R E S E A R C H A R T I C L E Open Access
Pao Pereira extract suppresses benign
prostatic hyperplasia by inhibiting
inflammation-associated NFκB signaling
, Jiakuan Liu
, Zesheng Xue
, Jingya Sun
, Zhengnan Huang
, Yifeng Jing
, Bangmin Han
, Jun Yan
and Ruimin Huang
Background: Our previous study revealed the extract from the bark of an Amazonian tree Pao Pereira can suppress
benign prostatic hyperplasia (BPH) in a rat model. Herein, we examined its inhibitory effects on human BPH cells
and dissect its molecular mechanism.
Methods: We applied Pao extract to human BPH epithelial BPH-1 and prostate myofibroblast WPMY-1 cells. Cell
viability, apoptosis and immunoblotting were performed, followed by gene expression profiling and gene set
enrichment analysis (GSEA) to detect the differentially expressed genes and signaling pathway induced by Pao
extract. Human ex vivo BPH explant organ culture was also used to examine the effects of Pao extract on human
Results: Pao extract treatment inhibited viability and induced apoptosis in human BPH-1 and WPMY-1 cells. Gene
expression profiling and the following validation indicated that the expression levels of pro-apoptotic genes (eg.
PCDC4,CHOP and FBXO32) were induced by Pao extract in both two cell lines. GSEA further revealed that Pao
extract treatment was negatively associated with the activation of NFκB signaling. Pao extract suppressed the
transcriptional activity of NFκB and down-regulated its target genes involved in inflammation (CXCL5,CXCL6 and
CXCL12) and extracellular matrix (ECM) remodeling (HAS2, TNC and MMP13) in both cultured cells and human
ex vivo BPH explants.
Conclusion: In both BPH epithelial and stromal cells, Pao extract induces apoptosis by upregulating the pro-
apoptotic genes and inhibiting the inflammation-associated NFκB signaling via reducing phosphorylation of NFκB
subunit RelA. Our data suggest that Pao extract may be a promising phytotherapeutic agent for BPH.
Keywords: Pao Pereira extract, BPH, NFκB, Inflammation, Extracellular matrix
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* Correspondence: firstname.lastname@example.org;email@example.com;
Yu Dong, Jiakuan Liu and Zesheng Xue contributed equally to this work.
Department of Urology, Shanghai General Hospital, Shanghai Jiaotong
University, 100 Haining Road, Shanghai 200080, China
Department of Laboratory Animal Science, Fudan University, 130 Dong’an
Road, Shanghai 200032, China
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555
Zuchongzhi Road, Shanghai 201203, China
Full list of author information is available at the end of the article
Medicine and Therapie
Dong et al. BMC Complementary Medicine and Therapies (2020) 20:150
Benign prostatic hyperplasia (BPH) is a non-malignant
enlargement of the prostate gland that is common in
older males. About 70% of men over 70 will develop
BPH . Due to the excessive proliferation of the epithe-
lial and stromal cells in the transition zone and periure-
thral glands, the enlarged prostate ultimately induces
lower urinary tract symptoms (LUTS) including urgency
and difficulty of urination [2,3]. Moderate-to-severe
LUTS have significant influences on the patients’quality
of life. The etiology of BPH is multi-factorial, including
sex hormones, smooth muscle and inflammation .
Currently, medications for BPH/LUTS primarily target
sex hormone synthesis and relief of tension in smooth
muscle. However, alpha-blockers and 5α-reductase in-
hibitors (5-ARIs) induce non-trivial adverse side effects
including asthenia and ejaculatory dysfunction .
Hence, more effective agents with fewer side effects are
Chronic inflammation has been implicated in BPH de-
velopment, as manifested by the infiltration of immune
cells, including activated T cells and macrophages into
human BPH tissues . It was reported that chronic
prostatic inflammation was associated with a larger pros-
tate volume and a higher International Prostate Symp-
tom Score (IPSS) .
