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\begin{document}

\title{Lycorine destabilizes lipid rafts to inhibit inflammation via LXR\selectlanguage{greek}α
\selectlanguage{english}signal in the lung of mice\selectlanguage{english}}



\author[1]{Fuhan Wang}%
\author[1]{Qirui Zhang}%
\author[1]{Kan Li}%
\author[1]{Yao Xue}%
\author[1]{Ziyi Li}%
\author[1]{Xuejiao Gao}%
\affil[1]{Northeast Agricultural University}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background 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-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}and IL-6
in mice. Lycorine dose-dependently inhibited the production of TNF-\selectlanguage{greek}α,
\selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}and IL-6. It also inhibited the transmission of TLR4/NF-\selectlanguage{greek}κ\selectlanguage{english}B
passway in LPS-stimulated A549 cells. Lycorine increased cholesterol
efflux through the activated LXR\selectlanguage{greek}α-\selectlanguage{english}ABCA1/ABCG pathway. Lycorine has a
good binding ability with LXR\selectlanguage{greek}α. \selectlanguage{english}After adding the LXR\selectlanguage{greek}α \selectlanguage{english}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 LXR\selectlanguage{greek}α-\selectlanguage{english}ABCA1/ABCG pathway, which
inhibits TLR4-mediated inflammation by increasing cholesterol efflux and
reducing TLR4 transport to lipid rafts.%
\end{abstract}%



\sloppy


\selectlanguage{greek}\textbf{Lycorine destabilizes lipid rafts to inhibit inflammation via
LXRα signal in the lung of mice}\selectlanguage{english}

Fuhan Wang \textsuperscript{a}, Qirui Zhang\textsuperscript{a}, Kan
Li\textsuperscript{a}, Yao Xue \textsuperscript{a}, Ziyi
Li\textsuperscript{a}, Xue-jiao Gao \textsuperscript{a,} *

\textsuperscript{a} College of Veterinary Medicine, Northeast
Agricultural University, Harbin 150030, People's Republic of China.

*Corresponding author.

Address: College of Veterinary Medicine, Northeast Agricultural
University, Harbin 150030, People's Republic of China.

E-mail address:\emph{xuejiaogao@126.com}

Declarations of interest: none

\textbf{Abstract}

\textbf{Background 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).

\textbf{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.

\textbf{Key Results:} The results showed that lycorine reduced
histopathological changes in lung, myeloperoxidase (MPO) activity, and
the production of inflammatory cytokines such as TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}and IL-6
in mice. Lycorine dose-dependently inhibited the production of TNF-\selectlanguage{greek}α,
\selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}and IL-6. It also inhibited the transmission of TLR4/NF-\selectlanguage{greek}κ\selectlanguage{english}B
passway in LPS-stimulated A549 cells. Lycorine increased cholesterol
efflux through the activated LXR\selectlanguage{greek}α-\selectlanguage{english}ABCA1/ABCG pathway. Lycorine has a
good binding ability with LXR\selectlanguage{greek}α. \selectlanguage{english}After adding the LXR\selectlanguage{greek}α \selectlanguage{english}inhibitor, the
anti-inflammatory effect of lycorine was eliminated.

\textbf{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 LXR\selectlanguage{greek}α-\selectlanguage{english}ABCA1/ABCG pathway,
which inhibits TLR4-mediated inflammation by increasing cholesterol
efflux and reducing TLR4 transport to lipid rafts.

\textbf{Keywords:} Lycorine; ALI; Lipid raft; TLR4; LXR\selectlanguage{greek}α

\selectlanguage{english}\textbf{Introduction}

Acute 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-\selectlanguage{greek}κ\selectlanguage{english}B 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-\selectlanguage{greek}κ\selectlanguage{english}B 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 \selectlanguage{greek}α \selectlanguage{english}and \selectlanguage{greek}β\selectlanguage{english}) 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-1\selectlanguage{greek}α \selectlanguage{english}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.

