Effect of strength training versus raloxifene on bone weight, blood glucose, lipid and antioxidant profile in ovariectomized rats

. We compared the effect of the treatment with strength training (ST) and raloxifene (RALOX) on bone weight, blood glucose, lipid, and antioxidant profile in ovariectomized rats. Twenty-four Wistar rats were distributed into four groups: ovariectomy + VEHICLE (control); ovariectomy + RALOX; ovariectomy + ST; ovariectomy + RALOX + ST. Thirty days after ovariectomy, the animals underwent the treatment with RALOX (750 μ cg day -1 ) and/or ST (three sessions week -1 ). Thirty days after, all groups were scarified, tibia and femur were weighed, and the blood was collected for analysis of the lipid profile, glucose, and antioxidants catalase (CAT) and glutathione (GSH). The ST group showed greater femur weight (0.82 ± 0.18 g) and RALOX + ST had greater tibia weight (0.61 ± 0.17 g) than CONTROL with femur weight of 0.65 ± 0.08 g and tibia of 0.49 ± 0.08 g with no differences between treatments (p > 0.05). ST group showed significantly higher catalase (181.7 ± 15.4 μ M g -1 ) compared to the other groups. In contrast, the GSH value was lower in ST group (89.2 ± 8.1 μ M g -1 ) compared to RALOX (175.9 ± 17.1 μ M g -1 ) and RALOX + ST (162.8 ± 12.1 μ M g -1 ), but the values of these two groups did not differ from CONTROL (115.3 ± 21.1 μ M g-1). Total cholesterol did not differ between groups (p > 0.05), but exercise alone (54.3 ± 2.5 mg dL -1 ) or with RALOX (53.0 ± 1.5 mg dL -1 ) resulted in higher HDL cholesterol than CONTROL (45.5 ± 2.5 mg dL -1 ). Only RALOX+ST presented lower glucose (140.3 ± 9.7 mg dL -1 ) values than CONTROL (201.7 ± 30.6 mg dL -1 ). In conclusion, ST promotes similar benefits on bone and metabolic parameters compared to pharmacological treatment in ovariectomized rats.


Introduction
Menopause is characterized as a progressive systemic change, of which stands out weight gain, increase in abdominal fat, dyslipidemia (Cardoso et al., 2018), and loss of bone microarchitecture associated to potential risk to fractures (Lupsa & Insogna, 2015).Genetics, hormonal status, eating habits, and physical activity level influence the rate of progression of this disorder (Marques & Liberali, 2012).There is already well-established positive association between these factors and chronic diseases, including hypertension and diabetes (Kabodi, Ajami, Zakiei, Zangeneh, & Saeidi, 2019).
Pharmacological hormone replacement therapies prevent further bone loss, reduce likelihood of future fractures, prevent the onset of cardiovascular disorders, and psychic disorders, resulting from hormone deficiency in postmenopausal women (Sousa, Alves, Rocha, Moraes, & Carvalho, 2007).Besides hormonal treatment, there has been several attempts to increase the effectiveness of estrogens through the selective estrogen receptor modulators.These second-generation drugs induced bone mineral gain from 2 to 4% at the lumbar spine, proximal femur, and total body in 602 newly menopause women, according to a European multicenter study (Delmas et al., 1997) and induces osteoblast activity with decreased osteoclast activity (Yogui et al., 2018).In particular the raloxifene (RALOX), a second-generation selective estrogen receptor modulator (SERM), is an example of a class of pharmacological compounds with agonist action on bone.
Concomitantly, studies claim that physical exercises aiming hypertrophy and muscle strength gain have positive correlation with bone mineral density (Moreira et al., 2014;Watson et al., 2018).Moreover, it is well Acta Scientiarum.Biological Sciences, v. 45, e59835, 2023 established that dynamic aerobic exercise training improves lipid and glucose profile, and further improves the antioxidant activity, thus preventing effects associated with aging and oxidative stress that are increased after menopause (Marques et al., 2011;Tobias et al., 2014).However, the effect of strength training (ST) on metabolic profile is still little studied.
This scenario allows us to assume that strength training can help improve postmenopausal bone health and promote other metabolic benefits not granted by estrogen, contributing to the overall health of menopausal females.However, in the absence of previous research evaluating the influence of RALOX and ST on metabolic profile, the present study was initially carried out with an animal model in order to subsidize future interventions in humans.
Therefore, the aim of this study was to verify the effect of treatments with RALOX and ST alone or in association on bone weight, antioxidant activity, lipid profile, and blood glucose in ovariectomized rats.

