Visible-light-initiated Sonogashira coupling reactions over CuO/TiO2 nanocomposites

Article abstract of DOI:10.1039/c8cy02333f

CuO/TiO₂ nanocomposites with different weight ratios of CuO were obtained by immersing TiO₂ in aqueous solution of Cu(NO₃)₂, followed by heat treatment at 400 °C. The resultant CuO/TiO₂ nanocomposites were fully characterized and their catalytic performance for light induced Sonogashira coupling reactions was investigated. CuO/TiO₂ nanocomposites show catalytic activity for the Sonogashira coupling between aryl alkynes and aryl iodides under visible light, with optimum performance realized over the 20 wt% CuO/TiO₂ nanocomposite. UV-vis DRS reveals that metal-metal charge transfer (MMCT) of Ti(iii)/Cu(ii) exists in the CuO/TiO₂ nanocomposite. When irradiated, accompanied by an electron transfer from Ti(iii) to Cu(ii), the Ti(iii)/Cu(ii) moiety is excited to generate Ti(iv)/Cu(i). The as-formed Cu(i) coordinates with the deprotonated phenylacetylene to form copper phenylacetylide, which acts as an active intermediate for Sonogashira coupling reactions. This study not only provides an efficient method to realize Sonogashira coupling reactions under visible light, but also highlights the great potential of photocatalysis in light induced organic syntheses.

Full text of DOI:10.1039/c8cy02333f

Author’s Accepted Manuscript
Magnesium lithospermate B prevents phenotypic
transformation of pulmonary arteries in rats with
hypoxic pulmonary hypertension through
suppression of NADPH oxidase
Tao Li, Xiu-Ju Luo, E-Li Wang, Nian-Sheng Li,
Xiao-Jie Zhang, Feng-Lin Song, Jin-Fu Yang, Bin
Liu, Jun Peng
www.elsevier.com/locate/ejphar
PII:
S0014-2999(19)30028-7
https://doi.org/10.1016/j.ejphar.2019.01.020
EJP72161
DOI:
Reference:
To appear in:
European Journal of Pharmacology
Received date: 8 September 2018
Revised date: 8 January 2019
Accepted date: 15 January 2019
Cite this article as: Tao Li, Xiu-Ju Luo, E-Li Wang, Nian-Sheng Li, Xiao-Jie
Zhang, Feng-Lin Song, Jin-Fu Yang, Bin Liu and Jun Peng, Magnesium
lithospermate B prevents phenotypic transformation of pulmonary arteries in rats
with hypoxic pulmonary hypertension through suppression of NADPH oxidase,
European
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of
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https://doi.org/10.1016/j.ejphar.2019.01.020
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1.
Magnesium lithospermate B prevents phenotypic transformation of pulmonary arteries
in rats with hypoxic pulmonary hypertension through suppression of NADPH oxidase
Tao Li1, Xiu-Ju Luo2, E-Li Wang1, Nian-Sheng Li1, Xiao-Jie Zhang1, Feng-Lin Song3, Jin-Fu
Yang3, Bin Liu1,4*, Jun Peng1,5*
1Department of Pharmacology, Xiangya School of Pharmaceutical Sciences, Central South
University, Changsha 410078, China.
2Department of Laboratory Medicine, the third Xiangya Hospital, Central South University,
Changsha 410013，China.
3Department of cardiovascular surgery, the second Xiangya Hosptial, Central South
University, 410011 Changsha China
4Department of Pharmacy, Xiangya Hospital, Central South University, Changsha 410008,
China.
5Hunan Provincial Key Laboratory of Cardiovascular Research, School of Pharmaceutical
Sciences, Central South University, Changsha 410078, China
liubin@csu.edu.cn
Junpeng@csu.edu.cn
*Correspondence to: Department of Pharmacy, Xiangya Hospital, Central South University,
No. 87 Xiangya Road, Changsha, 410008, China.
*Correspondence to: Department of Pharmacology, School of Pharmaceutical Sciences,
Central South University. No.110 Xiang-Ya Road, Changsha, 410078, China. Tel:
0086-731-82355080；Fax: 0086-731-82355078.
