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Astragalus membranaceus Extract Attenuates Inflammation and Oxidative Stress in Intestinal Epithelial Cells via NF-κB Activation and Nrf2 Response

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Astragalus membranaceus , dried root extract, also known asAstragali radix, is used in traditional Chinese medicine as a tonic remedy. Moreover, it has been reported thatAstragalus membranaceuscould attenuate intestinal inflammation; however, the underlying mechanism for its anti-inflammatory activity in intestinal epithelial cells (IECs) remains unclear. In this study, we evaluatedAstragalus membranaceusextract (5-100 µg/mL) in a model of inflammation and oxidative stress for IECs. We showed thatAstragalus membranaceusextract reduced the inflammatory response induced by lipopolysaccharide fromE. coli(LPS) plus interferon-γ (IFN), decreasing tumor necrosis factor-α (TNF-α) release, cycloxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, nitrotyrosine formation, nuclear factor-κB (NF-κB) activation, and reactive oxygen species (ROS) release in the non-tumorigenic intestinal epithelial cell line (IEC-6). The antioxidant potential ofAstragalus membranaceusextract was also evaluated in a model of hydrogen peroxide (H₂O₂)-induced oxidative stress in IEC-6, indicating that this extract reduced ROS release and increased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation and the expression of antioxidant cytoprotective factors in these cells. The results contributed to clarify the mechanisms involved inAstragalus membranaceusextract-reduced inflammation and highlighted the potential use of this extract as an anti-inflammatory and antioxidant remedy for intestinal diseases.
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International Journal of
Molecular Sciences
Article
Astragalus membranaceus Extract Attenuates
Inflammation and Oxidative Stress in Intestinal
Epithelial Cells via NF-κB Activation and
Nrf2 Response
Simona Adesso 1, Rosario Russo 2ID , Andrea Quaroni 3, Giuseppina Autore 1
and Stefania Marzocco 1, *ID
1Department of Pharmacy, University of Salerno-Via Giovanni Paolo II, 132-84084 Fisciano-Salerno, Italy;
sadesso@unisa.it (S.A.); autore@unisa.it (G.A.)
2
Giellepi S.p.A. Health Science Department, Via Benvenuto Cellini 37, 20851 Lissone (Monza Brianza), Italy;
rosario.russo@giellepi.it
3Department of Biomedical Sciences, Cornell University, Veterinary Research Tower, Cornell University,
Ithaca, NY 14853-6401, USA; aq10@cornell.edu
*Correspondence: smarzocco@unisa.it; Tel.: +39-08996-9250
Received: 11 February 2018; Accepted: 9 March 2018; Published: 10 March 2018
Abstract:
Astragalus membranaceus, dried root extract, also known as Astragali radix, is used in
traditional Chinese medicine as a tonic remedy. Moreover, it has been reported that Astragalus
membranaceus could attenuate intestinal inflammation; however, the underlying mechanism for
its anti-inflammatory activity in intestinal epithelial cells (IECs) remains unclear. In this study,
we evaluated Astragalus membranaceus extract (5–100
µ
g/mL) in a model of inflammation and
oxidative stress for IECs. We showed that Astragalus membranaceus extract reduced the inflammatory
response induced by lipopolysaccharide from E. coli (LPS) plus interferon-
γ
(IFN), decreasing tumor
necrosis factor-
α
(TNF-
α
) release, cycloxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS)
expression, nitrotyrosine formation, nuclear factor-
κ
B (NF-
κ
B) activation, and reactive oxygen species
(ROS) release in the non-tumorigenic intestinal epithelial cell line (IEC-6). The antioxidant potential of
Astragalus membranaceus extract was also evaluated in a model of hydrogen peroxide (H
2
O
2
)-induced
oxidative stress in IEC-6, indicating that this extract reduced ROS release and increased nuclear factor
(erythroid-derived 2)-like 2 (Nrf2) activation and the expression of antioxidant cytoprotective factors
in these cells. The results contributed to clarify the mechanisms involved in Astragalus membranaceus
extract-reduced inflammation and highlighted the potential use of this extract as an anti-inflammatory
and antioxidant remedy for intestinal diseases.
Keywords: Astragalus membranaceus; intestinal epithelial cells; inflammation; oxidative stress
1. Introduction
Inflammatory bowel disease (IBD) is one of the most prevalent gastrointestinal disorders, and
it includes ulcerative colitis (UC) [
1
] and Crohn’s disease (CD) [
2
]. IBD pathogenesis results from
a multifactorial process involving genetic, environmental, and immunogenic factors [
3
]. Over the
last few decades, our understanding of IBD aetiology has increased, yet its exact mechanisms remain
unclear. Nevertheless, it is well known that IBD is characterized by inflammation and abnormalities in
epithelial barrier function [
4
]. Current knowledge shows that intestinal epithelial cells (IECs), which
under physiological conditions are indispensable to maintain a selective barrier between the host and
harmful substances present in the lumen, have emerged as key players in the generation and persistence
of intestinal inflammation during IBD [
5
]. Different noxious agents, such as chemical, physical,
Int. J. Mol. Sci. 2018,19, 800; doi:10.3390/ijms19030800 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2018,19, 800 2 of 14
infectious, and inflammatory injuries can damage the intestinal epithelial integrity. This damage
can lead to increased penetration and absorption of toxic substances, the activation of immunogenic
responses, and a final disequilibrium in the host’s homeostasis. In response to these external factors,
IECs and immune cells are activated and can trigger an inflammatory response [
6
]. The release of
pro-inflammatory factors, such as cytokines and chemokines, as well as oxidative stress with the release
of reactive oxygen species (ROS), are the main events taking place in intestinal inflammation during the
active phase. In particular, by influencing transcription factors and redox-sensitive signaling pathways,
ROS and their oxidized products can further sustain inflammation within the intestinal layer.
Plants are considered a potential source of antioxidant and anti-inflammatory molecules. The genus
Astragalus belongs to the legume family (Fabaceae). This genus, the largest of flowering plants in
the world, includes 2500–3000 species mainly found in Central and Southwestern Asia [
7
,
8
]. Different
Astragalus species are used in traditional medicine, mostly Chinese, and the dried roots of A. membranaceus
(Fisch.) Bge and A. membranaceus (Fisch.) var. mongholicus (Bge) Hsiao are included in the drug
Huangqi (Radix Astragali), which is present in the pharmacopoeia of the People’s Republic of China [
9
].
Astragalus membranaceus is used as a tonic and has many effects, such as enhancing defensive energy
and inducing diuresis to treat edema [
10
]. It is widely used in East Asia to prevent some severe
chemotherapy side effects [
11
] and liver fibrosis [
12
]. Moreover, recent pharmacological studies and
clinical evidence centered on Astragalus membranaceus have reported a wide spectrum of biological
activities for this plant [
13
,
14
], including at an intestinal level [
15
17
]. Astragali radix, the dried root of
Astragalus membranaceus, is a popular health-promoting herb, and its use as a crude drug is one of the
oldest and most frequently used remedies in oriental medicine [
18
]. Pharmacological studies have
demonstrated that the water extract of Astragali radix possesses many biological functions [
19
23
]. Also,
Astragalus polysaccharides, which are major constituents of Astragali radix, possess many biological
effects and pharmacological properties, including at intestinal levels [
24
,
25
]. Despite this, there
is little mechanistic knowledge regarding the molecular action(s) of Astragalus membranaceus root
extract on IECs [
26
] and especially during inflammatory conditions. Considering the pivotal role of
IECs in maintaining and regulating intestinal homeostasis, in this study, we evaluated the effects of
Astragalus tmembranaceus extract (5–100
µ
g/mL) on inflammation and oxidative stress in the intestinal
epithelial cell line (IEC-6) in order to elucidate the effect of Astragalus membranaceus extract during
intestinal inflammation at the cellular level.
2. Results
2.1. Astragalus membranaceus Extract Did Not Have Any Antiproliferative Activity on IEC-6 Cells
To evaluate the antiproliferative potential of Astragalus membranaceus extract on IEC-6, cells were
treated with the extract (5–100
µ
g/mL) for 24 h. The results indicated that the extract did not have
any significant antiproliferative activity on IEC-6 cells (mean
±
SEM of % antiproliferative activity
vs. control: 1.12
±
1.04, 3.38
±
1.20, 4.06
±
1.15, 6.16
±
2.03, respectively for Astragalus membranaceus
extract 5, 10, 50, 100 µg/mL).
