3’-hydroxypuerarin mitigates LPS-induced acute lung injury by inhibiting TLR4 activation-mediated NF-κB p65/NLRP3/GSDMD signaling

Introduction: (Willd.) Ohwi, possesses significant pharmacological activities, but its effects on ALI remain limited. Methods: LPS-induced RAW264.7 macrophage inflammatory injury were carried out using ELISA kits, RT-qPCR, immunofluorescence, Western blotting, molecular docking, and molecular dynamics simulation analyses. Results: 3HP significantly reduced IL6 and TNF-α levels in bronchoalveolar lavage fluid (BALF) and serum, attenuated pulmonary edema, inhibited the mRNA levels of chemokines and inflammatory factors in lung tissues, and suppressed the expression of IL1β, IL6, TNF-α, HMGB1, TLR4, MyD88, p-IκBα (S32/S36), p-NF-κB p65 (S536), COX2, iNOS, ICAM1, VCAM1, NLRP3, ASC, Caspase-1, Cleaved Caspase-1 (Ala317) p10, NEK7, Caspase-8, IL18, GSDMD, and GSDMD N-terminal both in lung tissues and in RAW264.7 cells, indicating that 3HP inhibited LPS-stimulated TLR4 activation, thereby reducing IκBα phosphorylation and degradation, and preventing NF-κB p65 nuclear translocation to mediate the transcription and expression of inflammatory mediators. It also inhibited NLRP3 inflammasome activation, decreased Caspase-1 cleavage of GSDMD, and lowered the release of the pore-forming GSDMD N-terminal structural domain, blocking the immune response to pyroptosis. Moreover, the NLRP3 inhibitor MCC950 enhanced the effect of 3HP on acute cellular inflammatory injury. Notably, molecular docking and dynamics simulation revealed that 3HP stably bound to TLR4. Resatorvid (TAK-242), a selective TLR4 signaling inhibitor, significantly enhanced 3HP's inhibitory effect on TLR4, further indicating that 3HP may block the NF-κB p65/NLRP3/GSDMD signaling pathway by inhibiting TLR4 activation in response to LPS stimulation. Conclusions: Our results demonstrated that 3HP mitigates LPS-induced ALI by inhibiting TLR4 activation-mediated NF-κB p65/NLRP3/GSDMD signaling. These findings provide scientific evidence for the clinical treatment of ALI and present new insights into the pharmacological role of 3HP in mitigating acute lung inflammatory diseases.