ABSTRACT
Magnesium lithospermate B (MLB) shows multiple biological activities including
anti-oxidation and anti-proliferation in various diseases. However, the function of MLB in
pulmonary arterial hypertension (PAH) is still unknown. This study aims to investigate the
effect of MLB on hypoxia-induced phenotypic transformation of pulmonary arterial smooth
muscle cells (PASMCs) and the underlying mechanisms. SD rats (or PASMCs) were exposed
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to 10% O2 for 3 weeks ( or 3% O2 for 48h) along with MLB or NADPH oxidase （NOX）
inhibitor intervention. The effects of MLB on hemodynamics, pulmonary vascular remodeling
and phenotypic transformation of PASMCs were observed first. Then, its effects on the
protein levels of NOX (NOX2 and NOX4), ERK and p-ERK were examined. The results
showed that MLB prevented the elevation in right ventricular systolic pressure and the
increase in ratio of wall thickness to vessel external diameter of pulmonary arteries in PAH
rats, and attenuated phenotypic transformation of PASMCs (decrease in α-smooth muscle
actin while increase in osteopontin), accompanied by downregulation of NOX (NOX2 and
NOX4) protein levels, decrease of ROS and H2O2 production, and suppression of the
phosphorylation of ERK. NOX inhibitor (VAS2870) achieved similar results to that of MLB
did in the hypoxia-treated PASMCs. Based on the observations, we conclude that MLB is able
to prevent phenotypic transformation of pulmonary arteries in hypoxic PAH rats through
suppression of NOX/ROS/ERK pathway, and MLB might have the potentials in PAH
therapy.
Graphical abstract
fx1
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Key Words: Magnesium lithospermate B, pulmonary arterial hypertension, phenotypic
transformation, pulmonary vascular remodeling, NADPH oxidase
1. Introduction
Pulmonary arterial hypertension (PAH) is a destructive and progressive disease
characterized by elevation of pulmonary vascular resistance, which leads to increase of
pulmonary arterial pressure and eventually right heart failure (Li et al., 2018). Pulmonary
vascular remodeling is the major pathological features of PAH and the excessive proliferation
of pulmonary artery smooth muscle cells (PASMCs) is an important factor contributing to
vascular remodeling (Abe et al., 2010; Zurlo et al., 2018). PASMCs may undergo phenotypic
transition from a differentiated phenotype (contractile phenotype) to a dedifferentiated
phenotype (synthetic phenotype) in response to different stimuli such as hypoxia (Charles et
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al., 2018; Xie et al., 2011). The contractile phenotype is characterized by high expression of
contractile genes with a low rate of proliferation, such as α-smooth muscle actin (α-SMA) and
smooth muscle 22α (SM22α). Conversely, synthetic phenotype expresses low level of
contractile genes with a high rate of proliferation, such as osteopontin (OPN) and cyclin D1
(Owens et al., 2004). Therefore, the phenotypic transformation of PASMCs from contraction
to synthesis is recognized as a key step for vascular remodeling in the development of PAH
(Zhang et al., 2018b). However, the mechanisms responsible for phenotypic transformation of
PASMCs are not fully understood.
There is evidence that reactive oxygen species (ROS) are involved in various aspects of
pulmonary vasculature under physiological or pathological conditions, and NADPH oxidases
(NOX) - derived products are considered as the primary source of ROS (Jin et al., 2016;
Schroder et al., 2012). Reports from other labs and ours have repeatedly demonstrated that
NOX are up-regulated in animals or humans with PAH, suggesting that NOX-derived ROS
may contribute to the development of PAH (Jin et al., 2016; Liu et al., 2014; Schroder et al.,
2012). For example, NOX-derived ROS can either cause endothelial dysfunction through
oxidative injury or function as a signaling molecule to promote PASMCs proliferation and
migration (Liu et al., 2016). Since phenotypic transformation from contraction to synthesis
leads to acceleration of PASMCs proliferation, we thus hypothesize that NOX-derived ROS
may contribute to phenotypic transformation of PASMCs in the development of PAH, and
targeting NOX might be a useful strategy for PAH therapy.
Actually, NOX inhibitors, such as apocynin and diphenyleneiodonium, have been
reported to be effective on PAH (Peng et al., 2017). However, most inhibitors used for NOX
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intervention are not specific and they lack clinical value. Recently, we have found that
magnesium lithospermate B (MLB), an active component of the water-soluble fraction of
Salvia miltiorrhiza, might be a novel inhibitor of NOX because its protective effect on
cerebral ischemia/reperfusion injury in rats was related to inhibition of NOX (Lou et al.,
2015). In another report, MLB was able to prevent both injury-induced neointimal formation
in vivo and platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell
proliferation in vitro (Hur et al., 2008). Based on these reports, we predict that MLB can
prevent the development of PAH though mechanisms involving blocking the expression of
NOX and the phenotypic transformation of PASMCs.
