Lotus seed (Nelumbo nucifera Gaertn.) extract at low dose ameliorates reproductive dysfunction in l-NAME-induced hypertension and oxidative stress in male rats

Abstract

Background and aim

Nelumbo nucifera Gaertn. (Lotus) seeds have been traditionally used to treat hypertension and are reported to have antioxidant effects. The role of lotus seeds in reproductive dysfunction associated with hypertension has not yet been investigated. Therefore, we aimed to evaluate lotus seed extract (LSE) on reproductive functions in N^ω-nitro-l-arginine methyl ester hydrochloride (l-NAME) induced hypertension and oxidative stress in rats.

Experimental procedure

Male Sprague–Dawley rats were allocated into six groups: i) normotensive rats, ii) rats treated with l-NAME (40 mg/kg), iii-vi) rats treated with l-NAME + either 10, 100, 300 mg/kg LSE or 5 mg/kg captopril for 5 weeks. Systolic blood pressure (SBP), sperm concentrations, viability and motility and the serum testosterone were measured. The morphology, malondialdehyde (MDA) and superoxide dismutase (SOD) activities were evaluated in testis and epididymis.

Results and conclusion

l-NAME-treated rats exhibited high SBP (181.9 ± 6.5 mmHg, P < 0.01) and reduced SOD activities in comparison to the normal group. These were significantly improved by the administration of 10, 100, 300 mg/kg LSE or captopril (P < 0.05). l-NAME also led to a significant increase in MDA, and reductions in sperm count, viability and motility, testosterone levels, as well as the diameter and epithelial thickness of the seminiferous tubules. Treatment with 10 mg/kg LSE or captopril significantly attenuated these effects (P < 0.05), whereas 100 and 300 mg/kg LSE showed no significant differences compared to the l-NAME group. Thus, our study demonstrates that a low dose of LSE has therapeutic potential in alleviating reproductive dysfunction associated with hypertension.

Graphical abstract

Highlights

• •LSE was effective in restoring systolic blood pressure and suppressing hypertension.
• •LSE treatment is beneficial for both hormone production and sperm formation in the hypertensive rats.
• •LSE enhanced histopathological damage in the testis and epididymis of the l-NAME-induced hypertensive rats.
• •LSE improved the antioxidant properties and suppression of ROS in the testis and epididymis of l-NAME hypertensive rats.

1. Introduction

Hypertension is a major risk factor that contributes to cardiovascular disease, which is a major cause of global morbidity and mortality, and it also creates risk factors for reproductive dysfunction in male patients.^1,2 Clinical studies have indicated that hypertension in men is associated with a decrease in the circulatory concentrations of both testosterone and sex hormone-binding globulin.^3,4 This hormonal imbalance could lead to alterations in testicular morphology⁵ and the spermatogenesis process.⁶ Hypertension is linked to an increased generation of reactive oxygen species (ROS), which leads to the decline of total antioxidant status⁷ and tissue damage.⁸ An increase of ROS results in reduced sperm count, sperm motility, sperm DNA damage, and apoptosis.⁹

The N^ω-nitro-l-arginine methyl ester (l-NAME) induced hypertension model, is a widely used experimental animal model that helps in the understanding of the pathophysiology of hypertension, as well as the testing of potential antihypertensive drugs.¹⁰ l-NAME is a non-specific inhibitor of nitric oxide synthase (NOS), which is the enzyme responsible for the production of nitric oxide (NO), and a crucial regulator of blood pressure (BP). When NOS is inhibited, it leads to vasoconstriction and increased vascular resistance, which then induces hypertension.¹¹ In the male reproductive system, the deficiency of NO can contribute to erectile dysfunction due to impaired blood flow to the penis.¹² It can also negatively affect spermatogenesis, which potentially leads to a decreased sperm count and motility.¹³ In the animal model of l-NAME-induced hypertension, it exhibits reduced testosterone levels, changes in testicular morphology, decreased sperm count and motility, and impaired antioxidant status.^5,14,15

The use of certain antihypertensive medications has been associated with impaired semen quality¹⁶ and sexual dysfunction.¹⁷ Thus, this study focuses on the search for new and effective herbal medicines that can alleviate the reproductive damage caused by hypertension. Nelumbo nucifera Gaertn., is the scientific name of the lotus plant which has been widely used as a traditional medicine in Asia for the treatment of various conditions, such as skin diseases,¹⁸ tissue inflammation,¹⁹ hypertension, and spermatorrhea.²⁰ The lotus seeds are composed of three distinct parts: the seed epicarp, cotyledons, and the embryo. The seeds are associated with various beneficial properties, which include antioxidants,²¹ anti-diabetic,²²cardiovascular protection,²³ hepato-protection²⁴ and anti-inflammatory effects.²⁵ In traditional Thai medicine, lotus seeds soaked in hot water are utilized for treating hypertension, diarrhea, and spermatorrhea.²⁶