Pao pereira (Geissospermum vellosii) is an Amazon
rainforest tree, and its bark extract is used to treat mal-
aria, digestive disorders and cancers. Pao extract is rich
in β-carboline alkaloids  and exhibited anti-
proliferative activities against melanoma and glioblast-
oma, ovarian cancer and pancreatic cancer [9–13]. Our
previous study showed that Pao extract decreased hu-
man prostate cancer cell growth via inducing apoptosis
. Recently we found that Pao extract can attenuate
BPH development in a rat model . However, the mo-
lecular mechanism remains largely unclear.
In this study, we investigated the effects of Pao extract
on human BPH epithelial cells (BPH-1) and stromal cells
(WPMY-1). We found that Pao extract suppressed the
growth of BPH-1 and WPMY-1 cells, and induced apop-
tosis via inhibition of NFκB signaling pathway. An
ex vivo human BPH explant was also exploited to test
the effects of Pao extract. These findings suggest that
Pao extract may be a very promising therapeutic agent
Cell lines, human BPH tissues and reagents
Human BPH-derived prostate epithelial BPH-1 cell line
was kindly provided by Dr. Simon Hayward (Vanderbilt
University Medical Center, Nashville, TN, USA). Human
prostate myofibroblast WPMY-1 cell line was purchased
from the Cell Bank of Type Culture Collection of
Chinese Academy of Sciences (Shanghai, China). BPH-1
and WPMY-1 cells were cultured in RPMI 1640 (Corn-
ing, New York, NY, USA) and DMEM medium (Corn-
ing), respectively, containing 10% fetal bovine serum
(FBS; Life Technologies, Carlsbad, CA, USA) and
penicillin-streptomycin (S110JV, BasalMedia, Shanghai,
China). Passage number of BPH-1 and WPMY-1 cell
lines was less than 20 for all the cell-based experiments.
The study protocol using human BPH tissues was ap-
proved by the Ethics Committee of Shanghai General
Hospital, Shanghai Jiaotong University. Human BPH tis-
sues were collected also with patients’consent. Pao ex-
tract was from Natural Source International (New York,
NY, USA). Briefly, the extract was prepared with aque-
ous alcoholic extraction of the bark of the plant Pao Per-
eira, which was then transformed into a free-flowing
powder by spray drying. It contained 54% β-carboline al-
kaloids, including flavopereirine, by high-performance li-
quid chromatography [13,15]. The Pao extract was
dissolved in DMSO and diluted with sterile phosphate
buffered saline (PBS) to 50 mg/ml as a stock solution
and stored at −80 °C until use. The final concentration
of DMSO was less than 6% (v/v).
Primary antibodies targeting the following proteins were
used: Caspase-3 (#9662, Cell Signaling Technology
(CST), Danvers, MA, USA, 1:1000), Cleaved Caspase-3
(#9661, CST, 1:1000), PARP (#9532, CST, 1:1000),
Cleaved PARP (#5625, CST, 1:1000), NFκB/p65 (#8242,
CST, 1:1000), Phospho-NFκB/p65(Ser536) (#3033, CST,
1:1000), PDCD4 (12587–1-AP, Proteintech, Rosemont,
IL, USA, 1:1000), β-Actin (A2228, Sigma-Aldrich, St
Louis, MO, USA, 1:5000), and CHOP (#2895, CST, 1:
1000). Peroxidase AffiniPure goat anti-mouse IgG (H +
L) (115–035-003, 1:2500) and peroxidase AffiniPure goat
anti-rabbit IgG (H + L) (111–035-003, 1:2500) were pur-
chased from Jackson ImmunoResearch (West Grove,
PA, USA) and used as the secondary antibodies.
