AbstractBackground and Purpose:Lycorine is an alkaloid that was in the bulb of the genus Lycoris. It has properties of anti-inflammatory. This study aimed to investigate the molecular mechanism by which lycorine can reduce acute lung injury (ALI).Experimental Approach: ALI model was established by intranasal injection of lipopolysaccharide (LPS). In vitro, A549 cells were treated with LPS and pretreated with lycorine for 1 hour.Key Results: The results showed that lycorine reduced histopathological changes in lung, myeloperoxidase (MPO) activity, and the production of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in mice. Lycorine dose-dependently inhibited the production of TNF-α, IL-1β, and IL-6. It also inhibited the transmission of TLR4/NF-κB passway in LPS-stimulated A549 cells. Lycorine increased cholesterol efflux through the activated LXRα-ABCA1/ABCG pathway. Lycorine has a good binding ability with LXRα. After adding the LXRα inhibitor, the anti-inflammatory effect of lycorine was eliminated.Conclusion and Implications: Lycorine can reduce ALI that was induced by lipopolysaccharide. The anti-inflammatory mechanism of lycorine is related to the up-regulation of the LXRα-ABCA1/ABCG pathway, which inhibits TLR4-mediated inflammation by increasing cholesterol efflux and reducing TLR4 transport to lipid rafts.Keywords: Lycorine; ALI; Lipid raft; TLR4; LXRαIntroductionAcute lung injury (ALI) is a high morbidity and mortality disease. In recent years. Many people were died of ALI due to influenza and COVID-19(J. G. Zhang et al., 2021). ALI is the damage of alveolar epithelial cells and capillary endothelial cells caused by multiple direct or indirect injury factors. Alveolar epithelial cells are widely present in lung tissue. The damage of alveolar epithelial cells will cause diffuse pulmonary interstitial fibrosis and swelling(Shi et al., 2014). Lipopolysaccharide (LPS) is one of the important causes of ALI(Hu et al., 2021). LPS expressed its effects through the TLR family in cell membranes. The TLR family was related to inflammatory cytokines(Gross et al., 2020; Vitiello et al., 2021). TLR4 played an important role in natural immunity. LPS can promote the formation of lipid rafts. After LPS stimulation, TLR4 was recruited into the lipid raft(Kaelberer, Caceres, & Jordt, 2020). It interacted with some molecules on the lipid raft. It could activate the NF-κB signaling pathways and promoted the production of cytokines(Liao, Liu, & Zhang, 2021; S. Xu et al., 2023). Lots of cytokines caused the body’s inflammatory response, leading to multiple organ failures in severe cases(H. C. Wang, Wu, & Kong, 2021).The production of inflammatory factors required regulating signaling pathways(Alikiaii, Bagherniya, Askari, Johnston, & Sahebkar, 2021; Novoselova et al., 2015). Most signals were passed through something on the cell membrane, and lipid rafts were one of them(D’Aprile, Prioni, Mauri, Prinetti, & Grassi, 2021). Lipid rafts were the platform for protein docking, closely related to membrane signal transduction and protein sorting(Holani et al., 2020; Suzuki, 2012). The stability of lipid rafts played an important role in the TLR4/NF-κB pathway(Adebiyi, Soni, John, & Yang, 2014; B. Liu et al., 2018; Zhang et al., 2023). It was showed that the destruction of lipid rafts in lung epithelial cells could inhibit inflammatory response by LPS-induced(Colardo et al., 2021; Singh et al., 2021). The destruction of lipid rafts was associated with cholesterol metabolism on the cell membrane. Previous studies had found that cholesterol metabolism was related to nuclear receptors, liver X receptor (LXR), and had a regulatory effect on lipid metabolism. LXRs (Liver X Receptor α and β) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors (Hammer et al., 2021). Activation of LXRs could induce gene expression related to cholesterol excretion, such as ABCG(Song, Yan, Wang, & Lou, 2021; Wan et al., 2021). LXRs regulated the cholesterol metabolism of cells, affected the inflammatory response, and inhibited the expression of inflammatory genes(Kongkwamcharoen, Itharat, Pipatrattanaseree, & Ooraikul, 2021). It may be related to the pathogenesis of ALI. Currently, there is no very effective way to treat this disease. Some herbal medicines have been found to have anti-inflammatory effects and may be used to treat ALI.Lycorine is an alkaloid in the bulb of Lycoris Radiata, a plant of the family Lycoris Radiata. It has effective anti-inflammatory, anti-viral and anti-tumor effects (H. Chen et al., 2020; Li et al., 2022; M.