Magnesium Lithospermate B Derived from Salvia miltiorrhiza Ameliorates Right Ventricle Remodeling in Pulmonary Hypertensive Rats via Inhibition of NOX/VPO1 Pathway

• Introduction
• Results
• Discussion
• Limitations of the study
• Materials and Methods
• Animals
• Experiments in animals
• Experiments in cells
• Morphological observations
• Measurement of ROS levels
• Measurement of H2O2 levels
• Measurement of HOCl levels
• Assay of cell viability
• Real-time PCR analysis
• Western blot analysis
• Statistical analysis
• References

Abstract

Right ventricle (RV) remodeling is a major pathological feature in pulmonary arterial hypertension (PAH). Magnesium lithospermate B (MLB) is a compound isolated from the roots of Salvia miltiorrhiza and it possesses multiple pharmacological activities such as anti-inflammation and antioxidation. This study aims to investigate whether MLB is able to prevent RV remodeling in PAH and the underlying mechanisms. In vivo, SD rats were exposed to 10% O₂ for 21 d to induce RV remodeling, which showed hypertrophic features (increases in the ratio of RV weight to tibia length, cellular size, and hypertrophic marker expression), accompanied by upregulation in expression of NADPH oxidases (NOX2 and NOX4) and vascular peroxidase 1 (VPO1), increases in hydrogen peroxide (H₂O₂) and hypochlorous acid (HOCl) production and elevation in phosphorylation levels of ERK; these changes were attenuated by treating rats with MLB. In vitro, the cultured H9c2 cells were exposed to 3% O₂ for 24 h to induce hypertrophy, which showed hypertrophic features (increases in cellular size and hypertrophic marker expression). Administration of MLB or VAS2870 (a positive control for NOX inhibitor) could prevent cardiomyocyte hypertrophy concomitant with decreases in NOX (NOX2 and NOX4) and VPO1 expression, H₂O₂ and HOCl production, and ERK phosphorylation. Based on these observations, we conclude that MLB is able to prevent RV remodeling in hypoxic PAH rats through a mechanism involving a suppression of NOX/VPO1 pathway as well as ERK signaling pathway. MLB may possess the potential clinical value for PAH therapy.

Introduction

Right ventricle (RV) remodeling is characterized by right heart dilatation and myocardial hypertrophy [1], [2], [3], which occurs in many diseases, such as pulmonary arterial hypertension (PAH), ventricular arrhythmias, and hypertrophic cardiomyopathy. PAH is a progressive disease that affects both the pulmonary vasculature and the heart. The initial insult in PAH mainly involves the pulmonary vasculature; however, the survival of PAH patients is closely related to RV function. In contrast to the treatment of pulmonary vasculature in PAH, the right heart is a viable therapeutic target [4]. In the process of PAH development, the RV adapts to the elevated pulmonary artery pressure by increasing its wall thickness and contractility. Besides the pressure overload, multiple factors including neurohumoral activation [5], oxidative stress [6], and inflammation [7] actually contribute a lot to the pathophysiology of RV remodeling in PAH. Among them, oxidative stress attracts intensive attention recently.

Oxidative stress is usually caused by the overproduction of reactive oxygen species (ROS) and NADPH oxidases (NOX), particularly NOX2 and NOX4, which are considered as the major source of ROS in cardiovascular system. Studies from other labs and ours have repeatedly demonstrated that NOX-derived ROS contributes greatly to myocardial/cerebral ischemic injury [8], [9], [10], endothelial dysfunction [11], [12], and vascular remodeling [13], [14]. There is growing evidence that NOX is activated in animals or humans with PAH, indicating that NOX-derived ROS may contribute to the development of PAH [15], [16], [17]. Furthermore, our previous studies have demonstrated that NOX coordinates with vascular peroxidase 1 (VPO1) to amplify the role of NOX-derived ROS in mediation of oxidative injury in myocardial ischemia/reperfusion injury and vascular remodeling in PAH [6], [18] because VPO1 can catalyze NOX-derived hydrogen peroxide (H₂O₂) to produce more powerful oxidant hypochlorous acid (HOCl). Based on these reports, we postulated that targeting NOX/VPO1 pathway might be a valuable strategy for the prevention of RV remodeling in PAH.

Although NOX inhibitors, such as apocynin and diphenyleneiodonium (DPI), showed therapeutic effects on PAH animals [17], they lacked clinical value due to the nonspecific effect and unacceptable toxicity. Actually, the efforts to seek ideal NOX inhibitors, particularly from Chinese traditional herbs, for prevention and therapy of PAH have never stopped. Magnesium lithospermate B (MLB) is a compound isolated from the roots of Salvia miltiorrhiza Bunge, which belongs to a plant family of Lamiaceae. It possesses pharmacological activities of anti-inflammation, antioxidation, anti-excitotoxicity, and anti-apoptosis [19], [20], [21]. Danshen salvianolate injection, containing more than 90.0% of MLB, is commonly used in clinics as an adjunctive therapy of ischemic heart disease [22]. Recently, we have found that salvianolate is able to protect the rat brain from ischemia/reperfusion injury through a mechanism involving inhibition of NOX [9]. As the major component of salvianolate injection, MLB might function as a novel NOX inhibitor because it achieved similar effect as VAS2870 (a specific NOX inhibitor) did [9].

