CS often occurs during periods of systemic infection such as sepsis(28). Plague infections lead to major epidemics and trigger alveolar macrophages to produce excessive amounts of cytokines, leading to CS(29). The main feature of CS is increased levels of pro-inflammatory cytokines such as IL-6 and TNF-α(30). A dramatic increase in these pro-inflammatory factors can lead to multi-organ failure and ultimately death(31, 32). Anti-IL-6 receptor antibodies, as one of the therapeutic tools for treating CS, can effectively inhibit IL-6 secretion and reduce the inflammatory response(33). However, side effects such as infection, hepatotoxicity, hypertriglyceridemia and diverticulitis limit the therapeutic effect of anti-IL-6 receptor antibodies(34). Anti- TNF-α drugs are theoretically effective in inhibiting TNF-α levels, but there are insufficient data on the therapeutic(35, 36). Current means of treating cytokine storms have limitations, and the search for new therapeutic agents is particularly urgent. Traditional Chinese medicine may have potential in the treatment of inflammatory diseases.
As a natural phytosterol, β-sitosterol has abundant pharmacological effects. Haria and colleagues found that β-sitosterol can regulate the expression of chemokine genes, attenuate excessive inflammatory responses, and have good anti-inflammatory effects(37). However, the detailed mechanism of β-sitosterol's anti-inflammatory effects remains unclear and needs to be further explored. In our study, we demonstrated that β-sitosterol exerted anti-inflammatory effects by regulating macrophage function. Specifically, β-sitosterol prevented the overactivation of RAW264.7 macrophages by activating autophagy.
IGF1R is a transmembrane tyrosine kinase that is widely expressed in almost all mammalian cells and widely expressed in macrophages(38). IGF1R is mainly involved in proliferation, differentiation and metabolism. Therefore, more and more studies are focusing on IGF1R as a potential therapeutic target(39). IGF1R deficiency reduces inflammation, tumor proliferation and growth(40). In this study, the preinvestigation by network pharmacology and molecular docking revealed that β-sitosterol binds very stably to IGF1R protein. The results suggested that the anti-inflammatory effect of β-sitosterol may be related to the inhibition of IGF1R protein activity. The mechanism of action is similar to that of the IGF1R protein inhibitor Linsitinib.
There is growing evidence that autophagy is involved in clearing invading pathogens and maintaining the balance of the inflammatory response after induction by various pathogens or their associated substances, such as viral DNA or LPS. Inadequate autophagy does lead to uncontrolled infection and excessive inflammation, both of which are major contributors to the development of sepsis(41). Indeed, autophagy is considered a potential therapeutic target in sepsis because of its great capacity to eliminate abnormal intracellular proteins and its role in the quality control of multiple organelles(42). In this study, we went through network pharmacology to predict potential targets of β-sitosterol for CS treatment, and the results suggested that β-sitosterol may manage CS through autophagy. Following this, we observed a significant increase in the autophagy level of cells treated with β-sitosterol through MDC and TEM experiments. Western blot analysis further indicated that β-sitosterol augmented the conversion rate of LC3 I to LC3 II and concurrently hindered the degradation of p62. All these results suggested that β-sitosterol could manage CS by inducing autophagy.
Based on the results of the appeal study, we hypothesized that these anti-inflammatory mechanisms of β-sitosterol may contribute to the clinical application of β-sitosterol in the treatment of inflammatory diseases. LPS is a bacterial endotoxin found in gram-negative bacteria and commonly use to induce inflammation in vivo and in vitro experiments(43, 44). Mice were injected intraperitoneally with LPS to induce a sepsis model. In our study, we observed that treatment with β-sitosterol attenuated lung tissue congestion, edema, exudation, and fibrosis, and reduced lung injury, as indicated by the histological results of H & E staining of lung tissue in the β-sitosterol-treated group compared with the LPS group. In addition, β-sitosterol treatment effectively reduced the expression levels of pro-inflammatory cytokines (IL-6, TNF-α, IL-1β) in BALF of the LPS group. In addition, β-sitosterol treatment reduced the secretion of pro-inflammatory cytokines (IL-6, TNF-α) and ROS production by macrophages after LPS stimulation.
In conclusion, this study suggested that β-sitosterol could inhibit LPS-induced inflammation and oxidative stress and alleviate LPS-induced sepsis. Mechanistically, β-sitosterol may activate autophagy and reduce inflammation by inhibiting IGF1R activity. The findings provide partial support for the potential candidacy of β-sitosterol in sepsis treatment. However, the current study possesses certain limitations, notably the absence of an in-depth exploration of the underlying mechanism and the unclear impact on relevant pathways, highlighting areas for future investigation.