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Citation: Ahn, J.-H.; Choi, J.-M.;
Kang, E.-S.; Yoo, J.-H.; Cho, Y.-J.;
Jang, D.S.; Choi, J.-H. The Anti-
Endometriotic Effect of Cyperi
Rhizoma Extract, Inhibiting Cell
Adhesion and the Expression of
Pain-Related Factors through Akt
and NF-kB Pathways. Medicina 2022,
58, 335. https://doi.org/10.3390/
medicina58030335
Academic Editor: Iveta Yotova
Received: 20 January 2022
Accepted: 19 February 2022
Published: 23 February 2022
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4.0/).
medicina
Article
The Anti-Endometriotic Effect of Cyperi Rhizoma Extract,
Inhibiting Cell Adhesion and the Expression of Pain-Related
Factors through Akt and NF-kB Pathways
Ji-Hye Ahn 1, 2, * , Jun-Myeong Choi 2,3, Eun-Sol Kang 2, Jae-Hyeon Yoo 2, Yoon-Jin Cho 2, Dae Sik Jang 3
and Jung-Hye Choi 2, 3, *
1Department of Oriental Pharmacy, Woosuk University, Jeonju 55338, Korea
2Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University,
Seoul 02447, Korea; tkarjawhfh@gmail.com (J.-M.C.); amorfati8147@naver.com (E.-S.K.);
gaaon@hanmail.net (J.-H.Y.); cyjin422@khu.ac.kr (Y.-J.C.)
3Department of Biomedical and Pharmaceutical Sciences, Kyung Hee University, Seoul 02447, Korea;
dsjang@khu.ac.kr
*Correspondence: jihyeahn20@woosuk.ac.kr (J.-H.A.); jchoi@khu.ac.kr (J.-H.C.);
Tel.: +82-63-290-1580 (J.-H.A.); +82-2-961-2172 (J.-H.C.);
Fax: +82-63-290-1576 (J.-H.A.); +82-2-961-9580 (J.-H.C.)
Abstract:
Rhizomes of Cyperus rotundus have been widely used as a traditional medicine in Asia for
the treatment of gynecological diseases. However, there is no scientific evidence demonstrating the
effect of C. rotundus rhizomes on endometriosis, which is characterized by the adhesion of endometrial
tissues outside the uterus, resulting in chronic and severe pelvic pain. The aim of this study was
to investigate the effects of Cyperi rhizoma extract (CRE) on cell adhesion and the expression of
pain-related factors (neurotrophins) in endometriotic cells, and to elucidate the underlying molecular
mechanisms. CRE inhibited the adhesion of human endometriotic 12Z cells to peritoneal mesothelial
Met5A cells using by adhesion assays. The mRNA expression of adhesion molecules [P-cadherin
and matrix metalloproteinase (MMP)-2] was downregulated by CRE treatment. In addition, CRE
significantly inhibited the mRNA expression of neurotrophins (BDNF, NGF, NT-3 and NT-4/5) in
12Z cells. Moreover, Akt overexpression markedly neutralized the inhibition of cell adhesion by CRE
and expression of neurotrophins in 12Z cells. Furthermore, it was found that CRE suppressed NF-kB
activation through the Akt pathway. These data suggest that CRE exerts anti-endometriotic activities
by the inhibition of cell adhesion and neurotrophin expression, through the negative regulation of
the Akt and NF-kB pathways in endometriotic cells.
Keywords: Cyperi rhizome; endometriosis; adhesion; neurotrophins; Akt; NF-kB
1. Introduction
Endometriosis is a chronic gynecological disease causing chronic pelvic pain, dys-
menorrhea, dyspareunia and infertility [
1
]. It is characterized by the presence and growth
of endometrial tissue outside the uterus in the peritoneal cavity. It affects approximately
6–10% of women of reproductive age and 30–50% of women with chronic pelvic pain [
2
].
Although the exact etiology of endometriosis remains unclear, retrograde menstruation
and implantation theories are widely accepted for endometriosis [2–4].
The adhesion of retrograde endometrial fragments onto the pelvic mesothelium is a
key step in the initiation of endometriosis formation [
5
]. Several adhesion molecules, includ-
ing P-cadherin (a predominant cadherin subtype) and matrix metalloproteinase-2 (MMP-2)
are upregulated in ectopic endometrial lesions, and have been suggested to be critical
factors in regulating the adhesion of ectopic endometrium to the peritoneal mesothelium
and/or extracellular matrix (ECM) [
6
,
7
]. Recently, an endometriotic implant surrounded
Medicina 2022,58, 335. https://doi.org/10.3390/medicina58030335 https://www.mdpi.com/journal/medicina
Medicina 2022,58, 335 2 of 14
by an inflammatory environment has also been reported to be involved in the genera-
tion of endometriosis-associated pelvic pain, by releasing pro-nociceptive mediators [
8
,
9
].
Emerging studies have demonstrated that neurotrophins, as a family of nerve growth
factors, including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF),
neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5), are observed in endometriotic
lesions, resulting in pelvic pain in endometriosis via neurogenic inflammation [10,11].
Cyperus rotundus Linn. (Cyperaceae), also known as “nut grass” or “purple nut sedge”
is a species of sedge native to Asia, southern and central Europe, and Africa, and it grows
naturally in tropical, sub-tropical and temperate regions [
12
]. In Asia, the rhizome of
C. rotundus (Cyperi rhizoma, CR) has been traditionally used as a herbal medicine for
the treatment of stomach and bowel disorders, bacterial infection and inflammatory dis-
eases [
13
]. In traditional Chinese medicine, CR is one of the oldest known medicinal
plants used for the treatment of dysmenorrhea, a symptom of endometriosis [
14
]. Modern
pharmacological studies have demonstrated that CR has several pharmacological and bio-
logical properties, including anti-inflammatory, anti-diabetic, anti-diarrheal, cytoprotective,
anti-microbial, anti-bacterial, anti-oxidant, anti-pyretic, anti-analgesic and neuroprotective
activities [
15
–
22
]. However, there is no scientific evidence demonstrating the effect of
CR on endometriosis, a chronic gynecological disease characterized by the growth and
adhesion of endometrial tissues outside the uterus, resulting in chronic and severe pelvic
pain [
23
,
24
]. In this study, we aimed to investigate the effects of Cyperi rhizoma extract
(CRE) on endometriosis, and to elucidate the underlying molecular mechanisms.
2. Materials and Methods
2.1. Plant Material and Preparation of the Extract
The rhizomes of Cyperus rotundus L. were obtained from a domestic Korean market
(Kyungdong Crude Drugs Market, Seoul, Korea) in June 2011. The origin of the herbal
material was identified by one of the authors (D.S. Jang), and a voucher specimen (CYRO1-
2011) was deposited in the lab of Natural Product Medicine, College of Pharmacy, Kyung
Hee University. The dried and milled plant material (2.8 kg) was extracted with 10 L of 80%
ethanol (EtOH) three times by maceration. The extract was combined and concentrated in
vacuo at 40
◦
C to give an 80% EtOH extract (CRE; 399 g). For
in vitro
experiments, CRE
powder was dissolved in dimethyl sulfoxide (DMSO) (v/v) and filtered through a 0.22
µ
m
disk filter.