2.2. Astragalus membranaceus Extract Reduced Tumor Necrosis Factor-
α
(TNF-
α
) Levels in Lipopolysaccharide
from E. coli (LPS) + Interferon-γ(IFN)-Stimulated IEC-6
The effect of Astragalus membranaceus extract on TNF-
α
levels in IEC-6 cellular medium
was evaluated using an enzyme-linked immunosorbent assay (ELISA). Our results showed that
Astragalus membranaceus extract (5–100
µ
g/mL) significantly inhibited TNF-
α
release, induced by
LPS + IFN, in IEC-6 cells medium (p< 0.05 vs. LPS + IFN; Figure 1A).
Int. J. Mol. Sci. 2018,19, 800 3 of 14
2.3. Astragalus membranaceus Extract Reduced Cycloxygenase-2 (COX-2) and Inducible Nitric Oxide
Synthase (iNOS) Expression and Nitrotyrosine Formation in LPS + IFN-Stimulated IEC-6
Expression of COX-2 and iNOS were evaluated by a cytofluorimetric technique. Our results
showed that Astragalus membranaceus extract (5–100
µ
g/mL) inhibited COX-2 and iNOS expression
in IEC-6 cells at all tested concentrations (p< 0.05 vs. LPS + IFN; Figure 1B,C). Under the same
experimental conditions, the extract (5–100
µ
g/mL) also inhibited nitrotyrosine formation in IEC-6
cells (p< 0.01 vs. LPS + IFN; Figure 1D).
Int. J. Mol. Sci. 2018, 19, 800 3 of 14
2.3. Astragalus membranaceus Extract Reduced Cycloxygenase-2 (COX-2) and Inducible Nitric Oxide
Synthase (iNOS) Expression and Nitrotyrosine Formation in LPS + IFN-Stimulated IEC-6
Expression of COX-2 and iNOS were evaluated by a cytofluorimetric technique. Our results
showed that Astragalus membranaceus extract (5–100 µg/mL) inhibited COX-2 and iNOS expression in
IEC-6 cells at all tested concentrations (p < 0.05 vs. LPS + IFN; Figure 1B,C). Under the same
experimental conditions, the extract (5–100 µg/mL) also inhibited nitrotyrosine formation in IEC-6
cells (p < 0.01 vs. LPS + IFN; Figure 1D).
Figure 1. Inhibitory and concentration related effect of Astragalus membranaceus extract (5–100 µg/mL)
in LPS + IFN-stimulated IEC-6 on (A) tumor necrosis factor-α (TNFα) levels, evaluated using an
ELISA, (B) cyclooxygenase-2 (COX-2) expression, (C) inducible nitric oxide synthase (iNOS)
expression, and on (D) nitrotyrosine formation, evaluated by the cytofluorimetric technique. Data are
expressed as mean ± SEM. ***, **, * indicate p < 0.001, p < 0.01 and p < 0.05 vs. LPS + IFN.
2.4. Astragalus membranaceus Extract Reduced p65 Nuclear Factor-κB (NF-κB) Translocation in LPS +
IFN-Stimulated IEC-6
To evaluate NF-κB activation, p65 NF-κB was labeled with a green fluorescent marker.
Astragalus membranaceus extract alone did not induce p65 nuclear translocation in IEC-6 cells (Figure
2). However, at a concentration of 50 µg/mL, the extract inhibited p65 NF-κB nuclear translocation
when compared to LPS + IFN treatment alone (Figure 2).
Figure 1.
Inhibitory and concentration related effect of Astragalus membranaceus extract (5–100
µ
g/mL)
in LPS + IFN-stimulated IEC-6 on (
A
) tumor necrosis factor-
α
(TNF
α
) levels, evaluated using an ELISA,
(
B
) cyclooxygenase-2 (COX-2) expression, (
C
) inducible nitric oxide synthase (iNOS) expression, and on
(
D
) nitrotyrosine formation, evaluated by the cytofluorimetric technique. Data are expressed as
mean ±SEM. ***, **, * indicate p< 0.001, p< 0.01 and p< 0.05 vs. LPS + IFN.
2.4. Astragalus membranaceus Extract Reduced p65 Nuclear Factor-κB (NF-κB) Translocation in LPS +
IFN-Stimulated IEC-6
To evaluate NF-
κ
B activation, p65 NF-
κ
B was labeled with a green fluorescent marker. Astragalus
membranaceus extract alone did not induce p65 nuclear translocation in IEC-6 cells (Figure 2). However,
at a concentration of 50
µ
g/mL, the extract inhibited p65 NF-
κ
B nuclear translocation when compared
to LPS + IFN treatment alone (Figure 2).
Int. J. Mol. Sci. 2018,19, 800 4 of 14
Int. J. Mol. Sci. 2018, 19, 800 4 of 14
Figure 2. Effects of Astragalus membranaceus extract (50 µg/mL) alone and with LPS + IFN on nuclear
factor-κB (NF-κB) p65 nuclear translocation, evaluated by immunofluorescence analysis. The blue
fluorescence identified the nuclei, while the green fluorescence indicated the p65 NF-κB subunit.
2.5. Astragalus membranaceus Extract Reduced ROS Release by IEC-6 Cells
The antioxidant potential of Astragalus membranaceus extract was evaluated by measuring the
intracellular ROS production in LPS + IFN-stimulated IEC-6 cells. It was found that the root extract
(5–100 µg/mL) significantly inhibited ROS production in IEC-6 cells (p < 0.01 vs. LPS + IFN; Figure 3
A,B). To further evaluate its antioxidant potential, Astragalus membranaceus extract (5–100 µg/mL) was
also evaluated in IEC-6 cells treated with the pro-oxidant stimulus H
2
O
2
(1 mM). Again, under these
different experimental conditions, as assessed during inflammatory conditions, Astragalus
membranaceus exhibited significant antioxidant activity by inhibiting ROS release (p < 0.01 vs. H
2
O
2
;
Figure 3 C,D).
Figure 2.
Effects of Astragalus membranaceus extract (50
µ
g/mL) alone and with LPS + IFN on nuclear
factor-
κ
B (NF-
κ
B) p65 nuclear translocation, evaluated by immunofluorescence analysis. The blue
fluorescence identified the nuclei, while the green fluorescence indicated the p65 NF-κB subunit.
2.5. Astragalus membranaceus Extract Reduced ROS Release by IEC-6 Cells
The antioxidant potential of Astragalus membranaceus extract was evaluated by measuring the
intracellular ROS production in LPS + IFN-stimulated IEC-6 cells. It was found that the root extract
(5–100
µ
g/mL) significantly inhibited ROS production in IEC-6 cells (p< 0.01 vs. LPS + IFN; Figure 3A,B).
To further evaluate its antioxidant potential, Astragalus membranaceus extract (5–100
µ
g/mL) was also
evaluated in IEC-6 cells treated with the pro-oxidant stimulus H
2
O
2
(1 mM). Again, under these
different experimental conditions, as assessed during inflammatory conditions, Astragalus membranaceus
exhibited significant antioxidant activity by inhibiting ROS release (p< 0.01 vs. H2O2; Figure 3C,D).
Int. J. Mol. Sci. 2018, 19, 800 4 of 14
Figure 2. Effects of Astragalus membranaceus extract (50 µg/mL) alone and with LPS + IFN on nuclear
factor-κB (NF-κB) p65 nuclear translocation, evaluated by immunofluorescence analysis. The blue
fluorescence identified the nuclei, while the green fluorescence indicated the p65 NF-κB subunit.
2.5. Astragalus membranaceus Extract Reduced ROS Release by IEC-6 Cells
The antioxidant potential of Astragalus membranaceus extract was evaluated by measuring the
intracellular ROS production in LPS + IFN-stimulated IEC-6 cells. It was found that the root extract
(5–100 µg/mL) significantly inhibited ROS production in IEC-6 cells (p < 0.01 vs. LPS + IFN; Figure 3
A,B). To further evaluate its antioxidant potential, Astragalus membranaceus extract (5–100 µg/mL) was
also evaluated in IEC-6 cells treated with the pro-oxidant stimulus H
2
O
2
(1 mM). Again, under these
different experimental conditions, as assessed during inflammatory conditions, Astragalus
membranaceus exhibited significant antioxidant activity by inhibiting ROS release (p < 0.01 vs. H
2
O
2
;
Figure 3 C,D).
Figure 3. Cont.