Cell cytotoxicity assay
BPH-1 cells (1.5 × 10
cells/well) and WPMY-1 cells
(2.5 × 10
cells/well) were seeded into 96-well plates in
triplicate. The next day Pao extract with different con-
centrations was added into culture medium. 24, 48 and
72 h later, the cells were fixed with 10% trichloroacetic
acid (TCA; Sigma-Aldrich) over 4 h and stained with 4
mg/ml sulforhodamine B (SRB; Sigma-Aldrich) in 1%
acetic acid. 10 mM Tris base solution was used to dis-
solve the protein-bound dye. Optical density (OD) at
560 nm was determined by a microplate reader (Spectra-
Max M5, Molecular Devices, Sunnyvale, CA, USA).
Dong et al. BMC Complementary Medicine and Therapies (2020) 20:150 Page 2 of 10
Apoptosis assay was performed using FITC Annexin V
Apoptosis Detection Kit I (#556547, BD Biosciences,
Franklin Lakes, NJ, USA). BPH-1 and WPMY-1 cells
were seeded into 6-well plates at densities of 5 × 10
cells/well and 1 × 10
cells/well, respectively. The next
day, the medium was replaced with fresh medium and
the cells were treated with Pao extract or cisplatin
(5 μM, a positive control) for 72 h. Then the floating and
adherent cells were collected, washed with PBS twice
and re-suspended in 1 × Binding Buffer as 1 × 10
ml. 100 μl cell suspension was stained with 5 μl of FITC-
Annexin V and 5 μl propidium iodide (PI) for 15 min at
room temperature in the dark. Flow cytometry analysis
was performed with a FACS Calibur flow cytometer (BD
Biosciences) and cell death patterns were quantified
(Figure S1) with FlowJo software (version 10.0.7r2, Ash-
land, OR, USA).
Gene expression profiling analysis
BPH-1 and WPMY-1 cells were treated with Pao extract
for 24 h, and total RNA was extracted with TRIzol re-
agent (Life Technologies). Affymetrix GeneChips (Hu-
man Transcriptome Array 2.0) were used for the gene
expression profiling analysis (Shanghai Baygene Biotech-
nologies, Shanghai, China). The raw and normalized
microarray data from this study can be accessed at
GSE128856 in NCBI GEO Datasets. Gene Set Enrich-
ment Analysis (GSEA) was performed using the GSEA
software (v3.0, http://software.broadinstitute.org/gsea/
index.jsp) to determine the association between the
priori defined gene sets in the Molecular Signatures
Database (MSigDB) and the different changes of genes
induced by Pao extract treatment. The number of per-
mutations was 1000. Enrichment statistic was weighted
and the ranking metric was the difference of class means
(Diff_of_Classes). Normalized enrichment score (NES) ≥
1 and the false discovery rate (FDR) < 0.25 were used as
Cells were lysed with 2% SDS lysis buffer and total pro-
tein was quantified with the Enhanced BCA Protein
Assay Kit (P0009, Beyotime, Shanghai, China). 10 ~
20 μg total protein was separated on an SDS-PAGE gel
and transferred onto a polyvinylidene difluoride (PVDF)
membrane (Millipore, Billerica, MA, USA). The mem-
branes were blocked with 5% bovine serum albumin
(BSA; FA016, Gen-view Scientific, Houston, TX, USA)
and incubated with the primary antibodies overnight at
4 °C. Then the membranes were incubated with horse-
radish peroxidase (HRP)-conjugated secondary anti-
bodies for 1 h at room temperature. The signals were
developed with SuperSignal West Pico PLUS
Chemiluminescent Substrate (Thermo Fisher Scientific,
Waltham, MA, USA) and detected by the Mini Chemilu-
minescent Imaging and Analysis System (Beijing Sage
Creation Science, Beijing, China).