2.2. Materials
Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium, Medium 199 (M199),
Opti-modified Eagle’s medium (Opti-MEM), fetal bovine serum (FBS), penicillin and
streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, USA). Phos-
phate buffered saline (PBS), DMSO, RNase A, leupeptin, aprotinin and phenylmethyl-
sulfonylfluoride (PMSF) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from
Molecular Probes Inc. (Eugene, OR, USA). Antibodies against anti-phospho-Akt, anti-
phospho-IkB
α
and anti-phospho-IKK
α
/
β
were purchased from Cell Signaling Technology
(Beverly, MA, USA). The antibodies against anti-Akt, anti-p65, anti-IkB
α
, anti-IKK
α
/
β
,
anti-PARP and anti-ß-actin were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA, USA). CellTrackerTM was obtained from Invitrogen (Grand Island, NY, USA).
2.3. Cell Culture
Immortalized human endometriotic epithelial cells (12Z) were established from active
endometriotic lesions from women with endometriosis. In addition, the cell line displayed
the
in vivo
properties of the active phase of endometriosis as well as the phenotypic
characteristics [
25
]. The endometriotic 12Z cells were generously provided by Dr. Starzinski-
Powitz (Johann-Wolfgang-Goethe-Universitaet, Frankfurt, Germany) and maintained in
DMEM/F12 medium supplemented with 5% FBS, 100 U/mL penicillin and 100 mg/mL
Medicina 2022,58, 335 3 of 14
streptomycin (Life Technologies, Grand Island, NY, USA) in a humidified atmosphere of
5% CO
2
–95% air at 37
◦
C. The human mesothelial cell line Met5A were originally from the
American Type Culture Collection. The cells were cultured in M199 supplemented with
10% FBS, penicillin (100 U/mL) and streptomycin sulfate (100 µg/mL).
2.4. Transfection
The pcDNA3-Akt-Myr (the constitutively active form of Akt) or the empty vector were
obtained from Addgene (Cambridge, MA, USA). After cells were cultured to
60–70%
con-
fluence, the cells were transfected with plasmid DNA constructs (2
µ
g/mL) by polyethylen-
imine (PEI) in serum-free OPTI-MEM (Invitrogen, CA, USA), according to the manufac-
turer’s instructions. After 24 h, the medium containing the DNA plasmids was replaced
with a fresh DMEM/F12 medium with 5% FBS, and the cells were treated with CRE.
2.5. Cell Viability Assay
Cell viability was assessed using an MTT assay. Briefly, human endometriotic cells
(12Z) were seeded at a density of 9
×
10
4
cells/mL in each well containing 50
µ
L of
DMEM/F12 medium in a 96-well plate. After 24 h, various concentrations of CRE were
added. After 24 h, 30
µ
L of MTT (1 mg/mL stock solution) was added, and the plates were
incubated for an additional 4 h. The medium was discarded, and the formazan blue, which
was formed in the cells, was dissolved in 50
µ
L DMSO. The optical density was measured at
540 nm using a microplate spectrophotometer (Spectra Max; Molecular Devices, Sunnyvale,
CA, USA).
2.6. Adhesion Assay
The mesothelial Met5A cells were grown to confluence on 96-well plates. Endometri-
otic cells were detached by trypsinization, washed with phosphate-buffered saline (PBS),
and probed with 10
µ
M CellTracker
TM
for 45 min at 37
◦
C. CellTracker
TM
-labeled cells
were washed with a DMEM/F12 medium containing 0.1% FBS to remove the free dye, and
were added (2
×
10
4
cells/well) to the mesothelial cells. After incubation at 37
◦
C for the
indicated time, the non-adherent cells were removed by gentle washing, the fluorescence
in each well was imaged, and the fluorescence was quantified in pixels using Scion Image
Software (Scion Corp, Frederick, MD, USA).
2.7. Nuclear Extraction
Cells were washed with PBS, scraped into 1 mL of ice-cold PBS, and pelleted by
centrifugation. The cell pellets were resuspended in hypotonic buffer (10 mM HEPES,
pH 7.9, 1.5 mM MgCl
2
, 10 mM KCl, 0.2 mM PMSF, 0.5 mM DTT, 10
µ
g/mL aprotinin) and
incubated on ice for 15 min. The cells were then lysed by adding 0.1% Nonidet P-40 and
vortexed vigorously for 10 s. Nuclei were pelleted by centrifugation at 12,000
×
gfor 1 min
at 4
◦
C and resuspended in a high salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 400 mM
KCl, 1.5 mM MgCl
2
, 0.2 mM EDTA, 0.5 mM DTT, 1 mM NaF, 1 mM Sodium orthovanadate).
2.8. Western Blot Analysis
Cells were washed with ice-cold PBS and extracted in a protein lysis buffer (Intron
Biotechnology, Seoul, Korea). Protein concentrations were determined by the Bradford
assay. The cell lysates were mixed with 5 X SDS sample buffer, boiled for 4 min, and
then separated on 10% SDS-PAGE. After electrophoresis, proteins were transferred to
polyvinylidene difluoride membranes. The membranes were blocked in 2% bovine serum
albumin (BSA) for 30 min, washed, and incubated overnight at 4
◦
C with specific primary
antibodies in Tris-buffered saline 2% BSA and 0.1% Tween-20 (TBS-T). The membranes
were washed three times to remove the primary antibodies and incubated for 2 h with a
horseradish peroxidase-conjugated secondary antibody (1:1000–2000). After washing three
times with TBS-T, immuno-positive bands were visualized using the ECL chemiluminescent
Medicina 2022,58, 335 4 of 14
system (Amersham Pharmacia Biotech, ON, Canada) and analyzed using ImageQuant
Las-4000 (GE Healthcare Life Science, WI, USA).
2.9. RNA Isolation and Real-Time RT–PCR Analysis
Total RNA was extracted from cells using the Easy Blue
®
kits (Intron Biotechnology,
Seoul, Korea), according to the manufacturer’s instructions. The total RNA was reverse
transcribed into first-strand cDNA (Amersham Pharmacia Biotech, ON, Canada) follow-
ing the manufacturer’s procedure. The synthesized cDNA was used as a template for
polymerase chain reaction (PCR) amplification. Real-time PCR was performed using the
Thermal cycler dice real-time PCR system (Takara, Japan). The primers used for SYBR
Green real-time RT-PCR were purchased from Bioneer technology (Daejon, Korea) and are
listed in Table S1. A dissociation curve analysis of P-cadherin, MMP-2, BDNF, NGF, NT-3,
NT-4/5, and GAPDH showed a single peak. PCRs were carried out for 45 cycles using
the following conditions: denaturation at 95
◦
C for 5 s, annealing at 57
◦
C for 10 s, and
elongation at 72
◦
C for 20 s. The results for P-cadherin, MMP-2, BDNF, NGF, NT-3 and
NT-4/5 mRNA were normalized to a control gene GAPDH.
2.10. Statistical Analysis
All the data were expressed as mean
±
SD. The data obtained in experiments with
multiple treatments were subjected to one-way ANOVA analysis of variance followed by a
post hoc Tukey test of significance. Under all circumstances, p< 0.05 was considered to
be significant.
3. Results
3.1. Cyperi Rhizoma Extract Inhibits Endometriotic Cell Adhesion to Mesothelial Cells
Since the implantation of endometrial fragments at the peritoneum, which is covered
by a mesothelial cell layer, is a key step in the establishment of endometriosis [
24
,
26
], we
investigated the effect of CRE on the adhesion of endometriotic cells to mesothelial cells.
As shown in Figure 1A, pre-treatment of human endometriotic 12Z cells with CRE (25,
50, and 100
µ
g/mL) significantly reduced the adhesion of 12Z cells to human peritoneal
mesothelial Met5A cells. The inhibitory effect of CRE on the adhesion of 12Z cells to Met5A
cells was not attributed to cytotoxic effects when the endometriotic cells were exposed to
CRE up to 100
µ
g/mL for 24 h (Figure 1B). Furthermore, we investigated the expression of
P-cadherin and MMP-2, which have been well known as potent determinants in peritoneal
adhesion formation [
5
], after treatment with CRE in endometriotic 12Z cells. The results of
real-time RT-PCR showed that CRE at concentrations of 50 and 100
µ
g/mL significantly
decreased the mRNA expression of P-cadherin and MMP-2 in 12Z cells (Figure 1C).
