Pyroptosis was suppressed by 20-hydroxyecdysone through Lin28b-mediated let-7d in vascular endothelial cells.

20-hydroxyecdysone (20E), a natural polyhydroxylated steroid, has exhibited anti-inflammatory effects across various diseases. This study investigates the potential connection between 20E's anti-inflammatory properties and the RNA-binding protein Lin28b, which is notably upregulated in TNF-α-stimulated endothelial cells. Herein, we discovered that 20E can selectively downregulate Lin28b expression without affecting its paralog Lin28a. Notably, 20E treatment could significantly attenuate pyroptosis, an inflammatory form of programmed cell death, as evidenced by reduced IL-1β and LDH release, and fewer propidium iodide (PI)-positive cells. Subsequent protein analysis revealed that 20E inhibited the enhanced expressions of key pyroptosis-associated proteins, GSDMD, GSDMD-N, and GSDME-N. Besides, this suppression of Lin28b and pyroptosis may be partially mediated through TNFR1. Additionally, 20E upregulated let-7 miRNA, particularly let-7d, a critical downstream target of Lin28b. To elucidate the role of Lin28b in 20E-mediated pyroptosis attenuation, we performed Lin28b overexpression and silencing experiments. Overexpressing Lin28b negated 20E's inhibition of LDH release and PI-related fluorescence, exacerbating GSDMD and GSDME cleavage. Conversely, Lin28b knockdown augmented the suppressive effect of 20E on pyroptosis, which was reversed by a let-7d inhibitor. Co-transfection with let-7d mimic and Lin28b plasmid demonstrated let-7d's role in mitigating pyroptosis aggravated by Lin28b overexpression. Overall, this study demonstrates that 20E may mitigate GSDMD and GSDME-mediated pyroptosis in HUVECs through the Lin28b/let-7d-dependent signaling pathway. These results highlight the potential of 20E as a promising inhibitor of pyroptosis, offering new insights into its anti-inflammatory mechanisms.