Two resveratrol analogs, pinosylvin and 4,4′-dihydroxystilbene, improve oligoasthenospermia in a mouse model by attenuating oxidative stress via the Nrf2-ARE pathway

Article abstract of DOI:10.1016/j.bioorg.2020.104295

Two synthesized resveratrol analogs from our laboratory, namely pinosylvin (3,5-dihydroxy-trans-stilbene, PIN) and 4,4′-dihydroxystilbene (DHS), have been carefully evaluated for treatment of oligoasthenospermia. Recent studies have demonstrated that PIN and DHS improved sperm quality in the mouse. However, the mechanism of action of PIN and DHS on oligoasthenospermia remains unknown. Herein, we investigated the mechanistic basis for improvements in sperm parameters by PIN and DHS in a mouse model of oligoasthenospermia induced by treatment with busulfan (BUS) at 6 mg/kg b.w. Two weeks following busulfan treatment, mice were administered different concentrations of PIN or DHS daily for 2 consecutive weeks. Thereafter, epididymal sperm concentration and motility were determined, and histopathology of the testes was performed. Serum hormone levels including testosterone (T), luteinizing hormone (LH), and follicle stimulating hormone (FSH) were measured using corresponding specific enzyme-linked immunosorbent assay (ELISA) kits. Testicular mRNA expression profiles were determined by RNA sequencing analysis. These findings were validated by quantitative real-time PCR, western blotting and ELISA. Both PIN and DHS improved the epididymal sperm concentration and motility, enhanced testosterone levels, and promoted testicular morphological recovery following BUS treatment. PIN treatment was found to significantly reduce oxidative stress via the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE)-dependent antioxidant, glutathione peroxidase 3. DHS treatment significantly reduced oxidative stress via the Nrf2-ARE-dependent antioxidants glutathione S-transferase theta 2 and glutathione S-transferase omega 2. In summary, PIN and DHS ameliorated oligoasthenospermia in this mouse model by attenuating oxidative stress via the Nrf2-ARE pathway.

Full text of DOI:10.1016/j.bioorg.2020.104295

BioorganicChemistry104(2020)104295
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Two resveratrol analogs, pinosylvin and 4,4′-dihydroxystilbene, improve
oligoasthenospermia in a mouse model by attenuating oxidative stress via
the Nrf2-ARE pathway
⁎
Cheng-niu Wanga,1, Meng-meng Sanga,1, Sheng-nan Gonga, Jin-fei Yanga, C. Yan Chengb, ,
⁎
Fei Suna,
a Medical School, Institute of Reproductive Medicine, Nantong University, Nantong 226001, Jiangsu, China
b The Mary M. Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, New York, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two synthesized resveratrol analogs from our laboratory, namely pinosylvin (3,5-dihydroxy-trans-stilbene, PIN)
and 4,4′-dihydroxystilbene (DHS), have been carefully evaluated for treatment of oligoasthenospermia. Recent
studies have demonstrated that PIN and DHS improved sperm quality in the mouse. However, the mechanism of
action of PIN and DHS on oligoasthenospermia remains unknown. Herein, we investigated the mechanistic basis
for improvements in sperm parameters by PIN and DHS in a mouse model of oligoasthenospermia induced by
treatment with busulfan (BUS) at 6 mg/kg b.w.. Two weeks following busulfan treatment, mice were ad-
ministered different concentrations of PIN or DHS daily for 2 consecutive weeks. Thereafter, epididymal sperm
concentration and motility were determined, and histopathology of the testes was performed. Serum hormone
levels including testosterone (T), luteinizing hormone (LH), and follicle stimulating hormone (FSH) were
measured using corresponding specific enzyme-linked immunosorbent assay (ELISA) kits. Testicular mRNA
expression profiles were determined by RNA sequencing analysis. These findings were validated by quantitative
real-time PCR, western blotting and ELISA. Both PIN and DHS improved the epididymal sperm concentration
and motility, enhanced testosterone levels, and promoted testicular morphological recovery following BUS
treatment. PIN treatment was found to significantly reduce oxidative stress via the nuclear factor erythroid 2-
related factor 2 (Nrf2)-antioxidant response element (ARE)-dependent antioxidant, glutathione peroxidase 3.
DHS treatment significantly reduced oxidative stress via the Nrf2-ARE-dependent antioxidants glutathione S-
transferase theta 2 and glutathione S-transferase omega 2. In summary, PIN and DHS ameliorated oligoasthe-
nospermia in this mouse model by attenuating oxidative stress via the Nrf2-ARE pathway.