3.2. Cyperi Rhizoma Extract Decreases the Expression of Neurotrophins in Human
Endometriotic Cells
Neurotrophins, such as BDNF, NGF, NT-3 and NT-4/5, are overexpressed in en-
dometriosis and are involved in the pathophysiology of pain generation in women with
endometriosis [
27
]. In this regard, we investigated whether CRE has an inhibitory effect on
the expression of neurotrophins in human endometriotic 12Z cells. When 12Z cells were
treated with CRE, the mRNA expression of BDNF, NGF, NT-3 and NT-4/5 was inhibited in
a dose-dependent manner (Figure 2). BDNF and NT-3 expression was markedly decreased
by CRE at concentrations of 25, 50 and 100
µ
g/mL. NGF expression was slightly reduced
by CRE stimulation at concentrations of 25, 50 and 100
µ
g/mL. NT-4/5 expression was
significantly inhibited only at the highest concentration tested (100
µ
g/mL). Consequently,
the highest dose (100
µ
g/mL) of CRE significantly inhibited the expression of all four tested
neurotrophins in this study in endometriotic 12Z cells.
Medicina 2022,58, 335 5 of 14
Medicina 2022, 58, x FOR PEER REVIEW 5 of 15
Figure 1. Effect of CRE on endometriotic cell adhesion to mesothelial cells and expression of
adhesion molecules in endometriotic cells. (A) Human endometriotic cells (12Z) were treated with
CRE at indicated concentration (25, 50 and 100 µg/mL) for 24 h. The 12Z cells labeled with
CellTracker
TM
(10 µM) were cultured on Met5A cell layers in a 96-well plate for 1 h. The total
fluorescence in each well was measured by fluorescence microphotography. (B) Cell viability was
determined by MTT assay. Representative images show the cell morphology of 12Z cells treated
with CRE at indicated concentration (25, 50 and 100 µg/mL) for 24 h. (C) The mRNA expression of
P-cadherin and MMP-2 was measured by real-time RT-PCR. All expression levels were normalized
to GAPDH. Results are the combined data (mean ± SD) from three independent experiments. * p <
0.05 compared with control group.
Figure 1.
Effect of CRE on endometriotic cell adhesion to mesothelial cells and expression of adhesion
molecules in endometriotic cells. (
A
) Human endometriotic cells (12Z) were treated with CRE at
indicated concentration (25, 50 and 100
µ
g/mL) for 24 h. The 12Z cells labeled with CellTracker
TM
(10
µ
M) were cultured on Met5A cell layers in a 96-well plate for 1 h. The total fluorescence in each
well was measured by fluorescence microphotography. (
B
) Cell viability was determined by MTT
assay. Representative images show the cell morphology of 12Z cells treated with CRE at indicated
concentration (25, 50 and 100
µ
g/mL) for 24 h. (
C
) The mRNA expression of P-cadherin and MMP-2
was measured by real-time RT-PCR. All expression levels were normalized to GAPDH. Results are
the combined data (mean
±
SD) from three independent experiments. * p< 0.05 compared with
control group.
Medicina 2022,58, 335 6 of 14
Medicina 2022, 58, x FOR PEER REVIEW 6 of 15
3.2. Cyperi Rhizoma Extract Decreases the Expression of Neurotrophins in Human
Endometriotic Cells
Neurotrophins, such as BDNF, NGF, NT-3 and NT-4/5, are overexpressed in
endometriosis and are involved in the pathophysiology of pain generation in women with
endometriosis [27]. In this regard, we investigated whether CRE has an inhibitory effect
on the expression of neurotrophins in human endometriotic 12Z cells. When 12Z cells
were treated with CRE, the mRNA expression of BDNF, NGF, NT-3 and NT-4/5 was
inhibited in a dose-dependent manner (Figure 2). BDNF and NT-3 expression was
markedly decreased by CRE at concentrations of 25, 50 and 100 µg/mL. NGF expression
was slightly reduced by CRE stimulation at concentrations of 25, 50 and 100 µg/mL. NT-
4/5 expression was significantly inhibited only at the highest concentration tested (100
µg/mL). Consequently, the highest dose (100 µg/mL) of CRE significantly inhibited the
expression of all four tested neurotrophins in this study in endometriotic 12Z cells.
Figure 2. Effect of CRE on the expression of neurotrophins in endometriotic cells. The 12Z cells were
treated with CRE (25, 50 and 100 µg/mL) for 24 h, and then the mRNA expression of neurotrophins
(BDNF, NGF, NT-3 and NT-4/5) was measured by real-time RT-PCR. All expression levels were
normalized to GAPDH. Results are the combined data (mean ± S.D.) from three independent
experiments. * p < 0.05 compared with control group.
3.3. The Akt Pathway Is Involved in Cyperi Rhizoma Extract-Induced Anti-Endometriotic
Effects in Human Endometriotic Cells
The Akt pathway is a critical regulator of cell adhesion and migration through the
regulation of actin organization and extracellular degradation [28]. Western blot analysis
demonstrated that CRE significantly decreased Akt phosphorylation in 12Z cells at 50 and
100 µg/mL. In addition, CRE treatment inhibited the Akt activation in a time-dependent
manner (Figure 3). To determine the involvement of the Akt pathway in the CRE-induced
anti-endometriotic effect, we performed a cell adhesion assay and real-time RT-PCR
analysis after transfection with a constitutively active form of Akt (Akt-Myr) in 12Z cells.
Figure 2.
Effect of CRE on the expression of neurotrophins in endometriotic cells. The 12Z cells were
treated with CRE (25, 50 and 100
µ
g/mL) for 24 h, and then the mRNA expression of neurotrophins
(BDNF, NGF, NT-3 and NT-4/5) was measured by real-time RT-PCR. All expression levels were
normalized to GAPDH. Results are the combined data (mean
±
S.D.) from three independent
experiments. * p< 0.05 compared with control group.
3.3. The Akt Pathway Is Involved in Cyperi Rhizoma Extract-Induced Anti-Endometriotic Effects
in Human Endometriotic Cells
The Akt pathway is a critical regulator of cell adhesion and migration through the
regulation of actin organization and extracellular degradation [
28
]. Western blot analysis
demonstrated that CRE significantly decreased Akt phosphorylation in 12Z cells at 50 and
100
µ
g/mL. In addition, CRE treatment inhibited the Akt activation in a time-dependent
manner (Figure 3). To determine the involvement of the Akt pathway in the CRE-induced
anti-endometriotic effect, we performed a cell adhesion assay and real-time RT-PCR analysis
after transfection with a constitutively active form of Akt (Akt-Myr) in 12Z cells. As shown
in Figure 4A,B, the overexpression of Akt partially reversed CRE-inhibited adhesion of 12Z
cells to Met5A cells as well as the expression of P-cadherin and MMP-2 in 12Z cells, which
was downregulated by CRE. In addition, the downregulation of neurotrophins by CRE
was significantly attenuated by Akt overexpression in 12Z cells (Figure 5). These results
suggested that CRE exerts anti-endometriotic effects through the downregulation of Akt
activation in endometriotic cells.
