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Study on the protective effect and mechanism of Dicliptera chinensis (L.) Juss (Acanthaceae) polysaccharide on immune liver injury induced by LPS


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The purpose of this study is to use Dicliptera chinensis (L.) Juss (Acanthaceae) polysaccharide (DCP) to act on the NF-κB inflammatory pathway and Fas/FasL ligand system, in order to find a new method to improve immune liver injury. Lipopolysaccharide (LPS) was used to establish an injury model in vivo (Kunming mice) and in vitro (LO2 cells). In this experiment, hematoxylin-eosin (H&E) staining and related biochemical indicators were used to observe the pathological changes of liver tissues, oxidative stress and inflammatory reactions. Immunohistochemistry, ELISA, RT-PCR and Western blot were used to detect protein or mRNA expressions associated with inflammation response and apoptosis. The experimental results show that the model group has obvious liver cell damage and inflammatory infiltration. After DCP intervention, it could significantly reduce the levels of ALT, AST, ALP, TBIL and MDA in serum, and increase the content of SOD and GSH-Px. In addition, DCP can reduce the expression level of NF-κB in the liver and reduce the release of downstream inflammatory factors TNF-α, IL-6 and IL-1β, thereby reducing the inflammation. At the same time, DCP can significantly inhibit the expression of Fas/FasL ligand system and apoptosis related-proteins and mRNA, which in turn can reduce cell apoptosis. In conclusion, DCP can alleviate liver injury by inhibiting liver inflammation and apoptosis, which provides a new strategy for clinical treatment of immune liver injury.
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Biomedicine & Pharmacotherapy 134 (2021) 111159
Available online 25 December 2020
0753-3322/© 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
Study on the protective effect and mechanism of Dicliptera chinensis (L.) Juss
(Acanthaceae) polysaccharide on immune liver injury induced by LPS
Qiongmei Xu
, Jie Xu
, Kefeng Zhang
, Mingli Zhong
, Houkang Cao
, Riming Wei
Ling Jin
**, Ya Gao
College of Pharmacy, Guilin Medical University, Guilin 541004, Guangxi, China
College of Pharmacy, Gansu University of Chinese Medicine, Lanzhou, 730000, Gansu, China
Polysaccharides from Dicliptera chinensis
Immunological liver injury
The purpose of this study is to use Dicliptera chinensis (L.) Juss (Acanthaceae) polysaccharide (DCP) to act on the
NF-κB inammatory pathway and Fas/FasL ligand system, in order to nd a new method to improve immune
liver injury. Lipopolysaccharide (LPS) was used to establish an injury model in vivo (Kunming mice) and in vitro
(LO2 cells). In this experiment, hematoxylin-eosin (H&E) staining and related biochemical indicators were used
to observe the pathological changes of liver tissues, oxidative stress and inammatory reactions. Immunohis-
tochemistry, ELISA, RT-PCR and Western blot were used to detect protein or mRNA expressions associated with
inammation response and apoptosis. The experimental results show that the model group has obvious liver cell
damage and inammatory inltration. After DCP intervention, it could signicantly reduce the levels of ALT,
AST, ALP, TBIL and MDA in serum, and increase the content of SOD and GSH-Px. In addition, DCP can reduce the
expression level of NF-κB in the liver and reduce the release of downstream inammatory factors TNF-
, IL-6 and
IL-1β, thereby reducing the inammation. At the same time, DCP can signicantly inhibit the expression of Fas/
FasL ligand system and apoptosis related-proteins and mRNA, which in turn can reduce cell apoptosis. In
conclusion, DCP can alleviate liver injury by inhibiting liver inammation and apoptosis, which provides a new
strategy for clinical treatment of immune liver injury.
1. Introduction
The liver is an important organ for the biotransformation and
detoxication of various harmful substances. In recent years, the pro-
portion of people suffering from liver injury has been increasing. Liver
injury is the common basis for diseases such as hepatitis, liver brosis
and even cirrhosis. However, there are many factors that cause liver
injury. The most common liver injury is immune liver injury caused by
alcohol, drugs, chemicals and other factors [1,2]. In the occurrence and
development of chemical, drug and (non-)alcoholic liver steatosis, the
excessive response of the bodys immune system is an important cause of
liver parenchymal damage [3]. Therefore, it is an inevitable choice to
nd a hepatoprotective drug or a new target with dual effects of regu-
lating the body immune function and liver protection.
Nuclear factor kappa-B (NF-κB) is a nuclear transcription factor
widely present in various cells in the body, and plays an important role
in regulating cellular inammation and apoptosis. In the resting stage,
NF-κB binds to the inhibitory protein IκB-
to form a complex in the
cytoplasm. When IκB-
is phosphorylated, NF-κB dissociates from IκB-
and enters the nucleus through the cell nuclear membrane. It binds to
the corresponding sites on the DNA, activates the transcription and
translation processes, induces the synthesis and release of inammatory
factors, and thus plays a key role in pathological processes such as cell
injury and apoptosis [4,5]. Some scholars reported that during the
process of liver inammation by lipopolysaccharide (LPS)-induced
activation, the NF-κB signaling pathway was signicantly activated [6].
The Fas/FasL ligand system is a signaling pathway that regulates
apoptosis. Fas antigen is an apoptosis-related molecule on the surface of
cell membranes and is widely expressed in a variety of tissue nuclear
cells, among which the immune system is the most abundantly expressed
[7]. FasL is a natural ligand of Fas, which can transmit death signals to
Fas through T cell-mediated cellular immunity, thereby inducing
* Corresponding author at: College of Pharmacy, Guilin Medical University, Guilin, 541199, Guangxi, China.
** Corresponding author.
E-mail addresses: (L. Jin), (Y. Gao).
These authors contributed equally to this work.
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
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Received 23 October 2020; Received in revised form 13 December 2020; Accepted 14 December 2020
Biomedicine & Pharmacotherapy 134 (2021) 111159
apoptosis or death [8,9]. After lung injury in mice induced by LPS,
alveolar cell Fas/FasL was up-regulated in a dose-dependent manner
[10]. Ansinoside can reduce the high expression of Fas and FasL caused
by Bacillus Calmette-Guerin Bacillus plus LPS, suggesting that it may
effectively protect the liver by affecting Fas/FasL ligand [11]. It can be
seen that effectively inhibiting the NF-κB pathway and the Fas/FasL
ligand system is a potential treatment for alleviating immune liver
The chemical constituents of Dicliptera chinensis (L.) Nees are mainly
polysaccharides, organic acids, amino acids and other substances, which
have the functions of clearing heat and detoxication, clearing liver and
improving eyesight. This medicine is a food that can be used for cooking
porridge, soup, etc. It is widely used in the folk. At the same time, it is
also a common folk medicine. Its whole herb can treat stomachache,
enteritis and diarrhea [12]. Dicliptera chinensis (L.) Juss (Acanthaceae)
polysaccharide (DCP) has been researched by our research group in the
previous period. In accordance with Procedures for toxicological Assess-
ment on Food Safety, we carried out a series of experiments, including
acute toxicity to mice, micronucleus experiment for bone marrow cell in
mice, sperm shape abnormality test in mice, Ames experiment, and rat
30 d feeding experiment, and the experimental results reect that DCP is
nontoxic [13]. We have found that DCP has a variety of anti-oxidant and
anti-inammatory biological activities, and can signicantly relieve
liver injury induced by carbon tetrachloride or D-galactosamine [14,15].
DCP also has a positive effect on mice with immune liver injury, but the
specic mechanism remains elusive. Base on the previous period study,
the purpose of our research is to use DCP to regulate the NF-κB and
Fas/FasL pathways and explore new strategies to improve liver injury.
First, an in vivo and in vitro model of immune liver injury was estab-
lished. Second, this model was used to study the effect of DCP on liver
injury caused by immune factors. Finally, this model was used to further
explore the effects of DCP on the NF-κB inammatory pathway and
Fas/FasL apoptotic pathway.
2. Materials and methods
2.1. Preparation of DCP
Dicliptera chinensis (L.) Juss is widely distributed in southern China.
