地高辛抗血清对缺氧损伤心肌组织内源性地黄样因子和心肌细胞膜ATP酶活性的影响
作者：柯永胜 杨浩 杨霆 程光华 汪佑霞
单位：皖南医学院弋矶山医院,芜湖 241001
关键词：心肌；缺氧；内源性洋地黄样因子；钠钾ATP酶；钙ATP酶；地高辛抗血清
中国临床药理学与治疗学000103
目的 评价地高辛抗血清对心肌缺氧损伤的保护作用与机制。方法 制备心肌组织缺氧损伤模型，观察不同剂量的地高辛抗血清对缺氧损伤心肌组织内源性洋地黄样因子水平和心肌细胞膜ATP酶活性的影响。结果 缺氧损伤可使心肌组织内源性洋地黄样因子水平明显升高，心肌细胞膜ATP酶活性明显下降；地高辛抗血清能明显拮抗缺氧对心肌细胞膜ATP酶活性的抑制作用，使酶活性得到恢复。结论 缺氧损伤所致心肌组织内源性洋地黄样因子水平升高是缺氧介导心肌损伤的分子生物学基础，地高辛抗血清通过拮抗内源性洋地黄样因子的作用，减轻缺氧所致心肌损伤，对缺氧损伤心肌具有保护作用。
, 百拇医药
中图分类号 R972.1
Effect of anti-digoxin antiserum on endogenous digitalis-like factor and membrane ATPase activity in hypoxic injury myocardium
KE Yong-Sheng YANG Hao YANG Ting
CHENG Guang-Hua WANG You-Xia
(Yijishan Hospital,Wannan Medical College，Wuhu 241001)
Aim To evaluate the protective effect of anti-digoxin antiserum on hypoxic injury myocardium and its mechanism.Methods It was observed that different concentration anti-digoxin antiserum effect on endogenous digitalis-like factor and cell membrane ATPase activity in hypoxic myocardium model.Results The level of endogenous digitalis-like factor was remarkably higher,cell membrane ATPase activity were remarkably lower in hypoxic group than those of normal group;anti-digoxin antiserum can resume membrane ATPase activity.Conclusion Rise of endogenous digitalis-like factor was basic of molecular biology of myocardial damage during myocardial hypoxia.Anti-digoxin antiserum has lightened myocardial injury and has protective effect on hypoxic myocardium by against effect of endogenous digitalis-like factor.
, 百拇医药
Key words myocardium；hypoxic injury；endogenous digitalis-like factor;Na+-K+-ATPase;Ca2+-ATPase；anti-digoxin antiserum
Intracellular Ca2+ overload is basis of molec- ular biology in myocardial hypoxic injury.Intracellular Ca2+ overload is related with some factors inhibited to cell membrane ATPase.Endogenous digitalis-like factor is a factor which is similar with digitalis.It may be endogenous medium of digitalis receptor and can remarkably inhibit cellular membrane Na+-K+-ATPase activity.It has effect of increase heart contraction,diuresis and contract vascular .It is remarkably risen during acute myocardial ischemia and may be concerned with process of myocardial ischemic happen and progress[1,2].It was observed that different concentration anti-digoxin antiserum effect on endogenous digitalis-like factor and cell membrane ATPase activity in hypoxic myocardi um model (the model is similar with myocardial ischemic injury model in vivo) to evaluate the protective effect of anti-digoxin antiserum on hypoxic injury myocardium and its effect mechanism.
, http://www.100md.com
1 Materials and methods
1.1 Animals Eight New Zealand white rabbits of either sex,weighting(2.5±0.5) kg,were purchased by experiment animal center of Wannan Medical College(China).
1.2 Reagent Anti-digoxin antiserum(rabbit against rabbit) was purchased by Beijing Biotinge Biomedicine Company(China).Radioimmunoassay kit with reagent of endogenous digitalis-like factor was purchased by Radioimmune Institute of Tongji University (China).The kit with rea- gent of ATPase and of protein were purchased by Nanjing Jiancheng Biological Engineering Institute (China).