Medicina 2022,58, 335 7 of 14
Medicina 2022, 58, x FOR PEER REVIEW 7 of 15
As shown in Figure 4A,B, the overexpression of Akt partially reversed CRE-inhibited
adhesion of 12Z cells to Met5A cells as well as the expression of P-cadherin and MMP-2
in 12Z cells, which was downregulated by CRE. In addition, the downregulation of
neurotrophins by CRE was significantly attenuated by Akt overexpression in 12Z cells
(Figure 5). These results suggested that CRE exerts anti-endometriotic effects through the
downregulation of Akt activation in endometriotic cells.
Figure 3. Effect of CRE on the phosphorylation of Akt in endometriotic cells. 12Z cells were treated
with CRE (25, 50 and 100 µg/mL) for 2 h or with CRE (100 µg/mL) for 0.5, 1 and 2 h. Phosphorylated
Akt and total Akt were measured by western blot analysis using specific antibodies. β-Actin was
used as an internal control. Data are shown as mean band density. Data are presented as the means
± SD of three independent experiments; * p < 0.05 as compared with the control group.
Figure 3.
Effect of CRE on the phosphorylation of Akt in endometriotic cells. 12Z cells were treated
with CRE (25, 50 and 100
µ
g/mL) for 2 h or with CRE (100
µ
g/mL) for 0.5, 1 and 2 h. Phosphorylated
Akt and total Akt were measured by western blot analysis using specific antibodies.
β
-Actin was used
as an internal control. Data are shown as mean band density. Data are presented as the means
±
SD
of three independent experiments; * p< 0.05 as compared with the control group.
Medicina 2022, 58, x FOR PEER REVIEW 8 of 15
Figure 4. Involvement of the Akt pathway in endometriotic cell adhesion to mesothelial cells and
expression of adhesion molecules in endometriotic cells. 12Z cells were transfected with an empty
vector (EV) or a constitutive active form (Akt-Myr), and then the cells were treated with CRE (100
µg/mL). After 24 h, (A) an adhesion assay and (B) a real-time RT-PCR were performed. The mRNA
expression of P-cadherin and MMP-2 was normalized to GAPDH. * p < 0.05 compared with EV-
transfected none treatment group and
#
p < 0.05 compared with EV-transfected CRE treatment
group.
Figure 4. Cont.
Medicina 2022,58, 335 8 of 14
Medicina 2022, 58, x FOR PEER REVIEW 8 of 15
Figure 4. Involvement of the Akt pathway in endometriotic cell adhesion to mesothelial cells and
expression of adhesion molecules in endometriotic cells. 12Z cells were transfected with an empty
vector (EV) or a constitutive active form (Akt-Myr), and then the cells were treated with CRE (100
µg/mL). After 24 h, (A) an adhesion assay and (B) a real-time RT-PCR were performed. The mRNA
expression of P-cadherin and MMP-2 was normalized to GAPDH. * p < 0.05 compared with EV-
transfected none treatment group and
#
p < 0.05 compared with EV-transfected CRE treatment
group.
Figure 4.
Involvement of the Akt pathway in endometriotic cell adhesion to mesothelial cells and
expression of adhesion molecules in endometriotic cells. 12Z cells were transfected with an empty
vector (EV) or a constitutive active form (Akt-Myr), and then the cells were treated with CRE
(100
µ
g/mL). After 24 h, (
A
) an adhesion assay and (
B
) a real-time RT-PCR were performed. The
mRNA expression of P-cadherin and MMP-2 was normalized to GAPDH. * p< 0.05 compared with EV-
transfected none treatment group and
#
p< 0.05 compared with EV-transfected CRE treatment group.
Medicina 2022, 58, x FOR PEER REVIEW 9 of 15
Figure 5. Involvement of the Akt pathway in the expression of neurotrophins in endometriotic cells.
The 12Z cells were transfected with an empty vector (EV) or a constitutive active form (Akt-Myr),
and then the cells were treated with CRE (100 µg/mL). After 24 h, real-time RT-PCR was performed
to measure the mRNA expression of BDNF, NGF, NT-3 and NT-4/5. The expression levels were
normalized to GAPDH. * p < 0.05 compared with EV-transfected none treatment group and
#
p < 0.05
compared with EV-transfected CRE treatment group.
3.4. Cyperi Rhizoma Extract Inhibits NF-kB Activation through the Akt Pathway
Since NF-kB activation is implicated in the pathogenesis of endometriosis and an
endometriosis-associated pro-inflammatory environment [29], we examined the effect of
CRE on NF-kB activation in endometriotic 12Z cells. As shown in Figure 6A, CRE
markedly reduced the nuclear translocation of p65 NF-kB in 12Z cells. In addition, the
phosphorylation of both IkBα and IKKα/β, a key step in the modulation of NF-kB
activation, was inhibited by CRE treatment in 12Z cells. To examine the involvement of
Akt signaling in CRE-induced NF-kB inactivation in endometriotic cells further, we
performed western blot analysis after transfection with Akt-Myr in 12Z cells. As shown
in Figure 6B, NF-kB inactivation by CRE was reversed by Akt overexpression. In addition,
Akt overexpression could recover the decrease in phosphorylation of IkBα and IKKα/β in
the presence of CRE in 12Z cells. These results suggested that NF-kB inactivation by CRE
is mediated by the Akt pathway in endometriotic 12Z cells.
Figure 5.
Involvement of the Akt pathway in the expression of neurotrophins in endometriotic cells.
The 12Z cells were transfected with an empty vector (EV) or a constitutive active form (Akt-Myr),
and then the cells were treated with CRE (100
µ
g/mL). After 24 h, real-time RT-PCR was performed
to measure the mRNA expression of BDNF, NGF, NT-3 and NT-4/5. The expression levels were
normalized to GAPDH. * p< 0.05 compared with EV-transfected none treatment group and
#
p< 0.05
compared with EV-transfected CRE treatment group.
3.4. Cyperi Rhizoma Extract Inhibits NF-kB Activation through the Akt Pathway
Since NF-kB activation is implicated in the pathogenesis of endometriosis and an
endometriosis-associated pro-inflammatory environment [
29
], we examined the effect of
Medicina 2022,58, 335 9 of 14
CRE on NF-kB activation in endometriotic 12Z cells. As shown in Figure 6A, CRE markedly
reduced the nuclear translocation of p65 NF-kB in 12Z cells. In addition, the phosphory-
lation of both IkB
α
and IKK
α
/
β
, a key step in the modulation of NF-kB activation, was
inhibited by CRE treatment in 12Z cells. To examine the involvement of Akt signaling in
CRE-induced NF-kB inactivation in endometriotic cells further, we performed western
blot analysis after transfection with Akt-Myr in 12Z cells. As shown in Figure 6B, NF-kB
inactivation by CRE was reversed by Akt overexpression. In addition, Akt overexpression
could recover the decrease in phosphorylation of IkB
α
and IKK
α
/
β
in the presence of CRE
in 12Z cells. These results suggested that NF-kB inactivation by CRE is mediated by the
Akt pathway in endometriotic 12Z cells.
Medicina 2022, 58, x FOR PEER REVIEW 10 of 15
Figure 6. Effect of CRE on the NF-kB activation in endometriotic cells. Nuclear (Nu) and whole
cellular proteins were isolated from the cells (A) treated with CRE (25, 50 and 100 µg/mL) for 8 h
and (B) transfected with EV or Akt-Myr. The levels of p65 (Nu), p-IkBα, IkBα, p-IKKα/β and IKKα/β
were detected by western blot analysis using specific antibodies. PARP and β-actin were used as an
internal control for nuclear fractions and whole cell lysates, respectively. Data are shown as mean
band density. Data are presented as the means ± SD of three independent experiments; * p < 0.05 as
compared with the control group, and
#
p < 0.05 as compared with EV-transfected CRE treatment
group.