However, the content of polysaccharide in Dicliptera chinensis (L.) Juss
originated from Guilin is the highest [16]. The herb of Dicliptera chinensis
(L.) Juss in this study was native to Guilin, and was purchased from the
Chinese herbal medicine market in Guilin, PR China and identied by
professor Kefeng Zhang. The chopped 1 kg dried herb was placed in a
reux device and defatted with 95 % ethanol under high temperature
conditions. The ratio of herb to ethanol was 1:18 (w/v), 1.5 h/time, and
repeated 5 times. Then, it was extracted with distilled water, and the
extracts were combined. The pure extract of Dicliptera chinensis was
concentrated to 2000 mL under reduced pressure at 50 C. The
concentrated solution was added with ethanol with a nal concentration
of 90 % (v/v) and placed at 4 C overnight. The next day, the poly-
saccharide in the concentrated solution was precipitated. The collected
polysaccharide was dissolved in 1600 mL of water, placed in a separa-
tory funnel and shaken with butanol/chloroform (1/5), and centrifuged
to remove denatured protein. The supernatant was lyophilized to obtain
a crude polysaccharide. The crude polysaccharide is dissolved with
distilled water, and is decolorized with the activated carbon and then
lyophilized to obtain a rened polysaccharide. In addition, the puried
polysaccharide was passed through a Sephadex G-75 column (2.5 cm ×
100 cm), and the liquid was collected after elution at a ow rate of 0.5
mL/min. The collected samples were mixed with sulfuric acid and
phenol to verify a characteristic color reaction of polysaccharide. The
collected solution was concentrated and lyophilized to obtain puried
light yellow polysaccharide. Our previous experiments have studied the
main components of DCP. The gel permeation chromatography (GPC)
was used to determine Mw and distribution of polysaccharides. The
weight average Mw of DCP-1 and DCP-2 were 9650 Da and 2273 Da.
Among them, the content of DCP2 is high, and its efcacy is obvious. The
component used in this study was DCP2. For specic chemical infor-
mation of DCP2, refer to literature [14]. The puried polysaccharide
was stored in a desiccator for further experiments.
2.2. Ethical approval
All procedures followed the National Institutes of Healths Guide for
the Care and Use of Laboratory Animals and approved by the Guilin
Medical University Animal Ethics Committee (Approved No. 2019-
2.3. Animals and experimental design
The experimental SPF-grade male Kunming mice were purchased
from Hunan Slake Jingda Experimental Animal Co., Ltd (SCXK [Xiang]
2016-0002, Hunan, CHN). All mice were kept in a temperature of 22 ±2
C, humidity of 45 %50 %, and 12 h of light/dark cycle. The mice were
access to food and water, and were acclimatized for one week before the
Sixty mice were randomly divided into Control group, Model (LPS)
group, Silymarin group (150 mg/kg) and DCP dose group (200, 100, 50
mg/kg), 10 mice in each group. After one week of adaptive feeding, the
Control group and the Model group were given saline and the other
groups were given drug by intragastric administration for 7 days, once a
day. After the last administration, except the Control group, mice were
injected intraperitoneally with LPS (5 mg/kg, purity>99 %, Solarbio
Science & Technology Co., Beijing, CHN) to establish an immune liver
injury model [17].After fasting for 16 h, blood was collected from the
eyeballs, and the liver tissues were collected and xed in 4 % para-
formaldehyde solution. The remaining liver tissues were stored in a
refrigerator at 80 C for liver tissue index detection. The brain, heart,
lung, spleen and kidney of each group of mice were xed at 4 %
2.4. Histopathological examination
2.4.1. Hematoxylin and eosin (H&E) staining
Liver tissues were xed for 48 h, and then dehydrated and embedded
in parafn. The sections were sequentially treated with xylene and
gradient ethanol, and then stained with hematoxylin and eosin staining
solution for 10 min and 5 min. After being soaked in 95 % alcohol and
absolute ethanol, the slides were mounted with neutral resin, under a
200×optical microscope to observe histopathological changes.
In this study, while observing the protective effect of DCP on mice
with immunological liver injury, we further investigated whether high-
dose DCP had an effect on brain, heart, spleen, lung, and kidney tissue
structures. Therefore, we collected brain, heart, lung, liver, spleen, and
kidney of the DCP (200 mg/kg) group for H&E staining, and observed
whether the tissue structure was damaged under an optical microscope.
2.4.2. Immunohistochemistry
The liver sections were dewaxed, rehydrated and placed in 1x citrate
buffer (ThermoFisher Scientic, Shanghai, CHN) for high pressure an-
tigen repair over 5 min. The sections were then allowed to cool natu-
rally. After the liver peroxidase was blocked by H
, the sections were
incubated with a p-NF-κBp65 (1:100; Abcam, Cambridge, UK) or an
/β (1:100, Abcam) antibody at 4 C overnight. The next day, after
incubation with the secondary antibody, DAB reagent was added for
hematoxylin counterstaining. Then, gradient ethanol and xylene were
dehydrated and transparent, neutral gum was mounted, and the sections
were observed under optical microscope at 200×.
Q. Xu et al.
Biomedicine & Pharmacotherapy 134 (2021) 111159
2.5. Analysis of serum samples
According to the kit instructions (Nanjing Jiancheng Bioengineering
Institute, Nanjing, CHN), biochemical method was used to detect the
activity of alanine aminotransferase (ALT), aspartate aminotransferase
(AST), alkaline phosphatase, (ALP) and the content of total bilirubin
(TBIL) in serum. At the same time, strictly follow the instructions
(Nanjing Jiancheng Bioengineering Institute, Nanjing, CHN) of the
malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione
peroxidase (GSH-Px) kits to add samples and related working solutions.
After the air bath, follow the kit instructions to detect absorbance on a
microplate reader and calculate the content of MDA, SOD and GSH-Px in
2.6. Analysis of liver samples
After the liver tissue was shredded, 9 times volume of saline was
added, which was fully ground to obtain a 10 % (v/w) liver tissue ho-
mogenate. The samples and corresponding working solutions were
added according to the ELISA kit instructions (Wuhan Elabscience
Biotechnology Co., Hubei, China). After standard procedures, detect and
calculate the levels of Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and
Tumor necrosis factor-
) in the liver tissue using a microplate
2.7. In vivo experiments with LO2 cells
2.7.1. LO2 cell culture and model establishment
LO2 cells were purchased from chunmai Biotechnology Co., Ltd
(Shanghai, China) and cultured in a 37 C, 5 % CO
incubator. The
complete medium contains RPMI-1640 medium, 10 % FBS (900108;
Gemini, CA, USA), 1 % penicillin-streptomycin. After the cells are fused
to 80 %, follow-up experiments are performed.
2.7.2. MTT experiment
LO2 cells at logarithmic growth stage were inoculated in 96-well
plates at a density of 1 ×10
cells/well and incubated with DCP at
different concentrations (01.5 mg/mL) with or without LPS (10
for 24 h [18]. Then 10
L MTT (5 mg/mL) was added to each well and
incubated for 4 h. The colored products were then dissolved in 150
DMSO and the absorbance at 490 nm was measured using a microplate
reader. The experiment was repeated three times and the survival rate of
the cells was calculated.
2.7.3. Immunouorescence
After DCP and LPS interfered with LO2 cells, the cells were washed
with phosphate-buffered saline (PBS) for three times, xed with 4 %
paraformaldehyde for 30 min and then washed. A permeable solution
containing 0.2 % (V/V) TritonX-100/PBS was added and incubated at
room temperature for 30 min. The cell was washed three times with PBS
and blocked with 5 % (m/v) BSA/PBST for 30 min. The blocking solu-
tion was discarded and incubated with NF-κB p65 primary antibody
(1:200, CST) at 4 C overnight. The next day, the mixture was reheated
for 1 h, washed with PBS three times, and added with uorescent sec-
ondary antibody (1:200, Jackson) to incubate for 1 h in dark. After
washing, DAPI was added for nuclear staining for 5 min, followed by
washing with PBS for three times. After anti-uorescence quencher was
added dropwise, the water-soluble sealing tablets were sealed and
observed under a uorescent microscope.
2.8. Real-time reverse transcription polymerase chain reaction
Trizol reagent (Beyotime Biotechnology Co., Shanghai, China) was
added to the liver and LO2 cells of each experimental group to obtain
total RNA. Following the reverse transcription kit instructions (Cwbio
Century Biotechnology Co., Beijing, China), total RNA was reverse
transcribed into cDNA. Using the reverse transcription reaction product
as a template, a real-time quantitative PCR amplication reaction was
performed, and cDNA was amplied on the CFX96 uorescence quan-
titative PCR instrument according to the reaction conditions of the
detection kit (Cwbio Century Biotechnology Co.). Under the conditions
of 95 C for 10 min, 95 C for 15 s, and 60 C for 1 min, the cycle was
repeated 40 times. Expressions of the target genes were carried out by a
comparative method (2
) using GAPDH as an internal reference.
The primer sequences (Huada Gene Research Institute, Shenzhen,
China) are shown in Tables 1 and 2.