Pinosylvin
DHS
Oligoasthenospermia
Oxidative stress
Nrf2-ARE pathway
1. Introduction
motility, and germ cell apoptosis, as well as DNA damage in testicular
tissues [4–6]. The nuclear factor erythroid 2-related factor 2 (Nrf2)-
About 13% of couples worldwide suffer from infertility [1], and
men contribute to about 50% of infertility [2]. Oligoasthenospermia is
one of the most notable causes of male-factor infertility. While there are
many reasons, but the molecular mechanism is still unclear. Oxidative
stress is considered to be an important contributing factor, caused by
the overproduction of reactive oxygen species (ROS) in the male re-
productive tissues [3], leading to sperm cell damage, reduced sperm
antioxidant response element (ARE) signaling pathway is considered to
be one of the most important pathways of defense against oxidative
stress [7–8]. Ubiquitination is inhibited after Nrf2 is stimulated by
noxious chemicals, when more Nrf2 is generated and accumulated.
Thereafter, Nrf2 enters into the cell nucleus to bind to ARE gene se-
quences and activates downstream genes encoding antioxidative en-
zymes and the glutathione redox system, inducing ROS degradation,
Abbreviations: PIN, pinosylvin; DHS, 4,4′-dihydroxystilbene; RES, resveratrol; BUS, busulfan; ELISA, enzyme-linked immunosorbent assay; Nrf2, nuclear factor
erythroid 2-related factor 2; ARE, antioxidant response element; ROS, reactive oxygen species; MDA, malondialdehyde; Gpx3, glutathione peroxidase 3; GSTT2,
glutathione S-transferase theta 2; GSTO2, glutathione S-transferase omega 2; GAPDH, glyceraldehyde-3- phosphate dehydrogenase; HE, hematoxylin and eosin; T,
testosterone; FSH, follicle stimulating hormone; LH, luteinizing hormone; GO, gene Ontology; PVDF, polyvinylidene difluoride
⁎ Corresponding authors.
E-mail addresses: ccheng@rockefeller.edu (C.Y. Cheng), sunfei@ntu.edu.cn (F. Sun).
1 Co-first author.
https://doi.org/10.1016/j.bioorg.2020.104295
Received 19 June 2020; Received in revised form 10 September 2020; Accepted 16 September 2020
Availableonline19September2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

C.-n. Wang, et al.
BioorganicChemistry104(2020)104295
Fig. 1. Chemical structure of Resveratrol, Pinosylvin and DHS. (A): Resveratrol (RES), (B): Pinosylvin (PIN), (C): 4,4′-dihydroxystilbene (DHS).
which in turn protects cells and tissues against ROS-mediated damage
[9–11]. Current therapeutic approch for the treatment of oligoasthe-
nospermia is ineffective [12]. At present, Traditional Chinese Medicine
is receiving increasing attention as it often offers more effective treat-
ment options [13].
Na2SO4. After filtration, the solvent was evaporated and the crude
product was purified by column chromatography (n-Hexane/
EtOAc = 40:1) to obtain the desired product 2 (2a, 80% yield; 2b, 73%
yield). 3a (PIN) and 3b (DHS): To a solution of product 2 (0.2 mmol)
in anhydrous DCM (6 mL) maintained under nitrogen at −20 °C with
stirring was added BBr3 dropwise (0.34 g, 1.34 mmol, 30% in DCM).
The mixture, maintained under nitrogen with stirring, was allowed to
warm up to room temperature for 4–5 h, and then it was poured into
water (10 mL) and extracted with DCM (3 × 10 mL). The collected
organic phases were washed with brine (10 mL) and then dried over
Na2SO4, filtered and concentrated in vacuo. The residue purified by
column chromatography (n-Hexane/Acetone = 6:4) to afford the de-
sired product 3. PIN: 83% yield, white solid, 1H NMR (400 MHz,
(CD3)2CO): δ 7.57 (d, J = 7.2 Hz, 2H), 7.35 (t, J = 7.2 Hz, 2H), 7.25 (t,
J = 7.2 Hz, 1H), 7.10 (s, 2H), 6.59 (d, J = 2.0 Hz, 2H), 6.31 (t,
J = 2.4 Hz, 1H); 13C NMR (100 MHz, (CD3)2CO): δ 159.56, 140.37,
138.36, 129.77, 129.50, 129.17, 128.35, 127.34, 105.94, 103.06. DHS:
77.8% yield, white solid, 1H NMR (400 MHz, (CD3)2CO): δ 8.40 (s, 2H),
7.40 (d, J = 8.8 Hz, 4H), 6.96 (s, 2H), 6.83 (d, J = 8.8 Hz, 4H); 13C
NMR (100 MHz, (CD3)2CO): δ 157.72, 130.47, 128.29, 126.46, 116.35.
Resveratrol (3,4′,5-trihydroxy-trans-stilbene, RES; Fig. 1A), a poly-
phenolic stilbene compound, has been used to treat oligoasthenos-
permia and attenuate testicular damage of mouse, mainly because of its
strong antioxidant activity [14–17]. However, the clinical application
of resveratrol has limitations because this drug is relatively unstable, it
has poor bioavailability and is cleared rapidly from the systemic cir-
culation [18–20]. Therefore, it is necessary to find more active anti-
oxidants based on RES to overcome these disadvantages. We removed
the 4′-hydroxyl group from RES to increase its lipophilicity. Also, we
altered the position of the hydroxyl group to make the compound more
symmetrical and stable. Based on these principles, two RES analogs,
pinosylvin (3,5-dihydroxy-trans-stilbene, PIN, Fig. 1B) and 4,4′-dihy-
droxy-stilbene (DHS, Fig. 1C), have been synthesized in our laboratory
[21].