The main purpose of this study is to explore the effect of MLB on RV remodeling in hypoxic PAH rats. By using a rat model of hypoxic PAH, we first evaluated the inhibitory effect of RV remodeling and its relevance to NOX/VPO1 pathway. To confirm the findings in vivo study, we established H9c2 cell model of hypoxia-induced hypertrophy in vitro to mimic the condition of hypoxia injury in vivo. Combining with VAS2870, a specific inhibitor of NOX, we confirmed that the inhibitory effect of MLB on cardiac cell hypertrophy is related to suppression of NOX/VPO1 pathway.

Results

PAH was developed in the Sprague-Dawley (SD) rats after continuous exposure to hypoxia (10% O₂) for 21 d. Compared to the normoxia group, the RV systolic pressure (RVSP) in the hypoxia group was significantly increased; these phenomena were attenuated by MLB at a dose of 15 mg/kg ([Fig. 1 A, B]). Continuous chronic hypoxia (10% O₂) for 3 wk resulted in RV remodeling in the rats, which exhibited increases in RV weight/body weight ([Fig. 1 C]), RV weight/(left ventricle [LV] + interventricular septum [IVS]) weight ([Fig. 1 D]), RV weight/tibia length ([Fig. 1 E]), and cellular size (cross-sectional area, [Fig. 1 F, G]); these increases were attenuated by MLB at a dose of 15 mg/kg. Consistently, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNA levels, 2 biomarkers of cardiac hypertrophy, were also significantly upregulated in the RV of PAH rats, which were suppressed in the presence of MLB at a dose of 15 mg/kg ([Fig. 1 H, I]).

The NOX (NOX2 and NOX4) protein levels were significantly upregulated in the RV of the PAH rats and the increase was blocked by MLB at a dose of 15 mg/kg ([Fig. 2 A, B]). Consistently, ROS and H₂O₂ levels (a NOX-derived product) in the RV from the PAH rats were strikingly elevated; this phenomenon was attenuated in the presence of MLB ([Fig. 2 C – E]).

In agreement with the upregulation of NOX2 and NOX4 in the RV from the PAH rats, the expression of VPO1 was also upregulated, which was reversed by MLB ([Fig. 3 A]). As expected, the HOCl levels (a product of VPO1) in the RV were increased in the PAH rats compared with that in the control rats; these changes were blocked by MLB at a dose of 15 mg/kg ([Fig. 3 B, C]). As shown in [Fig. 3 D], the phosphorylation of extracellular regulated protein kinase (ERK) in the RV was significantly enhanced in the PAH rats and the phenomenon was attenuated in the presence of MLB. However, the total ERK levels in the RV were not altered among all groups.

As shown in [Fig. 4 A], MLB did not exert significant effect on the viability of H9c2 cells under normoxia condition at 10 µM, 25 µM, and 50 µM, but it decreased the cellular viability at 100 µM. Thus, the concentration of 100 µM was excluded in the following experiments. Under hypoxic condition, MLB suppressed hypoxia-induced proliferation of H9c2 cells, and this suppression was reversed by MLB at a concentration of 50 µM, which was hence chosen for the subsequent experiments ([Fig. 4 B]).

Next, the effect of MLB on hypoxia-induced hypertrophy of H9c2 cells was evaluated. Consistent with the results in vivo, the hypoxia-treated H9c2 cells exhibited characteristics of hypertrophy, such as increase in cellular size ([Fig. 4 C]) and upregulation of ANP and BNP ([Fig. 4 D, E]); these increases were attenuated in the presence of MLB. Similarly, VAS2870, a specific inhibitor of NOX, also suppressed the hypoxia-induced hypertrophy. The vehicle of VAS2870 did not show such effects ([Fig. 4 C – E]).

Consistent with the results in vivo, the protein levels of NOX2 and NOX4 in H9c2 cells were significantly elevated under hypoxic condition ([Fig. 5 A, B]), concomitant with increases in the production of ROS and H₂O₂; these increases were blocked by MLB or NOX inhibitor, VAS2870 ([Fig. 5 C – E]). The vehicle of VAS2870 had no such effects.

Consistent with the upregulation of NOX2 and NOX4 in hypoxia-treated H9c2 cells, the expression of VPO1 was also upregulated, which was mitigated by MLB or VAS2870 ([Fig. 6 A]). As expected, the HOCl levels in hypoxia-treated H9c2 cells were significantly increased compared to those in normoxia cells; these changes were prevented by MLB or VAS2870 ([Fig. 6 B, C]). As shown in [Fig. 6 D], the phosphorylation levels of ERK in hypoxia-treated H9c2 cells were significantly elevated; this phenomenon was reversed in the presence of MLB or VAS2870. The vehicle of VAS2870 did not show such effects. The total ERK levels in the H9c2 cells were not changed among all groups.

