Punicalagin Efek

The American Journal of Chinese Medicine, Vol. 44, No.
4, 1–17
© 2016 World Scientific Publishing Company
Institute for Advanced Research in Asian Science and Medicine
DOI: 10.1142/S0192415X16500439
Punicalagin Prevents Hypoxic

Pulmonary Hypertension via
Anti-Oxidant Effects in Rats
by UNIVERSITY OF NEW ENGLAND on 06/05/16. For personal use only.
Jingyun Shao,*,† Peng Wang,* An Liu,* Xusheng Du,* Jie Bai* and Mingwei Chen†
Am. J. Chin. Med. Downloaded from www.worldscientific.com
*Department of Respiratory Medicine
Xi’an Central Hospital Xi’an 710003, China

†
Department of Respiratory Medicine
First Affiliated Hospital of Medical College of Xi’an Jiaotong University
Xi’an 710061, China
Published 24 May 2016
Abstract: Punicalagin (PG), a major bioactive ingredient in pomegranate juice, has been
proven to have anti-oxidative stress properties and to exert protective effects on acute lung
injuries induced by lipopolysaccharides. This study aimed to investigate the effects of PG
treatment on hypoxic pulmonary hypertension (HPH) and the underlying mechanisms re-
sponsible for the effects. Rats were exposed to 10% oxygen for 2 wk (8 h/day) to induce the
HPH model. PG (5, 15, 45 mg/kg) was orally administered 10 min before hypoxia each day.
PG treatments at the doses of 15 and 45 mg/kg/d decreased the mean pulmonary arterial
pressure (mPAP) and alleviated right ventricular hypertrophy and vascular remodeling in
HPH rats. Meanwhile, PG treatment attenuated the hypoxia-induced endothelial dysfunction
of pulmonary artery rings. The beneficial effects of PG treatment were associated with
improved nitric oxide (NO)-cGMP signaling and reduced oxidative stress, as evidenced by
decreased superoxide generation, gp91 phox expression and nitrotyrosine content in the
pulmonary arteries. Furthermore, tempol’s scavenging of oxidative stress also increased
NO production and attenuated endothelial dysfunction and pulmonary hypertension in
HPH rats. Combining tempol and PG did not exert additional beneficial effects compared
to tempol alone. Our study indicated for the first time that PG treatment can protect against
hypoxia-induced endothelial dysfunction and pulmonary hypertension in rats, which may be
induced via its anti-oxidant actions.
Keywords: Punicalagin; Hypoxic Pulmonary Hypertension; Endothelial Function; Nitric

Oxide; Oxidative Stress.
Correspondence to: Dr. Mingwei Chen, Department of Respiratory Medicine, First Affiliated Hospital of Medical
College of Xi’an Jiaotong University, 227 Yanta West Road, Xi’an 710061, China. Tel: (þ86) 29-8532-3112,
E-mail: mingwchen@126.com
1
2 J. SHAO et al.
Introduction
Pulmonary hypertension (PH) is a life-threatening disease characterized by increased

pulmonary artery pressure and vascular remodeling, which ultimately results in right
ventricular hypertrophy and subsequent right heart failure, with high mortality. Among the
various forms of pulmonary hypertension, hypoxic pulmonary hypertension (HPH) occurs
in diverse clinical settings, including among patients with respiratory diseases such as
chronic obstructive pulmonary disease (COPD) and residents living in plateau environ-
ments, which has aroused great interest from scientific researchers (Scherrer et al., 2013;
Voelkel et al., 2013). Hypoxia-induced oxidative stress is considered to play a pivotal role
in the development of HPH. Increased oxidative stress mediates hypoxia-induced endo-
thelial dysfunction in the pulmonary vascular endothelium (Hoshikawa et al., 2001;

