The cardiovascular protective effect and mechanism of calycosin and its derivatives

Cardiovascular disease (CVD) is one of the leading cause of death worldwide, especially in developing countries, where the prevalence has increased significantly ^[1]. The global number of deaths from CVD has increased during the past decade by 12.5% ^[2]. Between 1990 and 2020, the burden of CVD in the developing countries is growing ^[3]. These changes are driven by population growth and aging populations, with the largest number occurring in countries of South and East Asia because of their large and growing populations ^[4]. Elevated blood lipid levels, hypertension, elevated blood glucose levels, overweight, unhealthy dietary habits, cigarette smoking, and insufficient regular exercise are established primary causal factors of CVD ^[5]. Over 95% of all CVD deaths are attributable to 6 conditions: ischemic heart disease (IHD), stroke, hypertensive heart disease (which ultimately results in heart failure), cardiomyopathy, rheumatic heart disease (RHD), and atrial fibrillation (AF) ^{[4, 6, 7]}. The treatment methods of CVD mentioned above generally include drug therapy, interventional methods and surgical techniques that improve the health of patients from different perspectives and mechanisms ^[8]. Among them, drug therapy is one of the main ways to treat CVD. In recent years, with the wide application of high and new technologies and biotechnology in drug research, natural medicines show great potential in the application and discovery of cardiovascular drugs ^[9].

Astragalus membranaceus has been widely studied and has been shown to have important pharmacological effects, such as protecting injured myocardium, inhibiting ventricular remodelling and regulating blood pressure ^[10]. However, the components of Astragalus membranaceus are complex and varied and more than 200 compounds have been identified from Astragalus membranaceus ^[11]. The major active extracts include flavonoids, triterpene saponins and polysaccharides ^{[11, 12]}. Astragalus membranaceus are a rich source of flavonoids. Studies have shown that flavonoids from Astragalus membranaceus have multiple physiological effects, such as antioxidant, antitumor, anti-inflammatory, anti-osteoporotic effects, and so on ^[13]. Especially, flavonoids that serve as potent antioxidants can inhibit the production of reactive oxygen species (ROS) and free radicals that are responsible for many human diseases ^[14].

Currently, exploring the pharmacological effects of a single bioactive component for the treatment of cardiovascular diseases and validating the specific therapeutic target are hot research topics. Calycosin and its derivatives are the main bioactive flavonoids in Astragalus membranaceus ^[15]. Studies have confirmed that calycosin has multiple biological effects, such as antioxidant ^[16], pro-angiogenesis ^[17], anti-tumour ^[18], and anti-inflammatory ^{[19, 20]} effects. Based on the above biological effects, current research has been increasingly focused on the protective effects of calycosin on cells and organs. For example, a recent study showed that calycosin has a protective effect against high-fat diet-induced liver damage ^[21]. In the nervous system, calycosin can activate the transient receptor potential canonical 6 (TRPC6) pathway and other protective proteins to alleviate cerebral ischaemia-reperfusion injury and other injuries ^[22]. Furthermore, the protective effect of calycosin on the cardiovascular system has also received attention. It has been shown, preliminarily, that calycosin can antagonize hypoxic injury in myocardial ischaemia and increase the myocardial survival rate. However, existing studies have only focused on the preliminary role of calycosin in ischaemic diseases, myocardial hypertrophy and viral myocarditis, while comprehensive and in-depth experimental studies have not been carried out to elucidate its molecular mechanism and determine its effects on other types of myocardial damage.

In recent years, studies on the effects of calycosin on the cardiovascular system have been carried out, and calycosin has been shown to have good potential for cardiovascular protection. Therefore, this review focuses on the bioactivity of calycosin in cardiovascular diseases and reviews previous studies of calycosin to provide a theoretical basis for further research and clinical application.