Discussion

In this study, we evaluated the effects of MLB on RV remodeling in hypoxic PAH rats and the underlying mechanisms. Our results show that MLB ameliorated RV hypertrophy in PAH rats, accompanied by decreases in protein levels of NOX (NOX2 and NOX4) and VPO1, and production of H₂O₂ and HOCl. These findings were further confirmed in hypoxia-treated H9c2 cells in vitro. To the best of our knowledge, we are the first to provide evidence that MLB prevents the hypoxia-induced RV remodeling through inhibition of NOX/VPO1 pathway.

RV remodeling is an important pathological characteristic of PAH, which is characterized by myocardial hypertrophy [23]. In this study, we observed an elevation in the RVSP concomitant with a marked increase in RV weight, myocardial size and hypertrophic markers (ANP and BNP) in rats exposed to hypoxia for 21 d, suggesting that the PAH animal model was successfully established.

Actually, RV remodeling is a response to high pulmonary vascular resistance and vascular remodeling because they result in a striking increase in RV afterload. Thus, prevention or reverse of pulmonary vascular remodeling, which brings the decrease of pulmonary artery pressure, is considered the priority for PAH therapy. Besides intervention of pulmonary arteries, accumulating evidence suggests that directly targeting the RV with the aim of supporting and protecting the right side of the heart also possesses the potential to improve cardiac function in patients or animals with PAH [24], [25]. During the development of PAH, multiple mechanisms are believed to be responsible for the right ventricular remodeling in response to the chronically elevated pulmonary artery pressure. Among them, oxidative stress is a well-recognized factor that contributes to RV remodeling in PAH [26]. We thus focused on the role of oxidative stress in RV remodeling in this study.

It has been shown that NOX-derived ROS plays a key role in mediation of oxidative stress in cardiovascular system under pathological conditions. NOX generates ROS through transferring electrons from NADPH to molecular oxygen. To date, at least 7 isoforms of NOX have been identified in humans and 6 in rodents (rats and mice). Although NOX1, NOX2, and NOX4 are expressed in cardiovascular systems including the pulmonary vasculature, only NOX2 and NOX4 are considered the major sources for ROS production in the cardiovascular system under pathological conditions [27]. Under normal conditions, NOX constitutively generates low levels of ROS, but it can be activated acutely under condition of ischemia [28], hypoxia [29], hyperlipidemia [30], high glucose [31], or high blood pressure [32]. Consistent with the reports, NOX2 and NOX4 protein levels were evidently elevated in the RV of hypoxic PAH rats or in hypoxia-treated H9c2 cells. The direct product of NOX is superoxide anion (O₂ ^.- ), which can be rapidly converted to H₂O₂ by superoxide dismutase. As a substrate of VPO1, H₂O₂ can be further catalyzed by VPO1 to form a stronger oxidant product HOCl [33]. Through the coordination between NOX and VPO1, the oxidative stress is markedly enhanced. Thus, the NOX/VPO1 pathway-mediated oxidative stress is reported to be involved in smooth muscle cell proliferation [34], cardiomyocyte or endothelial cell apoptosis, or senescence [18], [35]. As expectedly, in the present study, with the upregulation of NOX2 and NOX4 in the RV of hypoxic PAH rats or hypoxia-treated H9c2 cells, VPO1 was also upregulated concomitant with an elevation in H₂O₂ and HOCl production, indicating that the NOX/VPO1 pathway-mediated oxidative stress contributes to the RV remodeling in PAH rats. Thereby, intervention of the NOX/VPO1 pathway could prevent or reverse the RV remodeling in PAH rats.

To date, several NOX inhibitors have been identified and some of them have been reported to be effective against PAH, such as apocynin and DPI. However, their clinical value was denied due to the nonspecific effects or unacceptable toxicities. Recently, great efforts have been made to develop novel NOX inhibitors, such as VAS2870, GK-136901 and NOX2ds, but their clinical values have not yet been determined [36]. Interestingly, some natural compounds exert therapeutic effect on PAH via targeting NOX. For example, a number of studies have reported that resveratrol was able to prevent the development of PAH through a mechanism involving a suppression of NOX [6]. As an active ingredient of S. miltiorrhiza, MLB attracts our special attention because its antioxidative effect is greater than that of α-lipoic acid, a super antioxidant. It is reasonable to speculate that MLB exerts its antioxidative effect through suppression of NOX. In fact, we have recently reported that MLB protected rat brain against ischemia/reperfusion injury through downregulation of NOX2 and NOX4 [9]. In this study, we have extended our previous findings that MLB exerted therapeutic effect on PAH via targeting NOX because MLB ameliorated the RV hypertrophy in PAH rats and cardiac cell hypertrophy in hypoxia-treated H9c2 cells concomitant with the downregulation of NOX2 and NOX4. Furthermore, we have confirmed that VPO1 was coupled with NOX because inhibition of NOX by MLB or VAS2870 (a positive control) also downregulated VPO1 and its product HOCl in the RV of PAH rats and hypoxia-treated H9c2 cells. Since reduction of the increased pulmonary pressure in PAH rats by MLB could also prevent RV remodeling, we could not rule out the possibility that the inhibitory effect of MLB on RV remodeling is due to, at least in part, by its hypotensive effect, although it has the direct effect on cultured cardiomyocytes. It is worth to point out that MLB has an extreme low oral bioavailability and requires parenteral routes of administration [37]. Thus, its clinical value is compromised. Modification of MLB structure or development of novel MLB preparation in future may solve this problem.