In the present study, by using a rat model of hypoxia-induced PAH, we investigated
whether MLB was able to prevent the phenotypic transformation of pulmonary arteries and
whether the preventive effect of MLB on PAH is related to suppression of NOX. By using an
in vitro model of hypoxia-induced phenotypic transformation of PASMCs as well as the
specific inhibitor of NOX (VAS2870), we verified the preventive effect of MLB on
hypoxia-induced phenotypic transformation and the possible mechanisms.
2. Materials and methods
2.1. Ethics statement
Male Sprague-Dawley (SD) rats (180-220g) were obtained from Laboratory Animal
Center, Xiangya School of Medicine, Central South University, China. All procedures
performed on the rats were conducted according to the guidelines from the National Institutes
of Health (NIH Publication, 8th edition, 2011) and the ARRIVE guidelines (Animal Research:
Reporting In Vivo Experiments). The experiments were approved by the Central South
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University Veterinary Medicine Animal Care and Use Committee.
2.2. Experimental protocol for animals
SD rats were randomly allocated to 4 groups as follows (n = 6 per group): a normoxia
group, a hypoxia group, a hypoxia plus MLB (L) group, and a hypoxia plus MLB (H) group.
For the normoxia group, rats were placed in a chamber with normobaric normoxia (21% O2);
for the hypoxia group, rats were placed in a chamber and exposed to 10% O2 continuously for
3 weeks (the door of the chamber was opened for a while every day for supplementary feed
and water as well as replacing cage bedding); for the hypoxia plus MLB (L) or MLB (H)
group, rats were given MLB (purity≥90.0%, provided by Greenvalley Pharmaceutical
Company, Shanghai, China) at dosage of 5 or 15 mg/kg/d (i.p., dissolved in normal saline)
and subjected to hypoxia for 3 weeks. No animal was dead in all experimental groups in the
whole process. At the end, the rats were anesthetized with pentobarbital (30 mg/kg, i.p.) and
subjected to measurement of right ventricle systolic pressure (RVSP). After killing the
anesthetized rats, the lungs were saved for hematoxylin-eosin, Masson staining and
biochemical analysis (ROS level), and the pulmonary arterial tissues were isolated for
molecular studies (relevant protein expression).
2.3. Experimental protocol for cultured cells
The in vitro experiments were designed to verify the effect of MLB on phenotypic
transformation of PASMCs. The rat primary PASMCs were isolated from the pulmonary
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arteries and the passage 3~6 cells were chosen for the following studies.
The first set of in vitro experiments was designed to optimize the condition for
hypoxia-induced phenotypic transformation of PASMCs, which mimics the hypoxic
treatment in vivo. The PASMCs were plated in 6-well plates and were quiescent for 24 h in
DMEM supplemented with 0.1% serum. Then the cells were subjected to normoxia or
hypoxia (3% O2, 92% N2 and 5% CO2) treatment for 6h, 12h, 24h or 48h. At the end of
experiments, cell viability and relevant protein levels for phenotypic transformation were
measured. Based on the results from time-course experiments, the condition of 48h-hypoxia
was chosen for the following cell culture studies. Under this time condition, a dose-effect
relationship between MLB and PASMCs cell viability was performed. Based on this
experiment, a concentration of 20 μM for MLB was chosen for the following cell culture
experiments.
The second set of experiments was designed to evaluate the effect of MLB on
hypoxia-induced phenotypic transformation of PASMCs and the potential mechanisms. The
cells were divided into 5 groups (6 individual experiments per group): the control group,
PASMCs were cultured under normal conditions; the hypoxia group, PASMCs were
subjected to hypoxia for 48h; the hypoxia plus MLB group, MLB was added to the culture
medium (20 M, final concentration) before the hypoxia treatment; the hypoxia plus
VAS2870 (a specific inhibitor of NOX, dissolved in DMSO) group, VAS2870 was added to
the culture medium (10 M, final concentration) before the hypoxia treatment; and the
hypoxia plus vehicle group, equal volume of vehicle (DMSO) was added to culture medium
before the hypoxia treatment. At the end of experiments, the cells were collected for
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biochemical analysis (ROS level and H2O2 concentration) and molecular studies (relevant
protein expression).
