Inhibition of Myocardial Cell Apoptosis Is Important Mechanism for Ginsenoside in the Limitation of Myocardial Ischemia/Reperfusion Injury

Ischemic heart disease has a high mortality, and the recommended therapy is reperfusion. Nevertheless, the restoration of blood flow to ischemic tissue leads to further damage, namely, myocardial ischemia/reperfusion injury (MIRI). Apoptosis is an essential pathogenic factor in MIRI, and ginsenosides are effective in inhibiting apoptosis and alleviating MIRI. Here, we reviewed published studies on the anti-apoptotic effects of ginsenosides and their mechanisms of action in improving MIRI. Each ginsenoside can regulate multiple pathways to protect the myocardium. Overall, the involved apoptotic pathways include the death receptor signaling pathway, mitochondria signaling pathway, PI3K/Akt signaling pathway, NF-κB signaling pathway, and MAPK signaling pathway. Ginsenosides, with diverse chemical structures, regulate different apoptotic pathways to relieve MIRI. Summarizing the effects and mechanisms of ginsenosides contributes to further mechanism research studies and structure–function relationship research studies, which can help the development of new drugs. Therefore, we expect that this review will highlight the importance of ginsenosides in improving MIRI via anti-apoptosis and provide references and suggestions for further research in this field.


INTRODUCTION
Ischemic heart disease (IHD) is characterized by insufficient blood flow to the cardiac tissue (Querio et al., 2021). In 2019, "The top 10 causes of death" presented that IHD was the world's biggest killer, responsible for 16% of total deaths worldwide (WHO, 2020). Myocardial blood flow blockage causes inflammatory reactions, energy metabolism disorders, micrangium damage, oxidative stress, calcium overload, and arrhythmia (Hao et al., 2021;Vilela and Fontes-Carvalho, 2021). Reperfusion therapy is the standard therapy for IHD; nevertheless, the restoration of blood flow to ischemic areas aggravates myocardial damage, namely, myocardial ischemia/reperfusion injury (MIRI) (Neri et al., 2017;Li et al., 2019). Evidence indicated that the death rate of acute myocardial infarction (AMI) patients treated with optimal reperfusion therapy was approximately 7% (Hausenloy and Yellon, 2016). MIRI involves multiple regulatory mechanisms, such as cell death, oxidative stress response, and mitochondrial dysfunction (Neri et al., 2015;Dong et al., 2019;Bugger and Pfeil, 2020). Apoptosis, which is a programmed cell death, is the critical factor in the development of MIRI (Charununtakorn et al., 2016). Apoptosis causes myocardial infarction, damages cardiac systolic/diastolic dysfunction and electrophysiological performance (Li CY. et al., 2020;Cai et al., 2020), and even leads to irreversible damage . Previous studies indicated that inhibiting apoptotic pathways could effectively alleviate MIRI (Zhai et al., 2017;Cai et al., 2020;Liao et al., 2021). Regulating apoptosis is a promising therapeutic strategy.
Ginsenosides are triterpenoid saponins, which are deemed as the main bioactive components of Panax ginseng (Sabouri-Rad et al., 2017). Panax means "all healing" in Greek (Im and Nah, 2013), and Panax ginseng has effects in improving arrhythmia, decreasing the myocardial ischemic area, suppressing oxidative stress response, enhancing immune regulation, and inhibiting apoptosis . Ginsenosides have positive effects on MIRI via regulating oxidative stress, inflammation, calcium overload, and cell deaths (Fan et al., 2020;. Previous evidence indicated that ginsenosides could improve myocardial cell (MC) apoptosis to promote cardiac functions and reduce infarct size in MIRI Li CY. et al., 2020). Ginsenosides are classified into three types: protopanaxadiol (PPD) type, oleanolic acid type, and protopanaxatriol (PPT) type . Ginsenosides inhibit MC apoptosis via different apoptotic pathways, owing to their distinct chemical structures (Kim et al., 2015).
In this review, PubMed, Embase, Web of Science, and China National Knowledge Infrastructure (CNKI) were searched from inception to 21 September 2021 by using the following terms: ginsenoside, myocardial reperfusion injury, etc. This research included and reviewed research studies addressing the antiapoptosis effects of ginsenosides on MIRI to provide references and suggestions for further research in this field.

