生理学报Acta Physiologica Sinica, June 25, 2003, 55(3): 284－289
研究论文
活性氧和线粒体ATP敏感钾通道介导肿瘤坏死因子α对缺氧/复氧心肌的保护作用
傅琛, 曹春梅, 夏强*, 杨俊, 陆源
浙江大学医学院生理学教研室, 杭州 310031
摘要: 在培养的乳鼠心肌细胞上, 研究肿瘤坏死因子α (TNF-α)对缺氧/复氧损伤心肌的保护作用的机制。结果发现: (1) 用TNF-α (10－500 U/ml)预处理, 缺氧/复氧后心肌细胞内锰超氧化物歧化酶(Mn-SOD)活性增高、乳酸脱氢酶(LDH)释放量减少(P<0.05); (2) 用抗氧化剂N-乙酰半胱氨酸 (NAC, 1 mmol/L)、抗霉素A (antimycin A, 50 μmol/L)、2-巯基丙酰氨基乙酸(2-MPG, 400 μmol/L)和铜/锌超氧化物歧化酸(Cu/Zn-SOD)抑制剂二乙基二硫代氨基甲酸盐(DDC, 100 nmol/L)预处理, 可取消TNF-α的抑制缺氧/复氧心肌细胞LDH释放和诱导Mn-SOD活性增高的作用; (3) mitoKATP通道抑制剂5-羟基酸(5-HD)预处理可阻断TNF-α对缺氧/复氧心肌细胞的保护作用; 选择性mitoKATP通道开放剂diazoxide (50 μmol/L)预处理可减少复氧后心肌细胞LDH的释放(P<0.01), 其作用可被5-HD (100 μmol/L)和NAC所抑制。上述结果表明, 活性氧和线粒体ATP敏感钾通道参与介导TNF-α对缺氧/复氧损伤的心肌保护作用。
关键词: 心肌细胞; 肿瘤坏死因子α (TNF-α); 缺氧; 复氧; 活性氧; 线粒体ATP敏感钾通道
中图分类号: Q463
Reactive oxygen
species and mitochondrial KATP-sensitive channels mediated cardioprotection induced by TNF-α during hypoxia and reoxygenation
FU Chen, CAO Chun-Mei, XIA Qiang*, YANG Jun, LU Yuan
Department
of Physiology,
Abstract: The aim of the present study was to testify whether the reactive oxygen species and mitochondrial ATP-sensitive potassium (KATP) channels were involved in the cardioprotection induced by tumor necrosis factor α (TNF-α) in the cultured neonatal ventricular myocytes suffered from 12 h of hypoxia and 6 h of reoxygenation. We tested the release of lactate dihydrogenase (LDH) and manganese superoxide dismutase (Mn-SOD) with spectrophotometry. It was shown that pretreatment with TNF-α (10, 50, 100, or 500 U/ml) significantly increased the Mn-SOD activity and reduced LDH release in the neonatal ventricular myocytes subjected to hypoxia and reoxygenation. Pretreatment with NAC (1 mmol/L), antimycin A (50 μmol/L), 2-MPG (400 μmol/L), DDC (100 nmol/L) or 5-HD (100 μmol/L), respectively, attenuated the increase in Mn-SOD activity and reduction of LDH level induced by TNF-α in ventricular myocytes. Diazoxide (50 μmol/L), a selective opener of the mitochondrial KATP channel, decreased the LDH release of the myocytes subjected to hypoxia and reoxygenation, which could be abolished by pretreatment with NAC (1 mmol/L) or 5-HD (100 μmol/L). These results suggest that oxygen radical signals and mitochondrial KATP channels are involved in the cardioprotection induced by TNF-α.
