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Protective Effect of Short-Term Treatment With Low Dose of L-Arginine in Postmenopausal Rats Due to Increased Serum Levels of Argipressin and Oestrogen
*Corresponding author: Dr. Manizheh Karami, Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran. karami@shahed.ac.ir
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Received: ,
Accepted: ,
How to cite this article: Lakzaei F, Karami M, Jalali-Nadoushan M. Protective Effect of Short-Term Treatment With Low Dose of L-arginine in Postmenopausal Rats Due to Increased Serum Levels of Argipressin and Oestrogen. J Health Allied Sci NU. 2025;15:528-35. doi: 10.25259/JHS-2024-10-2-(1600)
Abstract
Objectives
Serum levels of arginine vasopressin (AVP) or argipressin, oestrogen (E) and nitric oxide (NO) decrease in menopause, but follicular stimulating hormone (FSH), luteinizing hormone (LH), tumour necrosis factor alpha (TNF-α), and interleukin 1 beta (IL-1β) increase, which causes postmenopausal problems in women. These factors were measured in aged rats treated with L-arginine and/or NO synthase inhibitor over three weeks (different time intervals).
Material and Methods
Wistar rats (dioestrus, weighing 250 g and 36 weeks old) were randomly divided into two control and experimental groups. They were treated with L-arginine (5, 25, and 50 mg/kg) or/and L-NAME (NG-nitro-L-arginine methyl ester -5 and 25 mg/kg), i.p. (intraperitoneal) for 3-21 days, once a day. The control group received only saline (1 mL/kg). Serum levels of AVP, E, FSH, LH, TNF-α, and IL-1β were determined. The animals’ ovaries, uterus, and body weight were also studied to show biometric differences. Findings were analysed using ANOVA (Analysis of variance - α = 0.05).
Results
L-Arginine at the highest doses in the 3-day treatment increased AVP levels and at lower concentrations improved oestrogen levels in all periods. It reduced the number of follicular cysts at the lowest dose and decreased TNF-α and IL-1β levels in the short period. L-NAME pretreatment significantly reduced the effects of low-dose and short-term L-arginine treatment. Gonadotropin levels did not change significantly, but ovarian diameter and cytokine levels increased in long periods, and weight decreased.
Conclusion
L-Arginine at low dose and short-term treatment can improve menopausal problems by increasing serum levels of AVP and oestrogen and reducing inflammatory factors with the involvement of NO.
Keywords
Arginine vasopressin
L-Arginine
Menopause
Oesterogen
Rat
INTRODUCTION
Menopause is characterised by 12 months of amenorrhea due to permanent cessation of ovarian function. The average age of menopause in women is 51 years.[1] During menopause, the ovaries largely stop producing oestrogen and progesterone. In addition, ovulation does not occur, and levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) increase.[1-3]
Mechanistically, the decline in sex hormone levels trigger feedback loops in the pituitary gland to increase FSH and LH levels. FSH stimulates follicular development, and LH stimulates ovulation.[1] These events cease during menopause, a period of a woman’s life marked by problems such as depression, osteoporosis, and premature aging.[2] The ovaries also become cystic at this stage.[4] More interestingly, the consequences of menopause in rodents (Order: Rodentia, Family: Muridae) are similar to those in humans.[5] The most common treatment for menopausal problems is oestrogen therapy,[6,7] which, however, has side effects such as high risk of cardiovascular disease,[8] and increased serum cholesterol and triglyceride concentrations.[9]
Oestrogen exerts its effect on the activity of the nitric oxide (NO) system in target organs, a system that metabolically produces a free radical molecule called NO.[10] This gaseous molecule (NO) plays a role as an endogenous mediator in many physiological and pathological phenomena of the body, such as inflammation. This molecule is formed from the conversion of the amino acid L-arginine to L-citrulline, which is catalysed by the enzyme NO synthase (NOS). This enzyme can be inhibited by L-NAME (NG-nitro-L-arginine methyl ester).[11] NOS enzyme activity can be altered by steroid hormones, as steroid hormones have been shown to regulate NO production in women. Furthermore, previous studies have suggested that decreased oestrogen levels in postmenopausal women may contribute to decreased NO levels during this period.[12]