Animals
This is an experimental study with animal model.We used 24 virgin female Wistar rats (200-300 g), 90 days old, from the vivarium of the Center for Agricultural Sciences-FUPI, fed rodent chow/water ad libitum and maintained in standard conditions at room temperature 26 ± 2˚C, relative humidity: 44-56%; cycle 12h light: 12 hours dark).The animals were distributed into 4 groups (n = 6/group): 1-ovariectomy + VEHICLE (control); 2-ovariectomy + RALOX; 3-ovariectomy + ST; 4-ovariectomy + RALOX + ST.All procedures were performed according to ethical principles in animal experimentation by the National Council for the Control of Animal Experimentation (CONCEA n. 097/12) and national legislation for pet section in vigor (Federal Law 11794 of 08 October 2008).

Procedures
The experimental model of ovariectomy was based in previous studies (Gomes et al., 2018;Kawakami et al., 2018).The ovariectomy was performed at age of 50 days, following anesthesia with intraperitoneal ketamine at a dose of 40 mg kg -1 and xylazine at a dose of 5 mg kg -1 bodyweight solution.The ovaries were exposed and was then held ovarian pedicle ligation using 5-0 catgut chrome wire needle (ETHICON -chrome catgut 5-0) for excision of ovarian after proper hemostasis.This was followed by the intramuscular administration of antimicrobial 0.1 ml 100 g -1 and anti-inflammatory compound (benzylpenicillin G benzathine and procaine, dihydrostreptomycin and piroxicam), as well as appropriate packaging for post anesthesia care of the animal.
Thirty days after the ovariectomy, the animals of the drug-treated groups started to receive selective estrogen receptor modulators (RALOX, 750 μcg) diluted in 0.5 ml propylene glycol daily for 30 consecutive days accordingly.This dosage, as well as the period of 30 days enough to start the osteopenic phenomenon, was based on a previous study by Carvalho and Cliquet Jr. (2004).The animals of the exercise group started a strength training following a protocol previously adopted by Brito et al. (2018).Strength training consisted of jumping in a PVC cylinder, 30 cm in diameter and 70 cm high, containing water heated at about 32ºC.After a two-day period of adjustment in the liquid environment, one hour a day, the animals went through a protocol of three sessions (alternated days) per week of strength training for four-week.Each session consisted of four sets of ten jumps with one-minute interval between sets and with overhead of 50% of body weight.The load was placed on the animals' chests through a special vest that involved the neck and back, but kept their feet free to perform the movements.The body weight of the animals was determined at baseline and during the training protocol for adjusting the training load.
Twenty-four hours after training and drug protocols and after 12h fast, the animals were anesthetized and euthanized (Rocha & Massone, 2006).The right tibia and right femur were removed by knee and tibiatarsal disarticulation and the joint in the anteroposterior direction using an analog caliper for clinical assessment and measurement of bone weight.These bones were chosen because the femoral head and hip fractures are the most prevalent nonvertebral fractures (Söderqvist et al., 2009) and are associated with a higher risk of mortality after fractures caused by osteoporosis (Johnell et al., 2004), in addition to the fact that the animals underwent strength exercises for lower limbs.
It was collected 3 mL of blood sample from each animal through puncturing of the caudal vena cava in order to dose the serum levels of total cholesterol, HDL cholesterol triglycerides (TG), and glucose by using the automatic immunochemical analyzer Labmax 240 premium using kits of the Labtest (Minas Gerais, Brazil).Reduced glutathione (GSH) was determined by 1 ml of liver tissue supernatant (0.5 ml homogenate precipitated by 2 ml) of 5% trichloroacetic acid (TCA) and 0.5 ml of Ellman's reagent (0.0198%) DTNB in 1% sodium citrate and 3 ml of phosphate buffer (pH 8.0).The color developed was read at 412 nm.Catalase were quantified through the enzyme decomposition of hydrogen peroxide by the decrease in optical density at 230 nm, 37°C.For this, liver tissue homogenate was prepared; 100 mg ml -1 of tissue in 0.1 M phosphate buffer (pH 7.0) and then centrifuged at 5800 rpm for 10 min under cooling at 4°C.The supernatant was diluted 50-fold with 1.0 M Tris-HCl buffer and 5 mM EDTA (pH 8.0).Then, an aliquot of 10 L was subjected to a reaction medium containing H 2 O 2 in 10 mM Tris HCl 1.0 M and 5 mM EDTA (pH 8.0).The reduction of H 2 O 2 was monitored spectrophotometrically for 6 minutes (Beers Jr. & Sizer, 1952).