Makarenko et al., 2014; Tuleta et al., 2014). Moreover, impaired nitric oxide (NO) pro-
duction has long been considered a main pathogenesis of HPH (Hampl and Herget, 2000).
In the presence of oxidative stress, superoxide anion (O 2 ) can react with NO to generate a
highly toxic oxidant peroxynitrite (ONOO Þ, which may result in oxidative/nitrative
damage and enhanced NO destruction (Mikkelsen and Wardman, 2003; Lee et al., 2003).
Therefore, identifying novel and effective interventions that attenuate hypoxia-induced
oxidative stress is a potential strategy for the prevention of HPH.
Traditional herbs and foods such as Rhodiola and dietary nitrate have generated
increasing interest as possible interventions against HPH (Baliga et al., 2012; Kosanovic
et al., 2013). Pomegranates, the fruit of Punica granatum, are a healthy food because of
their well-known anti-oxidant actions. Pomegranate juice exerts cardiovascular protective
effects in human and murine models (Basu and Penugonda, 2009; Stowe, 2011; Shema-
Didi et al., 2012) and improves endothelial function in vitro (de Nigris et al., 2006).
Recent studies showed that pomegranate juice ameliorates oxidative stress and reduces
stimulus-induced cell death in human villous trophoblasts (Chen et al., 2012). Pome-
granate juice is rich in polyphenols, including ellagitannins, gallotannins, and flavonoids.
Importantly, punicalagin (PG) is a prominent ellagitannin isolated from pomegranate
juice, which has been reported to exert anti-oxidative effects similar to those of pome-
granate juice (de Nigris et al., 2007; Chen et al., 2013). PG has been reported to inhibit
lipopolysaccharide-induced oxidative stress in macrophages (Xu et al., 2015) and at-
tenuate cerebral ischemia/reperfusion-induced oxidative brain damage via its anti-oxidant
potential (Yaidikar et al., 2014). Moreover, it has been reported that PG ameliorates the
acute respiratory distress syndrome induced by lipopolysaccharides in mice (Peng et al.,
2015), suggesting PG may have beneficial effects in the lung. However, it is currently
unknown whether PG can prevent hypoxia-induced oxidative stress and pulmonary
hypertension.
In this study, we hypothesized that PG has anti-oxidant effects in the pulmonary
arteries and prevents the development of HPH. Thus, we aimed to investigate whether oral
PG treatment protects against chronic hypoxia-induced pulmonary hypertension, and if
so, to further explore whether the underlying mechanisms are related to its anti-oxidant
effects.
PUNICALAGIN PREVENTS PULMONARY HYPERTENSION 3
Materials and Methods
HPH Animal Models
This study was conducted in adherence to the National Institutes of Health Guidelines
for the Care and Use of Laboratory Animals and was approved by the Xi’an Jiaotong
University Ethics Committee on Animal Research. Male 6–8 week old Sprague-Dawley
rats were placed in transparent plastic chambers at about 350 m above the sea level (Xi’an,
China). A rat HPH model was induced as described previously (Li et al., 2009; Wu et al.,
2013). Briefly, rats were exposed hypobaric hypoxia (air pressure 50 kPa, oxygen
concentration 10%) for 8 h every day. The control normoxic group of animals was kept
in room temperature air.
The rats were divided into the following groups randomly: (i) Control group: normoxic
control group; (ii) Hypoxia group, the animals were exposed to hypoxic conditions for 2
wk and received the vehicle each day; (iii) Hypoxia þ PG group, PG (purity > 98%,
Dalian Meilun Biology Technology Co., Ltd, China) was dissolved in saline. The animals
were exposed to hypoxic conditions for 2 wk and PG (5, 15, 45 mg/kg) was orally ad-
ministered 10 min before hypoxia each day. The dose of PG was based on previous studies
(Lin et al., 1999; Yaidikar and Thakur, 2015) and was validated in our preliminary
experiments.
Hemodynamic Measurements and Evaluation of Right Heart Hypertrophy
The rats were anesthetized by an injection of 3% pentobarbital sodium (60 mg/kg)

