值(E/E′)增高(P0.01),而其與舒張晚期二尖瓣環(huán)運(yùn)動速度(A′)比值(E′/A′)降低(P0.01)。2.與對照組相比,BH_4干預(yù)對SHRs血壓和心率的影響無明顯差異(P0.05),而奈必洛爾干預(yù)可顯著降低血壓和心率(P0.01);與安慰劑對照組和BH_4單藥治療組相比較,二者聯(lián)合治療可使心率和血壓明顯降低(P0.01),然而與奈必洛爾單藥治療組相比血壓和心率沒有變化(P0.05);與對照組相比,BH_4和(或)奈必洛爾單藥干預(yù)有效減低左室舒張末壓力(LVEDP)和E/E′,而增加E′/A′(P均0.01),并縮短左室松弛時間時間常數(shù)(tau),使左心室壓力下降最大速率(-dP/dt_(max))與左室收縮壓力(LVSP)的比值(-dP/dt_(max)/LVSP)更低(P均0.01);二者聯(lián)合治療與單藥治療(BH_4或奈必洛爾)比較具有協(xié)同作用(P0.05)。3.與對照組比較,BH_4和(或)奈必洛爾單藥干預(yù)后,心肌eNOS m RNA的表達(dá)水平、eNOS二聚體的表達(dá)、eNOS活性、NO的生成量、p-PLN/SERCA2a、β_3-AR、cGMP和PKG的表達(dá)水平明顯增加(P0.05或P0.01),而3-NT和ROS表達(dá)量明顯降低(P0.05或P0.01),并且聯(lián)合治療組具有協(xié)同效應(yīng)(P0.05或P0.01)。結(jié)論:1.BH_4或奈必洛爾單獨(dú)治療均可明顯改善心臟舒張功能,其可能的機(jī)制為二者均促使心肌eNOS再耦聯(lián)、增加eNOS活性,提高心肌NO生成;2.奈必洛爾阻斷β_1-AR,降低心率和血壓,同時興奮β_3-AR,再耦聯(lián)eNOS,改善心臟舒張功能;3.BH_4與奈必洛爾相應(yīng)劑量的聯(lián)合治療對降低心肌氧化應(yīng)激、提高BH_4/BH2的比值、增加eNOS活性、增加NO的生成,具有協(xié)同效應(yīng),從而對心臟舒張功能不全產(chǎn)生更明顯的保護(hù)效應(yīng),其主效應(yīng)經(jīng)由再耦聯(lián)eNOS,并通過NO/c GMP/PKG途徑而發(fā)揮心臟保護(hù)作用。目的:腫瘤放射治療誘發(fā)的心肌損害在臨床較為常見,但其發(fā)生機(jī)制有待進(jìn)一步探討,治療手段有限。本研究旨在探討B(tài)H_4干預(yù)是否對X線輻射誘導(dǎo)抑制心肌細(xì)胞增殖、促進(jìn)凋亡、氧化應(yīng)激等損傷產(chǎn)生保護(hù)效應(yīng)及其相關(guān)的作用機(jī)制。方法:應(yīng)用克隆形成法實驗檢測X射線和X射線+BH_4干預(yù)對H9c2心肌細(xì)胞增殖活力的影響;測定X射線和X射線+BH_4干預(yù)對心肌細(xì)胞乳酸脫氫酶(LDH)含量、超氧化物歧化酶(SOD)和丙二醛(MDA)水平,以明確輻射對H9c2心肌細(xì)胞氧化應(yīng)激水平的變化情況;Hoechst 33342染色檢測細(xì)胞凋亡變化情況;碘化丙錠染色,流式細(xì)胞術(shù)檢測輻射對H9c2心肌細(xì)胞周期分布的影響;采用Western blot法檢測PI3K/Akt/P53信號通路蛋白的表達(dá)水平以及內(nèi)皮型一氧化氮合酶(e NOS)蛋白表達(dá)水平,同時也明確誘導(dǎo)型一氧化氮合酶(iNOS)表達(dá)變化情況。結(jié)果:X線輻射劑量依賴性地抑制H9c2心肌細(xì)胞的增殖、促進(jìn)細(xì)胞凋亡和細(xì)胞周期G_0/G_1期阻滯(P0.01);增加LDH釋放、MDA濃度和NO的產(chǎn)生,降低SOD在心肌細(xì)胞中濃度(P0.05或P0.01,而BH_4治療則顯著逆轉(zhuǎn)X射線輻射造成的損傷(P0.05或P0.01);X線輻射可顯著上調(diào)Bax、p53和caspase-3的表達(dá)水平,同時下調(diào)PI3K、Akt、p-Akt和Bcl-2的表達(dá)水平;BH_4治療顯著下調(diào)Bax、p53和caspase-3的表達(dá),而上調(diào)PI3K,Akt、p-Akt和Bcl-2的表達(dá)(P0.05或P0.01);BH_4上調(diào)2、4 Gy輻射組eNOS表達(dá),同時下調(diào)iNOS的表達(dá)(P0.05或P0.01),最終的凈效應(yīng)是增加eNOS來源NO的生成量。結(jié)論:BH_4干預(yù)可以改善X射線誘導(dǎo)的H9c2心肌細(xì)胞的損傷,其機(jī)制可能是通過增加eNOS的再耦聯(lián)而不是iNOS再耦聯(lián),同時降低由X線誘導(dǎo)心肌細(xì)胞氧化應(yīng)激水平及抑制細(xì)胞凋亡而發(fā)揮對放療心肌損傷的保護(hù)效應(yīng)。
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R96
本文編號：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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R96
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
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 郭繼鴻;;舒張性心衰的新理念[J];中國醫(yī)刊;2011年02期
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