Nobiletin attenuates LPS-induced acute lung injury via a STING-dependent signaling pathway

Previous studies have shown that Quzhou Fructus Aurantii extract (QFAEE) ameliorates lipopolysaccharide (LPS)-induced acute lung injury (ALI) by modulating the Stimulator of interferon genes (STING) pathway. Nevertheless, the mechanisms underlying the anti-inflammatory effects of nobiletin (Nob), the primary bioactive component of QFAEE, have not been clearly elucidated. The present study aimed to investigate the protective effects of Nob on a mouse model of LPS-induced ALI and elucidate its underlying mechanism. The pulmonary inflammatory response in mice was quantified by performing fluorescence-based RT‒qPCR, bronchoalveolar lavage fluid (BALF) cell counting, and hematoxylin‒eosin (HE) staining. Transcriptomic profiling was performed to explore the mechanisms by which Nob ameliorates LPS-induced ALI. The anti-inflammatory mechanisms were further elucidated using western blotting, immunohistochemical staining, cellular thermal shift assays (CETSAs), and microscale thermophoresis. Functional validation was conducted using the STING agonist vadimezan (DMXAA), the STING inhibitor SN-001, and STING-knockout (STING-KO) mice. Nobiletin-loaded liposomes (Nob-Lipo) significantly attenuated LPS-induced pulmonary inflammation in mice. The results of the transcriptomic analysis suggested that this protective effect was associated with the modulation of the type I interferon pathway. In RAW264.7 and THP-1 cells, pretreatment with Nob effectively attenuated the interferon regulatory factor 3 (IRF3)-mediated type I interferon response and nuclear factor kappa-B (NF-κB)-dependent proinflammatory cytokine expression following DMXAA-induced STING pathway activation. Furthermore, the STING inhibitor SN‑001 markedly suppressed LPS‑induced inflammatory responses, as evidenced by significantly reduced levels of proinflammatory cytokines and interferon‑stimulated genes, and effectively blocked the nuclear translocation of NF‑κB and IRF3. Notably, after inhibitor treatment, Nob no longer exerted its previously observed anti‑inflammatory effects, further supporting the critical regulatory role of STING in LPS‑mediated inflammation and indicating that the anti‑inflammatory activity of Nob is primarily dependent on the STING pathway. Moreover, in STING‑KO mice with LPS‑induced ALI, the loss of the protective effect of Nob further confirmed that its anti‑inflammatory activity depends on STING, highlighting the essential role of STING signaling in mediating its protective effects. In conclusion, the protective effect of Nob on an LPS-induced ALI model is mediated primarily through the modulation of STING signaling.