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-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; LXRα
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-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 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-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 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 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 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 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 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 °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 assay
A549
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 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 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 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-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 assay
The 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 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 ± 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.