广枣抗心肌缺血再灌注损伤的物质基础及作用机制研究
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摘要
缺血性心脏病是严重危害人类健康和生命的一类疾病,是导致人类死亡的主要病因之一。心肌缺血再灌注损伤是缺血性心脏病常见的病理生理过程,同时也是加重心肌损伤的重要原因。导致再灌注损伤的机制是多方面的,主要包括细胞内离子累积(细胞内钙超载、钠离子增多,以及再灌注过程中pH降低)；线粒体膜电位降低(线粒体通透性转换孔mPTP及线粒体钙转运体MCU的改变)；氧自由基大爆发(黄嘌呤氧化酶的生成,mPTP的开放,ROS诱导的ROS释放)；内皮细胞功能受损(粘附分子表达,血管舒张功能障碍)；凋亡和自噬；血小板聚集和微栓塞形成等。如何减轻心肌缺血再灌注损伤已经成为近年来心血管疾病研究领域的热点问题。从中药中寻找疗效确切,不良反应小的天然药物已成为当前缺血性心脏病治疗的迫切需要。
广枣为漆树科植物南酸枣Choerospondias axillaris (Roxb.) Burtt et Hill的干燥成熟果实,味甘、酸,性平,具有行气活血,养心安神的功效,用于气滞血瘀,胸痹作痛,心悸气短,心神不安等。广枣是蒙医治疗心血管疾病的主药,疗效确切。广枣中含有黄酮、有机酸、酚酸、鞣质及多糖等成分。目前有关广枣的药效物质研究主要集中在黄酮,认为黄酮具有保护缺血心肌、抑制血小板聚集,增强免疫等多种药理活性。然而,文献研究表明广枣果皮中含有丰富的有机酸类成分,高达5.22%-8.13%。本实验室化学研究也证实广枣药材中除了含有小量的黄酮之外,还含有大量的有机酸和酚酸类成分,并采用MRM总离子流图确认了7个化合物,分别是柠檬酸、苹果酸、原儿茶酸、没食子酸、原儿茶醛、鞣花酸和槲皮素。在广枣水提物中,柠檬酸含量约为15%,原儿茶酸约为0.13%,没食子酸约为0.08%,而槲皮素约为0.0003%。中药有机酸类成分具有广泛的药理作用,如抗炎症反应、抑制血小板聚集、抗血栓、抗氧化及诱导肿瘤细胞凋亡等,其中抗炎症反应、抑制血小板聚集、抗血栓及抗氧化的药理作用提示了小分子有机酸可能在广枣治疗缺血性心脏病过程中发挥了不可忽视的作用。因而,我们推测广枣治疗缺血性心脏病的药效物质可能不仅仅是广枣黄酮,还可能是小分子有机酸类及酚酸类成分,值得进一步深入探讨。
迄今,心肌缺血再灌注损伤的机制已经取得了很大的研究进展,但仍然没有完全得以阐明。最新研究发现心脏成纤维细胞的免疫激活与炎症分泌可能在心肌缺血再灌注损伤过程中扮演了重要角色。研究认为心脏成纤维细胞在心肌缺血再灌注损伤中扮演“哨兵”的角色,心肌缺血再灌注时心脏成纤维细胞ROS产生以及钾离子外流,心脏成纤维细胞炎症体激活,诱导了早期炎症介导者IL-1p产生,早期炎症反应刺激趋化因子释放,招募单核细胞、巨噬细胞及中性粒细胞至缺血心肌组织,加重了心肌缺血再灌注损伤。因此,我们认为干预调节心脏成纤维细胞的炎症分泌可以降低心肌缺血再灌注损伤。
本实验室前期研究发现广枣有机酸部位具有显著的抗心肌缺血再灌注损伤作用。本研究采用体外筛选结合体内验证的方法探讨广枣抗心肌缺血再灌注损伤的药效物质,并从心肌细胞的凋亡和心脏成纤维细胞的炎症旁分泌角度阐述其作用机制。主要内容包括如下：
(1)采用新生大鼠乳鼠原代心肌细胞培养体系,考察广枣粗提物及所含成分有机酸(柠檬酸、L-苹果酸、琥珀酸、酒石酸、熊果酸和香草酸)、酚酸(原儿茶酸和没食子酸)、黄酮(槲皮素和山柰酚)对原代心肌细胞活力的影响。在此基础上,建立新生大鼠乳鼠原代心肌细胞缺氧复氧损伤模型,筛选广枣抗心肌细胞缺氧复氧损伤的具体成分,初步明确广枣抗心肌缺血再灌注损伤的药效物质。
(2)建立大鼠心肌缺血再灌注损伤模型,考察广枣粗提物、广枣模拟总有机酸、广枣模拟总酚酸以及所选取的4种代表成分(有机酸部分：柠檬酸和L-苹果酸；酚酸部分：原儿茶酸；黄酮部分：槲皮素)对心肌缺血再灌注损伤的影响,进一步明确广枣抗心肌缺血再灌注损伤的药效物质。
(3)从心肌细胞凋亡的角度,采用流式细胞术分析心肌细胞的凋亡百分数,western blot考察cleaved-caspase3及p-Akt(Ser473)蛋白表达的改变,探讨广枣小分子有机酸、酚酸、黄酮对心肌缺血再灌注损伤的干预机制。
(4)建立心脏成纤维细胞炎症分泌模型,明确广枣成分对心脏成纤维细胞活力的影响,通过检测细胞增殖,ELISA测定炎症因子TNF-α、IL-1β、IL-6及IL-18,明确广枣干预心脏成纤维细胞炎症分泌的药效物质,并进一步考察其对p-NF-kB P65(S276)和p-Akt (S473)蛋白表达的影响,明确其作用机制。研究结果简述如下：