In addition to causing oxidative injury (such as cellular apoptosis and necrosis), NOX/VPO1 pathway-derived ROS can also function as a signal molecule regulating cellular differentiation, proliferation, and migration. Since ERK is one of the best studied signal molecules that involves in cellular proliferation in response to numerous stress stimuli, and H₂O₂ or HOCl is able to activate the ERK signaling pathway [38], we evaluated the relationship between NOX/VPO1 pathway and ERK. Our results show that the phosphorylation levels of ERK in the RV of PAH rats or hypoxia-treated H9c2 cells were significantly elevated. As expected, inhibition of NOX/VPO1 pathway by MLB or VAS2870 blocked hypoxia-induced phosphorylation of ERK in the RV of PAH rats or H9c2 cells, which confirmed the link between NOX/VPO1 pathway and ERK.

In summary, our findings demonstrated for the first time that MLB is able to prevent RV remodeling in hypoxic PAH rats through a mechanism involving suppression of NOX/VPO1 pathway as well as ERK signaling pathway. MLB may possess a potential value in clinics for PAH therapy.

Limitations of the study

There are 2 major limitations that need to be acknowledged and addressed regarding the animal experiments in the present study. First of all, only 2 dosages (5 or 15 mg/kg/d) of MLB is not enough to observe the dose-response relationship between MLB and RV remodeling in PAH rats. Thus, at least 1 more dosage (such as 45 mg/kg/d) should be added to the animal experiments. Secondly, although a positive control was included in the cell experiments, it was missing in the animal experiments. It is necessary to include a positive control (VAS2870) in animal study to verify the role of NOX/VPO1 pathway in the RV remodeling in PAH rats in our future study.

Materials and Methods

Animals

Male SD rats (180 ~ 220 g) were purchased from Laboratory Animal Center, Xiangya School of Medicine, Central South University, China. The experiments were approved by the Central South University Veterinary Medicine Animal Care and Use Committee (September 20, 2017, No. CSU2017009). All animal procedures were performed according to the guidelines of the National Institutes of Health (NIH Publication, 8th edition, 2011).

Experiments in animals

Rats were randomly allocated to 4 groups (n = 6 per group) and kept in separate chambers in groups: a normoxia group, a hypoxia group, a hypoxia plus MLB (L) (low dose, 5 mg/kg/d) group, and a hypoxia plus MLB (H) (high dose, 15 mg/kg/d) group. Rats in the normoxia group were placed in a chamber connect to atmosphere (21% O₂) via small holes for 21 d, while rats in the hypoxia group were placed in a similar chamber for 21 d, where the O₂ content (10%) was automatically controlled by a setup through filling nitrogen. MLB (purity ≥ 90.0%) was purchased from Greenvalley Pharmaceutical Company. The rats in the hypoxia plus MLB groups were intraperitoneally injected with MLB (dissolved in distilled H₂O) at 5 or 15 mg/kg once a day for 21 d. At the end of hypoxia treatment, all rats were anesthetized with sodium pentobarbital (30 mg/kg, i. p.) and then the RVSP was measured via a polyethylene catheter. The hearts were removed from the rats and then the RV, LV, and the IVS were carefully dissected. The weight of every part of the heart and the tibia length of the rats were measured. The ratios of RV to body weight, LV plus IVS or tibia length were calculated to evaluate the RV remodeling. Some RV samples were frozen at − 80 °C for molecular studies (measurements of ANP and BNP mRNA expression as well as NOX2, NOX4, VPO1, and ERK/p-ERK protein levels) and biochemical analysis (ROS, H₂O₂, and HOCl levels); some were fixed with 4% paraformaldehyde for hematoxylin-eosin (HE) staining.

Experiments in cells

H9c2 cells derived from rat heart (Cell Bank of Chinese Science Academy) were seeded at a density of 1 × 10⁶ cells/cm² and grown to 70 – 80% confluence in DMEM supplemented with 10% FBS. Cells were washed with PBS and then kept quiescent in serum free DMEM for 24 h before experiments.

The first set of in vitro experiments was designed to optimize the concentration of MLB. Cells were incubated with MLB at a concentration of 10, 25, 50, or 100 µM under normoxic condition for 24 h. Except MLB at 100 µM, the others did not show obviously toxic effect on cellular viability. Next, cells were incubated with MLB at a concentration of 10, 25, or 50 µM and exposed to hypoxia (3% O₂) for 24 h. Only 50 µM of MLB could prevent the hypoxia-induced proliferation of H9c2 cells. Thus, this concentration (50 µM) was chosen for the second set of experiments.

The cells were cultured under normal atmospheric pressure and they were randomly divided into 5 groups: (i) the normoxia group, cells were cultured under normoxic condition (21% O₂/74%N₂/5% CO₂); (ii) the hypoxia group, cells were cultured under hypoxic condition (3% O₂/92% N₂/5% CO₂) for 24 h; (iii) the hypoxia plus MLB group, 50 µM (final concentration) of MLB was added to the culture medium before the hypoxia treatment; (iv) the hypoxia plus VAS2870 group, 10 µM (final concentration) of VAS2870 (a specific inhibitor of NOX, served as a positive control, dissolved in DMSO) was added to the culture medium before the hypoxia treatment; and (iv) the hypoxia + vehicle group, an equal volume of VAS2870 vehicle (DMSO, 0.04%) was added to the culture medium before the hypoxia treatment. At the end, the cells or culture medium were collected for morphological, biochemical or molecular analysis.