\textbf{Materials and methods}

Reagents

Lycorine (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-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, and THF-\selectlanguage{greek}α \selectlanguage{english}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 groups

Fifty 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 \selectlanguage{greek}μ\selectlanguage{english}l 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 \selectlanguage{ngerman}°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 analysis

The 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 content

The 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 rafts

Dewaxing 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 protein

Add 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 \selectlanguage{greek}μ\selectlanguage{english}L 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 membrane

Measure 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 treatment

The 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 \selectlanguage{ngerman}°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. LXR\selectlanguage{greek}α \selectlanguage{english}gene assay

A549 cells were cultured in a medium supplemented with LXR luciferase
reporter plasmid (LXR-Luc) and \selectlanguage{greek}β-\selectlanguage{english}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
\selectlanguage{greek}β-\selectlanguage{english}galactosidase normal group. Cell culture supernatants were collected
and the levels of TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}and IL-6 were determined by ELISA after
transfection with A549.

2.10. Molecular docking analysis of LXR\selectlanguage{greek}α \selectlanguage{english}and lycorine

ChemBioDraw 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 LXR\selectlanguage{greek}α \selectlanguage{english}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 LXR\selectlanguage{greek}α \selectlanguage{english}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 PCR

Total 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-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, TLR4, NF-\selectlanguage{greek}κ\selectlanguage{english}B, LXR\selectlanguage{greek}α, \selectlanguage{english}ABCA1, ABCG,
specific primers were synthesized by reverse transcription design based
on a known sequence of \selectlanguage{greek}β-\selectlanguage{english}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 \selectlanguage{ngerman}°C for 15
s, 60 \selectlanguage{english} for 60 s, and 72 degC for 20 s. Each experiment was repeated three
times and each sample was repeated three times. The \selectlanguage{greek}β-\selectlanguage{english}actin was used as
an endogenous internal standard control.

2.12. ELISA assay

The double sandwich antibody method was used to detect the concentration
of IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, IL-10, and TNF-\selectlanguage{greek}α \selectlanguage{english}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 analysis

Total 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 analysis

SPSS Statistical 19 was used for statistical analysis. The statistics
are represented by an average of \selectlanguage{ngerman}± S.E.M. for three separate
experiments. Differences between groups were analyzed by one-way ANOVA
or Student t test. \# p \textless{} 0.01 vs. the control group, * p
\textless{} 0.05 vs. the LPS group, ** p \textless{} 0.01 vs. the LPS
group.

\textbf{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-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}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-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}and IL-6 in the LPS group were
significantly lower than in the LPS group. The binding capacity of
lycorine to LXR\selectlanguage{greek}α \selectlanguage{english}was calculated using the molecular docking technique.

3.2. Effect of lycorine on TLR4/NF-\selectlanguage{greek}κ\selectlanguage{english}B 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-\selectlanguage{greek}κ\selectlanguage{english}B 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, I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α, \selectlanguage{english}and NF-\selectlanguage{greek}κ\selectlanguage{english}B 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 I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}and NF-\selectlanguage{greek}κ\selectlanguage{english}B 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 LXR\selectlanguage{greek}α \selectlanguage{english}signal.

Lycorine interacts with LXR\selectlanguage{greek}α \selectlanguage{english}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 LXR\selectlanguage{greek}α \selectlanguage{english}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 LXR\selectlanguage{greek}α \selectlanguage{english}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 LXR\selectlanguage{greek}α, \selectlanguage{english}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 LXR\selectlanguage{greek}α \selectlanguage{english}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 LXR\selectlanguage{greek}α. \selectlanguage{english}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-\selectlanguage{greek}κ\selectlanguage{english}B signaling pathway

The mRNA levels of TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}and IL-6 in A549 cells and the
concentrations of TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}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-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β,
\selectlanguage{english}and IL-6 in the lycorine group (C20, C40, C80) were significantly lower
than those in the LPS group. The concentrations of TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}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-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}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, I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α, \selectlanguage{english}and NF-\selectlanguage{greek}κ\selectlanguage{english}B 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 I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}and NF-\selectlanguage{greek}κ\selectlanguage{english}B 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-\selectlanguage{greek}κ\selectlanguage{english}B signaling pathway.