RNA isolation and quantitative real-time PCR
The total RNA from cells was extracted using TRIzol,
according to the manufacturer’s instructions. RNA was
dissolved in DEPC-treated water (Sangon Biotech,
Shanghai, China). The concentration and purity of RNA
were measured with a NanoDrop One Microvolume
UV-Vis Spectrophotometer (Thermo Fisher Scientific),
and RNA was kept at −80 °C. Residual DNAs in total
RNAs were removed and cDNAs were synthesized by
Hifair II 1st Strand cDNA Synthesis SuperMix
(11123ES60, YEASEN, Shanghai, China) following the
manufacturer’s protocol. Quantitative real-time PCR was
performed by ChamQ Universal SYBR qPCR Master
Mix (Q711–02, Vazyme, Nanjing, China) using a CFX96
Touch Real-Time PCR Detection System (Bio-Rad,
Hercules, CA, USA) with the reaction conditions: Stage
1, 95 °C for 30 s; Stage 2, 40 cycles of 95 °C for 10 s and
60 °C for 30 s; Stage 3, 95 °C for 15 s, 60 °C for 60 s and
95 °C for 15 s. Data analysis was performed using the
ΔΔCt method. Fold change was determined in relative
quantification units using ACTB gene for normalization.
Primers were listed in Table S1.
Dual-luciferase reporter gene assay
BPH-1 and WPMY-1 cells were seeded into 24-well
plates (5 × 10
cells/well). 200 ng 6 × NFκB-Luc plasmid
and 50 ng pRL-CMV plasmid for each well were trans-
fected into the cells at ~ 70% confluency by Lipofecta-
mine 3000 (Invitrogen, Carlsbad, CA, USA) in the
presence of FBS. Eight hours after transfection, Pao ex-
tract was added for 36 h (BPH-1 cells) and 40 h
(WPMY-1 cells), respectively. Cell lysates were then
measured by Dual-Luciferase Reporter Assay System
(E1910, Promega, Madison, WI, USA). The ratio of fire-
fly luciferase activity versus Renilla luciferase activity was
determined for NFκB transcriptional activity.
BPH ex vivo explant culture
Human BPH tissues (n= 4) were obtained from patients
with a transurethral resection of the prostate at Shanghai
General Hospital, Shanghai Jiaotong University. The
BPH ex vivo explant culture was described previously
[17,18]. In brief, the BPH tissues were subdivided into
pieces and cultured on absorbable gelatin
sponges (HSD-B, HUSHIDA, Nanchang, China) in 6-
well plates containing 4 ml DMEM/F-12 (Corning) with
10% FBS, antibiotic /antimycotic solution (S120, Basal-
Media), 0.01 mg/ml insulin (I1882, Sigma-Aldrich) and
0.01 mg/ml hydrocortisone (H0135, Sigma-Aldrich).
Dong et al. BMC Complementary Medicine and Therapies (2020) 20:150 Page 3 of 10
Tissues were treated with Pao extract for 48 h at 37 °C.
The tissues were washed with PBS twice and then
grinded with the High Throughput Tissue Grinder
(Scientz-48, Ningbo Scientz Biotechnology, Ningbo,
China) in protein lysis buffer or TRIzol.
Statistical analyses were performed using GraphPad
Prism Software (version 8.0.1, GraphPad, San Diego, CA,
USA). Two-tailed Student’sttest was used to compare
the difference between two groups, and P< 0.05 was
considered statistically significant.
Pao extract inhibited proliferation of BPH-1 and WPMY-1
To evaluate the effect of Pao extract on the growth
of cells derived from the prostate, BPH-1 and
WPMY-1 cells were exposed to Pao extract with dos-
ages ranging from 125 to 500 μg/ml, and the cell pro-
liferation were assessed by SRB assay. Pao extract
treatment significantly reduced the adherent cell
number at 48 h (Fig. 1a) and viability within 72 h (p<
0.05) in a concentration-dependent manner in both
cell lines (Fig. 1b).