intraperitoneally (i.p.). A polyethylene micro-catheter filled with heparin solution (500
IU/mL, v/v) was inserted into the right ventricle and pulmonary artery as described pre-
viously (Wu et al., 2013). The mean pulmonary arterial pressure (mPAP) and right
ventricular systolic pressure (RVSP) were then measured and analyzed on a hemodynamic
analyzing system (RM-6240C, Chengdu Instrument Co., China). Upon completion of
hemodynamic measurements, the left ventricle (LV), septum (S), and right ventricle
(RV) were separated and weighed. The ratio of RV/(LVþS) was used as an index of RV
hypertrophy.
Assessment of Vascular Remodeling
The lung tissue was fixed in 10% formalin overnight and was embedded in paraffin.
The tissue sections (5 μm) were treated with HE (hematoxylin and eosin) stain for mor-
phometric analysis. The pulmonary arterioles (external diameters of 50–150 μm) were
examined at 400 magnification. The medial wall thickness (WT), medial wall cross-
sectional area (WA), external semidiameter (ES) and total arterial cross-sectional area
(TAA) were measured in a double-blind manner. The ratio of WA to TAA (WA%) and the
ratio of WT to ES (WT%) were calculated to evaluate the degree of pulmonary artery
remodeling.
4 J. SHAO et al.
Determination of Vascular Function
Vascular function was analyzed in vitro as previously described (Lee et al., 2011; Hwang
et al., 2012). The pulmonary arteries were carefully separated and quickly placed in ice-
cold Kerb’s solution. After being cleaned of perivascular fat connective tissue, the sec-
ondary branches of the pulmonary arteries were picked and cut into 3 mm segments. The
segments were mounted onto stainless steel hooks and were connected to the force
transducers. The tension of arterial segments was measured by the MacLab data acquisition
system. After equilibration, phenylephrine (PE) was added to the tissue bath to reach a final
concentration of 1 μmol/L. Once the contraction was stable, acetylcholine (ACh) was
added to the bath in cumulative concentrations (10 9 –10 6 mol/L) to determine endo-
thelial function. The rings were then washed with buffer and allowed to re-equilibrate to
baseline. PE was added to the tissue bath to reach a final concentration of 1 μmol/L. Once
the contraction was stable, acidified NaNO2 was added to the bath in cumulative con-
centrations (10 8 -10 5 mol/L) to determine vascular smooth muscle function.
Determination of NO and cGMP Content
For the NO assay, pulmonary artery tissues were minced with scissors and homogenized in
ice-cold lysis buffer. After centrifugation, the supernatants were collected to measured total
NOx production (a stable marker of tissue NO content) by a commercial NO assay kit
(Nanjing Jiancheng Bioengineering Institute) (Ding et al., 2015). For the cGMP assay,
pulmonary artery tissues were homogenized in a 6% trichloroacetic acid solution as pre-
viously reported (Murata et al., 2005). The supernatants were collected to measured cGMP
levels by a cGMP enzyme immunoassay kit (Amersham Pharmacia), and total protein
content was determined in the supernatants.
Quantification of Superoxide Production, Superoxide Dismutase Activity

and 3-Nitrotyrosine Content
Lucigenin-enhanced chemiluminescence was used to assess superoxide production in

pulmonary artery tissues as described previously (Cacanyiova et al., 2013). The results
were expressed as relative light units (RLU) per second per milligram of tissue weight
(RLU/mg/s). The activity of anti-oxidant enzyme SOD (superoxide dismutase) in pul-
monary artery homogenates was determined spectrophotometrically as previously de-
scribed (Xiong et al., 2011) with the use of commercial kits (Nanjing jiancheng
Bioengineering, China). 3-nitrotyrosine content in the pulmonary artery tissue, a footprint
of ONOO formation in vivo, was determined with the chemiluminescence method (EMD
Millipore, Billerica, MA, USA) as previously described (Fan et al., 2013).
Immunoblot Analysis
Pulmonary artery tissues were homogenized in lysis buffer and protein concentrations were
determined by using a BCA protein assay. After separation by electrophoresis on sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were

transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk
for 1 h at room temperature and then incubated with the appropriate primary antibodies
including anti-gp91 phox (Cell Signaling Technology), HIF-1α (Beyotime, China), VEGFA
(Boster, China), MMP-9 (Cell Signaling Technology) and anti-actin (Cell Signaling
Technology) overnight at 4 C. After three washes of phosphate buffer solution with tween-
20 (PBST), the membranes were incubated with corresponding horseradish peroxidase-
conjugated second antibodies. After additional PBST washes, the blots were evaluated by
using an enhanced chemiluminescent system.
Quantitative Real-Time PCR

Total RNA was extracted with Trizol reagent (Invitrogen, Shanghai, China) and was
subjected to reverse transcription using superscriptr jjj first-strand systhesis system

(Invitrogen, Shanghai, China) as described previously (Ding et al., 2015). Real-time PCR
was carried out using a Power SYBR green PCR master mix (Bio-rad). The following
primer sequences were used: NF-κB forward TCTGGCGCAGAAGTTAGGT, reverse
CCAGAGACCTCATAGTTGT, TaqMan probe sequence GGACTCAGCCGGAAGG-
CATT; GAPDH forward TGGTGCCAAAAGGGTCAT, reverse TCACACCCATCA-
CAAACATG, TaqMan probe sequence CCCCTTCCGCTGATGCCC. Data were
normalized relative to those for GAPDH expression using the 2 Ct method.
Determination of Serum TNF-α Levels
Serum TNF-α levels were measured using an ELISA kit (eBioscience) following the
manufacturer’s instructions. Quantitative concentrations were extrapolated from the rat
TNF-α recombinant protein standard curve. All samples and standards were measured in
duplicate.
Statistical Analysis
All values are presented as means SEM. Differences among comparisons were evaluated
with one-way ANOVA followed by Bonferroni corrected t test where appropriate. A level
of p < 0:05 was taken as statistically significant.
Results
PG Prevented Chronic Hypoxia-Induced Pulmonary Hypertension, Right Ventricular