4. Discussion
The rhizomes of C. rotundus have been traditionally considered one of the most
important herbs for the treatment of obstetric/gynecologic disorders in Asia. In Vietnam
and Taiwan, the decoction of C. rotundus is used for treating infertility and dysmenorrhea
in women with endometriosis [14,30]. For example, Qi Gong Wan composed of various
herbs including Cyperi rhizoma relieves mid-cycle pain, vaginal discharge and adhesions
[14]. Although numerous experiences have been reported to show the effect of C. rotundus
on gynecologic disorders [14,31], there is little scientific evidence of the effect of C.
rotundus on gynecological diseases and the underlying molecular mechanisms of action.
Choi et al. examined the effect of water-extracted tubers of C. rotundus on endometrial
receptivity both in vitro and in vivo [32]. It was found that the water-extracted tubers of
C. rotundus enhanced the adhesion of trophoblastic JAr cells to endometrial cancer
Ishikawa cells for blastocyst implantation and improved the number of implantation sites
in a mifepristone-induced implantation failure mice model. In a recent retrospective
cohort study using the Taiwan National Health Insurance reimbursement database, the
rate of endometriosis-related surgery, including hysterectomy and oophorectomy, was
significantly lower in patients with endometriosis who took the traditional Chinese
Figure 6.
Effect of CRE on the NF-kB activation in endometriotic cells. Nuclear (Nu) and whole
cellular proteins were isolated from the cells (
A
) treated with CRE (25, 50 and 100
µ
g/mL) for 8 h
and (
B
) transfected with EV or Akt-Myr. The levels of p65 (Nu), p-IkB
α
, IkB
α
, p-IKK
α
/
β
and
IKK
α
/
β
were detected by western blot analysis using specific antibodies. PARP and
β
-actin were
used as an internal control for nuclear fractions and whole cell lysates, respectively. Data are shown
as mean band density. Data are presented as the means
±
SD of three independent experiments;
*p< 0.05
as compared with the control group, and
#
p< 0.05 as compared with EV-transfected CRE
treatment group.
4. Discussion
The rhizomes of C. rotundus have been traditionally considered one of the most
important herbs for the treatment of obstetric/gynecologic disorders in Asia. In Vietnam
and Taiwan, the decoction of C. rotundus is used for treating infertility and dysmenorrhea in
women with endometriosis [
14
,
30
]. For example, Qi Gong Wan composed of various herbs
including Cyperi rhizoma relieves mid-cycle pain, vaginal discharge and adhesions [
14
].
Medicina 2022,58, 335 10 of 14
Although numerous experiences have been reported to show the effect of C. rotundus on
gynecologic disorders [
14
,
31
], there is little scientific evidence of the effect of C. rotundus
on gynecological diseases and the underlying molecular mechanisms of action. Choi et al.
examined the effect of water-extracted tubers of C. rotundus on endometrial receptivity
both
in vitro
and
in vivo
[
32
]. It was found that the water-extracted tubers of C. rotundus
enhanced the adhesion of trophoblastic JAr cells to endometrial cancer Ishikawa cells for
blastocyst implantation and improved the number of implantation sites in a mifepristone-
induced implantation failure mice model. In a recent retrospective cohort study using
the Taiwan National Health Insurance reimbursement database, the rate of endometriosis-
related surgery, including hysterectomy and oophorectomy, was significantly lower in
patients with endometriosis who took the traditional Chinese medicine formula Gui-zhi-
fu-ling-wan, which mainly comprises C. rotundus, than in patients with endometriosis
who did not take it [
33
]. Kim et al. has also demonstrated that Boheotang containing
Cyperi rhizoma has an inhibitory effect on recurrent endometriosis after laparoscopic
excision and hormone therapy [
34
]. However, to the best of our knowledge, there is no
direct experimental evidence supporting the effect of C. rotundus on endometriosis. In this
study, we investigated the effect of CRE on endometriosis using human endometriotic
12Z cells for the first time. The adhesive ability of endometriotic cells has been well
observed in endometriotic lesion formation [
26
]. In this study, we demonstrated that CRE
significantly inhibited the adhesion of endometriotic cells to peritoneal mesothelial cells,
without cytotoxicity.
Previous phytochemical reports revealed the presence of flavonoids, tannins, alkaloids,
glycosides, furochromones and sesquiterpenes in C. rotundus [
35
,
36
]. In fact, most of the
biological activities of the rhizomes of C. rotundus have been attributed to the sesquiter-
penes [
36
]. It is reported that isocyperol and
α
-cyperone, known as major sesquiterpene
components of the rhizomes of C. rotundus, have various pharmacological activities, such
as anti-inflammation, antivirulence, antigenotoxic, antibacterial and anti-depressant ef-
fects [
37
–
42
]. For example, isocyperol and
α
-cyperone have an inhibitory effect on in-
flammation through suppression of the NF-
κ
B pathway [
37
,
38
]. Zhang et al. showed the
inhibitory effects of
α
-cyperone on adhesion and invasion of avian pathogenic Escherichia
coli O78 to chicken type II pneumocytes [
43
]. However, to the best of our knowledge,
there is no evidence supporting the effect of active constituents of CRE in gynecologic
disorders including endometriosis. In a previous study, we found that
α
-cyperone is the
most abundant and active component of CRE [
44
–
47
]. In these regards, we have examined
the anti-endometriotic effect of
α
-cyperone in human endometriotic 12Z cells. Similar to
CRE,
α
-cyperone significantly inhibited the adhesive ability of 12Z cells to mesothelial
Met5A cells (Figure S1). This observation suggests a possibility that the anti-endometriotic
effects of CRE are mainly attributed to its major component,
α
-cyperone. However, further
experiments are still needed to draw this conclusion.
Cell surface proteins, including cadherin and MMPs, are expressed in the ectopic
endometrium and implicated in the regulation of cell-cell adhesion as well as cell-ECM
adhesion during the development of endometriosis [
5
]. Unlike E-cadherin and N-cadherin,
P-cadherin is found to be remarkably increased in peritoneal endometriotic lesions and di-
rectly involved in the interaction between endometrial cells and peritoneal cells [
6
]. MMP-2
is a collagenase-degrading collagen IV, the principal component of basement membranes,
and is significantly elevated in the urine of patients with endometriosis, compared to
healthy women [
48
]. A recent study demonstrated that physiological changes, including
adhesion, in endometriosis involve abnormal matrix remodeling, which is affected by
proteolytic enzymes, such as MMP-2 [
49
]. In addition, MMPs support the irreversible inac-
tivation of cadherin-catenin complexes [
50
]. In this study, we showed that CRE inhibited
the mRNA expression of P-cadherin and MMP-2 in endometriotic 12Z cells. These results
suggest that the downregulation of adhesion molecules, such as P-cadherin and MMP-2 by
CRE, may contribute to the inhibitory effect of CRE on the adhesion of endometriotic cells
to peritoneal mesothelial cells.
Medicina 2022,58, 335 11 of 14
Pelvic pain is one of the major symptoms in women with endometriosis, and is as-
sociated with the activation of nociceptive neurons [
51
]. It has been suggested that the
neurotrophins such as BDNF, NGF, NT-3 and NT-4/5 generate pelvic pain in endometriosis
by the direct alteration of neurological responsiveness, leading to hyperalgesia and allody-
nia stimuli [
1
]. A recent study demonstrated that the level of BDNF in circulation is found
to be elevated in women with endometriosis compared to healthy controls, and it decreased
after the surgical removal of lesions [
10
]. In addition, it has been reported in a recent study
that specific antibodies against neurotrophins, including NGF, may be used to treat and
prevent pain and symptoms associated with endometriosis [
52
]. In the present study, we
elucidated the inhibitory effect of CRE on the expression of BDNF, NGF, NT-3 and NT-4/5
in endometriotic cells. It was found that CRE significantly decreased the mRNA expression
of BDNF, NGF, NT-3 and NT-4/5 in 12Z cells. Taken together, CRE may have an inhibitory
effect on endometriosis-associated pain by modulating the expression of neurotrophins,
which is elevated in women with endometriosis.
