Cardioprotective effect of epigallocatechin gallate in myocardial ischemia/reperfusion injury and myocardial infarction: a meta-analysis in preclinical animal studies

This meta-analysis aims to determine the efficacy of Epigallocatechin gallate (EGCG) in the treatment of myocardial ischemia–reperfusion injury (MIRI) and summarize the mechanisms involved. Literature from six databases including Web of Science, PubMed, Embase, China National Knowledge Infrastructure (CNKI), Wan-Fang database, and VIP database (VIP) were systematically searched. All the analysis were conducted by R. Twenty-five eligible studies involving 443 animals were included in this meta-analysis. The results indicated that compared to controls, EGCG exerts a cardioprotective effect by reducing myocardial infarct size (SMD = −4.06; 95% CI: −5.17, −2.94; P < 0.01; I2 = 77%). The funnel plot revealed publication bias. Moreover, EGCG significantly improves cardiac function, serum myocardial injury enzyme, and oxidative stress levels in MIRI animal models. This meta-analysis demonstrates that EGCG exhibits therapeutic promise in animal models of MIRI. However, further validation is still needed in large animal models and large clinical studies.

The methodological quality of the included studies.The quality score of the included studies ranged from 3 to 7. Three studies scored 4 points, nine studies scored 5 points, and 11 studies scored 6 points.The remaining two studies scored 3 and 7, respectively.All studies were published in peer-reviewed journals.All animals were randomly allocated to the treatment or control groups with appropriate animal models (aged, diabetic, or hypertensive).However, no study describes the blinded induction of the model and sample size calculation.Blinded assessment of outcome was described in two studies.Nine studies mentioned control of temperature.A total of 21 studies used anesthetic without significant intrinsic cardioprotective activity.Animals in 21 studies complied with animal welfare regulations.Only five studies included a statement of potential conflict of interest.The methodological quality of the included studies is shown in Table 2.The molecular mechanism of included studies that EGCG protects cardiomyocytes from MIRI was summarized in Table 3.
We conducted subgroup analysis to explore the sources of heterogeneity further.As shown in Table 4, there are significant differences in the analysis between subgroups of species, drug delivery method, and duration, which may be an important source of heterogeneity.