2.9. Western blot analysis
In each experimental group, 1 mL of cultured cell protein lysate
(protein phosphatase inhibitor with 1 % PMSF, Beyotime Biotechnology
Co.) was added to the liver and LO2 cells of each experimental group,
and lysed on ice for 30 min. Centrifuge at 4 C for 15 min at a speed of 12
000 g/min, draw the supernatant, and determine the protein concen-
tration by BCA method (P0010S, Beyotime Biotechnology Co.). Take 20
g of denatured protein for each group and load the protein on 10 %
SDS-PAGE (concentrated gel voltage 80 V, separated gel voltage 120 V);
constant current (300 mA current, 1.5 h) to 0.45
m PVDF membrane
(Millipore, Billerica, MA, USA); add 5 % skim milk powder was shaken
for 1.5 h at room temperature and shaker. After washing the membrane
with TBST, incubate with the following antibodies at 4 C in a shaker
overnight: β-actin (1: 2000, Proteintech, Wuhan, China), NF-κBp65 (1:
1000, Cell Signaling Technology, Boston, MA, USA), p-NF-κBp65, TNF-
, IL-1β, IL-6 (1: 1000, Abcam), Caspase-8, Fas, FasL, Caspase-3, FADD,
Bax, Bcl2 (1: 1000, Cell Signaling Technology, Boston, MA, USA). After
washing the membrane with TBST, goat anti-mouse IgG and goat anti-
rabbit IgG secondary antibody (1:2000, Proteintech) were incubated
at room temperature for 1 h, place the image in a fully automated
chemiluminescence image analysis system (Tanon Technology Co.,
Shanghai, China), and analyze using Quantity One 4.6.2 software Strip
protein gray, calculate relative protein expression based on β-actin gray
Table 1
Primer sequences used in RT-PCR of Mice.
Genes Primer Mice Sequence(53)
Table 2
Primer sequences used in RT-PCR of LO2 cell.
Genes Primer Sequence(53)
Q. Xu et al.
Biomedicine & Pharmacotherapy 134 (2021) 111159
2.10. Statistical analysis
All analyses were carried out by Graph Pad Prism 5.0 software, the
data was expressed as mean ±SD. To assess the differences between
groups, one-way analysis of variance was used. Independent sample t-
test was used for comparison between two groups. P <0.05 indicated
statistical signicance.
3. Results
3.1. Establishment of immune liver injury model
ALT, AST, ALP, and TBIL are the most representative indicators of
liver function. After intraperitoneal injection of LPS, the serum levels of
ALT, AST, ALP, and TBIL in the LPS group were signicantly higher than
those in the Control group (Fig. 1A). The liver surface of the LPS group
was rough and dull, while the liver of the Control group was smooth and
bright red (Fig. 1B). The results of HE staining showed that the structure
of the liver lobule in the LPS group was fuzzy and unclear, the hepato-
cytes were disorderly arranged, and there were a large number of in-
ammatory cell inltrations near the central vein and the junction area,
showing obvious characteristics of liver injury. In the Control group, the
liver cells were evenly arranged and liver lobules were intact (Fig. 1C).
The above data indicates that we have successfully established a model
of immune liver injury in mice.
3.2. Effects of DCP on liver function
The results show that DCP intervention can signicantly reduce the
activity of ALT, AST, ALP, and the content of TBIL in the serum of mice
with liver injury caused by LPS (Fig. 1A). Compared with the LPS group,
the liver color of mice in each dose group of DCP returned to bright red
and shiny (Fig. 1B), and cell necrosis and inammatory inltration in
the manifold area were signicantly reduced (Fig. 1C). The results
indicate that DCP has a protective effect on immune liver injury in mice.
In order to investigate whether DCP is toxic to main organs while pro-
tecting the liver, H&E staining results of brain, heart, lung, liver, spleen,
and kidney in the DCP200 group showed no signicant pathological
differences in tissues (Fig. 1D), indicating that DCP has no obvious side
effects on major organs in mice.
3.3. Effects of DCP on oxidative stress and inammation in liver
Compared with the Control group, the content of MDA in LPS group
Fig. 1. The effect of DCP on liver function. (A) The activity of ALT, AST, ALP and the content of TBIL in serum, (B) liver morphology and (C) HE staining, (D) HE
staining of brain, heart, lung, liver, spleen and kidney in DCP200. All data are presented as the means ±SD (n =10). (*p <0.05, **p <0.01). Bar=100
Q. Xu et al.
Biomedicine & Pharmacotherapy 134 (2021) 111159
was increased signicantly, and the content of SOD and GSH-Px showed
a downward trend (Fig. 2A and B). ELISA and RT-PCR results showed
that the levels of inammatory factors TNF-
, IL-1β, IL-6 and mRNA in
liver tissue of LPS group mice were signicantly higher than that of
Control group, and the level of NF-κBp65 mRNA was higher than Con-
trol group (Fig. 2C and D). Immunohistochemistry showed that the liver
tissue p-NF-κBp65 and p-IKK
/β in the LPS group were signicantly
higher than those in the Control group (Fig. 2E). The above data in-
dicates that immune liver injury is closely related to oxidative stress and
After DCP intervention, the MDA content in the serum of DCP200
mice decreased signicantly, and the SOD and GSH-Px contents
increased (Fig. 2A and B), indicating that DCP can signicantly inhibit
oxidative stress. The levels of TNF-
, IL-1β, IL-6 and mRNA in the liver of
DCP group decreased, and the level of NF-κBp65 mRNA decreased, of
which the most signicant reduction was in the DCP200 group (Fig. 2C
and D). The expression of p-NF-κBp65 and p-IKK
/β in liver tissue of
DCP200 group was signicantly lower than that of LPS group (Fig. 2E).
The results show that DCP reduces liver damage by inhibiting oxidative
stress and inammatory response.
3.4. Effect of DCP on NF-κB and Fas/FasL pathway in vivo
Western blot results showed that after intraperitoneal injection of
LPS, the protein expression of iNOS, p-NF-κBp65, TNF-
, IL-6 and IL-1β
signicantly increased (Fig. 3AD), indicating that LPS stimulated the
liver inammatory response, activates the NF-κB pathway, and causes
liver damage. In addition, the protein expression levels of Bax, Fas, FasL,
FADD, Cleaved Caspase-3, and Cleaved Caspase-8 in the LPS group were
signicantly higher than those in the Control group, while the expres-
sion level of the inhibitory protein Bcl2 was signicantly reduced
(Fig. 3EI). The results show that liver injury is closely related to Fas/
FasL pathway.
The gray statistics results showed that the protein expression levels
of iNOS, p-NF-κBp65, TNF-
, IL-6, and IL-1β in the Silymarin group were
lower than the LPS group. After DCP administration, iNOS, p-NF-κBp65,
, IL-6 and IL-1β protein expression was signicantly down-
regulated (Fig. 3AD), indicating that DCP can inhibit the NF-κB
pathway to reduce inammation and improve liver injury. The expres-
sion of Bax, Fas, FasL, FADD, Cleaved Caspase-3, and Cleaved Caspase-8
in the DCP group was signicantly down-regulated, while the expression
Fig. 2. The effect of DCP on oxidative stress and inammation. Serum (A) The content of MDA, (B) The content of SOD and GSH-Px, liver (C) The levels of TNF-
, IL-
6 and IL-1β, (D) The expression of TNF-
, IL-6, IL-1β and NF-κBp65 mRNA, (E) The expression of p-NF-κBp65, p-IKK
/β in immunohistochemistry. All data are
presented as the means ±SD (n =10). (*p <0.05, **p <0.01). Bar=100
Q. Xu et al.
Biomedicine & Pharmacotherapy 134 (2021) 111159
level of the inhibitory apoptosis protein Bcl2 was signicantly up-
regulated (Fig. 3EI), indicating that DCP improves liver damage by
inhibiting cell apoptosis.
3.5. The effect of DCP and LPS in vitro
MTT results showed that treatment with DCP in the range of 01.5
mg/mL had no signicant effect on LO2 cell viability (Fig. 4A). How-
ever, after stimulation with LPS (10 ug/mL), the DCP dose showed a
downward trend in the cell viability of LO2 cells within a certain range
(Fig. 4B). After immunouorescence staining, the expression level of NF-
κBp65 in the LPS group was signicantly higher than that in the Control
group (Fig. 4C and D), indicating that we have successfully established
liver injury model in vitro. In addition, after LPS stimulation, the levels of
, IL-6, IL-1β, NF-κBp65, Fas and FasL mRNA were signicantly
higher than those in the Control group (Fig. 4E and F). After DCP
intervention, the results of immunouorescence showed that the
expression level of NF-κBp65 was signicantly lower than that of the LPS
group (Fig. 4C and D). At the same time, the levels of TNF-
, IL-6, IL-1β,
NF-κBp65, Fas and FasL mRNA were signicantly reduced (Fig. 4E and
3.6. Effect of DCP on NF-κB and Fas/FasL pathway in vitro
Western blot results showed that after LPS intervention, the protein
expression of iNOS, p-NF-κBp65, TNF-
, IL-6 and IL-1β was signicantly
Fig. 3. The effect of DCP on NF-κB and Fas/FasL pathway in vivo. Liver (A-D) the protein expression of iNOS, p-NF-κBp65, NF-κBp65, TNF-
, IL-6 and IL-1β, (E-I) the
protein expression of Bcl2, Bax, Fas, FasL, FADD, Cleaved Caspase-3, Cleaved Caspase-8. All data are presented as the means ±SD (n =3). (*p <0.05, **p <0.01).