Int. J. Mol. Sci. 2018,19, 800 5 of 14
Int. J. Mol. Sci. 2018, 19, 800 5 of 14
Figure 3. (A,B) Effect of a graded concentration of Astragalus membranaceus extract (5–100 µg/mL) in
the presence of LPS + IFN on reactive oxygen species (ROS) levels, detected by 2,7
dichlorofluorescein-diacetate (H
2
DCF-DA). (C,D) Effect of Astragalus membranaceus extract (50
µg/mL) in the presence of H
2
O
2
on ROS formation, evaluated with the probe H
2
DCF-DA. Data are
expressed as mean ± SEM. ***, ** denote p < 0.001 and p < 0.01 vs. LPS + IFN or vs. H
2
O
2
.
510 50 100
0
20
40
60
80
100
***
**
LPS + IFN
***
**
B
A. membranaceus
extract [g/mL]
% of inhibition ROS release
vs. LPS + IFN
510 50 100
0
20
40
60
80
100
***
***
H
2
O
2
***
**
D
A. mem branaceus
extract [g/mL]
% of inhibition ROS release
vs. H
2
O
2
Figure 3.
(
A
,
B
) Effect of a graded concentration of Astragalus membranaceus extract (5–100
µ
g/mL) in
the presence of LPS + IFN on reactive oxygen species (ROS) levels, detected by 2
0
,7
0
dichlorofluorescein-
diacetate (H
2
DCF-DA). (
C
,
D
) Effect of Astragalus membranaceus extract (50
µ
g/mL) in the presence of
H
2
O
2
on ROS formation, evaluated with the probe H
2
DCF-DA. Data are expressed as mean
±
SEM.
***, ** denote p< 0.001 and p< 0.01 vs. LPS + IFN or vs. H2O2.
Int. J. Mol. Sci. 2018,19, 800 6 of 14
2.6. Astragalus membranaceus Extract Induced Nuclear Factor (Erythroid-Derived 2)-Like 2 (Nrf2)
Activation and Heme Oxygenase 1 (HO-1), NAD(P)H Quinone Dehydrogenase 1 (NQO1) Expression in
IEC-6 Cells
Oxidative stress is due to a disequilibrium between pro-oxidant and antioxidant factors. To assess
the antioxidant potential of Astragalus membranaceus, the antioxidant Nrf2 response was also evaluated.
As shown in Figure 4A, Nrf2 nuclear translocation in IEC-6 cells was slightly increased one hour
after the addition of Astragalus membranaceus extract (50
µ
g/mL) when compared to the untreated
control cells. Moreover, Nrf2 activation was increased even more with the addition of the extract
under pro-oxidant conditions when compared to cells treated by H
2
O
2
alone (Figure 4A). Expression of
Nrf2-related cytoprotective factors, such as HO-1 and NQO1, was significantly increased in the presence
of H
2
O
2
(p< 0.001 vs. control; Figure 4B,C), and it was further increased by Astragalus membranaceus
extract (p< 0.001 vs. H2O2; Figure 4B,C), thus supporting the antioxidant potential of the latter.
Figure 4.
(
A
) Effect of Astragalus membranaceus extract (50
µ
g/mL) alone and in the presence of
H
2
O
2
on the translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) into the cellular nuclei,
evaluated by immunofluorescence analysis. The blue fluorescence identified the nuclei while the
green fluorescence indicated Nrf2. (
B
) Effect of Astragalus membranaceus extract (5–100
µ
g/mL) in
the presence of H
2
O
2
on heme oxygenase 1 (HO-1) expression, evaluated by the cytofluorimetric
technique (
C
) and on NAD(P)H quinone dehydrogenase 1 (NQO1) in IEC-6 cells, evaluated by the
cytofluorimetric technique. The dark grey bars (B, C) represent IEC-6 cells treated with H
2
O
2
alone.
Data are expressed as mean ±SEM.◦◦ ◦ denotes p< 0.001 vs. control. *** denotes p< 0.001 vs. H2O2.
Int. J. Mol. Sci. 2018,19, 800 7 of 14
3. Discussion
Astragalus membranaceus, also known as Astragali radix, is a popular health-promoting herb that
has been used by people in China to strengthen immunity for more than 2000 years. The effect
of Astragalus membranaceus has been investigated in models of intestinal injury. In particular,
Astragalus membranaceus has been reported to have a protective effect during intestinal mucosa
reperfusion injury [
16
]. It has also been reported that Astragalus membranaceus has protective effects on
small intestine villi and that it increases the level of antioxidant factors, such as superoxide dismutase
activity (SOD) [17].
While the potential activity of Astragalus membranaceus extract as an anti-inflammatory and
antioxidant agent has been widely demonstrated in
in vivo
studies, its effects on IECs, which have
a pivotal role in maintaining intestinal homeostasis, are not well understood. For this reason, the aim of
this study was to clarify the contribution of Astragalus membranaceus extract in the inflammatory process
in the intestinal cells and to highlight its potential use as a new anti-inflammatory and antioxidant
natural remedy for IBD treatment.
This study provided evidence that Astragalus membranaceus extract decreased inflammatory
response in IECs by reducing the following: (1) TNF-
α
production; (2) COX-2 and iNOS expression;
(3) nitrotyrosine formation; and (4) NF-
κ
B activation. Interestingly, the extract also exerted an appreciable
antioxidant effect by reducing ROS release and by activating antioxidant elements, such as Nrf2 activation
and HO-1 and NQO1 expression.
During the inflammatory process, several pro-inflammatory cytokines were produced by IEC-6
cells, among them TNF-
α
. TNF-
α
has a pivotal role in intestinal inflammation, such as in IBD, and
clinical results using anti-TNF-
α
drugs support its role in IBD pathogenesis. TNF-
α
promotes the
activation and recruitment of immune cells and induces apoptosis of IECs and abnormal expression
levels of tight junction molecules in these cells factors that ultimately lead to disruption of the intestinal
mucosal barrier and increased permeability [
27
29
]. In our experiments, Astragalus membranaceus extract
significantly reduced TNF-
α
levels in IEC-6 cells during inflammation. These data are in accordance with
previous findings indicating the protective effect of the root extract of Astragalus membranaceus in a model
of hapten-induced colitis mediated by TNF-
α
modulation [
30
] and with the fact that its administration
could regulate TNF-
α
in UC patients [
31
]. Cytokines and TNF-
α
lead to the activation and release of
other inflammatory mediators, such as iNOS and COX-2, amplifying and perpetuating the inflammatory
condition in IBD. COX-2 and iNOS are expressed mainly at the inflammation sites, affecting colon
integrity and contributing to the progress of intestinal damage [
32
], potentially leading to carcinogenesis
associated with IBD. In fact, it was also shown that both COX-2 and iNOS stimulated tumor angiogenesis
in colorectal cancer, a process mainly induced by vascular endothelial growth factor [
33
]. Our results
showed that COX-2 expression was significantly reduced by Astragalus membranaceus extract in
LPS + IFN-stimulated IEC-6 at all tested concentrations. The inhibition of COX-2 expression by
Astragalus membranaceus extract indicated that the extract positively modulated the arachidonic acid
cascade during inflammation, thus significantly contributing to its anti-inflammatory effects in IECs.
Our findings agree with other studies reporting that Astragalus membranaceus reduced COX-2 expression
and inflammatory response in general [
19
,
34
]. iNOS is the inducible isoform of the nitric oxide synthase,
responsible for the synthesis of pro-inflammatory nitric oxide (NO), mainly induced in inflammatory
cells, such as macrophages and mononuclear cells neutrophils by pro-inflammatory stimulants [
35
].
It has been reported that NO levels and iNOS activity increased in the colonic mucosa of UC and CD
patients [
36
]. NO has a pivotal role in many organs and tissues and can exert beneficial or harmful
effects depending on its levels and on the redox environment of the site. Its role as a modulator of key
signaling pathways and as a regulator of the physiological functions of the gut, such as gastrointestinal
motility, absorption, and secretion, is exerted at low NO concentration. The expression of iNOS
results in high levels of NO and for a longer time. Therefore, under these conditions, NO regulates
many pathophysiologic processes. It is also known that NO produced by the inducible isoform
iNOS generates free radicals such as peroxynitrite and hydroxyl radical. Peroxynitrite affects protein
Int. J. Mol. Sci. 2018,19, 800 8 of 14
function through its ability to nitrate tyrosine residues, thus inducing nitrotyrosine formation. Since
tyrosine nitration represents an alternative pathway to phosphorylation of key residues, this process
can affect the enzymatic activity of proteins and thus significantly affect intracellular processes [
37
].
Our results provided evidence for the ability of Astragalus membranaceus extract to inhibit iNOS
protein expression at all tested concentrations, and this inhibitory effect could lead to significant
reduction in NO overproduction and thus to nitrotyrosine formation in LPS + IFN-treated IEC-6
cells. These data are in accordance with previous studies reporting that aqueous extracts of Astragalus
membranaceus could modulate pro-inflammatory cytokine gene expression and nitric oxide production in
macrophages [
38
]. A nuclear pro-inflammatory activated factor common to all these mediators is NF-
κ
B.