The bioactive compounds of the lotus seeds are flavonoids, glycosides, phenolic compounds, and alkaloids.²⁷ Recently, it was discovered that the embryos of the lotus seeds could potentially be used to treat several cardiovascular diseases, such as hypertension.²⁸ It has been reported that the embryos from the lotus seeds have the potential to induce vascular smooth muscle relaxation in mice, which could be beneficial for managing hypertension and increasing blood flow to the penis.²⁹ A previous study demonstrated that the alkaloids found in the lotus seed embryos may alleviate erectile dysfunction by exerting a relaxant effect on the rabbit corpus cavernosum tissues.³⁰ This mechanism is suggested to function via the inhibition of the extracellular calcium influx and the suppression of intracellular calcium release.³⁰ Moreover, the phenolic compounds are also important active components in the lotus seeds that exhibit antioxidants³¹ and anti-inflammatory properties.²⁵ These two properties can protect the sperm DNA, improve its quality and enhance the reproductive efficiency of males.³²

This study aimed to investigate the influence of lotus seed extract on the reproductive toxicity caused by l-NAME-induced hypertension and oxidative stress in male rats. This was done by evaluating their BP, sperm parameters, testosterone levels, oxidative stress biomarkers, and histopathological alterations in the testis and epididymis of l-NAME-treated rats. To the best of our knowledge, this is the first study demonstrating the therapeutic potential of lotus seeds in treating hypertension and related reproductive dysfunction in hypertensive rats.

2. Materials and methods

2.1. Drugs and chemicals

l-NAME and captopril were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Hematoxylin and eosin (H&E) staining were obtained from C.V. Laboratories Co. (Bangkok, Thailand). Formaldehyde, xylene and absolute ethanol were obtained from RCI Labscan Ltd. (Bangkok, Thailand).

2.2. Plant material and preparation of extract

The lotus seed extract (LSE) was prepared by the Thailand Institute of Scientific and Technological Research, Bangkok, Thailand. Authentication of the plants was performed by the Plant Variety Protection Department of Agriculture, Bangkok, Thailand. The voucher specimen (BK No. 082574) was deposited in the herbarium of the Bangkok Herbarium, Bangkok, Thailand. Fresh lotus seeds were obtained, then dried in an oven at 50 °C for 24 h. After drying, the seeds were ground to a coarse powder using an electric grinder. The plant powder was extracted with 95 % ethanol by sonication for 30 min, and then the residue was removed using Whatman filter paper No.1. This extraction process was repeated twice, and then the supernatants were combined. After filtration, the extract was evaporated under reduced pressure using a rotary evaporator. The yields of the extracts were 3.82 ± 0.56 % (w/w) by the dry weight of the seed. The extract was frozen and stored at −20 ^οC until required for further studies.

2.3. High-performance liquid chromatography analysis

High-performance liquid chromatography (HPLC) analyses were conducted to ensure the quality of the herbs used in the study. The quality control of LSE was characterized by measuring the concentration of its main active components, such as gallic acid. The HPLC analysis was conducted using a Hypersil BDS C-18 column (100 × 4.6 mm i.d., 3 μm). The mobile phase comprised of 0.5 % acetic acid in water (solvent A) and methanol (solvent B) with the flow rate 1 mL/min. The gradient elution started with 100 % solvent A for 5 min, followed by a linear increase from 0 % to 35 % solvent B in solvent A over 3 min, which was then maintained for an additional 17 min. The ultraviolet detector was set to operate at a wavelength of 254 nm. The identify of peak in chromatogram was compared with the retention time of standard compound while the amount of gallic acid in extract was determined by a comparison of peak area with the standard curve of gallic acid.

2.4. Animals

Thirty-six male Sprague Dawley rats (6 weeks old, 180–220g) were purchased from Nomura Siam International Co. Ltd., Bangkok, Thailand. The rats were kept in an environment with a 12-h light and dark cycle, at a temperature of 22 ± 1 ^οC, at the Naresuan University Center for Animal Research (NUCAR). They were provided a standard rodent diet and tap water. All experiments complied with the guidelines for care and use of laboratory animals. The experimental protocol (number NU-AE 630804) was approved by the Naresuan University Animal Care and Use Committee (NUACUC). The sample size was calculated using G Power software (Heinrich-Heine-Universität, Düsseldorf, Germany), with an effect size of 0.72 derived from a previous study,¹⁵ a significance level of 0.05, and a power of 80 %. A total of 6 rats per group were considered necessary.

2.5. Experimental protocol

After a one-week acclimatization, the rats were weighed and assigned into groups using a completely randomized design (CRD), then divided into one normotensive and five hypertensive groups, each with 6 rats. The normal control group was given tap water, while the hypertensive groups received l-NAME (40 mg/kg/day) in their drinking water for 5 weeks. Concurrently, rats in both the control and l-NAME-treated groups were intragastrically administered either distilled water, LSE, or captopril for 5 weeks as follows:

• Group I (Control): Normotensive rats were administered distilled water.

Groups II (l-NAME alone): Hypertensive rats were administered distilled water.

Groups III (l-NAME + LSE10): Hypertensive rats were treated with lotus seed extract at 10 mg/kg.

Groups IV (l-NAME + LSE100): Hypertensive rats were treated with lotus seed extract at 100 mg/kg.

Groups V (l-NAME + LSE300): Hypertensive rats were treated with lotus seed extract at 300 mg/kg.

