Luteolin: A Flavonoid that Has Multiple Cardio-Protective Effects and Its Molecular Mechanisms

1Flavonoids are naturally occurring compounds that are universally present in the plant kingdom. The basic structure of flavonoids consists of an aromatic A-ring and a heterocyclic C-ring, which are connected with an aromatic B-ring through a carbon–carbon bridge. Based on heterocyclic C-ring variation, flavonoids are divided into six subclasses: flavones, flavonols, flavanones, catechins, or flavanols, anthocyanidins and isoflavones. Data sets from the Australian National Nutrition Survey (10,851 subjects>18 years) on flavonoid content of foods showed that the key sources of flavonoids included tea (black and green tea), vegetable (onion, celery, parsley, broccoli, English spinach, bean), fruits (apple, grape, coffee, oranges, lemon, mandarin, cherry, blueberry) and wine. Since discovery of flavonoids in 1936, more than 5,000 structures of flavonoids have been reported with a wide range of molecular diversity. The Women’s Health Study with 38,408females ≥ 45years showed that the median intake of total flavonoids ranging from 8.88 to 47.44 mg/day in 11.5 years follow-up. The Dutch National Food Consumption Survey indicated that people in Netherlands have an average mixed flavonoids intake of 23 mg/day. Epidemiological evidence shows that intake of these compounds does not only inhibit tumor formation and inflammation, but also effectively prevents cardiovascular diseases (CVD). In the Zutphen Elderly Study, reported that the intake of flavonoids was inversely related to mortality (p = 0.015) and myocardial infarction (p = 0.08). reported that main flavonoids from Crataegus species increased coronary flow and relaxation velocity Langendorff heart models. The greatest increases of coronary flow (186%) and relaxation velocity (104%) were found in the luteolin-7-glucoside group. Therefore, generous dietary intake of flavonoids may be an effective primary prevention of CVD.

2Luteolin (3′, 4′, 5′, 7′-tetrahydroxyflavone, Lut) is one of the most prevalent flavones. Molecular formula of Lut is C15H10O6 and the structure is showed in Figure 1. A variety of vegetables, fruits and herbs such as carrot, cabbage, artichoke, tea, celery and apple are rich in Lut. The median intake of Lut is 0.01–0.20 mg/day (0.0349–0.698 μmol/day). Lut is absorbed by the intestinal mucosa. After oral administration of 14.3 mg/kg Lut, the maximum plasma concentration (Cmax) was 1.97 ± 0.15g/ml. The time to reach Cmax was 1.02 ± 0.22 h and the half-life of Lut (t1/2) was 4.94 ± 1.2 h. It was converted to free Lut, glucuronide and sulfate-conjugates of Lut and of O-methyl Lut (diosmetin or chrysoeriol) in the plasma of rats. Free Lut is also presentin human plasma. Through intrinsic and extrinsic signaling pathways, Lut is an active compound with anti-oxidant, anti-tumor, anti-inflammatory, and anti-apoptotic activities. Due to its potent anti-tumor and anti-inflammatory flavonoid, earlier studies and clinical trials on Lut had thus focused on cancer and inflammation. Since the 1950s to date, there has been an increase in the number of reports on the cardiovascular effects of Lut., N reported that Lut has effects on the heart and vascular vessels. With current advancements in the understanding of oxidative stress and inflammatory mechanisms of the cardiovascular system, Lut exhibits strong cardiovascular protective activities via complex signal transduction pathways and target effectors. Indeed, high dietary intake of Lut is related to a decreased risk of acute myocardial infarction. The cardio protection mainly stems from decreased myocardial apoptosis, diminished myocardial infarct size and through enhanced left ventricular ejection fraction.

3It has been proposed that Lut exerts protective effects in a multitude of CVD, including coronary artery disease (CAD), heart failure (HF), and atherosclerosis (AS). Relevant studies on this topic have mainly focused on the target effectors of Lut using in vitro and in vivo models. Here we present an update and detailed overview of the molecular mechanisms that orchestrate in cardiac protection conferred by Lut, mainly on the anti-I/R injury, cardiac function in HF and mechanism of anti-AS.