The PI3K/Akt pathway is well known to regulate various cellular events, such as cell
growth, proliferation, apoptosis and differentiation stimulated by intra- and extracellular
signals [
53
]. Recently, the role of PI3K/Akt signaling in endometriosis has been impli-
cated. Yin et al. demonstrated that the over activation of the PI3K/Akt signaling pathway
contributes to the decrease in decidualization of stromal cells from endometriosis [
54
]. In
addition, rapid activation of PI3K/Akt mediated the growth factor- and estrogen-induced
migration of endometrial stromal cells [
55
]. Moreover, it has been suggested that the activa-
tion of Akt signaling is involved in the expression of P-cadherin and MMP-2 [
56
,
57
]. In the
present study, we found that the inhibitory effect of CRE on endometriotic cell adhesion and
neurotrophin expression in endometriotic cells is mediated by the Akt pathway. Notably, it
has been demonstrated that the activation of Akt affects the phosphorylation of IKK
β
and
the translocation of the NF-kB p65 subunit [
58
,
59
]. Many
in vitro
and
in vivo
evidences
show that the constitutive activation of NF-kB in endometriotic lesions from patients with
endometriosis promotes inflammation, cell proliferation, adhesion invasion and angio-
genesis in an endometriosis-associated pro-inflammatory environment [
60
–
63
]. In these
regards, we investigated the involvement of NF-kB activation in the effect of CRE on cell
adhesion and neurotrophin expression in endometriotic cells through the Akt pathway.
The results suggest that NF-kB signaling is likely to mediate the anti-endometriotic effect
of CRE through the Akt pathway.
5. Conclusions
In summary, we demonstrated the inhibitory effect of CRE on endometriosis via the
inhibition of cell adhesion and neurotrophin expression through negative regulation of Akt
and NF-kB pathways in human endometriotic cells.
These results provide scientific evidence that CRE could be a candidate for multidis-
ciplinary therapy, demonstrating a reduction of the adhesion of endometriotic fragments
and pain relief in patients with endometriosis. The
in vivo
effect of CRE on endometriosis
should be further investigated.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/medicina58030335/s1, Figure S1: Effect of
α
-cyperone on en-
dometriotic cell adhesion to mesothelial cells, Table S1: Primer sequences for real-time RT-PCR
analysis, Supplementary Material and Methods: Preparation of the α-cyperone.
Author Contributions:
Conceptualization, J.-H.A. and J.-H.C.; formal analysis, J.-M.C., E.-S.K.,
J.-H.Y.
, Y.-J.C.; resources, D.S.J.; writing—original draft preparation, J.-H.A.; writing—review and
editing, J.-H.A. and J.-H.C.; supervision, J.-H.A. and J.-H.C. All authors have read and agreed to the
published version of the manuscript.
Funding:
This work has been supported by the National Research Foundation of Korea(NRF) grant
funded by the Korean government (MSIT) (NRF-2020R1I1A3073660).
Institutional Review Board Statement: Not applicable.
Medicina 2022,58, 335 12 of 14
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors have declared no conflict of interest.
References
1. Burney, R.O.; Giudice, L.C. Pathogenesis and pathophysiology of endometriosis. Fertil. Steril. 2012,98, 511–519. [CrossRef]
2. Giudice, L.C.; Kao, L.C. Endometriosis. Lancet 2004,364, 1789–1799. [CrossRef]
3.
Czyzyk, A.; Podfigurna, A.; Szeliga, A.; Meczekalski, B. Update on endometriosis pathogenesis. Minerva Ginecol.
2017,69, 447–461
.
[CrossRef] [PubMed]
4.
Andres, M.P.; Arcoverde, F.V.L.; Souza, C.C.C.; Fernandes, L.F.C.; Abrao, M.S.; Kho, R.M. Extrapelvic Endometriosis: A Systematic
Review. J. Minim. Invasive Gynecol. 2020,27, 373–389. [CrossRef]
5.
Young, V.J.; Brown, J.K.; Saunders, P.T.; Horne, A.W. The role of the peritoneum in the pathogenesis of endometriosis. Hum.
Reprod. Update 2013,19, 558–569. [CrossRef]
6.
Chen, G.T.; Tai, C.T.; Yeh, L.S.; Yang, T.C.; Tsai, H.D. Identification of the cadherin subtypes present in the human peritoneum and
endometriotic lesions: Potential role for P-cadherin in the development of endometriosis. Mol. Reprod. Dev.
2002
,62, 289–294.
[CrossRef] [PubMed]
7.
Kim, J.H.; Yang, Y.I.; Ahn, J.H.; Lee, J.G.; Lee, K.T.; Choi, J.H. Deer (Cervus elaphus) antler extract suppresses adhesion and
migration of endometriotic cells and regulates MMP-2 and MMP-9 expression. J. Ethnopharmacol.
2012
,140, 391–397. [CrossRef]
8.
Morotti, M.; Vincent, K.; Becker, C.M. Mechanisms of pain in endometriosis. Eur. J. Obstet. Gynecol. Reprod. Biol.
2017
,209, 8–13.
[CrossRef]
9. Pezet, S.; McMahon, S.B. Neurotrophins: Mediators and modulators of pain. Annu. Rev. Neurosci. 2006,29, 507–538. [CrossRef]
10.
Wessels, J.M.; Kay, V.R.; Leyland, N.A.; Agarwal, S.K.; Foster, W.G. Assessing brain-derived neurotrophic factor as a novel clinical
marker of endometriosis. Fertil. Steril. 2016,105, 119–128.e5. [CrossRef]
11.
Streiter, S.; Fisch, B.; Sabbah, B.; Ao, A.; Abir, R. The importance of neuronal growth factors in the ovary. Mol. Hum. Reprod.
2016,22, 3–17. [CrossRef] [PubMed]
12.
Uddin, S.J.; Mondal, K.; Shilpi, J.A.; Rahman, M.T. Antidiarrhoeal activity of Cyperus rotundus.Fitoterapia
2006
,77, 134–136.
[CrossRef] [PubMed]
13.
Dang, G.K.; Parekar, R.R.; Kamat, S.K.; Scindia, A.M.; Rege, N.N. Antiinflammatory activity of Phyllanthus emblica, Plumbago
zeylanica and Cyperus rotundus in acute models of inflammation. Phytother. Res. 2011,25, 904–908. [CrossRef] [PubMed]
14.
Zhou, J.; Qu, F. Treating gynaecological disorders with traditional Chinese medicine: A review. Afr. J. Tradit. Complement. Altern.
Med. 2009,6, 494–517. [CrossRef] [PubMed]
15.
Gupta, M.B.; Palit, T.K.; Singh, N.; Bhargava, K.P. Pharmacological studies to isolate the active constituents from Cyperus rotundus
possessing anti-inflammatory, anti-pyretic and analgesic activities. Indian J. Med. Res. 1971,59, 76–82.
16.
Singh, P.; Khosa, R.L.; Mishra, G.; Jha, K.K. Antidiabetic activity of ethanolic extract of Cyperus rotundus rhizomes in streptozotocin-
induced diabetic mice. J. Pharm. Bioallied Sci. 2015,7, 289–292. [CrossRef]
17.