(1)①广枣粗提物、广枣有机酸部分(模拟总有机酸、柠檬酸、L-苹果酸、琥珀酸和酒石酸)、黄酮部分(槲皮素和山柰酚)对心肌细胞缺氧复氧损伤均具有直接的保护作用；②广枣酚酸部分(原儿茶酸和没食子酸)不能抑制原代心肌细胞缺氧复氧损伤所致的LDH漏出,未见明显的心肌细胞保护作用。
(2)广枣粗提物589.2mg·kg-1、广枣模拟总有机酸500mg·kg-1、广枣模拟总酚酸500mg·kg-1、有机酸部分代表成分柠檬酸250、500mg·kg-1、L-苹果酸250.500mg·kg-1,酚酸部分代表成分原儿茶酸250.500mg·kg-1以及黄酮部分代表成分槲皮素20、40mg·kg-1均可显著降低心肌缺血再灌注所致的心肌梗死。研究证实了除黄酮之外,小分子有机酸及酚酸也是广枣抗心肌缺血再灌注损伤的重要药效物质。
(3)①流式研究结果表明广枣有机酸部分(柠檬酸、L-苹果酸和琥珀酸),酚酸部分(原儿茶酸)、黄酮部分(槲皮素)可以抑制缺氧复氧损伤所致的心肌细胞凋亡百分数升高,其中有机酸部分的酒石酸成分有降低趋势,但无统计学差异；②广枣有机酸部分(柠檬酸、L-苹果酸、琥珀酸和酒石酸),酚酸部分(原儿茶酸)、黄酮部分(槲皮素)均可显著降低缺氧复氧所致的cleaved-caspase3蛋白表达增加,与模型组相比,有显著性差异；(3)广枣有机酸部分(柠檬酸、L-苹果酸、琥珀酸和酒石酸),酚酸部分(原儿茶酸)、黄酮部分(槲皮素)均可显著增加缺氧复氧所致的Akt (Ser473)磷酸化激活,p-Akt蛋白表达显著增加,与模型组相比较,有显著性差异。实验证实广枣有机酸、酚酸和黄酮均可以通过激活Akt的磷酸化而抑制心肌细胞的凋亡,对心肌细胞缺氧复氧损伤有直接保护作用。
(4) LPS100ng·mL-1作用3h而后ATP5mmol·L-1作用36h可刺激心脏成纤维细胞分泌炎症因子TNF-α、IL-1β、IL-6及IL-18,广枣黄酮部分(槲皮素和山柰酚)可通过抑制Akt/NF-kB信号途径的激活而抑制心脏成纤维细胞分泌上述炎症因子,减轻心肌缺血再灌注过程中的炎症反应,对心肌缺血再灌注损伤具有间接保护作用。
综上所述,本研究的主要发现和结论是：①广枣抗心肌缺血再灌注损伤的药效物质基础是小分子有机酸、酚酸和黄酮；②广枣有机酸、酚酸和黄酮三大类成分均可通过激活Akt的磷酸化、抑制心肌细胞的凋亡,对心肌缺血再灌注损伤具有直接保护作用；③广枣黄酮(槲皮素和山柰酚)可通过抑制Akt/NF-kB信号途径的激活而抑制心脏成纤维细胞分泌TNF-α、IL-1β、IL-6及IL-18等炎症因子,减轻心肌缺血再灌注过程中的旁分泌炎症因子的释放,对心肌缺血再灌注损伤具有间接保护作用。
Ischemic heart disease is a leading cause of mortality of the clinical cardiovascular diseases and remains a major public health threat worldwide. Myocardial damage in ischemic heart disease is likely due to ischemia/reperfusion injury. Myocardial ischemia/reperfusion can lead to cardiomyocyte loss by several pathological mechanisms, which contain intracellular ion accumulation, reduction in mitochondrial membrane potential, free radical formation, inflammatory response and endothelial dysfunction, apoptosis, necrosis and autophagy, platelet aggregation and microembolization, and so forth. Therefore, a pharmacologic approach to ischemia/reperfusion injury remains a longstanding challenge in medicine.