Key words: myocytes; tumor necrosis factor α (TNF-α); hypoxia/reoxygenation; reactive oxygen
species; mitochondrial ATP-sensitive potassium channels
肿瘤坏死因子α (tumor necrosis factor α, TNF-α)是一种具有多种生物学功能的细胞因子, 包括心肌细胞在内的大部分细胞都能够合成TNF-α[1]。研究已证明, TNF-α可通过诱导锰超氧化物歧化酶(manganese superoxide dismutase, Mn-SOD)的表达和提高Mn-SOD的活性, 而对遭受缺血或缺氧损伤的整体心脏或心肌细胞产生保护作用[2－4]。Mn-SOD存在于线粒体的基质中, 是线粒体内主要的氧自由基清除剂[5]。但是, TNF-α是通过哪些信号转导通路诱导Mn-SOD表达及活性提高而起到心肌保护作用, 尚未见到相关的报道。
研究发现, TNF-α与其受体TR55结合后, 除了诱导Mn-SOD表达之外[6], 还促发包括产生活性氧(reactive oxygen species, ROS)在内的多种信号级联反应[7,8], 而后者可作为胞内信号分子参与激活缺氧预处理的心肌保护作用[9]。此外, Forber等发现, 心肌细胞线粒体内膜上的ATP敏感型钾通道(mitochondrial ATP-sensitive potassium channel, mitoKATP)开放后, ROS合成增多[10], 参与阿片类物质引起的心肌保护作用[11], 提示细胞内ROS的产生与mitoKATP有关。因此, 本文通过建立培养大鼠心室肌细胞缺氧/复氧损伤模型, 研究ROS和mitoKATP通道是否介导TNF-α预处理诱导的缺氧心肌保护作用。
图1.实验方案示意图
Fig. 1.Experimental protocols. A: The myocytes were incubated with DMEM for 33 h. B: The myocytes were incubated with DMEM for 15 h followed by 12 h of hypoxia and 6 h of reoxygenation. C: The myocytes were incubated with DMEM for 3 h, then TNF-α was added (final concentrations: 10, 50, 100 and 500 U/ml) for 12 h prior to hypoxia and reoxygenation. D: The myocytes were treated with NAC (1 mmol/L) or antimycin A (50 μmol/L) or 2-MPG (400 μmol/L) or DDC (100 nmol/L) or 5-HD (100 μmol/L) for 15 h followed by 12 h of hypoxia and 6 h of reoxygenation, respectively. E: Pretreatment with NAC (1 mmol/L) or antimycin A (50 μmol/L) or 2-MPG (400 μmol/L) or DDC (100 nmol/L) or 5-HD (100 μmol/L) for 3 h, respectively, then TNF-α was added (100 U/ml) and co-incubated for 12 h prior to hypoxia and reoxygenation. F: The myocytes were incubated with DMEM for 3 h, then diazoxide (50 μmol/L) was added for 12 h prior to hypoxia and reoxygenation. G: Pretreatment with NAC (1 mmol/L) or 5-HD (100 μmol/L) for 3 h, respectively, then diazoxide (50 μmol/L) was added and co-incubated for 12 h prior to hypoxia and reoxygenation.
1 材料和方法
1.1 药品和试剂
Diazoxide、 5-hydroxydecanoate(5-HD)、 N-acetylcysteine (NAC)、 antimycin A、 2-mercaptopropionyl glycine(2-MPG)均为Sigma公司产品。DMEM培养液(高糖及无糖)购自Gibco公司。胎牛血清为杭州四季青生物工程材料有限公司生产。Diethyldithiocarbamic acid (DDC) 由ALEXIS公司生产。SOD测定试剂盒购自南京建成生物工程研究所。TNF-α由中国科学院上海生物工程研究中心研制。
1.2 心肌细胞制备和培养
