Based on Network Pharmacology and Experimental Validation: β-Sitosterol Inhibits Cytokine Storm by Inducing Autophagy

β-Sitosterol, a prominent phytosterol abundant in Chinese medicinal plants like Hemerocallis and Astragalus, possesses diverse beneficial properties, including anti-inflammatory, anti-tumor, and anti-oxidative effects. Despite these recognized benefits, its precise anti-inflammatory mechanism remains elusive. In our investigation, we explored β-Sitosterol's impact on immune responses. In vitro experiments utilizing lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages demonstrated significant suppression of interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) production alongside reduced reactive oxygen species (ROS) levels, indicating its potential as an anti-inflammatory agent. Transitioning to in vivo experiments, β-Sitosterol effectively attenuated cytokine storm (CS), characterized by pulmonary inflammatory infiltration and elevated IL-6, interleukin-1β (IL-1β), and TNF-α levels. Autophagy, a pivotal cellular process in anti-inflammatory responses, emerged as a potential mediator of β-Sitosterol's action, as indicated by network pharmacology analyses. Molecular investigations revealed that β-Sitosterol promoted autophagic activity by enhancing the conversion of microtubule-associated protein 1A/1B-light chain 3 I (LC3I) to LC3II and facilitating p62 degradation. Notably, inhibition of autophagy reversed β-Sitosterol's anti-inflammatory effects, highlighting its dependence on this process. Furthermore, molecular docking studies unveiled stable binding between β-Sitosterol and the insulin-like growth factor 1 receptor (IGF1R), suggesting its potential modulation of CS through IGF1R. In summary, β-Sitosterol could exert anti-inflammatory effects by inhibiting IGF1R activity and enhancing autophagy, offering insights into its therapeutic potential and paving the way for novel disease-targeting formulations.