Hypertrophy and Vascular Remodeling
After 2 wk of chronic hypoxia, rats exhibited a significant increase in mPAP (Fig. 1A,
29:4 2:2 mm Hg vs. 13:7 1:3 mm Hg of Control, n ¼ 8, p < 0:01) and RVSP
(Fig. 1B, 38:7 1:8 mm Hg vs. 27:6 1:4 mm Hg of Control, n ¼ 8, p < 0:01).
6 J. SHAO et al.
(A) (B)
Figure 1. Effects of different doses of PG treatment on hypoxia-induced pulmonary hypertension in rats. (A)
mPAP, mean pulmonary artery pressure. (B) RVSP, right ventricular systolic pressure Values are means SEM.
n ¼ 8. Hypoxia, the rats were exposed 10% oxygen for 2 wk (8 h/d) and received vehicle per day; PG, punicalagin
(5, 15, 45 mg/kg) was orally administered 10 min before hypoxia per day. **p < 0:01 vs. Control. # p < 0:05, ##
p < 0:01 vs. Hypoxia.
Treatment with PG at the dose of 15 and 45 mg/kg/d, but not 5 mg/kg/d elicited a
significant decrease in mPAP and RVSP (n ¼ 8, p < 0:05 or p < 0:01), which was still
higher than that of control group.
As shown in Fig. 2, compared with the controls, hypoxia-exposed rats showed right
ventricular hypertrophy and pulmonary artery remodeling as evidenced by increased
RV/(LVþS) (0:39 0:02 vs. 0:26 0:01 of Control, n ¼ 8, p < 0:01), WT% (62:4 3:9
vs. 21:1 2:1 of Control, n ¼ 8, p < 0:01) and WA%. These pathological changes were
attenuated by PG at the doses of 15 and 45 mg/kg/d, but not 5 mg/kg/d. These results
(A) (B)
Figure 2. Effects of different doses of PG treatment on hypoxia-induced right-ventricular hypertrophy and vas-
cular remodeling in rats. (A) RV/(LVþS), right ventricle weight/left ventricleþseptum weight (B) Representive
histopathological changes with the use of HE staining in pulmonary arterioles (400). The bar represents 20 μm.
(C) WT%, medial wall thickness/external semidiameter. (D) WA%, medial wall cross-sectional area/total
arterial cross-sectional area. Values are means SEM. n ¼ 8. Hypoxia, the rats were exposed 10% oxygen for
2 wk (8 h/d) and received vehicle each day; PG, punicalagin (5, 15, 45 mg/kg) was orally administered 10 min
before hypoxia each day. **p < 0:01 vs. Control. # p < 0:05, ## p < 0:01 vs. Hypoxia.
(C) (D)
Figure 2. (Continued )
suggested that medium-dose and high-dose PG could prevent the pulmonary hypertension
and vascular remodeling induced by hypoxia.
PG Attenuated Chronic Hypoxia-Induced Endothelial Dysfunction
Endothelial dysfunction has been demonstrated to contribute to the pathogenesis and de-
velopment of HPH. We then investigated whether PG treatment could improve endothelial
function ex vivo. As shown in Fig. 3A, concentration-dependent vasorelaxation of pul-
monary artery rings in response to ACh was impaired in HPH rats compared with controls.
Treatment with PG (15 and 45 mg/kg/d) markedly preserved ACh-induced vasorelaxation,
indicating that PG treatment improved endothelial function in HPH animals. Addition of
NaNO2, an exogenous donor of nitric oxide (NO), resulted in comparable relaxations in
(A) (B)
Figure 3. Concentration–relaxation curves for ACh (A) or NaNO2 (B) in pulmonary artery rings isolated
from normoxic (Control) or hypoxic rats treated with vehicle or PG. Results represent mean SEM of 10 vascular
segments from 5 rats per group. Hypoxia, the rats were exposed 10% oxygen for 2 wk (8 h/d) and received vehicle
each day; PG, punicalagin (15, 45 mg/kg) was orally administered 10 min before hypoxia each day. **p < 0:01
vs. Control. # p < 0:05, ## p < 0:01 vs. Hypoxia.
8 J. SHAO et al.
(A) (B)
Figure 4. PG treatment at doses of 15 and 45 mg/kg increased NO (A) and cGMP (B) content in the pulmonary
arteries of hypoxic rats. Values are means SEM. n ¼ 8. Hypoxia, the rats were exposed 10% oxygen for 2 wk (8
h/d) and received vehicle each day; PG, punicalagin was orally administered 10 min before hypoxia each day.
**p < 0:01 vs. Control. # p < 0:05, ## p < 0:01 vs. Hypoxia.
pulmonary artery rings in different treatment groups, indicating that relaxation of smooth
muscle in response to exogenous NO was unaffected in these vascular segments (Fig. 3B).
PG Increased NO and cGMP Content in HPH Rats
To reveal the potential mechanism of PG treatment on endothelial dysfunction, the contents

