Natural compounds regulate the PI3K/Akt/GSK3β pathway in myocardial ischemia-reperfusion injury

ABSTRACT

The PI3K/Akt/GSK3β pathway is crucial in regulating cardiomyocyte growth and survival. It has been shown that activation of this pathway alleviates the negative impact of ischemia-reperfusion. Glycogen synthase kinase-3 (GSK3β) induces apoptosis through stimulation of transcription factors, and its phosphorylation has been suggested as a new therapeutic target for myocardial ischemia-reperfusion injury (MIRI). GSK3β regulatory role is mediated by the reperfusion injury salvage kinase (RISK) pathway, and its inhibition by Akt activation blocks mitochondrial permeability transition pore (mPTP) opening and enhances myocardial survival. The present article discusses the involvement of the PI3K/Akt/GSK3β pathway in cardioprotective effects of natural products against MIRI.

Abbreviations: Akt: protein kinase B; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; Bad: bcl2-associated agonist of cell death; Bax: bcl2-associated x protein; Bcl-2: B-cell lymphoma 2; CK-MB: Creatine kinase-MB; CRP: C-reactive-protein; cTnI: cardiac troponin I; EGCG: Epigallocatechin-3-gallate; Enos: endothelial nitric oxide synthase; ER: endoplasmic reticulum; ERK ½: extracellular signal‑regulated protein kinase ½; GSK3β: glycogen synthase kinase-3; GSRd: Ginsenoside Rd; GSH: glutathione; GSSG: glutathione disulfide; HO-1: heme oxygenase-1; HR: hypoxia/reoxygenation; HSYA: Hydroxysafflor Yellow A; ICAM-1: Intercellular Adhesion Molecule 1; IKK-b: IκB kinase; IL: interleukin; IPoC: Ischemic postconditioning; IRI: ischemia-reperfusion injury; JNK: c-Jun N-terminal kinase; Keap1: kelch-like ECH-associated protein- 1; LDH: lactate dehydrogenase; LVEDP: left ventricular end diastolic pressure; LVP: left ventricle pressure; LVSP: left ventricular systolic pressure; MAPK: mitogen-activated protein kinase; MDA: malondialdehyde; MIRI: myocardial ischemia-reperfusion injury; MnSOD: manganese superoxide dismutase; mPTP: mitochondrial permeability transition pore; mtHKII: mitochondria-bound hexokinase II; Nrf-1: nuclear respiratory factor 1; Nrf2: nuclear factor erythroid 2-related factor; NO: nitric oxide; PGC-1α: peroxisome proliferator‑activated receptor γ coactivator‑1α; PI3K: phosphoinositide 3-kinases; RISK: reperfusion injury salvage kinase; ROS: reactive oxygen species; RSV: Resveratrol; SOD: superoxide dismutase; TFAM: transcription factor A mitochondrial; TNF-α: tumor necrosis factor-alpha; VEGF-B: vascular endothelial growth factor B

KEYWORDS:

• PI3K/Akt/GSK3β pathway
• ischemia-reperfusion
• injury
• cardiomyocyte
• natural compounds
• apoptosis

1. Introduction

Myocardial ischemia-reperfusion injury (MIRI) is a critical condition that leads to an inflammatory response, which causes further cellular damage and organ function impairment through induction of apoptosis by inducing oxidative stress, calcium overload, mitochondrial permeability transition pore (mPTP) opening, and hypercontracture [1,2].

The term ischemia denotes insufficient oxygen supply due to arterial inflow obstruction. During ischemia, anaerobic metabolism dominates and reduces cell pH and ATP levels [3]. Thus, membrane ATP-dependent ionic pump function is altered. Anaerobic metabolism deactivates sodium-potassium and calcium pumps that preserve sodium and calcium inside and potassium outside the cell, on the cell membrane. Also, this condition disrupts calcium pumps on the endoplasmic reticulum, limiting calcium reuptake. The cytosolic Ca²⁺ rise increases mitochondrial Ca²⁺ and inner membrane permeability [4]. In addition, a higher level of sodium in cells reduces sodium-hydrogen pump activity. Sodium and calcium ions retention causes hyperosmolarity and cell swelling, while hydrogen retention decreases cellular pH, leading to enzyme dysfunction and nuclear chromatin accumulation [5].

The process of blood returning to the ischemic myocardium is called reperfusion. The potentially detrimental aspect of myocardial reperfusion injury involves reactive oxygen species (ROS) production, microvascular and endothelial dysfunction, myocardial metabolism changes, and activation of neutrophils, platelets, and complement [6]. The ROS formation in reperfusion injury occurs through molecular oxygen reduction or other sources including the function of the enzymes, such as xanthine oxidase, cytochrome oxidase, and cyclooxygenase, and the oxidation of catecholamines [7]. In ischemia, ATP is catabolized to hypoxanthine, and xanthine dehydrogenase (XDH) is converted via limited proteolysis and sulfhydryl oxidation to xanthine oxidase (XO). Upon reperfusion, an influx of O₂ reacts with hypoxanthine (or xanthine) and XO to generate superoxide (O₂−) and hydrogen peroxide (H₂O₂), which increase ROS generation [8]. Superoxide anion induces lipid peroxidation, cell permeability disruption, and oxidation of proteins and DNA, leading to cell death [9,10]. Free radicals stimulate the endothelium to release platelet-activating factor (PAF), attracting more neutrophils and amplifying the production of oxidant radicals [11]. Notably, lack of cell antioxidants further augments ROS levels. At the same time, responses to endothelium-dependent vasoconstrictors, such as endothelin-1 and oxygen-free radicals increase and result in endothelial injury and microvascular dysfunction [12,13].