Morphological observations

Morphological changes in RV and H9c2 cells were observed by HE and immunofluorescence staining, respectively. For HE staining, the RV tissues were fixed in 4% paraformaldehyde and embedded in paraffin and cut into 5 µm-thickness slices. The slices were subjected to hematoxylin and eosin staining for 20 min and 2 min, respectively. At the end, the slices were imaged by microscopy to assess the cardiac morphological changes.

For immunofluorescence staining, H9c2 cells were fixed with 4% paraformaldehyde for 15 min. After washing with PBS, the cells were permeabilized with 0.25% Triton X-100 and blocked with normal goat serum for 30 min. Then the cells were incubated with α-SMA (BOSTER) at 4 °C for 18 h followed by incubation with the secondary antibody of Goat Anti-Mouse IgG (Beyotime). The cell morphological alterations were observed under a fluorescence microscopy (Olympus Corporation).

Measurement of ROS levels

The ROS level in RV was measured by dihydroethidium staining. In brief, the RVs were cut into 5 µm-thickness slices and were stained with dihydroethidium (10 µM) (Beyotime). They were incubated at 37 °C for 30 min in a dark humidified chamber. The fluorescence of ethidium was observed under a microscope (Olympus IX71). Arbitrary fluorescence units were measured with Image J and normalized against the control and expressed as fold change.

The ROS levels in cultured H9c2 cells were measured by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), a cell-permeable indicator of ROS (Beyotime). DCFH-DA is nonfluorescent until the acetate groups are removed by intracellular ROS. Briefly, H9c2 cells were washed with PBS and incubated in dark with DCFH-DA (10 µM) at 37°C for 20 min. Then the ROS-mediated fluorescence was observed under a fluorescence microscope with excitation set at 502 nm and emission set at 523 nm. Arbitrary fluorescence units were normalized against the control and expressed as fold change.

Measurement of H₂O₂ levels

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 and the xylenol orange dye can yield a purple product with an absorbance maximum at 560 nm. For measurement of H₂O₂ level, the RV tissues or H9c2 cells were homogenized in ice-cold lysis buffer and sonicated for ~ 1 min, then centrifuged at 12,000 g for 5 min. Fifty microliters of supernatant of RV or H9c2 cells homogenates or culture medium and 100 µL of work solution (0.25 mM ammonium ferrous II sulfate, 25 mM H₂SO₄, 100 mM sorbitol, 125 µM xylenol orange) were mixed and incubated at room temperature for 30 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).

Measurement of HOCl levels

The HOCl levels in the RV or H9c2 cells were measured by commercially available kits (GENMED), in which aminophenyl fluorescein (APF) is a staining probe for HOCl while hydroxyphenyl fluorescein (HPF) serves as a negative control. Briefly, the samples were incubated with APF or HPF for 2 h at 37°C and then washed with PBS. The fluorescence intensity of HOCl was observed under a fluorescence microscope (Olympus IX71). Arbitrary fluorescence units were normalized against the control and expressed as fold change.

Assay of cell viability

Cell viability was evaluated with an MTS Assay Kit (Promega). Briefly, H9c2 cells were seeded at 96-well plates and they were starved for 24 h. Then 10 µL of MTS was added to each well and the cells were incubated at 37 °C for 2.5 h. The absorbance was recorded at 490 nm with a spectrophotometer (SpectraMAX190, Molecular Devices). The cell viability for the control group was set as 100%, and the cell viability of other groups was normalized against the control.

Real-time PCR analysis

Real-time PCR was performed to quantify mRNA levels of ANP and BNP. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. Briefly, total RNA was extracted from the RV or H9c2 cells by TRIzol (TaKaRa) and the concentrations of RNA were determined spectrophotometrically. Five hundred ng of RNA from each sample was subjected to reverse transcription reaction according to the protocol provided by the manufacturer (TaKaRa). The cDNAs were amplified and detected by using SYBR Premix Ex Taq (TaKaRa) at ABI 7300 system (Applied Biosystems). Then 20 µL of reaction mixture containing 4 µL of cDNA template, 10 µL SYBR Master mix, 0.40 µL ROX, 5.2 µL H₂O, and 0.40 µL of each primer was amplified by the following thermal parameters: an initial incubation at 95°C for 15 s, followed by 38 cycles of denaturation at 95 °C for 5 s, annealing and extension at 60°C for 31 s. The PCR primers for ANP, BNP, and GAPDH are shown in [Table 1]. Data analysis was performed by comparative Ct method using the ABI software. Results were expressed as the ratio of ANP or BNP mRNA to GAPDH mRNA.