Arginine vasopressin (AVP) or argipressin, known as antidiuretic hormone (ADH), can control water balance in the body. The hypothalamus is the main source of AVP in the brain, and this neuropeptide is synthesised by neurons in the paraventricular (PV) and supraoptic (SO) nuclei.[13] Studies have shown that these neurons decrease in rats during menopause.[14] Furthermore, AVP content in the brain decreases with age, and hence, AVP has been used as an anti-aging agent.[14] The amino acid L-arginine is likely to be effective in restoring human health.[15] Therefore, we aimed to use this amino acid in a wide concentration range and for short to long periods to treat menopausal problems in aged rats. We also examined changes in TNF-α and IL-1β levels, as it has been previously reported that levels of these cytokines are very high in postmenopausal women.[16]
MATERIAL AND METHODS
Subject
In this experimental study, virgin Wistar rats weighing approximately 250 g and aged 36 weeks were obtained from the Institutional Animal Care and Breeding Center and maintained under standard conditions in accordance with international ethical protocols: 23 ± 2°C and 12-hour light/dark cycle with free access to water and food ad libitum. The local ethics committee approved all experiments, and an ethical code was granted to this research (IR.SHAHED.REC.1399.106).
Papanicolaou stain and female cycle
To determine the stages of the oestrous cycle of female rats, vaginal smears were taken using a sterile dropper (Pasteur pipette), and the samples were fixed on a slide with 3% ethanol. After this step, the fixed samples were washed with running water for 5 seconds and then stained with haematoxylin (Farzaneh Arman Co. Ltd., Iran) for about two minutes. The samples were immediately rinsed with running water and, after exposure to 3% ethanol (3 seconds), stained with Orange G6 dye (Sigma Aldrich, St. Louis, U.S.A.) to distinguish the cytoplasm. The samples were then immersed in 3% ethanol. They were then cleared in xylene. Finally, the samples were mounted on slides using Entellan glue (Merck, Germany) and examined with a light photomicroscope (Olympus, Japan). The diestrus cycle was marked by the presence of many small leukocytes, so this smear preparation confirmed that the virgin rats were indeed old at this age.
Treatments
Animals in the diestrus stage were randomly divided into two groups: control and experimental. The control group received saline (1 mL/kg) intraperitoneally (i.p.) at all stages (3, 5, 9, and 21 days). The treatment groups were subdivided into L-arginine, L-NAME, and pre-treatment with L-NAME groups. The L-arginine groups received L-arginine (5, 25, and 50 mg/kg, i.p.) for three weeks (different time intervals). The L-NAME groups were given L-NAME (5 and 25 mg/kg, i.p.) once daily for three weeks (different time intervals). Groups receiving a combination of L-NAME, a competitive inhibitor of nitric oxide synthase (NOS), and L-arginine were treated first with L-NAME and then with L-arginine 20 minutes later. This was done for each of two doses of L-NAME (5 and 25 mg/kg, i.p.) versus each of the different doses of L-arginine (5, 25, and 50 mg/kg, i.p.) and at different time intervals (3, 5, 9, and 21 days)
Serology
Twenty-four hours after treatment, rats were deeply anesthetised with ketamine 100 mg/kg and xylazine 20 mg/kg, and blood samples were taken via cardiac puncture and centrifuged at 3000 rpm for 15 minutes after half an hour. Serum samples were collected and stored in a deep freeze (-80°C). These samples were then analysed by measuring serum concentrations of AVP or argipressin (Abnova Co., Taiwan, Cat No: KA4582), oestrogen (Cortez Diagnostics Inc., U.S.A., Cat No: 9028-16), gonadotropins (G-Biosciences, Missouri, U.S.A., Cat No: IT7468), TNF-α (R&D Systems, Inc., U.S.A., Cat No: RTA00), and IL-1β (R&D Systems Inc., U.S.A., Cat No: RLB00) using ELISA (enzyme-linked immunosorbent assay) kits.