Q. Xu et al.
Biomedicine & Pharmacotherapy 134 (2021) 111159
Fig. 4. Effect of DCP on LPS-treated LO2 cells. (A) Effect of DCP on the viability of LO2 cells, (B) Effect of DCP on the viability of LO2 cells treated with LPS (10ug/
mL), (C-D) The expression of NF-κBp65 in immunouorescence, (E) Fas and FasL mRNA levels, (F) TNF-
, IL-6, IL-1β and NF-κBp65 mRNA levels. All data are
presented as the means ±SD (n =5). (*p <0.05, **p <0.01).
Q. Xu et al.
Biomedicine & Pharmacotherapy 134 (2021) 111159
higher than that in the Control group (Fig. 5AD). At the same time, in
the LPS-induced cell injury model, the expressions of apoptosis-related
proteins Fas, FasL, FADD, Cleared Caspase-3, Cleared Caspase-8, and
Bax were signicantly up-regulated, and the expression of the inhibitory
apoptosis protein Bcl2 was signicantly down-regulated (Fig. 5EI). The
results showed that the immune liver injury with LPS intervention was
closely related to inammatory response and apoptosis.
Compared with the LPS group, after DCP intervention, the protein
expressions of iNOS, p-NF-κBp65, TNF-
, IL-6 and IL-1β were signi-
cantly reduced (Fig. 5AD). At the same time, the expression of Fas,
FasL, FADD, Cleaved Caspase-3, Cleaved Caspase-8 and Bax in the DCP
dose group was signicantly down-regulated, and the expression of Bcl2
was up-regulated (Fig. 5EI). The results show that DCP reduces the
inammatory response by inhibiting the NF-κB pathway, and improves
liver injury by inhibiting cell apoptosis.
4. Discussion
The activation of the NF-κB signaling pathway is closely related to
the inammatory response [17]. At the same time, the Fas/FasL-related
apoptosis signaling pathway plays an important role in immune liver
injury [19]. Therefore, we use DCP to simultaneously regulate NF-κB
pathways and Fas/FasL related apoptotic signaling pathways to reduce
inammation, and also regulate apoptosis ligands to reduce apoptosis.
We used LPS-induced SPF-grade Kunming mice to establish an immune
liver injury model to evaluate the effects of DCP on oxidative stress,
inammation, and apoptosis-related proteins. To further verify the ef-
fect of DCP on NF-κB pathway and Fas/FasL related apoptosis signal,
Fig. 5. The effect of DCP on NF-κB and Fas/FasL pathway in vitro. In LO2 cells, (A-D) the protein expression of iNOS, p-NF-κBp65, NF-κBp65, TNF-
, IL-6 and IL-1β,
(E-I) the protein expression of Bcl2, Bax, Fas, FasL, FADD, Cleaved Caspase-3, Cleaved Caspase-8. All data are presented as the means ±SD (n =3). (*p <0.05, **p
Q. Xu et al.
Biomedicine & Pharmacotherapy 134 (2021) 111159
LO2 cells were cultured in vitro and an immune liver injury model was
We found that DCP interfered with the NF-κB signaling pathway to
inhibit liver inammation and regulated the expression of key genes and
proteins in the Fas/FasL signaling pathway. The results of this study
provide a new strategy for clinical application of DCP to reduce liver
injury. First, in this study, we successfully established a mouse immune
liver injury model induced by LPS, the main component of endotoxin,
which is intraperitoneally injected into mice to trigger systemic immune
response, inammation and damage liver structure [20]. ALT and AST in
damaged liver cells were spilled into the blood, resulting in increased
activity of ALT and AST in the blood [21]. Liver biomarker enzymes viz.
ALP, ALT, AST and TBIL were increased due to LPS/d-GalN [22]. In this
experiment, liver tissues of LPS-treated mice was signicantly damaged,
hepatic lobule structure was signicantly damaged, liver cells were ar-
ranged in disorder with more inammatory inltration, and the activ-
ities of ALT, AST, ALP, and TBIL in the serum were increased
signicantly (Fig. 1). In conclusion, the immune liver injury model
established by LPS can be used in the study of liver protection by DCP.
Studies have shown that most inammatory reactions are often
accompanied by varying degrees of oxidative stress, which is the com-
mon pathogenesis of a variety of liver diseases [23]. MDA is the most
important lipid peroxides, and its overexpression implies a state of
oxidative stress [24]. Enzyme antioxidant system is an important anti-
oxidant system of the body. Antioxidant enzymes mainly include SOD
and GSH-Px, which can effectively reduce the generation of peroxides
such as MDA and maintain the bodys normal redox reaction [25].
Studies have shown that in LPS-induced immunological liver injury
mice, SOD and GSH-Px contents in liver tissues were signicantly
decreased, and MDA contents were signicantly increased, indicating
that oxidative stress in LPS-induced mice appeared [26], which was
consistent with the results of this study. In addition, DCP intervention
can signicantly enhance the content of SOD and GSH-Px in serum of
mice with LPS-induced immunological liver injury, and reduce the
content of MDA (Fig. 2), suggesting that DCP can inhibit oxidative stress,
which may be one of the important mechanisms of DCP to protect the
NF-κB is the central regulator of inammatory cytokines and plays an
important role in hepatocyte injury, brosis and hepatocellular carci-
noma. After LPS stimulation, the NF-κB signaling pathway is activated to
produce downstream pro-inammatory cytokines and chemokines,
leading to increased inammation in liver tissue and liver injury [27].
Immunohistochemical results showed that DCP inhibited NF-κB
signaling pathway and reduced inammatory response, improving im-
mune liver injury in mice. DCP signicantly inhibited the regulation of
NF-κB signaling pathway and improved liver damage caused by immune
factors in mice. Inhibition of NF-κB activation is a key pathway to reduce
inammatory responses in liver injury [28]. For example, curcumin
signicantly down-regulates the expression of p-NF-κBp65 in liver, re-
duces TNF-
and IL-1β, and improves immune liver injury by inhibiting
NF-κB pathway [29]. This is consistent with the decrease in protein
expression of DCP in this experiment. TNF-
and IL-6 not only promote
liver inammation, but the pro-inammatory cytokines IL-1β also
initiate immune and inammatory responses, leading to hepatocyte
apoptosis or necrosis [30]. In this study, DCP inhibited the release of
, IL-6 and IL-1β (Fig. 2), reducing inammation and thereby
protecting the liver. Ginsenosides reduce the release of inammatory
cytokines TNF-
, IL-6 and IL-1β, thereby reducing liver injury [31].
Angelica polysaccharide can protect the liver by inhibiting the release of
inammatory cytokines TNF-
, IL-6 and IL-1β and alleviating
LPS-induced immunological liver injury in mice [32]. Their research is
similar to the effect of using DCP to inhibit TNF-
, IL-6 and IL-1β in the
improvement of liver injury.
Liver injury is usually accompanied by hepatocyte inammation,
apoptosis and necrosis [33], and the role of DCP may be related to the
inhibition of apoptotic pathways. Apoptosis, also known as programmed
cell death, is a process in which cells automatically die under the
regulation of their own genetic mechanism under different physiological
and pathological conditions, and is the main procedure to exclude
harmful cells and excess cells in the body [34]. At present, the pathways
Fig. 6. Proposed model depicting the underlying mechanisms of DCP in regulating liver inammation responses and apoptosis in immune liver injury.
Q. Xu et al.
Biomedicine & Pharmacotherapy 134 (2021) 111159
involved in apoptosis signaling pathways are death receptor pathway,
mitochondrial-dependent pathway and endoplasmic reticulum
pathway. Fas receptor ligand system is the main receptor-mediated
pathway to stimulate hepatocyte apoptosis. The extracellular mem-
brane region of hepatocytes contains a death domain associated with
apoptosis. When the cell surface contains a sufcient density of Fas
antigen, Fas and its ligand FasL combine to form oligomers, which
mediate downward transmission of Fas apoptosis signal [35]. FasI type
cells, after binding with their ligand FasL, activate Fas, attracting the
formation of another related protein (FADD) with the same death
domain in the cytoplasm. The FAS-FADD dimer activates FADD and
activates Caspase-8. The combination of FasII cells and ligands lead to an
increase in membrane permeability, thus convening and activating
Caspase-3, thereby triggering cascade reactions of the Caspases family,
which ultimately leads to apoptosis [36,37]. The Bcl-2 family protein is
often associated with hepatocyte apoptosis and necrosis in liver injury.
Bcl-2 protein is an important substance against cell apoptosis [38]. Bax,
a Bcl-2 family protein, binds to the Bcl-2 protein on the mitochondrial
membrane, thereby inhibiting the activity of Bcl-2 and promoting
apoptosis [39]. Both Bcl-2 protein and Bax can be widely positioned on
organelles such as mitochondrial membrane. Bcl-2 prevents the release
of pro-apoptotic protein cytochrome C into cytoplasm by stabilizing
mitochondrial membrane potential and maintaining mitochondrial
integrity [40]. Bax can form a heterodimer with Bcl-2 protein and
inactivate it, enhance the expression of Bax to form a homodimer, and
accelerate cell death [41]. In summary, Fas/FasL-mediated apoptosis
plays an important role in the occurrence and development of liver
injury. The experimental results showed that the DCP with different
concentration gradient could signicantly inhibit the protein expression
of Fas, FasL, FADD, Caspase-8, Caspase-3 and Bax, and increase the
expression level of Bcl2 (Figs. 3 and 5). This shows that the DCP has a
repairing effect on immune liver injury, which is related to the Fas/FasL
apoptosis pathway and its cascade reaction.
