Effect of HongJing I in Treating Erectile Function and Regulating RhoA Pathway in a Rat Model of Bilateral Cavernous Nerve Injury

Abstract

HongJing I (HJI), a traditional Chinese herbal formula, has been confirmed to be effective for the clinical treatment of erectile dysfunction (ED). However, the mechanism of action of HJI remains unclear. Here, we aimed to investigate the effect and underlying mechanisms of HJI against ED in a rat model of bilateral cavernous nerve injury (BCNI). Rats were divided into five groups: normal control (NC), BCNI-induced ED model (M), M + low-dose HJI (HL), M + medium-dose HJI (HM), and M + high-dose HJI (HH). All groups were treated with normal saline or the relevant drug for 28 consecutive days after inducing BCNI-ED. At the end of the treatment period, the intracavernous pressure (ICP) was recorded, and histological examination was conducted using Masson’s trichrome staining. Immunofluorescence staining and western blotting were applied to detect the changes in fibrosis protein and Ras homolog A (RhoA), Rho-associated protein kinase 1 (ROCK1), and ROCK2 expression. We found that HJI effectively improved the ICP in the treatment groups. In addition, RhoA, ROCK1, and ROCK2 expression levels were increased upon BCNI-ED induction, and HJI successfully inhibited cavernosum fibrosis and the activation of RhoA/ROCK2 signaling. Overall, these results suggest that the effects of HJI in attenuating ED may be caused, at least in part, by the suppression of RhoA/ROCK2 signaling and alleviation of fibrosis. However, the precise mechanism surrounding this requires further investigation in future studies.

Full text

Research Article
Effect of HongJing I in Treating Erectile Function and Regulating
RhoA Pathway in a Rat Model of Bilateral Cavernous Nerve Injury
Miao-yong Ye
,
1
Fan Zhao
,
2
Ke Ma,
1
Kang Zhou,
1
Wen-Jie Huang,
3
Yin-feng Ma,
3
Jian-feng Zhao
,
3
Hui-ying Fu
,
4
Zeng-bao Xu,
5
and Bo-dong Lv
3
,
5
,
6
1
e Second Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou 310053, China
2
Department of Urology and Andrology, Affiliated Hospital of Nantong University, Nantong 226001, China
3
Department of Urology and Andrology, e Second Affiliated Hospital of Zhejiang Chinese Medical University,
Hangzhou 310053, China
4
Research Institute of Urology and Andrology, Zhejiang Chinese Medical University, Hangzhou, 310053, China
5
Department of Urology, Huzhou Hospital of Traditional Chinese Medicine, Huzhou 313000, China
6
Andrology Laboratory on Integration of Chinese and Western Medicine,
Zhejiang Provincial Key Laboratory of Traditional Chinese Medicine, Hangzhou 310053, China
Correspondence should be addressed to Bo-dong Lv; bodonglv0571@163.com
Received 22 May 2019; Revised 5 August 2019; Accepted 26 August 2019; Published 16 September 2019
Academic Editor: Raffaele Pezzani
Copyright ©2019 Miao-yong Ye et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
HongJing I (HJI), a traditional Chinese herbal formula, has been confirmed to be effective for the clinical treatment of erectile
dysfunction (ED). However, the mechanism of action of HJI remains unclear. Here, we aimed to investigate the effect and
underlying mechanisms of HJI against ED in a rat model of bilateral cavernous nerve injury (BCNI). Rats were divided into five
groups: normal control (NC), BCNI-induced ED model (M), M + low-dose HJI (HL), M + medium-dose HJI (HM), and M + high-
dose HJI (HH). All groups were treated with normal saline or the relevant drug for 28 consecutive days after inducing BCNI-ED.
At the end of the treatment period, the intracavernous pressure (ICP) was recorded, and histological examination was conducted
using Masson’s trichrome staining. Immunofluorescence staining and western blotting were applied to detect the changes in
fibrosis protein and Ras homolog A (RhoA), Rho-associated protein kinase 1 (ROCK1), and ROCK2 expression. We found that
HJI effectively improved the ICP in the treatment groups. In addition, RhoA, ROCK1, and ROCK2 expression levels were
increased upon BCNI-ED induction, and HJI successfully inhibited cavernosum fibrosis and the activation of RhoA/ROCK2
signaling. Overall, these results suggest that the effects of HJI in attenuating ED may be caused, at least in part, by the suppression
of RhoA/ROCK2 signaling and alleviation of fibrosis. However, the precise mechanism surrounding this requires further in-
vestigation in future studies.