Molecular mechanisms.
Numerous studies have shown that MIRI is closely associated with various pathological processes such as inflammation, oxidation stress, cardiomyocyte apoptosis, autophagy, etc 40 .An indepth understanding of the underlying mechanisms of EGCG will serve to better understand the cardioprotective effects of EGCG.Therefore, we summarize the potential mechanisms involved in the myocardial protective effects of EGCG as follows: Anti-inflammation.During MIRI, necrotic cardiomyocytes activate inflammatory responses and exacerbate myocardial injury by inducing oxidative stress, triggering the complement cascade, and releasing dangerassociated molecular patterns (DAMPs).Myeloperoxidase (MPO) is a neutrophil-specific heme peroxidase that is closely related to the inflammatory process of ischemia-reperfusion injury 41 .EGCG plays an anti-inflammatory role by reducing MPO activity and the infiltration of neutrophils 17 .Mitochondrial DNA (mtDNA), a naked circular or linear DNA, has been identified as a damage-associated molecular pattern (DAMP) that can trigger a series of inflammatory responses 42,43 .In MIRI, the expression of mtDNA was positively correlated with the expression of TNF-a, IL-6, and IL-8 44 .The PI3K/Akt pathway reduces myocardial injury by negatively regulating the inflammatory response 45 .The activation of the PI3K/Akt pathway is a critical pathway of cardioprotective and is associated with preservation of mitochondrial function during MIRI 46 .EGCG inhibits inflammation response to reduce myocardial IS by activating the PI3K/Akt pathway to www.nature.com/scientificreports/reduce the release of pro-inflammatory mediator mtDNA and the secretion of inflammatory factors in plasma 28 .
In addition, EGCG exerts anti-inflammatory effects and protects cardiomyocytes by inhibiting IKK/NF-kB and c-Jun/AP-1 pathways 17 .
Antioxidant.Oxidative stress caused by increased reactive oxygen species (ROS) is one of the main pathological mechanisms for the occurrence and development of MIRI.Due to the presence of multiple phenolic hydroxyl groups that are easily oxidized to quinones in the structure, EGCG exerts powerful antioxidant properties in the treatment of MIRI by regulating the redox system to scavenge free radicals and inhibiting lipid peroxidation, thereby alleviating myocardial damage 47,48 .Specifically, EGCG plays an antioxidant effect by significantly increasing the endogenous antioxidant activity of antioxidant enzymes (SOD, CAT, GPx, GRx, GST) and antioxidants (vitamin C, vitamin E, ceruloplasmin), and inhibiting the accumulation of MDA, thiobarbituric acid reactive substances, and uric acid 26,49 .SIRT1 (Sirtuin 1) is a nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase involved in regulating oxidative stress resistance under ischemic and hypoxic conditions 50 .In diabetic MIRI rats, EGCG increases the expression of the antioxidant enzyme manganese superoxide dismutase (MnSOD) by activating SIRT1 and reducing oxidative stress damage 33 .Calcium overload is a major cause of cellular damage during MIRI 51 .Activation of KATP channels protects cardiomyocytes from MIRI-induced Ca 2+ overload 52 .It has been found that the reduction in infarct size produced by myocardial ischemic preconditioning and pre-ischemic drug therapy is partly attributed to mitochondrial potassium ATP (mKATP) channels 53,54 .In addition, in Ossabaw minipigs, activating KATP channels by the ischemic preconditioning (IPC) process can delay the magnitude of ST-segment elevation 55 , further suggesting that opening mitochondrial KATP channels can play a cardioprotective role.During myocardial ischemia in isolated mice hearts, EGCG opens mKATP channels by activating A1 and A2B adenosine receptors to relieve Ca+ overload pressure and increase NAD+ expression, reduce the opening time of mitochondrial permeability transition (mPTP) pore and finally reduce the amount of ROS 14,23,30,56 .

