Atractylenolide I alleviates LPS-induced pulmonary cell injury relevant to pediatric pneumonia via the ERK/USP9X/Mcl-1 and Akt/Survivin axes.

Pediatric pneumonia remains a leading cause of childhood mortality worldwide, and lipopolysaccharide (LPS)-induced pulmonary cell injury plays a critical role in its pathogenesis. Atractylenolide I (ATL-1), a sesquiterpene lactone derived from Atractylodes macrocephala Koidz., possesses well-documented anti-inflammatory properties. However, its protective effects against LPS-induced lung cell injury remain unclear. In this study, WI-38 human embryonic lung fibroblasts and BEAS-2B human bronchial epithelial cells were stimulated with LPS to establish complementary in vitro models of pulmonary cell injury relevant to pediatric pneumonia. ATL-1 dose-dependently restored cell viability, inhibited apoptosis, and suppressed the production of IL-6, TNF-α, and IL-1β. Mechanistically, ATL-1 upregulated both Mcl-1 and Survivin through distinct regulatory mechanisms. ATL-1 restored ERK signaling to upregulate the deubiquitinase USP9X, which inhibited Mcl-1 ubiquitination and enhanced its protein stability. Meanwhile, ATL-1 restored Akt activity to promote Survivin expression at the transcriptional level. Knockdown of Mcl-1, Survivin, or USP9X abolished the protective effects of ATL-1. Similarly, pharmacological inhibition of ERK or Akt reversed ATL-1-mediated cytoprotection. Upstream, ATL-1 reduced MD2-TLR4 association and attenuated MD2/TLR4/MyD88/TRAF6/NF-κB signaling. CETSA and SPR analyses supported the interaction between ATL-1 and MD2. Time-course and NF-κB loss-of-function experiments further showed that suppression of sustained NF-κB signaling preceded and contributed to the restoration of ERK- and Akt-dependent pro-survival pathways. MD2 depletion mimicked and largely occluded the downstream effects of ATL-1. These findings demonstrate that ATL-1 alleviates LPS-induced cell injury and inflammatory responses through the coordinated restoration of the ERK/USP9X/Mcl-1 and Akt/Survivin axes. This study provides mechanistic insight into ATL-1's cytoprotective effects and supports further evaluation in additional models of pneumonia-associated lung injury.