2.4. Hematoxylin-eosin (HE) and Masson’trichrome staining
To compare the morphological changes in pulmonary arteries among the experimental
groups, HE staining was performed. Briefly, lung tissues were fixed with 4%
paraformaldehyde and embedded in paraffin and then cut into 5μm sections. The slices were
stained with HE (Beyotime, Jiangsu, China) and a minimum of 10 microscopic fields from
each slide were randomly selected for observation under a microscope (Olympus Corporation,
Tokyo, Japan). For quantitation of pulmonary arterial wall thickness, the lumen diameter (or
area at the level of the basement membrane) and total vascular diameter (or area at the
adventitial border) in 10 muscular arteries with diameter of 50-100 μm per lung section were
outlined. The diameters and area sizes were measured with Image J (NIH, USA). The
vascular wall thickness was calculated as follows: wall thickness (WT) = (total vascular
diameter – lumen diameter)/ total vascular diameter; wall area (WA) = (total vascular area –
lumen area)/ total vascular area.
To determine the extent of collagen deposition in pulmonary arterioles, Masson’
trichrome staining was performed following the instruction provided by the kit supplier (Baso
Biotech, Zhuhai, China).
2.5. Cell viability assay
Cell viability was measured with a MTS Assay Kit according to the protocol provided by
the manufacturer (Promega, USA). Briefly, PASMCs were seeded at the 96-well plates and
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starved for 24 h. Then MTS was added to culture medium DMEM [MTS: DMEM (v/v) =
1:10 per well]. The cells were incubated at 37°C for 2.5 h and the absorbance was examined
at 490nm by a spectrophotometer.
2.6. Cell proliferation assay
The proliferation of PASMCs was evaluated with EdU Incorporation Assay Kit
following the protocol provided by the manufacturer (RiboBio Co. Ltd., Guangzhou, China).
In brief, EdU (50 μM) was added to the culture medium and incubated at 37°C for 2 h. Then
the cells were fixed, stained, and imaged by fluorescent microscopy (Olympus IX71, Japan).
The EdU-positive cells were counted and normalized to the total number of DAPI-stained
cells.
2.7. Detection of reactive oxygen species (ROS) level
The ROS level in lung tissues was detected by using dihydroethidium staining assay. In
brief, lung tissues were cut into 5μm-thick sections. The slices were stained with
dihydroethidium (10 μM) (Beyotime, Jiangsu, China) and then were incubated at 37 °C for 30
min in a dark humidified chamber. The fluorescence of ethidium were randomly selected for
observation under a microscope (Olympus IX71, Japan).
The measurement of intracellular ROS levels was dependent on the fluorescent signal of
2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), a cell-permeable indicator of ROS
(Beyotime, Jiangsu, China). DCFH-DA is nonfluorescent until the acetate groups are
removed by intracellular ROS. Briefly, PASMCs were washed with PBS and incubated with
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DCFH-DA (10 M) at 37℃for 20 min. Then, the ROS-mediated fluorescence was observed
under a fluorescent microscope with excitation set at 502 nm and emission set at 523 nm.
2.8. Measurement of hydrogen peroxide (H2O2) content
The detection of H2O2 is based on oxidation of ferrous (Fe2+) to ferric ion (Fe3+) in the
presence of xylenol orange. In a sulfuric acid solution, the Fe3+ complexes with the xylenol
orange dye to yield a purple product with maximum absorbance at 560 nm. For measurement
of H2O2 level, 50 μL of supernatant of pulmonary tissue or PASMCs homogenates and 100 μL
of work solution (0.25mM ammonium ferrous II sulfate, 25mM H2SO4, 100 mM sorbitol, 125
μM xylenol orange) were mixed and incubated at room temperature for 20 min. The change
of absorbance at 560 nm was monitored and the levels of H2O2 were calculated according to a
standard curve made from the standard solutions provided by the supplier (Beyotime, Jiangsu,
China).