Fig. 1 Pao extract inhibited the proliferation and induced apoptosis in BPH-1 and WPMY-1 cells. aMorphological changes of BPH-1 and WPMY-1
cells upon Pao extract treated for 48 h. bEffects of Pao extract on cell viability by the SRB assay. BPH-1 and WPMY-1 cells were treated with Pao
extract at the indicated concentrations for 0, 24, 48 and 72 h. Data are presented as mean ± SD; * p< 0.05, ** p< 0.01, and *** p< 0.001
Dong et al. BMC Complementary Medicine and Therapies (2020) 20:150 Page 4 of 10
Pao extract induced apoptosis in BPH-1 and WPMY-1 cells
Flow cytometry analyses were performed to determine
whether Pao extract could induce apoptosis in BPH-1 and
WPMY-1 cells. After 72 h treated with Pao extract, both
cells showed that the percentages of apoptotic cells were
significantly increased in a concentration-dependent man-
ner by Annexin V-FITC/PI double staining (Fig. 2a). Not-
ably, apoptosis was detected in ~ 43% BPH cells and ~
24% WPMY-1 cells under the Pao extract treatment at
500 μg/ml (Fig. 2b). Moreover, the levels of cleaved
Caspase-3 and cleaved PARP were consistently increased
in the Pao extract-treated cells (Fig. 2c).
Pao extract regulated the pro-apoptotic and
inflammation-associated genes in BPH-1 and WPMY-1 cells
To investigate which genes were regulated by Pao extract
in both BPH-1 and WPMY-1 cells, gene expression profil-
ing was performed using the Affymetrix microarray chips.
Using 1.4-fold as the cutoff, 106 up-regulated genes and 68
down-regulated genes were identified in Pao extract-treated
BPH-1 cells, comparing to the vehicle-treated cells (Fig. 3a,
left panel); 212 up-regulated genes and 511 down-regulated
genes were identified in Pao extract-treated WPMY-1 cells
(Fig. 3a, right panel). Among them, several pro-apoptotic
and inflammation-associated genes were induced by
250 μg/ml Pao extract treatment (Fig. 3b). We further con-
firmed the induction of the pro-apoptotic genes (PDCD4,
FBXO32 and DDIT3) in both BPH-1 and WPMY-1 cells by
Pao extract at mRNA level (Fig. 3c). Western blotting data
also indicated that Pao extract increases PDCD4 and
DDIT3/CHOP at protein level (Fig. 3d).
Pao extract inhibited NFκB signaling pathway in BPH-1
and WPMY-1 cells
We also used the aforementioned microarray data to in-
vestigate which signaling pathways were regulated by
Fig. 2 Pao extract induced apoptosis in BPH-1 and WPMY-1 cells. aFlow cytometry analysis on BPH-1 and WPMY-1 cells treated with Pao extract
using an Annexin V-FITC/PI double staining kit. Representative plots were shown. bQuantification of cell death rates in BPH-1 and WPMY-1 cells
treated with Pao extract. Annexin V-positive cells were defined as apoptotic cells. cEffects of Pao extract on the apoptosis-related proteins by
Western blotting assay. Data are presented as mean ± SD; * p< 0.05, ** p< 0.01, and *** p< 0.001
Dong et al. BMC Complementary Medicine and Therapies (2020) 20:150 Page 5 of 10
Pao extract in both BPH-1 and WPMY-1 cells by GSEA.
The association between the down-regulation in gene
set of NFκB signaling pathway and Pao extract treatment
was identified in both BPH-1 cells (NES = 2.17, FDR <
0.001) and WPMY-1 cells (NES = 1.24, FDR = 0.12), re-
spectively (Fig. 4a). The phosphorylation levels of RelA
(a subunit NFκB transcription complex) were also shown
to be reduced in BPH-1 and WPMY-1 cells by Pao ex-
tract using Western blotting (Fig. 4b). Moreover, NFκB-
reporter was transiently transfected into BPH-1 and
WPMY-1 cells, followed by Pao extract treatment. The
luciferase activity from NFκB-reporter was suppressed
by 60% in BPH-1 cells (p< 0.01) and by 62% in WPMY-
1 cells (p < 0.01) using 500 μg/ml Pao extract (Fig. 4c).