By blocking the NF-κB signaling pathway and inhibiting the activity
of Fas/FasL apoptotic pathway, we found that DCP alleviates LPS-
induced liver injury. DCP inhibits the NF-κB signaling pathway, result-
ing in the reduction of downstream inammatory factors TNF-
, IL-6, IL-
1β, reducing the inammatory response. On the other hand, it inhibits
the activity of Fas/FasL apoptosis pathway, reduces the proteins
expression of Fas, FasL, FADD, Caspase-8, Caspase-3 and Bax, increases
the expression level of Bcl2, and thus reduces apoptosis to protect the
liver (Fig. 6). The results of this study provide a new strategy for the
clinical treatment of immune liver injury by DCP.
Ling Jin and Ya Gao designed the study; Qiongmei Xu, Jie Xu, Mingli
Zhong, performed experiments, Jie Xu, Riming Wei drew the gure and
the table; Qiongmei Xu, Kefeng Zhang, Houkang Cao, Ya Gao wrote the
manuscript. All authors read and approved the nal manuscript.
Declaration of Competing Interest
The authors report no declarations of interest.
This study was supported by National Natural Science Foundation of
China (81960779, 81760114, 81660104, 81860673), National Science
Foundation of Guangxi Province of China (2017GXNSFAA198218,
2017GXNSFAA198326 and 2018GXNSFAA281040), and Special fund-
ing for 2017 Guangxi BaGui Scholars.
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... And the results of liver histological observation also showed the polysaccharides could make resistance to liver injury induced by LPS. Xu et al. [18] found that Dicliptera chinensis (L.) Juss (Acanthaceae) polysaccharide could resist the liver injury induced by LPS. Li et al. [19] found that the Plantago seed polysaccharide could resist the liver injury induced by LPS. ...
Full-text available
To analyze the intervention mechanism of polysaccharides from charred Angelica sinensis (CASP) on the liver injury caused by Ceftiofur sodium (CS) and lipopolysaccharide (LPS) from the perspective of the intestine. Ninety-four one-day-old laying chickens underwent free feeding and drinking water for three days. Then, fourteen laying chickens were randomly selected as the control group, and sixteen laying chickens were selected as the model group. Sixteen laying chickens in the rest were randomly selected as the intervention group of CASP. Chickens in the intervention group were given CASP by the oral administration (0.25 g/kg/d) for 10 days, the control and model groups were given the same amount of physiological saline. During the 8th and 10th days, laying chickens in the model and CASP intervention group were subcutaneously injected with CS at the neck. In contrast, those in the control group were subcutaneously injected with the same amount of normal saline simultaneously. Except for the control group, the layer chickens in the model and CASP intervention groups were injected with LPS after CS injection on the 10th day of the experiment. In contrast, those in the control group were injected with the same amount of normal saline at the same time. 48 h after the experiment, the liver samples of each group were collected, and the liver injury was analyzed by hematoxylin-eosin (HE) staining and transmission electron microscopy. And the cecum contents of six-layer chickens in each group were collected, and the intervention mechanism of CASP on the liver injury from the perspective of the intestine was analyzed by the 16S rDNA amplicon sequencing technology and the short-chain fatty acids (SCFAs) detection of cecal contents based on Gas Chromatography-Mass Spectrometry (GC-MS), and their association analysis was carried out. The results showed that the structure of chicken liver in the normal control group was normal, while that in the model group was damaged. The structure of chicken liver in the CASP intervention group was similar to the normal control group. The intestinal floras in the model group were maladjusted compared to the normal control group. After the intervention of CASP, the diversity, and richness of chicken intestinal floras changed significantly. It was speculated that the intervention mechanism of CASP on the chicken liver injury might be related to the abundance and proportion of Bacteroidetes and Firmicutes. Compared with the model group, the indexes of ace, chao1, observed species, and PD whole tree of chicken cecum floras in the intervention group of CASP were significantly increased (p < 0.05). The contents of acetic acid, butyric acid, and total SCFAs in the intervention group of CASP were significantly lower than those in the model group (p < 0.05), and the contents of propionic acid and valeric acid in the intervention group of CASP were significantly lower than those in the model group (p < 0.05) and normal control group (p < 0.05). The correlation analysis showed that the changes in the intestinal floras were correlated with the changes in SCFAs in the cecum. It is confirmed that the liver-protecting effect of CASP is indeed related to the changes in the intestinal floras and SCFAs content in the cecum, which provides a basis for screening liver-protecting alternative antibiotics products for poultry.
... Our results are in line with those of Khan et al. [39], who reported the serum levels of ALT and AST in Sprague Dawley rats; compared with the control group, the levels of AST and ALT in the LPS-treated group were higher. Xu et al. [40] found that liver tissue was significantly damaged and serum ALT, AST, and ALP activities were significantly increased in LPS-treated mice. Similarly, Xu et al. [41] reported that an LPS challenge increased the serum AST activity and the AST/ALT ratio of weaned piglets within 24 h of LPS induction, and it reached the peak at 8 h. ...
Full-text available
Due to imperfections in their immune and digestive systems, weaned piglets are susceptible to invasions of the external environment and diseases, especially bacterial infections, which lead to slow growth, tissue damage, and even the death of piglets. Here, a model of weaned piglets induced by Escherichia coli lipopolysaccharide (LPS) was established to explore the effects of continuous low-dose LPS induction on the mechanism of liver injury. A total of forty-eight healthy 28-day-old weaned piglets (weight = 6.65 ± 1.19 kg) were randomly divided into two groups: the CON group and LPS group. During the experimental period of thirteen days, the LPS group was injected intraperitoneally with LPS (100 μg/kg) once per day, and the CON group was treated with the same volume of 0.9% NaCl solution. On the 1st, 5th, 9th, and 13th days, the serum and liver of the piglets were collected for the determination of serum biochemical indexes, an antioxidant capacity evaluation, and histopathological examinations. In addition, the mRNA expression levels of the TLR4 pathway and inflammatory cytokines were detected. The results showed that the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in the serum increased after LPS induction. The activities of total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) in the serum and liver homogenate of the LPS group were lower than those of the CON group, while the malondialdehyde (MDA) content in the serum and the activities of catalase (CAT) and superoxide dismutase (SOD) in the liver of the LPS group were higher than those in the CON group. At the same time, morphological impairment of the livers occurred, including hepatocyte caryolysis, hepatocyte vacuolization, karyopycnosis, and inflammatory cell infiltration, and the mRNA expression levels of TLR4, MyD88, NF-κB, TNF-α, IL-6, and IL-10 were upregulated in the livers after LPS induction. The above results were more obvious on the 1st and 5th days of LPS induction, while the trend during the later period was not significant. It was concluded that the oxidative stress and liver injury occurred at the early stage of LPS induction, while the liver damage weakened at the later stage. The weaned piglets probably gradually developed tolerance to the endotoxin after the continuous low-dose induction of LPS.
... Currently, CCl4, alcohol, high-fat diet, paracetamol, LPS, and D-GalN are commonly used agents to modulate acute liver damage [7][8][9] . LPS and D-GalN effectively kill hepatocytes, seriously impairing physiological liver function [10] and producing a stable and reproducible pattern of liver injury. The hepatoprotective effects of natural products such as schisandra polysaccharide, emodin, diterpenoid fenugreek lactone, and quercetin have been extensively studied [11,12] . ...
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Background: NLRP3 inflammasome activation results in liver inflammation and injury. Curcumin, a polyphenol from Curcumin longa, exhibits anti-inflammatory properties. The effects and anti-inflammatory mechanisms of curcumin were explored on acute liver injury induced by LPS/D-GalN. Methods and Results: An in vitro acute liver injury model was established on L-02 cells by treatment with10 μg/mL lipopolysaccharide (LPS); An in vivo model was established on SD rats by intraperitoneal injection of 0.02 mg/kg LPS and 500 mg/kg D-galactosamine (D-GalN). Biochemical index detection, histopathology, Western blotting, and qPCR were used to explore the effects and anti-inflammatory mechanisms of curcumin on acute liver injury induced by LPS/D-GalN in vitro and in vivo. Our results showed that curcumin significantly reduced the damage of L-02 cells induced by LPS, reduced the levels of transminase, inhibited oxidative stress induced by LPS in rats, and alleviated liver pathological injury. These effects accompanied by the downregulation of the expression of TLR4, NF-кB, and pyrotosis related proteins NLRP3 and caspase 1. Conclusions: Curcumin ameliorated acute liver injury induced by LPS and D-GalN in L-02 cells and SD rats by inhibiting TLR4/NF-кB/NLRP3 pathway.