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3 β (GSK3β) pathway is one of the vital intracellular signaling pathways, which controls cell metabolism, growth, and proliferation, and stress response [14,15]. This pathway activates PI3K, which then phosphorylates Phosphatidylinositol-4,5-bisphosphate (PIP2), forming phosphatidylinositol-3,4,5-trisphosphate (PIP3) as a membranous second messenger. Upon binding PIP3, Akt undergoes a conformational change, facilitating its phosphoinositide-dependent kinase-1 (PDK1)-dependent phosphorylation [16]. Once phosphorylated, Akt becomes active, moves from the membrane, and phosphorylates/inactivates GSK3β [17]. GSK3β participates in some apoptotic signaling transduction by phosphorylating transcription factors that regulate apoptosis; GSK3β induces activation of pro-apoptotic factors, such as p53 [18] and suppression of survival-promoting factors, so its phosphorylation/inactivation enhances cell survival [19]. Within this intracellular signaling, PI3K/Akt phosphorylates several downstream targets, including NF-KB, to upregulate cell survival signaling pathways. When NF-kB forms a complex with IkB, it remains in the cytoplasm. Akt phosphorylates IkB and degrades it [20]. For instance, it was shown that in human osteosarcoma cells, the liberated NF-kB enters the nucleus to increase cell survival [21]. PI3K/Akt also regulates the Fas/Fas-ligand system, an essential pathway for cellular apoptosis. Fas and Fas-ligand are members of the Tumor necrosis factor (TNF) family. Activation of this system leads to recruitment of Fas-associated death domain (FADD) and triggers cell apoptosis [22]. PI3K/Akt blocks forkhead protein, which promotes Fas-ligand [23]. Furthermore, PI3K/Akt blocks the pro-apoptotic protein Bad to increase the anti-apoptotic protein bcl-2 level [24] and reduces cytosolic calcium [25]. PI3K/Akt activation induces endothelial nitric oxide synthase (eNOS) phosphorylation, which leads to NO secretion [26] and suppression of c-Jun N-terminal kinase (JNK) thus enhancing cell survival [27,28]. GSK3β phosphorylation inhibits mPTP opening and stabilizes peroxisome proliferator‑activated receptor γ coactivator‑1α (PGC-1α) which increases mitochondrial respiration by activating PI3K/Akt and ERK1/2 involved in the reperfusion injury salvage kinase (RISK) pathway [29–31]. Moreover, GSK3β phosphorylation results in cell protection against oxidation and inflammation through Nrf2 stimulation by superoxide dismutase (SOD), heme oxygenase-1 (HO-1), manganese superoxide dismutase (MnSOD), and catalase activation [32]. The present article discusses the involvement of PI3K/Akt/GSK3β pathway in cardioprotective effects of natural products against MIRI (Figure 1).

Figure 1. The protective mechanisms mediated through regulation of PI3K/Akt/GSK3β against myocardial ischemia-reperfusion injury. ATP, adenosine triphosphate; Bcl‐2, B-cell lymphoma 2; eNOS, endothelial nitric oxide synthase; HO-1, heme oxygenase-1; mPTP, mitochondrial permeability transition pore; NO, Nitric oxide; ROS, reactive oxygen species; SOD, superoxide dismutase; and TNF-α, tumor necrosis factor-alpha.

2.1. Syringic acid

Syringic acid is a phenolic acid found in olives, grapes, and dates, and it exerts antioxidant, anti-inflammatory, and anti-endotoxic properties [33]. Recent studies have revealed that syringic acid protects hippocampal neuronal and H9C2 cells in hypoxia/reoxygenation (HR), and in other organs the spinal cord and kidney under ischemia-reperfusion (IR) [34–37]. In a rat model of MIRI, syringic acid administration (50 mg/kg for three consecutive days before reperfusion) diminished infarct size, decreased apoptosis, and enhanced cardiac function through PI3K/Akt/GSK3β phosphorylation. This pathway increases the ratio of Bcl-2/Bax and reduces the contents of cleaved-9 and 3. In this pathway, GSK3β is phosphorylated by activated Akt on its serine 9 residue. The phosphorylated GSK3β suppresses mPTP opening and inhibits cytochrome c release from the mitochondria (Table 1 and Figure 2) [38].