Busulfan (BUS), an anticancer drug, can cause male reproductive
system damage because of its toxic side effects [22]. Different con-
centrations of BUS can cause different degrees of testicular tissue da-
mage. For instance, high concentrations of BUS can cause aspermia, and
lower concentrations can cause oligoasthenospermia [23–24]. Our pilot
study demonstrated that PIN and DHS improved sperm quality of
mouse in a concentration-dependent manner (Supplementary Fig. S1;
note: Supplemental data can be found at: https://figshare.com/articles/
2.2. PIN and DHS treatments improved epididymal sperm quality
In this report, we elected to use RES, PIN and DHS at a concentra-
tion of 100 mg/kg b.w. because results of pilot experiments as noted in
Fig. S1 had indicated that this is the most effective dose to improve
oligospermia. Herein, it was shown that at the end of the experiment,
there were no significant differences in body weight among the five
groups of mice (Fig. 2A). However, BUS treatment considerably re-
duced testis weight compared with the normal control group. There was
a significant (P = 0.011) increase in testicular weight in the PIN
treatment group after BUS administration, while there were no sig-
nificant increases in testicular weights after the RES and DHS treat-
ments (Fig. 2B). The epididymal sperm concentration and motility in
the BUS group were lower than in the normal group, demonstrating that
our mouse model for oligoasthenospermia had been established suc-
cessfully. Treatment with RES, PIN, and DHS significantly improved
epididymal sperm concentration (P = 0.036, 0.001, 0.001) and motility
(P = 0.001, 0.001, 0.007). PIN and DHS treatments improved sperm
concentrations better than RES, while the DHS treatment group had a
lower improvement in sperm motility compared with the RES and PIN
groups (Fig. 2C, D).
figure/Supplemental_Data_BIOORG-D-20–00181-R1/12930947).
To
further understand the roles and mechanisms of PIN and DHS in
treating oligoasthenospermia, we investigated the mechanistic basis for
improving epididymal sperm parameters by treatment with high con-
centrations of PIN (100 mg/kg b.w.) and DHS (100 mg/kg b.w.) in a
mouse model of oligoasthenospermia induced by treatment of adult
mice with BUS. We found that PIN and DHS ameliorated BUS-induced
oligoasthenospermia by attenuating oxidative stress via the Nrf2-ARE
pathway.
2. Results
2.1. Synthesis of PIN and DHS
The synthesis of PIN and DHS is noted in Scheme 1. To a 15 mL
pressure tube were added diarylacetylene 1 (0.20 mmol), [Ir(cod)Cl]2
(5 μmol, 3.6 mg), DPPE (0.04 mmol, 15.9 mg) under N2, and then EtOH
(4 mmol, 232 μL) and THF (1.5 mL) were also added. The resulting
solution was stirred at 120 °C for 22 h. Then, the solution was cooled to
room temperature, and diluted with ethyl acetate (10 mL). The com-
bined organic phases were washed with saturated saline, and the aqu-
eous phase was extracted with ethyl acetate and dried over anhydrous
2.3. PIN and DHS treatments reversed testicular damage
Histological analysis by HE staining of testis sections from the
normal group indicated normal spermatogenesis, including regular ar-
rangements of primary/secondary spermatocytes, spermatids, and
spermatogenic epithelial cells across the epithelium of seminiferous
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BioorganicChemistry104(2020)104295
Scheme 1. Synthesis of resveratrol analogs, pinosylvin and 4,4′-dihydroxystilbene. Reagents and conditions: (i) EtOH (4.0 eq), [Ir(cod)Cl]2 (2.5 mol %), DPPE
(0.2 eq), THF, N2, 120 °C, 22 h; (ii) BBr3, DCM (dry), N2, −20 °C-rt, 4–5 h.
tubules (Fig. 3A, A′), whereas BUS induced significant tissue damage
including disorganization and loss of spermatogenic cells (Fig. 3B, B′).
Administration of RES, PIN, and DHS all reversed the BUS-induced
histopathology damage, but PIN and DHS treatments were more ef-
fective in this than RES (Fig. 3C, C′, D, D′, E, E′).
2.5. Differentially expressed mRNAs between the BUS, PIN, and DHS
treatment groups
To investigate the mechanism of action of PIN and DHS on oli-
goasthenospermia, expression profiling studies were performed on the
mRNA from two independent samples in the normal, BUS, PIN, and
DHS groups for RNA-Seq and transcriptome profiling. GO classification
(Fig. 5A, B) analyses demonstrated the expression patterns of mRNAs.
As PIN and DHS are both polyphenolic compounds acting as anti-
oxidants, we also studied antioxidant-related genes. GO analysis in-
dicated that the differentially expressed mRNAs were enriched in terms
of peroxidase activity, oxidoreductase activity (acting on peroxide as an
acceptor), and antioxidant activity (Fig. 5A, B; yellow background).
