Ameliorative Effects of Beetroot Juice Supplementation on Monocrotaline-Induced Pulmonary Hypertension in Rats

Abstract

Beetroot is a nitrate-rich vegetable with cardiovascular benefits. This study examined whether ingestion of beetroot juice (BRJ) protects against pulmonary hypertension (PH). Rats were injected subcutaneously with 60 mg/kg monocrotaline (MCT) and randomized to receive either drinking water, low-dose BRJ (BRJ-L, nitrate content: 1.4 mmol/L), or high-dose BRJ (BRJ-H, nitrate content: 3.5 mmol/L), which was started 1 week after MCT injection and continued until the end of the experiment. Four weeks after MCT injection, right ventricle (RV) hypertrophy, right ventricular systolic pressure (RVSP) elevation, and pulmonary vascular remodeling were observed. These PH symptoms were less severe in rats supplemented with BRJ-L (Fulton index, p = 0.07; RVSP, p = 0.09, pulmonary arterial medial thickening, p < 0.05), and the beneficial effects were more pronounced than those of BRJ-H supplementation. Plasma and RV nitrite and nitrate levels did not change significantly, even when BRJ-L and BRJ-H were administered. There were no differences in plasma thiobarbituric acid reactive substances (TBARS), a biomarker of oxidative stress, among the groups. BRJ-L supplementation significantly decreased RV TBARS levels compared to MCT alone (p < 0.05), whereas BRJ-H supplementation did not. These findings suggest that starting BRJ supplementation from an early stage of PH ameliorates disease severity, at least partly through the inhibition of local oxidative stress. Habitual ingestion of BRJ may be useful for the management of PH.

1. Introduction

Pulmonary hypertension (PH) is a progressive disease with a poor prognosis [1]. As this disease is characterized by decreased bioavailability of nitric oxide (NO), a multifunctional signaling molecule in the pulmonary circulation, drugs that target the NO pathway are beneficial in the treatment of PH [2,3,4]. Over the last decade, inorganic nitrates and nitrites have attracted increasing interest as sources of NO in the body [5]. Inorganic nitrates are converted to nitrites in the oral cavity by bacterial nitrate reductase. Nitrites are absorbed from the small intestine and transferred into the circulation, where they are reduced to NO by certain proteins and enzymes such as deoxyhemoglobin and xanthine oxidoreductase (XOR). Similar to NO donors, the pharmacological effects of inorganic nitrates and nitrites include lowering blood pressure and anti-tissue remodeling [6,7]. This evidence suggests that inorganic nitrates and nitrites are also useful in managing PH, and some animal studies have reported such results. For example, long-term treatment with sodium nitrate or sodium nitrite improved PH symptoms, including right ventricle (RV) hypertrophy, increased pressure in pulmonary circulation, and pulmonary vascular remodeling [8,9,10,11].

Beetroot (Beta vulgaris L.) is a root vegetable packed with vitamins, minerals, fiber, and antioxidants [12]. Notably, inorganic nitrates are also abundant in roots [13]. This is one of the reasons why beetroot and its products are widely used for the treatment of various diseases, including hypertension [14], heart failure [15], and chronic occlusive pulmonary disease [16]. We recently reported that beetroot juice (BRJ) supplementation from the day of monocrotaline (MCT) injection prevents the development of PH in a nitrate-dependent manner in a rat model of PH [17,18]. However, BRJ supplementation from 2 week (14 days) after MCT injection did not improve PH in the same model [17]. That is, these studies have not provided evidence that BRJ supplementation is effective for PH, even when the intervention is started with a delay after MCT injection. As treatment of disease cannot usually be started at the time of onset, it is very important from a medical point of view whether intervention after disease onset has a beneficial effect. To address this issue, we examined how starting BRJ supplementation from 1 week (7 days) after MCT injection affected the progression of PH in rats. In addition, we also examined whether the effects of BRJ supplementation were related to the increase in nitrate and/or the reduction of oxidative stress.