Pathology
After blood collection, a longitudinal incision was made on the rat’s abdominal surface, and the ovaries and uterus were examined biometrically. Finally, the tissues were collected in formalin (10%). After 72 hours, these tissues were cut into sections (3-4 µm) by a microtome (Leica, Italy) and then stained with haematoxylin-eosin (Farzaneh Arman Co. Ltd., Iran). The number of cysts was evaluated eventually, and primary, secondary, and Graafian follicles were determined. Results were analysed and compared with control data.
Statistical analysis
All findings were examined using the Kolmogorov-Smirnov test with SPSS software (version 22) and then analysed with analysis of variance (ANOVA). P<0.05 was considered significant. Tukey’s post hoc analysis was used to examine differences between groups. All results were shown as mean ± standard error of the mean. Images were also analysed by Image J software (Java, free).
RESULTS
Pap smear
Papanicolaou (Pap) smears were taken from all rats over four days to demonstrate the oestrous cycle. Animals in the dioestrus phase, which is indicative of ovarian senescence, were specifically selected, and reproductive animals were excluded from this study. It should be mentioned that small leukocytes predominate in the diestrus phase [Figure 1].
- Pap smear result. The figure under 100x magnification shows the diestrus phase because the leukocytes are the predominant cells (blue arrow). The scale bar is 10 µm.
Serum AVP and oestrogen levels
During the 3-day treatment, AVP levels increased relative to controls under the highest doses of L-arginine (50 mg/kg) and were not reversed by high-dose L-NAME intervention [Figure 2]. During this period, oestrogen levels were significantly increased by L-arginine doses [Figure 3]. However, hormone concentrations were decreased by L-NAME at the lowest L-arginine doses, suggesting the involvement of NO during this short-term treatment with low-dose L-arginine. At five days of treatment, AVP and oestrogen levels increased compared to the control group and were not affected by pretreatment with L-NAME, except for oestrogen levels at the lowest dose of L-arginine (5 mg/kg) [see Figure 2 and Figure 3]. In the 9- and 21-day treatments, a significant increase in AVP levels was obtained at the highest doses of L-arginine (50 mg/kg) and was not reversed under L-NAME treatment, but oestrogen levels did not change compared to the control group [see Figures 2 and 3].
- Levels of AVP. The control group (n=6) and the treatment group (n=6 in each dose) received saline (1 mL/kg, i.p.) and substances once a day for three consecutive days up to 21 days. The treatment groups included L-arginine; LA5, LA25 and LA50 representing different doses of L-arginine (5, 25 and 50 mg/kg). and L-NAME (at different doses- 5, 25 mg/kg). In the cumulative injection method, L-NAME was injected first, and L-arginine was injected 20 minutes later. AVP levels increased, particularly at higher doses of L-arginine, and were not blocked by pre-injection of L-NAME. AVP: Arginine vasopressin. L-NAME: N(G)-nitro-L-arginine methyl ester.
- Levels of oestrogen (E). The control group (n=6) and the treatment group (n=6/per dose) received saline (1 mL/kg, i.p.) or NO agent once a day for three up to 21 days. In the single L arginine group; LA5, LA25 and LA50 representing different doses of L-arginine (5, 25 and 50 mg/kg), oestrogen levels increased with L-arginine concentration in short-term treatment periods (3 to 5 days) and were not completely reversed by pre-injection of L-NAME (at different doses - 5, 25 mg/kg). except at very low doses in short treatment periods. Tukey’s post hoc test showed a difference between the experimental and control groups (*P<0.05). L-NAME: N(G)-nitro-L-arginine methyl ester.
Number of follicular cysts, ovarian and uterine dimensions, and animal weight
Regarding the number of follicular cysts, the number of cysts was significantly reduced at lower doses of L-arginine (5-25 mg/kg) (P<0.01) [Figure 4]. At this time, regardless of the NO precursor doses, the size of the ovary and uterus, as well as the weight of the animals, did not show significant changes [Figure 5]. During the 5-day treatment, a decrease in cyst number was observed at all doses of NO precursor except the highest doses (50 mg/kg) [Figure 4]. Ovarian and uterine size and animal weight similarly did not show significant differences. However, in the 9- and 21-day treatments, the number of cysts was reduced only at the lowest dose of L-arginine (5 mg/kg) [Figure 4]. Biometric data of ovaries and animal weight did not show significant changes at that dose. [see Figure 5].
