3.4 Leonurine inhibited ROS generation and lipid peroxidation and restored the expression of ferroptosis-related genes
Free iron can interact with ROS to form lipid radicals that rapidly react with oxygen to induce lipid peroxidation. We first investigated intracellular ROS using the sensitive fluorescent probe DCFH-DA, revealing that ROS generation was induced by RSL3 but reversed by leonurine (Fig. 4A). In addition, we used the Liperfluo probe to detect lipid peroxides and found that lipid peroxidation was increased by RSL3 treatment but decreased by leonurine (Fig. 4B). To further confirm that the leonurine-regulated ferroptosis blockade was associated with lipid peroxidation, we examined the cellular MDA and GSH levels. RSL3 treatment significantly downregulated GSH and upregulated MDA, and these trends were reversed by leonurine treatment (Fig. 4C to 4D). Next, we further evaluated the regulatory effect of leonurine on the expression of antioxidant and ferroptosis-related target molecules. RSL3 treatment significantly decreased the GPX4 and xCT expression compared to that in the control group, but leonurine restored these changes in a dose-dependent manner (Fig. 4E-F). Moreover, leonurine dose-dependently upregulated the protein expression of Nrf2, NQO1 and HO-1 (Fig. 4G).