3.5. Lycorine activates LXR\selectlanguage{greek}α \selectlanguage{english}signal by increasing LXR\selectlanguage{greek}α \selectlanguage{english}activity

In this study, qPCR and Western blot detected mRNA and protein levels of
LXR\selectlanguage{greek}α, \selectlanguage{english}ABCA1, and ABCG. Luciferase gene assay was used to determine
whether lycorine could enhance LXR\selectlanguage{greek}α \selectlanguage{english}activity. The result is shown in
Fig. 5. LXR\selectlanguage{greek}α \selectlanguage{english}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 LXR\selectlanguage{greek}α \selectlanguage{english}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 LXR\selectlanguage{greek}α, \selectlanguage{english}ABCA1,
and ABCG is shown in Fig. 5F-H. The results showed that lycorine
activated the LXR\selectlanguage{greek}α \selectlanguage{english}signal and promoted protein expression of ABCA1 and
ABCG by enhancing LXR\selectlanguage{greek}α \selectlanguage{english}transcriptional activity.

3.6. The anti-inflammatory effect of lycorine was weakened by inhibiting
LXR\selectlanguage{greek}α \selectlanguage{english}activity

In this study, we detected intracellular LXR\selectlanguage{greek}α \selectlanguage{english}activity, protein levels
of cholesterol, LXR\selectlanguage{greek}α \selectlanguage{english}signaling pathway, TLR4/NF-\selectlanguage{greek}κ\selectlanguage{english}B 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 LXR\selectlanguage{greek}α \selectlanguage{english}in
the lycorine group was significantly inhibited compared with CCG, but
the cholesterol content was increased considerably(Fig. 6A-B). The
protein level of LXR\selectlanguage{greek}α \selectlanguage{english}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 I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α \selectlanguage{english}and NF-\selectlanguage{greek}κ\selectlanguage{english}B p65
and phosphorylation were significantly increased. As shown in Fig. 7J,
the contents of TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β \selectlanguage{english}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
LXR\selectlanguage{greek}α \selectlanguage{english}was inhibited.

\textbf{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-\selectlanguage{greek}κ\selectlanguage{english}B 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 LXR\selectlanguage{greek}α. \selectlanguage{english}The
results showed that lycorine had an excellent combining ability with
LXR\selectlanguage{greek}α. \selectlanguage{english}The results showed that LXR\selectlanguage{greek}α \selectlanguage{english}plays a vital role in regulating
cholesterol homeostasis. LXR\selectlanguage{greek}α \selectlanguage{english}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-\selectlanguage{greek}κ\selectlanguage{english}B
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-\selectlanguage{greek}κ\selectlanguage{english}B 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-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}and IL-6 were significantly increased in ALI
of mice. We found that lycorine inhibits TNF-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}and IL-6
production in a dose-dependent manner. TLR4/NF-\selectlanguage{greek}κ\selectlanguage{english}B 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-\selectlanguage{greek}κ\selectlanguage{english}B signal pathway.

Therefore, we examined the effects of lycorine on the activation of
LXR\selectlanguage{greek}α, \selectlanguage{english}ABCA1, and ABCG. The results showed that lycorine induced the
expression of ABCA1 and ABCG by activating LXR\selectlanguage{greek}α. \selectlanguage{english}Is this pathway related
to the anti-inflammatory effects of lycorine? We performed an LXR\selectlanguage{greek}α
\selectlanguage{english}activity inhibition assay. The results showed that lycorine altered the
expression of ABCA1 and ABCG, membrane cholesterol levels, and
LPS-induced inflammatory when LXR\selectlanguage{greek}α \selectlanguage{english}activity was inhibited. It confirmed
that inhibition of LXR\selectlanguage{greek}α \selectlanguage{english}activity could reduce the anti-inflammatory
effect of lycorine. These results indicate that lycorine binding to LXR\selectlanguage{greek}α
\selectlanguage{english}may promote the activation of LXR\selectlanguage{greek}α. \selectlanguage{english}It confirmed that inhibition of LXR\selectlanguage{greek}α
\selectlanguage{english}activity could reduce the anti-inflammatory effect of lycorine. Lycorine
depletes cholesterol by promoting the activation of LXR\selectlanguage{greek}α. \selectlanguage{english}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-\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}and IL-6 in A549 cells stimulated by LPS. The
anti-inflammatory mechanism of lycorine is related to the up-regulation
of the LXR\selectlanguage{greek}α-\selectlanguage{english}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.

\textbf{5.Conflict of interest}

The authors declare that there are no conflicts of interest.