NF-
κ
B is a key regulator of inflammation and can be activated by a broad panel of stimuli, including
bacterial components such as LPS, proinflammatory cytokines such as TNF-
α
and interleukin-1, viruses,
and DNA-damaging agents [
39
]. Increased expression and activation of NF-
κ
B were observed in IBD
patients, especially in mucosal macrophages and epithelial cells, accompanied by enhanced production
of proinflammatory cytokines such as TNF-
α
, IL-1, and IL-6 [
40
]. The NF-
κ
B pathway functions as
a molecular link between inflammation and tumorigenesis due to its ability to stimulate the expression
of proinflammatory cytokines, antiapoptotic factors, angiogenesis factors, and proteases, which promote
tumor initiation and ensure the survival and proliferation as well as invasion of malignant cells. Here,
we reported that Astragalus membranaceus extract can inhibit NF-
κ
B activation, in accordance with
previous studies that demonstrated the protective effect of Astragalus spp. polysaccharide in inhibiting
NF-
κ
B signaling in a colitis animal model [
41
]. The roles of oxidative stress and the oxidant/antioxidant
balance in IBD development have recently received increasing attention. Oxidative stress has been
demonstrated to influence different gastrointestinal diseases, such as IBD, gastroduodenal ulcers, and
malignancies [
42
]. In our experimental model, Astragalus membranaceus extract exerted a significant
antioxidant effect by inhibiting ROS release induced both by LPS + IFN and by H
2
O
2
. These results
agree with previous data reporting the antioxidant potential of Astragalus membranaceus at the intestinal
level [
43
,
44
]. Oxidative stress is due to an imbalance between pro- and antioxidant factors; thus, cellular
antioxidant response plays a pivotal role in controlling oxidative stress. Nrf2 is a redox-sensitive
transcription factor that plays a key role in the antioxidant response. When cells were exposed
to oxidative stress, Nrf2 was released from Kelchlike ECH-associated protein 1 (Keap1), which
sequesters Nrf2 in the cytoplasm and then binds to Maf or Jun to form a heterodimer in the nucleus.
The heterodimer combines with the antioxidant response element (ARE) to promote the expression of
genes encoding many phase II detoxification and antioxidant enzymes, including HO-1 and NOQ1,
thereby improving the ability of the cell to remove ROS [
45
]. A Nrf2 deficiency has been shown to
exacerbate colonic injury in a mouse model of experimental colitis [
46
,
47
], whereas the pharmacological
activation of Nrf2 produced protective effects on the colon [
48
,
49
]. HO-1 and NQO1 enzymes also exert
a protective effect during intestinal inflammation [
50
]. In the presence of H
2
O
2
,Astragalus membranaceus
extract enhanced Nrf2 activation and HO-1 and NQO1 expression, thus increasing an antioxidant
response in IEC-6 cells. This evidence fits with other data reporting that the Astragalus constituents
activate Nrf2 cellular response [
51
]. Several bioactive compounds have been highlighted in the dried
root of Astragalus, such as polysaccharides, triterpene saponins, isoflavonoids, and various trace
elements [
52
,
53
]. The phytochemical characterization of the present extracts reveals the presence of
these active compounds [
52
,
53
]. Studies suggested that Astragalus polysaccharides have effects on the
activation of B cells and macrophages, promotion of humoral and immune responses, protection of
blood vessels, and prevention of inflammation and cancer [
54
,
55
]. Moreover, Astragalus polysaccharides
also have effects at theintestinal level; in particular, they attenuate murine colitis through inflammasome
inhibition [
25
]. The hepatoprotective properties have been widely studied, and it has been found that
the Astragalus polysaccharides’ antioxidant activity can prevent liver and intestinal damage [
13
,
56
,
57
].
Similarly, the anti-inflammatory effect of triterpene derivatives, also derived from Astagalus, has
been reported [
58
,
59
] and the antioxidant potential of Astragalus constituents has been reported and
Int. J. Mol. Sci. 2018,19, 800 9 of 14
reviewed [
60
62
]. However, despite the activity of Astragalus constituents, it is important to underscore
that the efficacy of 50% HA (Axtragyl®) is strongly related to the whole phytocomplex [53].
In conclusion, our results indicated that the Astragalus membranaceus extract exerted significant
anti-inflammatory and antioxidant effects in IECs, thus highlighting its potential application in
intestinal inflammatory conditions.
4. Materials and Methods
4.1. Reagents
Unless otherwise specified, all reagents and compounds were purchased from Sigma Chemicals
Company (Sigma, Milan, Italy).
4.2. Plant Material
Astragalus membranaceus dried root hydroalcoholic extract (Axtragyl
®
) was provided by Giellepi
Health Science Division (Lissone, Italy). The extract (50% HA) has already been characterized by
Di Cesare Mannelli et al. [
52
,
53
] and contains 70% polysaccharides and other bioactive molecules
(i.e., saponins and isoflavonoids).
4.3. Cell Culture
IEC-6 cells (CRL-1592) were purchased from the American Type Culture Collection (ATCC,
Rockville, MD, USA). These cells, derived from normal rat intestinal crypt cells [
63
], were grown in
Dulbecco’s modified Eagle’s medium (DMEM, 4 g/L glucose) with 10% (v/v) foetal bovine serum
(FBS), 2 mM L-glutamine, 1.5 g/L NaHCO3, and 0.1 unit/mL bovine insulin. Cells between the 17th
and 21st passages were used for these experiments, as previously reported [64].
4.4. Cell Treatment
IEC-6 cells were plated and, after adhesion, were treated with Astragalus membranaceus extract
(5–100
µ
g/mL) for 1 h and then co-exposed to Astragalus membranaceus extract and lipopolysaccharides
from E. coli (LPS; 10
µ
g/mL) plus interferon-
γ
(IFN; 10 U/mL) for different times, as outlined below.
In order to estimate specifically the antioxidant potential of Astragalus membranaceus extract, in other
experiments, the IEC-6 cells were incubated with Astragalus membranaceus extract (5–100
µ
g/mL) alone
for 1 h and then co-exposed to the extract and hydrogen peroxide (H2O2; 1 mM).
4.5. Antiproliferative Activity
IEC-6 cells (5.0
×
10
3
cells/well) were plated on 96-well plates and allowed to adhere for 72 h
at 37
C in a 5% CO
2
atmosphere. Thereafter, the medium was substituted with either a new
one alone or one containing serial dilutions of Astragalus membranaceus extract (5–100
µ
g/mL) and
incubated for 24 h. Cell antiproliferative activity was evaluated using the colorimetric assay of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), as formerly reported [
64
]. MTT
(5 mg/mL) was then added to IEC-6 cells. After 3 h, cells were lysed with 100
µ
L of a solution
containing 50% (v/v)N,N-dimethylformamide, and 20% (w/v) sodium dodecyl sulphate(SDS)
(pH = 4.5). A microplate spectrophotometer reader (Titertek Multiskan MCC/340-DASIT, Cornaredo,
Milan, Italy) was used to measure the optical density (OD) of released formazan in each well, as we
previously reported [
65
]. The antiproliferative activity was measured as % dead cells = 100
×
[(OD
treated/OD control) ×100].
4.6. TNF-αDetermination
TNF-
α
release in IEC-6 cells was measured with an Enzyme-Linked Immuno Sorbent Assay
(ELISA). IEC-6 cells (8.0
×
10
4
cells/well/24-well plates) were treated as previously reported for 24 h.
Supernatants from IEC-6 cells were then collected and a commercial kit (e-Biocscience, San Diego, CA,
Int. J. Mol. Sci. 2018,19, 800 10 of 14
USA) was used to perform the ELISA, according to the manufacturer’s instructions (e-Biosciences,
San Diego, CA, USA). The results were expressed as pg/mL, as formerly reported [66].
4.7. Measurement of COX-2, iNOS, HO-1, NQO1 Expression and Nitrotyrosine Formation by Cytofluorimetry
IEC-6 cells were plated into 96-well plates (1
×
10
4
cells/well) and treated as previously indicated
for 24 h in order to evaluate COX-2 and iNOS expression and nitrotyrosine formation in IEC-6 cells.
