Resveratrol inhibiting LPS-induced ferroptosis in HT-29 cells by activating Sirt1/Nrf2 pathway

Objective: To investigate the regulatory effect and potential mechanism of resveratrol (RSV) on ferroptosis in ulcerative colitis (UC). Methods: This study was divided into two parts. The first part was network pharmacology analysis of RSV. Protein-protein interaction (PPI), GO, KEGG and GVAS enrichment analysis were used to explore the internal relationship among RSV, UC and ferroptosis. Reverse transcription-quantitative PCR (RT-qPCR) was used to verify the selected common target genes at the cell level. The second part was cell experiments. HT-29 cells were treated with 10 μg/mL lipopolysaccharide (LPS) for 24 h to establish an in vitro model of UC. In addition, the cells were pretreated with RSV (25 μmol/L or 50 μmol/L) for 6 h, and SIRT1 inhibitor EX527 (10 μmol/L) for 4 h in addition to RSV (50 μmol/L) intervention. HT-29 cells were divided into control group, LPS group, low dose RSV group, high dose RSV group and EX527 group. First, malondialdehyde (MDA), glutathione (GSH), total iron ion kit, RT-qPCR, enzyme-linked immunosorbent assay (ELISA), western blotting and other detection techniques were used to detect the effects of RSV on the expresion of oxidative stress (MDA, GSH), inflammatory factors (TNF-α, IL-6), ferroptosis related indicators (total iron ion, GPX4, ACSL4) and pathway factors (SIRT1, NRF2). Then western blotting was used to detect the effect of EX527 on the expression of GPX4, ACSL4, Sirt1, and Nrf2 proteins in cells. Results: The results of network pharmacology showed that RSV, ferroptosis and UC interacted at multiple biological levels, and RSV promoted the expression of SIRT1 mRNA. The results of cell experiments showed that compared with the LPS group, RSV down-regulated the expression of pro-oxidant index (MDA), inflammatory factors (TNF-α, IL-6), total iron ion and negative regulator of ferroptosis (GPX4), and up-regulated the expression of positive regulator of ferroptosis (ACSL4) and pathway indicators (SIRT1, NRF2). Compared with the RSV high concentration group, EX527 down-regulated the protein expression of pathways (Sirt1, Nrf2) and negative regulator of ferroptosis (GPX4), and up-regulated the protein expression of positive regulator of ferroptosis (ACSL4). Conclusion: RSV can inhibit ferroptosis and inflammation in HT-29 cells, and its mechanism may be related to the activation of Sirt1/Nrf2 pathway.