3. Results
3.1. Lycorine attenuates LPS-induced ALI in mice
In this study, HE staining was used to evaluate pathological changes. ELISA was used to detect MPO activity and inflammatory factors. As shown in Fig. 1A, CG histopathological analysis showed standard lung structure. The lung slices of mice after LPS stimulation showed typical histological changes, including inflammatory cell infiltration, thickening of alveolar walls, interstitial edema, and pulmonary congestion (Fig. 1E). However, 20, 40, and 80 groups significantly alleviated the historical changes caused by LPS administration (Fig. 1B-D). And the higher the concentration of lycorine, the more significant the effect. It is shown in Fig. 1F. MPO activity was significantly increased in the LPS group compared with CG. The content of MPO in Groups 20, 40, and 80 was lower than the LPS group but still higher than CG. The sandwich ELISA method measured the concentration of TNF-α, IL-1β, and IL-6 in lung tissue. Compared with CG, stimulating cells with LPS without lycorine treatment will significantly increase cytokine production (Fig. 1G-I). After the cells were treated with lycorine (20, 40, 80 mg/L), TNF-α, IL-1β, and IL-6 in the LPS group were significantly lower than in the LPS group. The binding capacity of lycorine to LXRα was calculated using the molecular docking technique.
3.2. Effect of lycorine on TLR4/NF-κB signaling pathway and lipid raft
We used the immunofluorescence method to analyze membrane lipid rafts and p65 protein. Western Blot and qPCR detected the level of proteins and mRNA related to the TLR4/NF-κB signaling pathway. The result is shown in Fig. 2. The level of lipid raft in lung tissue of mice in the LPS group was significantly higher than that in the CG. the degree of lipid raft in the group treated with lycorine was reduced considerably compared with the LPS group (Fig. 2A-F). As shown in Fig. 2G-I, mRNA levels of TLR4, IκBα, and NF-κB p65 were not entirely different among all groups. Fig. 2J-Q shows the protein expression of each group. Compared with CG, the protein expression of TLR4 in the LPS group was increased, and the phosphorylation levels of IκBα and NF-κB p65 were significantly increased. Compared with the LPS group, the phosphorylation level and TLR4 expression level of histones 20, 40, and 80 decreased successively. Translocation of p65 protein in lung tissue of mice in the LPS group was significantly higher than that in the CG (Fig. 2R-W). The translocation of p65 protein in the group treated with lycorine was considerably reduced compared with the LPS group. The results showed that lycorine could destabilize lipid rafts and reduce LPS-induced inflammatory response.
3.3. Effects of lycorine on membrane cholesterol and LXRα signal.
Lycorine interacts with LXRα mainly through hydrogen bond formation and hydrophobic force. It forms a hydrogen bond with His419 (B); the hydrogen bond length is 2.98 A. It has a hydrophobic effect with Met296 (B), Trp441 (B), Ile293 (B), Ala259 (B), Leu297 (B), Thr300 (B), Leu329 (B), Phe255 (B), Phe252 (B) and Val423 (B). The binding energy of lycorine to LXRα was -8.2kcal/mol, which proved that lycorine had a good binding effect (Fig. 3A). Cholesterol content was detected using the Nanjing Jiancheng cholesterol testing kit. Western Blot and qPCR were used to detect LXRα signal-related protein and mRNA levels. Cholesterol levels did not change significantly in the LPS group compared with CG but significantly decreased in the lycorine group (Fig. 3B). The protein expressions of LXRα, ABCA1 and ABCG in the lycorine group were significantly increased compared with CG and LPS groups (Fig. 3C). The higher the lycorine dose was, the more significant the effect was (Fig. 3D-F). As shown in Fig. 3G-I, the mRNA levels of LXRα increased in both LPS and lycorine groups (20, 40, 80) compared with CG. The mRNA of ABCA1 and ABCG were significantly increased in the lycorine group compared with CG and LPS. The results showed that lycorine up-regulated the expression of LXRα. And it activated ABCA1 and ABCG pathways and promoted the leakage of cholesterol.
3.4. Lycorine inhibits the secretion of inflammatory factors by regulating TLR4/NF-κB signaling pathway
The mRNA levels of TNF-α, IL-1β, and IL-6 in A549 cells and the concentrations of TNF-α, IL-1β, and IL-6 in cell culture supernatant were detected. LPS stimulation of cells without lycorine treatment resulted in a significant increase in mRNA levels and concentrations of cytokines compared to CCG. The mRNA levels and contents of TNF-α, IL-1β, and IL-6 in the lycorine group (C20, C40, C80) were significantly lower than those in the LPS group. The concentrations of TNF-α, IL-1β, and IL-6 in the supernatant of the A549 culture were determined by sandwich ELISA. LPS stimulation of cells without lycorine treatment resulted in a significant increase in cytokine production compared to CCG. The levels of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in the lycorine group (C20, C40, and C80) were significantly lower than those in the LPS group (Fig. 4A-B). As shown in Fig. 4C, there was no significant difference in mRNA expression of TLR4, IκBα, and NF-κB p65 among all groups in cells. The protein expression levels of each group are shown in Fig. 4D-K. Compared with CG, the expression of TLR4 protein and the phosphorylation levels of IκBα and NF-κB P65 were significantly increased in the LPS group. Compared with the LPS group, the phosphorylation level and TLR4 expression level of C20, C40 and C80 decreased successively. The results showed that lycorine could reduce the inflammatory response induced by lipopolysaccharide by regulating TLR4/NF-κB signaling pathway.
3.5. Lycorine activates LXRα signal by increasing LXRα activity
In this study, qPCR and Western blot detected mRNA and protein levels of LXRα, ABCA1, and ABCG. Luciferase gene assay was used to determine whether lycorine could enhance LXRα activity. The result is shown in Fig. 5. LXRα mRNA and protein levels in lycorine group were not significantly increased compared with CCG (Fig.s 5A and 5E). The mRNA and protein expressions of ABCA1 and ABCG were increased dramatically in a dose-dependent manner compared with the control group (Fig. 5B-C, 5E). The activity of LXRα in the lycorine group was significantly increased compared with that of CCG, and the effect was more significant with increasing dose (Fig.s 5D). The protein ratio analysis of LXRα, ABCA1, and ABCG is shown in Fig. 5F-H. The results showed that lycorine activated the LXRα signal and promoted protein expression of ABCA1 and ABCG by enhancing LXRα transcriptional activity.
3.6. The anti-inflammatory effect of lycorine was weakened by inhibiting LXRα activity
In this study, we detected intracellular LXRα activity, protein levels of cholesterol, LXRα signaling pathway, TLR4/NF-κB signaling pathway, and inflammatory factors after the addition of GSK2033. The result is shown in Fig. 6. After the addition of GSK2033, the activity of LXRα in the lycorine group was significantly inhibited compared with CCG, but the cholesterol content was increased considerably(Fig. 6A-B). The protein level of LXRα in lycorine group was significantly increased compared with CCG (Fig. 6C-I). Protein expression of ABCA1 and ABCG was reduced considerably compared with CCG. The levels of IκBα and NF-κB p65 and phosphorylation were significantly increased. As shown in Fig. 7J, the contents of TNF-α and IL-1β increased considerably, and the range of IL-6 was significantly increased. The results showed that the anti-inflammatory effect of lycorine was weakened after the activity of LXRα was inhibited.