\textbf{6. Acknowledgements}

The authors gratefully acknowledge Professor Guo from Northeast
Agricultural University for providing financial support and experimental
technical analysis for this study.

7. \textbf{Author contributions}

Fuhan Wang: Conceptualization, Methodology, Software, Writing- Original
draft preparation. Qirui Zhang: Data curation. Kan Li: Visualization,
Investigation. Yao Xue: Supervision. Ziyi Li: Software, Validation.
Xue-jiao Gao: Writing- Reviewing and Editing.

\textbf{8. Data accessibility statement}

All data, models, or code generated or used during the study are
available from the corresponding author by request.

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cells.\emph{Aging Cell, 20} (2)doi:ARTN e13307

10.1111/acel.13307

Zhu, S. M., Huang, S. Q., Xia, G. F., Wu, J., Shen, Y., Wang, Y.,
\ldots{} Xu, C. F. (2021). Anti-inflammatory effects of alpha
7-nicotinic ACh receptors are exerted through interactions with adenylyl
cyclase-6. \emph{British Journal of Pharmacology, 178} (11), 2324-2338.
doi:10.1111/bph.15412

Zhu, Y. B., Wang, Y. Y., Xing, S. G., \& Xiong, J. (2021). Blocking
SNHG14 Antagonizes Lipopolysaccharides-Induced Acute Lung Injury via
SNHG14/miR-124-3p Axis. \emph{Journal of Surgical Research, 263} ,
140-150. doi:10.1016/j.jss.2020.10.034

Table 1\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
TLR4 & Forward: 5'- GCCATCATTATGAGTGCCAATT -3'\tabularnewline
\midrule
\endhead
& Reverse: 5'- AGGGATAAGAACGCTGAGAATT -3'\tabularnewline
NF-\selectlanguage{greek}κ\selectlanguage{english}B p65 & Forward:5'- CCATAGCCATAGTTGCGGTCCTTC -3'\tabularnewline
& Reverse: 5'- CGTTCTTCCCTCCCTTTTCCTTTCC -3'\tabularnewline
I\selectlanguage{greek}κ\selectlanguage{english}B-\selectlanguage{greek}α & Forward:5'- GAATCACCAGAACATCGTGAAG -3'\tabularnewline
& Reverse: 5'- CAGTACTCCATGATTAGCACCT -3'\tabularnewline
LXR\selectlanguage{greek}α & Forward:5'- AGTTGTGGAAGACAGAACCTCAAGATG -3'\tabularnewline
& Reverse: 5'- TGCTGACTCCAACCCTATCCCTAAAG -3'\tabularnewline
ABCG & Forward:5'- CCTGACACATCTGCGAATCACCTC -3'\tabularnewline
& Reverse: 5'- AACAGCATGGAGAAGAACAGGAAGC -3'\tabularnewline
ABCA1 & Forward:5'- TTGAATGACGAGGATGAGGATGT -3'\tabularnewline
& Reverse: 5'- TTGTTGCCGCCACTGTAGTTA -3'\tabularnewline
IL-1\selectlanguage{greek}β & Forward: 5'- TTCCCA TTAGACAACTGC -3'\tabularnewline
& Reverse: 5'- CTGTAGTGTTGTATGTGATC -3'\tabularnewline
IL-6 & Forward: 5'- CAGAACCGCAGTGAAGAG -3'\tabularnewline
& Reverse: 5'- CAGAACCGCAGTGAAGAG -3'\tabularnewline
TNF-\selectlanguage{greek}α & Forward:5'- CTCA TTCCTGCTTGTGGC -3'\tabularnewline
& Reverse: 5'- CACTTGGTGGTTTGCTACG -3'\tabularnewline
\selectlanguage{greek}β-\selectlanguage{english}actin & Forward:5'- CTACCTCATGAAGATCCTGACC -3'\tabularnewline
& Reverse: 5'- CACAGCTTCTCTTTGATGTCAC -3'\tabularnewline
\bottomrule
\end{longtable}

\textbf{Fig. legends}

\textbf{Fig. 1:} Histological analysis and inflammatory factor
detection. (A-E) Histopathology of lung tissue. (A) Lung tissue control
group (CG). (B-D) Lycorine administration group (80, 40, 20 mg/kg,
respectively). (E) LPS treatment group (LPS). (F) Myeloperoxidase
activity in lung tissue. (G) The TNF-\selectlanguage{greek}α \selectlanguage{english}protein level in lung tissue. (H)
The IL-1\selectlanguage{greek}β \selectlanguage{english}protein level in lung tissue. (I) The IL-6 protein level in
lung tissue. Data represent the contents of 1 mL of supernatant of lung
tissue homogenate and are presented as mean \selectlanguage{ngerman}± SD (n = 10). *p
\textless{} 0.05, significantly different from the CG; \#p \textless{}
0.05, significantly different from the LPS group.