取生后1 － 2 d的Sprague-Dawley大鼠心室肌, 剪成1 mm3大小后用0.1%胰蛋白酶溶液(37℃)消化10 min, 收集心肌细胞悬液, 并用8 ml含15% 胎牛血清的DMEM(高糖)培养液中和胰酶作用。重复消化收集8－10次, 直到心肌组织完全解离, 以1200 r/min 离心6 min, 所得的沉淀用10 ml DMEM(15%胎牛血清)重悬; 然后置于二氧化碳培养箱(95%空气+5% CO2, 37℃)中预培养, 1 h后悬浮的心肌细胞用200目不锈钢网过滤, 将细胞密度调整到2×105个/ml, 分装于24孔板中培养。细胞培养至4－5 d, 细胞单层贴壁, 互相融合, 同簇细胞发生同步搏动, 每分钟搏动次数达到180次以上, 可用于实验。缺氧前15 h, 换为无血清DMEM培养液培养。
1.3 缺氧/复氧模型
吸去24孔板中的细胞培养液, 换95％ N2＋5％ CO2混合气体饱和的无糖DMEM培养液, 置于缺氧罐中(用95％ N2＋5％ CO2混合气体饱和, 37℃, O2<1%); 缺氧培养12 h后, 取出培养板, 放回5％二氧化碳培养箱继续培养6 h, 作为复氧处理。
1.4 实验分组
实验方案见图1。分组情况如下。对照组: 心肌细胞不作缺氧/复氧处理; 缺氧/复氧(H/R)组: 心肌细胞无血清DMEM培养15 h后, 再进行缺氧12 h和复氧6 h处理; TNF-α组: 心肌细胞孵育3 h后, 加入TNF-α (10、 50、 100、 500 U/ml)12 h后, 再进行缺氧12 h及复氧6 h处理; 抗氧化剂NAC (1 mmol/L)、antimycin A (50 μmol/L)、2-MPG (400 μmol/L)和Cu/Zn-SOD抑制剂DDC (100 nmol/L)以及mitoKATP通道阻断剂5-HD预处理对TNF-α (100 U/ml)作用影响各组及mitoKATP通道开放剂diazoxide组的方案(图1)将在结果部分描述。
单独使用TNF-α (10、 50、 100、 500 U/ml)、NAC (1 mmol/L)、antimycin A (50 μmol/L)和2-MPG (400 μmol/L)、5-HD (100 μmol/L)、DDC (100 nmol/L)和diazoxide (50 μmol/L)对正常细胞LDH和Mn-SOD在实验观察时间内无明显影响。
1.5 乳酸脱氢酶和Mn-SOD活性的测定
分别于缺氧处理前和复氧6 h两个时点取上清培养液50 μl, 利用分光光度法测定乳酸脱氢酶(lactate dehydrogenase, LDH)的活性[1]。复氧6 h后将心肌细胞超声粉碎, 制成细胞悬液, 采用黄嘌呤氧化酶法测定Mn-SOD的活性[12]。
1.6 统计学处理
所有数据均以mean±SD表示, 采用t检验和单因素方差分析进行统计学处理, P<0.05表示有显著差异。
2 结果
2.1
TNF-α预处理对H/R乳鼠心肌细胞的作用
如表1所示, 与对照组比较, 12 h缺氧及6 h复氧处理后, 心肌细胞LDH释放量显著增多(P<0.01), 细胞内Mn-SOD活性也明显升高(P<0.05)。与H/R组比较, 经过TNF-α (10、 50、 100和500 U/ml)预处理后的心肌细胞, LDH释放明显减少(P<0.01), 同时Mn-SOD活性显著升高(P<0.01)。
表1. TNF-α处理对缺氧/复氧后心肌细胞LDH释放量和Mn-SOD活性的影响
Table 1. Effect of TNF-α on the activity of Mn-SOD and the release of LDH in cardiomyocytes suffering from hypoxia/reoxygenation (n=15)
|
Group |
LDH release(fold increase) |
Mn-SOD activity(fold increase) |
|
Control |
1.15±0.08 |
1.04±0.06 |
|
H/R |
2.80±0.26** |
1.25±0.07* |
|
TNF-α |
|
|
|
10 U/ml |
2.09±0.14**## |
2.42±0.26*# |
|
50 U/ml |
1.76±0.07**## |
2.44±0.22**## |
|
100 U/ml |
1.58±0.04**## |
2.33±0.21**## |
|
500 U/ml |
1.63±0.08**## |
2.56±0.28**## |
Data are expressed as mean±SD of the fold increase in LDH release and Mn-SOD activity vs the value of pre-hypoxia. Isolated cardiac myocytes were pretreated with TNF-α before H/R. *P<0.05, **P<0.01 vs control group. #P<0.05, ##P<0.01 vs H/R group.