Fructus Choerospondiatis, a widely known Mongolian herb derived from the dried mature fruit of Choerospondias axillaris (Roxb.) Burtt et Hill, with efficacy of "activating vital energy and blood circulation" and "nourishing heart for tranquilization", according to traditional Chinese medicine theory, has been used extensively as a remedy for ischemic heart disease and achieved good clinical efficacy. It consists of several ingredients, including organic acids, phenolic acids, flavonoids, tannins and polysaccharides, etc. The flavonoids of Fructus Choerospondiatis have the effects of protecting the ischemic myocardium, inhibition of platelet aggregation and enhancing immune activities, and so forth. Previous studies have been focused on flavonoids, which were always considered to be the main active constituents responsible for the pharmacological actions of Fructus Choerospondiatis. However, recent pharmaceutical chemistry studies showed that the content of total flavonoids (mainly quercetin) in Fructus Choerospondiatis was very low and only accounted for0.0003%of its water-soluable extracts, whereas the content of total organic acids was in significant amounts, up to8.13%. And citric acid and L-malic acid are two main organic acids of Fructus Choerospondiatis, the content of which accounted for26.36%and22.95%of total organic acids, respectively.
Organic acids, which are also widely distributed in fresh fruits, vegetables and spices besides Chinese herbs, were always considered to be weak in activity and were usually discarded in the extraction process. Therefore, they have been long-neglected and their pharmacological actions have not been sufficiently studied. Recent research indicated that some organic acids have various pharmacological effects, including anti-inflammatory response, anti-platelet aggregation, anti-oxidant, and reducing cell apoptosis, and so on. It implies that organic acids may have protective effect on myocardial ischemia/reperfusion injury. Therefore, we hypothesize that the effective substances for Fructus Choerospondiatis against ischemic heart disease may not only flavonoids, but also organic acids and phenolic acids. It deserves to be explored in-depth.
To date, the research of the mechanism of myocardial ischemia/reperfusion injury has been made great progress, but still not completely elucidated. Recent study revealed that immune activation and inflammation secretion of cardiac fibroblasts might play an important role in the process of myocardial ischemia/reperfusion injury. Cardiac fibroblasts play an important role as "sentinel" cells in the process of myocardial ischemia/reperfusion injury. ROS production and potassium efflux lead to the activation of inflammasomes and the secretion of IL-1β in cardiac fibroblasts. Such proinflammatory response stimulates chemotactic factor to release and recruits monocytes, macrophages and neutrophils to the ischemic myocardium tissue, which eventually aggravate myocardial ischemia/reperfusion injury. Therefore, we speculate that regulating the secretion of inflammatory cytokine in cardiac fibroblasts can prevent myocardial ischemia/reperfusion injury.
Previous study in our lab revealed that organic acid part had protective effect on myocardial ischemia/reperfusion injury. Therefore, in the present study, we investigated the material basis of Fructus Choerospondiatis in in vitro primary neonatal rat cardiomyocyte model of hypoxia/reoxygenation and in vivo rat model of myocardial ischemia/reperfusion, and explored the possible mechanisms involved.
The main contents are as follows:
(1) Using primary neonatal rat cardiomyocyte culture system, the effects of Fructus Choerospondiatis crude extract and its ingredients contained, such as organic acids (citric acid, L-malic acid, succinic acid, tartaric acid, ursolic acid and vanillic acid), phenolic acids (protocatechuie acid and gallic acid), flavonoids (quercetin and kaempferol) were investigated on neonatal rat cardiomyocyte viability. On this basis, the material basis of Fructus Choerospondiatis was screened in in vitro primary neonatal rat cardiomyocyte model of hypoxia/reoxygenation.