2. Materials and Methods

2.1. Animals

This study was approved by the Experimental Animal Research Committee of the Osaka University of Pharmaceutical Sciences (Permit No: 6/2021, approved 31 March 2021), and the animals were handled according to ethical principles. Eight-week-old male Sprague Dawley rats were obtained from Japan SLC, Inc. (Shizuoka, Japan). They were housed 2 per cage in a light-controlled room with a 12 h light/dark cycle, and food and water were available ad libitum.

2.2. Experimental Design

The rats were randomly assigned to one of four groups: (1) sham group, rats were given a single subcutaneous injection of 0.9% saline, and drinking water was supplemented; (2) MCT group, rats were given a single subcutaneous injection of MCT (60 mg/kg; Sigma-Aldrich Co. LLC, St. Louis, MO, USA), and drinking water was supplemented; (3) low-dose BRJ (BRJ-L) group, rats were given MCT in the same way as the MCT group, and BRJ (containing 1.4 mmol/L nitrate with a dilution factor of 50) was supplemented; and (4) high-dose BRJ (BRJ-H) group, rats were given MCT in the same way as the MCT group, and BRJ (containing 3.5 mmol/L nitrate with a dilution factor of 20) was administered. Supplementations were started 1 week (7 days) after saline or MCT injection and were maintained for an additional three weeks (21 days). BRJ (Beet It Organic Shot) was obtained from James White Drinks Ltd. (Ipswich, UK); the dilution factors were determined based on our previous study [18]. The solutions were changed every 1–2 days. The average water intake per day throughout the intervention period was as follows: sham group, 48 mL; MCT group, 38 mL; BRJ-L group, 54 mL (nitrate intake, 213 µmol/kg/day); BRJ-H group, 54 mL (nitrate intake, 557 µmol/kg/day); the daily nitrate intake for each week is included in Figure S1. Incidentally, body weight was measured once per week to calculate this value.

2.3. Hemodynamic Measurement and Sample Collection

Four weeks (28 days) after saline or MCT injection, each rat was anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and subcutaneously injected with meloxicam (1.0 mg/kg; Virbac Japan Co., Ltd., Osaka, Japan). A polyethylene catheter (SP-31, Natsume Seisakusho Co., Ltd., Tokyo, Japan) was inserted into the right carotid artery to measure heart rate and mean arterial pressure, and another catheter (PE 60, Becton Dickinson, Sparks, NV, USA) was introduced into the right jugular vein and passed into the RV to measure right ventricular systolic pressure (RVSP). The catheter was filled with 0.9% saline containing heparin and connected to a pressure transducer (DX-360, Nihon Kohden, Tokyo, Japan). Hemodynamics were simultaneously recorded using a PowerLab data acquisition system (PowerLab/4sp, AD Instruments, Sydney, Australia). The rats were not deprived of food or drink before the experiments.

After hemodynamic measurement, blood samples were collected from the inferior vena cava, the rats were euthanized, and the heart and lungs were excised and weighed. Heparinized blood samples were centrifuged at 3000 rpm for 10 min at 4 °C, and the obtained plasma was stored at −80 °C until use. The RV was isolated from the collected heart and homogenized in 10 volumes (w/v) of 0.1 M Tris-HCl buffer (pH 7.4). The homogenate was centrifuged at 3000 rpm for 10 min at 4 °C, and the obtained supernatant was stored at −80 °C until use. The collected left lung was inflated by injecting 10% phosphate-buffered formalin and fixed in formalin.

2.4. Histological Examination

The formalinized lungs were embedded in paraffin and sectioned at a thickness of 4 µm. The sections were stained with Elastica van Gieson stain to examine vascular remodeling. The medial wall thickness in muscular arteries with an external diameter of 30–100 µm was evaluated by calculating the percentage wall thickness as {(external diameter minus internal diameter)/external diameter} × 100. The external and internal diameters were calculated from the circumferences of the external and internal elastic lamellae, respectively. At least 10–15 muscular arteries per lung section were examined using an image analyzer (cellSens, Olympus Co., Ltd., Tokyo, Japan). The values obtained were averaged per animal and then per group.