1. Introduction
Erectile dysfunction (ED) remains a common consequence
of radical pelvic surgeries such as radical prostatectomy,
despite the development of effective surgical techniques [1].
Cavernous nerve (CN) injury is a common result, which
leads to neuropraxia and the damage and dysfunction of the
corpora cavernosa [2]. Many studies have focused on
postsurgery penile rehabilitation through the application of
stem cell therapy, gene therapy, and even small-molecule
treatment [1, 3]. However, most of these remain in the
experimental stage and require further study. Currently, no
causal approaches exist to restore erectile function after
radical pelvic surgery [4].
It is well known that corporal fibrosis acts as a major
factor in the pathophysiology of ED caused by CN damage
[5]. us, the antipenile fibrosis properties of HJI have also
been studied. Chitaley et al. first discovered that Ras homolog
A (RhoA)/Rho-associated protein kinase (ROCK) signaling
plays an important role in cavernosal vasoconstriction to
Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2019, Article ID 1083737, 11 pages
https://doi.org/10.1155/2019/1083737

inhibit penile tumescence independent of the nitric oxide
(NO) pathway [6]. In recent years, RhoA/ROCK in post-
prostatectomy ED has become a major focus of investigation
[5, 7–11], which can act on smooth muscle to affect erectile
function and attenuate cavernous fibrosis.
In China, traditional herbs or formulas to treat ED are
widespread and recognized for their effectiveness [12, 13].
e basic pathogenesis of ED after radical prostatectomy, in
terms of traditional Chinese medicine, is considered a Qi
deficiency and dysregulated blood stasis [14]. e HJI recipe,
which consists of nine commonly known herbs, can effec-
tively invigorate the Qi and activate blood circulation. We
previously found that treatment with HJI combined with
tadalafil was more effective for treating moderate ED than
treatment with phosphodiesterase 5 inhibitors (PDE5i)
alone [14]. Furthermore, we previously found that HJI ef-
fectively alleviated corpus cavernous smooth muscle cell
fibrosis and phenotypic changes in rats kept in a hypoxic
environment, which is important [15, 16]. However, the
pharmacological activity through which HJI improves
erectile function is currently unknown. Clinical treatment
with HJI has been found to significantly improve erectile
function and attenuate cavernosal fibrosis and phenotypic
modulation. In this study, we investigated whether HJI could
protect the corpus cavernosum from CN injury in a rat
model. Expanding our understanding of the drug mecha-
nism will provide a useful strategy for the clinic. Here, we
explored the changes in RhoA pathway proteins expression
and the degree of fibrosis in the penis tissue of rats with
bilateral CN crush injury (BCNI) and the regulatory effects
of HJI on ED.
2. Materials and Methods
2.1. Animals and Grouping. A total of fifty adult male
Sprague-Dawley (SD) rats (12-week-old, 350–400 g) with
normal erectile function were included in this study and
were purchased from the Laboratory Animal Center of
Zhejiang Chinese Medical University, China. All animal
studies were performed according to the Guide for the Care
and Use of Laboratory Animals of the National Institutes of
Health. e protocol was approved by the Animal Experi-
mental Ethics Committee of Zhejiang Chinese Medical
University. e rats were weighed, randomly divided into
five groups, and labeled with picric acid. e rats were di-
vided into the following groups: normal control
(NC) +saline; BCNI model (M) + saline; M + low-dose of
HJI (HL) (2.835 g/kg/day); M + medium-dose HJI (HM)
(5.67 g/kg/day); and M + high-dose HJI (HH) (11.34 g/kg/
day). All rats were housed in cages at a temperature of
22 ±1°C with a 12-h light/dark cycle.