(2) Using rat model of myocardial ischemia/reperfusion injury, the cardioprotective effects of Fructus Choerospondiatis crude extract, simulated total organic acid, simulated total phenolic acid, as well as four representative ingredients (organic acids part:citric acid and L-malic acid; phenolic acids part:protocatechuie acid; flavonoids part:quercetin) were explored. The material basis of Fructus Choerospondiatis on myocardial ischemia/reperfusion injury were determined.
(3) To explore the mechanism of organic acids, phenolic acids and flavonoids on myocardial ischemia/reperfusion injury, the apoptosis percentage of cardiomyocytes was analysed by flow cytometry. The protein expressions of cleaved-caspase3and p-Akt (Ser473) were investigated by western blot.
(4) The model of inflammatory cytokine secretion in cardiac fibroblasts was established. The effects of the ingredients of Fructus Choerospondiatis were investigated on cardiac fibroblasts viability. To further explore the material basis of Fructus Choerospondiatis on cardiac fibroblasts inflammation secretion, The inflammatory cytokine levels of TNF-α, IL-1β, IL-6and IL-18were determined, and phospho-NF-kB P65(S276), NF-kB P65, phospho-Akt (Ser473) and Akt were investigated by western blot.
The results are summarized as follows:
(1)①The Fructus Choerospondiatis crude extract and its ingredients contained, such as organic acids (simulated total organic acid, citric acid, L-malic acid, succinic acid and tartaric acid), flavonoids (quercetin and kaempferol) had significant protective effects on primary neonatal rat cardiomyocytes hypoxia/reoxygenation injury.②The phenolic acids (protocatechuic acid and gallic acid) had no obvious effects in decreasing LDH leakage of cardiomyocytes induced by hypoxia/reoxygenation injury.
(2) The crude extract of Fructus Choerospondiatis589.2mg·kg-1, simulated total organic acids500mg·kg-1, simulated total phenolic acids500mg·kg-1, representative ingredients citric acid250,500mg·kg-1, L-malic acid250,500mg·kg-1, protocatechuic acid250,500mg·kg-1and quercetin20,40mg·kg-1significantly reduced myocardial infarction induced by myocardial ischemia/reperfusion injury. Based on these findings, we concluded that both organic acids and phenolic acids may also be the major material basis of Fructus Choerospondiatis responsible for its cardioprotective effect, but not only flavonoids.
(3)①The flow cytometry results showed that hypoxia/reoxygenation injury significantly increased the number of apoptotic cardiomyocytes. while treatment with organic acids part (citric acid, L-malic acid and succinic acid), phenolic acids part (protocatechuic acid) and flavonoids part (quercetin) significantly reduced the number of apoptotic cells, but tartaric acid not obvious.②Cleaved caspase-3was significantly upregulated by hypoxia/reoxygenation-induced cardiomyocytes injury, while significantly downregulated by treatment with organic acids part (citric acid, L-malic acid, succinic acid and tartaric acid), phenolic acids part (protocatechuic acid) and flavonoids part (quercetin) relative to the model control group.③Our results revealed that organic acids part (citric acid, L-malic acid, succinic acid, and tartaric acid), phenolic acids part (protocatechuic acid) and flavonoids part (quercetin) significantly increased the activation of Akt (Ser473) and upregulated the expression of phosphorylated Akt. All data demonstrated that organic acids, phenolic acids and flavonoids had direct protective effects on primary neonatal rat cardiomyocytes hypoxia/reoxygenation injury through the activation of Akt (Scr473) and inhibition of cardiomyocytes apoptosis.
(4) After LPS100ng·mL-13hand ATP5mmol·L-136h, the inflammatory cytokine levels of TNF-α, IL-1β, IL-6and IL-18secreted by cardiac fibroblasts were significantly increased, which were significantly decreased by the flavonoids part (quercetin and kaempferol) of Fructus Choerspondialis by inhibiting the activation of Akt/NF-kB signaling pathway. These results demonstrated that flavonoids (quercetin and kaempferol) had indirect protective effects on myocardial ischemia/reperfusion injury by inhibition inflammatory cytokines secretion from cardiac fibroblasts.