Oxidative stress is an unbalanced state of ROS production and abnormal regulation of the endogenous antioxidant mechanism ^[23]. ROS production is affected by multiple factors, including dysfunction of oxidases, such as xanthinoxidase and nicotinamide adenine dinucleotide phosphate (NADPH), transport dysregulation in mitochondria, microsomes and/or the nucleus, neutrophil activation, arachidonic acid metabolism, and auto-oxidation of catecholamines, flavonoids, quinones, and proteins, etc. ^[24]. Mitochondria are considered the main source of ROS production in high-metabolic organs such as the heart. Mitochondrial calcium activates the dehydrogenases in the Krebs cycle involved in oxidative phosphorylation and maintains a low level of nicotinamide adenine dinucleotide (NADH) accumulation, thus contributing to cellular energy balance and maintaining adequate cardiac function ^[25]. In cardiac diseases, especially in heart failure, this process is disrupted, and mitochondrial Ca²⁺ uptake disorders eventually lead to NADPH oxidation and ROS production and accumulation ^{[26, 27]}. In addition, ROS can also be produced through the metabolism of drugs and exogenous substances, such as anticancer drugs, among which doxorubicin, which is related to the development of cardiomyopathy, is the most representative ^[28]. Oxidative stress is an important common mechanism of myocardial injury caused by various aetiologies, such as hypertension, hyperglycaemia, ischaemia-reperfusion injury, and chemotherapy drugs, and is closely related to the subsequent occurrence and development of cardiac failure. Abnormally elevated ROS can cause many negative effects on cardiac function ^[29]. In myocardial tissues, ROS directly damages the electrophysiology and contractile structure of myocardial cells by modifying the core proteins of the excitation-contraction coupling mechanism, including L-type calcium channels, sodium channels, potassium channels and sodium/calcium exchangers, and causes an energy deficiency in cardiac myocytes by affecting the function of proteins involved in energy metabolism ^[23]. ROS leads to the activation of multiple signalling pathways related to cell death, cardiac fibroblast proliferation, matrix metalloproteinase activation, mitochondrial deoxyribonucleic acid (DNA) damage, mitochondrial dysfunction, impaired calcium regulation and cardiac hypertrophy ^[30-33]. These effects eventually lead to abnormal adaptive myocardial remodelling and cardiac dysfunction ^[34]. In addition, recent studies have proposed that ROS can directly modify micro ribonucleic acid (microRNA), leading to changes in protein expression levels and the disruption of downstream gene regulation in corresponding tissues, thus leading to proteome remodelling and metabolic changes in cardiac tissues ^[35-37]. In addition to abnormally regulating related biomolecules and signaling pathways, ROS can also trigger cascade reactions of oxidative stress and expand the oxidative stress injury mentioned above, through producing other types of bioactive molecules (aldehydes), ROS induced ROS release and ROS mediated paracrine signals ^{[36, 38]}.

The abnormal increase in ROS and clearance barriers are the main factors of oxidative stress injury, while flavonoids have been shown to be the main effective components in Astragalus membranaceus responsible for antioxidation and free radical elimination ^[12]. Recently, studies have adopted electron spin resonance, chemiluminescence and other methods to test the bioactivity of anti-superoxide anion radicals in a few major types of flavonoids. Studies have shown that the antioxidant activity of different flavonoids by the order of strength is quercetin > kaempferol > isorhamnetin > isoquercitrin > rutin > calycosin > vitamin C > (3R)-8, 2′-dihydroxy 7, 4′-dimethoxyisoflavanone > formononetin > ononin > 2′, 4′-dimethoxy-3′-hydroxyl-isoflavanone-6-O-β-glucoside > (6aR, 11aR)-10-hydroxyl-3, 9-dimethylnissolin > (6aR, 11aR) 9, 10-dimethylnissolin-3-O-β-D-glycoside ^[39]. These results suggest that calycosin has relatively strong antioxidant activity and is also superior to standard antioxidant vitamin C. Recent studies have more accurately discriminated, from the perspective of quantum chemistry, the antioxidant activity of four major flavonoids, from which it was confirmed that the antioxidant activity of calycosin is stronger than that of three other antioxidants (calycosin > calycosin-7-glucoside > formononetin > ononin) ^[15]. The antioxidant bioactivity of calycosin is related to its chemical structure. The C-ring Δ^{2, 3}-double bond and 4-carbonyl group in calycosin are all bioactive antioxidant groups. They can remove oxidative free radicals, reduce malondialdehyde (MDA) (lipid peroxidation product), protein carbonyl (amino acid oxidation product) and ROS levels, and increase superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px) levels. It can also enter the lipid bilayer of biological membranes to ameliorate lipid peroxidation induced by free radicals ^[40]. Further studies suggest that calycosin may stabilize the membrane structure and enhance membrane integrity, which can reduce the lipid fluidity of the cell membrane and hinder the diffusion of free radicals, thereby reducing oxidative damage in cells ^{[19, 41]}. Using biological experiments, it was also shown that calycosin has a good antioxidant effect and that its cellular protective effect in multiple pathological states is mainly antioxidative. Li et al. showed that calycosin has a significant inhibitory effect on H₂O₂-induced oxidative stress in human liver parenchymal cells and can significantly alleviate oxidative damage in hepatic cells ^[42]. Recent studies on the neuronal protective effects of calycosin indicate that calycosin can alleviate oxidative stress and the inflammatory response of neurons in the hippocampus of a mouse model of Alzheimer′s disease by activating the protein kinase C pathway, thereby improving cognitive function ^[43]. In addition, in myocardial injury caused by ischaemia and hypoxia, calycosin can activate the Akt/PI3K and Nrf2/HO-1 signalling pathways, with myocardial protective effects; these pathways are important in organism oxidation and antioxidative regulation ^[44]. Therefore, the antioxidative activity of calycosin may play an important role in its cardioprotective potential.