... According to the investigation by World Health Organization (WHO), ALT serves as the most sensitive parameter of liver structure injury, and the serum ALT mainly stems from the damage to the cell membrane [20]. A previous study displayed that the serum ALT and AST activities were remarkably increased in LPS-treated liver-injury mice, which is in agreement with this study [2]. Nevertheless, L. paracasei CCFM1223 pretreatment remarkably suppressed the serum AST and ALT activities compared with the LPS group, suggesting that L. paracasei CCFM1223 had a protective effect against ALI. ...
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Background: Lactobacillus paracasei CCFM1223, a probiotic previously isolated from the healthy people's intestine, exerts the beneficial influence of preventing the development of inflammation. Methods: The aim of this research was to explore the beneficial effects of L. paracasei CCFM1223 to prevent lipopolysaccharide (LPS)-induced acute liver injury (ALI) and elaborate on its hepatoprotective mechanisms. Results: L. paracasei CCFM1223 pretreatment remarkably decreased the activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mice with LPS treatment and remarkably recovered LPS-induced the changes in inflammatory cytokines (tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), interleukin (IL)-1β, IL-6, IL-17, IL-10, and LPS) and antioxidative enzymes activities (total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT)). Metagenomic analysis showed that L. paracasei CCFM1223 pretreatment remarkably increased the relative abundance of Catabacter compared with the LPS group but remarkably reduced the relative abundance of [Eubacterium] xylanophilumgroup, ASF356, LachnospiraceaeNK4A136group, and Lachnoclostridium, which is closely associated with the inflammation cytokines and antioxidative enzymes. Furthermore, L. paracasei CCFM1223 pretreatment remarkably increased the colonic, serum, and hepatic IL-22 levels in ALI mice. In addition, L. paracasei CCFM1223 pretreatment remarkably down-regulated the hepatic Tlr4 and Nf-kβ transcriptions and significantly up-regulated the hepatic Tlr9, Tak1, Iκ-Bα, and Nrf2 transcriptions in ALI mice. Conclusions: L. paracasei CCFM1223 has a hepatoprotective function in ameliorating LPS-induced ALI by regulating the "gut-liver" axis.
... (DMSO) was added to each well and the absorbance was measured at a wavelength of 490 nm (Xu et al., 2021). All experiments were repeated three times to calculate cell viability. ...
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The crude Hedysarum polysaccharides (HPS: HPS‐50 and HPS‐80) obtained from Radix Hedysari exhibited great pharmacological activities in our previous research. This study investigated the effects of HPS on lipopolysaccharide (LPS)/D‐galactosamine (D‐GalN)‐induced acute liver injury (ALI) in mice and LPS‐induced injury in LO2 cells, as well as the relationship between structural characteristics and hepatoprotective activities. The in vivo results showed that compared with HPS‐80, HPS‐50 showed stronger hepatoprotection, which improved histopathological changes to normal levels. HPS‐50 significantly decreased the levels of ALT, AST, MPO, and MDA, increased the activities of SOD, CAT, and GSH, and suppressed the LPS/D‐GalN‐triggered production of TNF‐α, IL‐1β, and IL‐6 (p < .05). The results in vitro showed that HPS‐50‐P (HPS‐50‐1, HPS‐50‐2, and HPS‐50‐3) purified from HPS‐50 played significant protective roles against LPS‐induced injury in LO2 cells by reducing cell apoptosis and relieving cell cycle arrest. HPS‐50‐2 restored the percentage of normal cells from 54.8% to 94.7%, and reduced the S phase cells from 59.40% to 47.05% (p < .01). By analyzing the structure of HPS‐50‐P, including monosaccharide composition, molecular weight, chain conformation, and surface morphology, we speculated that the best protective effect of HPS‐50‐2 might be attributed to its beta configuration, highest molecular weight, and high glucose and galactose contents. These findings indicate that HPS‐50 might be a promising source of functional foods for the protection and prevention of ALI. Practical applications In this study, the protective effect of HPS on ALI was evaluated from multiple perspectives, and HPS‐50‐2 was screened as a potential active ingredient. This study has two practical applications. First, it provides a new way to improve ALI, and a new option for patients to prevent and treat ALI. Second, this work also complements the pharmacological activity of Radix Hedysari and provides a basis for the development of Radix Hedysari as a functional food.
... factor kappa-B (NF-kB) signaling pathway, produce downstream proinflammatory cytokines and chemokines, and enhance liver tissue inflammation and significant expansion of hepatic sinusoids (Chen et al., 2021;Xu et al., 2021). There were no reports similar to the substantial increase in the organ indices of the glandular stomach caused by multiple injections of LPS. ...
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This study aimed to determine whether the challenge from Escherichia coli (E. coli) lipopolysaccharide (LPS) affects the pharmacokinetics of danofloxacin in broilers. Twenty 1-day-old Arbor Acres (AA) broilers were equally and randomly divided into two groups. When the chickens were 23, 25, 27, and 29 days old, E. coli LPS (1 mL; 0.5 mg/kg body weight (BW)) and sterile saline (1 mL) were intraperitoneally injected into the two groups. After the last injection, danofloxacin was given to all chickens by gavage at the dose of 5 mg/kg BW. Then serum and plasma samples at each time point were collected through the wing vein. Danofloxacin concentrations in plasma were detected through the high-performance liquid chromatography (HPLC) method and subjected to non-compartmental analysis using Phoenix software. The levels of chicken interleukin-1β (IL-1β) and corticosterone (CORT) in serum were measured by the Enzyme-linked immunosorbent assay (ELISA) kit. In addition, after the collection of plasma or serum samples, seven chickens (31 days of age) in each group were killed to calculate the organ indices. Compared with the control group, the challenge of LPS significantly decreased the parameters of AUC0-∞, Cmax, and t1/2λz and increased the parameters of Tmax and λz. Additionally, in the LPS group, the absorption time of danofloxacin was prolonged; however, the elimination was accelerated, which resulted in reduced internal exposure.
... The IL-6 and IL-8 proteins in the cell supernatant were very small and difficult to collect, so the expression of IL-6 and IL-8 in the cells was measured (32). The expression levels of IL-6 and IL-8 in cells have been detected in numerous previous studies, in which the expression of IL-6 and IL-8 in the cell supernatant was not detected (32)(33)(34). ...
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Psoralen (PSO) exerts anti‑inflammatory pharmacological effects and plays an important role in a variety of inflammatory diseases. However, the effects of PSO with allergic rhinitis (AR) are yet to be reported. In the present study, an in vitro AR model was generated by inducing JME/CF15 human nasal epithelial cells with IL‑13, after which MTT was used to assess the cytotoxicity of PSO. The expression levels of inflammatory cytokines (granulocyte‑macrophage colony‑stimulating factor and Eotaxin) were determined by ELISA. Furthermore, the expression of inflammatory IL‑6 and ‑8, as well as mucin 5AC, was assessed by reverse transcription‑quantitative PCR and western blotting, and cellular reactive oxygen species were detected using a 2',7'‑dichlorodihydrofluorescein diacetate fluorescent probe. Western blotting was also used to detect the expression and phosphorylation of c‑Fos and c‑Jun in the activator protein 1 (AP‑1) pathway, as well as the expression of cystatin‑SN (CST1). PSO inhibited the inflammatory response and mucus production in IL‑13‑induced JME/CF15 cells. Furthermore, the levels of c‑Fos and c‑Jun phosphorylation in the AP‑1 pathway were decreased in IL‑13‑induced JME/CF15 cells following PSO treatment. The expression of pathway proteins was activated by the addition of PMA, an AP‑1 pathway activator, which concurrently reversed the inhibitory effects of PSO on the inflammatory response and mucus formation. The addition of an AP‑1 inhibitor (SP600125) further inhibited pathway activity, and IL‑13‑induced inflammation and mucus formation was restored. In conclusion, PSO regulates the expression of CST1 by inhibiting the AP‑1 pathway, thus suppressing the IL‑13‑induced inflammatory response and mucus production in nasal mucosal epithelial cells.