In summary, we demonstrated that organic acids and phenolic acids may also be the material basis for Fructus Chocrospondiatis responsible for its cardioprotective effect, but not only flavonoids. Our results revealed that organic acids, phenolic acids and flavonoids had direct protective effects on myocardial ischemia/reperfusion injury through the activation of Akt (Ser473) and inhibition of cardiomyocytes apoptosis, and flavonoids also had indirect protective effects on myocardial ischemia/reperfusion injury by inhibition inflammatory cytokines secretion from cardiac fibroblasts.
广枣为漆树科植物南酸枣Choerospondias axillaris (Roxb.) Burtt et Hill的干燥成熟果实,味甘、酸,性平,具有行气活血,养心安神的功效,用于气滞血瘀,胸痹作痛,心悸气短,心神不安等。广枣是蒙医治疗心血管疾病的主药,疗效确切。广枣中含有黄酮、有机酸、酚酸、鞣质及多糖等成分。目前有关广枣的药效物质研究主要集中在黄酮,认为黄酮具有保护缺血心肌、抑制血小板聚集,增强免疫等多种药理活性。然而,文献研究表明广枣果皮中含有丰富的有机酸类成分,高达5.22%-8.13%。本实验室化学研究也证实广枣药材中除了含有小量的黄酮之外,还含有大量的有机酸和酚酸类成分,并采用MRM总离子流图确认了7个化合物,分别是柠檬酸、苹果酸、原儿茶酸、没食子酸、原儿茶醛、鞣花酸和槲皮素。在广枣水提物中,柠檬酸含量约为15%,原儿茶酸约为0.13%,没食子酸约为0.08%,而槲皮素约为0.0003%。中药有机酸类成分具有广泛的药理作用,如抗炎症反应、抑制血小板聚集、抗血栓、抗氧化及诱导肿瘤细胞凋亡等,其中抗炎症反应、抑制血小板聚集、抗血栓及抗氧化的药理作用提示了小分子有机酸可能在广枣治疗缺血性心脏病过程中发挥了不可忽视的作用。因而,我们推测广枣治疗缺血性心脏病的药效物质可能不仅仅是广枣黄酮,还可能是小分子有机酸类及酚酸类成分,值得进一步深入探讨。
迄今,心肌缺血再灌注损伤的机制已经取得了很大的研究进展,但仍然没有完全得以阐明。最新研究发现心脏成纤维细胞的免疫激活与炎症分泌可能在心肌缺血再灌注损伤过程中扮演了重要角色。研究认为心脏成纤维细胞在心肌缺血再灌注损伤中扮演“哨兵”的角色,心肌缺血再灌注时心脏成纤维细胞ROS产生以及钾离子外流,心脏成纤维细胞炎症体激活,诱导了早期炎症介导者IL-1p产生,早期炎症反应刺激趋化因子释放,招募单核细胞、巨噬细胞及中性粒细胞至缺血心肌组织,加重了心肌缺血再灌注损伤。因此,我们认为干预调节心脏成纤维细胞的炎症分泌可以降低心肌缺血再灌注损伤。
本实验室前期研究发现广枣有机酸部位具有显著的抗心肌缺血再灌注损伤作用。本研究采用体外筛选结合体内验证的方法探讨广枣抗心肌缺血再灌注损伤的药效物质,并从心肌细胞的凋亡和心脏成纤维细胞的炎症旁分泌角度阐述其作用机制。主要内容包括如下：
(1)采用新生大鼠乳鼠原代心肌细胞培养体系,考察广枣粗提物及所含成分有机酸(柠檬酸、L-苹果酸、琥珀酸、酒石酸、熊果酸和香草酸)、酚酸(原儿茶酸和没食子酸)、黄酮(槲皮素和山柰酚)对原代心肌细胞活力的影响。在此基础上,建立新生大鼠乳鼠原代心肌细胞缺氧复氧损伤模型,筛选广枣抗心肌细胞缺氧复氧损伤的具体成分,初步明确广枣抗心肌缺血再灌注损伤的药效物质。
(2)建立大鼠心肌缺血再灌注损伤模型,考察广枣粗提物、广枣模拟总有机酸、广枣模拟总酚酸以及所选取的4种代表成分(有机酸部分：柠檬酸和L-苹果酸；酚酸部分：原儿茶酸；黄酮部分：槲皮素)对心肌缺血再灌注损伤的影响,进一步明确广枣抗心肌缺血再灌注损伤的药效物质。
(3)从心肌细胞凋亡的角度,采用流式细胞术分析心肌细胞的凋亡百分数,western blot考察cleaved-caspase3及p-Akt(Ser473)蛋白表达的改变,探讨广枣小分子有机酸、酚酸、黄酮对心肌缺血再灌注损伤的干预机制。