2.2. Surgical Procedure. Rats in the M and HJI treatment
groups were subjected to bilateral cavernous nerve crush
injury under aseptic conditions. Rats were anesthetized by
intraperitoneal injection with 3% sodium pentobarbital
solution (50 mg/kg). e procedure was performed as pre-
viously described [17]. Before the crush injury, the CN was
electrically stimulated while the penis was erect to make sure
the correct nerve was isolated. e injury was induced by
applying hemostatic forceps to the nerve at ∼3–5 mm distal
to the major pelvic ganglion (Figure 1(b)). Forceps were held
closed twice for 60 s each to induce a moderate nerve crush
injury. ere was a washout period of 3 d after the end of the
experimental period.
2.3. Preparation and Application of Drugs. HJI was com-
posed of Rhodiola rosea (Hong-Jing-Tian, batch no.
18061253) 15 g, Radix astragali (Zhi-Huang-Qi, batch no.
18081763) 20 g, Codonopsis pilosula (Chao-Dang-shen,
batch no. 18091823) 15 g, Angelica sinensis (Quan-Dang-
Gui, batch no. 18111803) 12 g, Salvia miltiorrhiza (Dan-
shen, batch no. 18100083) 15 g, Raidix Paeoniae Alba (Bai-
Shao, batch no. 18120903) 15 g, Lycium chinense Miller
(Gou-Qi, batch no. 18120903) 15 g, Epimedium brevicornu
(Yin-Yang-Huo, batch no. 18091753) 10 g, Cyathula offici-
nalis (Chuan-Niu-Xi, batch no. 18101783) 12 g, which were
listed in Table 1. All granulates were purchased from
Jiangyin Tianjiang Pharmaceutical Co., LTD. All the tra-
ditional Chinese medicine formula granules of HJI are
weighed and added to a clean 2 L beaker. e boiling pure
water is used as a solvent to dissolve the granules so that the
final concentration of the HJI formula granule solution is set
at 0.55 g/ml. In order to get the best dissolution of all the
granules, a glass rod is used for stirring for 10 minutes. Each
time, 2 liters of the red ginseng formula granule solution was
prepared, and then, it was used up and then stored in a
refrigerator at 4°C. According to the ratio of rat to human
body surface area, the dosage required for each kilogram of
rats was converted (the rat/human coefficient was 6.3 as the
concentration of HJI low-dose group). en, according to
the coefficients 12.6 and 25.2, the dosage concentrations of
medium and high dosage of HJI were calculated, which were
2.835 g/kg, 5.67 g/kg, and 11.34 g/kg, respectively.
2.4. ICP/Mean Arterial Pressure Examination. Rats were
anesthetized by intraperitoneal injection of 3% sodium
pentobarbital (50 mg/kg), and the CN below the crushed
segment was stimulated with a silver bipolar electrode.
Subsequently, a 25 G needle containing 100 U/ml heparin
solution was cannulated into the right shaft of the penis
(Figure 1(c)). Polyethylene-50 tubing was used to connect
the needle to an MP160 pressure transducer (Biopac Sys-
tems, Inc., Goleta, CA, USA). e following stimulus pa-
rameters were used: voltage, 5 V; frequency, 20 Hz; and pulse
width, 5 ms. e duration of each stimulation was 60 s with a
5–10 min rest period between stimulations, and each rat
underwent three stimulations. To record the mean arterial
pressure (MAP), the same tubing was cannulated into the
unilateral common carotid artery after making a medium
incision in the neck. Erectile function was evaluated based
on baseline ICP, maximal ICP, the ratio of maximal ICP to
MAP (ICP/MAP), and the ratio of the area under the curve
to MAP (AUC/MAP). e ICP operator tested the animals
in a blinded fashion.
2Evidence-Based Complementary and Alternative Medicine

2.5. Collection of Penile Tissue. After measuring ICP in vivo,
the penises were harvested. e middle region of the skin-
denuded penile shaft was fixed in 4% paraformaldehyde and
then embedded in paraffin for histological studies. e
remaining tissue was stored at −80°C for western blot analysis.
2.6. Masson’s Trichrome Staining and Immunofluorescence.
Paraffinized tissue was sliced to a thickness of 5 μm for im-
munofluorescence staining. For immunohistochemical stain-
ing, the slides were incubated overnight with rabbit primary
antibodies against RhoA (1 : 200), ROCK2 (1 : 250), and ROCK1
(1 : 200; Abcam, Cambridge, UK). Next, the sections were
immersed in a 1 : 500 dilution of Alexa Fluor 488-conjugated
secondary antibody (Immunoway, Plano, TX, USA) for 1 h at
room temperature. Nuclear staining was performed with 4′, 6-
diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China).
