Tianxiangdan Alleviates Myocardial Ischemia Reperfusion-Induced Ferroptosis through the Activation of ERα

Tianxiangdan (TXD) is a conventional Chinese herbal remedy with effective properties to reduce myocardial ischemia-reperfusion (I/R)-induced damage. This study employed a network pharmacology method to assess the therapeutic targets and pharmacological mechanisms of TXD for treating I/R. A total of 86 compounds in TXD were identified using HPLC-MS. Furthermore, a network pharmacological analysis was conducted to predict potential target genes and their modes of action. Moreover, cardiac function, ischaemic ST changes, lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD) activity, myocardial fiber, and infarct size were assessed using in vivo and in vitro I/R injury models. Additionally, estrogen receptor alpha (ERα) protein expression and estradiol (E2) levels were also measured to confirm the impact of TXD on estrogen levels and ERα expression. To examine if TXD reduces I/R injury through ERα, the AZD group (consisting of 300 nM AZD9496 and 15% TXD serum) was compared to the TXD group (comprising only 15% TXD serum). It was hypothesized that TXD upregulates the ERα-mediated iron metamorphosis pathway. Therefore, the I/R injury-induced ferroptosis was identified using the Fer-1 group (treated with 1.0 μM Fer-1 and 15% TXD serum) to elucidate the potential association between ferroptosis and ERα proteins. Moreover, the DCFH-DA probe was used the detect reactive oxygen species (ROS) and Fe^{2 +} . Whereas, the expression of various target proteins was assessed via western blotting. The results of both in vitro and in vivo experiments demonstrated that TXD attenuated I/R injury by affecting several parameters, such as by reducing the elevated levels of ST-segment, improving cardiac injury biomarkers (including LDH, MDA, and SOD), and alleviating pathological features, and by preventing I/R-induced loss of cell viability in vitro. The effects and potential mechanisms of TXD on I/R injury-associated ferroptosis were investigated using I/R-induced H9c2 cells. The TXD group showed a significant decrease in ROS and Fe^{2 +} levels, whereas the AZ group (treated with AZD9496) exhibited a significant increase in these levels. Moreover, the TXD group had notably enhanced expression of ERα and glutathione peroxidase 4 (GPX4), whereas the levels of P53 protein and ferritin-heavy polypeptide 1 (FTH1) were reduced. Conversely, the AZ group exhibited contrasting effects on these expression levels. The literature indicated a novel connection between ERα and ferroptosis. The ERα signaling pathway is activated by TXD, promoting a protective effect against I/R-induced myocardial cell ferroptosis. Overall, this study provides evidence supporting the use of TXD for myocardial ischemia treatment, particularly in older female patients who may benefit from its therapeutic outcomes.