When myocardial injury occurs, oxidative stress, cell apoptosis, Ca2+ overload, and other conditions result in irreversible damage to myocardial cells [35]. Myocardial ischemic injury is a complex phenomenon associated with several independent etiologies, such as genetic factors, obesity, environmental factors, and gender factors. Clinical studies have found that the incidence of cardiovascular diseases in males is significantly higher than that in females, and there is no significant difference between the incidence of cardiovascular diseases in postmenopausal females and males [36]. Low estrogen levels and postmenopausal women are at higher risk of cardiovascular disease than normal women [37]. The gender differences in cardiovascular disease presentation are consistent with the network pharmacological prediction of the TXD against myocardial ischemia, which focuses our study on the mechanism of the TXD's anti-myocardial ischemia action on the estrogen signaling pathway. The molecular docking results of the TXD active components and genes on the estrogen pathway showed that the complex formed after the docking of isoflavones was the most stable, which suggested that the active components of the TXD against myocardial ischemia were isoflavones.
Cell death occurs after ischemic injury, resulting in an energy crisis in cells, swelling, and rupture of organelles, which is then followed by cell apoptosis that is characterized by chromatin concentration and caspase activation. Caspases are central regulators of apoptosis, and a member of this family (Caspase-3) is the critical mediator of neuronal cells apoptosis [38]. After Caspase-3 activation, apoptosis cannot be prevented. Activation of the apoptotic pathway and an imbalance in cell survival factors determine cell survival and death, and oxidative stress is the main stimulus that triggers apoptosis and death of necrotic cardiomyocytes. Oxidative stress also disrupts intracellular signals associated with ROS generation and activates apoptotic regulatory proteins to mediate a series of cellular responses causing increased rates of apoptosis [39]. After H2O2-induced cell injury, cell apoptosis rate and ROS level in the model group were significantly elevated. In contrast, TXD drug-containing plasma and E2 pretreatment group significantly suppressed cell apoptosis rate and ROS level. The results show that the TXD drug-containing plasma enhanced the activity of cardiomyocytes, improved the morphology of apoptosis, and reduced the rate of apoptosis and intracellular ROS levels against H2O2-induced oxidative stress injury.
Regardless of external or internal factors, Ca2+ accumulate following damage to myocardial cells. Intracellular Ca2+ is a crucial second messenger for excitation-contraction and excitation-transcriptional coupling of coronary smooth muscles, and regulates the proliferation, migration, and calcification of coronary smooth muscles [40]. Moreover, Ca2+ induces intercellular decoupling during myocardial ischemia, which marks the transition from reversible ischemic injury to irreversible ischemic injury. This leads to excessive intracellular and mitochondrial Ca2+ level, which causes cell contracture, protein lysis, and mitochondrial failure, and ultimately to necrosis or apoptotic death [41]. The fluorescence intensity of Ca2+ in the model group was significantly increased, while that of TXD drug-containing plasma and E2 pretreated cells was significantly decreased. The results revealed that TXD drug-containing plasma suppressed intracellular Ca2+ levels reduced oxidative stress damage to cells.
SOD plays an essential role in oxidation balance in the body. It catalyzes the reaction involved in reducing cell damage attributed to free radicals [42]. MDA is one of the lipid peroxidation products used to measure oxidative stress in vivo [43]. NO is a significant regulatory factor in the cardiovascular system that protects cardiomyocytes by regulating downstream protein cascade reactions [44]. We found that SOD content and NO level in the model group were suppressed, while MDA level was elevated. After TXD drug-containing plasma and E2 pretreatment, SOD content and NO level were found to be elevated, and MDA content was greatly suppressed. Therefore, TXD drug-containing plasma has antioxidant capacities and improves NO bioavailability.
The role of the estrogen signaling pathway is to activate related receptors and produce physiological effects. There are three cardiac estrogen receptors; ESR1, ESR2, and GPER1 [45]. ESR1 plays a more critical role in cardiovascular maintenance than ESR2 [46]. Estrogen regulates vascular function, inflammation, metabolism, and myocardial cell survival. It also mediates related effects in the cardiovascular system by activating estrogen receptors, altering gene transcription in the nucleus, and by activating kinase signal transduction in the cytoplasm. Besides, estrogen can also change the expression or activity of ion channels, contractile proteins, and ROS production to participate in the regulation of complex diseases. Studies have shown that women who use postmenopausal hormone replacement therapy are less predisposed to cardiovascular diseases when compared to women not using the therapy [47]. Hormonal therapy can be used for cardiovascular disease management [48], however, hormonal therapy alone is not ideal and even causes risks to the human body [49]. Besides, reducing the risk of cardiovascular diseases, phytoestrogens in alternative therapies do not cause adverse effects on the cardiovascular system [50]. E2 has been shown to regulate the PI3K/Akt signaling pathway by activating ESR1, thus affecting the expression level of sub-factors and increasing microvascular permeability [51]. In this study, the ESR1 level was significantly elevated in the TXD drug-containing plasma and E2 groups compared to the model group. This suggests that the TXD drug-containing plasma may activate the ESR1 receptor. Estrogen receptor blockers were not added in cell experiments due to the adverse effects of multi-drug acts on cell growth. The arrangement of estrogen receptor blockers is more reasonable in the later animal experimental model. Combined with the previous molecular docking results, the main components with strong binding ability in TXD are isoflavones. This implying that TXD isoflavone active ingredients may exhibit estrogen-like effects, which can activate estrogen receptors, thus directly regulating the estrogen signaling pathway to resist myocardial ischemic.
PI3K is a phosphatidylinositol kinase composed of the regulatory subunit P85 and the catalytic subunit P110. PI3K plays a key role in Akt, activating signaling cascades involved in cell growth, survival, effort, movement, and morphology. In cells, AKT is the main effector target gene downstream of the PI3K/Akt signaling pathway. It affects many different signal substrates, thus significantly impacting on cell growth, cell cycle, and metabolism. Activation of the PI3K/Akt signaling pathway regulates cell apoptosis, angiogenesis, and endothelial cell growth [52]. The TXD drug-containing plasma protects myocardial cells by inhibiting activation of the PI3K/Akt signaling pathway in H2O2-induced damaged cardiac cells. When the PI3K/Akt signaling pathway is inhibited, it affects cell apoptosis by regulating the expression of the subordinate target protein eNOS and improves cellular injury following oxidative stress. eNOS is one of the many downstream targets of AKT and plays an important role in NO production and NO-mediated angiogenesis. Activation of the PI3K/Akt/eNOS pathway leads to the continuous production of NO, which is important in angiogenesis. We found that, compared to the model group, eNOS expression level in myocardial cells was elevated, PI3K and AKT expression levels in myocardial cells were significantly suppressed after TXD drug-containing plasma and E2 pretreatment. These results imply that TXD drug-containing plasma and E2 reduced oxidative stress induced myocardial cell injury by regulating the PI3K/Akt/eNOS protein expression levels. Therefore, TXD isoflavone active ingredients may exhibit estrogen-like effects, activates the ESR1 receptor, and plays a therapeutic role in myocardial ischemia through the estrogen signaling pathways.