丹酚酸B预适应的心脏细胞保护作用及机制研究
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摘要
为研究丹酚酸B预适应的心脏细胞保护作用及机制，我们基于缺氧预适应(HPC)机制，通过心脏微血管内皮细胞(CMEC)和心肌细胞体外培养技术，建立缺O_2／复O_2损伤的细胞模型，以细胞活力、乳酸脱氢酶(LDH)、一氧化氮(NO)、超氧化物歧化酶(SOD)、内皮素(ET)等生化指标及超微结构变化为评价标准，观察了丹酚酸B预适应的细胞保护效应；并采用以蛋白激酶C(PKC)抑制剂chelerythrine、K_(ATP)通道阻滞剂优降糖为工具药的方式和对内源性保护物质PKC、热休克蛋白70(HSP70)的mRNA表达影响角度，探讨了丹酚酸B预适应的细胞保护机制，这是本文研究内容的核心。
另外，为对中药特定的药效寻求最佳配伍比例，进一步提高临床疗效，丰富方剂配伍理论的科学内容，推进中医药现代化进程，我们又从饮片全成分(丹参和三七药对)、有效成分(丹酚酸B和丹参酮ⅡA)两个层次，借助缺O_2／复O_2损伤的CMEC细胞模型，进行了方剂配比的药效物质构成变化与生物效应差异的相关性研究。
结果表明：
(1)在正常培养条件下，丹酚酸B可明显提高CMEC和心肌细胞的细胞存活率和SOD活性、降低LDH活性、维持正常的形态超微结构，有降低CMEC的ET分泌和NO释放趋势。
(2)缺O_2预适应可增强细胞对随后较长时间缺O_2／复O_2损伤的耐受性，表现为可提高细胞存活率和SOD活性，降低LDH活性，而且能明显降低CMEC因缺O_2／复O_2导致的ET和NO水平升高，同时明显改善其形态超微结构变化。
（3）丹酚酸B预适应具有与缺OZ预适应相似的细胞保护效应，对于
CMEC表现为早期保护和延迟保护两个时相。
＠N）Ke通道阻滞剂优降糖不能消除 HPC的 CMEC保护作用，选择性
PKC抑制剂chele咖ne消除了HPC的CMEC保护效应，丹酚酸B预适应
可以使CmC被PKC抑制剂阻断的IIPC效应重现。
．
（5）在早期预适应中，丹酚酸 B预适应可促进缺 OZ／复 OZ损伤 C＼IEC
的HSP70 InRNA、PKC mRNA表达增强，且明显优于HPC；在晚期预运应
中，丹酚酸 B预适应同样促使 HSP70 mRNA表达增强，且与 HPC具有叠加
效应。
（6）在正常培养条件下或缺 OZ损伤条件下，丹参促使 CMEC增殖，三
七抑制增殖，各配比组中单纯丹参组促增殖作用最强（与其他组比较均P＜0．01
或0刀5），尤其在缺OZ损伤条件下，各配比组均可显著降低CMEC的NO释
放和 ET分泌，在降低 ET分泌方面以 10＊和 10：6效果最佳。
（）无论正常培养或缺 OZ损伤条件下，丹酚酸 B与丹参酮＊ A各配比
组均可显著提高CMEC活力，降低LDH释放，降低分泌ET和NO水平，以
2：8效果最佳，尤其在缺 OZ损伤条件下，单纯丹酚酸 B和单纯丹参酮＊ A作
用不如二者相互配伍明显。
结论：
（）丹酚酸B预适应与缺OZ预适应具有相类似的细胞保护效应，可增
＠强细胞对随后较长时间缺 O。l复 O。损伤的耐受力，从而激发了细胞的内源性
保护机制，在CMEC预适应中显示出双相保护时程即早、晚期双重保护效应。
忿 ＊）K＾TI，通道阻滞剂不能消除 HI，C的 CmC保护作用，丹酚酸 B预适
应可以使被PKC抑制剂Chel…阻断的HPC效应重现。
（3）丹酚酸 B预适应的 CMEC保护作用可能的机制主要是通过增强
199
HPC的级联反应的关键环节PKC的mANA表达以及HSP70InRNA表达，促
进PKC活化，诱导应激蛋白HSP70合成，促进内源性抗氧化因子的增加等。
N）丹参作用的主要靶点可能在 CMEC，而丹参、三七对于 CMEC作＠
用的最佳配比是10＊和10爪 丹酚酸B与丹参酮IIA抗CMEC缺OZ损伤，
保护＊＊＊C的最佳配比是二8。
In order to study the protective effect and mechanism of preconditioning with cinnabar phenic acid B on the cardiac cell, based on the mechanism of anoxic-preconditioning, we establish the damaging model of anoxic-reoxygenation on the cell by the technique of cultivating cardiac microvascular endothelial cell (CMEC) and cardiac cell in vitro. To observe the protective effect on the cell with the cinnabar phenic acid B - preconditioning, we use the biochemistry examination such as the rate of live cell(MTT), lactate dehydrogenase(LDH^ superoxide dismutase(SOD^ nitric oxide(NO^ endothelin (ET) ect and ultrastructure as the evaluating criterion. By the means of medicine such as chelerythrine, a kind of an inhibitor of protein kinase C (PKC), glibenclamide, a kind of an inhibitor of the ATP-sensitive potassium channel (KATp), and from the point of view of the intrinsic protective substance, PKC, and the express of mRNA of the heat shock protein 70 (HSP70) we probe into the protective mechanism on the cell with t