Groups VI (l-NAME + Captopril): Hypertensive rats were treated with a reference antihypertensive drug Captopril at 5 mg/kg.

In this study, the dosage of LSE was based on the human recommendation (1.5–6 g) for hypertension treatment in traditional Thai medicine textbook²⁶ and the Pharmacopoeia of the People's Republic of China.³³ This human dosage translates to a rat dosage of ∼155–620 mg/kg.³⁴ Therefore, we selected 300 mg/kg as the highest dose and also examined 10 mg/kg and 100 mg/kg to assess dose-dependent responses. The doses of l-NAME (40 mg/kg) and captopril (5 mg/kg) used in the present study were selected based on the previously published data.^5,35 LSE and captopril were dissolved in distilled water for administration. The LSE, captopril, or vehicle were administered orally via gavage daily for a duration of 5 weeks. The body weight of all rats was monitored daily, and the doses of LSE or captopril were adjusted accordingly based on these measurements. Additionally, the stock solution of LSE was freshly prepared every other day to maintain its efficacy. Twenty-four hours after the final treatment, animals were sacrificed and their blood and reproductive organs were collected for analysis (Supplementary Fig. 1).

2.6. Blood pressure measurement

The SBP of all rats were measured using non-invasive tail-cuff plethysmography method after the initial acclimatization period (week 0), and then subsequently in the second and fifth weeks. (NIBP, AD Instrument, Sydney, Australia). The conscious rats were placed in a restraining apparatus maintained at a temperature of 32–35 °C, their tails inserted into an automatically inflating and deflating cuff. SBP for each rat was calculated as the average of three readings, taken at 15 min intervals.

2.7. Surgical procedures

Following BP measurement, the final body weights of the rats were taken. All the animals were sacrificed under isoflurane anesthesia, and then their blood was collected from carotid artery. The blood samples were centrifuged at 3000 rpm for 10 min to obtain the serum,³⁶ which was subsequently used to determine serum testosterone. Reproductive organs including testes, epididymis, vas deference and seminal vesicle were dissected, and any surrounding fat pads were removed prior to determining their absolute weights. In this study, the relative weight of these organ were calculated according to previously published study.³⁷ Subsequently, their testis and epididymis were prepared for biochemical evaluations and histology. The right side of these organs were immediately preserved with 10 % neutral buffered formalin (10 % NBF) for histopathological examination via hematoxylin and eosin (H&E) staining, whereas, the left side of the dissected tissues was snap-frozen with liquid nitrogen for the subsequent analysis of oxidative stress indices.

2.8. Evaluation of the epididymal sperm count and viability

After the rats were sacrificed, the caudal epididymis was excised. Semen was then collected from the caudal epididymis and diluted in 1 mL of prewarmed phosphate-buffered saline (PBS, pH 7.4, at 37 °C). The sperm suspension was then resuspended and fixed with trypan blue,³⁸ followed by further dilution to a 1:20 ratio with PBS. A 10 μL sample was placed on a Neubauer counting chamber. Mature spermatozoa were observed and counted under 10× magnification using a light microscope (OLYMPUS BH Series, Pennsylvania, USA). This count was used to calculate the sperm concentration (x10⁶ cells/mL), following a previously described method.³⁷ To assess sperm viability, the unstained sperm were counted as live, while those stained dark blue were counted as dead. The number of live sperms were then calculated to determine the percentage of sperm viability, as previously described.³⁹

2.9. Sperm motility assays

The cauda epididymis of the rats was dissected, and then the epididymal semen was extracted and diluted with phosphate buffer (PBS, pH 7.4, 37 °C). Following this, 10 μL of the sperm suspension was placed onto a Makler counting chamber. Sperm concentrations and motility were then observed under a light microscope at 10× magnification (OLYMPUS BH Series). Sperm motility was categorized based on the proportion of progressive (both rapid and slow) and non-progressive spermatozoa. The immotile sperm were then counted, followed by the motile sperm. Data was subsequently expressed as a percentage representing the progressive motility of the sperm.³⁹

2.10. Serum testosterone

Total testosterone levels were measured by assessing serum obtained from the blood samples. This assessment was conducted using the enzyme-linked immunosorbent assay (ELISA) method at the Biolab Medical Technology Clinic, Phitsanulok, Thailand.

2.11. Assay of oxidative stress and antioxidant defense system

The level of lipid peroxidation in the testis and epididymis homogenates was determined by measuring the concentration of malondialdehyde (MDA) using an MDA assay kit (Sigma-Aldrich, Germany), following the manufacturer's instructions. The left testis and epididymis from each rat were homogenized in a microtube and incubated with thiobarbituric acid (TBA) for 60 min at 95 °C, then rapidly cooled to −20 °C for 10 min. The pink color that developed, indicative of MDA formation, was measured at an absorbance of 532 nm. The MDA concentration was then expressed as nmol/mg protein. The total protein concentration in tissue samples was determined using the Bradford method.⁴⁰

The activities of antioxidant enzymes, specifically superoxide dismutase (SOD), were measured using a commercial SOD assay kit (Sigma-Aldrich, Germany). The testis and epididymis were homogenized in a 50 mM Tris-HCl buffer (pH 7.4). This mixture was then centrifuged at 13,000×g for 10 min at 4 °C. The supernatant was collected and used for the antioxidant estimations. The absorbance of the resulting yellow color was measured at 450 nm. SOD activity was subsequently expressed as units of enzyme per mg of protein (U/mg protein).