of NO and its downstream effector cGMP were measured. As shown in Fig. 4, the content
of NO and cGMP in the pulmonary arteries was significantly reduced in HPH rats com-
pared with the content in control animals. PG treatment (15 and 45 mg/kg/d) elevated the
levels of NO in the pulmonary arteries of chronic hypoxic rats. Moreover, the cGMP
content was significantly higher in the HPH rats receiving PG treatment. These results
indicate that PG treatment restored pulmonary arterial NO-cGMP signaling in HPH rats.
PG Attenuated Hypoxia-Induced Oxidative Stress in HPH Rats
Considerable evidence suggests that increased superoxide production and subsequent NO

destruction is the major cause of hypoxia-induced endothelial dysfunction and pulmonary
hypertension. To determine whether PG treatment may attenuate superoxide generation in
hypoxia-induced pulmonary arteries, thus improving endothelial function, superoxide
generation was measured directly. As shown in Fig. 5A, superoxide generation was
significantly increased in the pulmonary arteries from HPH rats compared to those of
control animals (165:4 20:9 RLU/mg/s vs. 51:3 7:9 RLU/mg/s in Control, n ¼ 8,
p < 0:01). Treatment with PG at the doses of 15 and 45 mg/kg/d markedly reduced su-
peroxide production (92:3 12:9 RLU/mg/s in 15 mg/kg, 81:9 10:9 RLU/mg/s in
45 mg/kg, p < 0:01 vs. Hypoxia group). gp91 phox is a major component of NADPH ox-
idase, which is the most important superoxide anion (O
2 Þ producing source. PG treatment
(15 and 45 mg/kg/d) significantly reduced gp91 phox expression in HPH rats (Fig. 5B,
(A) (B)
(C) (D)
Figure 5. PG treatment at doses of 15 and 45 mg/kg attenuated hypoxia-induced oxidative stress in rats.
(A) Superoxide generation in pulmonary arteries. (B) gp91 phox expression. Top: representative blot images. (C) 3-
nitrotyrosine level. (D) Superoxide dismutase (SOD) activity in pulmonary arteries. Values are means SEM.
n ¼ 4–8. Hypoxia, the rats were exposed 10% oxygen for 2 wk (8 h/d) and received vehicle each day; PG,
punicalagin was orally administered 10 min before hypoxia each day. **p < 0:01 vs. Control. # p < 0:05, ##
p < 0:01 vs. Hypoxia.
n ¼ 4, p < 0:05 or p < 0:01 vs. Hypoxia group). Peroxynitrite (ONOO ), a biradical
reaction product of superoxide anion (O 2 ) and NO, was determined via measurement of
3-nitrotyrosine. As shown in Fig. 5C, 3-nitrotyrosine content was significantly increased in
hypoxia-induced pulmonary arteries. Treatment with PG markedly reduced 3-nitrotyrosine
content in HPH rats. Moreover, anti-oxidant capacity was measured by the activity of SOD
(O
2 scavenger) in pulmonary arteries. Hypoxia-induced reduction in SOD activity was
significantly preserved after PG treatment (Fig. 5D).
Increased reactive oxygen species (ROS) production influences redox-sensitive sig-
naling pathways in pulmonary vascular cells, leading to altered gene expression, the
activation of proteinases, and the increased production of inflammatory cytokines (Freund-
Michel et al., 2013). The downstream molecules or cytokines regulated by ROS, including
NF-κB, MMP-9 and TNF-α, were then measured. As shown in Fig. 6, chronic hypoxia
10 J. SHAO et al.
(A) (B) (C)