To detect the ratio of rat cavernosum smooth muscle to
collagen, cavernosum tissue sections were stained with Mas-
son’s trichrome (Solarbio, Beijing, China). Smooth muscle cells
were stained red, whereas collagen fibrils were stained blue.
Histomorphometric analyses of both Masson’s trichrome
staining and immunofluorescence (IF) staining were performed
using ImagePro Plus 6.0.
2.7. Western Blot Analysis. Western blotting was performed
as described in our previous study [18, 19]. In brief, penis
tissues were pulverized using a T10 basic homogenizer (IKA,
Staufen, Germany). e tissue fragments were then lysed in
cell lysis buffer for at least 0.5 h on ice and centrifuged at
12,000 ×g for 15 min at 4°C. Total tissue protein from rat
penises was determined using a bicinchoninic acid (BCA)
protein assay kit (Beyotime, Jiangsu, China) following the
manufacturer’s protocol. Next, 10 μg protein was separated
by ∼6–10% SDS-PAGE and transferred onto polyvinylidene
(a)
(b) (c)
Model group
Control group
Treatment groups
28 days
Crush operation None
None
None
Hongjing I recipe
Crush operation
Analysis
Analysis
Analysis
Figure 1: Experimental protocol for model establishment and treatment processes. (a) First, each group of rats was subjected to cor-
responding modeling treatment, followed by 28 days of drug treatment or saline treatment. (b) Hemostatic forceps clips the CN. (c) Blood
reflux can be seen after intubation of the corpus cavernosum.
Table 1: Herbal ingredients of Hongjing I recipe.
Chinese name Full scientific name Part used Proportion (%)
Hong-Jing-Tian (紅景天)Rhodiola rosea Dried root 4.8
Zhi-Huang-Qi (炙黃芪)Radix astragali Dried root 19.1
Chao-Dang-shen (炒黨參)Codonopsis pilosula Dried root 14.3
Quan-Dang-Gui (全當歸)Angelica sinensis Dried root 15.3
Dan-shen (丹參)Salvia miltiorrhiza Dried root 9.6
Bai-Shao (白芍)Raidix paeoniae Alba Dried root 4.8
Gou-Qi (枸杞)Lycium chinense Miller Dried fruit 19.1
Yin-Yang-Huo (淫羊藿)Epimedium brevicornu Dried leaf 1.6
Chuan-Niu-Xi (川牛膝)Cyathula officinalis Dried root 11.5
Evidence-Based Complementary and Alternative Medicine 3

HH
0
30
60
90
120
ICP (mmHg)
HM
0
30
60
90
120
ICP (mmHg)
HL
0
30
60
90
120
ICP (mmHg)
M
0
30
60
90
120
ICP (mmHg)
NC
0
30
60
90
120
ICP (mmHg)
(a)
#
#
##
NC
M
HL
HM
HH
∗∗∗∗
MHMHHNC HL
0
10
20
30
40
50
AUC/MAP
NC
M
HL
HM
HH
∗∗∗∗
####
####
M HM HHNC HL
0
0.2
0.4
0.6
0.8
1.0
Max ICP/MAP (mmHg)
∗∗∗∗
####
####
NC
M
HL
HM
HH
M HM HHNC HL
0
50
100
150
Max ICP (mmHg)
∗∗
##
NC
M
HL
HM
HH
MHMHHNC HL
0
5
10
15
20
25
Baseline ICP (mmHg)
(b)
Figure 2: Erectile function of all rats after 4 weeks of treatment was measured by electric stimulation of cavernous nerves. (a) Representative
intracavernous pressure (ICP) values in the normal control (NC), bilateral cavernous nerve crush injury model M, low-dose HJI (HL),
medium-dose HJI (HM), and high-dose HJI (HH) groups; blue lines means pressure curves, and red lines means 1 min electrical
stimulation. (b) Baseline ICP, max ICP, max ICP/mean arterial pressure (MAP) ratio, and area under the curve (AUC)/MAP ratio in
response to electric stimulation of the CN, respectively, in all rats. Data are expressed as the mean ±SD. ∗∗P<0.01 and ∗∗∗∗P<0.0001 vs NC
group; #P<0.05, ##P<0.01, and ####P<0.01 vs M group.