2.2 NAC、antimycin A、2-MPG和DDC对TNF-α预处理的H/R心肌细胞的影响
为了观察活性氧是否参与TNF-α对H/R处理后心肌细胞的作用, 心肌细胞分别用抗氧化剂NAC (1 mmol/L)、antimycin (50 μmol/L)和2-MPG (400 μmol/L)预处理3 h后, 加TNF-α (100 U/ml)共同作用12 h后, 再进行H/R处理。结果如图2所示, NAC、antimycin A和2-MPG预处理心肌细胞后, 均能阻断TNF-α (100 U/ml)抑制心肌细胞LDH释放及诱导Mn-SOD活性增加的作用。
为了观察过氧化氢是否参与TNF-α对H/R处理后心肌细胞的作用, 心肌细胞用Cu/Zn-SOD抑制剂DDC (100 nmol/L)预处理3 h后, 加TNF-α (100 U/ml)共同作用12 h后, 再进行H/R处理。图2结果表明, DDC预处理心肌细胞后, 阻断了TNF-α (100 U/ml)对H/R心肌细胞的作用。单独用NAC、antimycin A、2-MPG和DDC处理心肌细胞, 对H/R诱导心肌细胞LDH释放量和Mn-SOD活性的改变无明显影响。
图2.NAC (1 mmol/L)、antimycin A (50 μmol/L)、2-MPG (400 μmol/L)和DDC (100 nmol/L)对TNF-α (100 U/ml)引起的缺氧/复氧乳鼠心肌细胞LDH释放量(A)和Mn-SOD (B)活性改变的影响
Fig. 2.Effects of TNF-α (100 U/ml) on the release of LDH (A) and the activity of Mn-SOD (B) in cultured cardiomyocytes suffering from hypoxia/reoxygenation (H/R) in the presence or absence of NAC (1 mmol/L), antimycin A (50 μmol/L), 2-MPG (400 μmol/L), or DDC (100 nmol/L). n=15 per group. *P<0.05, **P<0.01 vs H/R group;#P<0.05, ##P<0.01 vs TNF-α group．
2.3 5-HD对TNF-α预处理的H/R心肌细胞的影响
为了观察mitoKATP是否参与TNF-α对H/R处理后心肌细胞的作用, 用选择性mitoKATP通道抑制剂5-HD(100 μmol/L)预处理心肌细胞3 h后, 加入TNF-α (100 U/ml)共同作用12 h后, 再进行H/R处理。图3结果表明, 用5-HD预处理后, 阻断了TNF-α (100 U/ml)抑制心肌细胞LDH释放及诱导Mn-SOD活性增加的作用。单独使用5-HD (100 μmol/L)处理, 与单独H/R组相比, 对H/R处理后心肌细胞LDH释放及Mn-SOD活性无明显作用。
图3.5-HD (100 μmol/L)对TNF-α (100 U/ml)引起的缺氧/复氧乳鼠心肌细胞LDH释放量和Mn-SOD活性改变的影响
Fig. 3.Effects of TNF-α (100 U/ml) on the release of LDH (A) and the activity of Mn-SOD (B) in cultured cardiomyocytes suffering from hypoxia/reoxygenation (H/R) in the presence or absence of 5-HD (100 μmol/L). n= 15 per group． **P<0.01 vs H/R group； #P<0.05, ##P<0.01 vs TNF-α group．
我们同时观察了mitoKATP通道开放剂diazoxide(50 μmol/L)对H/R心肌细胞LDH和Mn-SOD的影响。Diazoxide可显著减少心肌细胞LDH的释放量(P<0.01), 其作用还可被mitoKATP通道抑制剂5-HD或抗氧化剂NAC逆转, 但diazoxide对缺氧/复氧后心肌细胞内Mn-SOD活性无显著影响(P>0.05)(图4)。
