A systematic search of four databases (Pubmed, Web of Science, Embase, and Cochrane Library) was conducted to identify PEs that have been validated in animal studies or clinical studies for interventions in MIRI.

Four databases (Pubmed, Web of Science, Embase, and Cochrane Library) were searched using “myocardial ischemia-reperfusion injury”, “myocardial reperfusion injury”, and the PE retrieved in 2.1 as keywords. Then manually search and add any literature that may have been missed. Timespan: January 2016 - September 2021. Only published articles written in the English language were considered in the current meta-analysis. The specific search process was shown in Figure 1.

The following criteria were set in advance. Inclusion criteria: 1) Rats or mice were used as research subjects; 2) methods for establishing animal models of MIRI: ligation and loosening surgery of the left anterior descending branch of coronary artery (in vivo), or Langendroff perfusion (ex vivo); 3) the treatment group received any dose of PE, while the control group received vehicle or no treatment; 4) The myocardial infarction size was taken as the main outcome index, with or without other indexes such as myocardial enzymes, heart rate (HR), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS); 5) the size of the myocardial infarction is reported as a percentage (i.e. TTC staining or Evan’s blue/TTC staining). Exclusion criteria: 1) animals with other cardiovascular comorbidities (e.g. diabetes, hyperlipidemia); 2) animals are treated with PE analogs or given additional drugs. 3) Studies with incomplete and inaccessible data; 4) duplicate publications.

Two authors (YM W and XT S) extracted the study characteristics independently, and discrepancies were resolved by the corresponding author. Information extracted included: first author, year of publication, anesthetics, animal information (animal species, number, sex, and weight), I/R duration, interventions (PE type, dose, method of administration) for the treatment and control groups, outcome indicators, and staining method of infarct size. The data were pooled using the formula as follows when different doses of pharmacological interventions were employed in the treatment group (Zhang and Chen, 2016). When data are represented graphically, every effort will be made to contact the author for more information or to measure from the graph. ∑ i = 1 m ( n i − 1 ) S D i 2 + ∑ i = 1 m n i ( x ¯ i − x ¯ T ) 2 ∑ i = 1 m ( n i − 1 )

The SYRCLE’s RoB tool (Hooijmans et al., 2014) was used to evaluate the quality of the studies by two authors independently. The SYRCLE’s RoB tool contains 10 entries: sequence generation, baseline characteristics, allocation concealment, random housing, blinding, random outcome assessment, blinding, incomplete outcome data, selective outcome reporting, and other sources of bias. A third person ruled in case of disagreement.

Data were analyzed by STATA 16.0 software. Outcomes were expressed as standardized mean difference (SMD) with a 95% confidence interval (95%CI). In the forest plot, the dark squares represent the standardized mean difference (SMD) for each study, the diamonds represent the pooled SMD, and 95% of the CIs are indicated by lines. p values ≤0.05 were considered statistically significant. Statistics were analyzed using a fixed-effects model (I² ≤ 50%, p ≥ 0.10) or a random-effects model (I² > 50%, p < 0.10). Sensitivity analysis, stratification analysis, or univariable meta-regression should be performed to deal with high heterogeneity if necessary. We performed sensitivity analysis by removing each study in turn to assess the impact of this study. We conducted a stratified analysis and meta-regression of myocardial infarction size by study type, route of administration, staining method, PE type, animal species, and anesthetics type. The Egger’s test and Begg’s test for publication bias were performed using STATA 16.0.

The following PEs were retrieved that have been experimentally proven to be effective against MIRI. They are divided into four main categories: 1) Isoflavones: genistein, quercetin, biochanin A, formononetin, daidzein, kaempferol, icariin, puerarin, rutin, et al.; 2) Terpenoids: notoginsenoside R1, tanshinone IIA, ginsenoside Rb1, ginsenoside Rb3, ginsenoside Rg1, ginsenoside Re, et al.; 3) stilbenoids: resveratrol, polydatin, et al.; 4) miscellaneous classes: bakuchiol. We collected the chemical structure of these PEs and the representative herbs. The specific information is shown in Table 1.

A total of 337 articles were obtained from various databases. After removing duplicate articles, we reviewed the abstracts and full text sequentially according to strict inclusion and exclusion criteria. Finally, a total of 20 studies from 17 articles (Li et al., 2016b; Li et al., 2016c; Gu et al., 2016; Ling et al., 2016; Liu et al., 2016; Yang et al., 2016; Cui et al., 2017; Wang et al., 2017; Li et al., 2018; Lin et al., 2018; Wu et al., 2018; Bai et al., 2019; Li et al., 2020a; Wang et al., 2020a; Wang et al., 2020b; Kazemirad and Kazerani, 2020; Jiang et al., 2021) were included in our analysis (Figure 1).