, http://www.100md.com
1.3 Methods of experiment The model of hypoxic were established by literature[3].After rabbits were anesthetized,the hearts were taken out.The hearts were perfused by aorta with cool 0.9% NaCl liquid.Water of the hearts were absorbed with filter paper.Two gram left ventricular tissues of each rabbit was taken out and mixed with 10 ml cool 0.9% NaCl liquid.Homogenate of myocardial tissues were executed.The homogenate were centrifuged with 4 ℃ 3 000 r.min-1 for 5 min .The super liquids 0.9 ml were parked into each test tube and divided into 5 groups:(Group A) normal contrast group: the test tubes were gassed with 95%O2 and 5%CO2 for 20 min;(Group B) hypoxia group: the test tubes were gassed with 95%N2 and 5%CO2 for 20 min;(Group C) hypoxia add rabbit itself serum group (negative contrast group):after test tubes were added rabbit itself serum 0.1 ml,hypoxia were taken out;(Group D)hypoxia add low concentration anti-digoxin antiserum group:after test tubes were added 1∶30 000 anti-digoxin antiserum 0.1ml,hypoxia were taken out;(Group E)hypoxia add high concentration anti- digoxin antiserum group: after test tubes were added 1∶10 000 anti-digoxin antiserum 0.1 ml,hypoxia were taken out.
, http://www.100md.com
1.4 Methods of endogenous digitalis-like factor and ATPase analysis The endogenous digitalis-like factor of myocardial tissues were assayed with radioimmunoassay.ATPase activity were determined by colormeter.The content of protein in myocardial tissues were determined by protein-dye binding method[4].
1.5 Statistic analysis All data were shown by means±standard deviation(
±s)and treat with t-test.Interrelation of two factors were adopted with simple beeline correlation analysis.Differences are statistically significant at P<0.05.
, 百拇医药
2 Results
2.1 Effect of anti-digoxin antiserum on endoxin and membrane ATPase activity in hypoxic injury myocardium See tab 1.
2.2 Dose-effect relationships between anti-digoxin antiserum and endogenous digitalis-like factor,ATPase activity in hypoxic injury myocardium There were remarkably dose-ef- fect relationships between anti-digoxin antiserum concentration and the level of endogenous digitalis-like factor,ATPase activity in hypoxic injury myocardial tissues.That is that the higher anti-digoxin antiserum concentration,the higher endogenous digitalis-like factor,higher the membrane Na+-K+-ATPase and Ca2+-ATPase activity in hypoxic injury myocardial tissues.By correlation analysis,r is respectively 0.6725,0.6843 and 0.7966,P<0.01.The level of endogenous digitalis-like factor was remarkably positive correlated with membrane Na+-K+-ATPase and Ca2+-ATPase activity in hypoxic injury myocardial tissues.By correlation analysis,r is respectively 0.9457,0.9561,P<0.01.
, 百拇医药
Tab 1 Effect of anti-digoxin antiserum on endogenous digitalis-like factor andmembrane ATPase activity in hypoxic injury myocardium(±s,n=8) Groups
EDLF
/ng.ml-1
Na+-K+-ATPase
/μmol.mgPro-1.h-1
, http://www.100md.com
Ca2+.ATPase
/μmol.mgPro-1.h-1
GroupA
0.11±0.04
3.04±0.28
2.79±0.43
GroupB
0.16±0.04*
1.43±0.24**
, 百拇医药
1.78±0.37**
GroupC
0.18±0.04**
1.28±0.16**
1.56±0.33**
GroupD
0.56±0.05**##
3.08±0.15**##
2.39±0.39**##
GroupE
, 百拇医药
0.61±0.13**##
3.22±0.39**##
2.67±0.50**##
copmpared with Group A*P<0.05,**P<0.01;compared with Group B,group C ##P<0.01
3 Discussion
The experiments demonstrated that intracellular Ca2+ concentration rise was one of the main causes of irreversible damage during myocardial ischemic injury[4].Therefore,it has protective effect on myocardium by inhibited intracellular Ca2+ concentration rise during ische- mia[5].The mechanism of intracellular Ca2+ concentration rise was related with membrane ATPase activity inhibited during myocardial ischemia.That is that myocardial membrane Na+-K+-ATPase and Ca2+-ATPase activity were inhibited,the ability that they transferred and pump out intracellular Ca2+ was decreased,therefore,intracellular Ca2+ accumulated.But what is mechanism that membrane ATPase were inhibited+ Whether through some substances intervention+
, 百拇医药
Endogenous digitalis-like factor is a factor with digitalis-like biological activity.It is Na+pump inhibitor and may be endogenous medium of digitalis receptor.Because it can cross-react with digitalis antibody,it can be measured by radioimmunoassay and anti-digitalis antibody was successfully used to treat hypertension by against endogenous digitalis-like factor[6].