4Coronary artery disease is a serious problem affecting human health. The most efficient method used to reduce mortality in these patients is to reperfuse the myocardium by interventional treatment or pharmacological therapy. However, myocardial ischemia/reperfusion (I/R) injury followed by reperfusion can damage the myocardium, inducting reperfusion arrhythmia, no-reflow phenomenon, and systolic/diastolic dysfunction, which affects the outcomes of reperfusion therapy. Therefore, many researchers are seeking more effective drugs that can treat I/R injury in CAD patients. Emerging evidence has revealed that Lut regulates vital effectors involved in the process of I/R injury and has displayed satisfactory therapeutic value (Figure 2).

5The phosphoinositide 3-kinase (PI3K)/AKT pathway is a key pathway mediating in cell growth, metabolism and survival. AKT has also been confirmed to regulate the apoptotic pathological process of I/R injury. In cardiac-protection of Lut against I/R injury, AKT is widely and thoroughly studied: First, Lut suppresses apoptosis by up-regulating AKT in a simulated ischemia/reperfusion (sI/R) model. Pretreatment with Lut significantly inhibited apoptosis, increased BCL-2 expression and the BCL-2/BAX ratio as well as decreased the expression of BAX. Moreover, PI3K inhibitor LY294002 which downregulates AKT expression markedly diminished the function of Lut-induced positive contraction and attenuated the Lut protection against apoptosis in sI/R cardiomyocytes. Second, previous studies have shown that Ca2+ overload is another major mechanism of myocardial I/R injury. Sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) is a key protein involved in Ca2+ uptake from the cytosol into the SR and its dysfunction impairs myocardial systolic/diastolic function. Our previous study found that Lut improved SERCA2a activity partially through activation of the PI3K/AKT signaling pathway and increased the phosphorylation of AKT (p-Akt) at amino acids 308 and 473 levels, but did not change the SERCA2a expression at protein levels. The infarct size, pro-apoptosis proteins, and lactate dehydrogenase release (LDH) release were reduced by Lut during I/R injury in vivo, and the same effects were seen in diabetic rats.

6Heme oxygenase-1 (HO-1) is an integral membrane protein located in the smooth endoplasmic reticulum. It is under oxidant-responsive transcription factors NF-E2-related factor 2 (Nrf2) regulation, a pathway that promotes cytoprotection against oxidative stress. When Kelch-like ECH-associated protein 1(Keap1) binds Nrf2, transcriptional activation of Nrf2 is repressed. After exposure to oxidative stress, Nrf2 is released from Keap1 and translocates into nuclei, binding to the antioxidant responsive elements (ARE), which in turn activates Nrf2 target gene HO-1. It appears, therefore, that blocking the oxidative stress through the HO-1 is another possible cytoprotective mechanism by which Lut ameliorates I/R injury. In addition, the anti-oxidative activity of Lut has been confirmed by many researchers. reported that Lut is the most effective of the five flavonoids isolated from Ixeris sonchifolia in regulating antioxidant activities. In a middle cerebral artery occlusion-induced ischemia rat model, 4 mg/kg Lut reduced the infarct area, protected neuronal cells from death and increased neuroprotective effects. HO-1 expression was significantly up-regulated by Lut pretreatment. They also found that the neuroprotective and anti-apoptotic effects of Lut were abolished by a selective HO-1 inhibitor, tin protoporphyrin IX. To clarify the exact mechanisms of cardiac-protection, used hydrogen peroxide (H2O2)-induced cytotoxic model in the H9c2 cell line to show that Lut provided an adaptive survival response via a mechanism that involved alleviating H2O2-derived oxidative cytotoxicity by increasing HO-1 expression, enhanced the binding affinity of Nrf2 to the ARE and preventing of apoptosis in H9c2 cells. Taken together, these results indicated the vital role of HO-1 on Lut-induced cardio-protective effects.