(4)建立心脏成纤维细胞炎症分泌模型,明确广枣成分对心脏成纤维细胞活力的影响,通过检测细胞增殖,ELISA测定炎症因子TNF-α、IL-1β、IL-6及IL-18,明确广枣干预心脏成纤维细胞炎症分泌的药效物质,并进一步考察其对p-NF-kB P65(S276)和p-Akt (S473)蛋白表达的影响,明确其作用机制。研究结果简述如下：
(1)①广枣粗提物、广枣有机酸部分(模拟总有机酸、柠檬酸、L-苹果酸、琥珀酸和酒石酸)、黄酮部分(槲皮素和山柰酚)对心肌细胞缺氧复氧损伤均具有直接的保护作用；②广枣酚酸部分(原儿茶酸和没食子酸)不能抑制原代心肌细胞缺氧复氧损伤所致的LDH漏出,未见明显的心肌细胞保护作用。
(2)广枣粗提物589.2mg·kg-1、广枣模拟总有机酸500mg·kg-1、广枣模拟总酚酸500mg·kg-1、有机酸部分代表成分柠檬酸250、500mg·kg-1、L-苹果酸250.500mg·kg-1,酚酸部分代表成分原儿茶酸250.500mg·kg-1以及黄酮部分代表成分槲皮素20、40mg·kg-1均可显著降低心肌缺血再灌注所致的心肌梗死。研究证实了除黄酮之外,小分子有机酸及酚酸也是广枣抗心肌缺血再灌注损伤的重要药效物质。
(3)①流式研究结果表明广枣有机酸部分(柠檬酸、L-苹果酸和琥珀酸),酚酸部分(原儿茶酸)、黄酮部分(槲皮素)可以抑制缺氧复氧损伤所致的心肌细胞凋亡百分数升高,其中有机酸部分的酒石酸成分有降低趋势,但无统计学差异；②广枣有机酸部分(柠檬酸、L-苹果酸、琥珀酸和酒石酸),酚酸部分(原儿茶酸)、黄酮部分(槲皮素)均可显著降低缺氧复氧所致的cleaved-caspase3蛋白表达增加,与模型组相比,有显著性差异；(3)广枣有机酸部分(柠檬酸、L-苹果酸、琥珀酸和酒石酸),酚酸部分(原儿茶酸)、黄酮部分(槲皮素)均可显著增加缺氧复氧所致的Akt (Ser473)磷酸化激活,p-Akt蛋白表达显著增加,与模型组相比较,有显著性差异。实验证实广枣有机酸、酚酸和黄酮均可以通过激活Akt的磷酸化而抑制心肌细胞的凋亡,对心肌细胞缺氧复氧损伤有直接保护作用。
(4) LPS100ng·mL-1作用3h而后ATP5mmol·L-1作用36h可刺激心脏成纤维细胞分泌炎症因子TNF-α、IL-1β、IL-6及IL-18,广枣黄酮部分(槲皮素和山柰酚)可通过抑制Akt/NF-kB信号途径的激活而抑制心脏成纤维细胞分泌上述炎症因子,减轻心肌缺血再灌注过程中的炎症反应,对心肌缺血再灌注损伤具有间接保护作用。
综上所述,本研究的主要发现和结论是：①广枣抗心肌缺血再灌注损伤的药效物质基础是小分子有机酸、酚酸和黄酮；②广枣有机酸、酚酸和黄酮三大类成分均可通过激活Akt的磷酸化、抑制心肌细胞的凋亡,对心肌缺血再灌注损伤具有直接保护作用；③广枣黄酮(槲皮素和山柰酚)可通过抑制Akt/NF-kB信号途径的激活而抑制心脏成纤维细胞分泌TNF-α、IL-1β、IL-6及IL-18等炎症因子,减轻心肌缺血再灌注过程中的旁分泌炎症因子的释放,对心肌缺血再灌注损伤具有间接保护作用。
Ischemic heart disease is a leading cause of mortality of the clinical cardiovascular diseases and remains a major public health threat worldwide. Myocardial damage in ischemic heart disease is likely due to ischemia/reperfusion injury. Myocardial ischemia/reperfusion can lead to cardiomyocyte loss by several pathological mechanisms, which contain intracellular ion accumulation, reduction in mitochondrial membrane potential, free radical formation, inflammatory response and endothelial dysfunction, apoptosis, necrosis and autophagy, platelet aggregation and microembolization, and so forth. Therefore, a pharmacologic approach to ischemia/reperfusion injury remains a longstanding challenge in medicine.