Cardiovascular diseases are a large group of pathophysiological states with multiple risk factors and aetiologies, but at the cellular level, various cardiovascular diseases often have a common basis of cell damage, such as oxidative stress in cardiomyocytes or endothelial cells, substrate metabolism and energy consumption, cell survival and apoptosis, and autophagy ^[45]. Therefore, the treatment of cardiovascular diseases mainly includes two directions: intervention for the aetiologies of cardiovascular diseases and protective measures for the injury effects (organ level, cellular level or molecular level). Based on the multiple biological effects of calycosin and its derivatives, the protective effect of calycosin on cardiovascular cells has been studied.

For in vitro cardiomyocytes affected by different injury factors (H₂O₂, hypoglycaemia, hypoxia, and viruses, etc.), calycosin and its derivatives exhibit a common effect, that is, to reduce cell damage, predominantly indicated by the release of myocardial enzymes, and to reduce apoptosis-dominated cardiomyocyte death levels; furthermore, calycosin may play an anti-injury role through multiple mechanisms. For example, in H9C2 cells under hypoxic and hypoglycaemic conditions, pretreatment with calycosin can reduce the released level of the cell damage indicator lactate dehydrogenase (LDH) (Table 1), improve the cell survival rate, and increase the protein expression levels of phosphorylated Akt/PI3K, Nrf-l, Nrf-2, and HO-1, which suggests that the myocardial protective effect of calycosin in this process is related to the direct activation of the PI3K/Akt and Nrf-2/HO-1 pathways ^[44]. In addition, these pathways are important in cell proliferation and in the balance of oxidation and antioxidation. In another study, the cellular protective effect of calycosin is considered to be related to its phytoestrogen-like effects and the study first demonstrates that calycosin can inhibit the oxidative stress-induced apoptosis of cardiac muscle cells (H9C2 cells) in vitro and the protective effect on the heart occur through oestrogen receptor α (ERα) binding to ERβ and upregulating its expression, thereby activating the Akt signalling pathway ^[46]. For infective myocardial injury, calycosin can inhibit coxsackie virus replication in African green monkey kidney heteroploid cell (Vero cells), and its antiviral activity and specificity are all superior to the traditional antiviral drug ribavirin (antiviral activity analysis showed that its half-maximal inhibition (IC₅₀) was 25 μg·mL⁻¹ and that the ratio of cytotoxicity to antiviral activity was 5.7), suggesting that it plays a role in cell protection through direct antiviral effects ^[47].

In recent years, to further explore the myocardial protective effect and mechanism of calycosin, increasingly more in vivo experiments have provided more accurate and reliable evidence from different aspects.

Myocardial ischaemia-reperfusion injury is the main pathological of ischaemic heart disease develops and is associated with the development of myocyte necrosis, arrhythmia, myocardial stunning, endothelial dysfunction, and microvascular complications, etc ^[48-50]. Calycosin has been shown to have a good protective effect on ischaemic and reperfused myocardium: On the one hand, calycosin can improve cardiac function after ischaemia. For example, it increases the ejection fraction (EF), fractional shortening (FS), and left ventricular end systolic pressure (LVESP) and decreases left ventricular end diastolic pressure (LVEDP) (Table 2). Furthermore, it can induce the expression of vascular endothelial growth factor (VEGF) and CD31 and promote the angiogenesis of ischaemic myocardium ^[51]. On the other hand, oxidative stress is an important mechanism for myocardial ischaemia-reperfusion injury. Calycosin can significantly reduce the level of MDA, the lipid oxidation product of the cell membrane. It also increases the level of the protective antioxidant product SOD, thus inhibiting the release of myocardial enzymes including creatine kinase (CK) and LDH, etc ^[52-54]. The P13K/Akt signalling pathway is considered an important pathway involved in the regulation of cardiomyocyte apoptosis and myocardium tissue protection. In addition, calycosin can increase the protein phosphorylation level of P13K and Akt during ischaemia-reperfusion injury, suggesting that calycosin may exert a myocardial protective effect through activating the P13K/Akt signalling pathway in ischaemia-reperfusion injury ^[52].

