肌生、红花等8种中药注射液用于心脏保存的实验及机理研究
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摘要
1967年南非Barnard首次在临床上成功地为1例病人施行了原位心脏移植术，其后美国及欧洲也相继开展。目前，该法已从一个激进而有争议的外科手段变成一种治疗终末期心脏病的重要方法。供心不足限制了心脏移植的发展，1994年后心脏移植例数不断下降。延长心脏保存时间，提高心脏保存效果，是扩大供心来源的有效途径。
现代研究证实，很多中药及中药有效成分具有抗缺血再灌注损伤和心肌保护作用。将中药及中药有效成分作为已有保存液的添加剂，以延长心脏的存活时间，开拓了心脏保存的研究思路，扩大了中药的应用范围。
本研究旨在对相关中药进行筛选，以期寻找到效果显著的中药添加剂，突破心脏的有效保存时限。
第一部分，为了能用最有限的经费，筛选大量的中药。寻找到保存离体心脏效果显著的中药，我们建立了小鼠Langendorff离体心脏灌注模型。该模型可观察离体心脏的收缩、舒张功能，心率及测取冠脉流量。实验表明，该模型稳定，能满足离体心脏保存研究中对大量药物进行药效筛选的需要。
采用小鼠Langendorff离体心脏灌注模型。将肌生(赤芝孢子粉加工而成)、红花、刺五加、血塞通(三七皂甙加工而成)和灯盏花等8种中药注射液添加到改良的细胞内液型保存液Euro-Collins(mEC)液中，以单纯mEC液为对照组，保存小鼠离体心脏3h、7.5h、9h。
保存3h的实验结果表明，5种中药均可不同程度的改善保存心脏的心功能、冠脉流量和再灌注结束后的心肌含水量。比较而言，肌生、红花和刺五加的保存效果较血塞通、灯盏花好。
以复温后心脏的复活率为主要指标，观察肌生、红花、刺五加、血塞通和灯盏花作为mEC液添加剂保存小鼠离体心脏7.5h的效果。结果显示，红花组心脏复活率明显高于对照组(p＜0.01)，刺五加组明显高于对照组，具有显著性差异(p＜0.05)，肌生组高于对照组，但无统计学意义。
以复温后心脏的复活率为主要指标，观察肌生、红花、刺五加、黄芪、川芎嗪和当归注射液保存小鼠离体心脏9h的效果。实验结果显示，红花组心脏复活率明显高于对照组具有极显著性差异(p＜0.01)，肌生组心脏复活率明显高于对照组，具有显著性差异(p＜0.05)。药物组之间比较，红花的复活率最高，肌生次之，当归的复活率最低，刺五加、黄芪、川芎嗪三组复活率相同。复跳需时：红花组最低，川芎嗪组次之。心肌含水量：刺五加、黄芪的心肌含水量明显低于mEC组，具有极显著性差异(P＜0.01)。
第二部分，采用大鼠Langendorff离体心脏灌注模型。将保存小鼠心脏效果较好的肌生、红花、刺五加、黄芪等4种中药注射液添加到改良的细胞内液型保存液Euro-Collins(mEC)液中，以单纯mEC液为对照组，保存大鼠离体心脏20h，
4 肌生、红花等8种中药注射液用十心脏保仟的实验及机理研究
实验结果表明，保存后心脏收缩功能，红花、黄茬及肌生组明显高于mEC组，具
有极显著性差异（P＜O．OI）。红花组明显高于黄蔑和刺五加组；具有极显著性差异
（P1．01）；红花组高于肌生组，具有显著性差异（P叩．05）。保存后心脏舒张功
能；红花、黄茂及肌生组明显高于mBC组；具有极显著性差异（p＜0．OI）。红花组
明显高于刺五加组，具有极显著性差异（p＜0．01），高于肌生组；具有显著性差异
（P叩．05）。保存后冠脉流量＊F），红花及肌生组明显高于mEC组，具有极显著
性差异（P＜0．01），黄芭组高于mEC组，具有显著性差异（P＜0．05）。红花组明显
高于其它三个组，具有极显著性差异（p＜0．01）。保存后黄民组的心肌含水量明显
低于mEC组；具有极显著性差异（P＜0．01）。
第三部分，对肌生、红花注射液的心脏保护作用机制进行了探计。
（1）对心脏保存效果较好的肌生、红花注射液对大鼠心脏心肌酶漏出及抗氧
化作用的影响进行了研究。结果显示：
保存后 mEC组冠脉流液中的 LDH活性为 145．26 t 35．67，肌生组为 43．77 i
13．25，红花组为 2 8．7 7 ti 4．2 5。肌生、红花组明显低于帕C组，具有极显著性
差异（P＜0．01）。保存后 mEC组冠脉流液中的 CK活性为 36．68，14．93，肌生组为
1仁 89 t 3．5乙 红花组为 7．89 t 3．3人 肌生、红花组明显低于 mEC组，具有极显
著性差异hq．01）。
保存后心肌组织中超氧化物歧化酶侣m的活性：二m组为305．66。兆．u，
肌生组为344．71 37．75，红花组为346．76 i 38．89。肌生、红花组明显高于fllEC
组；具有显著性差异（p＜0．05）。保存后心肌组织中丙H醛（MDA）的含量：llEC
为8．6O．41；肌生组为6．刀t 0 24，红花组为6．53 t 0．27。肌生、红花组明
显高于m＊C组，具有显著性差异（p＜0．05）。
（2）对红花注射液对大鼠心肌组织能荷值及抗细胞凋亡基因 bC－2表达的影
响，进行了研究。实验结果表明，红花厂）加入 mEC液中；40C保存大鼠心脏
2 oh后；心肌组织能荷值与对照组相似；心肌细胞凋亡基因 bC－2的蛋白表达上
调。
本课题研究表明，中药在心脏保存领域具有广阔的应用前景。红花具有较好
的心脏保护作用，其机制可能与其改善
