Baicalin alleviates intestinal ischemia-reperfusion injury by regulating ferroptosis mediated by nuclear factor E2-related factor 2/Glutathione peroxidase 4 signaling pathway.

Abstract

Baicalin, a predominant bioactive flavonoid derived from the traditional Chinese medicinal herb Scutellaria baicalensis Georgi, has garnered significant attention. Ferroptosis, a relatively novel form of programmed cell death, implicates critical signaling pathways, notably those involving nuclear factor E2-related factor 2 (Nrf2) and glutathione peroxidase 4 (Gpx4). Consequently, this study aims is to elucidate whether baicalin mitigates intestinal tissue damage by modulating the Nrf2-Gpx4 signaling pathway in the context of intestinal ischemia-reperfusion (II/R) injury, thereby influencing iron deposition. For this purpose it was established an II/R rat model and a cellular hypoxia-glucose deficiency/reoxygenation (OGD/R) model and administered BA to the II/R rats and OGD/R model cells. It was utilized HE staining and probe staining techniques to assess intestinal injury and iron overload, respectively and employed RT-qPCR to measure the mRNA expression levels of inflammation-related genes (interleukin-1β, interferon-γ, interleukin-4, and interleukin-10) and iron deficiency-related genes (Nrf2, Gpx4, and xCT). The cell counting kit-8 (CCK-8) assay is employed to assess cell viability, while fluorescent probes are utilized to evaluate mitochondrial membrane potential. Colorimetric methods are applied to quantify intracellular oxidative stress-related indicators (reactive oxygen species (ROS) and malondialdehyde (MDA)) levels. Flow cytometry is used to determine cell membrane lipid ROS levels. Immunofluorescence techniques are implemented to examine intestinal tight junction proteins, such as ZO-1 and Occludin. Additionally, protein immunoblotting is conducted to measure markers of iron deposition (Gpx4 and xCT). In in vivo studies, BA treatment mitigated intestinal damage in II/R rats, inhibited intestinal iron ion overload, and elevated inflammatory levels and oxidative stress. Furthermore, BA treatment reduced the loss of tight junction proteins ZO-1 and Occludin expression levels induced by II/R. In vitro studies demonstrated that BA significantly attenuated OGD/R-induced iron overload, decreased cell viability, and mitigated mitochondrial membrane potential loss. Mechanistically, BA exerts its protective effect against iron overload-induced cellular damage by activating the Nrf2-Gpx4 signaling pathway. However, these effect was significantly counteracted by the use of Nrf2-GPX4 pathway inhibitors. The positive regulation of Nrf2-Gpx4 by BA can reduce ferroptosis and alleviate II/R in rats.