Total Pulsatilla saponins attenuate early LPS-induced lung inflammation in association with modulation of cGAS–STING signaling and ferroptosis-related responses

Background: Total Pulsatilla saponins (PSS), a saponin-enriched preparation derived from Pulsatilla chinensis (Bunge) Regel, have shown anti-inflammatory potential. Here, we investigated the effects of PSS on early LPS-induced lung inflammation and characterized associated changes in cGAS–STING signaling and ferroptosis-related responses in vivo and in LPS/erastin-stimulated BEAS-2B cells. Methods: Male BALB/c mice were challenged intratracheally with LPS and subsequently treated with PSS (10, 20, or 40 mg/kg) or dexamethasone; lung and serum samples were collected 6 h after LPS administration. Histopathological changes, inflammatory cytokines, lung transcriptomic profiles, serum metabolomic profiles, and selected molecular and biochemical parameters were evaluated. BEAS-2B cells were pretreated with PSS and then exposed to LPS plus erastin. Pharmacological STING inhibition with H-151, siRNA-mediated STING knockdown, and ferrostatin-1 treatment were used as complementary approaches to characterize the relationship between STING signaling and ferroptosis-related responses under these experimental conditions. Results: PSS attenuated early LPS-induced pulmonary inflammatory changes, as reflected by less pronounced histopathological alterations and reduced IL-6, IL-1β, and TNF-α levels in lung tissue and serum. Transcriptomic and metabolomic analyses identified changes in innate immune signaling and circulating lipid profiles associated with PSS treatment. PSS also reduced cGAS abundance and p-STING, p-TBK1, and p-IRF3 signals, together with lower CXCL10 and ISG15 expression. PSS partially restored SLC7A11, GPX4, and FTH1 expression, decreased Fe²⁺ and MDA levels, and preserved GSH, consistent with attenuation of oxidative changes associated with ferroptosis. In LPS/erastin-challenged BEAS-2B cells, PSS and Fer-1 reduced lipid ROS accumulation, increased GPX4 abundance, and attenuated the loss of cell viability. These findings support a ferroptosis-sensitive component of cellular injury under the co-challenge condition. However, the cellular experiments do not distinguish the individual contributions of LPS and erastin and therefore do not establish that LPS alone induces ferroptotic epithelial injury. Conclusions: In a short-duration LPS mouse model, PSS attenuated early pulmonary inflammatory responses, while in LPS/erastin-challenged BEAS-2B cells it modulated ferroptosis-related responses. These findings support an association between PSS treatment, cGAS–STING signaling, and ferroptosis-related changes, but do not establish a STING-dependent ferroptosis mechanism or imply therapeutic efficacy in human ALI/ARDS. Graphical Abstract: https://links.lww.com/AHM/A246