4Evidence-Based Complementary and Alternative Medicine

difluoride (PVDF) membranes (Bio-Rad Laboratories,
Hercules, CA, USA). After blocking with Tris buffer so-
lution containing 5% nonfat milk for 1 h at 25–30°C,
samples were incubated with the indicated primary anti-
bodies in phosphate-buffered saline supplemented with
Tween-20 at 4°C overnight. e primary antibodies used
were anti-RhoA (1 : 5000), anti-ROCK1 (1 : 5000), anti-
ROCK2 (1 : 1000; Abcam), anti-Collagen I (1 : 1000;
Abcam), and anti-β-actin (1 : 1000; Immunoway, China).
β-Actin was used as a loading control. Samples were then
incubated with horseradish peroxidase-conjugated sec-
ondary antibodies at room temperature for 1 h. Next, the
membranes were treated with an enhanced chem-
iluminescence reagent (Beyotime). Images were captured
with the FluorChem R imaging system and analyzed using
ImagePro Plus 6.0.
2.8. Statistical Analysis. Statistical analysis was performed by
GraphPad Prism Version 6 (GraphPad Software, Inc., San
Diego, CA, USA). All experimental data were collected in a
blinded fashion; the operator and designer were blinded to
each other. All data were expressed as the mean ±standard
deviation (SD) and analyzed by one-way ANOVA with
Tukey’s post hoc test for multiple-group comparison. A value
of P<0.05 was considered statistically significant.
3. Results
3.1. Effects of HJI on Erectile Function in Rats with BCNI-ED.
Erectile function was evaluated by measuring baseline ICP,
maximal ICP, maximal ICP/MAP, and the ratio of the area
under the ICP curve to MAP at stimulating voltage of 5V
(Figure 2). All erectile function variables were significantly
lower in the M group than those in the NC group (P<0.01).
Compared to that in the M group, the baseline ICP was
significantly improved in the HM and HH groups (P<0.05).
In addition, the maximal ICP and maximal ICP/MAP in the
HM and HH groups were significantly increased compared to
those in the M group (P<0.0001). e AUC/MAP was also
significantly improved in the HM and HH groups (P<0.05).
No statistically significant differences were found between the
M and HL groups in terms of baseline ICP, maximal ICP, or
maximal ICP/MAP. However, the HL group did show an
NC
200µm
HMMHL HH
(a)
SM/collagen ratio
∗∗∗
###
###
0
0.2
0.4
0.6
MHLHMHHNC
Smooth muscle
%∗∗∗∗
####
####
0
10
20
30
HMHLMHHNC
Collagen
%
∗∗
#
#
0
20
40
60
80
HMHLMHHNC
(b)
Figure 3: Masson’s trichrome staining of midshaft penile tissues (100x magnifications). (a) Representative Masson’s trichrome
staining of the normal control (NC), bilateral cavernous nerve crush injury model M, low-dose HJI (HL), medium-dose HJI (HM),
and high-dose HJI (HH) groups (scale bar �100 μm). (b) Effect of HJI treatment on the proportion of smooth muscle, the proportion
of collagen, and the ratio of SM to collagen in the corpus cavernosum. ∗∗P<0.01,∗∗∗ P<0.001, and ∗∗∗∗ P<0.0001 vs NC group;
#P<0.05,###P<0.001, and ####P<0.0001 vs M group.
Evidence-Based Complementary and Alternative Medicine 5

increase in the AUC/MAP (P<0.05). Furthermore, ICP
cannot restore to normal level in the HM and HH groups.
3.2. Effects of HJI on Smooth Muscle and Collagen Levels in the
Midshaft Penile Tissues of Rats with BCNI-ED.Cavernosal
fibrosis is an important pathological process leading to ED [20]
and can be determined by Masson’s trichrome staining.