The expression levels of several well-known NFκB target
genes involved in inflammation (CXCL5,CXCL6, and
CXCL12) and extracellular matrix (ECM) remodeling
(HAS2,TNC, and MMP13) were further tested.
Fig. 3 Pao extract regulated the pro-apoptotic and inflammation-associated genes in BPH-1 and WPMY-1 cells. aDownstream target genes of
Pao extract detected by gene expression profiling analysis. BPH-1 and WPMY-1 cells were treated with Pao extract (250 μg/ml) or vehicle for 48 h.
Heatmap of differentially expressed genes under the treatment of Pao extract by microarray (1.4-fold as the cutoff), compared with vehicle-
treatment. Rows corresponded to genes and were ordered by hierarchical clustering. The values used for clustering were the expression levels
normalized to average of all samples. bHeatmap of the pro-apoptotic and inflammation-associated genes from panel a.cThe mRNA expression
levels of pro-apoptotic and inflammation-associated genes (PDCD4,DDIT3 and FBXO32) in BPH-1 and WPMY-1 cells by qRT-PCR. dThe protein
expression levels of pro-apoptotic and inflammation-associated genes (PDCD4 and DDIT3) in BPH-1 and WPMY-1 cells by Western blotting. * p<
0.05, ** p< 0.01, and *** p< 0.001
Dong et al. BMC Complementary Medicine and Therapies (2020) 20:150 Page 6 of 10
Consistently, HAS2,CXCL6,CXCL5, and MMP13 were
down-regulated in BPH-1 cells and HAS2,CXCL6,
CXCL12,TNC were down-regulated in WPMY-1 cells
by Pao extract (Fig. 4d). Altogether, it was suggested that
Pao extract suppresses the activation of NFκB signaling
in both BPH epithelial and stromal cells.
Pao extract inhibited NFκB signaling pathway in human
To validate the effects of Pao extract on human BPH, an
ex vivo explant culture system was established for the
tissues from BPH patients (Fig. 5a). BPH explants were
treated with 0, 250 and 500 μg/ml Pao extract for 48 h,
following the assessment of NFκB signaling. Consistent
with the data from cultured cell lines, the phosphoryl-
ation levels of RelA were markedly decreased and the
protein levels of PDCD4 and DDIT3/CHOP were in-
creased in Pao extract-treated BPH explants (Fig. 5b). In
addition, the mRNA expression levels of the downstream
target genes of NFκB signaling, including CXCL6,HAS2,
CXCL5,CXCL12,MMP13 and TNC, were also signifi-
cantly reduced (p< 0.01), as well as the up-regulation of
the apoptosis-associated genes PDCD4,DDIT3 and
FBXO32 (p< 0.05), in BPH explants treated with Pao ex-
tract (Fig. 5c). Taken together, it was demonstrated that
Pao extract inhibited the NFκB signaling pathway in hu-
man BPH tissues.
Our previous study demonstrated that Pao extract sup-
presses testosterone-induced BPH in a rat model. To
examine its effects on human BPH sample and delineate
its molecular mechanism, herein we found that Pao ex-
tract inhibited the viabilities of BPH epithelial cells and
stromal cells in a dose-dependent manner, due to the in-
crease of apoptosis and suppression of NFκB signaling.