2.2. Berberine

Berberine, an isoquinoline alkaloid, is the main active ingredient of Rhizoma coptidis, a famous traditional Chinese herb with hypoglycemic and anti-inflammatory properties [39–41]. This compound could attenuate endothelial dysfunction and cardiovascular disorders in diabetic rats and reduce mortality in patients with severe congestive heart failure [42,43]. In pressure overload-induced cardiac hypertrophy models, berberine treatment reduced infarct size in nondiabetic rats with ischemia-reperfusion injury (IRI) and reduced left ventricular myocardium size [44,45]. During cardiac ischemia, AMPK is activated to increase myocardial glucose uptake, glycolysis, and fatty acid oxidation for energy production and heart protection [46]. It is noticeable that although crosstalk between Akt and AMPK signaling is still not apparent in cardiac myocytes and vascular endothelial cells, Akt activation appears to be regulated by the AMPK pathway [47]. Berberine pretreatment was shown to induce AMP/ATP and ADP/ATP ratio, AMPK activation, phosphorylation of AKT, and inhibition of GSK3β to save mitochondria. Also, berberine alleviated malondialdehyde (MDA) levels and cardiac arrhythmia in diabetic rats with IRI (Table 1 and Figure 2) [48].

2.3. Epigallocatechin-3-gallate

Epigallocatechin-3-gallate (EGCG), the primary catechin of green tea, is known to have cardioprotective effects [49] mediated through its anti-inflammatory property [50]. EGCG administration before [51] or after [52] ischemic insult limited infarct size in the heart. It mitigated regional MIRI by activating RISK, a group of pro-survival protein kinases, and a combination of two parallel cascades, PI3K-Akt and MEK1-ERK1/2 [53], and preventing cell death through JNK suppression. The PI3K/Akt pathway seems to be involved in EGCG-induced GSK3β inactivation. It was indicated that EGCG phosphorylates GSK3β through Akt activation and inhibits mPTP opening [54]. In addition, while MAPKs cause cell death following IRI [55], EGCG suppressed p38/JNK phosphorylation while increasing Akt/GSK3β phosphorylation (Table 1 and Figure 2) [54].

2.4. Hydroxysafflor yellow A

Hydroxysafflor Yellow A (HSYA) is one of the active components of Honghua (Carthamus tinctorius L.) which is used for coronary heart disease treatment [56–58]. It is evident that apoptosis plays a crucial role during MIRI and involves mitochondrial permeabilization leading to cytochrome c release and caspase-3 activation [59,60]. Mitochondrial function is associated with the RISK pathway [61,62]. It was demonstrated that mitochondrially bound hexokinase II (mtHKII) is a crucial element of ischemic preconditioning. Hexokinase II could be activated by Akt, thus restoring mitochondrial energy, reducing ROS generation, and inhibiting apoptosis [63]. HSYA was shown to significantly reduce lactate dehydrogenase (LDH), oxidative stress, caspase 3 levels, and cell apoptosis, while increasing cell viability, mitochondrial energy metabolism, Akt activation, and GSK3β suppression in H9c2 cells (Table 1 and Figure 2) [63].

2.5. Resveratrol

Resveratrol is a polyphenolic compound extracted from berries, peanuts, and particularly grape skins with various health-beneficial effects [64–68]. Its biological activities include lifespan extension [69], antidiabetic [70,71], and anticancer properties [72,73]. Its anti-apoptotic effect is mediated through the vascular endothelial growth factor B (VEGF-B) up-regulation in pancreatic cancer cells [74]. One of the most well-known effects of resveratrol is cardioprotection [75,76]. Resveratrol improves heart functions, upregulates MnSOD and catalase, reduces infarct size and CK-MB release, and inhibits cell death during MIRI. In addition, resveratrol showed a lowering effect on ROS generation in H9c2 cells. It was reported that resveratrol upregulated VEGF-B, which induces Akt and GSK3β phosphorylation, and showed cardioprotection against IRI. Furthermore, resveratrol reduced Bax protein expression in heart tissue (Table 1 and Figure 2) [77].

2.6. Kaempferol

Kaempferol is a flavonoid commonly found in kale, beans, tea, and spinach. Numerous preclinical studies have shown kaempferol pharmacological functions including antioxidant, anti-inflammatory, and anticancer activities [78–80]. In an IRI model of isolated rat heart, kaempferol improved the recovery phase and reduced intracellular oxidation, myocardial ischemia, and apoptosis through GSK3β phosphorylation. Also, kaempferol enhanced SOD activity and glutathione/oxidized glutathione (GSH/GSSG) ratio while decreasing MDA level and preventing myocardial membrane peroxidation. During MIRI, a large amount of TNF-α is produced, and kaempferol could reduce TNF-α levels by PI3K stimulation [81]. This decrease has been speculated to partially contribute to reduced myocardial infarction size and prevention of caspase-3 and −9 cleavage [82]. Kaempferol induced all these effects by PI3K/Akt/GSK3β pathway phosphorylation and, consequently, mPTP opening suppression (Table 1 and Figure 2) [81].