Western blot analysis

The RV tissues or H9c2 cells were homogenized in ice-cold lysis buffer containing 1% phenylmethanesulfonyl fluoride (PMSF), sonicated for 1 m, and then centrifuged for 15 min at 15,000 g. The protein concentration was determined using a BCA Protein Assay kit (Beyotime). Samples containing 20 – 60 µg of protein were separated by SDS-PAGE (10% gel) and then the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). Blots were blocked with 5% nonfat milk in tris-buffered saline tween (TBST) at room temperature for 1 h. The membranes were then incubated with primary antibodies against NOX2 (Santa Cruz, 1 : 200), NOX4 (Santa Cruz, 1 : 200), VPO1 (Abcam, 1 : 500), ERK/p-ERK (Cell Signaling Technology, 1 : 2000), or GAPDH (Beyotime, 1 : 1000) overnight at 4 °C. After incubation with horseradish peroxidase-labeled Goat Anti-Mouse or Rabbit IgG (Beyotime, 1 : 1000), the membranes were visualized by enhanced chemiluminescence (ECL kit, Amersham Biosciences) through Molecular Imager ChemiDoc XRS System (Bio-Rad). The densitometric quantification was performed with Image J 1.43. GAPDH (Beyotime) served as a loading control. Arbitrary optical density units of the targeting proteins were normalized against control, and the results were expressed as fold change.

Statistical analysis

SPSS software (version 20.0) was used for statistical analysis and the results were presented as means ± standard error of the mean (SEM). Statistical analyses were carried out by 1-way analysis of variance (ANOVA). Differences in values among the multiple groups were analyzed by 1-way ANOVA with Bonferroniʼs multiple comparisons test. The data were considered statistically significant when p-value < 0.05.

Conflict of Interest

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81703516 to Bin Liu; No. 81872873 to Jun Peng; No. 81573430 to Xiu-Ju Luo) and the Major Research Plan of the National Natural Science Foundation of China (No. 91439104 to Jun Peng; No. 91539111 to Jin-Fu Yang).