-H. Li et al., 2021). Does lycorine have a similar anti-inflammatory effect on ALI? Could it be used as a medicine in the clinical treatment of ALI? Previous studies had shown that lycorine can induce HSC-3 cell apoptosis and inhibit cell proliferation (W.-y. Liu et al., 2019). In addition, it has been found that lycorine can increase the production of reactive oxygen species (ROS) and trigger mitochondrial membrane potential (MMP) disorders(Jing, Zhang, Li, & Gao, 2020; Shang, Jang, Shi, & Ma, 2021). It was also found that lycorine significantly inhibited the expression of CXCL1 and IL-1α in the senescence-associated secretion phenotype (SASP) of SIPS cells and slowed down senescence(Y. Xu, Li, Li, Deng, & Gao, 2023; W. N. Zhang et al., 2021). However, few reports show lycorine can reduce lung injuries caused by inflammation and oxidative stress. Whether lycorine has a protective effect on lung injury needs further study. Lycorine could reduce the inflammatory response of LPS-induced by destroying the lipid rafts was not yet clear. This mechanism remains to be explored.Materials and methodsReagentsLycorine (L812279) was purchased from MACKLIN reagent, China. LPS (S11060) was purchased from Yuanye biotech, Shanghai. UItraRIPA kit for Lipid Raft (KA6023) was purchased from Bioleaf, Shanghai. The primers were purchased from Sangon Biotech, Shanghai. The antibodies were purchased from CST, USA. CCK-8 (BS350B) kit was purchased from Biosharp, China. MPO assay kit (EHJ-45871m) was purchased from HUIJIA BIOTECHNOLOGY, China. The ELISA kit of IL-1β, IL-6, and THF-α was purchased from LunChangShuo Biotech, China. LXR-Luc(11515ES) was purchased from Yeasen Bio, China. A549 cells were donated by Zhang Naisheng’s team at Jilin University. GSK2033 (SML1617) was purchased from Sigma Aldrich, USA.2.2. Animals and groupsFifty C57 mice (6 weeks of age, 18-25 g) were divided equally between male and female. All mice were randomly divided into 5 groups: control group (CG), LPS group (LPS), and LPS+lycorine (20, 40, 80 mg/kg) group (20, 40, 80). LPS-induced acute lung injury and administration were as follows: In brief, ALI was induced by inhalation of 50 μl 2 mg/mL LPS through the nose in each mouse after injected lycorine (20, 40, 80 mg/kg) or saline intraperitoneally(F. Wang et al., 2009). The drug was injected twice in total intraperitoneally at 12-hour intervals. The control group was injected with saline. The lycorine group was injected with different doses of lycorine hydrochloride solution (20, 40, 80 mg/kg). All mice survived within 36 h after LPS intranasal infusion. All mice were raised at room temperature and anaesthetized with sodium pentobarbital 24 hours after the second intraperitoneal injection. They died of cervical dislocation and were quickly sampled and store in the -80 °C refrigerator. All the procedures in the present study were carried out following the Animal Care and Use Committee of Northeast Agricultural University (SRM-16).2.3. Histology analysisThe fresh lung tissue of the mice was cut into 2mm-3mm tissue pieces, fixed with 10% neutral phosphate-buffered formalin, dehydrated and transparent, and then immersed in paraffin and cut into 3-5micron paraffin tissue sections. After staining with hematoxylin and eosin, the pathological changes in lung tissues were observed with a light microscope.2.4. Detection of myeloperoxidase contentThe double antibody sandwich method was used to determine the content of myeloperoxidase (MPO) in the sample. Coat the microtiter plate with purified mouse MPO antibody to make a solid phase antibody. Add MPO to the microporous of the coated monoclonal antibody, and then combine with the HRP-labeled MPO antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, add substrate TMB for color development. We calculated the content of MPO according to the OD value.2.5. Immunofluorescence analysis of lipid raftsDewaxing paraffin sections to water. The tissue sections were placed in a repair box filled with EDTA antigen repair buffer (PH8.0) and repaired in a microwave oven. Excessive evaporation of buffer solution should be prevented during this process. BSA was dropped onto the tissue sections and incubated for 30min. The BSA was gently removed, and the prepared flotillin-1 antibody was dropped onto the sections. The sections were placed flat in a wet box at 4°C and incubated overnight. Add secondary antibody and incubate at room temperature for 50min. After the slices were shaken dry, DAPI dye was added to the ring and incubated for 10min at room temperature, away from light. The self-quenching agent was added for 5min, and the water was rinsed for 10min. Tablets were sealed with anti-fluorescence quenching tablets. The sections were photographed under a fluorescence microscope.2.6. Extraction of cell membrane proteinAdd appropriate volume of ice-cold A-buffer to tissue samples (final concentration, 5 mg/ml total protein). Sonication is recommended to completely disrupt tissue debris and avoid contamination of the nucleus. Transfer tissue lysate to 1.5 mL tubes. Centrifuge samples at 10000 rpm for 5 min. Transfer supernatant to another tube. Add 0.5 mL of ice-cold A-buffer into the pellet (RIPA-insoluble fraction) and vigorously re-suspend the pellet with pipetting or voltas. Centrifuge samples at 10000 rpm for 5 min. Remove supernatant and add 100 μL of B-buffer into the pellet and vigorously re-suspended the pellet with pipetting or voltas at room temperature (option: sonication can be available on ice). Incubate for 5 min at room temperature. Centrifuge samples at 10000 rpm for 5 min. Collect the supernatant into a new tube.2.7. Cholesterol levels assay in cell membraneMeasure the absorbance values of the calibration standard tube and the sample tube at 510nm respectively, and calculate the cholesterol content. Cholesterol esters are broken down into cholesterol fatty acids under the action of cholesterol esterase. Cholesterol and oxygen generate hydrogen peroxide under the action of cholesterol oxidase. Hydrogen peroxide, 4-AAP, and phenol will produce red quinone under the action of peroxidase, and its color is directly proportional to the content of cholesterol. Measure the absorbance values of the calibration standard tube and the sample tube at 510nm respectively, and calculate the cholesterol content.2.8. cell culture and treatmentThe A549 cells were cultured adherently in a DME／F-12 medium. This medium contains 10% heat-inactivated fetal bovine serum (FBS) or 1% 100 units of penicillin and 100ug/ml streptomycin. The cells were cultured in a sterile incubator containing 5% CO2 at 37 °C. When the cell coverage in the culture flask is 80%-90%, cell passaging is required. When passaging, discard the dead cells and the original medium. Added 2 ml trypsin for digestion, until the cells do not adhere to the wall. Then add the medium to terminate the digestion quickly, transfer the cells to a 14 ml centrifuge tube with a dropper, and centrifuge at 1000 rpm for 5 min. Discard the supernatant, add 2 ml of medium, and gently pipette to mix the cells. Divide into two new culture flasks to prepare the culture medium. After the passage to the third generation, when the cells had expanded to a sufficient number, they were cultured in 6-well plates and treated separately.2.9. LXRα gene assayA549 cells were cultured in a medium supplemented with LXR luciferase reporter plasmid (LXR-Luc) and β-galactosidase control vector for 24 h. Others were cultured in a medium supplemented with LXR inhibitor GSK2033. A549 cells were pretreated with lycorine (20,40,80 mg/L). After 1 hour, the culture medium was poured out and LPS (3mg/kg) was added for 24 hours. Luciferase activity was detected. The transcriptional activity of the LXR-Luc assay system was compared with that of the β-galactosidase