Background: Ginseng polysaccharides (GP) have been found to exhibit significant immune regulatory effects, making them a promising candidate for treating immune-related diseases. However, their mechanism of action in immune liver injury is not yet clear. The innovation of this study lies in exploring the mechanism of action of ginseng polysaccharides (GP) in immune liver injury. While GP has been previously identified for its immune regulatory effects, this study aims to provide a clearer understanding of its therapeutic potential for immune-related liver diseases. Purpose: The purpose of this study is to characterize low molecular weight gingeng polysaccharides (LGP), investigate their effect on ConA-induced autoimmune hepatitis (AIH), and identify their potential molecular mechanisms. Methods: LGP was extracted and purified using water-alcohol precipitation, DEAE-52 cellulose column, and Sephadex G200. And its structure was analyzed. It was then evaluated for anti-inflammatory and hepatoprotective effects in ConA-induced cells and mice, assessing cellular viability and inflammation with Cell Counting Kit-8 (CCK-8), Reverse Transcription-polymerase Chain Reaction (RT-PCR), and Western Blot, and hepatic injury, inflammation, and apoptosis with various biochemical and staining methods. Results: LGP is a polysaccharide composed of glucose (Glu), galactose (Gal), and arabinose (Ara), with a molar ratio of 12.9:1.6:1.0. LGP has a low crystallinity amorphous powder structure and is free from impurities. LGP enhances cell viability and reduces inflammatory factors in ConA-induced RAW264.7 cells and inhibits inflammation and hepatocyte apoptosis in ConA-induced mice. LGP inhibits Phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Toll-like receptors/Nuclear factor kappa B (TLRs/NF-κB) signaling pathways in vitro and in vivo to treat AIH. Conclusions: LGP was successfully extracted and purified, exhibiting potential as a treatment for ConA-induced autoimmune hepatitis due to its ability to inhibit the PI3K/AKT and TLRs/NF-κB signaling pathways and protect liver cells from damage.
Salecan, a natural β-glucan and one of the novel food ingredients approved in China, has been shown a variety of positive health effects, yet the mechanism of liver injury remains poorly understood. In addition, β-glucan could induce the shifts in gut microbiota, however, whether modulation of gut microbiota by β-glucan is associated with their positive health effects remain elusive. Here, the anti-inflammatory effects and the underlying mechanism of Salecan supplementation in CCl4-induced liver injury were investigated. After 8 weeks of treatment, we observed that Salecan alleviated liver injury by regulating inflammatory response and M2 macrophage polarization. In addition, Salecan treatment modulated the composition of gut microbiota and antibiotic cocktail treatment indicated that the hepatoprotective effect of Salecan was dependent on the gut microbiota. Fecal microbiota transplantation was used to further verify the mechanism, and we confirmed that microbial colonization partially alleviated liver injury. Besides, microbiota-derived metabolites of Salecan also contributed to the hepatoprotective and anti-inflammatory effect of Salecan against liver injury. These findings supported that Salecan intervention attenuated liver injury by regulating gut microbiota and its metabolites.
Background Blepharis maderaspatensis is an ethnomedicinal plant used by the Mavilan and Koraga tribes of Kerala state, India for the treatment of liver diseases. Thus, the present study aims to evaluate the liver protective activity of defatted ethanolic extract of B. maderaspatensis on lipopolysaccharide-induced acute liver inflammation and oxidative stress in Wistar rat model. Methods Preliminary phytochemical evaluation and high-performance thin-layer chromatography fingerprint validation were performed. The total phenolic and flavonoid contents were measured using the Folin-Ciocalteu method and the aluminium chloride method, respectively. The acute oral toxicity study was conducted in Swiss albino mice in accordance with OECD 423 guideline. Effect of defatted ethanolic extract of the whole plant of B. maderaspatensis (BmE) on liver inflammation was evaluated in LPS-induced Wistar rat model. The rats were treated with BmE (100, 250 and 500 mg/kg body weight) once daily for 7 days prior to the LPS (single dose i.p., 10 mg/kg b.w.) challenge. Liver tissue biochemicals (ALT, AST and ALP), and antioxidant status (SOD, CAT, GSH, MDA, NO and MPO) in euthanised experimental rats were carried out using commercial kits/standard procedures. Haematology (RBC, WBC, Hb and PLT; 4 and 24 h), and histopathology analysis were also performed. Serum IL-1β, IL-6, TNF-α and PGE2 production were determined by ELISA. The expressions of COX-2, iNOS, IL-1β, IL-6, TNF-α and NF-κB were analysed by qRT-PCR. Phosphorylation of IκB-α was measured by Western blotting. LC-MS analysis of BmE was also performed. Results The LD50 value of BmE was found to be >5000 mg/kg b.w. BmE500 showed significant protection from LPS-induced liver injury as evidenced by reduced serum enzyme levels (AST, ALT and ALP; p ≤ 0.001) and markedly improved the liver antioxidant status (MDA, GSH, SOD and CAT; p ≤ 0.001), when compared to the LPS alone treated group. Haematology (24 h, RBC, WBC, Hb and PLT; p ≤ 0.001) and histopathology also supported the above results. BmE500 pre-treatment significantly suppressed the LPS-induced expression of iNOS (p ≤ 0.001) and COX-2 (p ≤ 0.001), and the subsequent release of NO (p ≤ 0.001) and PGE2 (p ≤ 0.001). Moreover, BmE500 inhibited the gene expression of pro-inflammatory cytokines, including IL-1β (p ≤ 0.001), IL-6 (p ≤ 0.001), and TNF-α (p ≤ 0.01), which is supported by ELISA results. In addition, BmE500 markedly attenuated the activation of transcription factor NF-κB (p ≤ 0.001) as well as phosphorylation of IκBα, as evidenced from the qRT-PCR and Western blot analysis, respectively. The silymarin (100 mg/kg b.w.) drug standard also showed significant improvement in all parameters analysed. Conclusion Results of the present investigation suggest that BmE has a significant protective effect on LPS-induced acute liver inflammation via attenuating inflammatory reactions, evidenced by the inhibition of NF-κB signalling cascade, and also through its antioxidant effects. Thus, the pharmacological data generated provide experimental evidence that clearly justifies the use of B. maderaspatensis as a liver protective agent in tribal medicine.
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The purpose of this study was to alleviate liver disturbance by applying polysaccharides from Dicliptera chinensis (DCP) to act on the adenosine monophosphate–activated protein kinase/ nuclear factor erythroid 2‐related factor 2 (AMPK/ Nrf2) oxidative stress pathway and the Toll‐like receptor 4 (TLR‐4)/ nuclear factor kappa‐B (NF‐κB) inflammatory pathway and to establish an in vivo liver disturbance model using male C57BL/6J and TLR‐4 knockout (−/−) mice. For this, we evaluated the expression levels of SREBP‐1 and Nrf2 after silencing the expression of AMPK using siRNA technology. Our results show that with regard to the TLR‐4/ NF‐κB inflammatory pathway, DCP inhibits TLR‐4, up‐regulates the expression of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), reduces the expression of phospho(p)‐NF‐κB and leads to the reduction of downstream inflammatory factors, such as tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐6 and IL‐1β, thereby inhibiting the inflammatory response. Regarding the AMPK/ Nrf2 oxidative stress pathway, DCP up‐regulates the expression of p‐AMPK and Nrf2, in addition to regulating glucose and lipid metabolism, oxidative stress and ameliorating liver disturbance symptoms. In summary, our study shows that DCP alleviates liver disturbances by inhibiting mechanisms used during liver inflammation and oxidative stress depression, which provides a new strategy for the clinical treatment of liver disturbance.
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Sepsis caused by infection is one of the most important problems of clinical medicine. This study aimed to determine the effect of Apilarnil (API), a bee product, on lipopolysaccharide (LPS) induced liver injury. In the study, 64 adult Sprague-Dawley rats were divided into eight groups; control, 0.2, 0.4 and 0.8 g / kg apilarnil (API) treated groups, LPS (30 mg / kg) group, LPS + 0.2, LPS + 0.4 and LPS + 0.8 g / kg API. At tissues obtained from rats, histopathological evaluation, biochemical analysis by ELISA (Catalase-CAT, malondialdehyde-MDA, superoxide dismutase-SOD, xanthine oxidase-XOD, and testican 1-TCN-1), immunohistochemical evaluation (Toll-like receptor 4 (TLR4), High Mobility Group Box Protein 1 (HMGB-1), nuclear factor kappa B (NF-κB), Tumor necrosis factor-alpha (TNF-α), Interleukin 1 beta (IL-1β), Interleukin 6 (IL-6) and Inducible nitric oxide (iNOS)), TUNEL analysis to determine the number of apoptotic cells and Comet test as an indicator of DNA damage were performed. Histopathological examination revealed dilated blood vessels, inflammatory cell infiltration, and pyknotic nuclei damaged hepatocytes in the liver tissues of the LPS group. It was found that tissue damage was decreased significantly in LPS + API treatment groups compared to the LPS group. The number of TUNEL positive cells observed in the LPS group in liver samples increased compared to control and API-treated groups only (p
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Background: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and has become a public health concern worldwide. The hallmark of NAFLD is hepatic steatosis. Therefore, there is an urgent need to develop new therapeutic strategies that are efficacious and have minimal side effects in hepatic steatosis and NAFLD treatment. The present study aimed to investigate the effect of dietary supplement of curcumin on high-fat diet (HFD)-induced hepatic steatosis and the underlying mechanism. Methods: ApoE-/- mice were fed a normal diet, high-fat diet (HFD) or HFD supplemented with curcumin (0.1% w/w) for 16 weeks. Body and liver weight, blood biochemical.parameters, and liver lipids were measured. Intestinal permeability, hepatic steatosis and mRNA and protein expressions of TLR4-related inflammatory signaling molecule were analyzed. Results: The administration of curcumin significantly prevented HFD-induced body weight gain and reduced liver weight. Curcumin attenuated hepatic steatosis along with improved serum lipid profile. Moreover, curcumin up-regulated the expression of intestinal tight junction protein zonula occluden-1 and occludin, which further improved gut barrier dysfunction and reduced circulating lipopolysaccharide levels. Curcumin also markedly down-regulated the protein expression of hepatic TLR4 and myeloid differentiation factor 88 (MyD88), inhibited p65 nuclear translocation and DNA binding activity of nuclear factor-κB (NF-κB) in the liver. In addition, the mRNA expression of hepatic tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) as well as the plasma levels of TNF-α and IL-1β were also lowered by curcumin treatment. Conclusion: These results indicated that curcumin protects against HFD-induced hepatic steatosis by improving intestinal barrier function and reducing endotoxin and liver TLR4/NF-κB inflammation. The ability of curcumin to inhibit hepatic steatosis portrayed its potential as effective dietry intervention for NAFLD prevention.
