In our study, we present ostarine’s effects on structural and chemical parameters, biomechanical stability, and gene expression at two skeletal sites, the lumbar spine and femur. This study provides new insights into the important topic of male osteoporosis. In the present study, Ostarine Proph. treatment prevented bone deterioration in male rats after Orx by maintaining the BMD of L4 at a higher level than in Orx rats, as was determined via in vivo pQCT analysis at weeks 12 and 18 after Orx. After 18 weeks had passed since Orx, this treatment even improved BMD as comparing to Non-Orx rats. The favorable effect of Ostarine Proph. was supported by a detailed micro-CT analysis of the bone structure. Enhanced cortical density and volume were measured via 3D micro-CT analysis in the femur, whereas in both L4 and the femur, trabecular density was higher in this group than in the Orx rats based on 2D micro-CT analysis. In the femur, the effect was more pronounced than in L4, with additionally elevated trabecular thickness and number of nodes in the Ostarine Proph. group. In contrast, mineral content in the Orx rats, as determined by ashing analysis, was maintained under Ostarine Proph. in L4, whereas in the femur, it was at the lower level seen in Orx rats. This could be explained by the heterogenous changes that occur in various skeletal parts during osteoporosis (Amling et al. 1996; Ritzel et al. 1997; Chen et al. 2013). Bone loss during osteoporosis depends on bone localization, and even anti-osteoporotic treatment varies between femora and vertebrae samples(Kavuncu et al. 2003; Chen et al. 2013). Ostarine applied as a therapeutic treatment for 6 weeks was effective solely in improving the cortical density of the femur. Perhaps, a prolonged treatment could have a stronger effect on osteoporotic bone tissue. The osteoanabolic effects of SARMs have been previously described in the literature; e.g., studies on SARM drugs such as S-4 or LGD-3303 have shown their positive effects on bone tissue. Kearbey et al.(Kearbey et al. 2007; Kearbey et al. 2009) showed that S-4 treatment maintained trabecular BMD, cortical content, and increased bone strength after 120 days of treatment in ovariectomized rats. Vajda et al. (2009) used another SARM, LGD-3303, which was orally administered for 14 days in osteopenic female rats, and found increased bone density at cortical and cancellous bone sites (Vajda et al. 2009).
In addition to the osteoanabolic effects of SARMS, inhibitory effects on bone resorption are described. S-4 showed antiresorptive effects by decreasing TRAP-positive multinucleated cells in an in vitro study (Kearbey et al. 2007). In addition to their effects on bone tissue, SARMs also improve muscle tissue. Beneficial effects have been observed on muscle structure and vascularization in previous studies (Roch et al. 2020; Roch et al. 2022), which can indirect improve bone tissue. Ostarine Therapy treatment did not change bone’s structural and chemical parameters, likely having been too short to ameliorate osteoporotic changes in bone. Testosterone treatments showed less effect than ostarine on bone structure and quality in the femur and vertebral body, irrespective of application regime (Proph. or Therapy). The cortical density of the femur was solely enhanced in the Test. Proph. group. In contrast, these testosterone treatments had a positive effect on bone healing for osteotomized tibiae, which was stronger than the effect of ostarine treatments (Komrakova et al. 2020). This can be explained by the aromatization of testosterone to estrogen, which is absent in ostarine (Narayanan et al. 2018), and testosterone could act not only through ARs but also through ERs (Komrakova et al. 2020). Apparently, the dosage and oral application of testosterone propionate were sufficient to improve bone healing in male rats (Komrakova et al. 2020) but failed to affect non-osteotomized femur and vertebral body in the present study. This likely occurred due to the differences in metabolic processes at the fracture site and in intact bone. Bone turnover during fracture healing is elevated (Ivaska et al. 2007), whereas in uninjured osteoporotic bone, the remodeling processes slow down with time (Eastell and Szulc 2017).
Further analysis of bone minerals showed that testosterone prophylaxis treatment increased the magnesium levels in femur samples and decreased the Ca/PO4 ratio in L4 as compared to Non-Orx and Orx animals. In serum, the phosphorus level after both prophylaxis treatments (Ostarine and Testosterone) was significantly increased as compared to that in Orx animals. Nevertheless, the significance of calcium, magnesium, and phosphorus in bone and serum is limited due to the kinetics of bone metabolism, their ubiquitous occurrence, and their diverse involvements in general metabolic processes (Rico 1991; Shaker, Deftos 2000). For instance, increased parathyroid activity can lead to increased serum magnesium levels and lower calcium Ca/PO4 ratios (Clark 1969). Therefore, serum was also analyzed for alkaline phosphatase (ALP), osteocalcin (OC), and collagen 1 degradation product (CTX-I). These peptides are biomarkers of bone turnover and can provide insights into the remodeling processes of bone. ALP and OC are markers of bone formation and products of active osteoblast metabolism, while CTX-I is a product of active osteoclasts and, therefore, a marker of bone resorption (Rosen et al. 2000). OC was reduced in both ostarine groups as compared to the Orx and Testosterone Therapy groups, suggesting reduced bone turnover after ostarine treatment. These data support antiresorptive activity on the part of ostarine, which was also reported after the treatment of orchiectomized rats with another SARM, andarine (Gao et al. 2005). ALP and CTX-I levels did not change between the groups, implying low protein degradation in terms of, e.g., collagen I.