7Three members of the mitogen-activated protein kinase (MAPK) family are extracellular signal-regulated kinases (ERKs), C-Jun N terminal kinase/stress-activated protein kinases (JNK/SAPKs) and p38. Cardiomyocyte function can be regulated by MAPKs after I/R injury. reported that Lut reduced the JNK, ERK and p38 protein expression to which prevented apoptosis associated neuronal death in rat primary cortical cultures. also found Lut inhibited the ROS-activated MAPK pathway in myocardial I/R injury. In the cardiovascular system, our research group found that the ERK/PP1a/PLB/SERCA2a and JNK pathways participated in Lut mediated cardiac-protection both in vitro and in vivo after I/R. Lut or JNK inhibitor SP600125 significantly enhanced the contraction of cardiomyocytes, improved cardiac function, reduced infarct size and decreased LDH activity. It was noteworthy that, ERK1/2 was not activated during I/R, and ERK1/2 inhibitor PD98059 had no effect on the above indices. However, Lut pretreatment concomitantly increased the phosphorylation of ERK1/2 (p-ERK1/2) and the protective effects of Lut were abrogated by PD98059.

8The p38 is another downstream effector of the MAPK pathway. It has been demonstrated that inhibition of p38 alleviated cardiomyocytes I/R injury and afforded cardiac-protection. Our experiments also showed that p38 expression was increased during I/R process and that Lut decreased it. On the other hand, the p38 MAPK pathway inhibitor SB203580 and Lut inhibited the phosphorylation of PLB (p-PLB), hence increased the activity of SERCA2a, alleviated the calcium overload which promoted the recovery of the mitochondrial membrane potential (Δψm) and reduced the calcium overload-induced apoptosis in I/R injury.

9The isozymes of nitric oxide (NO) synthase have been identified in humans: isozyme I (nNOS) that mainly exist in neuronal and epithelial cells, isozyme II (iNOS) that is primarily found in cytokine-induced cells, and isozyme III (eNOS) that is mainly expressed in endothelial cells. NO and NO synthases isozymes closely interact with superoxide radicals. In rats I/R injury model, the cardiac-protective capacity of Lut is partly mediated by down-regulation of NO production and its antioxidant activities. detected that pretreatment with Lut decreased the plasma NO levels so as to prevent NO from interacting with superoxide radicals. iNOS mRNA and protein level were down-regulated, but nNOS and eNOS expression levels were not significantly altered. On the contrary, another group found that, in diabetic patients and animals, the NO-eNOS pathway was down-regulated, and that Lut protected diabetic hearts against I/R injury by activating the myocardial eNOS pathway including the enhancing manganese superoxide dismutase (MnSOD) activity and inhibiting of mitochondrial permeability transition pore (mPTP) opening. Despite the fact that eNOS up-regulated MnSOD activity and increased mPTP opening, further studies are needed to elucidate the underlying regulatory mechanism.

10As a major complication in the CAD treatment I/R injury, I/R injury is associated with high mortality. The evidences reviewed above indicate that Lut is a promising anti-I/R agent that impacts a wide range of proteins that are involved in the protective pathways during I/R injury. It is clear that Lut works on AKT and MAPKs to alleviate cardiomyocyte apoptosis and to improve cardiomyocyte function, respectively. Lut also blocks oxidative stress and prevents injuries arising from free radicals through the HO-1 and NO signaling pathways as illustrated in Table 1. The success of investigations on the mechanisms of I/R injury in CAD are based on successful modeling of I/R injury in vivo or sI/R in vitro. Previous I/R injury models in rats or mice were constructed by left anterior descending (LAD) ligation. The silk suture slipknot was used to ligate the distal 1/3 of LAD, and then the slipknot was released after 30 min of ischemia to allow for reperfusion of the myocardium. As an alternative to these classical methods, we opted for endovascular balloon occlusion (2.5 × 8 mm balloon size for mice and 3.5 × 15mm balloon size for rats) to induce I/R models. This outer balloon ligation method is more efficient and cause less damage to the tissue than conventional approaches. One limitation of the available studies on I/R models is that many studies were performed on non-cardiac cell lines such as H9C2 cells and HL-1 cells. These cell lines might not model cardiac cells with high fidelity and hence isolated cardiomyocytes using Langendorff system or enzyme digestion methods may be a more reliable choice to study I/R mechanism.