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The current study was conducted to investigate the hepatoprotective effects of Salvia miltiorrhiza polysaccharides (SMP) against lipopolysaccharide (LPS) induced liver injury. The mice were treated with SMP first and LPS later. Results showed that SMPs significantly reduced the activities of serum alanine aminotransferase (ALT), aspartate transaminase (AST), total bilirubin (TBIL), malondialdehyde (MDA) and contents of inflammation factors and mRNA expressions in LPS induced liver injury mice. However, SMP significantly increased the contents of glutathione (GSH), Superoxide dismutase (SOD) and Total antioxidant capacity (T-AOC). SMP also downregulated the expressions of p-p65, p-IκBα, inducible nitricoxide synthase (iNOS) and improved the expression of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and Heme Oxygenase-1 (HO-1). The study indicates SMP protects the liver via attenuating inflammatory reactions and antioxidant effects.
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Acetaminophen (APAP)-induced acute hepatotoxicity is the leading cause of drug-induced acute liver failure. The aim of this study was to evaluate the effects of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol) on the protection of APAP-induced hepatotoxicity in mice. Mice were pretreated with a single dose of tempol (20 mg/kg per day) orally for 7 days. On the seventh day, mice were injected with a single dose of APAP (300 mg/kg) to induce acute hepatotoxicity. Our results showed that tempol treatment markedly improved liver functions with alleviations of histopathological damage induced by APAP. Tempol treatment upregulated levels of antioxidant proteins, including superoxide dismutase, catalase, and glutathione. Also, phosphorylation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) and protein expression of nuclear factor erythroid 2-related factor (Nrf 2) and heme oxygense-1 (HO-1) were all increased by tempol, which indicated tempol protected against APAP-induced hepatotoxicity via the PI3K/Akt/Nrf2 pathway. Moreover, tempol treatment decreased pro-apoptotic protein expressions (cleaved caspase-3 and Bax) and increased anti-apoptotic Bcl-2 in liver, as well as reducing apoptotic cells of TUNEL staining, which suggested apoptotic effects of tempol treatment. Overall, we found that tempol normalizes liver function in APAP-induced acute hepatotoxicity mice via activating PI3K/Akt/Nrf2 pathway, thus enhancing antioxidant response and inhibiting hepatic apoptosis.
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Aim of the study: In this study we investigated Fas, FasL and Foxp3 expression in relation to liver graft rejection and its severity in autoimmune hepatitis (AIH) patients. Material and methods: Twenty-three AIH patients including five post-transplant patients with acute rejection (AR) and 18 patients without AR (non-AR) were studied for Fas, FasL and Foxp3 gene expression in peripheral blood mononuclear cells on days 1, 3 and 7 after transplantation by real-time PCR. The relationships between gene expression and clinical features were determined. Results: Real-time PCR showed various Fas gene expression levels with no significant difference between the days in AR patients (p = 0.52). In non-AR patients, Fas level increased from 0.98 ±0.24 fold on the first day to 1.89 ±0.42 fold on day 3 after transplantation (p < 0.01). In this group of patients, we also found a significant increase in FasL expression on day 7 (29.91 ±6.89 fold) compared to day 1 (13.50 ±7.44 fold, p < 0.05). Foxp3 gene expression in both groups showed decreased levels during the first week after transplantation. The decreased Foxp3 expression in AR patients was correlated with rejection activity index (r = 0.86, p < 0.0001). Conclusions: Increased Fas and FasL gene expression levels in non-AR patients and decreased Foxp3 gene expression in both groups suggested the important role of these molecules in the alloreactive response after liver transplantation in AIH patients. Foxp3 expression might be useful for monitoring rejection severity.
Background: A mouse model in which diabetes mellitus was induced by low-dose streptozotocin (STZ) injection combined with a high fat diet was used to study the effect of two water cress (Lepidium savitum) preparations. Diabetic mice were treated with dried cress powder or with water-soluble extracts (tested at two doses), together with proper control groups. The mice were evaluated after 4 weeks of continuous intervention for type 2 diabetic and associated markers. We determined blood glucose, body weight, total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), serum insulin levels, and DNA integrity of hepatic cells. The concentrations of malondialdehyde (MDA) and lipid peroxide (LPO) and the activities of four enzymes that are part of the antioxidant defense system were determined in liver samples, as well as gene expression (by semi-quantitative RT-PCR) and enzyme activity of IRS-1, IRS-2, PI3K, AKT-2 and GLUT4. Results: After 4 weeks of intervention, the levels of TC, TG and LDL-C were significantly (P<0.5) decreased, while HDL-C was significantly increased. Enzyme activity of liver SOD, GSH, GSH-PX and CAT was significantly increased, while concentrations of MDA and LPO were significantly reduced. The transcription level of the 5 assessed genes was increased, with corresponding increases in protein expression. Conclusion: Oral uptake of garden cress can significantly reduce the blood glucose and improve the blood lipid metabolism of diabetic mice. Considerable improvements in the activity of antioxidant defense enzymes were observed in type 2 diabetic mice that improved the body's antioxidant emergency response. This article is protected by copyright. All rights reserved.
Peroxisomes are essential organelles for maintaining the homeostasis of lipids and reactive oxygen species (ROS). While oxidative stress-induced endoplasmic reticulum (ER) stress plays an important role in nonalcoholic fatty liver disease (NAFLD), the role of peroxisomes in ROS-mediated ER stress in the development of NAFLD remains elusive. We investigated whether an impaired peroxisomal redox state accelerates NAFLD by activating ER stress by inhibiting catalase, an antioxidant expressed exclusively in peroxisomes. Wild-type (WT) and catalase knockout (CKO) mice were fed either a normal diet or a high-fat diet (HFD) for 11 weeks. HFD-induced phenotype changes and liver injury accompanied by ER stress and peroxisomal dysfunction were accelerated in CKO mice compared to WT mice. Interestingly, these changes were also significantly increased in CKO mice fed a normal diet. Inhibition of catalase by 3-aminotriazole in hepatocytes resulted in the following effects: (i) increased peroxisomal H2O2 levels as measured by a peroxisome-targeted H2O2 probe (HyPer-P); (ii) elevated intracellular ROS; (iii) decreased peroxisomal biogenesis; (iv) activated ER stress; (v) induced lipogenic genes and neutral lipid accumulation; and (vi) suppressed insulin signaling cascade associated with JNK activation. N-acetylcysteine or 4-phenylbutyric acid effectively prevented these alterations. These results suggest that a redox imbalance in peroxisomes perturbs cellular metabolism through the activation of ER stress in the liver.
Tumour necrosis factor-stimulated gene 6 (TSG6) is a protective inflammatory reaction gene which is upregulated by inflammatory processes. Recent studies suggest that TSG-6 exhibits anti-scarring effects. However, the mechanism of TSG-6 action in the scar formation remains poorly understood. We investigated whether TSG-6 affects growth of the human hypertrophic scar fibroblasts (HSFs) via Fas/FasL signalling pathway. Cultured HSFs were transfected with a vector carrying the TSG6 gene (pLVX-Puro-TSG-6) or with a vector not containing the TSG6 gene (pLVX-Puro). Untransfected HSFs served as a control group to both transfected HSFs. The expressions level of TSG-6 was up-regulated in the pLVX-Puro-TSG-6 group at the protein and mRNA level. MTT and flow cytometry were used to assess the effect of TSG-6 on the growth and apoptotic status of HSFs. Finally, qRT-PCR and western blot were used to measure the expression levels of Fas, FasL, FADD, caspase-3 and caspase-8 in each group. The apoptosis rate was significantly enhanced and the growth rate reduced in the HSFs transfected with the TSG6 gene vector. The expression levels of Fas, FasL, FADD, caspase-3 and caspase- 8 were significantly raised in the TSG-6 overexpressing HSFs. It is concluded that increased expression of TSG-6 may induce apoptosis of human hypertrophic scar fibroblasts via activation of the Fas/FasL signalling pathway.