In another set of experiments, the IEC-6 cells were incubated with the extract and H
2
O
2
as previously
indicated, in order to measure HO-1 and NQO1 expression. For this analysis, IEC-6 cells were then
collected and washed with phosphate buffered saline (PBS). Fixing solution was added to cells for
20 min and then IEC-6 cells were incubated in fix perm solution for a further 30 min. Anti-COX-2
(BD Transduction Laboratories, Milan, Italy), anti-iNOS (BD Transduction Laboratories, Milan, Italy),
anti-nitrotyrosine (Merck Millipore, Milan, Italy), anti-HO-1 (Santa Cruz Biotechnologies, Dallas, TX,
USA), or anti-NQO1 (Santa Cruz Biotechnologies, Dallas, TX, USA) antibodies were then added for
1 h. The secondary antibody, in fixing solution, was added to IEC-6 cells and cell fluorescence was
then evaluated by a fluorescence-activated cell sorter (FACSscan; Becton Dickinson, Milan, Italy) and
analyzed by Cell Quest software (version 4; Becton Dickinson, Milan, Italy), as formerly reported [
67
].
4.8. Immunofluorescence Analysis for NF-κB and Nrf2 by Confocal Microscopy
IEC-6 cells were plated on coverslips in a 12-well plate (2
×
10
5
cells/well) and allowed to adhere
for 24 h. After the adhesion time, the cellular medium was substituted with a new one, either alone or in
the presence of Astragalus membranaceus extract (50
µ
g/mL), and treated for 1 h. It was then co-exposed
to the extract and LPS (10
µ
g/mL) plus IFN (10 U/mL) for 1 h in order to evaluate NF-
κ
B activation in
IEC-6 cells. In another set of experiments, the IEC-6 cells were incubated with Astragalus membranaceus
extract (50
µ
g/mL) alone for 1 h and then exposed simultaneously to Astragalus membranaceus extract
and H
2
O
2
(1 mM) for 1 h more in order to measure Nrf2 activation. Cells were then fixed with 4%
paraformaldehyde in PBS and permeabilized with 0.1% saponin in PBS. The blocking was performed
with bovine serum albumin (BSA) and PBS. Cells were then incubated with rabbit anti-phospho p65
NF-
κ
B antibody (Santa Cruz Biotechnologies, Dallas, TX, USA) or rabbit anti-Nrf2 antibody (Santa Cruz
Biotechnologies, Dallas, TX, USA) for 1 h at 37
C. Then, PBS was used to wash the slides. Next,
we added the fluorescein-conjugated (FITC) secondary antibody for 1 h. 4
0
,6-diamidine-2
0
-phenylindole
dihydrochloride (DAPI) was used for the nuclei, as formerly reported [
68
]. At the end, we mounted the
coverslips in mounting medium and images were taken using the laser confocal microscope (Leica TCS
SP5, Leica, Wetzalar, Germany), as formerly reported [68].
4.9. Intracellular ROS Release Measurement
ROS intracellular production was evaluated by the probe 2
0
,7
0
-dichlorofluorescein-diacetate
(H
2
DCF-DA) [
69
]. The IEC-6 cells were seeded in 24-well plates (8
×
10
4
cells/well) and allowed
to adhere for one day. After adhesion, we incubated cells with Astragalus membranaceus extract
(5–100
µ
g/mL) alone for 1 h and then co-exposed to the extract and LPS (10
µ
g/mL) plus IFN (10 U/mL)
for 24 h. In another set of experiments, the IEC-6 cells were treated with Astragalus membranaceus extract
(5–100
µ
g/mL) alone for 1 h and then exposed simultaneously to the extract and H
2
O
2
(1 mM) for
1 h more. After cellular treatment, IEC-6 cells were collected, washed with PBS, and then incubated
in PBS containing H
2
DCF-DA (10
µ
M). Cell fluorescence was evaluated after 15 min at 37
C, using
a fluorescence-activated cell sorter (FACSscan; Becton Dickinson, Franklin Lakes, NJ, USA), and was
analyzed by Cell Quest software version 4 (Becton Dickinson, Milan, Italy), as formerly reported [69].
4.10. Data Analysis
We reported data as mean
±
standard error mean (SEM) of at least three independent experiments.
Each experiment was conducted in triplicate. For the statistical analysis, we used the analysis of
Int. J. Mol. Sci. 2018,19, 800 11 of 14
variance test. Bonferroni’s test was used to make multiple comparisons. We considered significant
ap-value less than 0.05.
5. Conclusions
Our results indicated that Astragalus membranaceus root extract significantly reduced the inflammatory
response and the pro-oxidant status in IEC-6 cells. Our study provides evidence that might further the
development of Astragalus membranaceus root extract as a therapeutic agent for IBD treatments.
Acknowledgments:
The authors acknowledge the financial support of Giellipi S.p.A. “Ricerche sull’attivitàdi
integratori alimentari e dispositivi medici”.
Author Contributions:
Study conception and design: Stefania Marzocco and Simona Adesso. Acquisition,
analysis, and interpretation of data: Stefania Marzocco and Simona Adesso. Drafting of manuscript: Stefania
Marzocco and Simona Adesso. Critical revision: Giuseppina Autore, Rosario Russo, and Andrea Quaroni. English
editing: Andrea Quaroni. All authors have seen and approved the manuscript.
Conflicts of Interest:
Rosario Russo is an employee of Giellepi S.p.A. He had no influence on the interpretation
of study results nor the decision to submit the manuscript for publication. The other authors had no conflicts
of interest.
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... The beneficial effects of Astragalus membranaceus include immunomodulatory, anti-inflammation, antioxidation, antiautophagy, and it is reportedly that it can preserve cardiac function. [19][20][21] Radix Salviae can reduce the levels of tumor necrosis factor-alpha (TNF-a), IL-6, and MDA, enhance capacities of antioxidant enzymes and prevent myocardium cell apoptosis, activate ERK and PKB and downregulate the levels of TNF-a and angiotensin II, and inhibit H2O2-induced ROS production. [22,23] Chinese patent medicines is widely used in TCM practice. ...
... Inhibits the activity of ET-1 and NO; inhibits calcium mobilization from both the extracellular medium and intracellular stores, inhibits the vWF-mediated process of platelet thrombus formation; inhibition of calcineurin activities and the reduction in e-fos and proliferation cell nuclear antigen expression; promotes the activity of SOD and GSH-Px and decreases that of MDA, LDH, creatinine kinase, tumor necrosis factors-a, and IL-6. [17,18] Astragalus membranaceus Astragaloside Anti-inflammation; antioxidation; immunomodulatory; antiautophagy; preserve cardiac function Decrease TNF-a release, cycloxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, nitrotyrosine formation, NF-kB activation, and reactive oxygen species (ROS) release; induce the activation and migration, and monocyte maturation of peripheral blood mononuclear cells [19][20][21] Radix Salviae Tanshinone Antiapoptosis; antioxidation; antiinflammation ...
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Background: Modern clinical trials and experimental researches of traditional Chinese medicine (TCM) have been conducted for decades and provided support for the prevention and treatment of acute coronary syndrome (ACS). However the level of evidence and the proper application of TCM were still barely satisfactory. Methods: In this study, we divided ACS into 3 different stages, including unstable angina, acute myocardial infarction, and post myocardial infarction. Then we systematically reviewed and meta-analyzed the existing randomized controlled trials on both clinical manifestations and objective indicators, in these 3 aspects. Results: The results indicate that TCM can both improve the clinical manifestations and ameliorate the objective parameters in different courses of ACS, including C-reactive protein in unstable angina, left ventricular ejection fraction in acute myocardial infarction and post myocardial infarction. And the incidence of short-term cardiovascular events are lower in TCM intervention group. Some of the improvements lead to potential long-term benefits. Conclusion: TCM treatment is beneficial to different courses of ACS. To acquire more solid and comprehensive evidence of TCM in treating ACS, more rigorously designed randomized controlled trials with longer follow-up duration are warranted.
... In a study by Adesso et al. (2018), performed with rat intestinal crypt cell line (IEC-6 cells) co-exposed to the interferon-γ, lipopolysaccharide derived from E. coli (LPS) and A. membranaceus extract (5-100 µg/ml) showed that the extract of A. membranaceus root produced anti-inflammatory effect that was reported by reducing the activation of nuclear factor-κB (NF-κB), the formation of nitrotyrosine, the expression of inducible nitric oxide synthase (iNOS) and of cycloxygenase-2 (COX-2), as well as the release of tumor necrosis factor-α (TNF-α). Adesso et al. (2018) also showed the antioxidant properties of A. membranaceus extract against hydrogen peroxide-induced oxidative stress by reducing ROS levels and by increasing expression of antioxidant cytoprotective factors, the activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). ...