In addition to its protective effect on acute ischaemic injury, calycosin also has a protective effect on some maladaptive changes in myocardial tissue. In a mouse model of myocardial hypertrophy constructed by isoproterenol induction and aortic constriction, calycosin significantly reduced markers of cardiac hypertrophy (atrial natriuretic peptide and myosin heavy chain-β) and inhibited myocardial remodelling. Additionally, the activation of the mitogen-activated protein kinase (MAPK) and AKT signalling pathways in mouse myocardium tissue was significantly attenuated, suggesting that calycosin may reduce cardiac hypertrophy induced by isoproterenol or aortic coarctation by inhibiting AKT/GSK3β and extracellular signal-regulated kinase (ERK) signalling ^[55].

For infective myocardial injury, calycosin showed multiple positive effects, such as anti-viral, anti-apoptosis and anti-inflammatory properties. In a mouse model of coxsackie virus B3 infection, calycosin-7-O-β-D-glucopyranoside (CCGR) treatment increased myocardial contractile force and cardiac EF. In addition, the progression of heart failure in infected mice was inhibited by increasing the systolic ventricular septal thickness, left ventricular posterior wall thickness and left ventricular volume. Overall, the symptoms of viral myocarditis were reduced, and the survival rate was improved (77.7% in the treatment group and 44.4% in the non-treatment group). This study further suggested that CCGR could play a protective role in myocardial tissue in viral myocarditis by significantly reducing viral titres, reducing myocardial cell oedema and inhibiting myocardial cell necrosis and monocyte infiltration caused by viral infection ^[47].

The influence of calycosin on vascular endothelial cells has multiple effects and multiple mechanisms. First, calycosin, as a phytoestrogen, can bind to ER-positive cells in the vascular endothelium and exert its role as a selective oestrogen receptor modulator (SERM) by increasing ERK1/2 phosphorylation and activating the MAPK signalling pathway, thereby promoting vascular endothelial cell proliferation ^[57]. Second, calycosin can interfere with ion channels. It has been shown that calycosin has a vasodilating effect, which is not endothelium-dependent and independent of intracellular calcium ion release. Calycosin is a non-competitive calcium channel blocker that primarily blocks voltage-gated calcium ion channels and receptor-gated calcium channels ^[58]. Tseng ^[59] et al. demonstrated that calycosin could activate the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels of human umbilical vein endothelial cells, thereby increasing the hyperpolarization of endothelial cells and the production of nitric oxide and mediating endothelium-dependent vasodilation. In addition, calycosin also has protective effects on endothelial cell injury induced by various factors. For endothelial cell injury induced by bacterial endotoxin, calycosin can increase the production of nitric oxide, reduce phosphorylated myosin light chain, reduce Rho/Rock signalling pathway activation-induced cytoskeleton remodelling by inhibiting the AKT signalling pathway, and exert protective effects on endothelial cells ^[60]. Calycosin can also reduce the cytotoxicity and apoptosis of human umbilical vein endothelial cells induced by vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor II (VRI), which is related to the activation of the PI3K/Akt/Bad and BRAF/MEK1/2/ERK1/2 signalling pathways ^[61]. However, for human umbilical vein endothelial cell apoptosis induced by glycation end products, calycosin can directly increase the expression of anti-apoptotic protein Bcl-2 and simultaneously reduce the expression levels of pro-apoptotic proteins Bax and Bad, thereby inhibiting endothelial cell apoptosis ^{[62, 63]}.

In addition to these protective effects on endothelial cells, calycosin can also directly affect endothelial cells (Fig.1). Calycosin-7-glucoside has therapeutic effect on angiotensin II (Ang II)-induced renin-angiotensin-aldosterone system (RAAS) disorder in human umbilical vein endothelial cells by down-regulation of angiotensin-converting enzyme (ACE) expression and increased ACE2 expression ^[64]. Calycosin can inhibit the synthesis of Ang II-induced thromboxane A2 (TXA2) and prostacyclin (PGI2) in endothelial cells and reduce Ang II-induced endothelial cell injury. In addition, calycosin can inhibit the secretion and expression levels of endothelial cell inflammatory factors induced by tumor necrosis factor-α (TNF-α), such as intercellular adhesion molecule-1 (ICAM-1) and its receptor lymphocyte function-associated antigen 1 (LFA-1), thus protecting endothelial cells ^{[65, 66]}.