Fructus Choerospondiatis, a widely known Mongolian herb derived from the dried mature fruit of Choerospondias axillaris (Roxb.) Burtt et Hill, with efficacy of "activating vital energy and blood circulation" and "nourishing heart for tranquilization", according to traditional Chinese medicine theory, has been used extensively as a remedy for ischemic heart disease and achieved good clinical efficacy. It consists of several ingredients, including organic acids, phenolic acids, flavonoids, tannins and polysaccharides, etc. The flavonoids of Fructus Choerospondiatis have the effects of protecting the ischemic myocardium, inhibition of platelet aggregation and enhancing immune activities, and so forth. Previous studies have been focused on flavonoids, which were always considered to be the main active constituents responsible for the pharmacological actions of Fructus Choerospondiatis. However, recent pharmaceutical chemistry studies showed that the content of total flavonoids (mainly quercetin) in Fructus Choerospondiatis was very low and only accounted for0.0003%of its water-soluable extracts, whereas the content of total organic acids was in significant amounts, up to8.13%. And citric acid and L-malic acid are two main organic acids of Fructus Choerospondiatis, the content of which accounted for26.36%and22.95%of total organic acids, respectively.
Organic acids, which are also widely distributed in fresh fruits, vegetables and spices besides Chinese herbs, were always considered to be weak in activity and were usually discarded in the extraction process. Therefore, they have been long-neglected and their pharmacological actions have not been sufficiently studied. Recent research indicated that some organic acids have various pharmacological effects, including anti-inflammatory response, anti-platelet aggregation, anti-oxidant, and reducing cell apoptosis, and so on. It implies that organic acids may have protective effect on myocardial ischemia/reperfusion injury. Therefore, we hypothesize that the effective substances for Fructus Choerospondiatis against ischemic heart disease may not only flavonoids, but also organic acids and phenolic acids. It deserves to be explored in-depth.
To date, the research of the mechanism of myocardial ischemia/reperfusion injury has been made great progress, but still not completely elucidated. Recent study revealed that immune activation and inflammation secretion of cardiac fibroblasts might play an important role in the process of myocardial ischemia/reperfusion injury. Cardiac fibroblasts play an important role as "sentinel" cells in the process of myocardial ischemia/reperfusion injury. ROS production and potassium efflux lead to the activation of inflammasomes and the secretion of IL-1β in cardiac fibroblasts. Such proinflammatory response stimulates chemotactic factor to release and recruits monocytes, macrophages and neutrophils to the ischemic myocardium tissue, which eventually aggravate myocardial ischemia/reperfusion injury. Therefore, we speculate that regulating the secretion of inflammatory cytokine in cardiac fibroblasts can prevent myocardial ischemia/reperfusion injury.
Previous study in our lab revealed that organic acid part had protective effect on myocardial ischemia/reperfusion injury. Therefore, in the present study, we investigated the material basis of Fructus Choerospondiatis in in vitro primary neonatal rat cardiomyocyte model of hypoxia/reoxygenation and in vivo rat model of myocardial ischemia/reperfusion, and explored the possible mechanisms involved.
The main contents are as follows:
(1) Using primary neonatal rat cardiomyocyte culture system, the effects of Fructus Choerospondiatis crude extract and its ingredients contained, such as organic acids (citric acid, L-malic acid, succinic acid, tartaric acid, ursolic acid and vanillic acid), phenolic acids (protocatechuie acid and gallic acid), flavonoids (quercetin and kaempferol) were investigated on neonatal rat cardiomyocyte viability. On this basis, the material basis of Fructus Choerospondiatis was screened in in vitro primary neonatal rat cardiomyocyte model of hypoxia/reoxygenation.
(2) Using rat model of myocardial ischemia/reperfusion injury, the cardioprotective effects of Fructus Choerospondiatis crude extract, simulated total organic acid, simulated total phenolic acid, as well as four representative ingredients (organic acids part:citric acid and L-malic acid; phenolic acids part:protocatechuie acid; flavonoids part:quercetin) were explored. The material basis of Fructus Choerospondiatis on myocardial ischemia/reperfusion injury were determined.
