| 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. |
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