The characteristics of included 20 studies are provided in Table 2. The main PEs involved are genistein (n = 1), quercetin (n = 1), biochanin A (n = 1), formononetin (n = 1), kaempferol (n = 1), icariin (n = 2), puerarin (n = 1), rutin (n = 1), tanshinone IIA (n = 1), ginsenoside Rb1 (n = 6), ginsenoside Rg1 (n = 1), resveratrol (n = 2), and polydatin (n = 1). A total of 17 studies were conducted in vivo using ligation followed by the release of LAD to simulate MIRI, and the remaining three studies were conducted ex vivo using Langendroff perfusion. The author Wu B conducted both in vivo and ex vivo studies, which we labeled and distinguished with “Wu B 2018 (1)” and “Wu B 2018 (2).” All studies used infarct size as the primary efficacy index, with 12 studies using TTC single staining and 8 using Evan’s blue/TTC double staining. A total of six kinds of anesthetics were used in the studies included in this analysis: pentobarbital sodium (n = 12), thiopental sodium (n = 1), urethane (n = 1), chloral hydrate (n = 2), isoflurane (n = 3), and a mixture of xylazine and ketamine (n = 1). A total of 355 animals were enrolled for our study, including SD rats, Wistar rats, and C57BL/6J mice. Several studies have included markers of myocardial injury such as levels of cardiac troponin I (cTnI) (n = 5) and creatine kinase (CK) (n = 2) as outcome indicators in their analyses. Seven studies performed echocardiography on animals and reported LVEF and LVFS. All articles were written in English. Only one study is from Iran, the rest were conducted in China.

We used the SYRCLE’s RoB tool to score the quality of each study. Table 3 shows the information on methodological quality. As shown in Table 3, six studies scored five points, and four studies scored only two points, which indicate reliable data but lower quality of studies. Ten studies mentioned randomized groupings, but all were silent on the specific randomization method. All studies did not describe how allocation concealment is performed.

A total of 355 animals were included in our study, 222 of which were treated with PE. As a class of cardioprotective agent, PEs exerted significant anti-MIRI effects in animal models, mainly in reducing infarct size (p < 0.001), mitigating myocardial injury (p < 0.001), improving cardiac function (p < 0.001) and lowering heart rate (p < 0.001). Moreover, this cardioprotective effect was not limited by factors such as phytoestrogen species, rodent species, methods of I/R, I/R duration, or anesthetics, which were found by stratified analysis, sensitivity analysis, and regression analysis of different outcome indicators.

Prior to conducting a clinical trial, animal models can be used to determine the effectiveness of a drug or procedure and to explore its mechanisms. Through a comprehensive search of various databases, the specific mechanisms of PE against MIRI can be summarized as follows.

1. Mitochondrial pathway. The primary mechanism we found is a disturbance in mitochondrial structure, function, and quantity. Mitochondrial structure and function are impaired by ischemia and aggravated by reperfusion (Giedt et al., 2012; Consolini et al., 2017; Ham and Raju, 2017; Anzell et al., 2018). MIRI causes impairment of the mitochondrial respiratory chain leading to abnormal energy metabolism (Leist et al., 1997; Tahrir et al., 2019), and also causes excessive accumulation of reactive oxygen species (ROS) producing oxidative stress (Li and Jackson, 2002; Lesnefsky et al., 2017). PE can suppress mitochondrial swelling and reduce the number of fragmented mitochondria in terms of retaining mitochondrial structure (Zhai et al., 2001). As for the involvement in energy metabolism, PEs has been shown to inhibit the activation RhoA/ROCK signaling (He et al., 2014; Cui et al., 2017; Yan et al., 2021) as well as activate the mK_ATP channels ((Colareda et al., 2020), (Colareda et al., 2021)), thus promote ATP production. PE also directly or indirectly regulates the activity of mitochondrial complex. For example, PE has been found not only to reduce NADH dehydrogenase activity and inhibit mitochondrial complex I activity, but also to maintain mitochondrial complex V activity. (Jiang et al., 2021; Yan et al., 2021). Meanwhile, PE can further ameliorate oxidative stress injury in cardiomyocytes by regulating silent information regulator 1 (SIRT1) and thus downstream targets such as peroxlsome proliferator-activated receptor-γ coactlvator-1α (PGC-1α) (Feng et al., 2016; Zhong et al., 2019) and forkhead box O 1 (FOXO1) (Wu et al., 2018). Furthermore, it has been suggested that PE’s effect on mitochondrial function is dosage-dependent (Barton, 2013). It works as an antioxidant and enhances mitochondrial biogenesis at low dosages, but it also functions as a pro-oxidant and impairs mitochondrial function at high dosages (Roca et al., 2014). For example, Genistein has been reported to promote cell proliferation at low concentrations (0.1–10M) and inhibit cell proliferation at high concentrations (above 10M) (Matsumura et al., 2005; Seo et al., 2006).

In summary, in addition to their estrogen-like cardioprotective effects as hormone replacement therapy, PEs also act through other pathways unrelated to estrogen. PE can maintain mitochondrial function and structure, alleviate oxidative stress, improve Inflammation and immune responses, and mitigate calcium overload by regulating multiple signaling pathways. The signaling pathways involved are engaged in a variety of cross-talk. The mechanisms involved are equally interactive and causal (Figure8).