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• 15 Frazziano G, Al Ghouleh I, Baust J, Shiva S, Champion HC, Pagano PJ. Nox-derived ROS are acutely activated in pressure overload pulmonary hypertension: indications for a seminal role for mitochondrial Nox4. Am J Physiol Heart Circ Physiol 2014; 306: H197-H205
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• 16 Chen F, Li X, Aquadro E, Haigh S, Zhou J, Stepp DW, Weintraub NL, Barman SA, Fulton DJR. Inhibition of histone deacetylase reduces transcription of NADPH oxidases and ROS production and ameliorates pulmonary arterial hypertension. Free Radic Biol Med 2016; 99: 167-178
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• 17 Peng JJ, Liu B, Xu JY, Peng J, Luo XJ. NADPH oxidase: its potential role in promotion of pulmonary arterial hypertension. Naunyn Schmiedebergs Arch Pharmacol 2017; 390: 331-338
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• 18 Zhang YS, He L, Liu B, Li NS, Luo XJ, Hu CP, Ma QL, Zhang GG, Li YJ, Peng J. A novel pathway of NADPH oxidase/vascular peroxidase 1 in mediating oxidative injury following ischemia-reperfusion. Basic Res Cardiol 2012; 107: 266
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• 19 Park CH, Shin SH, Lee EK, Kim DH, Kim MJ, Roh SS, Yokozawa T, Chung HY. Magnesium lithospermate B from Salvia miltiorrhiza Bunge ameliorates aging-induced renal inflammation and senescence via NADPH oxidase-mediated reactive oxygen generation. Phytother Res 2017; 31: 721-728
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• 20 Yang ZB, Luo XJ, Ren KD, Peng JJ, Tan B, Liu B, Lou Z, Xiong XM, Zhang XJ, Ren X, Peng J. Beneficial effect of magnesium lithospermate B on cerebral ischemia-reperfusion injury in rats involves the regulation of miR-107/glutamate transporter 1 pathway. Eur J Pharmacol 2015; 766: 91-98
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• 21 Quan W, Wu B, Bai Y, Zhang X, Yin J, Xi M, Guan Y, Shao Q, Chen Y, Wu Q, Wen A. Magnesium lithospermate B improves myocardial function and prevents simulated ischemia/reperfusion injury-induced H9c2 cardiomyocytes apoptosis through Akt-dependent pathway. J Ethnopharmacol 2014; 151: 714-721
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• 22 Du CS, Yang RF, Song SW, Wang YP, Kang JH, Zhang R, Su DF, Xie X. Magnesium lithospermate B protects cardiomyocytes from ischemic injury via inhibition of TAB1-p 38 apoptosis signaling. Front Pharmacol 2010; 1: 111
  PubMedSearch in Google Scholar
• 23 Choudhary G, Troncales F, Martin D, Harrington EO, Klinger JR. Bosentan attenuates right ventricular hypertrophy and fibrosis in normobaric hypoxia model of pulmonary hypertension. J Heart Lung Transplant 2011; 30: 827-833
  CrossrefPubMedSearch in Google Scholar
• 24 Campos-Carraro C, Turck P, de Lima-Seolin BG, Tavares AMV, Dos Santos Lacerda D, Corssac GB, Teixeira RB, Hickmann A, Llesuy S, da Rosa Araujo AS, Bello-Klein A. Copaiba oil attenuates right ventricular remodeling by decreasing myocardial apoptotic signaling in monocrotaline-induced rats. J Cardiovasc Pharmacol 2018; 72: 214-221
  CrossrefPubMedSearch in Google Scholar
• 25 Taran IN, Belevskaya AA, Saidova MA, Martynyuk TV, Chazova IE. Initial riociguat monotherapy and transition from sildenafil to riociguat in patients with idiopathic pulmonary arterial hypertension: influence on right heart remodeling and right ventricular-pulmonary arterial coupling. Lung 2018; 196: 745-753
  CrossrefPubMedSearch in Google Scholar
• 26 Rawat DK, Alzoubi A, Gupte R, Chettimada S, Watanabe M, Kahn AG, Okada T, McMurtry IF, Gupte SA. Increased reactive oxygen species, metabolic maladaptation, and autophagy contribute to pulmonary arterial hypertension-induced ventricular hypertrophy and diastolic heart failure. Hypertension 2014; 64: 1266-1274
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• 27 Konior A, Schramm A, Czesnikiewicz-Guzik M, Guzik TJ. NADPH oxidases in vascular pathology. Antioxid Redox Signal 2014; 20: 2794-2814
  CrossrefPubMedSearch in Google Scholar
• 28 Yu L, Yang G, Zhang X, Wang P, Weng X, Yang Y, Li Z, Fang M, Xu Y, Sun A, Ge J. Megakaryocytic leukemia 1 bridges epigenetic activation of NADPH oxidase in macrophages to cardiac ischemia-reperfusion injury. Circulation 2018; 138: 2820-2836
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• 29 Adesina SE, Kang BY, Bijli KM, Ma J, Cheng J, Murphy TC, Michael Hart C, Sutliff RL. Targeting mitochondrial reactive oxygen species to modulate hypoxia-induced pulmonary hypertension. Free Radic Biol Med 2015; 87: 36-47
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• 30 Choi SH, Gonen A, Diehl CJ, Kim J, Almazan F, Witztum JL, Miller YI. SYK regulates macrophage MHC-II expression via activation of autophagy in response to oxidized LDL. Autophagy 2015; 11: 785-795
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• 31 Manea SA, Antonescu ML, Fenyo IM, Raicu M, Simionescu M, Manea A. Epigenetic regulation of vascular NADPH oxidase expression and reactive oxygen species production by histone deacetylase-dependent mechanisms in experimental diabetes. Redox Biol 2018; 16: 332-343
  CrossrefPubMedSearch in Google Scholar
• 32 Wang Y, Dong J, Liu P, Lau CW, Gao Z, Zhou D, Tang J, Ng CF, Huang Y. Ginsenoside Rb3 attenuates oxidative stress and preserves endothelial function in renal arteries from hypertensive rats. Br J Pharmacol 2014; 171: 3171-3181
  CrossrefPubMedSearch in Google Scholar
• 33 Zhang YZ, Wang L, Zhang JJ, Xiong XM, Zhang D, Tang XM, Luo XJ, Ma QL, Peng J. Vascular peroxide 1 promotes ox-LDL-induced programmed necrosis in endothelial cells through a mechanism involving beta-catenin signaling. Atherosclerosis 2018; 274: 128-138
  CrossrefPubMedSearch in Google Scholar
• 34 You B, Liu Y, Chen J, Huang X, Peng H, Liu Z, Tang Y, Zhang K, Xu Q, Li X, Cheng G, Shi R, Zhang G. Vascular peroxidase 1 mediates hypoxia-induced pulmonary artery smooth muscle cell proliferation, apoptosis resistance and migration. Cardiovasc Res 2018; 114: 188-199
  CrossrefPubMedSearch in Google Scholar
• 35 Liu SY, Yuan Q, Li XH, Hu CP, Hu R, Zhang GG, Li D, Li YJ. Role of vascular peroxidase 1 in senescence of endothelial cells in diabetes rats. Int J Cardiol 2015; 197: 182-191
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• 36 Altenhofer S, Radermacher KA, Kleikers PW, Wingler K, Schmidt HH. Evolution of NADPH oxidase inhibitors: selectivity and mechanisms for target engagement. Antioxid Redox Signal 2015; 23: 406-427
  CrossrefPubMedSearch in Google Scholar
• 37 Zhang Y, Akao T, Nakamura N, Duan CL, Hattori M, Yang XW, Liu JX. Extremely low bioavailability of magnesium lithospermate B, an active component from Salvia miltiorrhiza, in rat. Planta Med 2004; 70: 138-142
  Thieme ConnectPubMedSearch in Google Scholar
• 38 Tang Y, Xu Q, Peng H, Liu Z, Yang T, Yu Z, Cheng G, Li X, Zhang G, Shi R. The role of vascular peroxidase 1 in ox-LDL-induced vascular smooth muscle cell calcification. Atherosclerosis 2015; 243: 357-363
  CrossrefPubMedSearch in Google Scholar

