Ginsenoside Rh4 Triggers Ferroptosis in Lung Cancer: Targeting KEAP1/NRF2/HO-1 and Remodeling Gut Microbiota for Butyrate-Mediated ATF3 Activation

Lung cancer progression is regulated by multiple factors, including ferroptosis and gut microbiota-mediated butyrate metabolism. This study investigates the anti-tumor effects of ginsenoside Rh4 on lung cancer cells via ferroptosis mechanisms in vitro and in vivo. In vitro, ginsenoside Rh4 inhibited the proliferation of Lewis lung carcinoma (LLC) and A549 cells and triggered ferroptosis, effects that were suppressed by the ferroptosis inhibitor Ferrostatin-1 (Fer-1). In vivo, tumor-bearing mouse models were established and treated with 100 mg/kg ginsenoside Rh4 for 21 days. Tumor growth, ferroptosis markers, gut microbiota, and butyrate were analyzed, with in vitro validation of butyrate's pathway effects. Ginsenoside Rh4 induced ferroptosis in LLC cells both in vitro and in vivo, inhibiting tumor growth. It promoted ferroptosis by disrupting iron homeostasis through elevated Fe²⁺ and transferrin receptor (<i>TFRC</i>), and impaired antioxidant defense via depletion of glutathione (<i>GSH</i>) and reduction in ferritin heavy chain 1 (<i>FTH1</i>), solute carrier family 40 member 1 (<i>SLC40A1</i>), solute carrier family 7 member 11 (<i>SLC7A11</i>), and glutathione peroxidase 4 (<i>GPX4</i>). Additionally, ginsenoside Rh4 enhanced lipid peroxidation, indicated by increased lipid peroxides (<i>LPO</i>) and malondialdehyde (<i>MDA</i>). In vivo, it suppressed the <i>KEAP1</i>/<i>NRF2</i>/<i>HO-1</i> pathway, reducing antioxidant enzyme activity. Gut microbiota modulation and butyrate production further amplified ferroptosis by activating transcription factor 3 (<i>ATF3</i>)-mediated <i>GPX4</i> suppression. Ginsenoside Rh4 induces ferroptosis by inhibiting the <i>KEAP1</i>/<i>NRF2</i>/<i>HO-1</i> pathway and remodeling the gut microbiota to increase butyrate levels, which synergistically enhance tumor cell ferroptosis sensitivity through <i>ATF3</i> activation and suppression of <i>GPX4</i>.