Figure 1.  Schematic chart of the effects and mechanisms of calycosin on vascular endothelial cells

The pathophysiological mechanism of many cardiovascular diseases is closely related to the inflammatory response. Previous studies have shown that the inflammatory response plays an important role in the occurrence and progression of cardiovascular diseases ^[67]. In recent years, it has been found that calycosin and its derivatives have potential anti-inflammatory effects. Recent studies have revealed that the inhibition of the NF-κB signalling pathway may be the main mechanism of calycosin’s anti-inflammatory effects ^{[68, 69]}. By inhibiting the expression of NF-κB and MAPK signalling pathway-related proteins, calycosin-7-glucoside can reduce the production of pro-inflammatory factors including Prostaglandin E2 (PGE2), TNF-α, interleukin-1β (IL-1β) and interleukin-6 (IL-6) in endotoxin-induced macrophagocytes and inhibit the mRNA expression levels of inflammatory mediators inducible Nitric Oxide Synthase (iNOS) and cyclooxygenase-2 (COX-2) ^[70]. However, studies on the anti-inflammatory effects of calycosin mainly focus on the diseases of the haematologic system, immune system and neurological system. There is no definitive research on the effects of calycosin on the inflammatory response in cardiovascular diseases.

Ischemic heart disease and thromboembolic disease are common types of cardiovascular diseases. Platelets play a crucial role in the development of these diseases by participating in thromboembolism development ^[71]. Thus, anti-platelet is often a crucial part of cardiovascular disease therapy ^[72]. Studies have shown that calycosin and its derivatives are the main active ingredients in many traditional Chinese medicine compounds widely used in cardiovascular diseases ^[73-76]. For example, calycosin-7-O-β-D-glucoside as one of main active ingredients of BuyangHuanwu decoction (BHD) can inhibit adenosine diphosphate (ADP)-induced platelet aggregation in vitro ^[73]. In addition, the core bioactive components of compound xueshuantong capsule (CXC) act on different aspects of the vascular system to promote blood circulation. Among them, calycosin-7-O-β-D-glucoside has been proved to mainly affect extrinsic clotting activity and to have negative effects on red blood cell (RBC) aggregation, RBC deformability, intrinsic clotting activity and platelet aggregation ^[74]. Although the above studies reveal the effect of calycosin on vascular system, the mechanism underlying it functional activity requires further systematic research.

Calycosin is characterized by low toxicity and diverse biological effects ^[77] and is a monomer compound with a definitive chemical structure, making it more conducive to research and application in pharmacology. Calycosin and its derivatives have been shown to have multiple effects, such as antioxidant, anti-tumour, angiogenesis regulation and anti-inflammation properties. Therefore, calycosin has attracted much attention in many fields, such as tumour treatment ^[78] and nervous system rehabilitation ^[22], but there have been few studies on its cardiovascular pharmacological effects. In fact, in recent years, related research has suggested that calycosin and its derivatives have good cardiovascular protective potential. However, existing studies have only focused on the role of calycosin in ischaemic disease, myocardial hypertrophy and viral myocarditis, while the pathological conditions including drug-induced myocardial injury and metabolism-related myocardial injury have not been studied. Second, in the intervention studies of calycosin on various types of myocardial injury, the drug routes and dosages are different; therefore, the pharmacokinetics of calycosin and the correlation between dosage and myocardial protective strength deserve further study. In addition, research on the mechanisms by which calycosin exerts its effects are mostly limited to changes in protein expression level and lack more accurate and in-depth research on signalling pathways and regulatory mechanisms. For example, the main pharmacological effect of calycosin on cardiovascular protection is reducing oxidative stress; however, the specific molecular mechanism of its antioxidative effect is not clear. In addition, calycosin has shown pro-apoptotic effects in tumour studies and anti-apoptotic effects in myocardial protection. Calycosin may have different effects on the corresponding signalling pathways in different pathological conditions. Therefore, the biological effects of calycosin should still be further studied, which could help to further understand its pharmacological characteristics and improve the value of its clinical application.