Correspondence

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• 23 Choudhary G, Troncales F, Martin D, Harrington EO, Klinger JR. Bosentan attenuates right ventricular hypertrophy and fibrosis in normobaric hypoxia model of pulmonary hypertension. J Heart Lung Transplant 2011; 30: 827-833
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• 24 Campos-Carraro C, Turck P, de Lima-Seolin BG, Tavares AMV, Dos Santos Lacerda D, Corssac GB, Teixeira RB, Hickmann A, Llesuy S, da Rosa Araujo AS, Bello-Klein A. Copaiba oil attenuates right ventricular remodeling by decreasing myocardial apoptotic signaling in monocrotaline-induced rats. J Cardiovasc Pharmacol 2018; 72: 214-221
  CrossrefPubMedSearch in Google Scholar
• 25 Taran IN, Belevskaya AA, Saidova MA, Martynyuk TV, Chazova IE. Initial riociguat monotherapy and transition from sildenafil to riociguat in patients with idiopathic pulmonary arterial hypertension: influence on right heart remodeling and right ventricular-pulmonary arterial coupling. Lung 2018; 196: 745-753
  CrossrefPubMedSearch in Google Scholar
• 26 Rawat DK, Alzoubi A, Gupte R, Chettimada S, Watanabe M, Kahn AG, Okada T, McMurtry IF, Gupte SA. Increased reactive oxygen species, metabolic maladaptation, and autophagy contribute to pulmonary arterial hypertension-induced ventricular hypertrophy and diastolic heart failure. Hypertension 2014; 64: 1266-1274
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 28 Yu L, Yang G, Zhang X, Wang P, Weng X, Yang Y, Li Z, Fang M, Xu Y, Sun A, Ge J. Megakaryocytic leukemia 1 bridges epigenetic activation of NADPH oxidase in macrophages to cardiac ischemia-reperfusion injury. Circulation 2018; 138: 2820-2836
  CrossrefPubMedSearch in Google Scholar
• 29 Adesina SE, Kang BY, Bijli KM, Ma J, Cheng J, Murphy TC, Michael Hart C, Sutliff RL. Targeting mitochondrial reactive oxygen species to modulate hypoxia-induced pulmonary hypertension. Free Radic Biol Med 2015; 87: 36-47
  CrossrefPubMedSearch in Google Scholar
• 30 Choi SH, Gonen A, Diehl CJ, Kim J, Almazan F, Witztum JL, Miller YI. SYK regulates macrophage MHC-II expression via activation of autophagy in response to oxidized LDL. Autophagy 2015; 11: 785-795
  CrossrefPubMedSearch in Google Scholar
• 31 Manea SA, Antonescu ML, Fenyo IM, Raicu M, Simionescu M, Manea A. Epigenetic regulation of vascular NADPH oxidase expression and reactive oxygen species production by histone deacetylase-dependent mechanisms in experimental diabetes. Redox Biol 2018; 16: 332-343
  CrossrefPubMedSearch in Google Scholar
• 32 Wang Y, Dong J, Liu P, Lau CW, Gao Z, Zhou D, Tang J, Ng CF, Huang Y. Ginsenoside Rb3 attenuates oxidative stress and preserves endothelial function in renal arteries from hypertensive rats. Br J Pharmacol 2014; 171: 3171-3181
  CrossrefPubMedSearch in Google Scholar
• 33 Zhang YZ, Wang L, Zhang JJ, Xiong XM, Zhang D, Tang XM, Luo XJ, Ma QL, Peng J. Vascular peroxide 1 promotes ox-LDL-induced programmed necrosis in endothelial cells through a mechanism involving beta-catenin signaling. Atherosclerosis 2018; 274: 128-138
  CrossrefPubMedSearch in Google Scholar
• 34 You B, Liu Y, Chen J, Huang X, Peng H, Liu Z, Tang Y, Zhang K, Xu Q, Li X, Cheng G, Shi R, Zhang G. Vascular peroxidase 1 mediates hypoxia-induced pulmonary artery smooth muscle cell proliferation, apoptosis resistance and migration. Cardiovasc Res 2018; 114: 188-199
  CrossrefPubMedSearch in Google Scholar
• 35 Liu SY, Yuan Q, Li XH, Hu CP, Hu R, Zhang GG, Li D, Li YJ. Role of vascular peroxidase 1 in senescence of endothelial cells in diabetes rats. Int J Cardiol 2015; 197: 182-191
  CrossrefPubMedSearch in Google Scholar
• 36 Altenhofer S, Radermacher KA, Kleikers PW, Wingler K, Schmidt HH. Evolution of NADPH oxidase inhibitors: selectivity and mechanisms for target engagement. Antioxid Redox Signal 2015; 23: 406-427
  CrossrefPubMedSearch in Google Scholar
• 37 Zhang Y, Akao T, Nakamura N, Duan CL, Hattori M, Yang XW, Liu JX. Extremely low bioavailability of magnesium lithospermate B, an active component from Salvia miltiorrhiza, in rat. Planta Med 2004; 70: 138-142
  Thieme ConnectPubMedSearch in Google Scholar
• 38 Tang Y, Xu Q, Peng H, Liu Z, Yang T, Yu Z, Cheng G, Li X, Zhang G, Shi R. The role of vascular peroxidase 1 in ox-LDL-induced vascular smooth muscle cell calcification. Atherosclerosis 2015; 243: 357-363
  CrossrefPubMedSearch in Google Scholar