Seo, W.G.; Pae, H.O.; Oh, G.S.; Chai, K.Y.; Kwon, T.O.; Yun, Y.G.; Kim, N.Y.; Chung, H.T. Inhibitory effects of methanol extract
of Cyperus rotundus rhizomes on nitric oxide and superoxide productions by murine macrophage cell line, RAW 264.7 cells.
J. Ethnopharmacol. 2001,76, 59–64. [CrossRef]
18.
Ahn, J.H.; Lee, T.W.; Kim, K.H.; Byun, H.; Ryu, B.; Lee, K.T.; Jang, D.S.; Choi, J.H. 6-Acetoxy Cyperene, a Patchoulane-type
Sesquiterpene Isolated from Cyperus rotundus Rhizomes Induces Caspase-dependent Apoptosis in Human Ovarian Cancer Cells.
Phytother. Res. 2015,29, 1330–1338. [CrossRef]
19.
Zhu, M.; Luk, H.H.; Fung, H.S.; Luk, C.T. Cytoprotective effects of Cyperus rotundus against ethanol induced gastric ulceration in
rats. Phytother. Res. 1997,11, 392–394. [CrossRef]
20.
Kamala, A.; Middha, S.K.; Gopinath, C.; Sindhura, H.S.; Karigar, C.S.
In vitro
Antioxidant Potentials of Cyperus rotundus L.
Rhizome Extracts and Their Phytochemical Analysis. Pharmacogn. Mag. 2018,14, 261–267. [CrossRef]
21.
Mannarreddy, P.; Denis, M.; Munireddy, D.; Pandurangan, R.; Thangavelu, K.P.; Venkatesan, K. Cytotoxic effect of Cyperus
rotundus rhizome extract on human cancer cell lines. Biomed. Pharmacother. 2017,95, 1375–1387. [CrossRef]
22.
Jia, H.; Liu, Y.; Yu, M.; Shang, H.; Zhang, H.; Ma, L.; Zhang, T.; Zou, Z. Neuroprotective Effect of Cyperi rhizome against
Corticosterone-Induced PC12 Cell Injury via Suppression of Ca2+ Overloading. Metabolites 2019,9, 244. [CrossRef] [PubMed]
23. Jiang, L.; Yan, Y.; Liu, Z.; Wang, Y. Inflammation and endometriosis. Front. Biosci. 2016,21, 941–948.
24.
Saunders, P.T.K.; Horne, A.W. Endometriosis: Etiology, pathobiology, and therapeutic prospects. Cell
2021
,184, 2807–2824.
[CrossRef]
25.
Zeitvogel, A.; Baumann, R.; Starzinski-Powitz, A. Identification of an invasive, N-cadherin-expressing epithelial cell type in
endometriosis using a new cell culture model. Am. J. Pathol. 2001,159, 1839–1852. [CrossRef]
26.
Vercellini, P.; Vigano, P.; Somigliana, E.; Fedele, L. Endometriosis: Pathogenesis and treatment. Nat. Rev. Endocrinol.
2014,10, 261–275. [CrossRef] [PubMed]
27.
Kobayashi, H.; Yamada, Y.; Morioka, S.; Niiro, E.; Shigemitsu, A.; Ito, F. Mechanism of pain generation for endometriosis-
associated pelvic pain. Arch. Gynecol. Obstet. 2014,289, 13–21. [CrossRef] [PubMed]
Medicina 2022,58, 335 13 of 14
28.
Fabi, F.; Grenier, K.; Parent, S.; Adam, P.; Tardif, L.; Leblanc, V.; Asselin, E. Regulation of the PI3K/Akt pathway during
decidualization of endometrial stromal cells. PLoS ONE 2017,12, e0177387. [CrossRef] [PubMed]
29.
Gonzalez-Ramos, R.; Van Langendonckt, A.; Defrere, S.; Lousse, J.C.; Colette, S.; Devoto, L.; Donnez, J. Involvement of the nuclear
factor-kappaB pathway in the pathogenesis of endometriosis. Fertil. Steril. 2010,94, 1985–1994. [CrossRef] [PubMed]
30.
Hung, Y.C.; Kao, C.W.; Lin, C.C.; Liao, Y.N.; Wu, B.Y.; Hung, I.L.; Hu, W.L. Chinese Herbal Products for Female Infertility in
Taiwan: A Population-Based Cohort Study. Medicine 2016,95, e3075. [CrossRef] [PubMed]
31. Bhattarai, N.K. Folk herbal remedies for diarrhoea and dysentery in central Nepal. Fitoterapia 1993,64, 243–250. [CrossRef]
32.
Choi, H.J.; Chung, T.W.; Park, M.J.; Jung, Y.S.; Lee, S.O.; Kim, K.J.; Ha, K.T. Water-extracted tubers of Cyperus rotundus L. enhance
endometrial receptivity through leukemia inhibitory factor-mediated expression of integrin αVβ3 and αVβ5. J. Ethnopharmacol.
2017,208, 16–23. [CrossRef]
33.
Su, S.Y.; Muo, C.H.; Sung, F.C.; Morisky, D.E. Reduction of surgery rate in endometriosis patients who take Chinese medicine:
A population-based retrospective cohort study. Complement. Ther. Med. 2014,22, 632–639. [CrossRef]
34.
Kim, H.W.; Yoo, J.E. Inhibitory effect of traditional Korean medicine on the recurrent endometriosis after laparoscopic excision:
A case report. Integr. Med. Res. 2018,7, 296–301. [CrossRef]
35.
Xu, X.T.; Deng, Z.P.; Zhong, H.; Yao, Q.Q. Research progress on chemical constituents and pharmacological activities of
Cyperus rotundus L. Qilu Pharm. Aff. 2012,31, 473–475.
36.
Gamal, M.A.; Hani, K.M.K.; Sabrin, I.R.M. A Review: Compounds Isolated from Cyperus Species (Part II):Terpenoidal. Int. J.
Pharmacogn. Phytochem. Res. 2015,7, 83–99.
37.
Seo, Y.J.; Jeong, M.; Lee, K.T.; Jang, D.S.; Choi, J.H. Isocyperol, isolated from the rhizomes of Cyperus rotundus, inhibits LPS-induced
inflammatory responses via suppression of the NF-kappaB and STAT3 pathways and ROS stress in LPS-stimulated RAW 264.7
cells. Int. Immunopharmacol. 2016,38, 61–69. [CrossRef]
38.
Jung, S.H.; Kim, S.J.; Jun, B.G.; Lee, K.T.; Hong, S.P.; Oh, M.S.; Jang, D.S.; Choi, J.H. alpha-Cyperone, isolated from the rhizomes of
Cyperus rotundus, inhibits LPS-induced COX-2 expression and PGE2 production through the negative regulation of NFkappaB
signalling in RAW 264.7 cells. J. Ethnopharmacol. 2013,147, 208–214. [CrossRef]
39.
Luo, M.; Qiu, J.; Zhang, Y.; Wang, J.; Dong, J.; Li, H.; Leng, B.; Zhang, Q.; Dai, X.; Niu, X.; et al. alpha-cyperone alleviates
lung cell injury caused by Staphylococcus aureus via attenuation of alpha-hemolysin expression. J. Microbiol. Biotechnol.
2012,22, 1170–1176. [CrossRef]
40.
Singh, N.; Kulshrestha, V.K.; Gupta, M.B.; Bhargava, K.P. Apharmacological study of Cyperus rotundus.Indian J. Med. Res.
1970,58, 103–109.
41.
Jin, J.H.; Lee, D.U.; Kim, Y.S.; Kim, H.P. Anti-allergic activity of sesquiterpenes from the rhizomes of Cyperus rotundus.Arch.