11Lut has not only been demonstrated to increase myocardial contractility during I/R injury, but also has received considerable attention from many researchers regarding its long-term application in HF treatment. reported that long-term Lut treatment of doxorubicin-induced HF in rats was able to improve the cardiac function, in part, by reversing ventricular remodeling. In diabetes mellitus-induced cardiac dysfunction, Lut attenuated myocardial oxidative stress and reversed the reduction of cardiac function. Studies that have explored the effectiveness and mechanisms of Lut in improving cardiac function in HF have mainly focused on its regulation of cardiomyocytes contractility, enhancement of autophagy in cardiomyocytes and limitation of cardiac remodeling (Figure 3).

12Calcium (Ca2+) recycling is a pivotal process for effective cardiomyocyte contraction and relaxation. SERCA2a is an ATP-dependent enzyme that regulates SR Ca2+ stores by pumping Ca2+ into the SR. It is important to modulate cardiac cytosolic Ca2+ levels. Prior investigations have revealed that the expression and activity of SERCA2a were altered in many heart diseases, such as HF and cardiomyopathies. SERCA2a dysfunction causes Ca2+recycling disorders resulting in severe impairment of cardiomyocyte systolic and diastolic functions. SERCA2a has become a promising target of pharmacological or gene therapy in HF. Our previous work indicated that Lut improved the contractility of cardiomyocytes by modulatingSERCA2a during I/R injury. In a HF rat model, we also found that Lut improved cardiac dysfunction and significantly increased SERCA2a expression, activity and stability, which improved Ca2+ transient and cardiomyocyte contractility. The levels of AKT, phospholamban (PLB), and specificity protein 1 (Sp1), which are capable of upregulating SERCA2a expression and activity, were also increased in the study. Based on another study, small ubiquitin-related modifier (SUMO) 1, a post-translationally modified protein that can enhance SERCA2a activity is concurrently increased by Lut. To confirm the function of SERCA2a in Lut-mediated cardiac protection, inhibition of SERCA2a activity markedly abolished Lut-induced benefits, and inhibition of PI3K/AKT and the MAPKs pathway (ERK, p38) produced the same results.

13Autophagy is a self-process cell constituents through the lysosomal degradative pathway. This physiological process is essential for protecting cells against excessive or dysfunctional organelles and for maintaining cellular homoeostasis. In HF or ischemic cardiomyopathy disease, autophagy acts as a self-protective mechanism in response to stress. Inefficient autophagy leads to poor myocardial performance. Mammalian sterile 20-like kinase 1 (Mst 1) inhibits autophagy by impairing protein quality control mechanisms in the heart cells. reported that Lut up-regulated autophagy through Mst1 inhibition which alleviated post-infarction cardiac dysfunction in the Lut pretreatment group, enhanced autophagic flux, and lessened the aggresomes accumulation in neonatal cardiomyocytes in hypoxia model. Furthermore, in Mst1+/- murine model, the protective effects of Lut were abolished. Taken together, the interaction between Lut and Mst1 is a crucial pathway that regulates autophagy in cardiac cells. These findings are important as autophagy is a hallmark of many CVD. Hence, how Lut impacts autophagy at different stages awaits further investigation in order to better exploit its cardiac benefits.

14Cardiac remodeling is a pathological process associated with HF. It is characterized by neurohormonal activation, compromised the heart’s response to a hemodynamic load and/or injury and alteration to the structure (dimensions, mass, and shape). Cardiac fibrosis and hypertrophy are two types of remodeling patterns, which impair cardiac function and metabolism over time. Oxidative stress has been confirmed as a risk factor for cardiac remodeling. Anti-oxidative compounds may have protective effects against cardiac remodeling. Blockade of the Ang II-TGFβ1 signaling pathway has been demonstrated as an effective strategy to prevent cardiac fibrosis in animal models.

15Lut has been reported as an anti-fibrotic compound in the lung and liver. The renin-angiotensin system (RAS) participates in the pathological process of cardiac remodeling; Ang II is the initial factor of RAS and the pro-fibrotic activator of cardiac fibroblasts. It stimulates pro-fibrotic cytokine transforming growth factor β (TGFβ1) expression. clearly demonstrated that in Ang II-induced hypertension rats, TGF β1, CTGF, Nox2, and Nox4 gene expression were increased and oral pretreatment with a high-luteolin diet restored the above indices, and also suppressed H2O2-induced TGFβ1 expression. Food-derived Lut may be a therapeutic strategy through the inhibition of Ang II-induced cardiac remodeling.

