Urocortin I后處理抗心肌缺血再灌注損傷的線粒體機制研究
發(fā)布時間：2018-07-18 10:49
【摘要】：目的:觀察UrocortinⅠ后處理對缺血/缺氧大鼠心臟功能、線粒體結(jié)構(gòu)及功能的影響,探討UrocortinⅠ后處理產(chǎn)生心肌保護(hù)作用的線粒體機制。方法:1.采用Langendorff離體心臟灌注裝置建立大鼠心肌缺血再灌注損傷模型,SPF級健康雄性SD大鼠48只隨機分為正常組(Nor組)、缺血再灌組(I/R組)、UrocortinⅠ后處理組(UcnⅠ組)、5-羥葵酸拮抗UrocortinⅠ組(5-HD+UcnⅠ組),每組12例。其中Nor組:Kerbs-Henseleit(K-H)液平衡20min后持續(xù)灌注135min。I/R組:平衡20min后灌注4℃停跳液,于32℃下全心缺血40min,再復(fù)灌95min。UcnⅠ組:缺血后復(fù)灌含UcnⅠ的K-H液30min,再續(xù)灌K-H液65min。5-HD+UcnⅠ組:UcnⅠ后處理前先給予含5-HD的K-H液5min,其余處理同UcnⅠ組。各組于平衡末及復(fù)灌末:(1)Powerlab/8SP數(shù)據(jù)采集系統(tǒng)記錄:心率(HR)、左室發(fā)展壓(LVDP)、左室舒張末壓力(LVDEP)及最大dp/dt(+dp/dtmax)等心功能指標(biāo);(2)取左室心肌組織提取線粒體,漢莎氧電極測定線粒體3態(tài)呼吸、呼吸控制比(RCR)等呼吸功能及煙酰胺腺嘌呤二核苷酸氧化酶(NADH-OX)、琥珀酸氧化酶(Suc-OX)等呼吸酶活性變化;(3)透射電子顯微鏡(TEM)觀察心肌超微結(jié)構(gòu)改變。2.離體心臟灌注裝置(MPA)分離成年大鼠心肌細(xì)胞,培養(yǎng)24h后進(jìn)行細(xì)胞計數(shù)并隨機分為正常組(Nor組)、缺氧/復(fù)氧組(I/R組)、UrocortinⅠ后處理組(UcnⅠ組)、5-羥葵酸拮抗UrocortinⅠ組(5-HD+UcnⅠ組)。其中Nor組:37℃培養(yǎng)箱中細(xì)胞持續(xù)培養(yǎng)135min;I/R組:細(xì)胞缺氧40min后復(fù)氧95min;UcnⅠ組:缺氧后復(fù)氧時先在含UcnⅠ的培養(yǎng)基中處理30min后再復(fù)氧60min;5-HD+UcnⅠ組:在給予UcnⅠ處理前先用特異性線粒體ATP敏感性鉀通道(mito-KATP)拮抗劑5-HD處理5min,余處理同UcnⅠ組。各組于復(fù)氧末對比觀察(1)激光共聚焦顯微鏡(LSCM)下線粒體膜電位(MMP)的變化;(2)CCK-8試劑盒檢測細(xì)胞活力的改變。結(jié)果:1.心功能指標(biāo)變化:與復(fù)灌末相比各組HR、LVDP、LVEDP、+dp/dtmax心功能缺血前均好于復(fù)灌末(P0.05);而缺血前各組心臟功能差異無統(tǒng)計學(xué)意義(P0.05);在復(fù)灌末:Nor組均優(yōu)于其余各組(P0.05),而UcnⅠ組比I/R組和5-HD+UcnⅠ組心功能好(P0.05);I/R組LVEDP、+dp/dtmax較5-HD+UcnⅠ組差(P0.05),但兩組間HR、LVDP比較差異無統(tǒng)計學(xué)意義(P0.05)。2.心肌線粒體呼吸功能和酶活性變化:缺血前各組呼吸功能及酶活性改變無統(tǒng)計學(xué)意義(P0.05);與復(fù)灌末相比,各組呼吸控制比(RCR)、煙酰胺腺嘌呤二核苷酸氧化酶(NADH-OX)、琥珀酸氧化酶等酶活性變化缺血前均好于復(fù)灌末(P0.05);在復(fù)灌末:Nor組線粒體呼吸功能及酶的活性均優(yōu)于其余各組(P0.05),而UcnⅠ組呼吸酶活性及3態(tài)呼吸、呼吸控制比(RCR)較I/R組和5-HD+UcnⅠ組要好(P0.05),但I(xiàn)/R組較5-HD+UcnⅠ組差(P0.05);關(guān)于4態(tài)呼吸(State4)缺血前及復(fù)灌末組間、組內(nèi)比較差異均無統(tǒng)計學(xué)意義(P0.05)。3.線粒體膜電位的變化:Nor組心肌細(xì)胞膜電位比例均高于其余各組(P0.01),而UcnⅠ組膜電位比例高于I/R組和5-HD+UcnⅠ組(P0.05),但5-HD+Ucn I組與I/R組比差異無統(tǒng)計學(xué)意義(P0.05)。4.心肌細(xì)胞活力測定:Nor組細(xì)胞活力高于其余各組(P0.05);UcnⅠ組細(xì)胞活力較5-HD+UcnⅠ組和I/R組高(P0.05),而5-HD+UcnⅠ組與I/R組比差異無統(tǒng)計學(xué)意義(P0.05)。結(jié)論:1.UcnⅠ后處理可改善離體心臟心肌收縮力、降低舒張末壓,恢復(fù)缺血再灌注后的心臟功能。2.UcnⅠ后處理可通過保護(hù)缺血心肌線粒體3態(tài)呼吸、RCR呼吸功能及Suc-OX、NADH-OX、Cyt-OX呼吸酶的活性,維持呼吸鏈電子傳遞及氧化磷酸化正常進(jìn)行,并穩(wěn)定線粒體膜電位,減輕線粒體結(jié)構(gòu)的損害而產(chǎn)生心肌保護(hù)效應(yīng)。且該心肌保護(hù)效應(yīng)與UcnⅠ后處理能開放mito-KATP通道有關(guān)。
[Abstract]:Objective: To observe the effects of Urocortin I post treatment on cardiac function, mitochondrial structure and function in ischemic / anoxic rats, and to explore the mitochondrial mechanism of myocardial protection after Urocortin I treatment. Methods: 1. the model of myocardial ischemia reperfusion injury in rats was established by Langendorff isolated cardiac perfusion device, and 48 healthy male SD rats of SPF grade were established. Only randomly divided into normal group (group Nor), ischemia-reperfusion group (group I/R), Urocortin I post treatment group (group Ucn I), 5- hydroxyl acid antagonistic group Urocortin I (group 5-HD+Ucn I), 12 cases in each group. Group 95min.Ucn I: 30min of K-H solution containing Ucn I after ischemia-reperfusion, and then reperfusion K-H liquid 65min.5-HD+Ucn I group: Ucn I was given K-H liquid 5min with 5-HD before reprocessing, and the rest treatment was in the same Ucn group. Each group was at the end of