2.9. Western blot analysis
Pulmonary arterial tissues or PASMCs were homogenized in ice-cold lysis buffer (250
mmol/L Tris-HCl, pH 6.8; 4% SDS, 20% vol/vol glycerol and Roche EDTA-free protease
inhibitor cocktail) and sonicated for 1 min, then centrifuged for 15 min at 15000g. The
protein concentration in homogenate was determined by a BCA Protein Assay kit (Beyotime,
Jiangsu, China). Western blot were performed according to standard techniques. Briefly,
samples containing 20 - 60 μg of protein were subjected to SDS-PAGE (10% gel) and they
were transferred to polyvinylidene fluoride (PVDF) membranes. The blots were then
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incubated with primary antibodies against NOX2 (Santa Cruz, CA, USA), NOX4 (Santa Cruz,
CA, USA), SM22α (Abcam, Cambridge, MA, USA), α-SMA (BOSTER, Wuhan, China),
OPN (Abcam, Cambridge, MA, USA), cyclin D1 (BOSTER, Wuhan, China) or ERK/p-ERK
(Cell Signaling Technology, Danvers, USA) followed by HRP-conjugated secondary
antibodies (Beyotime, Jiangsu, China). The signals were detected by Luminata™ Creseendo
Western HRP substrate through Molecular Imager ChemiDoc XRS System (Bio-Rad,
Philadelphia, PA). The densitometric quantification was conducted with Image J (NIH, USA).
Antibodies against -actin (Beyotime, Jiangsu, China) were used as loading controls.
2.10. Statistical analysis
SPSS software (Version 20) was used for statistical analysis. Data were expressed as
mean ± S.E.M.. Differences in values among the multiple groups were determined by the
analysis of variance with Bonferroni’s multiple comparison tests. Differences were
considered significant when P < 0.05.
3. Results
3. 1. MLB prevented the development of hypoxia-induced pulmonary hypertension
Pulmonary hypertension was developed after the rats continuously exposed to hypoxia
(10% O2) for 3 weeks. Compared with the normoxia group, the right ventricular systolic
pressure (RVSP) in the hypoxia group were significantly elevated; these phenomena were
markedly attenuated by MLB in a dose-dependent manner (5 and 15 mg/kg) (Fig. 1 A and B).
As shown in Fig. 1 C-E, the ratio of wall thickness to vessel external diameter (WT %)
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and the ratio of wall area to total vessel area (WA %) in the small pulmonary arteries were
significantly increased in the hypoxia-treated rats as compared with that in the normoxia
group. The hypoxia-induced pulmonary vascular remodeling was reduced by MLB in a
dose-dependent manner. Consistent with the vascular remodeling, Masson’ trichrome staining
showed that the increased collagen deposition around pulmonary vessels in hypoxia-treated
rats, these phenomena were attenuated in the presence of MLB (Fig. 1 F).
3.2. MLB suppressed hypoxia-induced phenotypic transformation of pulmonary arteries
As shown in Fig.2, pulmonary arteries from hypoxia-treated rats exhibited phenotypic
transformation from a contractile to a synthetic type, as evidenced by down-regulation of
contractile proteins α-smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α) while
up-regulation of the synthetic protein osteopontin (OPN) and proliferative protein cyclin D1.
The hypoxia-induced phenotypic transformation of pulmonary arteries was suppressed by
MLB in a dose-dependent manner.
3.3. MLB blocked hypoxia-induced NOX expression and ERK phosphorylation in pulmonary
arteries
Compared to the normoxia group, hypoxia treatment caused a significant increase in
NOX (NOX2 and NOX4) protein levels in the pulmonary arteries of rats, which was blocked
by MLB in a dose-dependent manner (Fig.3 A and B). Consistently, ROS level and H2O2
content (a NOX - derived product) in the hypoxia group was significantly increased (Fig.3 C
and D); this phenomenon was reversed in the presence of MLB. Moreover, the
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phosphorylation level of ERK (p-ERK) in pulmonary arteries was apparently elevated in
hypoxia-treated rats, which was blocked in the presence of MLB (Fig.3 E). However, there
was no change in total ERK level in all groups.
3.4. Establishment of hypoxia-induced phenotypic transformation of PASMCs in vitro
Primary PASMCs were cultured under hypoxic condition for 6h, 12h, 24h or 48h to
establish an in vitro model of phenotypic transformation. As shown in Fig. 4, compared to the
normoxia group, the cell viability was increased in a time course-dependent manner
concomitant with a down-regulation of the contractile proteins α-SMA and SM22α while
up-regulation of the synthetic protein OPN and proliferative protein cyclin D1, suggesting the
establishment of hypoxia-induced phenotypic transformation of PASMCs in vitro was
successful. Based on the results, the 48h-hypoxia was chosen for the following cell culture
studies. Under this time condition, MLB suppressed hypoxia-induced PASMCs proliferation
in a dose-dependent manner with IC50 at ~ 60.75 μM (Figure S1). Based on this experiment,
we chose MLB at 20 μM for the following cell culture experiments.
