Abstract
ObjectiveIn this study, we mainly explored the mechanism and target of the anti-inflammatory effects of Aureusidin (Aur) in acute liver injury.MethodsLipopolysaccharide (LPS) was used to induce inflammatory injury in Kupffer cells (KCs) in vitro. After Aur treatment with gradient concentration, flow cytometry, propidium iodide (PI) staining, and Hoechst 33342 staining were used to detect the apoptotic level of KCs, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of inflammatory factors, including Interleukin-1β (IL-1β), Interleukin-18 (IL-18), and tumor necrosis factor alpha (TNF-α). Western blot was used to detect the expression of toll-like receptor 4 (TLR4), myeloid differentiation protein-2 (MD2), MyD88, and p-P65. Aur was labeled with biotin, followed by a pull-down assay to detect its binding with MD2. Moreover, D-GalN/LPS was used to induce acute liver injury in mice in vitro, followed by Aur treatment by gavage. H&E staining was used to detect the pathological changes of liver tissue, an IF assay was used to detect the expression of MD2, Western blot was used to detect the expression of relevant proteins.ResultsAur pretreatment could significantly inhibit LPS-induced KC injury, downregulate the apoptotic level, inhibit the expression of inflammatory factors, decrease the level of MDA, and downregulate the expression of MD2 in cells. Aur could inhibit the activation level of TLR4/MD2-NF-κB in a dose-dependent pattern, a high dose of Aur had a superior effect compared to low-dose Aur. In the case of MD2 deletion, the effects of Aur were suppressed. Additionally, pull-down and co-immunoprecipitation assays show that Aur can bind with the MD2 protein to inhibit the activation of TLR4/MD2-NF-κB. Results of mice experiments also showed that Aur could relieve liver injury, decrease the levels of ALT and AST, and simultaneously downregulate the levels of inflammatory factors in tissues and peripheral blood.ConclusionWe found that Aur exerted an anti-inflammatory effect by directly targeting the MD2 protein, further inhibiting the expression of TLR4/MD2-NF-κB, thereby relieving acute liver injury. Therefore, Aur might be a potential inhibitor for MD2.
Highlights
The lipopolysaccharide (LPS)-mediated inflammatory response plays an important role in the pathogenesis and progression of various diseases, but without a currently effective control approach (Santos et al, 2018; Yang et al, 2018)
The detection of superoxide dismutase (SOD) and MDA revealed that LPS can decrease the level of SOD and increase the level of MDA; while Aur can increase the SOD level and decrease the MDA level, which were significantly different compared with the LPS group, with significantly superior effects in the high-dose group than the low-dose group (P
propidium iodide (PI) staining and Hoechst 33342 staining showed that LPS treatment could significantly induce the apoptosis of KCs, with an upregulated level of positive cells; the number of positive cells was significantly downregulated in the Aur group than the LPS group (P
Summary
The lipopolysaccharide (LPS)-mediated inflammatory response plays an important role in the pathogenesis and progression of various diseases, but without a currently effective control approach (Santos et al, 2018; Yang et al, 2018). Myeloid differentiationprotein 2 (MD2) is an important accessory protein of toll-like receptor 4 (TLR4); MD2 can assist the binding of LPS and TLR4 along with TLR4 and MyD88, thereby activating inflammatory signaling pathways (Fitzgerald et al, 2004; Schnabl et al, 2008). Under the assistance of CD14, TLR4 and LPS form a complex with MD2. The dimerization of the complex on the cell membrane and conformational changes of the intracellular segment can cause intracellular signals, eventually leading to the conversion transcription factors, including NF-kB and AP-1 (Kurt-Jones et al, 2000), thereby inducing the expression of downstream inflammatory factors, chemokines, and adhesion factors. Macrophages are important effector cells for the TLR4 signalingmediated inflammatory response (Oak et al, 2006; Zhang et al, 2008). How to inhibit the inflammatory response of macrophages is the focus of research
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