2.5. Nitrite and Nitrate Measurement

Plasma and RV samples were mixed with methanol (1:1, v/v) and centrifuged at 10,000 rpm for 10 min at 4 °C. The resultant supernatant was diluted (1:9, v/v) with a mobile phase (NOCARA, Eicom, Kyoto, Japan) and injected into an ENO-20 NOx Analyzer (Eicom). Nitrite and nitrate levels were calculated by comparison with the results obtained from the standard solutions (NO-STD, Eicom).

2.6. TBARS Measurement

Thiobarbituric acid reactive substances (TBARS) levels in the plasma and RV samples were quantified using a commercially available assay kit (Oxford Biomedical Research, Inc., Rochester Hills, MI, USA) according to the manufacturer’s instructions. The absorbance of the prepared samples was measured at 532 nm using a multilabel plate reader (EnSpire 2300, PerkinElmer Japan, Co., Ltd., Kanagawa, Japan). TBARS levels were calculated by interpolation of a standard curve of malondialdehyde.

2.7. Statistics

All values are expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 7.0 software (GraphPad Software Inc., San Diego, CA, USA). Data were compared using one-way analysis of variance (ANOVA) followed by the Holm–Sidak multiple comparisons test. Differences were considered statistically significant at p < 0.05.

3. Results

3.1. Morphological and Hemodynamic Parameters

The main data are summarized in

Table 1. Morphological and hemodynamic parameters.

Parameters	Sham (n = 6)	MCT (n = 9)	BRJ-L (n = 6)	BRJ-H (n = 6)
BW (g)	416 ± 10 **	348 ± 5	366 ± 9	337 ± 10
HW/BW (g/kg)	2.84 ± 0.14 **	3.57 ± 0.11	2.98 ± 0.11 **	3.70 ± 0.19
(LV + S)W/BW (g/kg)	1.94 ± 0.02	2.16 ± 0.04	2.01 ± 0.06	2.18 ± 0.10
LW/BW (g/kg)	3.32 ± 0.19 **	6.39 ± 0.17	6.10 ± 0.38	6.98 ± 0.33
HR (bpm)	405 ± 20 *	324 ± 18	353 ± 22	325 ± 32
MAP (mmHg)	119 ± 7	112 ± 7	112 ± 5	105 ± 4

Data are presented as mean ± SEM of 6–9 experiments. One-way analysis of variance with the Holm–Sidak post hoc test was performed to determine the significance; * p < 0.05 and ** p < 0.01, compared to the MCT group. Abbreviations: MCT, monocrotaline; BRJ-L, low-dose beetroot juice; BRJ-H, high-dose beetroot juice; BW, body weight; HW, heart weight; (LV + S)W, left ventricle plus septum weight; LW, lung weight; HR, heart rate; MAP, mean arterial pressure.

. MCT injection resulted in body weight loss and lung edema, which are general characteristics of this model. Regardless of the dose administered, BRJ supplementation did not improve these parameters. In addition, MCT injection caused cardiac hypertrophy, as evidenced by the increased heart weight/body weight ratio, which was suppressed by BRJ-L supplementation but not by BRJ-H supplementation. Left ventricle (LV) hypertrophy, evidenced by the LV plus septum weight/body weight ratio, was not observed in any of the MCT-injected groups.

Regarding systemic hemodynamics, the heart rate was lower in the MCT group than in the sham group; the values in the BRJ-L and BRJ-H groups were similar to those in the MCT group. There were no significant differences in the mean arterial pressure among the sham, MCT, BRJ-L, and BRJ-H groups.

3.2. Right Ventricle Hypertrophy

MCT injection led to a marked increase in RV weight/body weight ratio, which was suppressed by BRJ-L supplementation. Such beneficial effects on MCT-induced RV hypertrophy were not observed with BRJ-H supplementation (Figure 1a). The Fulton index (RV weight/LV plus septum weight ratio) in the BRJ-L group was somewhat lower than that in the MCT group (p = 0.07) (Figure 1b).

3.3. Right Ventricle Dysfunction

Representative images of RVSP in the four groups are shown in Figure 2a. RVSP was significantly elevated in the MCT group compared with that in the sham group. The elevation was less pronounced in the BRJ-L group, although this difference did not reach statistical significance (p = 0.09); RVSP in the BRJ-H group was comparable to that in the MCT group (Figure 2b).