(D) (E) (F)
Figure 6. PG treatment reduced downstream molecule expression and cytokine production regulated by ROS
in HPH rats. (A) mRNA expression of NF-κB. (B) Representative blot images of MMP-9, HIF-1α and VEGFA.
(C) Protein expression of MMP-9. (D) serum TNF-α level. (E) Protein expression of HIF-1α. (F) Protein
expression of VEGFA. Values are means SEM. n ¼ 3–6. Hypoxia, the rats were exposed 10% oxygen for 2 wk
(8 h/d) and received vehicle each day; PG, punicalagin was orally administered 10 min before hypoxia each day.
**p < 0:01 vs. Control. # p < 0:05, ## p < 0:01 vs. Hypoxia.
significantly upregulated the mRNA expression of NF-κB and increased the protein ex-
pression of MMP-9. Moreover, serum TNF-α production was significantly increased in
HPH rats compared with control animals (Fig. 6D). Treatment with PG attenuated the rise
in NF-κB and MMP-9 expressions as well as TNF-α production in HPH rats (p < 0:05 or
p < 0:01). These data suggest that PG treatment reduced hypoxia-induced oxidative stress
in HPH rats.
PG Inhibited Chronic Hypoxia-Induced HIF-1α and VEGFA Expression
HIF-1α and VEGFA have been implicated in the pathogenesis of HPH and hypoxic pul-
monary artery remodeling (Irwin et al., 2009; Shimoda and Laurie, 2014) Consistent with
the results of previous studies (Irwin et al., 2009; Shimoda and Laurie, 2014), chronic
hypoxia significantly increased the protein expressions of HIF-1α and VEGFA (Figs. 6B,
6E and 6F). PG treatment prevented the hypoxia-induced rise in HIF-1α and VEGFA
expressions (p < 0:05 or p < 0:01).
Anti-oxidant Stress Effect of PG as a Critical Mechanism Responsible

for its Vascular Protection
To further ascertain the degree of involvement of anti-oxidant action in the vascular

protective effects of PG, male adult rats were exposed to hypoxic conditions for 2 wk as
(A) (B)
(C) (D)
Figure 7. Effects of tempol (Temp) treatment or tempol plus PG treatment on hypoxia-induced superoxide
generation (A), NO production (B), vasorelaxation-response to ACh (C) and mean pulmonary artery pressure (D).
Values are means SEM. n ¼ 6. H, the rats were exposed hypoxia for 2 wk (8 h/d) and received vehicle per day;
Temp, tempol was administered at the dose of 86 mg/kg/d in drinking water. PG, punicalagin was orally ad-
ministered 10 min before hypoxia each day. **p < 0:01 vs. H.
described above and treated with either tempol (86 mg/kg/d in drinking water), a super-
oxide cell-permeable scavenger, or tempol plus PG (45 mg/kg) each day. The dose of
tempol was based on previous studies (Elmedal et al., 2004; Rashid et al., 2013). As
summarized in Fig. 7, treatment with tempol significantly reduced superoxide generation,
increased NO production and improved endothelial function in HPH rats (n ¼ 6,
p < 0:01). Co-treatment with PG had no additional vascular protective effects (n ¼ 6,
p > 0:1 vs. HþTemp group). Tempol treatment also markedly attenuated hypoxia-induced
pulmonary hypertension (n ¼ 6, p < 0:01). Co-treatment with PG afforded no additional
effects. These results demonstrate that scavenging superoxide protected against hypoxia-
induced endothelial dysfunction and pulmonary hypertension and suggest that anti-oxidant
stress action plays a critical role in the vascular protection of PG.
PG Attenuated Acute Hypoxic Pulmonary Hypertension in Rats
To further confirm the protective effect of PG on HPH, we tested the effect of PG treatment
on acute hypoxic pulmonary hypertension. The rats were pretreated with PG (15 or
12 J. SHAO et al.
(A) (B)
Figure 8. Effects of PG treatment on acute hypoxia-induced pulmonary hypertension in rats. (A) RVSP, right
ventricular systolic pressure. (B) RV dp/dtmax , maximal rate of rise in right ventricular pressure. Values are
means SEM. n ¼ 6. The rats were pretreated with punicalagin (PG, 15 or 45 mg/kg) or vehicle 1 h before acute
hypoxic (Acute H) exposure for 30 min. **p < 0:01 vs. Control. # p < 0:05, ## p < 0:01 vs. Acute H.
45 mg/kg) or vehicle, as previously described, 1 h before acute hypoxic exposure. Right