- Ovarian cyst and H & E evidence. The number of cysts was examined in all groups. Low doses of L-arginine and short treatment periods reduced the number of cysts. The difference between the experimental and control groups was obtained by Tukey’s post hoc test (**P<0.01). (a and b) Based on haematoxylin-eosin (H & E)-stained images at 4x magnification, control ovaries showed many follicular cysts, which were reduced in low-dose L-arginine treatment. “C” and “GF” represent cyst and Graafian follicle, respectively. The scale bar is 50 µm. H & E: Haematoxylin and eosin stain. The numbers 5, 25 and 50 represent different doses of L-arginine (5, 25, and 50 mg/kg).
- Biometrical evidence. (a-c) Ovarian and uterine diameters and animal weight were examined in all groups. At the lowest dose of L-arginine and short treatment periods, no significant change was observed compared to the control group. Tukey’s post hoc test showed a difference between the experimental and control groups (*P<0.05). The numbers 5, 25 and 50 represent different doses of L-arginine (5, 25, and 50 mg/kg).
Serum LH and FSH levels
During the 3-day treatment, LH and FSH levels did not change significantly. During the 5-day to 21-day treatment, their levels similarly did not show significant differences [see Figure 6].
- FSH and LH levels. FSH and LH levels were measured in all groups. There was no significant change. The numbers 5, 25 and 50 represent different doses of L-arginine (5, 25, and 50 mg/kg). FSH: Follicular stimulating hormone, LH: Luteinizing hormone.
Serum levels of TNF-α and IL-1β
Interestingly, during 3 days of treatment, TNF-α and IL-1β levels were significantly reduced at the lowest dose of L-arginine (P<0.01) [Figure 7], indicating a relationship between NO and cytokine levels at the low dose of L-arginine during short-term treatment. However, cytokine levels increased at the highest doses of L-arginine during all treatment periods [Figure 7].
- (a and b) TNF and IL-1β levels. TNF and IL-1β levels were studied in all groups. At the lowest doses of L-arginine and short treatment periods, a highly significant decrease was observed compared to the control group (p<0.01). However, the result was different with increasing dose and especially with increasing treatment duration (increasing levels were observed). Tukey’s post hoc test showed the difference between groups (*p<0.05, **p<0.01, ##p<0.01, ###p<0.001). The number zero represents the control group and the numbers 5, 25 and 50 represent different doses of L-arginine (5, 25, and 50 mg/kg). TNF: Tumour necrosis factor, IL-1β: Interleukin 1 beta.
DISCUSSION
In order to overcome menopausal problems such as decreased AVP or argipressin and E, female aged Wistar rats that were in diestrus based on Pap smear test were administered L-arginine. In addition, they had follicular cysts in their ovaries, which were likely indicative of postmenopausal ovarian aging.
AVP or argipressin and oestrogen levels improved with L-arginine at low doses in short time courses, but no significant change was observed in gonadotropin levels. However, cytokine levels increased at high doses during long-term treatment. Pre-treatment with L-NAME only blocked the effects of the low dose in short-term periods. Reductions in follicular cysts were observed only in short-term periods, and weight loss and ovarian swelling were observed in the high dose of L-arginine in longer periods (9 and 21 days).