3.4. Pulmonary Vascular Remodeling

Representative images of the small pulmonary arteries in the four groups are shown in Figure 3a. Significant pulmonary arterial medial thickening was observed following MCT injection, which was significantly milder in rats supplemented with BRJ-L and BRJ-H (Figure 3b).

3.5. Nitrite and Nitrate Levels

Plasma nitrite levels were similar among the four groups (Figure 4a). The plasma nitrate levels in the MCT group did not differ from those in the sham group. Although BRJ-L supplementation did not affect these levels, BRJ-H supplementation tended to increase them (Figure 4b). Similar to the findings in plasma, no differences in RV nitrite and nitrate levels between the sham and MCT groups were observed; BRJ-L supplementation did not affect NO metabolite levels, and BRJ-H supplementation tended to increase nitrate levels but did not affect nitrite levels (Figure 4c,d).

3.6. Oxidative Stress

Plasma TBARS, a biomarker of oxidative stress, did not differ among the four groups (Figure 5a). In contrast, a significant increase in TBARS levels was observed in the RV of the MCT group, which was suppressed by BRJ-L supplementation (Figure 5b).

4. Discussion

This study demonstrated for the first time that PH progression can be alleviated by starting BRJ supplementation at a relatively early stage in MCT-injected rats. One week after MCT injection, when BRJ supplementation was started, is known to be before the appearance of pulmonary/RV pressure elevation and RV hypertrophy [19,20,21,22]. However, reactive oxygen species (ROS) production in the lung is already increased; that is, the lung is under oxidative stress, and morphological changes, such as parenchymal damage, pulmonary arterial remodeling, and pulmonary endothelial dysfunction are observed [19,22]. Importantly, MCT is metabolized in the liver to an active metabolite that induces such toxic effects [23]. Although we previously reported that starting BRJ supplementation at the same time as MCT injection improves PH symptoms, including RV hypertrophy, RVSP elevation, and pulmonary vascular remodeling [18], BRJ ingestion may have suppressed this metabolic conversion rather than prevented disease progression. Therefore, this study, which shows definite ameliorative effects of BRJ supplementation on PH, is of great significance.

BRJ supplementation did not mitigate the increased lung weight/body weight ratio. MCT-induced lung edema is attributed to elevated capillary permeability from pulmonary inflammation and develops within 1 week after MCT injection [24]. In line with our results, many studies have reported that treatment with vasodilators from an early stage of PH improves PH symptoms without affecting lung weight gain in MCT-injected rats [25,26,27]. Vasodilators are well acknowledged to induce lung edema in PH with pulmonary vein stenosis [28]. In this regard, MCT induces pulmonary vascular remodeling in arteries and veins [29]. Altogether, PH symptoms in an MCT model may be relieved more effectively by managing pulmonary edema.

The health-promoting effects of BRJ ingestion are now considered to be mainly due to increased NO production via the nitrate-nitrite pathway [30]. In cases where BRJ supplementation at a dose that exerted greater beneficial effects on the progression of PH, plasma nitrite and nitrate levels at the end of the experiment did not increase. As XOR, a key enzyme in the nitrate-nitrite-NO pathway, has been reported to be stimulated in the RV in MCT-injected rats [31], we hypothesized that these levels might be increased efficiently in the pulmonary circulation tissue. However, no increase was observed in the RV. In this regard, there is an interesting report by Zuckerbraun et al., who found that inhalation of nebulized nitrite was accompanied by increased nitrite levels in the lung in normoxic mice but not in hypoxic mice [8]. They argued that the reason for this difference is due to enhanced XOR enzymatic activity in the lung and the concomitant rapid conversion of nitrite to NO. Based on this theory, BRJ supplementation may have been able to supply sufficient NO even if there was no increase in nitrite and nitrate levels. In addition, as BRJ-H supplementation tended to increase their levels in both plasma and RV, it is reasonable to assume that more NO was supplied than with BRJ-L supplementation.