ventricular systolic pressure (RVSP) and right ventricular contractility (RV dP/dtmax Þ were
recorded while rats were subjected to acute hypoxia for 30 min. Compared with vehicle
treatment, PG treatment at doses of 15 and 45 mg/kg significantly prevented the hypoxia-
induced rise in RVSP (Fig. 8A, p < 0:01) and RV dP/dtmax (Fig. 8B, p < 0:05). Taken
together, these data imply that PG treatment is sufficient to attenuate both acute and chronic
hypoxic pulmonary hypertension.
Discussion
Several novel findings were made in the present study. First, we demonstrated for the first
time that PG treatment attenuates hypoxia-induced endothelial dysfunction and pulmonary
hypertension. Second, we demonstrated that PG treatment improves NO-cGMP signaling
in the pulmonary arteries. Third, we demonstrated that the major mechanisms of PG-
mediated vascular protection are mediated by inhibiting oxidative stress and preventing
subsequent NO destruction.
The pomegranate fruit has been employed as a folk medicine for a variety of ailments,
including wound healing, diarrhea, and microbial infections (Murthy et al., 2004; Ismail
et al., 2012). In the recent years, pomegranate juice has been widely consumed for its
health-promoting effects. Punicalagin (PG) has been identified as the major active com-
pound in pomegranate juice, which exerts beneficial effects similar to pomegranate juice
by preventing nonalcoholic fatty liver disease (Zou et al., 2014) or reducing hypoxia-
induced cell death in human placental syncytiotrophoblasts (Chen et al., 2012). Previous
studies showed that pomegranate fruit extract rich in PG could reduce oxidative stress
and atherogenesis and improve vascular function in the carotid arteries of hypercholes-
terolemic mice, which demonstrates that PG has vascular protective effects (de Nigris et al.,
2007). Recent studies have shown that PG treatment can attenuate pulmonary pathological
changes and inhibit lung edema in the mouse model of LPS-induced ARDS (Peng et al.,
2015). These studies suggest that PG may have protective effects on pulmonary circulation.
Toxicity evaluations indicate that repeated oral administration of PG to rats at a dose of
4.8 g/kg/day over 37 days did not provoke toxic effects (Cerda et al., 2003). In this study,
regular nontoxic doses of PG were administered to both acute and chronic hypoxic rats.
It was found that relatively medium-dose (15 mg/kg/d) and high-dose (45 mg/kg/d) PG
treatments attenuated acute and chronic hypoxia-induced pulmonary hypertension. More-
over, PG treatment mitigated the histological changes in pulmonary arteries and alleviated
the right ventricle hypertrophy induced by chronic hypoxia.
The vascular endothelium is impaired when exposed to hypoxic environments, which
can disturb the balance among various vasomotor factors. Among these vasomotor factors,
NO is of great importance in the pathogenesis of hypoxia-induced pulmonary hyperten-

sion. The reduced production of NO has been deemed as an important pathogenic factor of
HPH (Veyssier-Belot and Cacoub, 1999; Hampl and Herget, 2000). Consistent with pre-
vious studies, this study demonstrated that the level of NO in the pulmonary arteries of
chronic hypoxic rats was significantly reduced. Meanwhile, isolated vascular perfusion
results demonstrated that pulmonary endothelial dysfunction was induced by chronic
hypoxia. It is believed that impaired NO production and endothelial dysfunction induced
by hypoxia may share a reciprocal causation relationship. It was shown that PG treatment
reversed NO reduction in pulmonary arteries and ameliorated endothelial dysfunction in
HPH rats.
Accumulating evidence has confirmed that oxidative stress participates in chronic
hypoxic pulmonary hypertension and vascular remodeling (Hoshikawa et al., 2001;
Makarenko et al., 2014; Tuleta et al., 2014). NADPH oxidase is the most important source
of superoxide anion (O 2 ) in vascular tissues (Rueckschloss et al., 2003; Tao et al., 2003).
In hypoxia, O
2 reacts with NO at a very fast rate to form peroxynitrite (ONOO ), which