Table S1 shows the differentially expressed mRNAs of antioxidant-re-
lated genes. Most of these were enriched in the glutathione redox
system including Gpx3, GSTT2, and GSTO2. RNA-seq analysis showed
that, compared with the BUS group, the expression of Gpx3 was sig-
nificantly upregulated in the PIN group with a > 1.5-fold change
2.4. Effects of PIN and DHS treatments on serum levels of T, FSH, and LH
Serum hormone assays showed that the levels of T and FSH were
significantly (P = 0.001, 0.001) reduced by BUS administration com-
pared with the normal group, the levels of LH were also reduced by BUS
administration (P = 0.049). The results showed that PIN treatments
markedly increased the levels of T, FSH, and LH (P = 0.001, 0.001,
0.001) after BUS administration, which was similar to RES. The serum T
levels significantly (P = 0.0025) increased with DHS treatment but the
levels of FSH and LH levels decreased (Fig. 4A–C).
Fig. 2. Effects on body, testicular organ index and
sperm parameters. (A): Body weight, (B): Testicular
weight, (C): Sperm concentration, (D): Sperm moti-
lity. **P < 0.01 vs. Normal group, ***P < 0.001
vs. Normal group, #P
< 0.05 vs. BUS group,
##P < 0.01 vs. BUS group, ###P < 0.001 vs. BUS
group. Each column represents the mean
n = 6.
SEM,
3

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Fig. 3. Histological examination of mice testes. (A–E): Scale bar = 50 μm, (A′–E′): Scale bar = 25 μm.
(P < 0.05), whereas it was downregulated in the DHS group with
a > 1.5-fold change (P < 0.05). Compared with the BUS treatment
group, the expressions of both GSTT2 and GSTO2 were significantly
upregulated in the DHS group with a > 1.5-fold change (P < 0.05).
normal group, and were significantly increased after DHS treatment
(Fig. 7E, F, H, I).
2.8. Effects of PIN and DHS treatments on the levels of ROS and MDA
The levels of ROS and MDA in testicular tissue were measured using
corresponding specific ELISA kits. The concentrations of ROS and MDA
in the BUS group were markedly (P = 0.001, 0.026) higher than in the
normal group, and were significantly decreased by PIN (P = 0.005,
0.001) and DHS (P = 0.009, 0.001) treatments (Fig. 7J, K). These
findings thus support that notion that PIN and DHS treatments ap-
peared to improve experimental oligoasthenospermia by attenuating
oxidative stress via the Nrf2-ARE pathway.
2.6. Validation of the expression of genes in the glutathione redox system by
RT-qPCR
The relative Gpx3 gene expression in the PIN group was higher than
in the BUS group, while it was lower in the DHS group. The relative
gene expression levels of GSTT2 and GSTO2 in the DHS group were
higher than in the BUS group (Fig. 6). These results of the qRT-PCR
analysis were consistent with the RNA-seq data, indicating that the
expressions of Gpx3, GSTT2, and GSTO2 might be related to the anti-
oxidative actions of PIN and DHS.
2.9. Interactions of PIN and DHS with Nrf2
Molecular docking studies demonstrated that PIN and DHS could
bind effectively with Nrf2 (Fig. 8A, D). The docking grid score for PIN
combined with Nrf2 was −22.248619, and for DHS with Nrf2 it was
−34.274078. Furthermore, we used MOE software to analyze the in-
teractions of PIN and DHS with the Nrf2 protein. This showed that PIN
might have hydrogen bonds that interacted with Nrf2 specifically at
Cys513 and Tyr520 (Fig. 8B, C), and that DHS might also have a hy-
drogen bond to interact with Nrf2 specifically at Val465 (Fig. 8E, F).
Based on these docking results, PIN and DHS are highly likely to be
potent activators of Nrf2. Further work is needed to test this hypothesis.
2.7. Validation of the Nrf2-ARE pathway
RT-qPCR was also used to detect the expression of Nfe2l2, an up-
stream gene in the glutathione redox system. The relative Nfe2l2 ex-
pression in the BUS group was obviously less than in the normal group,
but was induced in both the PIN and DHS groups, even higher than the
control group (Fig. 7A). The protein levels of Nrf2, Gpx3, GSTT2, and
GSTO2 were quantified by western blotting. This showed that the Nrf2
protein level was lower in the BUS group than in the normal group, and
higher in both the PIN and DHS groups than in the BUS group and also
the control group (Fig. 7B, C). The Gpx3 protein level in the BUS group
was significantly lower than in the normal group, and was significantly
increased after PIN treatment (Fig. 7D, G). The GSTT2 and GSTO2
protein levels in the BUS group were obviously lower than in the
3. Discussion
In Asia, and especially in China, Traditional Chinese Medicine
Fig. 4. Effect of PIN and DHS on hormone levels. (A): T concentration, (B): FSH concentration, (C): LH concentration. *P < 0.05 vs. Normal group, ***P < 0.001
vs. Normal group, #P < 0.05 vs. BUS group, ##P < 0.01 vs. BUS group, ###P < 0.001 vs. BUS group. Each column represents the mean
SEM, n = 6.