he cinnabar phenic acid B -preconditioning.
In addition, in order to search the best ratio of compatibility of Chinese medicine, enhance the clinical curative effect, enrich the scientific connotation of the description compatibility theory and advance the progress of the Chinese medical modernization, we studied the relationship between the varieties of the effective substance composition in a description and its bio-effect by the injury cell model of CMEC by anoxic-reoxygenation using all composition of the prepared herbs (the paired description elan-sen root and pseudo-ginseng ) and its effective composition.
Result:
(1) Under the normal culturing condition, cinnabar phenic acid B can
enhance the livability of both of cells and the activity of SOD significantly, reduce the release of LDH, keep the ultrastructure, have the tendency of decreasing the secret of ET and the release of NO of CMEC.
(2) Hypoxic preconditioning can strengthen the cell endurance of the later damage of longer time anoxic-reoxygenation, which can be observed through the increase of the cell livability and the SOD activity, and the decrease of the LDH activity. At the same time, it can obviously decrease the level of ET and NO of CMEC by anoxic-reoxygenation and improve the change of the ultrastructure.
( 3) The protective effect on the cell by cinnabar phenic acid B is similar with the effect by anoxic-reoxygenation. For CMEC, this function will be expressed as the early and delayed protection phases.
(4) Glibenclamide, a kind of an inhibitor of KATP, cannot eliminate the HPC protective effect of CMEC. Chelerythrine, a kind of selective inhibitor of PKC, can eliminate the HPC protective effect of CMEC that can be recurred by the preconditioning of cinnabar phenic acid B.
(5) During the early preconditioning, cinnabar phenic acid B can improve the mRNA espression of HSP70 and PKC on the anoxic-reoxygenation injured CMEC. The effect is better than HPC obviously. During the delayed preconditioning, cinnabar phenic acid B also can improve the mRNA expression of HSP70, and it has the plus effect with HPC.