In 1967, Christiaan Barnard led the team performed the world's first human-to-human heart transplantation, followed with America and Europe. Heart transplantation is currently the only clinical treatment for end stage heart disease patients. The number of the heart transplantation cases had decreased while 1994. One of the most important reasons is the lack of donor hearts because the preservation time of donor heart is too short. It is essential for successful heart transplantation to improve preservation donor heart safely and effectively. The prolonging of preservation time of donor heart could expand the donor hearts pool.
A lot of researches confirmed that many effective extracts of Chinese herbs had effects of resisting ischemia-reperfusion(IR) injury and protecting myocardial. It is feasible that the effective Chinese herbs or extracts of Chinese herbs are added into conventional preserve solution to improve the effects of heart preservation, prolong the heart preserve time, and to expand Chinese herbs application.
The objective of this research is: To select systematically Chinese herbs that have effects of resisting isochemia-reperfusion injury and protecting myocardial. To look for Chinese herbs that have the effects on heart preservation and prolong the preserve time of donor heart.
Firstly, We established mice Langendorff heart perfusion method for preliminary selection of Chinese herbs. It is possible to measure left ventricular function including LVPSP, ?dp/dtmax, HR, RPP and CF of isolated heart. Stableness of the model had been confirmed by experiments. It is possible to study for heart preservation.
Secondly, with mice Langendorff isolated heart perfusion model, we add the generally recognized intracellular cardial preservation solution-modified Euro-Col 1 ins solution (mECS) with: G. Lucidum Karst (Ji Sheng), Carthamas tinciorius L (Hong Hua), Acanthopanax Senticosus Harms (Ci Wu Jia), Saponins of panax notoginseng, PNS (Xue Sai Tong), Erigeron breviscapus Hand-Mazz, EbHM(Deng Zhan Hua), Huang Qi, radix angelicase sinensis(Dang Gui), Tetramethylpyrazine, TMP(Chang Xiong Qin), respectively. Mice hearts were preserved for 3hr, 7. 5hr, 9hr, respectively, under simple cold storage condition with mECS as control.