MCT-induced RV dysfunction and hypertrophy are closely associated with oxidative stress [32,33]. Treatment of MCT-injected rats with pentaerythritol tetranitrate, an organic nitrate, inhibits cardiac ROS production and suppresses pulmonary arterial pressure elevation and cardiac hypertrophy [34]. In addition, the physiological level of NO functions as an antioxidant, although excess NO acts as a pro-oxidant [35]. These findings suggest that a suitable amount of exogenous nitrate can alleviate oxidative stress and produce beneficial effects on RV. Therefore, we examined whether BRJ supplementation affects oxidative stress. The results, in line with other studies [36,37,38], showed that TBARS levels in the RV increased in MCT-injected rats; importantly, BRJ-L supplementation significantly suppressed this increase. Consequently, BRJ supplementation may prevent the development of PH, at least partly through the inhibition of oxidative stress. As beetroot is rich in antioxidants other than nitrate, including vitamin C and betanin [12], this conclusion is plausible. However, this study did not address the balance between anti- and pro-oxidant systems in detail. It will be interesting to see how BRJ supplementation may change the expression and activity of key enzymes and proteins such as endothelial NO synthase, XOR, catalase, glutathione peroxidase, and superoxide dismutase.

It is difficult to explain why BRJ-L supplementation was superior to BRJ-H supplementation in ameliorating the PH symptoms. Possible mechanisms for this have been discussed in detail in previous papers [11,17]. For example, although body blood pressure was unchanged in rats supplemented with BRJ-H, baroreflex-mediated sympathetic activation, a well-known counter-regulatory response to nitrate-induced hypotension [39], may have occurred. This scenario would be possible as BRJ-H supplementation tended to increase plasma nitrate levels. Noradrenaline, a sympathetic transmitter, is capable of causing RVSP elevation [40]. Interestingly, sympathectomy has been reported to suppress MCT-induced RV hypertrophy but not to prevent pulmonary vascular remodeling [41]. These findings are in line with the present study observations wherein the protective effects of BRJ-H supplementation on RV hypertrophy and RVSP elevation were weak. The important thing is that it is not uncommon for the effect to be reversed if the dose is increased, not only for BRJ [42,43,44]. No significant side effects or toxicity from BRJ ingestion have been reported thus far [45], and further research is needed on the dose-independent effects of BRJ observed in this study. In any case, excessive BRJ intake does not appear to be beneficial for the management of PH.

The ameliorating effects of BRJ supplementation on PH were not very strong, but this result is notable because BRJ is not a drug but a dietary supplement. Attempts to combine drug therapy and nutritional supplementation to treat PH have increased in recent years [46,47,48,49]. For example, a 16-week oral administration of tetramethylpyrazine, a constituent of natto (fermented soybeans), has been reported to improve exercise capacity (reflected by a 6 min walk distance) in patients with pulmonary arterial hypertension or chronic thromboembolic pulmonary hypertension treated with targeted drugs, including endothelin receptor antagonists, phosphodiesterase 5 inhibitors, soluble guanylate cyclase stimulators, and prostacyclin analogs [49]. Interestingly, RV hypertrophy, RVSP elevation, and pulmonary vascular remodeling were not completely normalized by tetramethylpyrazine monotherapy in PH animal models [49]. These findings imply that the effects of dietary supplements may be add-on to the effects of standard clinical therapy. That is, if any beneficial effects are observed in animal studies, it is worth considering their application in clinical setting. Regarding BRJ, the BEET-PAH study, an exploratory randomized, double-blind, placebo-controlled crossover trial, showed that 1-week (7-day) ingestion of BRJ (nitrate intake: approximately 250 µmol/kg/day) resulted in slight improvements in RV systolic function and exercise tolerance (reflected by W peak-VO₂ peak ratio) in patients with pulmonary arterial hypertension [50]. Although no significant improvement was observed in that study, the intervention period was relatively short and the number of participants was limited. Therefore, further studies are needed to determine whether BRJ ingestion has a clinical benefit in patients with PH.

In conclusion, this study demonstrated that starting BRJ supplementation from an early stage of PH ameliorates disease severity, at least partly through antioxidant effects. The finding that BRJ supplementation reduced oxidative stress in a PH model animal is a novel finding, further reinforcing the usefulness of BRJ ingestion on PH. Thus, BRJ ingestion is expected to be a useful dietary intervention for the treatment of PH.