EFFECTS AND MECHANISMS OF GINSENOSIDES ON MYOCARDIAL CELL APOPTOSIS IN MYOCARDIAL ISCHEMIA/ REPERFUSION INJURY
Panax ginseng, a medicinal plant, belongs to the Araliaceae family and has a long history of usage (Geng et al., 2010). The main active ingredients of Panax ginseng are ginsenosides , which inhibit oxidative stress, enhance immune regulation, promote physiological functions (Chen S. et al., 2019;, and are adopted to improve IHD, depression, diabetes, etc. (Zhang JH. et al., 2021;Jiang et al., 2021). Ginsenosides Rb1, Rb2, Rb3, Rd, Re, Rg1, Rg2, Rg3, Rh1, Rh2, Rh3, Rk3, and Rc were proved to alleviate MIRI. The chemical structures of ginsenosides determine their pharmacological effects, especially hydroxyl groups and sugar moieties (Kim et al., 2015). Based on the differences in the parent ring structure, ginsenosides are divided into PPD (Ra1/2/3, Rb1/2/3, Rc, Rd, Rg3, Rh2, F2, compound K), oleanolic acid (Rh3, Ro, Ri), and PPT (Re, Rf, Rg1/2, Rh1, F1) types . The parent ring structure of PPD type ginsenosides contains two hydroxyl groups at C-3 and C-12, and their sugar moieties attach to β-OH at C-3 and/or C-20 (Bai et al., 2014;Kim et al., 2015). The oleanolic acid type ginsenosides are comprised of a pentacyclic structure with the aglycone oleanolic acid (Choi, 2008;Kim et al., 2015). The parent ring structure of PPT type ginsenosides contains three hydroxyl groups at C-3, C-6, and C-12, and sugar moieties attach to β-OH at C-20 and/or α-OH at C-6 (Bai et al., 2014;Kim et al., 2015). It was demonstrated that the three types of ginsenosides were effective in inhibiting MC apoptosis and regulating different apoptotic pathways to relieve MIRI. According to the included studies, PPD type ginsenosides can trigger the death receptor-mediated signaling pathway; PPD and PPT type ginsenosides both regulate PI3K/Akt-mediated and NF-κB-mediated signaling pathways; furthermore, all three types of ginsenosides can affect mitochondria-and MAPK-mediated signaling pathways. The chemical structures of the ginsenosides included in this study are shown in Figure 2. The mechanisms of ginsenosides on MC apoptosis in MIRI are summarized in Supplementary Table S1.
Published studies addressing the death receptor-mediated MC apoptotic pathway of ginsenosides focus on the Fas/FasL signaling pathway. Fas/FasL binds to the FADD and transmits the apoptotic signal to procaspase-8, resulting in the formation of death-induced signal complex (DISC), which leads to caspase hydrolysis (Lee et al., 2012;Wang and Su, 2018). The combination of ginsenosides Rb3 and Rb2/Rb3 effectively regulates FasL and FADD to decrease the levels of caspase-8 and caspase-3 (Liu, 2014), while ginsenoside Rb1 improves MC apoptosis via the downregulation of caspase-8 and caspase-3 (Ai et al., 2015). Moreover, caspase-8 can trigger the mitochondria-mediated signaling pathway by cleaving Bid (Chae et al., 2007). Bid decreases the level of Bcl-2 and increases the level of Bax to increase the release of cyt-C, and the levels of caspase-9 and caspase-3 (Chen et al., 2001;Pasdois et al., 2011;Kim et al., 2021). Ai et al. reported that ginsenoside Rb1 downregulated caspase-8, bid, caspase-9, caspase-3, and cyt-C (Ai et al., 2015). The death receptor-mediated signaling pathway of ginsenosides in relieving MC apoptosis is shown in Figure 3.