16According to these reports, Lut appears to improve myocardial contractility by increasing the expression and function of SERCA2a, up-regulating autophagy through Mst1 inhibition, and preventing cardiac fibrosis by blocking the Ang II-TGFβ1 signaling pathway (Table 2). Moremore, different in vitro and in vivo models have been constructed using different Lut working concentrations; the optimal concentration of Lut should be defined by cytotoxic effect test and cell function detection. For example, in isolated ventricular myocytes, the shortening amplitude of single cardiomyocytes was detected after pretreating the myocytes with different Lut concentrations (4, 8, 16 μM). The maximum cell shortening was recorded at the concentration of 8 μM and without affecting cellular activities. This concentration of Lut is appropriate for application in isolated ventricular myocytes for HF research.

17Atherosclerosis is a chronic inflammatory artery disorder that can affect coronary arteries, the peripheral vasculature, and cerebrovascular structures. Genetic, environmental, psychosocial and pathological factors cause may contribute to the onset of this multi-stage, multi-factorial disease. The pathogenesis of AS comprises lipid accumulation, endothelial dysfunction, cell proliferation and migration, and inflammation. Lut has been demonstrated in many studies to have anti-inflammatory effects, and to inhibit cell proliferation and migration (;; Figure 4).

18AKT is essential for vascular smooth muscle cell (VSMC) proliferation and migration. Ablation of AKT caused severe atherosclerotic plaques. Using MTT and transwell chamber assays, Ang II-induced VSMC proliferation and migration were inhibited, and the levels of p-AKT (308) and p-AKT (473) levels were decreased in the Lut group compared to the Ang II group. These findings strongly suggest that Lut down-regulates the AKT signaling pathway and inhibits the proliferation and migration of VSMCs.

19The Src kinase signaling pathway is involved in the progression of AS in VSMC proliferation and migration. It also participates in reactive oxygen species (ROS)-induced pathophysiological processes of VSMCs. H2O2 is one type of ROS, which can elicit migration of VSMCs. Lut can block H2O2-triggered Src phosphorylation. It was shown that activation of Src in the Lut group was significantly lower compared with that of the H2O2 group. These results indicated that Lut can suppress H2O2-directed migration and proliferation in VSMCs. Furthermore, via blockade of Src and Syk, Lut suppressed nuclear translocation of NF-κB but did not suppress other MAPKs. In comparison with wild type and point mutant Src, Lut strongly suppressed the phosphorylation of Src, and ATP could compete with Lut at the ATP–binding pocket of Src. When the concentration of ATP was increased from 400 to 800 μg, the suppression of Src by Lut was abrogated, suggesting that the ATP-binding pocket may work as a binding site of Lut in Src.

20The pathogenesis of AS is also linked to a chronic inflammatory processes. mRNA stability is known to regulate inflammatory gene expression to impact inflammatory processes. Tristetraprolin (TTP) is regarded as a substrate of p38 MAPK-activated protein kinase 2 (MK2), which can directly target mRNA to the exosome for rapid degradation by binding to adenine-uridine-rich elements (AREs) in the 3′ UTR of target mRNAs. A previous study reported that activation of p38/MK2 pathway can abolish TTP-mediated repression of IL-6 3′-UTR reporter activity. However, it remained elusive whether mRNA stability is involved in the anti-AS mechanism of Lut. first found that Lut, in a dose-dependent manner, suppressed the expression of” tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in macrophages. TTP expression was enhanced in the Lut group, but another RNA binding protein, HuR, was not significantly altered. To examine whether Lut affects TNF-α and IL-6 mRNA stability through TTP activation, we compared the Lut and LPS groups and found that by promoting TTP expression, Lut inhibited the p38 and MK2 phosphorylation level.