3.5. MLB inhibited hypoxia-induced phenotypic transformation of PASMCs in vitro
Consistent with the results in vivo, the hypoxia-treated PASMCs exhibited phenotypic
transformation from a contractile to a synthetic type, as evidenced by down-regulation of the
contractile proteins α-SMA and SM22α while up-regulation of the synthetic protein OPN.
The hypoxia-induced phenotypic transformation of PASMCs was inhibited in the presence of
MLB. Similarly, VAS2870, a specific inhibitor of NOX, could also suppress the
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hypoxia-induced phenotypic transformation, but the vehicle of VAS2870 has no such effect
(Fig. 5 A-C).
In agreement with results of hypoxia-induced phenotypic transformation, PASMCs
proliferation was significantly accelerated under hypoxic condition, as revealed by an
increase in the EdU-positive cells and the proliferative protein cyclin D1; these phenomena
were attenuated in the presence of MLB or VAS2870, but the vehicle of VAS2870 did not
show such effect (Fig. 5 D-F).
3.6. MLB suppressed hypoxia-induced NOX expression and ERK phosphorylation in PASMCs
in vitro
Consistent with the results in vivo, the hypoxia-treated PASMCs exhibited up-regulation
of NOX2 and NOX4 (Fig.6 A and B), accompanied by an increase in ROS level in cells and
H2O2 contents in both culture medium and cells (Fig. 6 C-E); these increases were suppressed
in the presence of MLB or VAS2870. Furthermore, the levels of p-ERK in hypoxia-treated
PASMCs was apparently elevated, which was attenuated in the presence of MLB or
VAS2870 (Fig. 6 F). However, there was no change in total ERK level in all groups.
4. Discussion
In this study, we explored the effects of MLB on hypoxia-induced phenotypic
transformation of PASMCs and the underlying mechanisms. Our data showed that
administration of MLB significantly prevented phenotypic transformation of pulmonary
arteries in PAH rats, accompanied by a decrease in pulmonary vascular remodeling while an
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increase in NOX (NOX2 and NOX4) protein expression and H2O2 production; these in vivo
findings were further confirmed in hypoxia-treated PASMCs in vitro. To the best of our
knowledge, this is the first study to provide evidence that MLB may exert preventive effect on
PAH through prevention of phenotypic transformation of PASMCs; this effect is related to
down-regulation of NOX2 and NOX4.
Pulmonary artery remodeling is a basic pathological characteristic for PAH (Ranchoux et
al., 2015). In this study, rats continuously exposed to hypoxia for 3 weeks significantly
induced pulmonary artery remodeling, evidenced by an increase in the ratio of wall thickness
to vessel external diameter and the ratio of wall area to total vessel area in the small
pulmonary arteries, concomitant with an elevation in the right ventricular systolic pressure,
indicating the PAH animal model was successfully established.
It is well recognized that hypoxia-induced hyperproliferation of PASMCs is a major
initial factor for pulmonary vascular remodeling in PAH (Zhang et al., 2018a). Different from
cardiac cells and skeletal muscle cells, smooth muscle cells can undergo phenotypic changes
under pathological conditions. For example, under hypoxic condition, PASMCs accelerate the
phenotypic transformation from contractile to synthetic phenotype. This phenotypic switching
obviously alters the functions of PASMC because the synthetic PASMCs migrate and
proliferate more easily than contractile ones. Moreover, synthetic PASMCs can produce up to
25 to 46-fold more collagen than contractile ones, all of which contribute to pulmonary
vascular remodeling in PAH (Owens et al., 2004). Consistent with the reports, the results of
the present study demonstrated a down-regulation of contractile proteins and an up-regulation
of synthetic proteins in the pulmonary arteries from hypoxia-treated rats, accompanied by a
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phenotype of vascular remodeling. Based on these results, it naturally thinks that prevention
of phenotypic transformation of PASMCs by a drug may have a promising prospect for PAH
therapy. In the present study, our results indeed showed that MLB significantly prevented
phenotypic transformation of pulmonary arteries from contractile to synthetic phenotype in
PAH rats concomitant with improved pathological characteristics (decrease in RVSP, WT %
and WA %), supporting that MLB might be a potential candidate for the prevention and
treatment of PAH. However, the underlying mechanisms remain to be determined. It is
noteworthy that we only showed the preventive effect of MLB on PAH in the present study.