can effectively reduce NO bioavailability. Moreover, the formation of ONOO represents
a major mechanism of tissue injury, including substantial oxidation and potential damage
of host cellular constituents (Virag et al., 2003). In this study, it was shown that increased
superoxide generation, gp91 phox expression (a critical component of NADPH oxidase) and
3-nitrotyrosine content (a footprint of ONOO formation) were reduced in the pulmonary
arteries of HPH rats receiving PG treatment. Meanwhile, PG treatment enhanced vascular
anti-oxidant capacity as evidenced by increased anti-oxidant enzyme SOD activity in pul-
monary arteries of HPH rats. Moreover, PG treatment reduced the downstream molecule
expression or cytokine production regulated by ROS in HPH rats, which further confirms the
anti-oxidant effects of PG. The anti-oxidant action of PG has been studied on cerebral
ischaemia/reperfusion injury in vivo (Yaidikar et al., 2014) and lipopolysaccharide-stimu-
lated macrophage activation in vitro (Xu et al., 2015). This is the first study to indicate that
PG protects vascular tissues from hypoxia-induced oxidative stress in HPH rats.
To further clarify the role of oxidative stress in PG-induced vascular protective effects, a
superoxide scavenger tempol was applied in this study. Consistent with previous studies
(Simonsen et al., 2009; Bourgoin et al., 2013), tempol treatment significantly reduced
superoxide generation, increased NO production and improved endothelial function in
14 J. SHAO et al.
HPH rats, which further demonstrates that oxidative stress can lead to NO destruction and
endothelial dysfunction. Tempol, being a superoxide scavenger, also decreased pulmonary
arterial hypertension in HPH rats. More importantly, the co-treatment of PG and tempol
did not have additional beneficial effects compared to tempol treatment alone. These data
indicate that PG may exert its vascular protective effects against hypoxia mainly via its
anti-oxidant effects. In addition, HIF-1α and VEGFA play an important role in the path-
ogenesis of HPH and hypoxic pulmonary vascular remodeling, and PG inhibited hypoxia-
induced HIF-1α and VEGFA expression in HPH rats. It is necessary to point out that PG
may prevent against HPH also by suppressing HIF-1α/VEGF-dependent pathways.
In conclusion, we demonstrated for the first time that punicalagin treatment effectively
improves endothelial function and prevents hypoxic pulmonary hypertension via its anti-
oxidative stress property, which prevents NO destruction. Consequently, these results

suggest punicalagin as a protective nutrient against HPH, which may be a promising and
safe complementary medicine for the prevention of HPH in clinical practice.
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The American Journal of Chinese Medicine, Vol. 44, No.

Punicalagin Prevents Hypoxic

*Department of Respiratory Medicine

Xi’an Central Hospital Xi’an 710003, China

Published 24 May 2016

Keywords: Punicalagin; Hypoxic Pulmonary Hypertension; Endothelial Function; Nitric

Pulmonary hypertension (PH) is a life-threatening disease characterized by increased

thelial dysfunction in the pulmonary vascular endothelium (Hoshikawa et al., 2001;

Materials and Methods

HPH Animal Models

Hemodynamic Measurements and Evaluation of Right Heart Hypertrophy

The rats were anesthetized by an injection of 3% pentobarbital sodium (60 mg/kg)

Assessment of Vascular Remodeling

Determination of Vascular Function

Determination of NO and cGMP Content

Quantiﬁcation of Superoxide Production, Superoxide Dismutase Activity

Lucigenin-enhanced chemiluminescence was used to assess superoxide production in

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were

Quantitative Real-Time PCR

subjected to reverse transcription using superscriptr jjj ﬁrst-strand systhesis system

Determination of Serum TNF-α Levels

PG Prevented Chronic Hypoxia-Induced Pulmonary Hypertension, Right Ventricular

PG Attenuated Chronic Hypoxia-Induced Endothelial Dysfunction

PG Increased NO and cGMP Content in HPH Rats

To reveal the potential mechanism of PG treatment on endothelial dysfunction, the contents

PG Attenuated Hypoxia-Induced Oxidative Stress in HPH Rats

Considerable evidence suggests that increased superoxide production and subsequent NO

(A) (B) (C)

(D) (E) (F)

PG Inhibited Chronic Hypoxia-Induced HIF-1α and VEGFA Expression

Anti-oxidant Stress Effect of PG as a Critical Mechanism Responsible

To further ascertain the degree of involvement of anti-oxidant action in the vascular

PG Attenuated Acute Hypoxic Pulmonary Hypertension in Rats

45 mg/kg) or vehicle, as previously described, 1 h before acute hypoxic exposure. Right

NO is of great importance in the pathogenesis of hypoxia-induced pulmonary hyperten-

oxidative stress property, which prevents NO destruction. Consequently, these results

safe complementary medicine for the prevention of HPH in clinical practice.

Physiol. Rev. 80: 1337–1372, 2000.

and S. Fujimura. Generation of oxidative stress contributes to the development of pulmonary

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