4

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Fig. 5. The data analysis of RNA sequencing. (A): GO classification of differentially expressed mRNAs in the between the BUS and PIN treatment mouse testis. The x-
axis shows counts of genes enriched in GO and the y-axis shows the GO classification. (B): GO classification of differentially expressed mRNAs in the between the BUS
and DHS treatment mouse testis. The x-axis shows counts of genes enriched in GO and the y-axis shows the GO classification.
(TCM) has been used to treat human oligoasthenospermia for decades
with limited success [25]. The efficacy of RES for treating men with
oligoasthenospermia is also limited because of its chemical instability
and low bioavailability. The two RES analogs we have synthesized PIN
and DHS are both naturally occurring bisphenol-based compounds. PIN
is found in the wood and leaves of various Pinus species [26]. It pos-
sesses various biological activities and has been suggested as a potential
therapeutic agent against cancers and inflammatory diseases [27–28].
DHS is found in the bark of Yucca periculosa [29] and exhibits promising
antitumor, antioxidant, and anti-inflammatory activities [30–31]. To
our knowledge, this is the first study to investigate the effects of PIN
and DHS on oligoasthenospermia in a mouse model. Intratesticular
injections of BUS in male ICR mice resulted in tissue damage and in-
duced oligoasthenospermia in epididymal sperm suspensions. Treat-
ments with PIN and DHS could attenuate this damage to the testes and
ameliorate the impaired epididymal sperm parameters. We conclude
that PIN and DHS improved this oligoasthenospermia by attenuating
oxidative stress via the Nrf2-ARE pathway.
Fig. 6. qRT-PCR validated the expression of antioxidative-related glutathione
redox system. *p 0.05 and **p 0.01. Each column represents the
mean SEM, n = 3.
<
<
It is known that RES and its analogs can cross biological membranes
[32]. We hypothesize that reduction of the hydroxyl group at the 4′-
5

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BioorganicChemistry104(2020)104295
Fig. 7. The effects of of PIN and DHS on the expression of Nrf2-AREs pathway. (A): The relative Nfe2l2 expression in testes. (B and C): The expression of Nrf2 was
analyzed by western blot. Relative integrated density value analysis of Nrf2. (D and G):The expression of Gpx3 was analyzed by western blot. Relative integrated
density value analysis of Gpx3. (E and H):The expression of GSTT2 was analyzed by western blot. Relative integrated density value analysis of GSTT2. (F and I): The
expression of GSTO2 was analyzed by western blot. Relative integrated density value analysis of GSTO2. (J): ROS concentration, (K): MDA concentration. *P < 0.05
vs. Normal group, **P < 0.01 vs. Normal group, ##P < 0.01 vs. BUS group. Each column represents the mean
SEM, n = 6.
position of PIN might make it more lipophilic than RES, which would
help PIN to enter damaged cells more easily and improve its efficacy
[33]. We found that PIN treatment produced better improvements in
mouse epididymal sperm concentrations and motility compared with
RES treatment (Fig. 2C, D), and that PIN treatment also improves the
recovery from BUS-induced histopathological damage to the testes
more than RES treatment (Fig. 3). These results confirmed that the li-
pophilicity of PIN might be more helpful than RES in improving oli-
goasthenospermia. We also proposed that both PIN and DHS are more
stable and have better lipophilicity. The log P of RES, PIN and DHS is
6

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BioorganicChemistry104(2020)104295
Fig. 8. The interaction of PIN and DHS with Nrf2 protein. (A): Molecular docking of PIN with Nrf2 protein. Grid Score is −22.248619. (B): Diagrams of the
interactions between PIN with Nrf2 protein. (C): Interactive visualisation of PIN binding to Cys513 and Tyr520 with Nrf2 protein through hydrogen bonding. (D):
Molecular docking of DHS with Nrf2 protein. Grid Score is −34.274078. (E): Diagrams of the interactions between DHS with Nrf2 protein. (F): Interactive visua-
lisation of DHS binding to Val465 with Nrf2 protein through hydrogen bonding.
3.06, 3.45, and 3.45, respectively. We have also examined these analogs
for the drug-likeness on the Lipinski rule of 5 at https://chemicalize.
com/app/calculation, indicating that both PIN and DHS fulfill the Li-
pinski Rule of 5. We speculate that the symmetrical structure of the
hydroxyl group at the 4,4′ position of DHS makes it more stable with
more potent antioxidant activity, which would help DHS improve oli-
goasthenospermia. It has been shown that DHS exhibited more anti-
oxidant activity and cytotoxicity than RES [34]. Here, DHS treatment
had a better improvement in epididymal sperm concentration com-
pared with RES treatment, while the lower improvement in sperm
motility might be related to DHS being more toxic than RES (Fig. 2C,
D). These findings suggest that the symmetrical structure of DHS might
help improve oligoasthenospermia and increase sperm output.