( 6 ) Under the normal or the anoxic cultivating conditions, elan-sen root can improve the reproduce proliferation of CMEC while pseudo-ginseng inhibits it. Among all the compatibility groups, the proliferation effect of the group of only elan-sen root is the most obvious (compare with other groups p<0.01 p<0.05), especially under the anoxic condition. All the compatibility groups can obviously decrease the release of NO and the secret of ET of CMEC. The best groups decreasing the secret of ET is the compatibility of 10:1 and 10:6. No matter the normal conditions or the anoxic conditions, all groups greatly promote the activity of CMEC, decrease the release of LDH. The effect the group of 2:8 decreasing the
secret of ET and the level of NO is the best. Especially under the anoxic condition, the effect of only cinnabar phenic acid B and only tanshinone II -A is not better than the compatibilit
另外，为对中药特定的药效寻求最佳配伍比例，进一步提高临床疗效，丰富方剂配伍理论的科学内容，推进中医药现代化进程，我们又从饮片全成分(丹参和三七药对)、有效成分(丹酚酸B和丹参酮ⅡA)两个层次，借助缺O_2／复O_2损伤的CMEC细胞模型，进行了方剂配比的药效物质构成变化与生物效应差异的相关性研究。
结果表明：
(1)在正常培养条件下，丹酚酸B可明显提高CMEC和心肌细胞的细胞存活率和SOD活性、降低LDH活性、维持正常的形态超微结构，有降低CMEC的ET分泌和NO释放趋势。
(2)缺O_2预适应可增强细胞对随后较长时间缺O_2／复O_2损伤的耐受性，表现为可提高细胞存活率和SOD活性，降低LDH活性，而且能明显降低CMEC因缺O_2／复O_2导致的ET和NO水平升高，同时明显改善其形态超微结构变化。
（3）丹酚酸B预适应具有与缺OZ预适应相似的细胞保护效应，对于
CMEC表现为早期保护和延迟保护两个时相。
＠N）Ke通道阻滞剂优降糖不能消除 HPC的 CMEC保护作用，选择性
PKC抑制剂chele咖ne消除了HPC的CMEC保护效应，丹酚酸B预适应
可以使CmC被PKC抑制剂阻断的IIPC效应重现。
．
（5）在早期预适应中，丹酚酸 B预适应可促进缺 OZ／复 OZ损伤 C＼IEC
的HSP70 InRNA、PKC mRNA表达增强，且明显优于HPC；在晚期预运应
中，丹酚酸 B预适应同样促使 HSP70 mRNA表达增强，且与 HPC具有叠加
效应。
（6）在正常培养条件下或缺 OZ损伤条件下，丹参促使 CMEC增殖，三
七抑制增殖，各配比组中单纯丹参组促增殖作用最强（与其他组比较均P＜0．01
或0刀5），尤其在缺OZ损伤条件下，各配比组均可显著降低CMEC的NO释
放和 ET分泌，在降低 ET分泌方面以 10＊和 10：6效果最佳。
（）无论正常培养或缺 OZ损伤条件下，丹酚酸 B与丹参酮＊ A各配比
组均可显著提高CMEC活力，降低LDH释放，降低分泌ET和NO水平，以
2：8效果最佳，尤其在缺 OZ损伤条件下，单纯丹酚酸 B和单纯丹参酮＊ A作
用不如二者相互配伍明显。
结论：
（）丹酚酸B预适应与缺OZ预适应具有相类似的细胞保护效应，可增
＠强细胞对随后较长时间缺 O。l复 O。损伤的耐受力，从而激发了细胞的内源性
保护机制，在CMEC预适应中显示出双相保护时程即早、晚期双重保护效应。
忿 ＊）K＾TI，通道阻滞剂不能消除 HI，C的 CmC保护作用，丹酚酸 B预适
应可以使被PKC抑制剂Chel…阻断的HPC效应重现。
（3）丹酚酸 B预适应的 CMEC保护作用可能的机制主要是通过增强
199
HPC的级联反应的关键环节PKC的mANA表达以及HSP70InRNA表达，促
进PKC活化，诱导应激蛋白HSP70合成，促进内源性抗氧化因子的增加等。
N）丹参作用的主要靶点可能在 CMEC，而丹参、三七对于 CMEC作＠
用的最佳配比是10＊和10爪 丹酚酸B与丹参酮IIA抗CMEC缺OZ损伤，
保护＊＊＊C的最佳配比是二8。
In order to study the protective effect and mechanism of preconditioning with cinnabar phenic acid B on the cardiac cell, based on the mechanism of anoxic-preconditioning, we establish the damaging model of anoxic-reoxygenation on the cell by the technique of cultivating cardiac microvascular endothelial cell (CMEC) and cardiac cell in vitro. To observe the protective effect on the cell with the cinnabar phenic acid B - preconditioning, we use the biochemistry examination such as the rate of live cell(MTT), lactate dehydrogenase(LDH^ superoxide dismutase(SOD^ nitric oxide(NO^ endothelin (ET) ect and ultrastructure as the evaluating criterion. By the means of medicine such as chelerythrine, a kind of an inhibitor of protein kinase C (PKC), glibenclamide, a kind of an inhibitor of the ATP-sensitive potassium channel (KATp), and from the point of view of the intrinsic protective substance, PKC, and the express of mRNA of the heat shock protein 70 (HSP70) we probe into the protective mechanism on the cell with t