2.7. Safranal

Safranal, a monoterpene aldehyde isolated from saffron (Crocus sativus), has shown radical-scavenging and anti-apoptotic properties [83], and conferred protection against indomethacin-induced gastric ulcers [84], pentylenetetrazol-induced status epilepticus [85], and cancer [86]. Safranal also showed an anti-ischemic effect during renal IRI [87]. It was shown that this compound prevents MIRI progression and improves cardiac performance in rats by phosphorylation of Akt/GSK3β/eNOS and inhibition of IKK-b/NF-kB pathways. Also, it provides anti-inflammatory and anti-apoptotic effects by TNF-α suppression. Safranal upregulated the endogenous antioxidant defense system and reduced nitrotyrosine levels. Thus, safranal limited the infarct size and improved inotropic reserves and contractile function. Enhanced Akt/GSK3β phosphorylation negatively regulates MIRI by activating the sarcoplasmic reticulum Ca^{2 +}-ATPase2a which reduces cytosolic Ca²⁺ overload and improves contractile function [88]. Inhibition of IKK-b/NF-kB/caspase-3, Bax, and TNF-α improved heart function and decreased infarct size following MIRI [89,90]. Safranal indirectly upregulates Bcl-2, which leads to Ca²⁺ overload reduction [91], and normalized LDH and CK-MB activities in MIRI [92]. Also, Akt phosphorylates and activates eNOS [93], and oxidative stress augments IRI by promoting functional NO deficiency. Increased superoxide/ROS and NO/tyrosine produce nitrotyrosine, a marker for ROS-arbitrated NO oxidation [94]. Notably, safranal-suppressed nitrotyrosine levels were correlated with increased eNOS phosphorylation in MIRI. Therefore, one of the mechanisms by which safranal ameliorated MIRI, is Akt phosphorylation and decreased nitrotyrosine level (Table 1 and Figure 2) [95].

2.8. Astragaloside IV

Astragaloside IV is one of the effective constituents of Astragalus membranaceus, with various pharmacological effects, including immunity enhancement [96], anti-inflammation [97], antioxidation [98], and anti-virus [99] properties. It has been reported that astragaloside IV attenuates viral myocarditis [100], myocardial fibrosis [101], and heart failure [102]. In addition, previous studies have shown that it protects the myocardium against IRI with different mechanisms [103–106]. Besides, astragaloside IV improved left ventricular systolic pressure (LVSP), fractional shortening, and ejection fraction, and reduced LDH, CK-MB levels, heart-to-body weight ratio, and myocardial infarct size in MIRI through PI3K/Akt/GSK3β phosphorylation (Table 1 and Figure 2)[107].

2.9. Kaempferide

Kaempferide is a flavonoid obtained from Alpinia officinarum roots with excellent antioxidant [108], anticancer [109], and antihypertension properties [110]. Its pretreatment in MIRI improved LVSP, fractional shortening, and ejection fraction, while left ventricular end-diastolic pressure (LVEDP), CK-MB and LDH levels, and myocardial infarct size were decreased. Kaempferide treatment also decreased C-reactive-protein (CRP), interleukin (IL)-6, TNF-α, MDA, ROS, and apoptosis and enhanced SOD activity. So, according to these results, this compound’s cardioprotective effects depend on its anti-inflammatory and anti-oxidative features. The GSK3β/caspase-3-dependent pathway phosphorylation has been proven to protect cardiomyocytes from IRI and inhibit apoptosis [111]. So, GSK3β phosphorylation and caspase-3 inhibition in rats could be responsible for the kaempferide cardioprotection against IRI. Together, these results suggest that the potential benefits of kaempferide in MIRI are likely mediated via activation of PI3K/Akt/GSK3β/caspase-3 pathways (Table 1 and Figure 2) [112].

2.10. Ginsenoside Rd

Ginsenoside Rd is one of the active components of Panax ginseng that scavenges free radicals [113], inhibits Ca²⁺-influx via a receptor and store-operated Ca²⁺ channels [114], and protects against neuronal apoptosis [115]. It is a lipophilic compound that readily diffuses across biological membranes, so that it may be advantageous for the heart [116]. A study reported that ginsenoside Rd could attenuate infarct size and enhance myocardial structure in rats [117]. In a cell culture model, Panax notoginseng saponins prevented cardiomyocyte apoptosis induced by glucose and oxygen deprivation injury via PI3K/Akt signaling [118]. Ginsenoside Rd augmented cardiac function, increasing ±LVdP/dt max and decreasing LVEDP, and reduced cardiomyocytes intracellular ROS generation. Ginsenoside Rd also attenuated cellular damage in cultured neonatal rat cardiomyocytes (NRCs) subjected to IR through ROS, mitochondrial membrane potential, cytochrome c release mitigation, and Bcl-2/Bax ratio enhancement. Akt overexpression raises Bcl-2 levels, and phosphorylated GSK3β suppresses mPTP opening by binding to adenine nucleotide translocase; these findings support the involvement of the Akt/GSK3β signaling pathway in ginsenoside Rd cardioprotection (Table 1 and Figure 2) [119].