MAPK-Mediated Signaling Pathway
MAPK is an important signal transducing enzyme that has effects on the regulation of gene expression, cell proliferation, and cell death (Chang and Karin, 2001). MAPK kinase (MAPKK) is activated by MAPK kinase kinase (MAPKKK) to reactivate MAPK (Junttila et al., 2008). MAPK includes JNK, p38, and ERK, which are activated by special MAPKK and have different functions (Chang and Karin, 2001). The activation of JNK and p38 mediates apoptosis (Junttila et al., 2008). JNK promotes apoptosis through regulating c-Jun, which is its most classical substrate (Davis, 2000). JNK also effectively regulates proapoptotic protein, Bax (Syeda et al., 2019). The p38 MAPK pathway is related to the regulation of inflammation, gene expression, and energetic metabolism (Bassi et al., 2008). p38 participates in the promotion of apoptosis via its substrates, such as MAPKAP-2, MSK-1, and GADD153 (Zhang et al., 2019a;Ashraf et al., 2014;Das et al., 2006). Inhibition of the p38 MAPK pathway up-regulates the levels of Nrf2 and HO-1 to increase antioxidative proteins and improve apoptosis . Evidence indicated that ginsenosides Rb1, Rb3, Rg2, Rg3, Rh1, Rh2, and Rk3 had a function of down-regulating JNK, and ginsenosides Rb1 and Rk3 decreased the level of p38 (Supplementary Table S1). ERK can proliferate cells and regulate cell growth, and activated ERK inhibits the formation of DISC to relieve death receptor-mediated signaling pathway-induced apoptosis (Meloche and Pouysségur, 2007;Holmström et al., 2000). Additionally, ERK can increase Nrf2 to alleviate mitochondria damage (Ai et al., 2015). Ginsenosides Rb1, Rg1, and Rk3 can up-regulate ERK (Supplementary Table  S1). Notably, one study indicated that ginsenoside Rb1 downregulated ERK to inhibit apoptosis (Ai et al., 2015). In this study, MIRI increases the level of ERK. And over-expressed ERK leads to reversible or permanent cell cycle arrest (Meloche and Pouysségur, 2007); thus, ginsenoside Rb1 may decrease overexpressed ERK induced by apoptosis to protect MCs. Figure 7 showed MAPK-mediated signaling pathway of ginsenoside in relieving MC apoptosis.

CONCLUSION AND PERSPECTIVE
MIRI is functional and organic damage to the heart, which results from restoration of blood flow in ischemic areas (Wang K. et al., 2021). Through a number of studies addressing MIRI, the mechanisms of MIRI have not been fully revealed. Previous studies indicated that MC apoptosis was one of the fundamental pathogenic factors of MIRI, and the inhibition of MC apoptosis was effective in alleviating MIRI (Zhou et al., 2018;Xu et al., 2019;Fu D. et al., 2021). Ginsenosides can improve MIRI by relieving mitochondria damage, resisting oxidation, reducing inflammatory response, and inhibiting the generation of DISC (Wang and Roh, 2020;Shaukat et al., 2021). Ginsenosides can relieve MIRI via multiple signaling pathways, such as the death receptor signaling pathway, mitochondria signaling pathway, PI3K/Akt signaling pathway, NF-κB signaling pathway, and MAPK signaling pathway. The occurrence and development of MIRI is complex and multifactor interacted; thus, it is vital to investigate multi-target therapy in future studies. Ginsenosides, which are regarded as undoubtedly low-toxicity drugs (Xu JF. et al., 2021), have favorable safety profiles (Mancuso and Santangelo, 2017). Toxicity studies showed that most ginsenosides have no oral toxicity, such as Re, Rg2, and Rh2 (Wang et al., 2006;Lu et al., 2012;Gou et al., 2020). Undeniably, in vitro studies indicated that ginsenosides Rb1, Rg1, and Re had embryotoxic and teratogenic effects (Chan et al., 2004;Liu et al., 2005;Liu et al., 2006). However, results from in vitro animal studies may not reflect the true conditions in humans; thus, previous studies suggested that these ginsenosides need to be used with caution in clinics during the first trimester of gestation, before more data in humans are available Mancuso and Santangelo, 2017). Overall, the development and application of ginsenosides in improving MIRI are significant, and the toxicity data from in vivo studies and clinical studies are needed. Currently, multiple studies have explored the anti-apoptotic mechanism of ginsenosides; however, problems still exist, and further studies are needed. Firstly, the research studies about the structure-function relationship of ginsenosides in inhibiting MIRI are still needed to be conducted. The hydroxyl groups and sugar moieties have influences on the pharmacological effects of ginsenosides, which can interact with membrane lipids (Kim et al., 2015). Thus, the research of structure-function relationship of ginsenosides in anti-MIRI can contribute to developing safe and effective drugs via chemical modification. Secondly, the current results are mainly generated by ex vivo experiments or animal experiments. Rare clinical evidence has showed that ginsenoside Rb has protective effects on MIRI in patients undergoing mitral valve surgery (Zhan et al., 1994). Existing studies are in infancy, and more clinical research studies are needed to be designed and conducted to supply further clinical evidence. Thirdly, evidence showed that pyroptosis occurred during the development of MIRI (Xu XN. et al., 2021;Ji et al., 2021), and pro-apoptotic caspase-3 can cleave GSDME to induce pyroptosis (Wang Y. et al., 2017). However, no study has confirmed the anti-pyroptosis effect of ginsenosides in improving MIRI. Thus, the mechanisms of ginsenosides need to be further explored.
Overall, this review of anti-apoptotic mechanisms of ginsenoside in MIRI presents pharmacological mechanisms and lays the foundation for further research studies, hoping to contribute to the development of undiscovered mechanism and new drugs.