Our experiment demonstrated that the level of endogenous digitalis-like factor in myocardial tissues was remarkably higher than those of normal contrast group during hypoxic injury,moreover,membrane ATPase activity of myocardium were remarkably lower than those of normal contrast group.It showed that myocardial cells have function synthesizing and releasing endogenous digitalis-like factor in hypoxia.Elevatory endogenous digitalis-like factor inhibited myocardial membrane ATPase activity by combined with digitalis receptor and caused intracellular Ca2+ rise and induced consequently myocardial cells damage.Myocardial cells membrane ATPase activity restored completely after previous use anti-digoxin antiserum.The higher anti-digoxin antiserum con- centration,the more remarkable myocardial cells membrane ATPase activity restored.It illuminated that quantity that endogenous digitalis- like factor combined with myocardial cells membrane Na+-K+-ATPase were decreased by anti-digoxin antiserum combined with endogenous digitalis-like factor.Therefore,degree of ATPase activity inhibited was lightened and ATPase activity were restored.Although the level of endogenous digitalis-like factor of myocardial tissues was not decreased but increased after anti-digoxin antiserum were used,it may be related with effect that anti-digoxin antiserum divided endogenous digitalis-like factor from membrane Na+-K+-ATPase position.Because only free endogenous digitalis-like factor or antigen-antibody compounds can be measured,endogenous digitalis-like factor combined with cell membrane cannot be measured with radioimmunoassay,therefore,the high level of endogenous digitalis-like factor in myocardial tissues was false.
, http://www.100md.com
Our primary experiment approved that myocardial cells injury during hypoxia may be related with endogenous digitalis-like factor.Rise of endogenous digitalis-like factor was basic of molecular biology of myocardial damage during myocardial hypoxia.Anti-digoxin antiserum has lightened myocardial injury and has protective effect on hypoxic myocardium by against effect of endogenous digitalis-like factor.
Project supported by the Foundation of Anhui Education Committee，№ 99jl0219
, http://www.100md.com
Ke Yongsheng,male,associate professor,engaged in research of cardiovascular diseases.