Compared to the NC group, the M group exhibited a clear
decrease in corporal smooth muscle content and an increase in
collagen deposition (P<0.0001) (Figure 3(a)). Smooth muscle
content was significantly increased, and collagen area was
significantly decreased, in the HM and HH groups (P<0.01)
(Figure 3(b)). In addition, the smooth muscle/collagen ratio
was significantly decreased in the M group compared to that in
the NC group, and the HM and HH treatment groups showed
a significantly improved smooth muscle/collagen ratio
(P<0.001) (Figure 3(b)). Furthermore, there was no statistical
significance in the smooth muscle, collagen, and SM/collagen
content of the HH group and NC group.
3.3. Effects of HJI on the Expression of RhoA in BCNI Rats.
e expression of RhoA in the model group was significantly
increased after CN injury as shown by IF staining, and RhoA
expression decreased after treatment in a dose-dependent
manner. Accordingly, RhoA expression in the HH group
was the lowest (Figure 4).
3.4. Effects of HJI on the Expression of ROCK1 in BCNI Rats.
e expression of ROCK1 was significantly increased in the
model group after BCNI. However, its expression was not
majorly affected by increased drug administration
(Figure 5).
3.5. Effects of HJI on the Expression of ROCK2 in BCNI Rats.
e expression of ROCK2 was significantly increased in the
model group after BCNI. Notably, the expression of ROCK2
decreased, similar to our observations for RhoA, upon
treatment with increasing drug concentrations; the ex-
pression of ROCK2 showed the greatest decrease in the HH
group (Figure 6).
3.6. Effects of HJI on the Expression of RhoA, ROCK1, and
ROCK2 in the Penis Tissues of BCNI-ED Rats.RhoA,
ROCK1, ROCK2, and collagen I expression were quantified
by western blotting (Figure 7). Compared to that in the NC
100μm
DAPI RhoA Merge
NC
M
HL
HM
HH
Figure 4: Effects of HJI on the expression of RhoA in BCNI rats through immunofluorescence staining (200x magnifications). Immunofluorescent
staining shows the colocalization of RhoA (red) and neuronal cells (blue) in the corpora cavernosa. DAPI: 4′,6-diamidino-2-phenylindole; NC:
normal control; M: bilateral cavernous nerve crush injury model; HL: low-dose HJI; HM: medium-dose HJI; HH: high-dose HJI.
6Evidence-Based Complementary and Alternative Medicine

group, the relative expression of above proteins was in-
creased in the M group (P<0.05). In the HL treatment
group, there were no significant differences in RhoA,
ROCK1, ROCK2, and collagen protein expression. However,
RhoA, ROCK2, and collagen I protein expression was
markedly decreased in the HM and HH groups (P<0.05).
ROCK2 protein expression was significantly higher in the
HH group only. No significant differences in RhoA, ROCK1,
ROCK2, or collagen I expression were found in the HL
treatment group. Compared with the control group, RhoA
could not return to normal level in the treatment group.
Furthermore, there was no significant difference in collagen
I expression compared the NC group with the HH group.
4. Discussion
Apoptosis, collagen deposition, and the fibrosis of smooth
muscle and endothelial cells in the corpus cavernosum may
occur during neurapraxia mediated by CN injury. e re-
duced expression of nitric oxide synthase (NOS) and changes
in the signal intensity of transforming growth factor-β1
(TGF-β1) and RhoA are closely related to the above patho-
logical process [5, 9, 10, 21, 22]. Clinical studies suggest that
the erection induced by sexual stimulation and nonsexual
stimulation (spontaneous erection at night) in patients with
radical prostatectomy is significantly reduced [23], resulting
in a relatively ischemic/hypoxic state of the corpus cav-
ernosum during convalescence, which is an important factor
leading to organic changes such as penile fibrosis. e corpus
cavernosum is the final effector organ of the erectile response.
