Research code: مقاله مستخرج از رساله دکتری است
Ethics code: IR.IAU.TABRIZ.REC.1404.092
Clinical trials code: مقاله مستخرج از رساله دکتری است
Farzad Seifi1 
, Farzad Zehsaz *2 , MirAlireza Nourazar1 , Roghayeh Poozesh Jadidi1 , Mehri Ghahremani1
, Farzad Zehsaz *2 , MirAlireza Nourazar1 , Roghayeh Poozesh Jadidi1 , Mehri Ghahremani1
1- Islamic Azad University, Islamic Azad University
2- Islamic Azad University, Islamic Azad University ,Farzad.zehsaz@iau.ac.ir
2- Islamic Azad University, Islamic Azad University ,
Abstract: (222 Views)
Introduction
Essentially, apoptosis is the final stage of cellular damage, and researchers believe that menopause and aging are factors that contribute to apoptosis [5, 6]. Apoptosis is a physiological cell suicide program that may be involved in many clinical cardiac disorders [7].
Some studies have shown the positive effects of aerobic exercise and physical activity in preventing and minimizing the complications of estrogen deficiency during menopause [16]. Exercise has an anti-inflammatory effect that inhibits the production of TNF-α, a potential inducer of the extrinsic pathway of apoptosis. It has been reported that exercise reduced the ratio of caspase-3/caspase-3 and increased the expression of Bcl-2 [19].
Given the beneficial and abundant medicinal properties of the clover plant, and given that previous studies have not examined the combined effect of physical activity in the form of regular exercise along with a special diet on reducing the symptoms and complications of menopause, Therefore, the aim of the present study was to investigate the combined effect of eight weeks of aerobic exercise training combined with red clover extract on oxidative factors and some indicators of cardiac apoptosis in orchiectomized rats.
Method
For this study, 48 female Wistar rats weighing approximately 250±20 g and approximately 12-13 weeks of age were used. Feeding and housing conditions were the same for all groups, with a 12-hour light/dark cycle and a temperature of 21±2°C. The same diet and water were also freely available, and after one week of habituation to the new conditions, the experiment began. The rats were randomly divided into 6 groups of 8 (healthy control, sham, ovariectomized, ovariectomized + red clover, ovariectomized + training, and ovariectomized + red clover + training). Two weeks after ovariectomy surgery (mice were anesthetized intraperitoneally with ketamine (90 mg/kg) and xylazine (10 mg/kg). After complete anesthesia, bilateral surgery was performed and both ovaries were removed), administration of red clover extract began. 1 gram of clover plant extract and 1 gram of red clover seed extract, mixed with 8.5 cc of distilled water, will then be fed 0.5 ml per kilogram of body weight of the extract daily for 8 weeks via gavage system to each mouse. The red clover extract and aerobic exercise treatment group received the extract at a dose of 500 mg/kg of body weight, and they exercised on a treadmill for eight weeks, just like the exercise group. The sham group received 1 ml/kg of normal saline daily by intraperitoneal injection (28).
Finally, the mice were anesthetized and euthanized with ketamine (90 mg/kg) and xylazine (10 mn/kg). The chest region was incised and the heart was carefully removed from the animal's body. Immediately after removal and washing with saline solution, it was immediately placed in tubes and transferred to liquid nitrogen, and then stored in a -80°C refrigerator until measurement.
Results
In graph A, the results of the One-way ANOVA test showed that the expression of pro-Caspase 3 (Pro Cas-3) protein in the heart tissue of OVX group mice was significantly reduced compared to the Sham group (p<0.01). This protein did not differ significantly between the OVX+E, OVX+M, and OVX+E+M groups, while it was significantly increased compared to the OVX group (p<0.05 (compared to the OVX+E and OVX+M groups and p<0.01 compared to the OVX+E+M group). Also, no significant difference was observed between the Sham and OVX+E+M groups. The expression level of Cleaved Caspase-3 (Cleaved Cas-3) protein in graph B showed a significant increase in the OVX group compared to the other groups (p<0.001). The expression of this protein did not show a statistically significant difference between the Sham, OVX+E, OVX+M and OVX+E+M groups.
The expression levels of pro-Caspase 9 (Pro Cas-9) and Cleaved Caspase-9 (Cleaved Cas-9) proteins are shown in graphs C and D. According to these results, a slight decrease in the expression of proCas-9 protein was observed in the OVX group compared to the other groups, which was not statistically significant. No statistically significant differences were observed between the studied groups in terms of the expression of these proteins.
Discussion
In the present study, red clover and aerobic exercise training partially reduced caspase-3 activity, possibly exerting a protective effect on cells. Caspase-3 activation is observed after induction of reactive oxygen species by exposure to xanthine/xanthine oxidase, whereas no caspase-3 activity is induced by H2O2 treatment [34]. Studies suggest that physical activity is a key component of cardiac rehabilitation programs for patients with CVD, as it reduces the risk of CVD as a non-pharmacological treatment by protecting the heart from serious damage and improving heart function [18]. In a study investigating increased cardiac apoptosis in diabetic rats, 10 weeks of aerobic exercise on a treadmill was found to protect the heart from apoptosis [35]. Some studies have shown the positive effects of aerobic exercise and physical activity in preventing and minimizing the complications of estrogen deficiency during menopause [16]. Aerobic exercise and meditation are also effective in reducing anxiety and depression. Depression and anxiety are more pronounced in CVD patients than in healthy individuals (38). A recent meta-analysis found a small but significant increase in the risk of AH in women with premature menopause compared with women with menopause at age >45 years.[39] Possible pathogenetic mechanisms include the production of vasoconstrictor factors such as endothelin and angiotensinogen as a result of reduced estrogen concentrations and a lower estrogen-to-androgen ratio during menopause [40]. Epidemiological studies have shown a reduced cardiovascular risk in populations with high dietary isoflavone intake [41]. Additionally, a previous study provided evidence that two weeks of twice-daily supplementation with red clover reduced VCAM-1 expression and significantly improved vascular inflammation in postmenopausal women [42]. Studies have been conducted on the association between red clover supplementation and cardiovascular disease risk factors. Vascular endothelial function is not firmly linked to the development of atherosclerosis or hypertension, but it is hypothesized to play a role in the pathology of these diseases. Two published studies show that adhesion and platelet factors are not altered by red clover isoflavone supplementation. Since prostacyclin can inhibit platelet adhesion and aggregation to the endothelium, this may be a mechanism by which red clover isoflavones improve vascular health. Red clover isoflavones also increased endothelial nitric oxide synthase activity and nitrite levels in human umbilical vein endothelial cells after 48 hours of exposure. This is another potential vascular mechanism (non-immediate, genomic) of action of red clover isoflavone supplements. Arterial compliance is a measure of arterial stiffness that is associated with the presence of atherosclerotic plaques in major blood vessels. The effect of red clover isoflavones on this parameter has been investigated in two studies (43).
Essentially, apoptosis is the final stage of cellular damage, and researchers believe that menopause and aging are factors that contribute to apoptosis [5, 6]. Apoptosis is a physiological cell suicide program that may be involved in many clinical cardiac disorders [7].
Some studies have shown the positive effects of aerobic exercise and physical activity in preventing and minimizing the complications of estrogen deficiency during menopause [16]. Exercise has an anti-inflammatory effect that inhibits the production of TNF-α, a potential inducer of the extrinsic pathway of apoptosis. It has been reported that exercise reduced the ratio of caspase-3/caspase-3 and increased the expression of Bcl-2 [19].
Given the beneficial and abundant medicinal properties of the clover plant, and given that previous studies have not examined the combined effect of physical activity in the form of regular exercise along with a special diet on reducing the symptoms and complications of menopause, Therefore, the aim of the present study was to investigate the combined effect of eight weeks of aerobic exercise training combined with red clover extract on oxidative factors and some indicators of cardiac apoptosis in orchiectomized rats.
Method
For this study, 48 female Wistar rats weighing approximately 250±20 g and approximately 12-13 weeks of age were used. Feeding and housing conditions were the same for all groups, with a 12-hour light/dark cycle and a temperature of 21±2°C. The same diet and water were also freely available, and after one week of habituation to the new conditions, the experiment began. The rats were randomly divided into 6 groups of 8 (healthy control, sham, ovariectomized, ovariectomized + red clover, ovariectomized + training, and ovariectomized + red clover + training). Two weeks after ovariectomy surgery (mice were anesthetized intraperitoneally with ketamine (90 mg/kg) and xylazine (10 mg/kg). After complete anesthesia, bilateral surgery was performed and both ovaries were removed), administration of red clover extract began. 1 gram of clover plant extract and 1 gram of red clover seed extract, mixed with 8.5 cc of distilled water, will then be fed 0.5 ml per kilogram of body weight of the extract daily for 8 weeks via gavage system to each mouse. The red clover extract and aerobic exercise treatment group received the extract at a dose of 500 mg/kg of body weight, and they exercised on a treadmill for eight weeks, just like the exercise group. The sham group received 1 ml/kg of normal saline daily by intraperitoneal injection (28).
Finally, the mice were anesthetized and euthanized with ketamine (90 mg/kg) and xylazine (10 mn/kg). The chest region was incised and the heart was carefully removed from the animal's body. Immediately after removal and washing with saline solution, it was immediately placed in tubes and transferred to liquid nitrogen, and then stored in a -80°C refrigerator until measurement.
Results
In graph A, the results of the One-way ANOVA test showed that the expression of pro-Caspase 3 (Pro Cas-3) protein in the heart tissue of OVX group mice was significantly reduced compared to the Sham group (p<0.01). This protein did not differ significantly between the OVX+E, OVX+M, and OVX+E+M groups, while it was significantly increased compared to the OVX group (p<0.05 (compared to the OVX+E and OVX+M groups and p<0.01 compared to the OVX+E+M group). Also, no significant difference was observed between the Sham and OVX+E+M groups. The expression level of Cleaved Caspase-3 (Cleaved Cas-3) protein in graph B showed a significant increase in the OVX group compared to the other groups (p<0.001). The expression of this protein did not show a statistically significant difference between the Sham, OVX+E, OVX+M and OVX+E+M groups.
The expression levels of pro-Caspase 9 (Pro Cas-9) and Cleaved Caspase-9 (Cleaved Cas-9) proteins are shown in graphs C and D. According to these results, a slight decrease in the expression of proCas-9 protein was observed in the OVX group compared to the other groups, which was not statistically significant. No statistically significant differences were observed between the studied groups in terms of the expression of these proteins.
Discussion
In the present study, red clover and aerobic exercise training partially reduced caspase-3 activity, possibly exerting a protective effect on cells. Caspase-3 activation is observed after induction of reactive oxygen species by exposure to xanthine/xanthine oxidase, whereas no caspase-3 activity is induced by H2O2 treatment [34]. Studies suggest that physical activity is a key component of cardiac rehabilitation programs for patients with CVD, as it reduces the risk of CVD as a non-pharmacological treatment by protecting the heart from serious damage and improving heart function [18]. In a study investigating increased cardiac apoptosis in diabetic rats, 10 weeks of aerobic exercise on a treadmill was found to protect the heart from apoptosis [35]. Some studies have shown the positive effects of aerobic exercise and physical activity in preventing and minimizing the complications of estrogen deficiency during menopause [16]. Aerobic exercise and meditation are also effective in reducing anxiety and depression. Depression and anxiety are more pronounced in CVD patients than in healthy individuals (38). A recent meta-analysis found a small but significant increase in the risk of AH in women with premature menopause compared with women with menopause at age >45 years.[39] Possible pathogenetic mechanisms include the production of vasoconstrictor factors such as endothelin and angiotensinogen as a result of reduced estrogen concentrations and a lower estrogen-to-androgen ratio during menopause [40]. Epidemiological studies have shown a reduced cardiovascular risk in populations with high dietary isoflavone intake [41]. Additionally, a previous study provided evidence that two weeks of twice-daily supplementation with red clover reduced VCAM-1 expression and significantly improved vascular inflammation in postmenopausal women [42]. Studies have been conducted on the association between red clover supplementation and cardiovascular disease risk factors. Vascular endothelial function is not firmly linked to the development of atherosclerosis or hypertension, but it is hypothesized to play a role in the pathology of these diseases. Two published studies show that adhesion and platelet factors are not altered by red clover isoflavone supplementation. Since prostacyclin can inhibit platelet adhesion and aggregation to the endothelium, this may be a mechanism by which red clover isoflavones improve vascular health. Red clover isoflavones also increased endothelial nitric oxide synthase activity and nitrite levels in human umbilical vein endothelial cells after 48 hours of exposure. This is another potential vascular mechanism (non-immediate, genomic) of action of red clover isoflavone supplements. Arterial compliance is a measure of arterial stiffness that is associated with the presence of atherosclerotic plaques in major blood vessels. The effect of red clover isoflavones on this parameter has been investigated in two studies (43).
Type of Study: Research |
Subject:
Exercise Physiology
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