Fig. legends
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-α protein level in lung tissue. (H)
The IL-1β 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 ± SD (n = 10). *p
< 0.05, significantly different from the CG; #p <
0.05, significantly different from the LPS group.
Fig. 2: Effects of allicin on TLR4/NF-κB 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, IκBα, and NF-κB p65
were detected by qPCR and Western blot. (G) The mRNA level of TLR4; (H)
The mRNA level of IκBα; (I) The mRNA level of NF-κB p65. (J) The protein
levels of TLR4, IκBα, phosphorylated IκBα, p65, and phosphorylated p65
were detected. β-actin was used as a control; (K-Q) The relative
intensities of TLR4 and IκBα, p-IκB α, p65, and p-p65. (R-V)
Immunohistochemistry of NF-κB 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<0.05, **: p<0.01,***: p<0.001, ****:
p<0.0001, significantly different from CG; #: p
<0.05, ##: p<0.01,###: p<0.001,
####: p<0.0001. significantly different from LPS group.
Fig. 3: Effects of lycorine on membrane cholesterol and LXRα
signal. (A) The binding sites of lycorine and LXRα. (B) Cholesterol
levels of lipid raft in lung tissue. (C) The levels of LXRα, ABCA1, and
ABCG proteins in lung tissues were detected by Western blotting. β-actin
was used as a control. (D-F) The relative intensities of LXRα, ABCA1,
and ABCG; (G)The mRNA level of LXRα; (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<0.05, **: p<0.01,***: p<0.001, ****:
p<0.0001, significantly different from CG; #: p
<0.05, ##: p<0.01,###: p<0.001,
####: p<0.0001. significantly different from LPS group.
Fig. 4: Lycorine
inhibits the secretion of inflammatory factors by regulating TLR4/NF-κB
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, IκBα, phosphorylated IκBα, p65, phosphorylated p65 were
detected by Western blotting. β -actin was used as a control. (E-K) The
relative intensities of TLR4 and IκBα, p-IκB α, p65, and p-p65 (n = 3).
CCG, control group; lycorine administration groups (80, 40, and 20
mg/kg, respectively); LPS, LPS treatment group. *: p<0.05, **:
p<0.01,***: p<0.001, ****: p<0.0001,
significantly different from CCG; #: p <0.05, ##:
p<0.01,###: p<0.001, ####:
p<0.0001. significantly different from LPS group.
Fig. 5: Lycorine activates LXRα signal by increasing LXRα
activity. (A) The mRNA level of LXRα. (B) The mRNA level of ABCA1. (C)
The mRNA level of ABCG. (D) The activity of LXRα. (E) The levels of
LXRα, ABCA1 and ABCG were detected by Western blotting. β -actin was
used as a control. (F-H) The relative intensities of LXRα, ABCA1 and
ABCG. CCG, control group; lycorine administration groups (80, 40, and 20
mg/kg, respectively). *: p<0.05, **: p<0.01,***:
p<0.001, ****: p<0.0001, significantly different
from CG; #: p <0.05, ##: p<0.01,###:
p<0.001, ####: p<0.0001. significantly different
from LPS group.
Fig. 6: The
anti-inflammatory effect of lycorine was weakened by inhibiting LXRα
activity. (A) Lipid raft cholesterol level of A549 cells after the
addition of inhibitors. (B) Effects of lycorine on LXRα activity after
addition of inhibitor. (C) The protein levels of LXRα, ABCA1, ABCG,
TLR4, phosphorylated IκBα, and phosphorylated p65 by western blot
analysis in A549 cells after inhibitor addition. β-actin was used as the
control group. (D-I) The relative intensities of LXRα, ABCA1, ABCG,
TLR4,p-IκBα, 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<0.05,
**: p<0.01,***: p<0.001, ****: p<0.0001,
significantly different from CCG.
Graphical Abstract:Schematic illustration of the mechanism by which
Lycorine destabilizes lipid rafts to inhibit inflammation via LXRα
signal in the lung. ① LPS induced acute lung injury in mice. ② Mechanism
of lycorine’s anti-inflammatory effect.
③ Diagram of the mechanism of
LPS-induced inflammation. ④ GSK2033 inhibits the anti-inflammatory
effects of lycorin by inhibiting LXRα activity.
Table 1 : Primer sequence table