4. Discussion
Lycorine, an alkaloid found in the bulbs of Lycoris Radiata, has been proven to have anti-inflammatory effects(de Queiroz Souza et al., 2021; Ge et al., 2020). ALI is a respiratory disease with a high fatality rate that can be caused by a variety of reasons(L. Li et al., 2021; Zhu, Wang, Xing, & Xiong, 2021). In this study, we performed many tests to analyze whether lycorine has a protective effect on LPS-induced ALI in mice(Qiu, Luo, & Fang, 2021; Yao, Xu, Jia, Li, & Wei, 2021; S. M. Zhu et al., 2021). The results showed that lycorine significantly reduced the degree of LPS-induced ALI. The results of pathological analysis and molecular biological analysis proved that lycorine has a protective effect on LPS-induced acute lung injury(Cui, Xu, Lv, & Guo, 2023).
Lipid rafts are signal transduction platforms on cell membranes. The increase or decrease of lipid rafts will have a pronounced influence on TLR4 (Amine, Benomar, & Taouis, 2021; da Cruz et al., 2021; Park, Cha, Lee, Kim, & Choi, 2021)After LPS stimulation of lung tissues and cells, TLR4 is recruited into lipid rafts to induce downstream signal transduction(Y. Chen et al., 2020; Wei et al., 2016). It has shown that TLR4 recruitment into lipid rafts can be inhibited using drugs by interfering with lipid raft formation (cholesterol consumption)(Dolganiuc, Bakis, Kodys, Mandrekar, & Szabo, 2006). It reduces lipopolysaccharide-induced NF-κB activation and inflammatory cytokine production(Kang et al., 2020; R. Xu et al., 2023; T. Xu, Cui, Xu, Cao, & Guo, 2023). Our results show that lycorine can inhibit the transfer of TLR4 to lipid rafts by decreasing the formation of lipid rafts via consuming cholesterol. Next, we investigated the cause of lycorine lowering cholesterol levels in lipid rafts. We used AutoDock software to calculate the binding capacity of lycorine to LXRα. The results showed that lycorine had an excellent combining ability with LXRα. The results showed that LXRα plays a vital role in regulating cholesterol homeostasis. LXRα mediates the expression of ABCA1 and ABCG to regulate intracellular cholesterol levels.
We tested our hypothesis in cells that lycorine plays an anti-inflammatory role by inhibiting the activation of the TLR4/NF-κB signaling pathway through lipid rafts. The human non-small cell lung cancer cell line (A549) is the primary constituent cell of lung tissue. In this study, LPS stimulation of A549 induces the activation of the TLR4/NF-κB signal pathway and promotes the secretion of inflammatory cytokines, which play an essential role in the process of ALI. This is similar to previous studies (Sivanantham et al., 2019). The results showed that TNF-α, IL-1β, and IL-6 were significantly increased in ALI of mice. We found that lycorine inhibits TNF-α, IL-1β, and IL-6 production in a dose-dependent manner. TLR4/NF-κB signal pathway plays a crucial role in inducing cytokine production. Our results show that lycorine inhibits LPS-induced cytokine production by inhibiting the activation of the TLR4/NF-κB signal pathway.
Therefore, we examined the effects of lycorine on the activation of LXRα, ABCA1, and ABCG. The results showed that lycorine induced the expression of ABCA1 and ABCG by activating LXRα. Is this pathway related to the anti-inflammatory effects of lycorine? We performed an LXRα activity inhibition assay. The results showed that lycorine altered the expression of ABCA1 and ABCG, membrane cholesterol levels, and LPS-induced inflammatory when LXRα activity was inhibited. It confirmed that inhibition of LXRα activity could reduce the anti-inflammatory effect of lycorine. These results indicate that lycorine binding to LXRα may promote the activation of LXRα. It confirmed that inhibition of LXRα activity could reduce the anti-inflammatory effect of lycorine. Lycorine depletes cholesterol by promoting the activation of LXRα. Cholesterol depletion causes lipid raft instability. It inhibits TLR4 transport to lipid rafts and LPS-induced inflammation.
In summary, our research shows that lycorine can inhibit the expression of TNF-α, IL-1β, and IL-6 in A549 cells stimulated by LPS. The anti-inflammatory mechanism of lycorine is related to the up-regulation of the LXRα-ABCA1/ABCG pathway, which leads to the consumption of cholesterol and reduces the transfer of TLR4 to lipid rafts thereby inhibiting the inflammatory response induced by LPS.