balance and the end of reperfusion: (1) recording of heart rate, left ventricular development pressure and left ventricular end diastolic pressure. And the maximum dp/dt (+dp/dtmax) index of cardiac function; (2) extracting mitochondria from left ventricular myocardium, measuring the respiratory function of mitochondria 3 State respiration, respiratory control ratio (RCR), nicotinamide adenine dinucleotide oxidase (NADH-OX), succinic oxidase (Suc-OX) and other respiratory enzyme activities by the Lufthansa oxygen electrode; (3) observation of transmission electron microscope (TEM) Myocardial ultrastructure changes.2. isolated cardiac perfusion device (MPA) isolated adult rat cardiac myocytes, and then cultured 24h to carry out cell count and randomly divided into normal group (group Nor), hypoxia / reoxygenation group (I/R group), Urocortin I post treatment group (Ucn I group), 5- hydroxyl acid antagonistic Urocortin I group (5-HD+Ucn I group). Nor group: cell holding in 37 C incubator Continuous culture of 135min; group I/R: reoxygenation 95min after anoxic 40min; group Ucn I: reoxygenation 60min in the medium containing Ucn I first in the culture medium containing Ucn I after hypoxia; group 5-HD+Ucn I was treated with specific mitochondrial ATP sensitive potassium channel (mito-KATP) antagonist before the treatment of Ucn I. The final contrast observation (1) the change of the grain bulk membrane potential (MMP) under laser confocal microscopy (LSCM); (2) the change of cell viability by CCK-8 kit. Results: 1. changes of cardiac function index: HR, LVDP, LVEDP, +dp/dtmax in each group were better than that of the end of reperfusion (P0.05) before the end of reperfusion, but there was no difference in cardiac function before ischemia. Meaning (P0.05); at the end of reirrigation, group Nor was better than the other groups (P0.05), while group Ucn I was better than group I/R and 5-HD+Ucn I (P0.05); LVEDP in group I/R and 5-HD+Ucn (P0.05) in group I/R, but there was no significant difference in myocardial mitochondrial respiratory function and enzyme activity in the two groups: respiratory work before ischemia The changes of energy and enzyme activity were not statistically significant (P0.05). Compared with the end of reperfusion, the changes of respiratory control ratio (RCR), nicotinamide adenine dinucleotide oxidase (NADH-OX), succinic acid oxidase and other enzymes were better than those of the end of reperfusion (P0.05). At the end of reperfusion, the mitochondrial respiratory function and enzyme activity in the Nor group were better than those in the other groups (P0.05). In Ucn I group, respiratory enzyme activity and 3 State respiration, respiratory control ratio (RCR) were better than group I/R and 5-HD+Ucn I group (P0.05), but I/R group was less than 5-HD+Ucn I group (P0.05). There was no significant difference between group I/R and 5-HD+Ucn I group (State4) before and after reperfusion (P0.05).3. mitochondrial membrane potential change: the ratio of membrane potential ratio in the myocardial cell of Nor group Compared with the other groups (P0.01), the membrane potential ratio in group Ucn I was higher than that in group I/R and group 5-HD+Ucn I (P0.05), but there was no significant difference between 