\textbf{Fig. 2:} Effects of allicin on TLR4/NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling pathway and
lipid raft in lung tissue. (A-E) Immunohistochemistry of flotillin-1
protein was performed on paraffin sections, enabling lipid rafts to be
observed under electron microscopy. (A) Control group (CG); (B-D)
lycorine administration groups (80, 40 and 20 mg/kg, respectively); (E)
LPS treatment group (LPS). (F) Immunofluorescence relative intensity of
Flotillin-1. The mRNA and protein levels of TLR4, I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α, \selectlanguage{english}and NF-\selectlanguage{greek}κ\selectlanguage{english}B p65
were detected by qPCR and Western blot. (G) The mRNA level of TLR4; (H)
The mRNA level of I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α; \selectlanguage{english}(I) The mRNA level of NF-\selectlanguage{greek}κ\selectlanguage{english}B p65. (J) The protein
levels of TLR4, I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α, \selectlanguage{english}phosphorylated I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α, \selectlanguage{english}p65, and phosphorylated p65
were detected. \selectlanguage{greek}β-\selectlanguage{english}actin was used as a control; (K-Q) The relative
intensities of TLR4 and I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α, \selectlanguage{english}p-I\selectlanguage{greek}κ\selectlanguage{english}B \selectlanguage{greek}α, \selectlanguage{english}p65, and p-p65. (R-V)
Immunohistochemistry of NF-\selectlanguage{greek}κ\selectlanguage{english}B p65 protein was performed on paraffin
sections so that the nuclear translocation of p65 protein could be
observed by electron microscopy. (R) Control group (CG); (S-U) lycorine
administration groups (80, 40 and 20 mg/kg, respectively); (V) LPS
treatment group (LPS). (W) Immunofluorescence relative intensity of p65.
*: p\textless{}0.05, **: p\textless{}0.01,***: p\textless{}0.001, ****:
p\textless{}0.0001, significantly different from CG; \#: p
\textless{}0.05, \#\#: p\textless{}0.01,\#\#\#: p\textless{}0.001,
\#\#\#\#: p\textless{}0.0001. significantly different from LPS group.

\textbf{Fig. 3:} Effects of lycorine on membrane cholesterol and LXR\selectlanguage{greek}α
\selectlanguage{english}signal. (A) The binding sites of lycorine and LXR\selectlanguage{greek}α. \selectlanguage{english}(B) Cholesterol
levels of lipid raft in lung tissue. (C) The levels of LXR\selectlanguage{greek}α, \selectlanguage{english}ABCA1, and
ABCG proteins in lung tissues were detected by Western blotting. \selectlanguage{greek}β-\selectlanguage{english}actin
was used as a control. (D-F) The relative intensities of LXR\selectlanguage{greek}α, \selectlanguage{english}ABCA1,
and ABCG; (G)The mRNA level of LXR\selectlanguage{greek}α; \selectlanguage{english}(H) The mRNA level of ABCA1; (I)
The mRNA level of ABCG. CG, control group; lycorine administration
groups (80, 40, and 20 mg/kg, respectively); LPS, LPS treatment group.
*: p\textless{}0.05, **: p\textless{}0.01,***: p\textless{}0.001, ****:
p\textless{}0.0001, significantly different from CG; \#: p
\textless{}0.05, \#\#: p\textless{}0.01,\#\#\#: p\textless{}0.001,
\#\#\#\#: p\textless{}0.0001. significantly different from LPS group.