After preserved for 3hr, Ji Sheng, Hong Hua, Ci Wu Jia, Xue Sai Tong, Deng Zhan hua all improved left ventricular function, including LVPSP,
眃p/dtmax, HR, RPP, CF. Effectiveness of Ji Sheng, Hong Hua, Ci Wu Jia group was better than that of Xue Sai long and Deng Zhan Hua .
After preserved 7. 5hr for mice hearts: Resurrect rate of Hong Hua group was significantly higher than that of control group (p<0. 01). Resurrect rate of Ci Wu Jia group was also significantly higher than that of group (p<0. 05). Resurrect rates of Ji Sheng and Deng Zhan Hua groups were higher than that of control group, but no statistics sense. Resurrect rate of Mice heats of Xue Sai long group was lower than that of control group.
After preserved 9hr of mice hearts: Resurrect rates of Hong Hua and Ji Sheng groups were significantly higher than that of control group (p<0. 01, p<0. 05). Resurrect rates of Ci Wu Jia, Huang Qi and Chuan Xiong Qin groups were also higher than that of control group, but no statistics sense. As for resurrect rate of mice hearts, Hong Hua group was the highest, Ji Sheng group was the second highest, and Dang Gui group was lowest among the six groups. As for the heart beat regaining time, Hong Hua group was the lowest and Chuan Xiong Qin group was the second lowest among the six groups without statistical sense. Myocardial water contents of Ci Wu Jia and Hong Hua groups were significantly lower than that of mEC group(p<0. 01). CF of Dang Gui group was significantly higher than mECS group (p<0. 05).
Secondly, The experiments were done with rat hearts used Euro-Collins solution adding Ji Sheng, Hong Hua, Ci Wu Jia, Huang Qi, Chuan Xiong Qin, and Dang Gui, respectively. Rat hearts were preserved for 20hr under simple cold storage condition with mECS compare with the study s
现代研究证实，很多中药及中药有效成分具有抗缺血再灌注损伤和心肌保护作用。将中药及中药有效成分作为已有保存液的添加剂，以延长心脏的存活时间，开拓了心脏保存的研究思路，扩大了中药的应用范围。
本研究旨在对相关中药进行筛选，以期寻找到效果显著的中药添加剂，突破心脏的有效保存时限。
第一部分，为了能用最有限的经费，筛选大量的中药。寻找到保存离体心脏效果显著的中药，我们建立了小鼠Langendorff离体心脏灌注模型。该模型可观察离体心脏的收缩、舒张功能，心率及测取冠脉流量。实验表明，该模型稳定，能满足离体心脏保存研究中对大量药物进行药效筛选的需要。
采用小鼠Langendorff离体心脏灌注模型。将肌生(赤芝孢子粉加工而成)、红花、刺五加、血塞通(三七皂甙加工而成)和灯盏花等8种中药注射液添加到改良的细胞内液型保存液Euro-Collins(mEC)液中，以单纯mEC液为对照组，保存小鼠离体心脏3h、7.5h、9h。