e corpus cavernosum smooth muscle cells are the basis of
the corpus cavernosum; they also control the relaxation and
contraction of the corpus cavernosum. When tissue structures
are destroyed due to fibrosis, erectile function cannot be
restored, even after the improvement of neurological
symptoms. erefore, the prevention and treatment of cav-
ernous fibrosis is of great significance to regain normal
erectile function upon neurapraxia due to CNI. A major
purpose of this study was to analyze the effect of HJI on
fibrosis in the cavernous penile tissue of rats. We found that
BCNI caused significant cavernous corpus remodeling, as
shown by the observed increase in cavernous fibrosis and
collagen deposition in the model and HL treatment groups
compared to those in the sham, HM treatment, and HH
treatment groups. Medium-dose treatment and high-dose
DAPI ROCK1 Merge
NC
M
HL
HM
HH
100μm
Figure 5: Effects of HJI on the expression of Rho-associated protein kinase 1 (ROCK1) in BCNI rats through immunofluorescence staining
(200x magnification). Immunofluorescence staining shows the colocalization of ROCK1 (green) and neuronal cells (blue) in the corpora
cavernosa. DAPI: 4′,6-diamidino-2-phenylindole; NC: normal control; M: bilateral cavernous nerve crush injury model; HL: low-dose HJI;
HM: medium-dose HJI; HH: high-dose HJI.
Evidence-Based Complementary and Alternative Medicine 7

DAPI
100µm
ROCK2 Merge
NC
M
HL
HM
HH
Figure 6: Effects of HJI on the expression of Rho-associated protein kinase 2 (ROCK2) in BCNI rats through immunofluorescence staining.
Immunofluorescent staining shows the colocalization of ROCK2 (red) and neuronal cells (blue) in the corpora cavernosa (200x mag-
nification). DAPI: 4′,6-diamidino-2-phenylindole; NC: normal control; M: bilateral cavernous nerve crush injury model; HL: low-dose HJI;
HM: medium-dose HJI; HH: high-dose HJI.
NC M HL HM HH
ROCK2
ROCK1
RhoA
β-Actin
Collagen I
(a)
NC
M
HL
HM
HH
∗∗
##
∗
∗
#
Protein expression/β-Actin
∗
#
#
Collagen IROCK1RhoA ROCK2
0.0
0.5
1.0
1.5
2.0
(b)
Figure 7: Effect of HJI on RhoA, ROCK1, ROCK2, and collagen I activity in rat corpus cavernosum tissues. (a) Western blot analysis
showing the protein expression levels of RhoA, ROCK1, ROCK2, and collagen I in rat corpus cavernosum tissues. (b) Data are presented as
the relative density of RhoA, ROCK1, ROCK2, and collagen I expression with β-actin as the loading control. Values are presented as the
mean ±standard deviation. ∗P<0.05 and ∗∗P<0.01 vs NC group; #P<0.05 vs M group. NC: normal control; M: bilateral cavernous nerve
crush injury model; HL: low-dose HJI; HM: medium-dose HJI; HH: high-dose HJI.
8Evidence-Based Complementary and Alternative Medicine

treatment were found to increase smooth muscle content
while reducing the deposition of the fibrosis-related protein
collagen I compared to those in the model group, indicating
that HJI could reduce corpus cavernosum fibrosis and im-
prove erectile function.
e RhoA/ROCK pathway was discovered in the 1990s
and has since been proven to play an important role in
regulating erectile function [6]. e ROCK signaling
pathway maintains the contractile state of corpus cav-
ernosum smooth muscle, and phosphorylated ROCK allows
myosin light chain to stay phosphorylated and thus actin-
contracted [24]. Early studies on ED did not differentiate
between the two ROCK1 and ROCK2 subtypes, but in-
creasingly exhaustive research has shown that they play
distinctive roles in ED caused by different etiologies [7]. An
early view was that the upregulation of ROCK2 played a
major role in CNI-ED, but subsequent studies have con-
firmed that the high expression of ROCK1 is also closely
related to cavernous fibrosis after CNI-ED [5, 9, 10, 21].
Studies by Cho et al. [5], Song et al. [11], and Cho et al. [21]
found that ROCK1 expression was increased in SD rats after
the CN was crushed and transected. However, Gratzke et al.
[10] found that ROCK1 was not upregulated after CNI, and
whether the degree of damage caused by different CNI
methods affects the expression of ROCK1 remains to be
studied. erefore, we included both ROCK1 and ROCK2 in
the present study. Unlike the above studies, we found that
both isoforms showed an increase after 28 days of CN injury.