... In a study by Adesso et al. (2018), performed with rat intestinal crypt cell line (IEC-6 cells) co-exposed to the interferon-γ, lipopolysaccharide derived from E. coli (LPS) and A. membranaceus extract (5-100 µg/ml) showed that the extract of A. membranaceus root produced anti-inflammatory effect that was reported by reducing the activation of nuclear factor-κB (NF-κB), the formation of nitrotyrosine, the expression of inducible nitric oxide synthase (iNOS) and of cycloxygenase-2 (COX-2), as well as the release of tumor necrosis factor-α (TNF-α). Adesso et al. (2018) also showed the antioxidant properties of A. membranaceus extract against hydrogen peroxide-induced oxidative stress by reducing ROS levels and by increasing expression of antioxidant cytoprotective factors, the activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Flavonoids of Astragalus extract exhibited anti-inflammatory and immunomodulatory properties in LPS-induced RAW 264.7 cells by blocking the expression of nuclear NF-κBp65 and the phosphorylation of JNK and p38 in the NF-κB and MAPKs pathway, respectively, and by reducing the levels of COX-2 and iNOS . ...
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Medicinal plants always are part of folk medicine and are nowadays receiving worldwide attention for prophylaxis, management, and treatment of several diseases, as an alternative to chemical drugs. The current work provided a comprehensive overview and analysis of the Astragalus and health relationship in literature. The analysis of their therapeutic potential is thus instrumental to understand their bioactivity. Among these, the flowering medicinal plant Astragalus membranaceus has raised interest due to several beneficial health effects. This perspective review discussed the botanical, geographical, historical, and the therapeutic properties of A. membranaceus , with a special focus on its health improving effects and medicinal applications both in vitro and in vivo. Graphic abstract
... These anti-oxidation components make AM an effective antioxidant that protects cells and tissues against ROS damage. Herein, AWE exhibited significant ROS scavenging capability in the AAPH cell-free system, which was compatible with other studies that showed that AM could clear ROS in H 2 O 2 assay (Han et al., 2017;Adesso et al., 2018). Interestingly, we also found that AWE cleared neither the superoxide anion generated by xanthine oxidase nor the nitrogen-centered radicals, including DPPH and ABTS. ...
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Neutrophils are the primary immune cells in innate immunity, which are related to various inflammatory diseases. Astragalus mongholicus Bunge is a Chinese medicinal herb used to treat various oxidative stress-related inflammatory diseases. However, there are limited studies that elucidate the effects of Astragalus mongholicus Bunge in human neutrophils. In this study, we used isolated human neutrophils activated by various stimulants to investigate the anti-inflammatory effects of Astragalus mongholicus Bunge water extract (AWE). Cell-free assays were used to examine free radicals scavenging capabilities on superoxide anion, reactive oxygen species (ROS), and nitrogen-centered radicals. Imiquimod (IMQ) induced psoriasis-like skin inflammation mouse model was used for investigating anti-psoriatic effects. We found that AWE inhibited superoxide anion production, ROS generation, and elastase release in human neutrophils, which exhibiting a direct anti-neutrophil effect. Moreover, AWE exerted a ROS scavenging ability in the 2,2’-Azobis (2-amidinopropane) dihydrochloride assay, but not superoxide anion in the xanthine/xanthine oxidase assay, suggesting that AWE exhibited anti-oxidation and anti-inflammatory capabilities by both scavenging ROS and by directly inhibiting neutrophil activation. AWE also reduced CD11b expression and adhesion to endothelial cells in activated human neutrophils. Meanwhile, in mice with psoriasis-like skin inflammation, administration of topical AWE reduced both the affected area and the severity index score. It inhibited neutrophil infiltration, myeloperoxidase release, ROS-induced damage, and skin proliferation. In summary, AWE exhibited direct anti-inflammatory effects by inhibiting neutrophil activation and anti-psoriatic effects in mice with IMQ-induced psoriasis-like skin inflammation. Therefore, AWE could potentially be a pharmaceutical Chinese herbal medicine to inhibit neutrophilic inflammation for anti-psoriasis.
... As early as 3000 years ago, the classic Huangdi Neijing of traditional Chinese medicine recorded the physiological and pathological relationship between the lungs and the large intestine in detail. Xuanbai Chengqi decoction, Gegen Qinlian decoction, and other tonic Chinese medicines, such as Ginseng Radix et Rhizoma, Gardeniae Fructus Praeparatus, Angelicae Sinensis Radix, and Astragali Radix, can improve LPSinduced acute lung tissue damage and pathological colon tissue damage by adjusting the lung-gut mucosal immune function and are thus candidate drugs in innovative drug development based on the concept of treating the lung and intestine together [72][73][74]. ...
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The microbiota colonized in the human body has a symbiotic relationship with human body and forms a different microecosystem, which affects human immunity, metabolism, endocrine, and other physiological processes. The imbalance of microbiota is usually linked to the aberrant immune responses and inflammation, which eventually promotes the occurrence and development of respiratory diseases. Patients with chronic respiratory diseases, including asthma, COPD, bronchiectasis, and idiopathic pulmonary fibrosis, often have alteration of the composition and function of intestinal and lung microbiota. Gut microbiota affects respiratory immunity and barrier function through the lung-gut microbiota, resulting in altered prognosis of chronic respiratory diseases. In turn, lung dysbiosis promotes aggravation of lung diseases and causes intestinal dysfunction through persistent activation of lymphoid cells in the body. Recent advances in next-generation sequencing technology have disclosed the pivotal roles of lung-gut microbiota in the pathogenesis of chronic respiratory diseases. This review focuses on the association between the gut-lung dysbiosis and respiratory diseases pathogenesis. In addition, potential therapeutic modalities, such as probiotics and fecal microbiota transplantation, are also evaluated for the prevention of chronic respiratory diseases.
... It was mainly used to treat anorexia and dyspepsia in children with spleen and stomach de ciency syndrome [6]. Due to its good health care effect and high safety, it was also widely used in the adjuvant treatment of children with recurrent respiratory tract infections in clinical [5,7,8]. Moreover, modern pharmacological studies have shown that polysaccharides from Dendrobium, astragalus, Codonopsis pilosula and other major traditional Chinese medicines in QHZG could inhibit the in ammatory reaction caused by a variety of infections, and then have immunomodulatory effect [9,10,11,12]. ...
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Background Qihuzha granule (QHZG) is a Chinese patent medicine, composed of 11 kinds of edible medicinal plants, which is used to treat dyspepsia and anorexia in children caused by spleen and stomach deficiency syndrome. However, its role and mechanism in immunosuppression induced by cyclophosphamide remained unclear. The purpose of this study is to investigate the effect of QHZG on immunosuppression induced by cyclophosphamide in mice and its possible mechanism.Methods The immunosuppression injury model was induced by intraperitoneal injection of cyclophosphamide (100 mg/kg); the mRNA level of cytokines (IL-2/4/6, IFN-γ) and critical targets of signaling pathways related to immune regulation (JNK, ERK, P38, JAK2, SRC and STAT3) were tested by QPCR; related protein levels were detected by western blotting; hematoxylin-eosin (HE) staining was employed to observe the histological alterations; macrophages and neutrophils in the mouse spleen were examined by immunofluorescence analysis. ResultsQHZG significantly increased the spleen index and thymus index of mice with immunodeficiency induced by cyclophosphamide and up-regulated the mRNA expression of cytokines (IL-2/4/6, IFN-γ) and critical targets of signaling pathways related to immune regulation (JNK, ERK, P38, JAK2, SRC and STAT3), which were decreased by cyclophosphamide treatment. The results of immunofluorescence staining and histological analysis showed that QHZG could also protect mice from immunosuppressive injury caused by cyclophosphamide via keeping structural integrity of spleen, and partially restoring the production levels of macrophages and monocytes in the spleen. Further studies indicated that QHZG could significantly counter the decline of phosphorylated protein levels of JAK2/SRC-STAT3 axes (P-JAK2, P-SRC and P-STAT3), and MAPK pathways (P-JNK, P-ERK and P-P38) induced by cyclophosphamide, suggesting that the protective effects of QHZG on immunosuppressive injury triggered by cyclophosphamide were involved in JAK2/SRC-STAT3 axes, and MAPK pathways. Meanwhile, we also found that QHZG could partially restore the vital phosphorylated proteins of PI3K/Akt/mTOR signaling pathway (P-Akt, P-mTOR), which were reduced by cyclophosphamide. The data implied that PI3K signaling pathway was also responsible for the protection of QHZG against the immunosuppression induced by cyclophosphamide in mice. Conclusions Our study demonstrated that QHZG protected mice from cyclophosphamide-triggered immunosuppressive injury via IL-6 and its downstream signaling pathways including PI3K/Akt/mTOR signal pathway and JAK2-SRC/MAPK/STAT3 axes. These results suggested that QHZG might serve as a new drug for the treatment of the immunosuppression caused by cyclophosphamide therapy.