(3) To explore the mechanism of organic acids, phenolic acids and flavonoids on myocardial ischemia/reperfusion injury, the apoptosis percentage of cardiomyocytes was analysed by flow cytometry. The protein expressions of cleaved-caspase3and p-Akt (Ser473) were investigated by western blot.
(4) The model of inflammatory cytokine secretion in cardiac fibroblasts was established. The effects of the ingredients of Fructus Choerospondiatis were investigated on cardiac fibroblasts viability. To further explore the material basis of Fructus Choerospondiatis on cardiac fibroblasts inflammation secretion, The inflammatory cytokine levels of TNF-α, IL-1β, IL-6and IL-18were determined, and phospho-NF-kB P65(S276), NF-kB P65, phospho-Akt (Ser473) and Akt were investigated by western blot.
The results are summarized as follows:
(1)①The Fructus Choerospondiatis crude extract and its ingredients contained, such as organic acids (simulated total organic acid, citric acid, L-malic acid, succinic acid and tartaric acid), flavonoids (quercetin and kaempferol) had significant protective effects on primary neonatal rat cardiomyocytes hypoxia/reoxygenation injury.②The phenolic acids (protocatechuic acid and gallic acid) had no obvious effects in decreasing LDH leakage of cardiomyocytes induced by hypoxia/reoxygenation injury.
(2) The crude extract of Fructus Choerospondiatis589.2mg·kg-1, simulated total organic acids500mg·kg-1, simulated total phenolic acids500mg·kg-1, representative ingredients citric acid250,500mg·kg-1, L-malic acid250,500mg·kg-1, protocatechuic acid250,500mg·kg-1and quercetin20,40mg·kg-1significantly reduced myocardial infarction induced by myocardial ischemia/reperfusion injury. Based on these findings, we concluded that both organic acids and phenolic acids may also be the major material basis of Fructus Choerospondiatis responsible for its cardioprotective effect, but not only flavonoids.
(3)①The flow cytometry results showed that hypoxia/reoxygenation injury significantly increased the number of apoptotic cardiomyocytes. while treatment with organic acids part (citric acid, L-malic acid and succinic acid), phenolic acids part (protocatechuic acid) and flavonoids part (quercetin) significantly reduced the number of apoptotic cells, but tartaric acid not obvious.②Cleaved caspase-3was significantly upregulated by hypoxia/reoxygenation-induced cardiomyocytes injury, while significantly downregulated by treatment with organic acids part (citric acid, L-malic acid, succinic acid and tartaric acid), phenolic acids part (protocatechuic acid) and flavonoids part (quercetin) relative to the model control group.③Our results revealed that organic acids part (citric acid, L-malic acid, succinic acid, and tartaric acid), phenolic acids part (protocatechuic acid) and flavonoids part (quercetin) significantly increased the activation of Akt (Ser473) and upregulated the expression of phosphorylated Akt. All data demonstrated that organic acids, phenolic acids and flavonoids had direct protective effects on primary neonatal rat cardiomyocytes hypoxia/reoxygenation injury through the activation of Akt (Scr473) and inhibition of cardiomyocytes apoptosis.
(4) After LPS100ng·mL-13hand ATP5mmol·L-136h, the inflammatory cytokine levels of TNF-α, IL-1β, IL-6and IL-18secreted by cardiac fibroblasts were significantly increased, which were significantly decreased by the flavonoids part (quercetin and kaempferol) of Fructus Choerspondialis by inhibiting the activation of Akt/NF-kB signaling pathway. These results demonstrated that flavonoids (quercetin and kaempferol) had indirect protective effects on myocardial ischemia/reperfusion injury by inhibition inflammatory cytokines secretion from cardiac fibroblasts.
In summary, we demonstrated that organic acids and phenolic acids may also be the material basis for Fructus Chocrospondiatis responsible for its cardioprotective effect, but not only flavonoids. Our results revealed that organic acids, phenolic acids and flavonoids had direct protective effects on myocardial ischemia/reperfusion injury through the activation of Akt (Ser473) and inhibition of cardiomyocytes apoptosis, and flavonoids also had indirect protective effects on myocardial ischemia/reperfusion injury by inhibition inflammatory cytokines secretion from cardiac fibroblasts.
引文
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