In addition, no clear correlation has been shown for the
effect of age on ED in rats compared to that in humans. It is
also uncertain whether changes in the expression of ROCK1
and ROCK2 at different time points represent the changes of
ROCK subtypes in the acute, subacute, and chronic stages
after surgery.
Cavernous injury, diabetes, and other types of ED-as-
sociated disorders are not significant for first-line clinical
treatment with PDE5i, so more treatment regimens and
means are needed for Ed treatment [25]. At present, the
relationship between RhoA/ROCK signaling and ED has
attracted the attention of many researchers. RhoA/ROCK
pathway activation is closely related to erectile function, as
well as cavernosal and penile fibrosis [20, 26]. e inhibition
of ROCK1 or its downstream molecules, LIMK2 and Coflin,
has been shown to effectively attenuate penile fibrosis. e
current results showed that ROCK1 expression was de-
creased in the drug group, but not significantly; whether HJI
affects the downstream molecules of ROCK1 requires fur-
ther study. ROCK2 plays an important role in fibrosis in
organs such as the heart, kidney, and lung [27–29]. e
degree of fibrosis is known to be reduced through ROCK2
gene knockout or inhibition [9, 30]. Moreover, ROCK2
expression is upregulated in ED; erectile function can be
markedly improved by knocking down the gene or treat-
ment with ROCK inhibitors. Fluorescence and western
blotting results showed that the activated ROCK2 signaling
pathway was significantly inhibited after drug treatment. e
relationship between ROCK2 and fibrosis in ED caused by
CN injury has not been researched often. Future studies
should investigate whether ROCK2 elevation can regulate
cavernous fibrosis. Based on the small sample population for
ED pathology [31], expression of ROCK2 was significantly
upregulated in tissue from men with ED. Drug can inhibit
ROCK2 efficiently, which may have important benefits for
the treatment of ED. In this study, activated RhoA/ROCK2
signaling in CNI rats was significantly inhibited by drug
treatment. erefore, we speculate that the antifibrosis effect
of this drug could have been through the action of ROCK2 or
other pathways. Because RhoA signaling also plays an im-
portant role in regulating erectile function independent of
the classical NO signaling pathway, several key molecules in
the RhoA pathway, namely, RhoA, ROCK1, and ROCK2,
were also analyzed in this study. e results showed that
RhoA, ROCK1, and ROCK2 were highly expressed in the
spongy tissue of BCNI rats, consistent with the results of
previous studies. RhoA and ROCK2 in rat cavernosum
tissues showed decreased expression after intervention with
HJI, but the expression of ROCK1 was not significantly
affected by our treatment. As the most commonly used index
for evaluating erectile function [32], the ICP indicated that
HM and HH treatments did not fully improve erectile
function to normal levels. HJI plays a role in the partial
pathogenesis of BCNI-induced ED; however, HJI may
achieve better therapeutic effects if it is combined with
PDE5i drugs or other clinical treatments. is study showed
that the improvement of CNI-ED by HJI treatment may be
related to the inhibition of RhoA/ROCK2 signaling acti-
vation and amelioration penis fibrosis, and that might be the
mechanisms through which HJI protects erectile function.
5. Conclusions
Our results demonstrated that the Chinese medicine HJI
ameliorated erectile function and inhibited penis fibrosis in
BCNI rats. Furthermore, the effects of HJI on erectile
function may be caused, at least in part, by the suppression of
RhoA/ROCK2 signaling and cavernosum fibrosis. However,
the precise mechanism of action of this medicine must be
clarified through further investigations.
Data Availability
e data used and analyzed in the present article are
available from the corresponding author on reasonable
request.
Conflicts of Interest
e authors do not have any possible conflicts of interest.
Authors’ Contributions
Miao-yong Ye and Fan Zhao contributed equally to this
study.
Acknowledgments
he experiment was funded by the National Natural Science
Foundation of China (grant nos. 81571431 and 81603620).
is research has been partially supported by Zhejiang
Evidence-Based Complementary and Alternative Medicine 9

grants funded by Provincial Natural Science Foundation of
China (grants nos. LY19H270011, LY18H270002, and
LQ19H040001), the major Research Projects of Traditional
Chinese Medicine in Zhejiang Province (grant no.
2018ZY007), and the Zhejiang “New Seeding Talent Plan”
Project (grant no. 2018R410048).
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