Fig. 4 Pao extract inhibited NFκB signaling pathway in BPH-1 and WPMY-1 cells. aGene set enrichment analysis identified the association
between the down-regulation in gene set of NFκB signaling pathway and Pao extract treatment using microarray data from vehicle- and Pao
extract-treated BPH-1 and WPMY-1 cells. In the enrichment plot, genes were ranked by signal/noise ratio according to their differential expression
between vehicle- and Pao extract-treated cells. bPao extract decreased phosphorylation of NFκB p65/RelA subunit in BPH-1 and WPMY-1 cells. c
Pao extract inhibited transcriptional activities of NFκB after BPH-1 and WPMY-1 cells were treated with Pao for 36 h and 40 h, respectively. dPao
extract down-regulated the mRNA levels of NFκB target genes, including CXCL5,CXCL6,CXCL12,HAS2,TNC, and MMP13 in BPH-1 and WPMY-1
cells by qRT-PCR. * p< 0.05, ** p< 0.01, *** p< 0.001, and ns p≥0.05
Dong et al. BMC Complementary Medicine and Therapies (2020) 20:150 Page 7 of 10
Downregulation of inflammation- and ECM remodeling-
related genes via NFκB signaling was further demon-
strated in human BPH cell lines in vitro and in human
BPH explants ex vivo (Fig. 6).
The canonical NFκB pathway is frequently up-
regulated during the progression of BPH, and the sever-
ity of BPH is correlated with activation of NFκB,
which makes it an interesting target for BPH. For ex-
ample, Elocalcitol was reported to inhibit BPH stromal
cells proliferation by targeting NFκB/p65 nuclear trans-
location . Here, we showed that Pao extract could
inhibit the phosphorylation level of RelA (Ser536), lead-
ing to the decrease of transcriptional activity of NFκB
complex. In addition, Pao extract increased the mRNA
and protein expression levels of two pro-apoptotic
proteins, PDCD4 and DDIT3/CHOP. PDCD4 was re-
ported to directly bind with NFκB/p65 and suppressed
NFκB-dependent transcription in human glioblastoma
. Hence, it is suggested that Pao extract may target
NFκB activity in BPH at different levels.
As an age-related disease, BPH progression is accom-
panied by chronic inflammation and ECM remodeling
around BPH nodules. The inflammatory BPH micro-
environment contains various secreted cytokines and
chemokines, including CXCL5, CXCL6 and CXCL12,
which are the direct NFκB target genes [22–24] and can
promote the proliferation of both prostatic epithelial
cells and stromal fibroblasts [25–27]. For the ECM-
associated genes, such as HAS2,MMP13 and TNC, they
are also target genes of NFκB signaling. HAS2 gene
Fig. 5 Pao extract inhibited NFκB signaling pathway in human BPH tissues. aSchematic diagram of the ex vivo BPH explant culture process. BPH
explants were treated with 0, 250, 500 μg/ml Pao extract for 48 h. bThe phosphorylation of RelA and protein expression levels of pro-apoptotic
and inflammation-associated genes survival-associated proteins (PDCD4 and DDIT3/CHOP) were analyzed by Western blotting in BPH explants
with Pao extract treatment. cThe mRNA expression levels of target genes from NFκB signaling pathway, pro-apoptotic and inflammation-
associated genes in BPH explants with Pao extract treatment by qRT-PCR. * p< 0.05, ** p< 0.01, *** p< 0.001, and ns p≥0.05
Dong et al. BMC Complementary Medicine and Therapies (2020) 20:150 Page 8 of 10
encodes hyaluronan synthase 2, an enzyme that synthe-
sizes hyaluronan (HA) in BPH tissues [28,29]. When
BPH-1 cells were cultured in 3D gel containing collagen,
they proliferated faster in the collagen from aged mice
(high level of HA) than that from young mice (low level
of HA). Previous studies also showed that MMP13 pro-
moted ECM degradation, and the elevated ECM glyco-
protein Tenascin-C was associated with myofibroblast in
BPH tissues [30,31]. Here, we observed that Pao extract
downregulates the expression levels of CXCL5, CXCL6,
CXCL12,HAS2,MMP13 and TNC in BPH-1 and
WPMY-1 cells, thus indicating that Pao extract attenu-
ates the inflammation and ECM-remodeling via inhib-
ition of NFκB signaling in BPH.