2.12. Histopathological examination of the testis and epididymis

The right testis and epididymis were fixed in a solution of 10 % NBF and processed. Then, they were inserted in a paraffin block, cut into 4 μm sections, which were stained with H&E.³⁹ These sections were observed under a light microscope and images were recorded using Zeiss AxioCam microscope cameras (Carl Zeiss Inc., Germany).

2.13. TUNEL assay

TUNEL staining was performed using a TUNEL Assay Kit-HRP-DAB (ab206386, Abcam), in accordance with the manufacturer's protocol. The process began with formalin-fixed, paraffin-embedded sections being deparaffinized in xylene and then rehydrated through a series of graded alcohols. Following this, the sections were permeabilized. The final step involved inactivating endogenous peroxidases by incubating the sections with 3 % H₂O₂. To label the exposed 3′-OH ends of the DNA in apoptotic cells with biotin, the tissue sections were incubated with the Terminal deoxynucleotidyl transferase (TdT) enzyme and TdT labeling reaction mixture for 90 min at 37 °C in a humidified chamber. The reaction was halted by applying a stop buffer. Negative controls were established by excluding TdT from the reaction mixture. For positive controls, sections were treated with DNAse I for 20 min prior to DNA end labeling. The biotinylated DNA was then identified by incubating the sections with a streptavidin-horseradish peroxidase (HRP) conjugate for 30 min at 37 °C within a humidified chamber. The sections were washed with Tris-buffered saline (TBS), and then developed with 3,3′-diaminobenzidine (DAB) substrate for 15 min. They were subsequently observed using a ZEISS OxiCam 105 color microscope camera. Under the microscope, the apoptotic cells appeared stained brown.

2.14. Statistical analysis

Data are presented as means ± standard error of the mean (SEM). Statistical comparisons were performed using one-way analysis of variance (ANOVA), followed by the Tukey–Kramer post hoc test with GraphPad Prism Software Inc. (San Diego, CA, USA). A P < 0.05 was considered statistically significant.

3. Results

3.1. HPLC fingerprint profiles of lotus seed extract

The chromatographic profile of LSE was analyzed using HPLC. The results demonstrated a symmetric peak for gallic acid, which had a retention time of approximately 3.9 min. The quantities of gallic acid detected in the LSE were approximately 0.37 ± 0.01 % w/w, as shown in Supplementary Figs. 2–3.

3.2. Effects of LSE on systolic blood pressure

The effect of LSE and captopril on SBP is shown in Fig. 1. Administration of l-NAME for 5 weeks markedly increased SBP (P < 0.01 vs control, Fig. 1). Treatment with 10, 100 or 300 mg/kg of LSE lowered SBP by around 26–29 mmHg (P < 0.05 vs l-NAME group, Fig. 1), whereas 5 mg/kg of captopril restored SBP back to normal (P < 0.01 vs l-NAME and not significant vs control, Fig. 1). No dose dependent effect was observed for LSE. These data indicate that LSE was effective in restoring BP in l-NAME hypertensive rats, but it was less potent than 5 mg/kg of midodrine.

Fig. 1.

Effects of lotus seed extract (10, 100, 300 mg/kg) or captopril (5 mg/kg) on the systolic blood pressures in l-NAME-induced hypertensive rats at 5 weeks. The data is presented as mean ± SEM (n = 6). *P < 0.05, **P < 0.01 versus control, ^#P < 0.05, ^##P < 0.01 versus l-NAME. CAP, captopril; l-NAME, LN, N^ω-nitro-l-arginine methyl ester hydrochloride; LSE, lotus seed extract; SBP, systolic blood pressure.

3.3. Effects of LSE on body weight, absolute and relative organ weights

After 5 weeks of experiments, there were no differences in the body weights or the absolute and relative organ weights of testes, epididymis, vas deference and seminal vesicle among the experimental rat groups (Table 1).

Table 1.

Effects of lotus seed extract (10, 100, 300 mg/kg) or captopril (5 mg/kg) on the body weights, male reproductive organs weights (testes, epididymis, vas deference and seminal vesicle) and testosterone level in l-NAME-induced hypertensive rats at 5 weeks.