normal group. Cell culture supernatants were collected and the levels of TNF-α, IL-1β, and IL-6 were determined by ELISA after transfection with A549.2.10. Molecular docking analysis of LXRα and lycorineChemBioDraw Ultra 14.0 was used to draw small molecules, and the small molecules were imported into ChemBio3D Ultra 14.0 for energy minimization. The Minimum RMS Gradient was set to 0.001, and the small molecules were saved in MOL2 format. The optimized small molecules were imported into AutoDockTools-1.5.6 for hydrogenation, charge calculation, charge distribution, and rotatable bond setting and then kept in ”PDBQT” format. Download the LXRα structure (PDB ID: 3FAL) from the PDB database. Pymol2.3.0 was used to remove the protein crystal water and original ligand. The protein structure was imported into AutoDocktools (V1.5.6) for hydrogenation, charge calculation, charge distribution, and atom type designation and saved in ”PDBQT” format. AutoDock Vina1.1.2 was used for docking, and LXRα parameters were set as: center_x = 64.711, center_Y = 37.098, center_z = 23.658; The search space: size_x: 50, size_y: 50, size_z: 50 each lattice spacing is 0.375 (A) and exhaustiveness: 10, the rest of the parameters as the default Settings. PyMOL2.3.0 and LIGPLOT V 2.2.4 were used to analyze the interaction mode of the docking results.2.11. Fluorescence quantitative PCRTotal RNA was extracted from mice lung tissue and A549 cells. The concentration and purity of the RNA solution were determined by ultraviolet spectrophotometry at 260 nm and 280 nm. A single cDNA template targeting TNF-α, IL-1β, IL-6, TLR4, NF-κB, LXRα, ABCA1, ABCG, specific primers were synthesized by reverse transcription design based on a known sequence of β-actin. Real-time quantitative PCR was performed using the ABI PRISM 7500 processing system. For each gene to be measured, a cDNA template and sample cDNA defining the expressed gene is selected for the PCR reaction. There are 40 cycles, such as 95 °C for 15 s, 60 ℃ for 60 s, and 72 °C for 20 s. Each experiment was repeated three times and each sample was repeated three times. The β-actin was used as an endogenous internal standard control.2.12. ELISA assayThe double sandwich antibody method was used to detect the concentration of IL-1β, IL-6, IL-10, and TNF-α in mouse tissues and A549 cells. The tissues were weighed and rinsed with pre-chilled PBS. The tissue was ground with PBS (Simple: PBS=1:9) thoroughly with a glass homogenizer. The supernatant was taken after being centrifuged at 5000 rpm for 10 min. The cell culture supernatant was 2000rpm for 20 min to remove impurities and cell debris. The supernatant was tested. The anti-mouse antibody is coated on the ELISA plate. The cell factor in the sample was combined with the anti-mouse antibody. Then the horseradish peroxidase-labeled antibody is added, and the chromogenic substrate TMB is added. After the stop reaction solution was added. To measure the OD value with a microplate reader at 450nm wavelength. We can calculate the cytokine concentration in the sample by drawing a standard curve.2.13. Western blot analysisTotal protein was extracted from mouse lung tissue and A549 cells. The protein concentration was determined by the BCA method. Separate the sample using an agarose SDS gel and transfer it to the NC membrane. The membrane was blocked with Tris-buffered saline (TBST) containing 5% skim milk at room temperature for 2 hours and then incubated with a specific primary antibody (1:1000) overnight. Subsequently, the membrane was washed with TBST and then incubated with a secondary antibody at room temperature for 1 hour. Wash the batch with TBST again, and then use BCL luminescent color developing solution to take pictures under the imaging system to analyze the brightness.2.14. Statistical analysisSPSS Statistical 19 was used for statistical analysis. The statistics are represented by an average of ± S.E.M. for three separate experiments. Differences between groups were analyzed by one-way ANOVA or Student t test. # p < 0.01 vs. the control group, * p < 0.05 vs. the LPS group, ** p < 0.01 vs. the LPS group.