\textbf{Fig. 4:} Lycorine inhibits the secretion of inflammatory factors
by regulating TLR4/NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling pathway. (A) The mRNA level of
inflammatory factors; (B) The inflammatory factor levels; (C) mRNA level
of TLR4/p65 pathway; (D) The levels of TLR4, I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α, \selectlanguage{english}phosphorylated I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α,
\selectlanguage{english}p65, phosphorylated p65 were detected by Western blotting. \selectlanguage{greek}β -\selectlanguage{english}actin was
used as a control. (E-K) The relative intensities of TLR4 and I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α,
\selectlanguage{english}p-I\selectlanguage{greek}κ\selectlanguage{english}B \selectlanguage{greek}α, \selectlanguage{english}p65, and p-p65 (n = 3). CCG, control group; lycorine
administration groups (80, 40, and 20 mg/kg, respectively); LPS, LPS
treatment group. *: p\textless{}0.05, **: p\textless{}0.01,***:
p\textless{}0.001, ****: p\textless{}0.0001, significantly different
from CCG; \#: p \textless{}0.05, \#\#: p\textless{}0.01,\#\#\#:
p\textless{}0.001, \#\#\#\#: p\textless{}0.0001. significantly different
from LPS group.

\textbf{Fig. 5:} Lycorine activates LXR\selectlanguage{greek}α \selectlanguage{english}signal by increasing LXR\selectlanguage{greek}α
\selectlanguage{english}activity. (A) The mRNA level of LXR\selectlanguage{greek}α. \selectlanguage{english}(B) The mRNA level of ABCA1. (C)
The mRNA level of ABCG. (D) The activity of LXR\selectlanguage{greek}α. \selectlanguage{english}(E) The levels of
LXR\selectlanguage{greek}α, \selectlanguage{english}ABCA1 and ABCG were detected by Western blotting. \selectlanguage{greek}β -\selectlanguage{english}actin was
used as a control. (F-H) The relative intensities of LXR\selectlanguage{greek}α, \selectlanguage{english}ABCA1 and
ABCG. CCG, control group; lycorine administration groups (80, 40, and 20
mg/kg, respectively). *: p\textless{}0.05, **: p\textless{}0.01,***:
p\textless{}0.001, ****: p\textless{}0.0001, significantly different
from CG; \#: p \textless{}0.05, \#\#: p\textless{}0.01,\#\#\#:
p\textless{}0.001, \#\#\#\#: p\textless{}0.0001. significantly different
from LPS group.

\textbf{Fig. 6:} The anti-inflammatory effect of lycorine was weakened
by inhibiting LXR\selectlanguage{greek}α \selectlanguage{english}activity. (A) Lipid raft cholesterol level of A549
cells after the addition of inhibitors. (B) Effects of lycorine on LXR\selectlanguage{greek}α
\selectlanguage{english}activity after addition of inhibitor. (C) The protein levels of LXR\selectlanguage{greek}α,
\selectlanguage{english}ABCA1, ABCG, TLR4, phosphorylated I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α, \selectlanguage{english}and phosphorylated p65 by
western blot analysis in A549 cells after inhibitor addition. \selectlanguage{greek}β-\selectlanguage{english}actin
was used as the control group. (D-I) The relative intensities of LXR\selectlanguage{greek}α,
\selectlanguage{english}ABCA1, ABCG, TLR4,p-I\selectlanguage{greek}κ\selectlanguage{english}B\selectlanguage{greek}α, \selectlanguage{english}and p-p65. (J) The inflammatory factors
levels after the addition of inhibitors. CCG, control group; Lycorine
administration group (80, 40, 20 mg/kg); LPS, LPS treatment group. *:
p\textless{}0.05, **: p\textless{}0.01,***: p\textless{}0.001, ****:
p\textless{}0.0001, significantly different from CCG.

Graphical Abstract:Schematic illustration of the mechanism by which
Lycorine destabilizes lipid rafts to inhibit inflammation via LXR\selectlanguage{greek}α
\selectlanguage{english}signal in the lung.  LPS induced acute lung injury in mice.  Mechanism
of lycorine's anti-inflammatory effect.  Diagram of the mechanism of
LPS-induced inflammation.  GSK2033 inhibits the anti-inflammatory
effects of lycorin by inhibiting LXR\selectlanguage{greek}α \selectlanguage{english}activity.

\textbf{Table 1} : Primer sequence table\selectlanguage{english}
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