Parameters	Group
	Control	l-NAME	LN + LSE10	LN + LSE100	LN + LSE300	LN + CAP
Body weight (g)	531.92 ± 8.99	531.71 ± 12.87	535.86 ± 6.59	541.53 ± 12.25	550.28 ± 7.91	537.39 ± 5.63
Testis
Absolute (g)	1.78 ± 0.04	1.78 ± 0.03	1.81 ± 0.01	1.85 ± 0.04	1.86 ± 0.02	1.85 ± 0.02
Relative (per BW%)	0.33 ± 0.01	0.34 ± 0.00	0.35 ± 0.01	0.34 ± 0.00	0.34 ± 0.01	0.35 ± 0.01
Epididymis
Absolute (g)	0.78 ± 0.03	0.77 ± 0.01	0.75 ± 0.02	0.81 ± 0.08	0.80 ± 0.02	0.72 ± 0.02
Relative (per BW%)	0.15 ± 0.01	0.15 ± 0.01	0.13 ± 0.01	0.15 ± 0.01	0.14 ± 0.00	0.13 ± 0.00
Vas deference
Absolute (g)	0.22 ± 0.02	0.21 ± 0.01	0.19 ± 0.01	0.21 ± 0.02	0.18 ± 0.01	0.17 ± 0.01
Relative (per BW%)	0.04 ± 0.00	0.04 ± 0.00	0.03 ± 0.01	0.03 ± 0.00	0.03 ± 0.00	0.03 ± 0.00
Seminal vesicle
Absolute (g)	0.97 ± 0.05	0.92 ± 0.03	1.02 ± 0.03	1.07 ± 0.07	1.05 ± 0.04	0.94 ± 0.03
Relative (per BW%)	0.18 ± 0.01	0.17 ± 0.01	0.19 ± 0.01	0.19 ± 0.01	0.17 ± 0.01	0.17 ± 0.01
Testosterone (ng/ml)	8.71 ± 1.09	3.89 ± 0.49b	8.02 ± 0.87c	6.27 ± 1.25	5.50 ± 0.92ad	7.46 ± 0.66c

CAP, captopril; l-NAME, LN, Nω-nitro-l-arginine methyl ester hydrochloride; LSE, lotus seed extract.

^aP < 0.05.

^bP < 0.01 versus control.

^cP < 0.05 versus l-NAME.

^dP < 0.05 versus LSE10.

3.4. Effects of LSE on testosterone levels in the serum

To assess whether l-NAME-induced hypertension is associated with changes in testosterone levels, we investigated hormone production in hypertensive rats and evaluated the potential protective effects of LSE. Oral administration of l-NAME for 5 weeks decreased serum testosterone levels in the rats (P < 0.01 vs control), whereas treatment with 10 mg/kg of LSE or 5 mg/kg of captopril elevated the serum testosterone levels (P < 0.05 vs l-NAME group, Table 1). The higher doses (100 and 300 mg/kg) of LSE showed no effect. The administration of 10 mg/kg of LSE or captopril ameliorated these reductions in the hormonal levels.

3.5. Effects of LSE on sperm concentration, viability, and motility

The reduction in testosterone levels is related to decreased testicular sperm production and quality. In this study, the concentrations, viability, and motility of epididymal spermatozoa were reduced in the l-NAME-induced hypertensive rats (P < 0.01 vs control, Table 2) and these were restored by the administration of 10 mg/kg LSE or 5 mg/kg captopril (P < 0.01 vs l-NAME group, Table 2). l-NAME increased the percentage of immotile sperm (P < 0.01 vs control, Table 2) and this effect could be counteracted by treatment with either the 10 or 100 mg/kg LSE, or with 5 mg/kg captopril (P < 0.05, P < 0.01 vs l-NAME group). These findings suggest that a low dose of LSE (10 mg/kg) and 5 mg/kg of captopril were effective in enhancing sperm functionality in rats with l-NAME-induced hypertension.

Table 2.

Effects of lotus seed extract (10, 100, 300 mg/kg) or captopril (5 mg/kg) on sperm concentrations, viability, and motility in l-NAME-induced hypertensive rats at 5 weeks.

Sperm parameters	Group
	Control	l-NAME	LN + LSE10	LN + LSE100	LN + LSE300	LN + CAP
Sperm concentration (x106/ml)	82.76 ± 0.98	45.87 ± 4.05b	75.79 ± 2.97d	62.57 ± 3.61b	55.03 ± 5.89be	74.13 ± 3.09d
Sperm viability (%)	80.59 ± 1.33	47.26 ± 3.65b	74.98 ± 4.06d	59.74 ± 3.09be	52.86 ± 3.09bf	71.91 ± 2.07d
Sperm motility (%)	79.10 ± 2.74	48.97 ± 3.86b	68.68 ± 1.76d	62.06 ± 4.63a	50.41 ± 3.15be	70.91 ± 4.27d
Rapid progressive (%)	35.58 ± 1.59	7.98 ± 1.78b	32.00 ± 2.87d	22.99 ± 3.09bc	15.20 ± 1.76be	29.89 ± 2.40d
Slow progressive (%)	17.85 ± 0.87	5.67 ± 1.10b	10.50 ± 0.89bc	11.85 ± 2.12ac	7.54 ± 1.17b	13.19 ± 1.04d
Non progressive (%)	25.67 ± 1.79	35.33 ± 1.75b	26.02 ± 2.03c	27.28 ± 1.99c	27.70 ± 1.41c	27.85 ± 1.64c
Immotile (%)	20.91 ± 1.94	51.03 ± 2.37b	31.51 ± 1.95d	37.95 ± 1.94bc	49.60 ± 3.54bf	29.10 ± 2.85d

CAP, captopril; l-NAME, LN, N^ω-nitro-l-arginine methyl ester hydrochloride; LSE, lotus seed extract.

