Determining the Effect of L-arginine and Physical Activity on the Plasma Levels of Endothelin-1 and Tissue Inhibitor of Metalloproteinase-1 in Postmenopausal Women with Hypertension

Background and Objectives Exercise and L-arginine supplementation are known as effective strategies for treating hypertension. The aim of the present study was to determine the effect of aerobic exercise and L-arginine consumption on tissue inhibitor of metalloproteinase-1 (TIMP-1) and endothelin-1 (ET-1) levels in postmenopausal women with hypertension. Subjects and Methods In this quasi-experimental research, 40 postmenopausal women with hypertension were divided into four equal groups including exercise, exercise + L-arginine, L-arginine, and placebo groups. Interventions of aerobic exercise (60-75% of maximum heart rate) and L-arginine supplementation (6 g/day) were completed for 12 weeks. Forty-eight hours after the last intervention session, blood samples similar to the pre-test were collected to measure TIMP-1 and ET-1 levels using ELISA method. Results Plasma ET-1 levels in the training and training + L-arginine groups were significantly reduced compared to the placebo and L-arginine groups. In addition, plasma TIMP-1 increased significantly in the exercise group and exercise + L-arginine group compared to the placebo group. No synergistic effect was observed on TIMP-1 and ET-1 levels, despite the greater effect of exercise combined with L-arginine supplementation. Conclusion According to the results of the present research, improved blood pressure in adaptation to aerobic exercises is associated with reduced ET-1 and increased TIMP-1. L-arginine supplement, either alone or in combination with aerobic exercise, has no significant effect on TIMP-1 and ET-1 changes.


Extended Abstract
Introduction igh blood pressure conditions are defined as systolic blood pressure equal to or greater than 140 mm Hg and diastolic blood pressure equal to or greater than 90 mm Hg (2).Obesity is the main risk factor for hypertension and accounts for 65-78% of hypertension cases (4).In addition, some cases report an increased risk of hypertension among postmenopausal women compared to premenopausal women (5).Various mechanisms have been identified for the increase in blood pressure, but endothelial dysfunction has attracted much scholarly attention as a factor contributing to the development of hypertension (7).Endothelial cells can synthesize anti-atherosclerotic substances, a well-known example of which is nitric oxide (NO), which is produced from the metabolism of L-arginine by endothelial NO synthase (endotheli Nitric Oxide Synthase: eNOS) (8).NO is an important dilator that lowers blood pressure, and NOdependent dilation plays an important role in the pathogenesis of hypertension (9).Following NO research, dysfunctional endothelium can be a source of other mediators, including endothelin-1 (endothelin-1: ET-1), prostaglandin H2, and reactive oxygen species (ROS), which are harmful to arterial walls (10).ET-1, known as a peptide with 21 amino acids, plays a role in the regulation of the healing system, and its upregulation has been reported in patients with high blood pressure (11).In addition to ET-1, the levels of matrix metalloproteinases (MMP-2 and MMP-9) were increased in patients with hypertension (13).It has been suggested that circulating MMPs predict the risk of increased complications of hypertension-related diseases, while the tissue abilities of MMPs (Tissue Inhibitors of Metalloproteinases: TIMPs), including TIMP-1, can counteracts adverse effects of MMPs (14).Therefore, targeting MMPs can be considered a strategy for the management of hypertension.According to the literature, the use of sports exercises and related supplements on physical performance, including the L-arginine pathway and vasodilation, guides pathways for the antihypertensive effect of exercise and L-arginine in particular, which indicates the need for more research on this topic.Therefore, the present study investigated the changes in endothelin-1 and TIMP-1 levels after 12 weeks of aerobic training and L-arginine consumption among postmenopausal women with high blood pressure.

Methods
In this quasi-experimental research, 40 postmenopausal women with high blood pressure were divided into four equal groups including exercise, exercise + L-arginine, Larginine, and placebo.Forty-eight hours before the intervention, anthropometric variables and fasting blood samples were measured.Exercise intervention in exercise groups was performed for 12 weeks (three sessions per week) which involved running on a treadmill.In each session of aerobic training, five minutes of warm-up with 40% of maximum heart rate (low intensity) was performed, and the main part of the training program consisted of 20 minutes of walking or jogging with moderate intensity (60-75% of the maximum heart rate).At the end of the training session, a five-minute cool-down at 40% of maximum heart rate (low intensity) was performed by the participants (19).L-arginine supplementation was considered at a dose of six grams per day (three 2-gram servings).The placebo group received the same amount of placebo (white flour capsules).The positive effects of the defined dose of L-arginine supplementation in patients with high blood pressure observed in this study had been confirmed in previous studies without any side effects (20).Forty-eight hours after the last intervention session, blood samples similar to the pre-test were collected to measure TIMP-1 and ET-1 levels using ELISA method.Plasma was kept in a freezer at -80°C.
The plasma levels of TIMP-1 (Elabscience Co, Cat No: E-EL-H0184) and endothelin-1 (Elabscience Co, Cat No: E-EL-H0064) were measured using ELISA method according to the kit manufacturer's instructions.In addition, the participant's body fat percentage was measured by body composition analysis (BOCA-X1, South Korea).All data of this research were analyzed using SPSS statistical software version 24 and using paired t-test for intra-group comparison and analysis of covariance test.ANCOVA and Bonferroni's post hoc test were used to determine the between-group difference, and the significance level was set at P < 0.05.

Results
In the exercise and exercise + L-arginine groups, analysis of covariance test showed a significant change in the levels of ET-1 (P < 0.001) and TIMP-1 (P = 0.002) after 12 weeks of intervention.The results of post hoc Bonferroni test showed a significant decrease in ET-1 levels in the exercise group compared to the placebo (P < 0.001) and supplement (P = 0.021) groups.Similarly, a significant decrease in ET-1 plasma levels was observed in the exercise + L-arginine group compared to the placebo and supplement groups (P < 0.001).On the other hand, paired t-test showed that ET-1 decreased in the exercise group ((P = 0.001) and the exercise + L-arginine group (P < 0.001), which was significant after 12 weeks.TIMP-1 changes in placebo (P = 0.482) and supplement (P = 0.380) groups were not significant.However, the results showed that TIMP-1 levels increased significantly in the exercise (P = 0.039) and the exercise + L-arginine groups (P = 0.002) compared to the P group.Within-group analysis showed that TIMP-1 levels were significantly increased in the supplement (P = 0.005), exercise (P < 0.001) and exercise + L-arginine groups (P < 0.001).

Conclusion
The findings of the present study showed that aerobic exercise is an effective strategy to reduce ET-1, which can ultimately lead to the improvement of systolic and diastolic H blood pressure.According to the results of the present study, the improvement of blood pressure in adaptation to aerobic exercise in terms of a decrease in ET-and L-arginine supplementation either alone or in combination with aerobic exercises does not have a significant effect on the changes of TIMP-1 and ET-1.