2.11. Curcumin

Curcumin is a yellow pigment from Curcuma longa (turmeric) commonly used as a spice/food coloring agent. It is relatively safe and nontoxic and exerts diverse biological effects such as anti‐inflammatory [120], antidiabetic, antioxidant [121,122], immunomodulatory [123], and anti-carcinogenic functions [124]. Also, evidence suggests that curcumin has cardioprotective effects against MIRI [125]. Curcumin reduces the phosphorylation of JNK and p38-MAPKs and infarct size by the PI3K/Akt and ERK1/2 pathway phosphorylation. It was indicated that curcumin administration before left anterior descending coronary artery occlusion protects against focal MIRI through induction of PI3K/Akt and ERK1/2 [75]. While in IR, the pro-survival kinases PI3K/Akt and ERK1/2 may be overwhelmed by p38-MAPK and JNK [126], resulting in myocardial apoptosis and necrosis, curcumin suppresses p38 and JNK phosphorylation. Curcumin administration resulted in myocardial protection by inhibiting GSK3β and mPTP opening (Table 1 and Figure 2) [75].

2.12. Fisetin

Fisetin is a flavonoid found in many plants, such as strawberries, apples, persimmons, onions, and cucumbers. This compound has antioxidant, anti-inflammatory, and anti-apoptotic effects, exhibited neuroprotective properties, and inhibited cancer cells in several preclinical studies [127,128]. Also, cardioprotective potentials of fisetin were shown in vitro and in vivo [129]. The compound caused a marked improvement in cardiac function, decreased myocyte injury markers, such as LDH and CK, and attenuated mitochondrial swelling. Fisetin also maintained mitochondria hyperpolarized and boosted their antioxidant capacity; these observations suggest that fisetin inhibits mPTP opening and improves mitochondrial function, thereby preventing IR-induced myocardial tissue injury. In addition, fisetin blunted excessive oxidative stress in lysosomes and microsomes obtained from MIRI-affected hearts [130]. Fisetin could activate Nrf-2 and augment the expression of HO-1 to protect against oxidative insults [131]. It was indicated that though MIRI reduced the level of PGC-1α, nuclear respiratory factor 1 (Nrf-1), and transcription factor A mitochondrial (TFAM), fisetin marginally enhanced their expression [130]. GSK3β suppresses PGC-1α activation through its phosphorylation and thus prepares PGC-1α for degradation via the ubiquitin-proteasome pathway [132]. In a mouse model of MIRI, GSK3β activity in myocardial tissues was raised, and this increase was suppressed by fisetin treatment. In summary, fisetin confers cardioprotection against MIRI by bolstering mitochondrial physiology, suppressing oxidative stress, and augmenting mitochondrial biogenesis, and these effects are mediated via inhibition of GSK3β activity (Table 1 and Figure 2) [130,133].

2.13. Leonurine

Leonurine is an alkaloid obtained from Herba leonuri and it has an extensive range of biological activities, including anti-inflammatory [134], antioxidant [135], anti-tumor [136], and cardiovascular protective effects [137]. It also induced Akt phosphorylation and hypoxia-inducible factor 1 expression, survival, and VEGF expression in rats with myocardial ischemia. Besides, leonurine inhibited apoptosis in H9c2 cells under hypoxia by increasing the Bcl‑2/Bax ratio [138]. This compound also diminished the infarct size, alleviated collagen deposition, inhibited cardiomyocyte apoptosis, prevented left ventricular dilation, and improved cardiac function by activating the PI3K/Akt/GSK3β signaling pathway. Together, leonurine improved cardiac function in MIRI through up‑regulation of anti‑apoptotic protein Bcl‑2 and down‑regulation of pro‑apoptotic protein Bax and cleaved caspase-3 (Table 1 and Figure 2).

2.14. Lycopene

Lycopene is a carotenoid present in tomatoes and other red fruits and vegetables, and it exerts antioxidant properties and reduces the risk of coronary heart disease [139]. Some studies demonstrated that lycopene protects against IRI by inhibiting the development of endoplasmic reticulum (ER) stress [140] and blocking mPTP opening [141]. It was found that lycopene and Ischemic postconditioning (IPoC) activated Akt and ERK1/2, subsequently inhibiting GSK3β and mPTP opening in hypercholesterolemic rats heart. This finding suggests that restoration of IPoC by lycopene may be partly attributed to the reactivation of the RISK pathway. ER stress has been demonstrated to impair RISK pathway activation, suppress Akt and ERK1/2 phosphorylation, and further phosphorylated GSK3β-mediated suppression of mPTP opening [142]. Notably, the effect of lycopene administration in IPoC on the RISK pathway was similar to that of an ER stress inhibitor. Thus, it may be inferred that lycopene reactivates Akt and ERK1/2 in hypercholesterolemic rats heart partly through inhibition of ER stress (Table 1 and Figure 2) [143].

2.15. Rhein

Rhein, an anthraquinone isolated from rhubarb, has been used for many years in China to treat constipation, gastrointestinal hemorrhage, and ulcers [144]. Also, rhein protects the pancreatic β cells from hyperglycemia-induced cell apoptosis [145]. Rhein was shown to protect H9c2 cells from HR injury by reducing ROS production via PI3K/Akt signaling pathway. Rhein was found to increase phosphorylated Akt and GSK3β, and GSK3β silencing abolished rhein’s anti-apoptotic effects, suggesting that rhein protects the myocardium against IRI through Akt/GSK3β pathway. In addition, rhein inhibited apoptosis by suppressing P38 phosphorylation (Table 1 and Figure 2) [27].

2.16. Salvianolic acid A

Salvianolic acid A, the main ingredient of Salvia miltiorrhiza, has various pharmacological activities, such as preventing myocardial ischemia and regulating the immune system [146,147]. It was indicated that salvianolic acid A preserved cardiac function against IRI by reducing the myocardial infarct area and enzyme leakage [148]. The compound could attenuate cardiomyocyte apoptosis and decrease the expression of cleaved caspase-3 and Bax/Bcl-2 ratio, suggesting that it inhibits MIRI by suppressing apoptosis [149]. Also, salvianolic acid A enhanced ATP levels, reduced ROS production, and protected cardiomyocytes against H₂O₂-induced injury [150]. It preserved the mitochondrial membrane potential and blocked mPTP opening through Akt/GSK3β phosphorylation, which boosts cardiomyocyte tolerance to HR. The salvianolic acid A protective effects were attributed to GSK3β phosphorylation and, consequently, mitochondria survival (Table 1 and Figure 2) [149].

2.17. Shikonin

Shikonin is a natural naphthoquinone pigment purified from the root of Lithospermum erythrorhizon [151]. It alleviates brain and liver IR outcomes [152,153] and protects against isoproterenol-induced heart damage [154]. Shikonin protection against HR injury in H9c2 cells was suggested to be mediated through attenuating excessive ROS generation and apoptosis. It prevented mitochondrial membrane potential collapse and cytochrome c release to the cytosol to inhibit apoptosis during HR associated with mitochondria dysfunction through Akt/GSK3β phosphorylation (Table 1 and Figure 2) [155].

2.18. Spinosin and 6‴-feruloylspinosin

Spinosin and 6‴-feruloylspinosin, two C-glycoside flavonoids obtained from Semen Ziziphi spinosae (Suanzaoren), have shown anxiolytic and hypnotic effects [156]. In addition, spinosin ameliorated neurogenesis, memory deficit, cognitive performance, and Alzheimer’s disease in mice [157–159]. These compounds reduced myocardial damage and cellular apoptosis, promoted autophagy flux, and upregulated PGC-1α, Nrf2, and HO-1 which were associated with GSK3β inhibition [160]. Overproduction of ROS and free radicals in the mitochondria triggers myocardial damage in the reperfusion phase. Increased ROS levels open mPTP which boosts ROS production and causes myocardial damage [161]. Approaches such as GSK3β inhibition, which limit mPTP induction by increasing the mPTP-ROS threshold, protect myocardial cells [162]. Spinosine and 6‴-feruloylspinosin restrain GSK3β activity and protect myocardial cells. GSK3β inhibition induces multiple cell survival pathways, including pro-survival autophagy [163] and PGC-1α/Nrf2/HO-1 [164]. It was further identified that both spinosin and 6‴-feruloylspinosin could enhance the expression of LC3B-II, while 6’’‘‑feruloylspinosin could reduce the level of p62, indicating activation of autophagy. PGC-1α is an inductive transcription activator in repairing ROS-induced mitochondrial damage and biogenesis and regulating energy metabolism. GSK3β can phosphorylate PGC-1α and promote its ubiquitin-dependent degradation; however, its inhibition induces PGC-1α accumulation to protect cells [165]. The cytoprotective effect of PGC-1α is related to Nrf2 upregulation and subsequently, induction of the expression of HO-1 gene. This enzyme catalyzes the degradation of pro-oxidant heme into three important antioxidant products, bilirubin, carbon monoxide, and ferrous ion [166]. Besides, Nrf2 induces the expression of superoxide dismutase 2 (SOD-2). Overall, the cardioprotective effects of spinosin and 6‴-feruloylspinosin were shown to be mediated by inhibition of GSK3β and upregulation of PGC-1α, Nrf2, and HO-1 proteins level (Table 1 and Figure 2) [160].

Table 1. Natural compounds that protect against myocardial ischemia-reperfusion injury via phosphorylation of PI3K/Akt/GSK3β.

Natural compound	Experimental model	NCs Dose/Route/Time	Findings	Reference
Syringic acid	Rats	50 mg/kg gavage for 3 days, 30-minute ischemia followed by 120-minute reperfusion	↑LVEF, LVFS, ↑p-PI3K, p-Akt, p-GSK3β, Bcl-2 ↓MI size,caspase-3 and −9, Bax	[38]
Berberine	Type 2 diabetic rats	100 mg/kg gavage for 7 days	↑p-Akt, p-GSK3β ↓MI size, arrhythmia, TG, cholesterol, MDA level	[48]
Epigallocatechin-3-gallate	Rats	10 mg/kg i.p. 5 minutes before the onset of reperfusion	↓MI size, p38, and JNK ↑p-Akt, p-GSK3β	[54]
Hydroxysafflor yellow A	H9c2 cells	1.25, 5, and 20 mM during reoxygenation for 4 hours	↓LDH, Caspase 3, oxidative stress, apoptosis ↑Mitochondrial energy metabolism	[63]
Resveratrol	Rats	10 μmol/L 15 minutes prior to the ischemia period	↑VEGF-B mRNA, Akt expression ↓CK-MB release, GSK3β expression, MI size, apoptosis, ROS generation by MnSOD up-regulation	[77]
Kaempferol	Rats	15 mmol/L for 10 min after being subjected to global ischemia for 15 minutes and reperfusion for 85 minutes	↑SOD, P-GSK3β, GSH/GSSG ratio levels. ↓MI size, caspase-3, cytoplasm cytochrome C, LDH, MDA, and TNF-α.	[81]
Safranal	Rats	0.1–0.5 ml/kg/day, i.p. for14 days, and on the 15th day, one-stage ligation of LAD for 45 minutes was performed, followed by 60-minute reperfusion	↓MI size, CK-MB, TNF-a, IKK-b/NF-κB, apoptosis ↑LV function, p-Akt, p-GSK3β, p-eNOS, myocardial antioxidant, nitrotyrosine levels.	[95]
Astragaloside IV	Rats	2.5, 5 and 10 mg/kg, intragastrical for 7 days	↓ LVEDP, LDH, CK, MI size ↑ LVSP, FS, EF ↑p-Akt, p-GSK3β	[107]
Kaempferide	Rats	0.1, 0.3 and 1 mg/kg injection 30 minutes prior to a 30-minute LAD coronary artery ligation followed by a 2 hour reperfusion	↑p-Akt, p-GSK3β, SOD ↓MI size, TNF-α, IL-6, CRP ↓MDA, ROS, caspase-3	[112]
Ginsenoside Rd	Rats Neonatal rat cardiomyocytes	50 mg/kg i.p. for 30 minutes prior to coronary IR 10 mM for 30 minutes followed by I/R treatment	↑LVEF, p-Akt, p-GSK3β, Bcl-2/Bax ratio. ↓MI size, apoptosis, CK-MB, LDH, ROS, caspase-9/3	[119]
Curcumin	Rats	100 mg/kg 20 minutes before LAD occlusion	↓MI size, p38, JNK expression ↑p-Akt/p-ERK1/2/p-GSK3β	[75]
Fisetin	Rats	20 mg/kg i.p. 1 hour before ischemia induction	↓IR injury, oxidative stress, mitochondrial dysfunction ↑p-GSK3β	[130]
Leonurine	Rats	15 mg/kg/day oral gavage following the onset of MI	↓Collagen deposition, caspase3, Bax expression, MI size, apoptosis ↑Myocardial function, p-PI3K/p-AKT/p-GSK3β, Bcl2,	[178]
Lycopene	Rats	5 mg/kg/day i.p. for 5 consecutive days before ischemia and reperfusion	↓MI size, cytochrome c, caspase‑9/3, mPTP opening, apoptosis ↑p-AKT/p-ERK1/2/p-GSK3β	[143]
Rhein	H9c2 cells	1 h incubation with 1 μg/mL rhein prior to 6 h hypoxia followed by 2 h reoxygenation	↓Apoptosis, ROS, p-P38 ↑p-AKT/p-GSK3β	[27]
Salvianolic Acid A	Rat Cardiomyocytes	12.5, 25, and 50 μg/mL salvianolic Acid A at the beginning of hypoxia and reoxygenation	↓ROS, apoptosis, caspase 3, Bax/Bcl-2 ratio, and mPTP activation ↑ p-AKT/p-GSK3β	[149]
Shikonin	H9C2 cells	10, 20 and 40 μM of shikonin 2 hours prior to HR	↑ p-AKT/p-GSK3β ↓LDH, apoptosis ↓cytochrome c release	[155]
Spinosin and6‘‑Feruloylspinosin	Rats	5 mg/kg i.p. 30 minutes before LAD ligation.	↓MI size, apoptosis ↑ p-GSK3β ↑PGC‑1α, Nrf2, HO‑ 1	[160]
Urolithin B	Sprague Dawley rats H9c2 cardiomyocytes	0.7 mg/kg UB 0, 24 and 48 h before 30 min LADA ligation followed by 120 min reperfusion 20 μM 12 h prior to 3 h hypoxia followed by 3 h reoxygenation	↓MI size, CK-MB, LDH, MDA ↑LVEF, p62 level ↓Autophagy, ROS, oxidative stress, caspase 3, apoptosis ↑Nrf2, HO-1, NQO1,GSTP, p-Akt	[175]
Urolithin A	Male adult C57BL/6 mice neonatal rat cardiomyocytes (NRCs)	1 mg/kg UA in 0.5 ml dimethylsulfoxide i.p., 24 and 1 h before 45 min ischemia followed by 24 h reperfusion. 10 μM UA at 24 and 1 h prior to 3h hypoxia followed by 3 h reoxygenation	↓MI size, apoptosis, CK-MB, LDH ↑LVFS, EF ↓ ROS, MDA content Bcl-2/Bax ratio, caspase 3 ↑SOD activity, p-Akt	[177]

Abbreviations: Bax, bcl2-associated x protein; Bcl-2, B-cell lymphoma 2; CK-MB, creatine kinase-MB; CRP, C-reactive-protein; EF, ejection fraction; eNOS, endothelial nitric oxide synthase; ERK1/2, extracellular signal‑regulated protein kinase 1/2; FS, fractional shortening; GSH, glutathione; GSSG, glutathione disulfide; GSTP1, glutathione S-transferase P1; HO‑ 1, heme oxygenase-1; IKK, IκB kinase; JNK, c-Jun N-terminal kinase; LADA, left anterior descending artery; LDH, lactate dehydrogenase; LV, left ventricular; LVEDP, left ventricular end diastolic pressure; LVEF, Left ventricular ejection fraction; LVFS, Left ventricular function measurements; LVSP, left ventricular systolic pressure; MDA, malondialdehyde; MI, myocardial infarction; mPTP, mitochondrial permeability transition pore; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NQO1, NAD(P)H dehydrogenase [quinone] 1; Nrf2, nuclear factor erythroid 2-related factor; p-Akt, phosphorylated Ak strain transforming; PGC‑1α, peroxisome proliferator‑activated receptor γ coactivator‑1α; p-GSK3β, phosphorylated glycogen synthase kinase 3 beta; p-PI3K, phosphorylated phosphoinositide 3-kinases; ROS, reactive oxygen species; SOD, superoxide dismutase; TG, triglycerides; TNF-α, tumor necrosis factor-alpha; VEGF-B, vascular endothelial growth factor B.

2.19. Urolithin A, B

The link between gut microbiota and its metabolites and MIRI has been indicated, and it was reported that different diets might influence MIRI consequences [167–169]. Ellagitannins (ETs) are a class of polyphenols that exist in pomegranates, walnuts, and berries [170]. Gut microbiota hydrolyzes ETs to ellagic acid (EA) which is further hydrolyzed into Urolithin A (UA) and Urolithin B (UB) with antioxidant and anti-inflammatory properties [171,172]. UB was found to protect against colonic fibroblast inflammation [173], lipopolysaccharide-induced microglia and macrophage activation [174], and high glucose-induced cardiomyocyte injury. Interestingly, UB could decrease myocardial infarction (MI) size, elevate cardiac function in rats after IR, and protect against HR injury in H9C2 cardiomyocytes. UB inhibited autophagy by activating Akt/mTOR/ULK1 pathway and protected against oxidative stress and caspase 3-dependent cell apoptosis [175]. UA health benefits include neuroprotection effects against Alzheimer’s disease and attenuating oxidized-LDL-induced endothelial dysfunction by up-regulation of NO expression and eNOS mRNA expression [176]. UA also protected the heart against IRI by significantly improving cardiac function and reducing myocardial apoptosis. It prevented ROS generation, decreased MDA levels and apoptosis, while increased SOD activation in an HR model, and inhibited myocardial apoptosis following IR through PI3K/Akt pathway activation in mice (Table 1 and Figure 2) [177].

Figure 2. The regulatory effects of natural compounds on the PI3K/Akt/GSK3β signaling pathway. Ischemia-reperfusion in cardiomyocytes induces apoptosis, and some natural products enhance cell survival through PI3K/Akt/GSK3β regulation (phosphorylation). Akt, Ak strain transforming; ARE, antioxidant response element; BAX, Bcl2‐associated X protein; Bcl‐2, B-cell lymphoma 2; eNOS, endothelial nitric oxide synthase; GSK3β, glycogen synthase kinase 3 beta; HO-1, heme oxygenase-1; IKK, IκB kinase; JNK, c-Jun N-terminal kinase; mPTP, mitochondrial permeability transition pore; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; Nrf2, nuclear factor erythroid 2-related factor; NO, Nitric oxide; PGC‑1α, peroxisome proliferator‑activated receptor γ coactivator‑1α; PI3K, Phosphoinositide 3-kinases; PKC, Protein kinase C; PKG, protein kinase G; ROS, reactive oxygen species; TNF-α, tumor necrosis factor-alpha; VEGF-B, vascular endothelial growth factor B.

3. Conclusion

Myocardial ischemia-reperfusion injury (MIRI) is a tissue damage that occurs when the blood returns to tissue after a period of ischemia. It can increase the risk of developing congestive heart failure and arrhythmias, and mortality. Based on the literature, cardiotoxic compounds induce heart damage via various mechanisms, such as inducing ROS generation, oxidative stress, and apoptosis following dysregulation of PI3K/Akt/GSK3β signaling [179,180], so, phosphorylation of this pathway may be a promising therapeutic approach for cardiac IRI. It has been reported that MIRI is accompanied by induction of apoptosis via increasing the level of Bcl-2/Bax, ROS generation, and mPTP opening while reducing NO production. The phytochemicals discussed in the present article have been shown to alleviate MIRI through phosphorylation of the PI3K/Akt/GSK3β signaling pathway, thus preventing untoward consequences and cell death.

Data availability statement

Data sharing is not applicable to this article as no datasets were generated or analyzed.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by Mashhad University of Medical Sciences