^aP < 0.05.

^bP < 0.01 versus control.

^cP < 0.05.

^dP < 0.01 versus l-NAME.

^eP < 0.05.

^fP < 0.01 versus LSE10.

3.6. Effects of LSE on MDA level and SOD activity in the testis and epididymis

Increased ROS production can lead to lipid peroxidation and impaired antioxidant defense, resulting in reduced sperm production and damage to testicular and epididymal structures. We measured MDA levels and SOD activities in the testis and epididymis to assess these effects. Elevated MDA and reduced SOD activity of testis and epididymis were observed in l-NAME hypertensive rats (P < 0.01 vs control, Fig. 2A and B). In testis, treatment with only low dose (10 mg/kg) of LSE or 5 mg/kg captopril reduced MDA (P < 0.01, 0.05 vs the l-NAME group), whereas in the epididymis, all doses of LSE or the 5 mg/kg captopril reduced the MDA level to near control values (P < 0.01 vs the l-NAME group). The increase in testicular and epididymal SOD activity caused by l-NAME were prevented by all doses of LSE and captopril (P < 0.01 vs l-NAME group, Fig. 2B).

Fig. 2.

Effects of lotus seed extract (10, 100, 300 mg/kg) or captopril (5 mg/kg) on MDA levels (A) and the SOD activities (B) in the testis and epididymis in l-NAME-induced hypertensive rats at 5 weeks. The data is presented as mean ± SEM (n = 6). **P < 0.01 versus control, ^#P < 0.05, ^##P < 0.01 versus l-NAME. CAP, captopril; l-NAME, LN, N^ω-nitro-l-arginine methyl ester hydrochloride; LSE, lotus seed extract; MDA, malondialdehyde; SOD, superoxide dismutase.

3.7. Effects of LSE on testicular and epididymal morphology

The study investigated the association between increased oxidative stress and histopathological alterations in hypertensive rats. We observed histopathological damage in the testis and epididymis of rats with l-NAME-induced hypertension. In these rats, the diameter and epithelial thickness of the seminiferous tubules were reduced (P < 0.01 vs control, Fig. 3, Fig. 4B), whereas the lumen of the seminiferous tubules were enlarged (P < 0.05 vs control, Fig. 3, Fig. 4C) and a loss of sperm mass in the cauda segment of the epididymis was observed (Fig. 3D). Only low dose (10 mg/kg) of LSE or 5 mg/kg captopril ameliorated l-NAME induced testicular morphological changes (P < 0.01, Fig. 3, Fig. 4B). Furthermore, treatment with 10 and 100 mg/kg of LSE or 5 mg/kg of captopril prevented epididymal damages (Fig. 3I, J, 3L and 4D). The higher doses (300 mg/kg) of the LSE showed no effect. Interestingly, the low dose of LSE (10 mg/kg) and 5 mg/kg of captopril could improve on testicular and epididymal damages caused by l-NAME.

Fig. 3.

Effects of lotus seed extract (10, 100, 300 mg/kg) or captopril (5 mg/kg) on the histopathological examination of the testis (A–F) and epididymis (G–L) in l-NAME-induced hypertensive rats (x20 magnification). CAP, captopril; l-NAME, LN, Nω-nitro-l-arginine methyl ester hydrochloride; LSE, lotus seed extract.

Fig. 4.

Effects of lotus seed extract (10, 100, 300 mg/kg) or captopril (5 mg/kg) on seminiferous tubules diameter (A), seminiferous epithelial thickness (B), seminiferous luminal diameter (C), and epididymal epithelial height (D) in the l-NAME -induced hypertensive rats at 5 weeks. The data is presented as mean ± SEM (n = 6). *P < 00.05, **P < 00.01 versus control, ^#P < 00.05, ^##P < 00.01 versus l-NAME. CAP, captopril; l-NAME, LN, Nω-nitro-l-arginine methyl ester hydrochloride; LSE, lotus seed extract.

3.8. Effects of LSE on apoptosis in rat testis

Besides observing histopathological damage and increased oxidative stress, we examined the increase in apoptotic spermatogenic cells within the testes of hypertensive rats and evaluated the protective role of LSE against these changes. The l-NAME-induced hypertensive group demonstrated an increase in the number of apoptotic spermatogenic cells in the testis compared to the control (P < 0.01 vs control, Fig. 5B and G). Treatment with the 10 and 100 mg/kg of LSE or 5 mg/kg captopril enhanced testicular cell apoptosis (P < 0.01 vs l-NAME group, Fig. 5C, D, 5F and 5G), whereas 300 mg/kg of LSE had no effect (Fig. 5E and G).

Fig. 5.

Effects of lotus seed extract (10, 100, 300 mg/kg) or captopril (5 mg/kg) on TUNEL assay of germ cell apoptosis in l-NAME induced hypertensive rats at 5 weeks (x20 magnification); (A) Control group, (B) l-NAME group, (C) LN + LSE10 group, (D) LN + LSE100 group, (E) LN + LSE300 group, (F) LN + CAP group and (G) quantification of % apoptotic cell per file. CAP, captopril; l-NAME, LN, Nω-nitro-l-arginine methyl ester hydrochloride; LSE, lotus seed extract.