... Similarly, the main biologically active components of Radix astragalus (e.g., polysaccharides, astragaloside, flavonoids, and saponins) have been revealed to show antioxidant effects (Gong et al., 2018). It has been reported that extracts of R. astragalus can decrease the inflammatory response induced by lipopolysaccharide in Escherichia coli, while decreasing the release of reactive oxygen species (ROS) and increasing the activation of nuclear factor and the expression of antioxidant cytoprotective factors in cells (Adesso et al., 2018). As the other ingredient in the mixture of Z. Tk, Radix isatidis is well-known for its broad antiviral activities and antioxidant properties. ...
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To explore the feasibility of using fermented Chinese herbal mixture Zhihuasi Tk (Z. Tk) supplementation to increase the swine production, the protective effect of dietary supplementation with Z. Tk on the intestinal oxidative stress model and the regulation of both growth performance and intestinal microbiota of weaned piglets were investigated in vitro. Our results showed that the addition of Z. Tk increased the cell viability, prevented the decrease of glutathione peroxidase, and significantly increased the total antioxidant capacity and reduced the damage caused by H2O2 to the tight junction proteins of the porcine small intestinal epithelial cell line (IPEC-J2). Furthermore, weaned piglets supplemented with either 2 kg/ton zinc oxide (ZnO) or 4 kg/ton of Z. Tk in the diet increased body weight as well as average daily feed intake and daily gain, while the feed conversion rate and diarrhea rate decreased within 0–35 days. Results of the taxonomic structure of the intestinal microbiota showed that, in 21 days after weaning, the Firmicutes/Bacteroidetes ratio in experimental group was increased, while the abundance of beneficial bacteria such, as Lactobacillus, was increased by Z. Tk, showing inhibitory effect on pathogenic bacteria such as members of Proteobacteria. In summary, dietary supplementation with Z. Tk maintained the intestinal microbiota in a favorable state for the host to effectively reduce the abnormal changes in the intestinal microbial structure and improved growth performance of weaned piglets. Therefore, Z. Tk may potentially function as a substitute for ZnO in feed additives for weaned piglets in modern husbandry.
... Frontiers in Microbiology | www.frontiersin.org officinalis, bark extract, and Astragalus membranaceus activate the Nrf2 pathway to protect against oxidative stress in cells (Rajgopal et al., 2016;Adesso et al., 2018). We found that dietary HEM regulated the Nrf2-Keap1 pathway through decreasing the mRNA expression of Keap1 in the jejunum and the protein level of Keap1 in the ileum. ...
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Recently, herbal extracts have been applied in multiple aspects, such as medicine and animal feed. Different compositions of herbal extract mixture (HEM) have various components and diverse functions. This study aimed to evaluate the effects of HEM (Lonicera japonica, Astragalus membranaceus, Eucommia folium, and Codonopsis pilosula) on intestinal antioxidant capacity and colonic microbiota in weaned pigs. A total of 18 piglets [Duroc × (Landrace × Yorkshire)] with the initial body weight of 5.99 ± 0.13 kg (weaned at 21 days) were randomly divided into two groups (n = 9): the control group (CON, basal diet) and the HEM treatment group (HEM, 1,000 mg/kg HEM + basal diet). The experiment period lasted for 14 days. Our results showed that dietary supplementation with HEM modulated the antioxidant capacity through decreasing the activity of superoxide dismutase (SOD) in the ileum and glutathione peroxidase (GSH-PX) in the serum, and decreasing the mRNA expression of Kelch like-ECH-associated protein 1 (Keap1) in the jejunum and the protein level of Keap1 in the ileum. Moreover, the HEM group modified the composition of colonic microbiota with affecting relative abundances of the Firmicutes and Bacteroidetes at the phylum level. Taken together, supplementation of HEM can regulate the antioxidant capacity and modify the composition of colonic bacteria in weaning piglets. This study provides new insights into the combination effects of herbal extracts on weaning piglets.
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Objective Urinary tract infections (UTI) among women form a substantial part of medical practice and both patients and medical professionals have an interest in non-antibiotic treatments and preventative measures. This research provides preliminary data on a multi-functional composition, DAPAD, which explored several biologic activities of relevance to UTI. Study Design: This formulation included D-mannose, citric acid, three prebiotic compounds, and extracts of dandelion and astragalus. Studies performed employed 4 bacterial strains that have relevance to UTI including E. coli, Proteus mirabilis, Streptococcus agalactiae and Enterococcus faecalis. Results Key findings from in vitro studies included: DAPAD at full- and half-strength inhibited growth of all UTI bacteria. Evidence for D-mannose agglutination of E. coli was demonstrated. D-mannose also showed unexpected effects on bacterial membrane integrity with vital staining and modest growth restriction. We did not demonstrate growth inhibition by dandelion or astragalus extracts but the latter showed diminished cytokine elaboration by bladder epithelial cells. Conclusion DAPAD is a multifunctional composition that may warrant further development as a UTI treatment or preventive if supported by clinical evaluation.
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Background : Cancer is the most dreadful disease increasing rapidly causing an economic burden globally. A standardized chemotherapy regimen planned with curative intent weakens the immune system and damages healthy cells making the patient prone to infections and severe side effects with pain and fatigue. Purpose : Astragalus membranaceus (AM) has a long history of use in the treatment of severe adverse diseases. For thousands of years, it has been used in mixed herbal decoctions for the treatment of cancer. Due to growing interest in this plant root for its application to treat various types of cancers and tumors, has attracted researcher's interest. Method : The literature search was done from core collections of electronic databases such as Web of Science, Google Scholar, PubMed and Science Direct using keywords given below and terms like pharmacological and phytochemical details of this plant. Outcome : Astragalus membranaceus has demonstrated the ability to modulate the immune system during drug therapy making the patient physically fit and prolonged life. It has become a buzzword of herbalists as it is one of the best of seven important adaptogenic herbs with a protective effect against chronic stress and cancer. It demonstrated significant amelioration of the perilous toxic effects induced by concurrently administered chemo onco-drugs. Conclusion : The natural phytoconstituents of this plant formononetin, astragalus polysaccharide, and astragalosides which show high potential anti-cancerous activity are studied and discussed in detail. One of them are used in clinical trials to overcome cancer related fatigue. Overall, this review aims to provide an insight into Astragalus membranaceus status in cancer therapy.
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Ethnopharmacological relevance Qihuzha granule (QHZG), is one of traditional Chinese patent medicines composed of eleven edible medicinal plant, which has been used in the clinic for the treatment of indigestion and anorexia in children caused by deficiency of the spleen and stomach. Yet it is noteworthy that QHZG has therapeutic effect on recurrent respiratory tract infection (RRTI) in children. However, its potential molecular mechanisms remained unclear. Aim of the study The aim of this study was to investigate the therapeutic effect and potential mechanism of QHZG on lipopolysaccharide (LPS) induced acute spleen injury. Materials and methods The acute spleen injury model was induced by intraperitoneal injection of LPS (10 mg/kg) and safe doses of QHZG was administered by gavage once a day for 23 days before LPS treatment. Serum inflammatory cytokines including interleukin-2 (IL-2), IL-1β, IFN-γ, and tumor necrosis factor-α (TNF-α) were tested by ELISA. Related protein levels were detected by Western blotting. Hematoxylin-eosin (HE) staining was employed to observe the histological alterations. The distribution of macrophages and neutrophils in the mouse spleen was examined by immunofluorescence analysis. Results QHZG pretreatment significantly abolished the increased secretion of cytokines such as interleukin-2 (IL-2), IL-1β, IFN-γ, and tumor necrosis factor-α (TNF-α), which were attributable to LPS treatment. Immunofluorescence staining and Histological analysis of spleen tissue revealed the protective effect of QHZG against LPS-induced acute spleen injury in mice. Further study indicated that pretreatment with QHZG significantly inhibited LPS-induced phosphorylation of Src. Accordingly, the increased phosphorylation of Src downstream components (JNK, ERK, P38 and STAT3) induced by LPS was remarkably diminished by QHZG, suggesting the involvement of Src/MAPK/STAT3 pathway in the inhibitory effects of QHZG on spleen injury in mice. Conclusion Our study demonstrated that QHZG protected mice from LPS-induced acute spleen injury via inhibition of Src/MAPK/Stat3 signal pathway. These results suggested that QHZG might serve as a new drug for the treatment of LPS-stimulated spleen injury.