Nrf2 activators. Our molecular docking studies have shown that PIN has
a hydrogen bond to the Nrf2 protein specifically at Cys513 and Tyr520,
and that DHS has a hydrogen bond specifically at Val465 (Fig. 8).
Different binding sites with Nrf2 might cause the differences in re-
sponse between the PIN and DHS treatments, including epididymal
sperm concentration and motility, hormone levels, and AREs. These
findings indicate that PIN might improve oligoasthenospermia by at-
tenuating oxidative stress via the Nrf2-ARE-dependent antioxidant
Gpx3, and that DHS might act similarly by attenuating oxidative stress
via the Nrf2-ARE-dependent antioxidants GSTT2 and GSTO2 (Fig. 9).
The development of novel potential candidates for the treatment of
oligoasthenospermia has very important practical significance. Even
though the protective effects of RES on testicular damage and sperm
function are known [14–17], its clinical application is limited due to the
relative unstability of RES, and its poor bioavailabaility, as well as its
rapid metabolic clearance from the systemic circulation [18–20]. The
two new analogs of RES, namely PIN and DHS, were obtained through
structural modification of resveratrol. Most importantly, PIN and DHS
were found to improve oligoasthenospermia in a mouse model by at-
tenuating oxidative stress via the Nrf2-ARE pathway. PIN and DHS may
be promising candidates for clinical treatment of oligoasthenospermia.
Oxidative stress is known to be involved in the pathophysiology of
oligoasthenospermia [35]. Oxidative stress is caused by an excessive
production of ROS. In the function of the male reproductive system,
seminal sperm parameters, including concentration and motility are
particularly vulnerable to ROS [36]. ROS cause sperm damage, and
ultimately damage nuclear DNA; factors associated with human male
infertility [35]. It has been suggested that Nrf2 plays a critical role in
the defense against oxidative stress by activating the expressions of
genes encoding antioxidative enzymes downstream of AREs to reduce
the production of ROS [10]. BUS testicular injection induces oli-
goasthenospermia with tissue damage and oxidative stress [22]. Our
results indicated that BUS caused a considerable reduction in Nrf2
protein expression in the mouse testis, and both PIN and DHS treat-
ments were capable of reversing this (Fig. 7A, C, D). RNA-seq, RT-qPCR,
and western blot analysis showed that PIN treatment enhanced the
expression of the gene encoding Gpx3, and DHS treatment enhanced
the expressions of the genes for GSTT2 and GSTO2 (Figs. 6, 7). We have
also shown that BUS increased the ROS and MDA levels in mouse testes,
and that either PIN or DHS treatment was able to reverse these in-
duction (Fig. 8). It has been shown that RES activates the transcription
factor Nrf2 [37], and, based on this, we identified PIN and DHS as novel
4. Experimental procedures
4.1. Chemicals
PIN and DHS were synthesized as described [21]. Their chemical
structures were identified fully using 1H NMR and 13C NMR spectral
analyses (Fig. S2 A-D). The purity (> 99%) of PIN and DHS was
checked using gas chromatography-mass spectroscopy (Supplementary
Fig. S3; Table S2, S3). RES, BUS, and Proteoprep® total extraction
sample kits were purchased from Sigma-Aldrich (Darmstadt, Germany).
Reverse transcription (RT) kits, BCA protein assay kits, and TB Green
Premix Ex Taq II (Tli RNaseH Plus) were purchased from Takara
7

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BioorganicChemistry104(2020)104295
Fig. 9. Schematic representation of proposed mechanism of PIN and DHS in improving oligoasthenospermia. Our findings indicated PIN may improve oligoasthe-
nospermia by attenuating oxidative stress via the Nrf2-ARE-dependent antioxidants Gpx3 and DHS may improve oligoasthenospermia by attenuating oxidative stress
via the Nrf2-ARE-dependent antioxidants GSTT2 and GSTO2.
Biotechnology (Dalian, China). Testosterone (T), follicle stimulating
hormone (FSH), luteinizing hormone (LH), ROS and malondialdehyde
(MDA) enzyme-linked immunosorbent assay (ELISA) kits were pur-
chased from Elabscience Biotechnology Inc. (Houston, USA). TRIzol
reagent was purchased from Life Science Products (ThermoFisher Sci-
entific, Waltham, MA, USA). Primary rabbit anti-mouse antibodies
against Nrf2, glutathione peroxidase 3 (Gpx3), glutathione S-trans-
ferase theta 2 (GSTT2) and glutathione S-transferase omega 2 (GSTO2),
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were pur-
chased from Proteintech (Rosemont, IL, USA). Horseradish peroxidase-
conjugated goat anti-rabbit secondary antibodies were purchased from
Cell Signaling Technology (Danvers, MA, USA).
receiving 100 mg/kg via intragastric administration daily as in group 2;
and (5) a DHS group receiving 100 mg/kg via intragastric administra-
tion daily as in group 2. After the final administration, all mice were
weighed, then euthanized under anesthesia; testes and epididymides
were then removed and weighed separately. Six testes of each group
were fixed in 4% paraformaldehyde solution, and the rest were snap-
frozen in liquid nitrogen and stored at −80 °C; the following experi-
ments were performed immediately.
