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).