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Background: Doronicum austriacum Jacq., Asteraceae, is a plant which is used in traditional alpine medicine. Historical sources describe the medical use of the root, but up until now only a few studies evaluated its pharmacological properties. The evaluation of the dichloromethane extract, and its major compounds for their anti-inflammatory and anti-oxidant potential was performed in macrophages J774A.1 and C6 astrocytes. Nitric oxide (NO) and reactive oxygen species (ROS) release, as well as nitrotyrosine formation, were evaluated. Moreover, in order to evaluate the potential anti-proliferative activity, under the same experimental conditions, 3-(4,5-dimethyltiazol-2yl)-2,5-phenyl-2H-tetrazolium bromide (MTT) assay was also performed. Our results indicate thatDoronicum austriacumhas a significant effect in inhibiting both pro-inflammatory and pro-oxidative mediators. All isolated compounds were able to significantly inhibit NO and ROS release both in macrophage and in astrocytes cells, even if the effect was more pronounced in macrophage. In particular, among the tested compounds, 6,12-dihydroxy-(-)-2S-tremetone exerted stronger activity. Both extract and single compounds did not affect cellular viability. This study provides evidence for the pharmacological anti-inflammatory and anti-oxidant potential ofDoronicum austriacumextract. These effects could be due to the activity of its major constituents and subsequent identification of benzofurans as a promising compound class to combat inflammation and related diseases.
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Neurotoxicity is a major side effect of platinum derivatives both during and after treatment. In the absence of effective pharmacological compounds, the opportunity to identify safe adjuvant treatments among medicinal plants seems appropriate. Astragali radix is an adaptogenic herbal product recently analyzed in platinum-treated cancer patients. With the aim of evaluating the anti-neuropathic profile of Astragali radix, a previously characterized aqueous (Aqu) and two hydroalcoholic (20%HA and 50%HA) extracts were tested in a rat model of oxaliplatin-induced neuropathy. Repeated administrations significantly reduced oxaliplatin-dependent hypersensitivity with 50%HA, the most effective, fully preventing mechanical and thermal hypersensitivity. Ex vivo, 50%HA reduced morphometric and molecular alterations induced by oxaliplatin in peripheral nerve and dorsal-root-ganglia. In the spinal cord and in brain areas, 50%HA significantly decreased activation of microglia and astrocytes. Furthermore, 50%HA prevented the nephro- and hepato-toxicity induced by the anticancer drug. The protective effect of 50%HA did not alter oxaliplatin-induced apoptosis in colon tumors of Pirc rats, an Apc-driven model of colon carcinogenesis. The hydroalcoholic extract (50%HA) of Astragali radix relieves pain and promotes the rescue mechanisms that protect nervous tissue from the damages triggering chronic pain. A safe profile strongly suggests the usefulness of this natural product in oxaliplatin-induced neuropathy.
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Endothelial cells of cerebral microvessels are one of the components of blood-brain-barrier (BBB), which are connected by tight junctions (TJs). BBB disruption in cerebral diseases such as ischemic stroke, Alzhemer's disease, multiple sclerosis and traumatic brain injury is implicated to exacerbate the disease progression. Astragaloside IV (ASIV) isolated from Astragalus membranaceus prevents BBB breakdown in rodents induced with cerebral edema and experimental autoimmune encephalomyelitis. However, its underlying molecular mechanism has not been elucidated yet. In present study, ASIV was found to prevent the leakage of BBB in LPS-induced mice, which was accompanied with increased zo-1 and occludin but reduced VCAM-1 in brain microvessels. Similarly, in brain endothelial cell line bEnd.3 cells, ASIV mitigated the increased permeability induced by LPS, as evidenced by increased TEER and reduced sodium fluorescein extravasation. ASIV also enhanced the expression of TJ proteins such as zo-1, occludin and claudin-5 in LPS stimulated bEnd.3 cells. Meanwhile, it inhibited the inflammatory responses and prevented the monocyte adhesion onto bEnd.3 cells upon LPS stimulation. Further study disclosed that ASIV could alleviate ROS level and activate Nrf2 antioxidant pathway in bEnd.3 cells. When Nrf2 was silenced, the protective effect of ASIV was abolished. In brain microvessels of LPS-induced mice, ASIV also enhanced the expression of Nrf2 antioxidant pathway related proteins. Collectively, our results demonstrated that ASIV protected the integrity of BBB in LPS-induced mice, the mechanism of which might be mediated via activating Nrf2 signaling pathway. The findings suggested that ASIV might be a potential neuroprotective drug acting on BBB.
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Astragalus polysaccharide (APS) is a bioactive extract of Astragalus membranaceus (AM), which possess a wide range of medicinal benefits, including anti-inflammatory, anti-oxidative, anti-tumor and anti-diabetic effects. The present work evaluated the therapeutic effect of APS and its potential mechanisms in a mouse model of dextran sulfate sodium (DSS)-induced colitis. The APS treatment led to significant improvements in colitis disease activity index (DAI) and histological scores, as well as significantly increased weight and colon length in mice as compared to the control group. Mechanically, reduced NF-κВ DNA phosphorylation activity and downregulated TNF-α, IL-1β, IL-6, IL-17 expressions and myeloperoxidase (MPO) activity were associated with improvement in colitis observed in APS-treated mice. These findings suggest that APS may represent a natural therapeutic approach for treating inflammatory bowel disease, such as ulcerative colitis.
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Background: Luteolin has a reputation for being a safe and effective natural antioxidant that has strong radical scavenging and cell protective properties. The role of oxidative stress in inflammatory bowel disease (IBD) has been well established and is increasingly highlighted. Thus, we studied the protective effect of luteolin administration in a mouse model of experimental colitis. Methods: Experimental acute colitis was induced by administering 3% dextran sulfate sodium (DSS) in the drinking water of mice for 7days. The disease activity index (DAI); colon length; histological assessment; mRNA levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), heme oxygenase-1 (HO-1), and NADP(H): quinone oxidoreductase 1 (NQO-1); protein expression of Nrf2 and inducible nitric oxide synthase (iNOS); colon malondialdehyde (MDA) levels; and the activity levels of colonic superoxide dismutase (SOD) and catalase (CAT) were examined. Results: Luteolin (20 and 50mg/kg) significantly attenuated the DAI, colon shortening and histological damage. In addition, luteolin administration effectively decreased the expression of inflammatory mediators, such as iNOS, TNF-α and IL-6. Luteolin also decreased the colonic content of MDA. The activities of colonic SOD and CAT and the levels of Nrf2 and its downstream targets, HO-1 and NQO1, were elevated by luteolin treatment. Conclusion: These observations indicate that luteolin may suppress experimental colitis through the Nrf2 signaling pathway.
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Astragalus membranaceus Bunge has been used to treat numerous diseases for thousands of years. As the main active substance of Astragalus membranaceus Bunge, Astragaloside IV (AS-IV) also demonstrates the potent protective effect on focal cerebral ischemia/reperfusion, cardiovascular disease, pulmonary disease, liver fibrosis, and diabetic nephropathy. Based on studies published during the past several decades, the current state of AS-IV research and the pharmacological effects are detailed, elucidated, summarized. This review systematically summarizes the pharmacological effects, metabolism mechanism and the toxicity of AS-IV. AS-IV has multiple pharmacologic effects, including anti-inflammatory, anti-fibrotic, anti-oxidative stress, anti-asthma, anti-diabetes, immunoregulation and cardioprotective effect via numerous signaling pathways. According to the existing studies and clinical practices, AS-IV possesses potential for broad application in many diseases. This article is protected by copyright. All rights reserved.
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Stracchino is a typical Italian soft cheese, widely consumed and appreciated for its flavour and freshness. With the aim to follow the release of health-promoting peptides, an in vitro gastro-intestinal digestion of Stracchino was carried out. The digest was fractionated by semi-preparative RPLC and tested for its antioxidant properties in H2O2-treated intestinal epithelial cell line (IEC-6). The most active fraction was characterized by UHPLC-MS/MS, showing the presence of two abundant β-casein hexapeptides: EAMAPK (f: 115–120) and AVPYPQ (f: 192–197), which were synthesized and tested, showing antioxidant activities in a wide concentration range (5–150 µg/mL), involving ROS reduction, SOD expression increase and Nrf2 antioxidant response activation. Hence, bioavailability studies were carried out through Caco-2 monolayers cells on the two hexapeptides, which revealed a significative concentration in the basolateral side. The results highlight the potential of Stracchino as health promoting food, and the possible employment of casein peptides in nutraceutical formulations.