21Oxidative stress is regarded as a contributing factor during the AS disease progression. NOX4 is widely expressed in the heart, and it is the major source of ROS generation. reported that Lut may be an effective agent for preventing AS by protecting ECs from TNF-α-induced inflammatory responses and oxidative damage. They found that Lut suppressed TNF-α-induced ROS generation, and it decreased NOX4, p22phoxexpression. In turn, down-regulated NOX4 reduced the expression of BCL-2, ICAM-1, and VCAM-1. They proposed the formation of a ternary complex involving NOX4 and ROS with NF-κB that regulated TNF-α-induced oxidative stress. This hypothesis was based on the following evidences: upon exposure to TNF-α in ECs, NF-κB inhibitor pyrrolidinedithiocarbamic acid (PDTC) inhibited Nox4 expression, while Nox inhibitor diphenyleneiodonium (DPI) deceased NF-κB activation. Moreover, the DCF-detected ROS fluorescence intensity induced by TNF-α are both attenuated by PDTC and DPI. However, the interactions between and structures of the three constituents are needed to be confirmed in the further.

22Anti-atherosclerosis properties of Lut are shown in Table 3. Lut inhibits the proliferation and migration of VSMCs by regulating AKT and Src, and decreases inflammation and oxidative damage through TTP activation related to mRNA stability and NOX4/ROS-NF-κB pathways. Inflammation and oxidative stress are the main accelerating factors of AS. As the capacities of Lut may be applied to prevent cardiovascular system from AS formation, especially in the early stage of AS or unstable plaques, well-designed studies are needed to explore therapeutic effects of Lut in different stages of AS and different types of plaques.

23A number of current studies seek to explore ways to reduce the incidence of CVD and improve the therapeutic effectiveness. This review summarizes the molecular mechanisms and cardiovascular effects of Lut and its role in HF, I/R injury, and AS. Lut alleviates cardiomyocyte apoptosis and blocks oxidative stress, all of which promote cytoprotection and reduce I/R injury. The data sets reviewed also provide evidence that Lut improves heart function by enhancing contractility, up-regulating autophagy, and preventing cardiac fibrosis. The anti-atherosclerosis properties of Lut stems from inhibiting the proliferation and migration of VSMCs, decreasing inflammation and ameliorating oxidative damage. Overall, Lut, which is a ubiquitous dietary flavonoid, is efficacious in cardiovascular protection, and shows promise as a preventive and treatment option for CVD.

24Although multiple mechanisms of the protective function of Lut on the heart have been addressed, limitations exist in the following aspects. First, previous studies mainly focused on basic research. Therefore, we lack large clinical trials designed to explore the clinical utilization of Lut, especially on the effective therapeutic dosage, and the safety of long-term administration. Based on the evidences of dosages in observational clinical trials or experimental animal studies, the human dietary intake of Lut appears to be much smaller than that used for rodent experimental studies, this result implies that intake of Lut from food may work as a preventative method for CVD, while this intake is insufficient to be an treatment dosage of CVD. Therefore, well-designed prospective clinical trials to observe the effective therapeutic dosages of Lut on patients with myocardium I/R injury, HF, and AS would greatly advance knowledge on its clinical application. In an open-label pilot study, Lut effectively reduced autism spectrum disorders symptoms on behavior in children, which was based on the antioxidant, anti-inflammatory, and neuroprotective effects of Lut. Major adverse effects had not been observed in children who took 10 mg/kg of Lut (ClinicalTrials.gov identifier: NCT01847521). Although it is not a cardiovascular clinical research, 10mg/kg may be used as a reference dosage of safety in future CVD trials. Second, in-depth studies of mechanism need to focus on the synthetic function of Lut on its target effectors and signaling pathways. Further in vitro and in vivo studies should aim to identify the relationship and interaction of these effectors. We hope this review generates the scientific awareness and interest in Lut and provokes that further research by scientists from different fields would boost understanding of the molecular functions of Lut. In conclusion, extensive studies in this area are needed, and appreciation of the molecular mechanisms of Lut may pave the way for development of Lut-based therapeutic agents for CVD.

25DL and YL outlined the review. YL drafted the manuscript. YL and PS edited and reviewed the manuscript. DL, YL, and PS re-reviewed it and all authors approved it.

26The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.