Anti-apoptosis.
Apoptosis, as one of the critical mechanisms in the MIRI process, plays a dual role in MIRI according to the degree of apoptosis.On the one hand, proper apoptosis reduces myocardial cell necrosis In the MIRI model, EGCG alleviated cardiomyocyte apoptosis/exerts antiapoptotic effects by up-regulating the expression of anti-apoptotic protein Bcl-2, down-regulating the expression of pro-apoptotic proteins p53, Bax, cleaved-caspase-3 and caspase-9 26,27 .Existing evidence has identified the PI3K/Akt signaling pathway played a protective role in protecting cardiomyocytes from the two aspects of promoting the survival of cardiomyocytes and inhibiting the apoptosis of cardiomyocytes by regulating the expression of apoptotic proteins and transcription factors 57 .In MIRI models, EGCG increases the survival rate of cardiomyocytes by activating PI3K/Akt signaling pathway, promoting the phosphorylation of eNOS, and increasing the NO content 35 .JNK and p38 are members of the mitogen-activated protein kinase (MAPK) family, and the JNK/p38 MAPK cascade plays a pivotal role in mediating apoptosis.EGCG inhibits p38 and JNK phosphorylation, decreases Caspase-3 expression, plays an antiapoptotic role in cardiomyocytes, and reduces the size of myocardial infarction 22,38 .STAT-1 is a signal transducer and transcriptional activator, and its phosphorylation can induce apoptosis by mediating the expression of apoptosis-related genes 58 .EGCG is an effective inhibitor of STAT1 phosphorylation, which can reduce the expression of caspase-3 and the degree of myocardial cell apoptosis by inhibiting the activation of the STAT-1/Fas pathway 59 .miR-30, a member of the MicroRNA family, regulates cell apoptosis by targeting the inhibition of the mitochondrial apoptosis activator p53 60 .In hypoxia-reoxygenation (H/R)-induced H9C2  www.nature.com/scientificreports/cells and I/R-induced rats, EGCG can inhibit mPTP opening and anti-apoptotic protein expression by activating the miR-30a/p53 signaling pathway 15 .
Autophagy.Autophagy plays opposite roles in different periods of myocardial injury.Autophagy in ischemia can supply energy and play a role in cardiac protection, while in reperfusion its excessive activation accelerates cardiomyocyte death 61 .During ischemia, EGCG trigger autophagy to protect cells from apoptosis by regulating mTOR negative feedback mechanism 24 .During reperfusion, EGCG protects against MIRI by activating PI3K/ Akt/mTOR signaling cascade, reducing Beclin-1 expression, and restoring autophagy flux to inhibit excessive autophagy 24,35 .Recent studies have shown that long non-coding RNA GM4419 can alleviate myocardial infarction by activating miR-682/TRF3 in I/R and H/R-induced myocardial injury models 62 .EGCG reduces the degree of myocardial injury by reducing Gm4419 expression and epigenetic silencing/inhibiting DUSP5/ERK1/2 signaling pathway-mediated autophagy in H 2 O 2 -induced cardiomyocytes and I/R-induced mouse models 63 .ATG4C, a cysteine enzyme, is involved in autophagy by regulating the functions of LC3 and ATG8 64,65 .EGCG can increase ATG4C expression and decrease LC3II expression, thus inhibiting H/R-induced apoptosis and autophagy of H9C2 cells 66 .In atherosclerotic diseases, miR-384 has been found to target and inhibit Beclin-1 to suppress macrophage autophagy 27 .In MIRI, EGCG can inhibit autophagy and alleviate the injury by activating PI3K/Akt pathway, increasing miR-384, and decreasing Beclin1 37 .EGCG can exert cardioprotective effects from three aspects of anti-inflammation, anti-apoptosis, and autophagy by regulating the PI3K/Akt pathway, suggesting an important role of the PI3K/Akt pathway in the cardioprotective process of EGCG.
Antiplatelet effect.During myocardial infarction, platelets play a dual role of promoting arterial thrombosis leading to cardiac injury and regulating cardiomyocyte secretion of factors leading to cardioprotection 67,68 .It has been found that EGCG can inhibit platelet aggregation induced by U46619, collagen, arachidonic acid, and toxic carotenoids and shear force-induced platelet adhesion dose-dependently by suppressing PLCγ2 and tyrosine phosphorylation of various platelet proteins, up-regulating the expression of intracellular PGD2, blocking the increase of intracytoplasmic free calcium ions and reducing the release of arachidonic acid (AA), thus delaying the formation of arterial thrombus and exerting potent antithrombotic effects.What's more, its combination with common antiplatelet therapeutic agents, aspirin (ASA), clopidogrel (CPD), and tiglitazarol (TCG), did not further inhibit platelet aggregation resulting in bleeding complications, demonstrating the potent antiplatelet www.nature.com/scientificreports/platelet activation by inhibiting microsomal cyclooxygenase-1 activity in platelets as well as platelet extracellular vesicle release 72,73 .During MIRI, platelets can exacerbate ischemia/reperfusion (IR) injury by promoting thrombosis, decreasing myocardial perfusion, secreting vasoconstrictors, and causing endothelial dysfunction 67,68 .Therefore, we speculated that EGCG may exert cardioprotective effects during MIRI by inhibiting platelet aggregation and thrombosis, but further experimental validation is needed.