he cinnabar phenic acid B -preconditioning.
In addition, in order to search the best ratio of compatibility of Chinese medicine, enhance the clinical curative effect, enrich the scientific connotation of the description compatibility theory and advance the progress of the Chinese medical modernization, we studied the relationship between the varieties of the effective substance composition in a description and its bio-effect by the injury cell model of CMEC by anoxic-reoxygenation using all composition of the prepared herbs (the paired description elan-sen root and pseudo-ginseng ) and its effective composition.
Result:
(1) Under the normal culturing condition, cinnabar phenic acid B can
enhance the livability of both of cells and the activity of SOD significantly, reduce the release of LDH, keep the ultrastructure, have the tendency of decreasing the secret of ET and the release of NO of CMEC.
(2) Hypoxic preconditioning can strengthen the cell endurance of the later damage of longer time anoxic-reoxygenation, which can be observed through the increase of the cell livability and the SOD activity, and the decrease of the LDH activity. At the same time, it can obviously decrease the level of ET and NO of CMEC by anoxic-reoxygenation and improve the change of the ultrastructure.
( 3) The protective effect on the cell by cinnabar phenic acid B is similar with the effect by anoxic-reoxygenation. For CMEC, this function will be expressed as the early and delayed protection phases.
(4) Glibenclamide, a kind of an inhibitor of KATP, cannot eliminate the HPC protective effect of CMEC. Chelerythrine, a kind of selective inhibitor of PKC, can eliminate the HPC protective effect of CMEC that can be recurred by the preconditioning of cinnabar phenic acid B.
(5) During the early preconditioning, cinnabar phenic acid B can improve the mRNA espression of HSP70 and PKC on the anoxic-reoxygenation injured CMEC. The effect is better than HPC obviously. During the delayed preconditioning, cinnabar phenic acid B also can improve the mRNA expression of HSP70, and it has the plus effect with HPC.
( 6 ) Under the normal or the anoxic cultivating conditions, elan-sen root can improve the reproduce proliferation of CMEC while pseudo-ginseng inhibits it. Among all the compatibility groups, the proliferation effect of the group of only elan-sen root is the most obvious (compare with other groups p<0.01 p<0.05), especially under the anoxic condition. All the compatibility groups can obviously decrease the release of NO and the secret of ET of CMEC. The best groups decreasing the secret of ET is the compatibility of 10:1 and 10:6. No matter the normal conditions or the anoxic conditions, all groups greatly promote the activity of CMEC, decrease the release of LDH. The effect the group of 2:8 decreasing the
secret of ET and the level of NO is the best. Especially under the anoxic condition, the effect of only cinnabar phenic acid B and only tanshinone II -A is not better than the compatibilit
引文
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