Tianxiangdan (TXD) alleviates myocardial ischemia reperfusion-induced ferroptosis through the activation of estrogen receptor alpha (ERα)

Abstract

Tianxiangdan (TXD), a traditional Chinese herbal remedy, demonstrates efficacy in mitigating myocardial ischemia-reperfusion (I/R)-induced damage. This study employed network pharmacology to evaluate the therapeutic targets and mechanisms of TXD in treating I/R. High-performance liquid chromatography-mass spectrometry (HPLC-MS) identified 86 compounds in TXD. Network pharmacological analysis predicted potential target genes and their modes of action. 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. Estrogen receptor alpha (ERα) protein expression and estradiol (E2) levels were measured to confirm TXD's impact on estrogen levels and ERα expression. To examine if TXD reduces I/R injury through ERα, an AZD group (300 nmol·L⁻¹ AZD9496 and 15% TXD serum) was compared to a TXD group (15% TXD serum). The study hypothesized that TXD upregulates the ERα-mediated iron metamorphosis pathway. I/R injury-induced ferroptosis was identified using a Fer-1 group (1.0 μmol·L⁻¹ Fer-1 and 15% TXD serum) to elucidate the potential association between ferroptosis and ERα proteins. A DCFH-DA probe detected reactive oxygen species (ROS) and Fe²⁺, while Western blotting assessed target protein expression. Both in vitro and in vivo experiments demonstrated that TXD attenuated I/R injury by reducing elevated ST-segment levels, improving cardiac injury biomarkers (LDH, MDA, and SOD), alleviating pathological features, and preventing I/R-induced loss of cell viability in vitro. The effects and mechanisms of TXD on I/R injury-associated ferroptosis were investigated using I/R-induced H9c2 cells. The TXD group showed significantly decreased ROS and Fe²⁺ levels, while the AZ group (treated with AZD9496) exhibited increased levels. The TXD group demonstrated enhanced expression of ERα and glutathione peroxidase 4 (GPX4), with reduced levels of P53 protein and ferritin-heavy polypeptide 1 (FTH1). The AZ group exhibited contrasting effects on these expression levels. The literature indicated a novel connection between ERα and ferroptosis. TXD activates the ERα signaling pathway, promoting protection against I/R-induced myocardial cell ferroptosis. This study provides evidence supporting TXD use for myocardial ischemia treatment, particularly in older female patients who may benefit from its therapeutic outcomes.