Statistical analyses
Data is presented as mean and standard error of the mean (SEM).After confirming data normality and homogeneity through Shapiro Wilk and Levene tests respectively, One-way ANOVA was performed followed by Tukey post-test for comparison between groups.The level of significance was set at p < 0.05.The statistical procedures were performed in GraphPad InStat version 3.03 software (GraphPad, San Diego, CA, USA).

Results
The animals in the four groups maintained their body weight without statistically significant changes, although descriptive reduction was noted in the trained (5.3%), trained + raloxifene (3.7%) and in the group without any treatment (4.5%).Moreover, the group treated only with raloxifene showed an increase of 1.3% in body weight.
ST group was the only which the femur weight was significantly higher than control.This was noted both in the analysis of absolute bone weight (Figure 1) and in the analysis of bone weight normalized by body weight (0.34 ± 0.07 g 100 g -1 of body weight for the ST group; 0.26 ± 0.06 g 100 g -1 of body weight for the CONTROL; 0.30 ± 0.05 g 100 g -1 of body weight for RALOX; 0.27 ± 0.03 g 100 g -1 of body weight for the RALOX + ST group).No difference was observed among treated groups.
Acta Scientiarum.Biological Sciences, v. 45, e59835, 2023 Animals treated with hormone therapy or exercise showed total cholesterol levels similar to those of the control group (Table 1).Strength training alone or associated with estrogen resulted in higher HDL cholesterol compared to control or estrogen treated animals.For glucose, only animals of the control group showed values higher than 200 mg dL -1 .Treated animals presented values around 25% smaller, but only RALOX + ST group presented statistically lower glycemia levels in relation to the control group.

Discussion
Data from this study showed that strength exercise results in higher bone weight and increased antioxidant activity of the catalase.Additionally, strength training promotes increase in HDL cholesterol, and associated with hormone therapy, promotes decrease in blood glucose.
Unlike our study, which used the ovariectomy model, Stringhetta-Garcia et al. ( 2016) conducted a strength training protocol in aged Wistar rats (physiologically menopaused) and found an increase in osteoblast cell activity and a decreased cell osteoclastic activity, resulting in increased bone strength and micro architecture increment in the neck of the femur.Souza, Rosa, Ricardo, Moraes and Rocha (2005) verified that estrogen repairs tibia of rats submitted to oophorectomy, with statistically significant increase in bone mineral density of 21.8% in twenty-eight days at dose of 3 mg kg -1 day -1 .Thus, our study corroborates to this previous data.It can be noted that in either model (aged or ovariectomized rats), both raloxifene and strength training treatment are effective according to data from our study (for physical training) and previous literature (for raloxifene).
Results of the clinical trials are more discrete.Reginster et al. (2004) showed a decrease in biochemical markers of bone turnover in women with osteoporosis after the use of estrogen.However, estrogen improved bone mineral density of the lumbar spine, femur and spine only by 2-4% (Sousa et al., 2007;Marques & Liberali, 2012).McClung (2005) found increased mineral density by 1.6% in both lumbar spine and total hip, as well as an increase of 1.2% in the femur neck after two years of treatment with raloxifene at a clinical dose of 60 mg per day.The data from our study indicate that the physical exercise associated with selective estrogen receptor modulators is more promising, at least in animal model, since strength training was the only treatment that resulted in greater weight femur, and selective estrogen receptor modulators was only able to result in higher weight of the tibia when associated with physical training.
In addition to demonstrating benefits of the association of strength exercise and RA LOX on tibia weight, the other finding of this study was that strength training has the additional benefit of improving aspects of the metabolic profile involved in menopause (antioxidant activity, lipidic profile and glycaemia).The beneficial influence on these metabolic profile indicators is relevant because the lack of estrogen production in the menopause is accompanied by disorders of the lipid and glycemic profile, in addition to systemic inflammation and oxidative stress (Wang et al., 2018).On the other hand, several studies support the benefits of physical activity on lipid metabolism and oxidative stress, thus preventing cardiovascular disease (Mann, Beedie, & Jimenez, 2014;Poblete-Aro et al., 2018;Abdelbasset, Tantawy, Kamel, Alqahtani, & Soliman, 2019).
This study provides a further indication that strength training combined with therapy of selective estrogen receptor modulators is better than just one or the other isolated intervention.
Although estrogen receptors are present in several other tissues besides bone tissue (Stringhetta-Garcia et al., 2016) and are reported to improve the lipidic profile (Reginster et al., 2004), our study corroborates the findings from Reginster et al. (2004), Martyn-St James e Carroll (2008) and Takada, Iba, Yoshizaki and Yamashita (2012), which failed to verify beneficial effect of RALOX on HDL cholesterol.In our study, only strength training was able to increase HDL, alone or associated to raloxifene.The training effect in increasing HDL lipoproteins is something well established.In their studies, Karolkiewicz et al. (2009), Costa et al. (2018), andNassef et al. (2019) indicated an effectivity of the aerobic training to increase HDL, but the training load should be relatively high and precise, so that it could result in energy expenditure of 1500 kcal week -1 .On the other hand, data with strength exercise are scarce, particularly in menopaused populations, which evidences that our finding is relatively unknown.
While Motahari-Tabari, Shirvani, Shirzad-E-Ahoodasthy, Yousefi-Abdolmaleki and Teimourzadeh (2014), Glouzon, Barsalani, Lagacé and Dionne (2015), and Mendoza et al. (2016) reported that aerobic training improved insulin sensitivity, data regarding strength training are scarcer, although Panveloski-Costa et al. (2011) have already reported that strength training reduces blood glucose and inflammatory markers in obese animals, but not under conditions of induced menopause.These findings suggest two important practical implications.The first is that increased blood glucose and diabetes are associated with menopause and exogenous estrogen and progestogens (Stuenkel, 2017), which is in accordance with the higher glycemic values of rats in the control group of our study.The second is that the data from this study reinforce the possibility that the combination of RALOX and strength training is effective in preventing post-menopausal blood sugar increase.
The main limitation of this study was that the effect of treatments on bone health was evaluated only by weight bone.Bone mineral density, as well as its architecture and biochemical markers of osteoblastic / osteoclast activities deserve to be evaluated in future studies, considering the paucity of these data in training programs with strength exercise.The main highlight of this study was the finding of improvement of important metabolic aspects resulting from strength training alone or associated with RALOX.
Taking into consideration the potentials and limitations of the present study, the data collaborate to strengthen a body of evidence indicating potential benefits of physical training in the prevention and treatment of osteopenia determined menopause.While Müller, Keiler, Kräker, Zierau and Bernhardt (2018) found an association between the motivation of rats to exercise with estrogen levels, Gomes et al. (2018) demonstrated that strength training reversed the increase in glycemia, insulin and prevented sarcopenia in ovariectomized rats.To end, Stunes et al. (2020), validated that strength training protocol alone or combined with metformin improved the trabecular microarchitecture of ovariectomized rats.Although aerobic training is the most studied modality, our data and these other studies are forming an amount of evidence for benefits promoted by strength training under experimental menopause.

Conclusion
In summary, this study shows that the combination of RALOX + ST ensures greater benefit in the bone weight and glycaemia of ovariectomized rats.Moreover, exercise promotes an additional improvement of the lipid profile and increase of the antioxidant activity.These results seem potentially relevant by the fact that the deterioration of the lipid profile, blood glucose and reduced antioxidant capacity are the effects of menopause occurring before the symptoms of osteopenia, so that combining these two forms of treatment is potentially more robust for health promotion after menopause.

Figure 1 .
Figure 1.Effect of strength exercise isolated and associated to hormone therapy (RALOX) on femur and tibia weight.n = 6 for all groups.* = p < 0.05 to control group (ANOVA one way).

Table 1 .
Biochemical characteristics of the groups at the post-intervention.