Our data have proved the inhibitory effect of Pao extract
on NFκB signaling pathway in two cell lines derived
from human BPH and ex vivo explants from human
BPH patients. Using Pao extract as a negative regulator
of NFκB signaling may be a promising phytotherapeutic
agent for BPH.
Supplementary information accompanies this paper at https://doi.org/10.
Additional file 1: Figure S1. Flow cytometry analysis on vehicle
treated-BPH-1 cells (a) and WPMY-1 cells (b) stained with negative control
buffer (Unstained), FITC-Annexin-V dye only (FITC only) and PI dye only
(PI only). Representative plots were shown.
Additional file 2: Table S1. The sequences of primers used in
quantitative real-time PCR assay.
BPH: Benign prostatic hyperplasia; GSEA: Gene set enrichment analysis;
LUTS: Lower urinary tract symptoms; 5-ARIs: 5α-reductase inhibitors;
IPSS: International Prostate Symptom Score; TCA: Trichloroacetic acid;
SRB: Sulforhodamine B; OD: Optical density; PI: Propidium iodide; BSA: Bovine
serum albumin; HRP: Horseradish peroxidase; ECM: Extracellular matrix;
We thank the Institutional Technology Service Center of Shanghai Institute of
Materia Medica for technical support.
JY and RH designed the work; YD, JL, ZX and JS performed the research and
analyzed the data; ZH, YJ, BH and BS provide the resource. YD, JL and ZX
Fig. 6 Schematic diagram of the working model for inhibitory effects of Pao extract on BPH tissues. BPH is characterized as the excessive
proliferation of both epithelial and stromal cells in prostate. In both cell populations, Pao extract not only up-regulates the pro-apoptotic genes
and induces apoptosis, but also inhibits the NFκB signaling and induces the inflammatory and ECM-associated genes expression via reducing
phosphorylation of NFκB subunit RelA
Dong et al. BMC Complementary Medicine and Therapies (2020) 20:150 Page 9 of 10
drafted the work; YD, JY and RH revised the paper. BS, JY and RH supervised
the study. All authors read and approved the final manuscript.
This study was supported by the National Natural Science Foundation
(81672873 and 81872373 to JY), the National Science & Technology Major
Project “Key New Drug Creation and Manufacturing Program”, China
(2018ZX09711002 to RH), One Hundred Talent Program of Chinese Academy
of Sciences (to RH), Wu Jieping Medical Foundation (320.6750.16051 to BS),
Shanghai Songjiang Municipal Science and Technology Commission Natural
Science Foundation (17SJKJGG10 to BS), Shanghai Specialized Research Fund
for Integrated Chinese and Western Medicine in General Hospitals (ZHYY-
ZXYJHZX-1-201705 to BS) and The Beljanski Foundation to JY. The founding
sponsor had no role in the design of the study; in the collection, analyses, or
interpretation of data; in the writing of the paper; and in the decision to
publish the results.
Availability of data and materials
All microarray files are available from the NCBI GEO Datasets (accession
number GSE128856). Other datasets used and/or analyzed during the current
study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study protocol using human BPH tissues was approved by the Ethics
Committee of Shanghai General Hospital, Shanghai Jiaotong University
(2018KY067). Human BPH tissues were collected with patients’oral consent.
Consent for publication
The authors declare that they have no competing interests.
Shanghai University, Shanghai, China.
Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203,
Model Animal Research Center of Nanjing University, Nanjing,
Department of Urology, Shanghai General Hospital, Shanghai
Jiaotong University, 100 Haining Road, Shanghai 200080, China.
of Laboratory Animal Science, Fudan University, 130 Dong’an Road, Shanghai
MOE Key Laboratory of Model Animals for Disease Study,
Model Animal Research Center of Nanjing University, Nanjing, Jiangsu, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Received: 27 January 2020 Accepted: 4 May 2020
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