4.4. Analysis of epididymal sperm concentration and motility
The whole epididymides were removed immediately after eu-
thanasia, then the cauda epididymidis cut into small pieces and in-
cubated in a tube containing 2 mL phosphate buffered saline at 37 °C for
15 min and gently shaken to allow the spermatozoa to swim out.
Aliquots of 200 μL of the suspension were diluted with 2 mL Tyrode’s
solution. Sperm concentration and motility parameters were measured
using a computer-assisted sperm analysis system: the Hamilton Thorne
CEROS Ⅱ for humans and animals.
4.2. Animals
Male Institute of Cancer Research (ICR) strain mice (n = 50,
8 weeks old, 40
2 g), were purchased from the Experimental Animal
Center of Nantong University (Nantong, P. R. China). Mice were housed
under standard laboratory environmental conditions (room tempera-
ture 22–24 °C on a 12/12-h light/dark cycle) with free access to food
and water. Our project was submitted to the ethics committee super-
vising animal experimentation in Nantong University, and all proce-
dures were approved according to the Jiangsu Province Animal Care
Ethics Committee with Approval ID: SYXK(SU)2007-0021.
4.5. Histology
Testes were fixed overnight in 4% paraformaldehyde and soaked
consecutively in 10% and 30% sucrose overnight. Frozen sections of 8-
μm thickness were performed using a cryotome. The sections were
stained with hematoxylin and eosin (HE) using standard procedures
and observed by light microscopy.
4.3. Experimental groups, treatment, and sample preparation
Fifty mice were divided randomly into five groups of 10 each as
follows: (1) a normal control group fed with normal diet and water; (2)
a BUS group with oligoasthenospermia induced by the intratesticular
injection of BUS at 6 mg/kg (3 mg/kg/side, all drugs were dissolved in
5% DMSO aqueous solution); (3) a RES group receiving 100 mg/kg
via intragastric administration daily for 2 consecutive weeks, 2 weeks
after testes had been injected with BUS as above; (4) a PIN group
4.6. Serum hormone levels
Collection of blood and preparation of serum were performed as
described [38]. Briefly, blood was collected by cardiac puncture with a
heparinized syringe and then serum collected immediately by cen-
trifugation at 3000g, 4 °C for 10 min. Samples were stored at −80 °C for
8

C.-n. Wang, et al.
BioorganicChemistry104(2020)104295
quantitative real-time PCR data analysis used the 2−△△CT method.
4.9. Western blotting
further analysis. Serum levels of T, FSH, and LH were measured by
ELISA kits according to the manufacturer’s instructions. The sensitiv-
ities of the mouse T, FSH, and LH assays were 0.10 ng/mL, 0.94 ng/mL,
and 0.28 ng/mL, respectively. The intra- and inter-assay coefficient of
variation were < 10% for all assays.
The protein levels of Nrf2, Gpx3, GSTT2, and GSTO2 were measured
by western blot analysis to validate the qPCR data. Total testicular
protein was extracted using Proteoprep® total extraction sample kits
(Merk-Shanghai, P. R. China). BCA protein assay kits were used to es-
timate protein concentrations. Proteins were separated by sodium do-
decyl sulphate-polyacrylamide gel electrophoresis, transferred to poly-
vinylidene difluoride (PVDF) membranes, then blocked with 5%
skimmed milk for 1 h at room temperature. PVDF membranes were
incubated with primary antibodies against Nrf2, Gpx3, GSTT2, GSTO2,
or GAPDH (served as a protein loading control) at 4 °C overnight.
Thereafter, they were incubated with the corresponding secondary
antibodies (Goat anti-rabbit IgG H&L (Alexa Fluor 790) and Goat anti-
mouse IgG H&L (Alexa Fluor 791)) for 2 h at room temperature.
Densitometry analysis was performed using an Odyssey infrared ima-
ging system. Image J software (https://imagej.nih.gov/ij/download.
html) was used for the analysis of relative signal intensity.