图4.5-HD (100 μmol/L)或NAC (1 mmol/L)对diazoxide (50 μmol/L)引起的缺氧/复氧乳鼠心肌细胞LDH释放量和Mn-SOD活性改变的影响
Fig. 4.Effects of diazoxide (50 μmol/L) on the release of LDH and the activity of Mn-SOD in cultured cardiomyocytes suffering from hypoxia/reoxygenation (H/R) in the absence or presence of 5-HD (100 μmol/L) or NAC (1 mmol/L). n=15 per group． *P<0.05 vs H/R group．
3 讨论
本实验在培养的乳鼠心肌细胞缺氧/复氧模型上发现, TNF-α可减少心肌标志酶LDH的漏出量, 保护心肌细胞, 此作用可能与其诱导Mn-SOD活性升高以及开放mitoKATP通道有关。
活性氧(ROS)是生物体内产生的超氧阴离子(O·〖TX-*8]2 )、过氧化氢(H2O2)、羟自由基(HO·)、一氧化氮(NO·)等活性含氧化合物的总称。研究表明, ROS在细胞稳态调节等生命活动中可作为第二信使参与多种细胞生物学效应的启动[13]。在本实验中,
经抗氧化剂NAC或antimycin A处理后, TNF-α诱导的Mn-SOD活性升高及LDH漏出量减少的作用减弱,
提示ROS可能参与了TNF-α心肌保护过程的信号转导。据报道, ROS作为胞内重要的信号调节分子,
参与缺血(氧)预处理、阿片类物质、乙酰胆碱等诱导的心肌保护作用[8,11,14]。Higuchi等通过使用对活性氧敏感的荧光染料发现, TNF-α处理后心肌细胞内的荧光信号增加, 而且该效应可被NAC阻断[15]。虽然本实验没有直接证明TNF-α诱导活性氧产生增多, 但抗氧化剂NAC或antimycin A对TNF-α效应的对抗作用提示活性氧在TNF-α的心肌保护中具有重要地位。
Tanaka等发现缺氧预处理过程中线粒体内产生的超氧化物进入胞质后,
被Cu/Zn-SOD氧化生成过氧化氢[9], Cu/Zn-SOD抑制剂DDC可阻断或削弱缺氧预处理和阿片类物质的心肌保护作用[11]。2-MPG通过保持胞质中还原型谷胱甘肽的水平而清除过氧化氢,
可部分对抗缺氧预处理[16]、阿片类物质[11]和乙酰胆碱[14]等的心肌保护作用。本研究发现TNF-α诱导的Mn-SOD活性升高及LDH漏出量减少的作用, 能够被DDC或2-MPG抑制, 说明胞质中形成的过氧化氢参与了TNF-α心肌保护作用中的信号转导。本研究结果在细胞水平证实了Yao等的整体实验结果, 他们发现2-MPG可完全阻断TNF-α诱导的Mn-SOD活性升高和心肌梗塞面积减小作用[17]。
近年来研究发现, mitoKATP通道开放可能是缺血心肌保护反应的触发因子[18]或最终效应分子[19]。目前认为有关mitoKATP通道开放后引起心肌保护效应的机制主要与线粒体肿胀、呼吸链优化[20]、线粒体内钙超载程度减轻[21]以及自由基生成的减少有关[10]。mitoKATP通道开放剂diazoxide可促进线粒体内活性氧的产生,
通过作用于对氧化还原敏感的信号机制发挥心肌保护作用, 其作用可被抗氧化剂NAC所逆转[10]。本研究发现, 选择性mitoKATP通道阻断剂5-HD减弱了TNF-α诱导的Mn-SOD活性升高及LDH漏出量减少的作用, 提示mitoKATP通道参与了TNF-α诱导的心肌保护作用。但是, mitoKATP通道开放剂diazoxide只引起LDH释放量的减少, 而对Mn-SOD活性无明显作用, 即不能完全模拟TNF-α的心肌保护作用。我们认为这可能是由于mitoKATP通道的活动强度在两种实验条件下的不一致而产生的。
Garban等认为在缺血预处理过程中, mitoKATP通道激活是ROS产生的下游途径[22], 即ROS诱导mitoKATP通道开放。但有研究表明, 5-HD可阻断乙酰胆碱[14,18]和阿片类物质[11]诱导ROS的产生, 提示mitoKATP通道为ROS的上游途径。因此, 有关mitoKATP通道开放究竟是ROS对TNF-α心肌保护作用的下游还是上游途径, 有待进一步的研究。
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