4. Discussion

Hypertension is a serious health problem with a high prevalence worldwide, and is associated with the impairment of the male reproduction function, which includes testicular dysfunction and morphological changes.³ This study provided scientific evidence that the LSE effectively ameliorated reproductive dysfunction in the l-NAME-induced hypertensive rats. Our findings demonstrated that the LSE was effective in restoring systolic blood pressure and suppressing hypertension. It also mediated an increase in the sperm quality, antioxidant properties, testosterone levels and spermatogenic functions, in the hypertensive rats.

In our study, gallic acid was identified as the major component of the LSE, constituting 0.37 % w/w. This was determined using HPLC analysis for quality control assessment. A previous study has indicated that gallic acid attenuates BP and oxidative stress in spontaneously hypertensive rats.⁴¹ Thus, it may also have beneficial effects on male fertility, as observed in both human⁴² and animal models.^43,44

l-NAME, a NOS inhibitor, is used to induce hypertension in animal models, mimicking the pathophysiology of human hypertension.^45,46 It's known to cause endothelial dysfunction and vasoconstriction, thereby increasing peripheral resistance and leading to hypertension.¹¹ In this study, a dose of 40 mg/kg of l-NAME was selected based on previous evidence of its efficacy in developing hypertension and its potential negative impact on various functions, including reproduction.^5,14, 15, 16 After 5 weeks of l-NAME administration in our study, the rats exhibited high BP, reproductive dysfunction via increased oxidative stress in testicular and epididymal tissues, decreased testosterone and sperm production, and other abnormalities potentially due to apoptosis were also observed. Nevertheless, consistent with previous studies, the body weight of the l-NAME treated rats, as well as the weight of their testes, epididymis, vas deference and seminal vesicle did not differ from those of normal ones.^5,14,15

The data from our study showed that l-NAME affected the quality of sperms by reducing their count, the percentage of their progressive motility and viability. These findings are in accordance with previous studies that reported a decrease in sperm production and quality, following the administration of l-NAME.^5,14,15 The impairment in sperm and its fertilizing ability may be associated with a subsequent reduction in testosterone secretion levels⁴⁷ and increased ROS production in the testis and epididymis.⁴⁸

Testosterone, secreted by Leydig cells, is crucial for initiating and maintaining sperm production. It directly influences Sertoli cells, which release several proteins, including growth factors that are important for sperm maturation.⁶ Our study demonstrated that administering l-NAME to rats significantly lowered serum hormonal levels of testosterone. This reduction is consistent with earlier findings, and could be attributed to diminished responsiveness of Leydig and Sertoli cells, possibly due to oxidative damage in the testes of hypertensive rats.^5,14,49 Our findings also showed that l-NAME treatment markedly decreased testicular sperm production and quality, which were related to the diminished circulatory concentrations of testosterone in the hypertensive rats. The administration of only 10 mg/kg of LSE or captopril ameliorated these reductions in the hormonal levels, and improved the sperm functionality in l-NAME-induced hypertensive rats. These results suggest that the effect of the LSE treatment is beneficial for both hormone production and sperm formation in the hypertensive rats.

This study revealed histopathological damage in the testis and epididymis of l-NAME-induced hypertensive rats. The testis consists of seminiferous tubules, which are formed by the Sertoli cells. Their function is to provide structural support for the development of the germinal cells.⁵⁰ In mammals, spermatogenesis occurs within these tubules, releasing spermatozoa into the rete testis, which is connected to the epididymis.^6,47 The testicular structure in the l-NAME group exhibited morphological changes and disruption of the spermatogenesis process, which included a reduction in the diameter of the tubulars, an enlarged lumen, and increased apoptosis in spermatogonia in the seminiferous tubules. Previous studies have also reported that hypertensive conditions induced morphological alterations in both the testis and epididymis.^5,14,15 However, our results showed that oral treatment with 10 mg/kg of LSE or captopril enhanced the structure and tubular diameter in the testis, while higher doses (100 and 300 mg/kg) of LSE did not improve the histopathological damage. These findings suggest that the positive effects of LSE on seminiferous tubule damage could explain the increase in sperm count, the percentage of progressive motility and viability in the hypertensive rats.

The histopathological alterations in the testicular and epididymal structures of the l-NAME-induced hypertensive rats in this study, may be attributed to the direct or indirect effects of increased ROS.^5,14 This can cause lipid peroxidation and at the same time impair the antioxidant defense system of the rats.⁵¹ Elevated levels of ROS can adversely impact sperm quality, which causes reduced function, decreased motility, and DNA damage, thus diminishes fertility.⁴⁸ The study observed an imbalance between the antioxidant defense system and ROS production in the testis and epididymis of hypertensive male rats. There was a significant increase in ROS production and a decrease in the SOD levels in these rats. The damage to the testis and epididymis of the rats was caused by oxidative stress, which resulted from the elevated production of ROS.⁹ LSE administration effectively prevented the decrease in SOD levels in the testis and epididymis of l-NAME hypertensive rats treated with 10, 100, and 300 mg/kg of LSE or 5 mg/kg of captopril. Additionally, significant reductions in ROS were observed in the testis with the treatment of 10 mg/kg of LSE or 5 mg/kg of captopril. However, this improvement was not observed with 100 and 300 mg/kg doses of LSE. This observation demonstrated that LSE improved the antioxidant properties and inhibition of ROS lead to restoration of testosterone levels, and spermatogenic function in these hypertensive rats.