It has previously been shown that a decrease in the number of follicles during the reproductive life span is associated with an increase in the percentage of lymphocytes[17] and a change in the macrophage population in the ovary.[18] In this study, lymphocytes and macrophages were not specifically studied, so we do not have a similar interpretation. However, we may attribute these findings to an inflammatory response due to the significant changes in ovarian size at higher doses of L-arginine. In addition, TNF-α and IL-1β levels were simultaneously elevated at those doses. As noted elsewhere, there is a relationship between women’s aging and increased levels of cytokines involved in inflammation.[16-19]
The aforementioned result is consistent with other cases that have evaluated inflammation during ovarian aging and reported an association between the levels of these factors and the occurrence of ovarian aging and the reduction of primary follicles.[20] Furthermore, the increased levels of cytokines may be association with high levels of NO (a proinflammatory factor), as NO has been reported to induce the production of proinflammatory cytokines including TNF-α, IL-1β, IL-1α, and IL-12, which cause tissue damage.[21]
No significant changes in animal weight were observed after short-term treatment periods. However, weight loss was still observed with increasing dose and duration of treatment (P<0.01). Previous studies have shown that inhibiting NO synthesis in rats can increase serum triglyceride levels and increase body fat mass. Conversely, they have reported that increasing NO levels in rats can lead to weight loss.[22] This laboratory has previously reported activation of NO synthase (NOS) in ovaries after L-arginine treatment,[23] so based on both results, weight change during high-dose L-arginine treatment periods may be due to an increase in NO levels, so NO levels should be measured in future studies.
E levels showed a significant increase compared to the control, especially during short periods (3-day and 5-day treatment). Based on previous achievements,[12] NO can increase oestrogen levels, which is consistent with current data. Moreover, there is an innovation in the present study, since the increase in oestrogen levels coincided with AVP secretion. According to the findings of other researchers, the oestrogen receptors are present in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) in the brain and can regulate AVP secretion.[13] To discuss, we may hypothesise that high oestrogen levels could activate NOS in the hypothalamic-pituitary-gonadal (HPG) axis, which could restore steroid-dependent AVP levels. This fact has previously been attributed to the NO donor [24], but according to our findings, treatment with low concentrations of L-arginine in short periods gave the best results, and also with this result, further data can be discussed more clearly. We know that AVP-secreting neurons are found in the PVN,[13] a part of the circumventricular system that senses changes in the concentration of substances in the circulation. Therefore, due to changes in extracellular NO levels in response to L-arginine treatment, PVN neurons are likely to be activated and trigger supraoptic and suprachiasmatic sites to produce AVP. We suggest studying this signalling pathway in the future.
With two doses of L-arginine (5 and 25 mg/kg), during three and five days of treatment, and with the lowest dose of L-arginine (5 mg/kg) in nine and 21-day treatments, a significant reduction in the number of cysts was observed (P<0.01). For interpretation, the presence of NO is essential for the development of follicles in the ovary,[25] however, increased concentrations of L-arginine can disturb female steroid production and cause increased androgen levels, which stimulate the production of ovarian cyst.[26] It has also been shown that excessive production of NO in the body can lead to cyst-genesis and inflammation.[23]
Gonadotropin levels did not show any significant changes in this study, whereas previous studies have shown that NO can increase gonadotropin levels,[27] which may challenge previous studies in this aspect. On the contrary, this study indicated a protective role of NO in ovarian aging, in agreement with other authors[28,29] who have demonstrated the efficacy of sex hormones in health and prevention of aging. We showed that short-term treatment with low-dose L-arginine protects against postmenopausal problems, which is associated with increased AVP and E levels.
Sex steroids regulate NOS and influence NO levels in humans and murids.[30,31] One of the strengths of this work was the use of a rat model that simulates the problems of human ovarian aging and menopause in terms of histology and neuroendocrine function. However, due to time constraints during this work, we did not explore all possible mechanisms and had to suggest them for future studies.
CONCLUSION
We found that L-arginine at low doses over short periods of three to five days can increase NO production in a rat model of menopause with low oestrogen levels and improve AVP and oestrogen levels. Therefore, the amino acid L-arginine may alleviate some of the problems of menopause by increasing AVP and oestrogen levels through NO intervention.
Acknowledgment
We thank our University for supporting this Ph.D program.
Ethical approval
The research/study approved by the Local Ethics Committee, at Shahed University, number IR.SHAHED.REC.1399.106, dated 27th October 2020.
Declaration of patient consent
Patient’s consent not required as patients identity is not disclosed or compromised.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Use of artificial intelligence (AI)-assisted technology for manuscript preparation
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
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