Sepsis-associated acute lung injury (ALI) is a major contributor to mortality in critically ill patients. Disruption of intracellular quality-control processes, notably impaired autophagic flux, has been implicated in the pathogenesis of sepsis-associated ALI. Salidroside (Sal), a naturally occurring glucoside, has documented anti-inflammatory and cytoprotective properties. The authors used computational and experimental approaches to evaluate whether Sal can protect against sepsis-associated ALI and to identify candidate molecular mechanisms, focusing on the ERK/FoxO3a signaling axis and autophagy.
The investigation combined network pharmacology and molecular docking with preclinical laboratory experiments. Two sepsis-associated ALI models were used: an in vitro model employing lipopolysaccharide (LPS)-treated RLE-6TN alveolar epithelial cells, and an in vivo rat model produced by cecal ligation and puncture (CLP). Key techniques included western blotting and immunofluorescence to assess protein signaling and localization; Ad-mCherry-GFP-LC3B assays and transmission electron microscopy to evaluate autophagic flux and autolysosome formation; and JC-1 staining to examine mitochondrial membrane potential. Phorbol 12-myristate 13-acetate (PMA), an ERK activator, was applied to test mechanistic dependence on ERK signaling.
Computational analyses suggested that the ERK/FoxO3a signaling axis is a plausible target through which Sal may exert biological effects relevant to sepsis-associated ALI. The docking and network results guided selection of the ERK/FoxO3a pathway for subsequent experimental validation in cell and animal models.
In both LPS-treated RLE-6TN cells and the CLP rat model, Sal treatment was associated with reduced inflammatory responses and attenuation of lung injury. The reported outcomes included reductions in markers consistent with inflammation and histologic evidence of less severe lung damage in treated models compared with untreated sepsis models. These findings support an anti-inflammatory and tissue-protective role for Sal in sepsis-associated ALI.
Sepsis models exhibited biochemical features consistent with impaired autophagic flux, specifically an increased LC3-II/LC3-I ratio and accumulation of the autophagy substrate p62. Following Sal administration, p62 levels decreased and assays showed increased autolysosome formation, including results from the Ad-mCherry-GFP-LC3B reporter and corroborating ultrastructural evidence from transmission electron microscopy. Collectively, these changes were interpreted as improvement of impaired autophagic flux rather than further autophagy inhibition.
Biochemical analysis indicated that Sal reduced phosphorylation of ERK and was associated with greater nuclear localization of FoxO3a. These observations align with modulation of the ERK/FoxO3a axis as part of Sal’s mechanism of action in the studied models. The shift in FoxO3a localization suggests potential downstream transcriptional effects relevant to autophagy and cell survival pathways.
To probe causality, the authors used PMA, an ERK activator. PMA partially attenuated Sal’s effects on ERK/FoxO3a-related changes, autophagy-associated markers, and mitochondrial function. This partial reversal supports involvement of ERK signaling in mediating Sal’s protective actions, while indicating that additional pathways or mechanisms may also contribute.
Mitochondrial assessments using JC-1 staining indicated that Sal helped maintain mitochondrial function in the sepsis models. The protective effects on mitochondrial membrane potential were diminished when ERK was activated by PMA, providing further linkage between ERK/FoxO3a modulation, autophagy status, and mitochondrial integrity in the context of sepsis-associated ALI.
In preclinical in vitro and in vivo models of sepsis-associated ALI, Salidroside demonstrated anti-inflammatory and cytoprotective effects that were associated with decreased ERK phosphorylation, increased nuclear localization of FoxO3a, restoration of autophagic flux (evidenced by decreased p62 and increased autolysosome formation), and preservation of mitochondrial function. Mechanistic experiments using an ERK activator partially reversed these benefits, supporting a role for the ERK/FoxO3a signaling axis in Sal’s effects. The results are derived from laboratory models; translational relevance to human sepsis and clinical dosing, safety, or efficacy requires further study. Details such as specific dosing regimens, quantitative outcome measures, and statistical results were reported in the full article but are not reproduced in this abstract source.