References
1，Ke YS,Tao YY,Yang SY,et al.Circadian change of endogenous digitalis-like factor in acute myocardial infarction.Chin J Cardiology,1990;18:360
2，Bagrov AY,Kuznetsova EA,Fedorova OV.Endoge-nous digoxin-like factor in acute myocardial infarction.J Intern Med,1994;235:63
, http://www.100md.com
3，Kirshenbaum LA,Singal PK.Changes in antioxidant enzymes in isolated cardiac myocytes subjected to hy-poxia-reoxygenation.Lab Invest,1992;67:796
4，Silverman HS,Stern MD.Zonic basis of ischemic car-diac injury:insights from cellular studies.Cardiovasc Res,1994;28:581
5，Van Gilst.Protection of the myocardium against postischemic reperfusion damage.J Cardiovasc Phar-macol 1989;14(Suppl 9):49
6，Huang CT,Smith RM.Lowering of blood pressure in chronic aortic coarctate hypertensive rats with anti-digoxin antiserum.Life Sci,1984;35:115
received：1999-12-10
accepted：1999-12-30, http://www.100md.com
单位：皖南医学院弋矶山医院,芜湖 241001
关键词：心肌；缺氧；内源性洋地黄样因子；钠钾ATP酶；钙ATP酶；地高辛抗血清
中国临床药理学与治疗学000103
目的 评价地高辛抗血清对心肌缺氧损伤的保护作用与机制。方法 制备心肌组织缺氧损伤模型，观察不同剂量的地高辛抗血清对缺氧损伤心肌组织内源性洋地黄样因子水平和心肌细胞膜ATP酶活性的影响。结果 缺氧损伤可使心肌组织内源性洋地黄样因子水平明显升高，心肌细胞膜ATP酶活性明显下降；地高辛抗血清能明显拮抗缺氧对心肌细胞膜ATP酶活性的抑制作用，使酶活性得到恢复。结论 缺氧损伤所致心肌组织内源性洋地黄样因子水平升高是缺氧介导心肌损伤的分子生物学基础，地高辛抗血清通过拮抗内源性洋地黄样因子的作用，减轻缺氧所致心肌损伤，对缺氧损伤心肌具有保护作用。
, 百拇医药
中图分类号 R972.1
Effect of anti-digoxin antiserum on endogenous digitalis-like factor and membrane ATPase activity in hypoxic injury myocardium
KE Yong-Sheng YANG Hao YANG Ting
CHENG Guang-Hua WANG You-Xia
(Yijishan Hospital,Wannan Medical College，Wuhu 241001)
Aim To evaluate the protective effect of anti-digoxin antiserum on hypoxic injury myocardium and its mechanism.Methods It was observed that different concentration anti-digoxin antiserum effect on endogenous digitalis-like factor and cell membrane ATPase activity in hypoxic myocardium model.Results The level of endogenous digitalis-like factor was remarkably higher,cell membrane ATPase activity were remarkably lower in hypoxic group than those of normal group;anti-digoxin antiserum can resume membrane ATPase activity.Conclusion Rise of endogenous digitalis-like factor was basic of molecular biology of myocardial damage during myocardial hypoxia.Anti-digoxin antiserum has lightened myocardial injury and has protective effect on hypoxic myocardium by against effect of endogenous digitalis-like factor.
, 百拇医药
Key words myocardium；hypoxic injury；endogenous digitalis-like factor;Na+-K+-ATPase;Ca2+-ATPase；anti-digoxin antiserum
Intracellular Ca2+ overload is basis of molec- ular biology in myocardial hypoxic injury.Intracellular Ca2+ overload is related with some factors inhibited to cell membrane ATPase.Endogenous digitalis-like factor is a factor which is similar with digitalis.It may be endogenous medium of digitalis receptor and can remarkably inhibit cellular membrane Na+-K+-ATPase activity.It has effect of increase heart contraction,diuresis and contract vascular .It is remarkably risen during acute myocardial ischemia and may be concerned with process of myocardial ischemic happen and progress[1,2].It was observed that different concentration anti-digoxin antiserum effect on endogenous digitalis-like factor and cell membrane ATPase activity in hypoxic myocardi um model (the model is similar with myocardial ischemic injury model in vivo) to evaluate the protective effect of anti-digoxin antiserum on hypoxic injury myocardium and its effect mechanism.
, http://www.100md.com
1 Materials and methods
1.1 Animals Eight New Zealand white rabbits of either sex,weighting(2.5±0.5) kg,were purchased by experiment animal center of Wannan Medical College(China).
1.2 Reagent Anti-digoxin antiserum(rabbit against rabbit) was purchased by Beijing Biotinge Biomedicine Company(China).Radioimmunoassay kit with reagent of endogenous digitalis-like factor was purchased by Radioimmune Institute of Tongji University (China).The kit with rea- gent of ATPase and of protein were purchased by Nanjing Jiancheng Biological Engineering Institute (China).
, http://www.100md.com
1.3 Methods of experiment The model of hypoxic were established by literature[3].After rabbits were anesthetized,the hearts were taken out.The hearts were perfused by aorta with cool 0.9% NaCl liquid.Water of the hearts were absorbed with filter paper.Two gram left ventricular tissues of each rabbit was taken out and mixed with 10 ml cool 0.9% NaCl liquid.Homogenate of myocardial tissues were executed.The homogenate were centrifuged with 4 ℃ 3 000 r.min-1 for 5 min .The super liquids 0.9 ml were parked into each test tube and divided into 5 groups:(Group A) normal contrast group: the test tubes were gassed with 95%O2 and 5%CO2 for 20 min;(Group B) hypoxia group: the test tubes were gassed with 95%N2 and 5%CO2 for 20 min;(Group C) hypoxia add rabbit itself serum group (negative contrast group):after test tubes were added rabbit itself serum 0.1 ml,hypoxia were taken out;(Group D)hypoxia add low concentration anti-digoxin antiserum group:after test tubes were added 1∶30 000 anti-digoxin antiserum 0.1ml,hypoxia were taken out;(Group E)hypoxia add high concentration anti- digoxin antiserum group: after test tubes were added 1∶10 000 anti-digoxin antiserum 0.1 ml,hypoxia were taken out.
, http://www.100md.com
1.4 Methods of endogenous digitalis-like factor and ATPase analysis The endogenous digitalis-like factor of myocardial tissues were assayed with radioimmunoassay.ATPase activity were determined by colormeter.The content of protein in myocardial tissues were determined by protein-dye binding method[4].
1.5 Statistic analysis All data were shown by means±standard deviation(
±s)and treat with t-test.Interrelation of two factors were adopted with simple beeline correlation analysis.Differences are statistically significant at P<0.05., 百拇医药
2 Results
2.1 Effect of anti-digoxin antiserum on endoxin and membrane ATPase activity in hypoxic injury myocardium See tab 1.
2.2 Dose-effect relationships between anti-digoxin antiserum and endogenous digitalis-like factor,ATPase activity in hypoxic injury myocardium There were remarkably dose-ef- fect relationships between anti-digoxin antiserum concentration and the level of endogenous digitalis-like factor,ATPase activity in hypoxic injury myocardial tissues.That is that the higher anti-digoxin antiserum concentration,the higher endogenous digitalis-like factor,higher the membrane Na+-K+-ATPase and Ca2+-ATPase activity in hypoxic injury myocardial tissues.By correlation analysis,r is respectively 0.6725,0.6843 and 0.7966,P<0.01.The level of endogenous digitalis-like factor was remarkably positive correlated with membrane Na+-K+-ATPase and Ca2+-ATPase activity in hypoxic injury myocardial tissues.By correlation analysis,r is respectively 0.9457,0.9561,P<0.01.
, 百拇医药
Tab 1 Effect of anti-digoxin antiserum on endogenous digitalis-like factor andmembrane ATPase activity in hypoxic injury myocardium(
±s,n=8) GroupsEDLF
/ng.ml-1
Na+-K+-ATPase
/μmol.mgPro-1.h-1
, http://www.100md.com
Ca2+.ATPase
/μmol.mgPro-1.h-1
GroupA
0.11±0.04
3.04±0.28
2.79±0.43
GroupB
0.16±0.04*
1.43±0.24**
, 百拇医药
1.78±0.37**
GroupC
0.18±0.04**
1.28±0.16**
1.56±0.33**
GroupD
0.56±0.05**##
3.08±0.15**##
2.39±0.39**##
GroupE
, 百拇医药
0.61±0.13**##
3.22±0.39**##
2.67±0.50**##
copmpared with Group A*P<0.05,**P<0.01;compared with Group B,group C ##P<0.01
3 Discussion
The experiments demonstrated that intracellular Ca2+ concentration rise was one of the main causes of irreversible damage during myocardial ischemic injury[4].Therefore,it has protective effect on myocardium by inhibited intracellular Ca2+ concentration rise during ische- mia[5].The mechanism of intracellular Ca2+ concentration rise was related with membrane ATPase activity inhibited during myocardial ischemia.That is that myocardial membrane Na+-K+-ATPase and Ca2+-ATPase activity were inhibited,the ability that they transferred and pump out intracellular Ca2+ was decreased,therefore,intracellular Ca2+ accumulated.But what is mechanism that membrane ATPase were inhibited+ Whether through some substances intervention+
, 百拇医药
Endogenous digitalis-like factor is a factor with digitalis-like biological activity.It is Na+pump inhibitor and may be endogenous medium of digitalis receptor.Because it can cross-react with digitalis antibody,it can be measured by radioimmunoassay and anti-digitalis antibody was successfully used to treat hypertension by against endogenous digitalis-like factor[6].