Coronary microcirculation.
Myocardium ischemia-reperfusion inevitably leads to myocardial cell and coronary microvascular injury.Among them, coronary microvascular injury includes various mechanisms such as coronary microembolism (CME), platelet activation, endothelial dysfunction, and increased permeability, which eventually lead to capillary injury, complications of no-reflow, intramyocardial hemorrhage, and adverse  www.nature.com/scientificreports/microvascular obstruction, which has been identified as one of the key factors affecting the prognosis of patients with acute myocardial infarction [74][75][76] .Coronary microcirculation has increasingly become an effective target for cardioprotection during the treatment of acute myocardial infarction.Cardiomyocyte apoptosis and myocardial inflammation induced by coronary microembolism, a common complication during ACS and PCI treatment, are considered to be one of the main mechanisms of myocardial injury and cardiac dysfunction 77 .Inhibition of cardiomyocyte apoptosis and myocardial inflammation attenuated CME-induced myocardial injury and improved cardiac dysfunction 78 .Although there is no report of EGCG improving coronary microcirculation by treating coronary microembolism, other flavonoids such as curcumin (CCM) have been shown to inhibit CMEinduced myocardial inflammation and cardiomyocyte apoptosis through the TLR4/MyD88/NF-kβ signaling axis 79 , implying that EGCG may also have the potential of improving coronary microcirculation, but the specific research still needs to be further studied.In addition, a study based on four different structures of theaflavins (TFs) found that chemicals containing only a single galloyl group improved coronary microcirculation, while none or two galloyl groups chemicals were ineffective 80 .Since the chemical structure of EGCG happens to contain a single galloyl group, we speculate that EGCG may also have the effect of improving coronary microcirculation.In addition, two clinical trials have found that EGCG supplementation improves endothelial dysfunction in the short term during the treatment of patients with coronary artery disease 81 .Therefore, we speculated that EGCG might also have the capability to improve coronary microcirculation according to both the effects of its structural analogs as well as its structural properties, but it remains to be further verified experimentally.