保存3h的实验结果表明，5种中药均可不同程度的改善保存心脏的心功能、冠脉流量和再灌注结束后的心肌含水量。比较而言，肌生、红花和刺五加的保存效果较血塞通、灯盏花好。
以复温后心脏的复活率为主要指标，观察肌生、红花、刺五加、血塞通和灯盏花作为mEC液添加剂保存小鼠离体心脏7.5h的效果。结果显示，红花组心脏复活率明显高于对照组(p＜0.01)，刺五加组明显高于对照组，具有显著性差异(p＜0.05)，肌生组高于对照组，但无统计学意义。
以复温后心脏的复活率为主要指标，观察肌生、红花、刺五加、黄芪、川芎嗪和当归注射液保存小鼠离体心脏9h的效果。实验结果显示，红花组心脏复活率明显高于对照组具有极显著性差异(p＜0.01)，肌生组心脏复活率明显高于对照组，具有显著性差异(p＜0.05)。药物组之间比较，红花的复活率最高，肌生次之，当归的复活率最低，刺五加、黄芪、川芎嗪三组复活率相同。复跳需时：红花组最低，川芎嗪组次之。心肌含水量：刺五加、黄芪的心肌含水量明显低于mEC组，具有极显著性差异(P＜0.01)。
第二部分，采用大鼠Langendorff离体心脏灌注模型。将保存小鼠心脏效果较好的肌生、红花、刺五加、黄芪等4种中药注射液添加到改良的细胞内液型保存液Euro-Collins(mEC)液中，以单纯mEC液为对照组，保存大鼠离体心脏20h，
4 肌生、红花等8种中药注射液用十心脏保仟的实验及机理研究
实验结果表明，保存后心脏收缩功能，红花、黄茬及肌生组明显高于mEC组，具
有极显著性差异（P＜O．OI）。红花组明显高于黄蔑和刺五加组；具有极显著性差异
（P1．01）；红花组高于肌生组，具有显著性差异（P叩．05）。保存后心脏舒张功
能；红花、黄茂及肌生组明显高于mBC组；具有极显著性差异（p＜0．OI）。红花组
明显高于刺五加组，具有极显著性差异（p＜0．01），高于肌生组；具有显著性差异
（P叩．05）。保存后冠脉流量＊F），红花及肌生组明显高于mEC组，具有极显著
性差异（P＜0．01），黄芭组高于mEC组，具有显著性差异（P＜0．05）。红花组明显
高于其它三个组，具有极显著性差异（p＜0．01）。保存后黄民组的心肌含水量明显
低于mEC组；具有极显著性差异（P＜0．01）。
第三部分，对肌生、红花注射液的心脏保护作用机制进行了探计。
（1）对心脏保存效果较好的肌生、红花注射液对大鼠心脏心肌酶漏出及抗氧
化作用的影响进行了研究。结果显示：
保存后 mEC组冠脉流液中的 LDH活性为 145．26 t 35．67，肌生组为 43．77 i
13．25，红花组为 2 8．7 7 ti 4．2 5。肌生、红花组明显低于帕C组，具有极显著性
差异（P＜0．01）。保存后 mEC组冠脉流液中的 CK活性为 36．68，14．93，肌生组为
1仁 89 t 3．5乙 红花组为 7．89 t 3．3人 肌生、红花组明显低于 mEC组，具有极显
著性差异hq．01）。
保存后心肌组织中超氧化物歧化酶侣m的活性：二m组为305．66。兆．u，
肌生组为344．71 37．75，红花组为346．76 i 38．89。肌生、红花组明显高于fllEC
组；具有显著性差异（p＜0．05）。保存后心肌组织中丙H醛（MDA）的含量：llEC
为8．6O．41；肌生组为6．刀t 0 24，红花组为6．53 t 0．27。肌生、红花组明
显高于m＊C组，具有显著性差异（p＜0．05）。
（2）对红花注射液对大鼠心肌组织能荷值及抗细胞凋亡基因 bC－2表达的影
响，进行了研究。实验结果表明，红花厂）加入 mEC液中；40C保存大鼠心脏
2 oh后；心肌组织能荷值与对照组相似；心肌细胞凋亡基因 bC－2的蛋白表达上
调。
本课题研究表明，中药在心脏保存领域具有广阔的应用前景。红花具有较好
的心脏保护作用，其机制可能与其改善
In 1967, Christiaan Barnard led the team performed the world's first human-to-human heart transplantation, followed with America and Europe. Heart transplantation is currently the only clinical treatment for end stage heart disease patients. The number of the heart transplantation cases had decreased while 1994. One of the most important reasons is the lack of donor hearts because the preservation time of donor heart is too short. It is essential for successful heart transplantation to improve preservation donor heart safely and effectively. The prolonging of preservation time of donor heart could expand the donor hearts pool.