Although it might be able to exert therapeutic effect on PAH, further studies are necessary
before drawing a firm conclusion.
MLB is a water-soluble active ingredient from Danshen. Early studies mainly focused on
the lipophilic compounds of Danshen while recent studies have turned to hydrophilic
compounds. So far, at least 50 chemical constituents have been isolated and identified from
the aqueous extracts of Danshen. Among them, MLB is a major hydrophilic compound of
Danshen and preserves its most pharmacological activities, such as anti-vasospasm,
anti-inflammation and anti-oxidation (Chang et al., 2011; Lou et al., 2015; Park et al., 2017).
There was evidence that MLB could inhibit PDGF-induced vascular smooth muscle cell
proliferation and migration by suppressing ROS production (Hur et al., 2008). Based on this
report, we hypothesize that MLB prevent phenotypic transformation of pulmonary arteries in
PAH rats through a mechanism involving its anti-oxidative property. As a major source of
ROS in cardiovascular system, NOX (particularly the NOX2 and NOX4 subunits) are
activated under multiple pathological conditions (Liu et al., 2014; Ma et al., 2013).
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NOX-derived ROS contribute to pulmonary artery remodeling, endothelial dysfunction,
myocardial or cerebral ischemia/reperfusion injury (Lou et al., 2015; Zhang et al., 2012). We
thus examined the correlation between MLB and NOX. We have found that MLB could
significantly block NOX2 and NOX4 expression in pulmonary arteries of PAH rats
concomitant with a decrease in H2O2 content, which confirmed our hypothesis before.
It is well known that ROS not only cause oxidative injury but also function as a second
messenger to regulate intracellular signaling pathways (Vara and Pula, 2014). Thus, we
focused on identification of the potential signaling molecules that involves in phenotypic
transformation of pulmonary arteries in PAH rats. As a member of MAPK, ERK plays a key
role in regulation of proliferation and migration in different type of cells (Sun et al., 2015).
Since H2O2 is able to activate ERK in human pulmonary artery smooth muscle cell under
hypoxic condition (Bijli et al., 2015), we therefore examined the effect of MLB on ERK
phosphorylation in rat pulmonary artery. The results from the present study really showed that
the levels of p-ERK in pulmonary arteries were apparently elevated in the hypoxia-treated rats,
which were reversed in the presence of MLB. These results indicate that MLB prevents
phenotypic transformation of pulmonary arteries in PAH rats through suppressing the
NOX/ROS/ERK pathway. Previous report showed that MLB was able to decrease
endothelin-1 through an NO-dependent mechanism, which led to reduction of experimental
vasospasm in rats (Chang et al., 2011). Since endothelin-1 plays an important role in PAH
development, thereby we could not rule out the possibility that the inhibitory effect of MLB
on phenotypic transformation of pulmonary arteries in PAH rats might be also related to
decrease of endothelin-1 production.
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To further confirm our findings in vivo, we performed cell experiments in primary
cultured PASMCs with MLB and VAS2870 (a NOX specific inhibitor). Here, VAS2870 was
used as the a positive control for NOX inhibition (Lou et al., 2015). Consistent with the
findings in vivo, the primary cultured PASMCs displayed phenotypic transformation from a
contractile to a synthetic type concomitant with an up-regulation of NOX(NOX2 and NOX4)
and an increase in H2O2 production as well as an elevation in p-ERK level; these phenomena
were attenuated in the presence of MLB or VAS2870.
In conclusion, the results from this study have demonstrated for the first time that MLB
exerts the preventive effect on PAH through blocking the phenotypic transformation of
pulmonary arteries; and this action is related to suppression of the NOX/ROS/ERK pathway.
Therefore, MLB may have the potential value in clinic for PAH prevention and/or therapy.
Acknowledgements
This work was supported by National Natural Science Foundation of China (No. 81703516 to
Bin Liu, No. 81573430 to Xiu-Ju Luo, No. 81872873 to Jun Peng), Major Research Plan of
the National Natural Science Foundation of China (No. 91439104 to Jun Peng; No. 91539111
to Jin-Fu Yang).
Author Agreement
We state here that all authors have seen and approved the final version of the manuscript
being submitted. They warrant that the article is the authors' original work, hasn't received
prior publication and isn't under consideration for publication elsewhere.