Clinical trial.
Although there is currently a dearth of clinical trials on the cardioprotective effects of EGCG against MIRI, several clinical trial studies have reported that EGCG protects against multiple aspects of a wide range of cardiovascular diseases.For example, two clinical studies found that EGCG reduced the risk of cardiovascular disease in obese subjects by significantly lowering plasma triglyceride levels, as well as blood  www.nature.com/scientificreports/pressure 82,83 .In normal male healthy volunteers, EGCG may delay the progression of oxidative stress-associated atherosclerotic disease by decreasing low-density lipoprotein (LDL) oxidizing capacity 84 .A clinical trial based on patients with mild to moderate hypertension also discovered that the administration of Benifuuki, whose active ingredient is an EGCG-O-methylated derivative, exerts a hypotensive effect by significantly inhibiting angiotensin I-converting enzyme activity 85 .Furthermore, in patients with amyloid transthyretin cardiomyopathy, EGCG may curb cardiac amyloidosis by reducing left ventricular myocardial mass 86,87 .In terms of antiplatelet drug therapy, EGCG was found to reduce adenosine diphosphate (ADP)-and collagen (COL)-induced platelet aggregation, as well as shear force-induced platelet adhesion dose-dependently, and its combination with common antiplatelet therapeutic agents, aspirin (ASA), clopidogrel (CPD), and tiglitazarol (TCG), did not further inhibit platelet aggregation resulting in bleeding complications, demonstrating the potent antiplatelet effect of EGCG and its favorable safety profile 71 .As for the effects of EGCG on endothelial dysfunction, clinical trials based on healthy subjects have shown that EGCG has no effect on improving endothelial dysfunction 88 ; whereas, in patients with atherosclerosis, EGCG can reduce the incidence of cardiovascular disease by improving endothelial function 89 ; and in patients with coronary artery disease, the acute administration of EGCG significantly reversed endothelial dysfunction in coronary patients in the short term.The ability of EGCG to improve endothelial dysfunction may be related to the subjects studied in the clinical trials, and EGCG may be effective in improving endothelial dysfunction in patients with cardiovascular disease in the short term, but not in healthy subjects, and the specific efficacy of EGCG remains to be further verified by trials in the future.The above clinical trial results suggest that EGCG may exert cardioprotective effects through multiple mechanisms, including lowering blood pressure, improving endothelial dysfunction, and inhibiting platelet aggregation and adhesion.
Implications.Reperfusion injury will inevitably lead to myocardial cell death and cardiac dysfunction during the treatment of acute myocardial infarction 90 , but until now there is no clinical intervention drug to improve MIRI 91 , so it is urgent to seek a new potential therapeutic drug to improve MIRI.Studies reported that EGCG reduces myocardial injury by reducing oxidative stress, inhibiting apoptosis and inflammatory response, and regulating autophagy and mitochondrial function during MIRI 47 .The results of this study showed that EGCG significantly reduces myocardial infarct size, improves cardiac function, down-regulates myocardial enzyme levels, and inhibits oxidative stress to play a cardioprotective role in MIRI animal models.
Studies have shown that meta-analysis and systematic review to evaluate the therapeutic efficacy of drugs for experimental animal research can help the translation of research results from animal experiments to clinical applications and narrow the gap between the two 92 .During the study inclusion process, we considered the relevance of the article to the research topic during the title and abstract selection process and the full-text review process and evaluated the quality of the article according to 10 criteria.These steps contributed to improving the stability and reliability of the results of this meta-analysis.However, the results of this study only used youthful and healthy small animal models and did not include aged small animal models with multiple coexisting diseases such as diabetes and hypertension as well as coadministration of medications, which may differ from the complex pathology of patients in the actual clinical setting.According to Improving the Preclinical Assessment of Cardioprotective Therapies (IMPACT) guidelines in the EU Cardiac Protection Cost Action Guidelines, the first step in reducing the risk of failure in the translation of preclinical research into clinical research requires the use of healthy, young animals for initial experiments, but in the future, small animal models with multiple confounding factors still need to be further verified 93 .This study only included young and healthy small animal models, so the actual efficacy of EGCG still needs to be further verified in small animal models with at least one confounding factor.In addition, the results of this study were not validated in large animal models, which are crucial for the translation of preclinical research to clinical research, because the anatomy and vascular dynamics of large animals, especially pigs, are much closer to the actual configuration of human beings so that the clinical model can better mimic that of MIRI under specific conditions, which can aide in clinical translation.Moreover, animal research itself has certain methodological flaws, design variations, and publication bias caused by the fact that negative results are more difficult to be published, further widening the gap between animal experiments and clinical applications 94 .Nevertheless, to further facilitate the translation of EGCG from animal studies to clinical practice, more small animal models with multiple confounding factors, high-quality large-scale animal studies, and randomized controlled trials are still needed for further discussion and validation.
Limitations.First, we only retrieved studies in Chinese and English databases, lacking studies in other language databases, which may cause a certain degree of selection bias.Secondly, negative results from drug studies are less likely to be published, which may lead to an overestimation of drug efficacy.Thirdly, all the animals included in the study did not adopt the disease model of myocardial injury and related comorbidities, such as advanced age, hypertension, hyperlipidemia, diabetes, etc., while patients with clinical myocardial injury often suffer from multiple diseases.

Conclusions
This meta-analysis demonstrates that EGCG exhibits therapeutic promise in animal models of MIRI.However, further validation is still needed in large animal models and large clinical studies.

Table 1 .
Baseline characteristics of included studies.

Table 2 .
Quality assessment of included studies.A, peer-reviewed publication; B, control of temperature; C, random allocation to treatment or control; D, blinded induction of model; E, blinded assessment of outcome; F, appropriate use of anesthetic; G, appropriate animal model; H, sample size calculation; I, compliance with animal welfare regulations; J, statement of potential conflict of interests.

Table 3 .
Molecular and cellular mechanisms of myocardial ischemia/reperfusion injury treated with epigallocatechin gallate.