4.7. RNA sequencing and data analysis
Total RNA was extracted from testes using Trizol reagent according
to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham,
MA, USA). Samples of ~60 mg were ground into powder with liquid
nitrogen and transferred into 2-mL tubes containing 1.5 mL Trizol. The
mix was centrifuged at 12,000g for 5 min at 4 °C. The supernatant was
transferred to a new 2.0-mL tube to which was added 0.3 mL of
chloroform/isoamyl alcohol (24:1) per 1.5 mL of Trizol reagent. After
the mix was centrifuged at 12,000g for 10 min at 4 °C, the aqueous
supernatant was transferred to a new 1.5-mL tube to which was added
an equal volume of isopropyl alcohol. The mix was centrifuged at
12,000g for 20 min at 4 °C, and the supernatant was removed. After
washing with 1 mL 75% ethanol, the RNA pellet was air-dried in a
biosafety cabinet and then dissolved by adding 25–100 μL of diethyl
pyrocarbonate treated water. Subsequently, total RNA was qualified
and quantified using a NanoDrop spectrophotometer (Thermo Fisher
Scientific) and 2100 Bioanalyzer (Agilent Technologies, San Diego, CA,
USA). The products were enriched by PCR to create the final cDNA
library. The libraries were assessed for quality and quantity using two
methods: first by checking the distribution of fragment sizes using an
Agilent 2100 bioanalyzer, and second by quantifying the library using
real-time qPCR (see below) and TaqMan probes. The libraries were
sequenced pair end-on using the BGISEQ-500/MGISEQ-2000 System
(BGI-Shenzhen, P. R. China). Fastp software (https://github.com/
OpenGene/fastp) was used to perform all-round quality control and
to clean up raw data, including linker processing, global trimming by
directly cutting off low-quality bases at the beginnings and ends of
sequences, sliding-window quality trimming, filtering short sequences,
base correcting for double-end sequences, and quality filtering. STAR
software was used to perform sequence alignment on the clean data,
with reference Mus musculus genome GRCm38, and each read was
aligned to the genome to obtain the mapping rate of each sample.
FeatureCounts software was used to count each gene in all samples.
Finally, DEseq2 software was used to analyze the differentially ex-
pressed genes of different samples, and the differential genes were
analyzed using Gene Ontology (GO) annotations.
4.10. Testicular levels of ROS and MDA
Testicular levels of ROS and MDA were measured using ELISA kits
according to the manufacturer’s instructions. The sensitivities of the
mouse ROS and MDA assays were 0.039 U/mL and 18.75 ng/mL, re-
spectively. The intraassay and interassay coefficient of variation
were < 10% for both assays.
4.11. Molecular docking
Molecular docking studies of PIN and DHS were carried out using
DOCK v. 6.7 (http://dock.compbio.ucsf.edu/DOCK_6/index.htm). The
grid score is defined as the total docking score of the DOCK 6 scheme.
Negative values indicate molecular combination, and positive values
indicate no binding. Molecular Operating Environment (MOE) software
(https://www.chemcomp.com/Products.htm) was used to determine
interactions between ligand and target proteins. The crystal structure of
Nrf2 (Protein Data Bank [PDB] entry 3WN7) in complex with Kelch-like
ECH-associated protein 1 was derived from the PDB (http://www.rcsb.
org/structure/3WN7) [39].
4.12. Statistical analysis
4.8. Quantitative real-time PCR
Statistical analyses were performed using GraphPad Prism 7.0
(GraphPad Software, Inc., San Diego, CA, USA). Student’s t-tests were
used to analyze differences between two experimental groups, and one-
way analysis of variance were used to analyze the differences between
multiple experimental groups Data are expressed as the mean
standard error of mean, of at least three independent experiments, and
P < 0.05 was considered to be statistically significant.
The expression of antioxidative-related genes involving the Nrf2-
ARE signal pathway were further evaluated by quantitative real-time
PCR. Total RNA was extracted from frozen mouse testes as above. The
RNA was used to synthesize cDNA using reverse transcription kits.
cDNA was estimated using NanoDrop 2000/2000C spectrophotometer.
Gene-specific primers were synthesized by Genewiz (USA) for the cor-
responding mRNA sequences: Nfe2l2 (Nrf2) (Forward) 5′-TCCTATGC
GTGAATCCCAAT-3′, (Reverse) 5′-GCGGCTTGAATGTTTGTCTT-3′;
Gpx3 (F) 5′-CCGGGGACAAGAGAAGTCTA- A-3′, (R) 5′-AGGCAAGCAG
ACCTGAGTTT-3′, GSTT2 (F) 5′-TGGAGCTCTACCTG- GACCTG-3′, (R)
5′-GCTGTGCTTTCGGTCAACAC-3′; GSTO2 (F) 5′-TGTACGGAT- CTGA
AGGTCGC-3′, (R) 5′-GGGCCAGACGAGGTAATCAA-3′; and β-actin (F)
5′-CTGTCCCTGTATGCCTCTG-3′, (R) 5′-TTGATGTCACGCACGATT-3′.
The β-actin gene served as an internal control. PCR reactions were run
in triplicate using TB Green Premix Ex Taq II (Tli RNaseH Plus) on a
Lightcycler 480 system (Roche, Mannheim, Germany). The reaction
conditions were as follows: 95 °C for 30 s, followed by 40 cycles of
denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s. Melting curves
were generated with a three-segment cycle of 95 °C for 5 s, 60 °C for
60 s, and 95 °C for 1 s in the continuous acquisition mode. The
Author contributions
F. S. and C.Y. C. designed this study. C. W. wrote the manuscript. C.
W. and S. G. performed the experiments. M. S. and J. Y. helped col-
lecting and analyzing the data. All the authors read and approved this
manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
9

C.-n. Wang, et al.
BioorganicChemistry104(2020)104295
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