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Figure 1. MLB prevented the development of hypoxia-induced pulmonary hypertension
A. Representative images for the recording of RVSP. B. The value of RVSP. C.
Representative images of HE staining for lung tissues (arrows indicate the pulmonary artery).
D. The ratio of wall thickness to total vessel external diameter for pulmonary artery. E. The
ratio of wall area to total vessel area for pulmonary artery. F. Representative images of
Masson’ trichrome staining for lung tissues. The data are presented as mean ± S.E.M.(n=6 per
group). MLB, Magnesium lithospermate B; RVSP, right ventricle systolic pressure; +MLB(L):
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hypoxia + MLB (5mg/kg/d); +MLB(H): hypoxia + MLB (15mg/kg/d). **P﹤0.01 vs
Normoxia; ##P＜0.01 vs Hypoxia.
Figure 2. MLB suppressed hypoxia-induced phenotypic transformation of pulmonary
arteries The protein levels of α-SMA (A), SM22α (B), osteopontin (OPN, C) and cyclin D1
(D). Top, optical density of protein band. Bottom, representative images of Western blot. All
values are expressed as mean ± S.E.M. (n=6 per group). MLB, Magnesium lithospermate B;
+MLB(L): hypoxia + MLB (5mg/kg/d); +MLB(H): hypoxia + MLB (15mg/kg/d). **P﹤0.01
vs Normoxia; #P＜0.05 vs Hypoxia.
Figure 3. MLB blocked hypoxia-induced NOX expression and ERK phosphorylation in
pulmonary arteries. The protein levels of NOX2 (A) and NOX4 (B) in pulmonary arteries.
Top, optical density of protein band. Bottom, representative images of Western blot. C.
Detection of ROS level in lung tissues by dihydroethidium staining. D. H2O2 content in lung
tissues. E. The protein levels of p-ERK and ERK in pulmonary arteries. Top, optical density
of protein band. Bottom, representative images of Western blot. All values are expressed as
mean ± S.E.M.(n=6 per group). MLB, Magnesium lithospermate B; +MLB(L): hypoxia +
**
MLB (5mg/kg/d); +MLB(H): hypoxia + MLB (15mg/kg/d). P﹤0.01vs Normoxia; #P＜0.05,
##P＜0.01vs Hypoxia.
Figure 4. Establishment of hypoxia-induced phenotypic transformation of PASMCs in
vitro A. Cell viability under hypoxic condition at different time points (n=5 per group). B-E.
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the protein levels of α-SMA, SM22α, osteopontin (OPN) and cyclin D1 in PASMCs under
hypoxic condition at different time points (n=3 per group). Top, optical density of protein
band. Bottom, representative images of Western blot. Data are presented as means ± S.E.M..
*P＜0.05, **P﹤0.01vs Normoxia.
Figure 5. MLB inhibited phenotypic transformation and proliferation in
hypoxia-treated PASMCs in vitro The protein levels of α-SMA (A), SM22α (B) and
osteopontin (OPN, C) in PASMCs. Top, optical density of protein band. Bottom,
representative images of Western blot. D. Representative images of EdU assay for PASMCs.
E. Percentage of EdU-positive cells. F. Cyclin D1 protein level in PASMCs. Top, optical
density of protein band. Bottom, representative images of Western blot. All values are
expressed as mean ± S.E.M.(n=3 per group). MLB, Magnesium lithospermate B; +MLB:
hypoxia + MLB; +VAS2870: hypoxia+VAS2870; +Vehicle: hypoxia +vehicle of VAS2870
(DMSO). **P < 0.01 vs Normoxia; #P＜0.05 vs Hypoxia.
Figure 6. MLB suppressed NOX expression and ERK phosphorylation in
hypoxia-treated PASMCs in vitro. The protein levels of NOX2 (A) and NOX4 (B). Top,
optical density of protein band. Bottom, representative images of Western blot. C.
Representative images for ROS detection with DCFH-DA in PASMCs. D. H2O2 level in
culture medium. E. H2O2 content in PASMCs. F. The protein levels of p-ERK and ERK in
PASMCs. Data are expressed as mean ± S.E.M. (n=3 per group). MLB, Magnesium
lithospermate B; +MLB: hypoxia + MLB; +VAS2870: hypoxia+VAS2870; +vehicle: hypoxia
+vehicle of VAS2870 (DMSO). **P﹤0.01 vs Normoxia; #P＜0.05 vs Hypoxia.
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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