5-HD+Ucn I group and I/R group (P0.05).4. cardiomyocyte viability measurement: Nor group cell viability was higher than that of other groups (P0.05). There was no significant difference in the comparison between the group and the I/R group (P0.05). Conclusion: the post-treatment of 1.Ucn I can improve the cardiac contractility of the isolated heart, reduce the end diastolic pressure, and restore the cardiac function after the ischemia-reperfusion after the.2.Ucn I treatment can protect the 3 State respiration of the mitochondria of ischemic myocardium, the activity of RCR breathing and the activity of Suc-OX, NADH-OX, Cyt-OX respiratory enzyme, and maintain the reactivation of the respiratory tract. The absorption of chain electron transfer and oxidative phosphorylation is normal, and the mitochondrial membrane potential is stabilized, and the damage of mitochondria structure can be reduced to produce myocardial protection effect, and the protective effect of the myocardium is related to the opening of the mito-KATP channel after Ucn I treatment.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R654.2
本文編號：2131670
[Abstract]:Objective: To observe the effects of Urocortin I post treatment on cardiac function, mitochondrial structure and function in ischemic / anoxic rats, and to explore the mitochondrial mechanism of myocardial protection after Urocortin I treatment. Methods: 1. the model of myocardial ischemia reperfusion injury in rats was established by Langendorff isolated cardiac perfusion device, and 48 healthy male SD rats of SPF grade were established. Only randomly divided into normal group (group Nor), ischemia-reperfusion group (group I/R), Urocortin I post treatment group (group Ucn I), 5- hydroxyl acid antagonistic group Urocortin I (group 5-HD+Ucn I), 12 cases in each group. Group 95min.Ucn I: 30min of K-H solution containing Ucn I after ischemia-reperfusion, and then reperfusion K-H liquid 65min.5-HD+Ucn I group: Ucn I was given K-H liquid 5min with 5-HD before reprocessing, and the rest treatment was in the same Ucn group. Each group was at the end of balance and the end of reperfusion: (1) recording of heart rate, left ventricular development pressure and left ventricular end diastolic pressure. And the maximum dp/dt (+dp/dtmax) index of cardiac function; (2) extracting mitochondria from left ventricular myocardium, measuring the respiratory function of mitochondria 3 State respiration, respiratory control ratio (RCR), nicotinamide adenine dinucleotide oxidase (NADH-OX), succinic oxidase (Suc-OX) and other respiratory enzyme activities by the Lufthansa oxygen electrode; (3) observation of transmission electron microscope (TEM) Myocardial ultrastructure changes.2. isolated cardiac perfusion device (MPA) isolated adult rat cardiac myocytes, and then cultured 24h to carry out cell count and randomly divided into normal group (group Nor), hypoxia / reoxygenation group (I/R group), Urocortin I post treatment group (Ucn I group), 5- hydroxyl acid antagonistic Urocortin I group (5-HD+Ucn I group). Nor group: cell holding in 37 C incubator Continuous culture of 135min; group I/R: reoxygenation 95min after anoxic 40min; group Ucn I: reoxygenation 60min in the medium containing Ucn I first in the culture medium