Our experiment demonstrated that the level of endogenous digitalis-like factor in myocardial tissues was remarkably higher than those of normal contrast group during hypoxic injury,moreover,membrane ATPase activity of myocardium were remarkably lower than those of normal contrast group.It showed that myocardial cells have function synthesizing and releasing endogenous digitalis-like factor in hypoxia.Elevatory endogenous digitalis-like factor inhibited myocardial membrane ATPase activity by combined with digitalis receptor and caused intracellular Ca2+ rise and induced consequently myocardial cells damage.Myocardial cells membrane ATPase activity restored completely after previous use anti-digoxin antiserum.The higher anti-digoxin antiserum con- centration,the more remarkable myocardial cells membrane ATPase activity restored.It illuminated that quantity that endogenous digitalis- like factor combined with myocardial cells membrane Na+-K+-ATPase were decreased by anti-digoxin antiserum combined with endogenous digitalis-like factor.Therefore,degree of ATPase activity inhibited was lightened and ATPase activity were restored.Although the level of endogenous digitalis-like factor of myocardial tissues was not decreased but increased after anti-digoxin antiserum were used,it may be related with effect that anti-digoxin antiserum divided endogenous digitalis-like factor from membrane Na+-K+-ATPase position.Because only free endogenous digitalis-like factor or antigen-antibody compounds can be measured,endogenous digitalis-like factor combined with cell membrane cannot be measured with radioimmunoassay,therefore,the high level of endogenous digitalis-like factor in myocardial tissues was false.
, http://www.100md.com
Our primary experiment approved that myocardial cells injury during hypoxia may be related with endogenous digitalis-like factor.Rise of endogenous digitalis-like factor was basic of molecular biology of myocardial damage during myocardial hypoxia.Anti-digoxin antiserum has lightened myocardial injury and has protective effect on hypoxic myocardium by against effect of endogenous digitalis-like factor.
Project supported by the Foundation of Anhui Education Committee，№ 99jl0219
, http://www.100md.com
Ke Yongsheng,male,associate professor,engaged in research of cardiovascular diseases.
References
1，Ke YS,Tao YY,Yang SY,et al.Circadian change of endogenous digitalis-like factor in acute myocardial infarction.Chin J Cardiology,1990;18:360
2，Bagrov AY,Kuznetsova EA,Fedorova OV.Endoge-nous digoxin-like factor in acute myocardial infarction.J Intern Med,1994;235:63
, http://www.100md.com
3，Kirshenbaum LA,Singal PK.Changes in antioxidant enzymes in isolated cardiac myocytes subjected to hy-poxia-reoxygenation.Lab Invest,1992;67:796
4，Silverman HS,Stern MD.Zonic basis of ischemic car-diac injury:insights from cellular studies.Cardiovasc Res,1994;28:581
5，Van Gilst.Protection of the myocardium against postischemic reperfusion damage.J Cardiovasc Phar-macol 1989;14(Suppl 9):49
6，Huang CT,Smith RM.Lowering of blood pressure in chronic aortic coarctate hypertensive rats with anti-digoxin antiserum.Life Sci,1984;35:115
received：1999-12-10
accepted：1999-12-30, http://www.100md.com