A lot of researches confirmed that many effective extracts of Chinese herbs had effects of resisting ischemia-reperfusion(IR) injury and protecting myocardial. It is feasible that the effective Chinese herbs or extracts of Chinese herbs are added into conventional preserve solution to improve the effects of heart preservation, prolong the heart preserve time, and to expand Chinese herbs application.
The objective of this research is: To select systematically Chinese herbs that have effects of resisting isochemia-reperfusion injury and protecting myocardial. To look for Chinese herbs that have the effects on heart preservation and prolong the preserve time of donor heart.
Firstly, We established mice Langendorff heart perfusion method for preliminary selection of Chinese herbs. It is possible to measure left ventricular function including LVPSP, ?dp/dtmax, HR, RPP and CF of isolated heart. Stableness of the model had been confirmed by experiments. It is possible to study for heart preservation.
Secondly, with mice Langendorff isolated heart perfusion model, we add the generally recognized intracellular cardial preservation solution-modified Euro-Col 1 ins solution (mECS) with: G. Lucidum Karst (Ji Sheng), Carthamas tinciorius L (Hong Hua), Acanthopanax Senticosus Harms (Ci Wu Jia), Saponins of panax notoginseng, PNS (Xue Sai Tong), Erigeron breviscapus Hand-Mazz, EbHM(Deng Zhan Hua), Huang Qi, radix angelicase sinensis(Dang Gui), Tetramethylpyrazine, TMP(Chang Xiong Qin), respectively. Mice hearts were preserved for 3hr, 7. 5hr, 9hr, respectively, under simple cold storage condition with mECS as control.
After preserved for 3hr, Ji Sheng, Hong Hua, Ci Wu Jia, Xue Sai Tong, Deng Zhan hua all improved left ventricular function, including LVPSP,
眃p/dtmax, HR, RPP, CF. Effectiveness of Ji Sheng, Hong Hua, Ci Wu Jia group was better than that of Xue Sai long and Deng Zhan Hua .
After preserved 7. 5hr for mice hearts: Resurrect rate of Hong Hua group was significantly higher than that of control group (p<0. 01). Resurrect rate of Ci Wu Jia group was also significantly higher than that of group (p<0. 05). Resurrect rates of Ji Sheng and Deng Zhan Hua groups were higher than that of control group, but no statistics sense. Resurrect rate of Mice heats of Xue Sai long group was lower than that of control group.
After preserved 9hr of mice hearts: Resurrect rates of Hong Hua and Ji Sheng groups were significantly higher than that of control group (p<0. 01, p<0. 05). Resurrect rates of Ci Wu Jia, Huang Qi and Chuan Xiong Qin groups were also higher than that of control group, but no statistics sense. As for resurrect rate of mice hearts, Hong Hua group was the highest, Ji Sheng group was the second highest, and Dang Gui group was lowest among the six groups. As for the heart beat regaining time, Hong Hua group was the lowest and Chuan Xiong Qin group was the second lowest among the six groups without statistical sense. Myocardial water contents of Ci Wu Jia and Hong Hua groups were significantly lower than that of mEC group(p<0. 01). CF of Dang Gui group was significantly higher than mECS group (p<0. 05).
Secondly, The experiments were done with rat hearts used Euro-Collins solution adding Ji Sheng, Hong Hua, Ci Wu Jia, Huang Qi, Chuan Xiong Qin, and Dang Gui, respectively. Rat hearts were preserved for 20hr under simple cold storage condition with mECS compare with the study s
引文
1 张海林，傅绍萱，李蕴山．家兔工作心脏及两种缺血模型的制备法及实验观察．中国药理学通报，1989，5(1)：53-56．
2 藏旺福，谢振华，夏求明，等．速尿对改善供心保存的作用．中华器官移植杂志，1999，20(2)：103-105．
2 藏旺福，谢振华，夏求明，等．速尿对改善供心保存的作用．中华器官移植杂志，1999，20(2)：103-105．