containing Ucn I after hypoxia; group 5-HD+Ucn I was treated with specific mitochondrial ATP sensitive potassium channel (mito-KATP) antagonist before the treatment of Ucn I. The final contrast observation (1) the change of the grain bulk membrane potential (MMP) under laser confocal microscopy (LSCM); (2) the change of cell viability by CCK-8 kit. Results: 1. changes of cardiac function index: HR, LVDP, LVEDP, +dp/dtmax in each group were better than that of the end of reperfusion (P0.05) before the end of reperfusion, but there was no difference in cardiac function before ischemia. Meaning (P0.05); at the end of reirrigation, group Nor was better than the other groups (P0.05), while group Ucn I was better than group I/R and 5-HD+Ucn I (P0.05); LVEDP in group I/R and 5-HD+Ucn (P0.05) in group I/R, but there was no significant difference in myocardial mitochondrial respiratory function and enzyme activity in the two groups: respiratory work before ischemia The changes of energy and enzyme activity were not statistically significant (P0.05). Compared with the end of reperfusion, the changes of respiratory control ratio (RCR), nicotinamide adenine dinucleotide oxidase (NADH-OX), succinic acid oxidase and other enzymes were better than those of the end of reperfusion (P0.05). At the end of reperfusion, the mitochondrial respiratory function and enzyme activity in the Nor group were better than those in the other groups (P0.05). In Ucn I group, respiratory enzyme activity and 3 State respiration, respiratory control ratio (RCR) were better than group I/R and 5-HD+Ucn I group (P0.05), but I/R group was less than 5-HD+Ucn I group (P0.05). There was no significant difference between group I/R and 5-HD+Ucn I group (State4) before and after reperfusion (P0.05).3. mitochondrial membrane potential change: the ratio of membrane potential ratio in the myocardial cell of Nor group Compared with the other groups (P0.01), the membrane potential ratio in group Ucn I was higher than that in group I/R and group 5-HD+Ucn I (P0.05), but there was no significant difference between 5-HD+Ucn I group and I/R group (P0.05).4. cardiomyocyte viability measurement: Nor group cell viability was higher than that of other groups (P0.05). There was no significant difference in the comparison between the group and the I/R group (P0.05). Conclusion: the post-treatment of 1.Ucn I can improve the cardiac contractility of the isolated heart, reduce the end diastolic pressure, and restore the cardiac function after the ischemia-reperfusion after the.2.Ucn I treatment can protect the 3 State respiration of the mitochondria of ischemic myocardium, the activity of RCR breathing and the activity of Suc-OX, NADH-OX, Cyt-OX respiratory enzyme, and maintain the reactivation of the respiratory tract. The absorption of chain electron transfer and oxidative phosphorylation is normal, and the mitochondrial membrane potential is stabilized, and the damage of mitochondria structure can be reduced to produce myocardial protection effect, and the protective effect of the myocardium is related to the opening of the mito-KATP channel after Ucn I treatment.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R654.2
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