The expression analysis of bone genes in L6 showed increased RANKL gene expression in the Orx group, which is in line with the literature and can be explained by increased osteoclast activity (Li et al. 2009). After orchiectomy, the bone mass and the absolute number of osteoclasts are reduced, although the activity of existing osteoclasts is increased (Rissanen et al. 2008). Nevertheless, none of the tested treatments showed a significant effect on RANKL expression. For OPG, no difference in gene expression was detected, whereas OPG/RANKL quotient was increased by ostarine prophylaxis treatment as compared to Orx rats, confirming the positive structural changes in bone observed in this study. ALP showed no significant difference in gene expression, which is in line with the non-significant changes in ALP observed in the serum analysis. OC expression did not differ significantly between the groups, but in the serum, it was elevated. The level of protein synthesis does not always correspond with mRNA expression (Komrakova et al. 2015). These parameters were measured at the end of the study, and the dynamics of their expression remains unknown. No changes were observed for ERα expression, while ERβ expression was significantly increased after ostarine therapy treatment as compared to Non Orx animals. ERβ plays an important role in regulating cellular mechanotransduction events in osteoblasts, e.g., in ERK phosphorylation and MAPK pathway activation, as well as being increased in COX-2 expression (Castillo et al. 2014). On the other hand, AR expression was only affected by both therapy treatments, while prophylaxis treatments did not change AR expression significantly. AR expression has been found in whole bone marrow obtained from mice, and AR is also widely expressed in human bone and bone marrow (Abu et al. 1997). Because therapy treatments solely influenced AR expression, this effect seems to be time dependent and was negated in prophylaxis treatments.
Despite significant improvements in bone structure under ostarine treatments, the biomechanical properties of bone were only slightly changed. In femora no significant biomechanical changes were observed, while the reduced stiffness in L4 due to Orx treatment could be rescued by all tested treatments.Similar results were observed in a female rat model, in which the bone stiffness of femora was not affected but the significant structural improvements in bone tissue were measured under ostarine treatment (Hoffmann et al. 2019). Ostarine treatment should likely be combined with other bone-sparing substances, e.g., SERMs (Komrakova et al. 2022). In previous studies, SARMs (S-101479 and ostarine), predominantly bone-anabolic substances, when applied in combination with the SERM raloxifene, known as an antiresorptive drug, improved bone parameters to a greater extent than single compounds in female and male rat models (Furuya et al. 2012; Komrakova et al. 2022).
At the end of experiment, Orx was checked visually via the absence of testis and confirmed based on an atrophied prostate. The detrimental effect on the part of Orx on bone tissue was confirmed by reduced bone structural parameters, e.g., decreased BMD, Ct.V, Tb.Nd., Tb.Wi., and Tb.Dn., as well as reduced biomechanical properties, like a reduction of stiffness in L4, while all treatments maintained the values at the level of the Non-Orx group. Orx caused also a significant decrease of BW, while the ostarine and testosterone treatments had no effect on the body weight of Orx rats, confirming the results of other studies in female rats (Kearbey et al. 2007; Hoffmann et al. 2019). As previously reported, BW did not directly reflect food intake (Gentry and Wade 1976), while metabolic changes (Wade and Gray 1979) and bone and muscle loss (Wink and Felts 1980) may reduce BW in Orx rats. Furthermore, the strong reduction in prostate weight observed in Orx rats was diminished by the Ostarine Proph. treatment. Similar results were obtained for the SARM S-4 (S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide), which affected prostate weight after Orx surgery (Gao et al. 2005). This indicates that the effect of ostarine applied for a prolonged time as a prophylaxis treatment reduced its selectivity for the musculoskeletal system to some extent and also affected the sex organs. Furthermore, prostate weight was not affected by oral treatment with testosterone, which can be explained by its limited bioavailability when using this administration route, which was chosen based on previous studies showing favorable effects on bone tissue (Seidlová-Wuttke et al. 2006; Stürmer et al. 2006; Stuermer et al. 2009). Other studies have applied testosterone at higher concentrations (100mg/kg BW) and used injections instead of oral intake (Jota-Baptista et al. 2022; Jayusman et al. 2018). The clinical application of testosterone as a hormone replacement therapy is limited due to its side effects (Gruenewald and Matsumoto 2003). However, testosterone treatments are usually included as controls in experimental designs, and therefore, we suggest higher doses and injections instead of oral administration in future studies.
In sum, Ostarine Proph. treatment showed positive effects in terms of preventing osteoporotic changes in cortical and trabecular bone, though the biomechanical parameters were not changed. Ostarine Therapy treatment had less effect, solely improving the cortical density of femur. Thus, in monotherapy, the effect of ostarine does not appear to be sufficient to significantly reduce the development and progression of osteoporosis. Combination therapies of SARM and SERM could be considered in future studies, e.g., a combination of ostarine and raloxifene (Roch et al. 2022). By applying this combination therapy, the anabolic influence on musculoskeletal tissue is maintained, whereas the androgenic effect on the prostate is reduced (Roch et al. 2022; Komrakova et al. 2022).
Test. Proph. treatment positively affected only cortical density in the femur, whereas Test. Therapy did not change any bony parameters, likely due to the low dosage and oral route of administration. In future studies, higher doses and administration via injection should be considered when applying testosterone treatments. To conclude, Ostarine Proph. treatment could be further investigated as a preventative treatment for osteoporosis in orchiectomized males, but the androgenic effect on the prostate should be taken into consideration, and combination therapy with other agents could be considered.