Pharm. Res. 2011,34, 223–228. [CrossRef]
42.
Xia, B.; Tong, Y.; Xia, C.; Chen, C.; Shan, X. alpha-Cyperone Confers Antidepressant-Like Effects in Mice via Neuroplasticity
Enhancement by SIRT3/ROS Mediated NLRP3 Inflammasome Deactivation. Front. Pharmacol. 2020,11, 577062. [CrossRef]
43.
Zhang, L.Y.; Lv, S.; Wu, S.C.; Guo, X.; Xia, F.; Hu, X.R.; Song, Z.; Zhang, C.; Qin, Q.Q.; Fu, B.D.; et al. Inhibitory effects of
alpha-cyperone on adherence and invasion of avian pathogenic Escherichia coli O78 to chicken type II pneumocytes. Vet. Immunol.
Immunopathol. 2014,159, 50–57. [CrossRef]
44.
Kim, S.J.; Kim, H.J.; Kim, H.J.; Jang, Y.P.; Oh, M.S.; Jang, D.S. New patchoulane-type sesquiterpenes from the rhizomes of Cyperus
rotundus.Bull. Korean Chem. Soc. 2012,33, 3115–3118. [CrossRef]
45.
Kim, S.J.; Ryu, B.; Kim, H.-Y.; Yang, Y.-I.; Ham, J.; Choi, J.-H.; Jang, D.S. Sesquiterpenes from the rhizomes of Cyperus rotundus and
their potential to inhibit LPS-induced nitric oxide production. Bull. Korean Chem. Soc. 2013,34, 2207–2210. [CrossRef]
46.
Ryu, B.; Kim, H.M.; Lee, J.-S.; Cho, Y.J.; Oh, M.S.; Choi, J.-H.; Jang, D.S. Sesquiterpenes from rhizomes of Cyperus rotundus with
cytotoxic activities on human cancer cells in vitro. Helv. Chim. Acta 2015,98, 1372–1380. [CrossRef]
47.
Sim, Y.; Choi, J.G.; Gu, P.S.; Ryu, B.; Kim, J.H.; Kang, I.; Jang, D.S.; Oh, M.S. Identification of Neuroactive Constituents of the Ethyl
Acetate Fraction from Cyperi Rhizoma Using Bioactivity-Guided Fractionation. Biomol. Ther. 2016,24, 438–445. [CrossRef]
48.
Osteen, K.G.; Yeaman, G.R.; Bruner-Tran, K.L. Matrix metalloproteinases and endometriosis. Semin. Reprod. Med.
2003,21, 155–164. [CrossRef]
49.
Yang, W.V.; Au, H.K.; Chang, C.W.; Chen, H.W.; Chen, P.H.; Chen, C.C.; Tang, Y.L.; Wang, I.T.; Tzeng, C.R. Matrix remodeling and
endometriosis. Reprod. Med. Biol. 2005,4, 93–99. [CrossRef]
50.
Ribeiro, A.S.; Albergaria, A.; Sousa, B.; Correia, A.L.; Bracke, M.; Seruca, R.; Schmitt, F.C.; Paredes, J. Extracellular cleavage and
shedding of P-cadherin: A mechanism underlying the invasive behaviour of breast cancer cells. Oncogene
2010
,29, 392–402.
[CrossRef]
51.
Howard, F.M. Endometriosis and mechanisms of pelvic pain. J. Minim. Invasive Gynecol.
2009
,16, 540–550. [CrossRef] [PubMed]
52.
Howe, D.C.; Pullen, N.; Shelton, D.L. Treatment of Endometriosis. 2010. Available online: https://patents.google.com/patent/
WO2010029497A1/zh (accessed on 1 October 2020).
53.
Hennessy, B.T.; Smith, D.L.; Ram, P.T.; Lu, Y.; Mills, G.B. Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat. Rev.
Drug Discov. 2005,4, 988–1004. [CrossRef] [PubMed]
54.
Yin, X.; Pavone, M.E.; Lu, Z.; Wei, J.; Kim, J.J. Increased activation of the PI3K/AKT pathway compromises decidualization of
stromal cells from endometriosis. J. Clin. Endocrinol. Metab. 2012,97, E35–E43. [CrossRef]
Medicina 2022,58, 335 14 of 14
55.
Gentilini, D.; Busacca, M.; Di Francesco, S.; Vignali, M.; Vigano, P.; Di Blasio, A.M. PI3K/Akt and ERK1/2 signalling pathways
are involved in endometrial cell migration induced by 17beta-estradiol and growth factors. Mol. Hum. Reprod.
2007
,13, 317–322.
[CrossRef] [PubMed]
56.
Wang, X.; Li, X.; Li, C.; He, C.; Ren, B.; Deng, Q.; Gao, W.; Wang, B. Aurora-A modulates MMP-2 expression via AKT/NF-kappaB
pathway in esophageal squamous cell carcinoma cells. Acta Biochim. Biophys. Sin. 2016,48, 520–527. [CrossRef]
57.
Vieira, A.F.; Ribeiro, A.S.; Dionisio, M.R.; Sousa, B.; Nobre, A.R.; Albergaria, A.; Santiago-Gomez, A.; Mendes, N.; Gerhard, R.;
Schmitt, F.; et al. P-cadherin signals through the laminin receptor alpha6beta4 integrin to induce stem cell and invasive properties
in basal-like breast cancer cells. Oncotarget 2014,5, 679–692. [CrossRef]
58.
Ozes, O.N.; Mayo, L.D.; Gustin, J.A.; Pfeffer, S.R.; Pfeffer, L.M.; Donner, D.B. NF-kappaB activation by tumour necrosis factor
requires the Akt serine-threonine kinase. Nature 1999,401, 82–85. [CrossRef]
59.
Sizemore, N.; Leung, S.; Stark, G.R. Activation of phosphatidylinositol 3-kinase in response to interleukin-1 leads to phosphoryla-
tion and activation of the NF-kappaB p65/RelA subunit. Mol. Cell. Biol. 1999,19, 4798–4805. [CrossRef]
60.
Cao, W.G.; Morin, M.; Sengers, V.; Metz, C.; Roger, T.; Maheux, R.; Akoum, A. Tumour necrosis factor-alpha up-regulates
macrophage migration inhibitory factor expression in endometrial stromal cells via the nuclear transcription factor NF-kappaB.
Hum. Reprod. 2006,21, 421–428. [CrossRef]
61.
Gonzalez-Ramos, R.; Donnez, J.; Defrere, S.; Leclercq, I.; Squifflet, J.; Lousse, J.C.; Van Langendonckt, A. Nuclear factor-kappa B is
constitutively activated in peritoneal endometriosis. Mol. Hum. Reprod. 2007,13, 503–509. [CrossRef]
62.
Gonzalez-Ramos, R.; Rocco, J.; Rojas, C.; Sovino, H.; Poch, A.; Kohen, P.; Alvarado-Diaz, C.; Devoto, L. Physiologic activation
of nuclear factor kappa-B in the endometrium during the menstrual cycle is altered in endometriosis patients. Fertil. Steril.
2012,97, 645–651. [CrossRef]
63.
Grund, E.M.; Kagan, D.; Tran, C.A.; Zeitvogel, A.; Starzinski-Powitz, A.; Nataraja, S.; Palmer, S.S. Tumor necrosis factor-alpha
regulates inflammatory and mesenchymal responses via mitogen-activated protein kinase kinase, p38, and nuclear factor kappaB
in human endometriotic epithelial cells. Mol. Pharmacol. 2008,73, 1394–1404. [CrossRef] [PubMed]
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