These results suggested that a low dose of LSE demonstrates therapeutic effectiveness, by exhibiting antioxidant properties and preventing lipid peroxidation in the testis and epididymis. Previous studies have associated lotus seeds with various beneficial properties, including antioxidant^31,52 and anti-inflammatory effects,²⁵ as well as the ability to inhibit the apoptotic signaling pathway.⁵³ This observation may be attributed to the protective roles that both phenolic compounds and flavonoids play in restoring male sex hormonal balance and spermatogenesis.^31,54,55 However, high doses of LSE may produce detrimental effects on the reproductive function in this hypertensive animal model. Several studies support anti-fertility activities due to high doses of LSE. The administration of the ethanol extract at dose levels of 50, 100, and 200 mg/day given orally to the rats for 60 days, resulted in decreased weights of the reproductive organs, suppressed testosterone levels, and reduced the sperm count and its motility.⁵⁶ These high doses also notably increased the testicular concentration of cholesterol, while significantly decreasing sialic acid, fructose, glycogen, and protein content.⁵⁶ Another study found that administering 100, 500, and 1000 mg/kg of lotus seed ethanolic extract led to a sustained decrease in the sexual behavior of the rats.⁵⁷ Additionally, previous research on gallic acid, which is the main bioactive compound found in LSE, suggested that high doses might have deleterious effects, while low doses could be beneficial in protecting against reproductive dysfunction in male rats.⁵⁸ Gallic acid has also been reported to exert a dose-dependent effect on the reproductive capability of male Brandt's voles, with low doses enhancing their reproductive functions,⁵⁹ thus providing support to our present observation.

Due to certain limitations in our study, we were unable to measure DBP and conduct the LSE experiment on normotensive rats. However, it is important to assess these effects in the future, as LSE may have an impact on health in control tissues. Additionally, the specific effects of the main component in LSE, which may contribute to its antihypertensive activity and influence reproductive dysfunction in l-NAME-induced hypertension, were not determined. Further research is required to evaluate the impact of LSEs on healthy tissues, and to identify the active compounds in LSE that alleviate reproductive dysfunction that are related to hypertension.

The administration of 10 mg/kg of LSE increased the serum total testosterone level and sperm parameters including count, individual motility, and viability. These findings suggest an enhancement in the antioxidant properties of LSE, as evidenced by improved reproductive parameters. A low dose of LSE demonstrates that its therapeutic effectiveness may be used in clinical practice to alleviate or prevent reproductive dysfunction associated with hypertension.

In this study, captopril was used as a positive control, due to it being a well-recognized medication for the treatment of hypertension. It has the ability to lower BP by inhibiting the angiotensin-converting enzyme.⁶⁰ Our results indicated that captopril reduced BP and improved the reproductive dysfunction in l-NAME-induced hypertensive rats. These findings are consistent with a previous pharmacological study, that showed that captopril enhanced sexual function in hypertensive rats by reducing BP and the modulation of sperm parameters, antioxidant properties and the reproductive hormones.⁶¹ Captopril has also been proven to protect against testicular damage and dysfunction in rats exposed to cadmium.⁶² Our study demonstrated that the administration of LSE at 10, 100, and 300 mg/kg was effective in restoring BP in l-NAME hypertensive rats, but it was less potent than 5 mg/kg of midodrine. Notably, treatment with 10 mg/kg of LSE showed the most potential in alleviating reproductive dysfunction associated with hypertension. The beneficial effects of LSE observed in our study suggest that it could be further tested in clinical settings for the prevention and treatment of hypertension. This is particularly relevant for male patients who present with reproductive dysfunction as a complication of hypertension.

5. Conclusions

This study is the first to demonstrate that LSE can ameliorate reproductive dysfunction in male rats with l-NAME-induced hypertension. LSE restored BP, testosterone levels and spermatogenic function in these hypertensive rats via the inhibition of ROS and enhancement of antioxidant status in the testis and epididymis. Therefore, our findings suggest that LSE at low dose could be utilized in functional foods to prevent hypertension-related male reproductive dysfunction. Further studies are recommended to investigate the therapeutic potential of low doses of LSE in clinical practice for alleviating male reproductive damage associated with hypertension.

Section: 1

Natural Products.

Taxonomy (classification by EVISE)

The experimental approach.

Traditional medicine, Reproductive dysfunction, Rat, Traditional herbal medicine.

Conflicts of interest

On behalf of all authors, I declare that there are no conflicts of interest.

Declaration of competing interest

All authors declare that they have no conflicts of interest to disclose.

Appendix A. Supplementary data

The following is the Supplementary data to this article: