- Diosgenin has preventive and therapeutic effect on osteoporosis.
- Diosgenin can prevent bone loss.
- Model group of osteoporosis are successfully induced by retinoic acid.
Methods: The rats induced by retinoic acid were used as rat model of osteoporosis. The femoral dry weight, bone calcium (Ca), phosphor (P) contents of femoral were measured; the biochemical index in serum of alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), estradiol and osteocalcin were determined after the rats were given diosgenin at the dose of 10, 30 and 90mg kg-1, respectively, and were compared among the model group, normal group and positive control group.
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Results: The osteoporosis rat model was successful induced by retinoic acid. Compared with the model group, the lessening of femoral weight, the short femoral transverse diameter and the bone mineral of diosgenin groups were improved in the diosgenin-treated group. The estradiol and osteocalcin levels in the middle and high dose groups were significantly higher than that of the model group, while the ALP and TRAP levels were lower than the model group.
Conclusions: Diosgenin can prevent the loss of bone in experimental rats. The mechanism may be that it improves the level of estrogenic hormone of estradiol and inhibits the high bone turnover.
Osteoporosis is a bone disease that leads to an increased risk of fracture with reduced bone mineral density (BMD), deteriorated bone microarchitecture, and altered amounts and types of proteins in bone [1, 2]. The purpose of treatment of osteoporosis is to prevent the bone fractures by decreasing bone loss or, preferably, by enhancing bone density and strength [3, 4]. Early detection and treatment of osteoporosis can sufficiently decrease the risk of future bone diseases, while it was difficult to cure the osteoporosis by rebuilding the bone. There was none of an available treatment to cure osteoporosis completely. Therefore, early prevention of osteoporosis is as important as treatment .
The model of retinoic acid induced osteoporosis in rats is used in several studies to evaluate the influence of substances on bone loss in human, for its easy operation, high successful rate, short time consumption and type symptoms of osteoporosis [6-9]. Early studies observed that large dose of vitamin A was toxic to the skeletal system of rats [10, 11]. Further studies also showed that retinoic acid causes constant decrease of BMD in a short period of 1-3 weeks . All these findings demonstrated short-term effects of retinoic acid could act as an appropriate revulsive of osteoroposis .
Diosgenin, asteroid sapogenin (Fig. 1), extracting from Dioscorea wild yam tubers, such as the Kokoro [14, 15], has been shown to inhibit proliferation, suppress inflammation, and induce apoptosis in tumor cells [16-18]. The aglycone (sugar-free) and diosgenin is used for the commercial synthesis of steroid products, such as pregnenolone, cortisone, progesterone, etc.Previous studies showed that diosgenin could be used to prevent and treat osteoporosis [19, 20]. All these studies indicate the safety and efficacy of diosgenin using as a certain alternative treatment modality for osteoporosis, and it is available for diosgenin being used therapeutically for postmenopausal women who attempt to reduce osteoporotic progression. However, the molecular mechanism of diosgenin activity in bone-derived cells, remains largely unknown.
In the present study, we investigated the influence and mechanisms of diosgenin activity in preventing and treating osteoporosis rat model induced by retinoic acid. Our study also provides further information to the possible therapeutic use of diosgenin on the treatment of bone-related diseases.
Materials and Methods
Rats (National Grade A experimental animal) aged 90 days old weighting between 190-260 g were offered by the Center of Experimental Animals, China Pharmaceutical University. All of the animal care and animal experimentations were provided in accordance with the Guide for the Care and Use of Laboratory Animals (National Research and Council, 1996).
Diosgenin was provided by the National Institute for the Control of Pharmaceutical and Biological Products. Retinoic acid, alkaline phosphatase (ALP) reagent kit, tartrate-resistant acid phosphatase (TRAP) reagent kit, and inorganic calcium (Ca) and phosphorus (P) assay kit were purchased from Nanjing Jiancheng Bioengineering Institute. Osteocalcin labeled by 125I were obtained from Aladdin reagent Co. Primary and secondary anti-estradiol and osteocalcin were purchased from Sigma Co. (Shanghai, China).
A total of 40 female rats were treated with the retinoic acid suspension (70 mg/kg) once daily for 14 days. The rat osteoporosis model induced by retinoic acid was examined by the cortex, size and beam of bone.
These rats were randomly allocated to one of four reagents for another 14 days, with 0.5% CMC-Na 70 mg/kg (as the model control), or three doses of diosgenin (10, 30 and 90 mg/kg) as the low, middle and high dose-treated groups, respectively. Additionally, another 10 healthy rats were supplemented as the healthy control with 0.5% 70 mg/kg CMC-Na once daily for 28 days. The dosing was adjusted according to the daily weight conditions. All rats were raised under consistent conditions during the study.
Bilateral femur bones were got from the sacrificed rats for histomorphology analysis. The right one was weighed to analysis the weight index (g/100 g body weight), and the left was cut at the mid-diaphysis to test bone density using dual energy X-ray bone densitometer. Both the diameter and the length of the femur bone were measured.
Bone mineral detection
For mineral detection of bone in different treatment groups of rats, the femur bone was first dehydrated and then carbonized by burning into ashes at 800 ºC for 8 hours. The levels of Ca and P (mmol/g) were determined by inorganic calcium and phosphorus assay kits when some ashes were dissolved in 6N HCl.
Biochemical indexes of serum
The serum samples were obtained from the rats after the last dose of the study drugs given for 24 hours. The levels of ALP and TRAP with their reagent kits were measured, respectively. The absorbance of serum samples was measured by ALP and TRAP kits at 400-415 nm under the base and acid conditions, respectively, to detect their enzymatic activity. The levels of estradiol and osteocalcin were measured by radial immunoassay.
Statistical comparison analysis was performed by students’ t-test. Results were expressed as mean ± SE with significance defined as P < 0.05.
Effect on bone histomorphology
The cortex of bone in normal control group was much thicker than osteoporosis model group treated by retinoic acid. The size of bone was tiny and the beam of bone was thin in the model group (Fig. 2 A & B), which was consistent with the literature. The cortex, size and beam of bone in diosgenin-treated groups (Fig. 2 C, D & E) were obviously improved compared with model group, especially in high dose group.
Effect on bone calcium (Ca) and phosphorus (P)
Bone Ca (P < 0.05) and P (P < 0.01) levels of model group and diosgenin-treated groups (low, middle and high) had been significantly increased compared with normal control group. As shown in Fig. 3, there was a significant difference in bone Ca content between high dose group and model group (P < 0.05).
* (P < 0.05) and ** (P < 0.01) indicate siginificant difference of bone Ca and P level between model group, diosgenin-treated groups (low, middle and high) and model group, respectively; # indicates significant difference in bone Ca content between high dose group and model group (P < 0.05).
Effect on ALP and TRAP
The colorless para-nitrophenyl phosphate (pNPP) is converted to yellow encounter with ALP and TRAP at the base condition and acid condition, respectively. Thus, the enzymatic activities of ALP and TRAP could be evaluated by colorimetric analysis of pNPP. As shown in Fig. 4, enzymatic activities of ALP and TRAP in serum of model group were much more than those of control group (P < 0.01). However, ALP and TRAP activities in serum of middle and high dose-treated groups were evidently less than those of model group (P < 0.05, P < 0.01).
* (P < 0.05) indicate siginificant difference in enzymatic activities of ALP and TRAP betetween model group and control group; # (P < 0.05) and ## (P < 0.01) indicates significant difference of ALP and TRAP activities between middle, high dose-treated groups and model group.
Effect on estradiol and osteocalcin
Fig. 5 showed estradiol and osteocalcin contents in model group were evidently lower than those of normal control group. There was a rising trend of estradiol content in diosgenin-treated groups. In addition, there was a significant difference of estradiol content between model group and middle, high dose-treated groups (P<0.05, P<0.01), indicating diosgenin could improve estradiol loss induced by retinoic acid. The osteocalcin contents in middle and high dose-treated groups were also distinctly more than that of model group (P<0.01), implying diosgenin could influence the activity of osteoblast and the regulation bone metabolism.
* and ** indicate significant difference of estradiol content between model group and middle, high dose-treated groups (P < 0.05, P < 0.01); ## indicate the osteocalcin contents in middle and high dose-treated groups were distinctly more than that of model group (P < 0.01).
In the present research, we investigated whether diosgenin intake inhibited osteoporosis in retinoic acid induced rats by regulating bone metabolism toward negative balance. Firstly, the retinoic acid induced rats showed a decreased femur length and estradiol and osteocalcin level, and increased level of ALP and TRAP, indicating a successful model. Indeed, significant promotion of bone formation and inhibition of bone absorption were observed in diosgenin-treated groups, indicating the improvement of bone metabolism and bone mineralization. All these changes closely imitate the human bone metabolism.
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The histomorphometric changes of bone tissue are an important index for the objective evolution of the state and condition of osteoporosis . The experimental results showed the reduced weight of femoral shaft, shortened width of bone, and decreased bone mineral substances and collagen in rats model group administered retinoic acid for 14 days, implying successfully induced osteoporosis-like changes. All the indexes have obvious improvement of middle, high dose of diosgenin-treated groups compared with model group, indicating the protective effect of diosgenin against osteoporosis induced by retinoic acid in rats. Nutritional factors also contribute to the development of osteoporosis. Contents of Ca and P in model group were higher than in control, the reason could be explained by the slow lost process of these mineral substances. The phases of bone resorption was much longer than bone formation when estrogen level declined caused by ovariectomization . Comparison of Ca and P contents between model and diosgenin-treated groups showed significant difference in our study, especially the bone Ca content between high dose group (90 mg/kg) and model group. This changes may be due to the botanical composition (e.g. flavonoids, phenolics) in diosgenin, which could exhibit strong bone protective properties in rat models of osteoporosis .
ALP is a biomarker of bone information, released from human osteoblast cells and reflect the activity of osteoblast . TRAP mainly expressed by osteoclast, having a pivotal role in many biological processes including bone mineralization, skeletal development, etc. can indicate the amount and activity of osteoclast . As bone metabolic markers, the serum ALP and TRAP level associated with bone formation was increased in osteporosis and other bone metabolic disorders . Similar changes happened in our study, the increased activity levels of ALP and TRAP under osteoporosis conditions was significantly decreased by diosgenin-mediated suppression, indicating an antagonistic effect of diosgenin on bone absorption and bone loss, and an enhancement of bone absorption and compensated bone formation diosgenin-treated group occurred.
Bone construction and reconstruction are two basic regulatory mechanisms that linked to an imbalance through bone formation and its resorption. Therefore, osteoporosis is a direct result of a disorder balance between bone formation and its resorption. In addition, Osteocalcin, secreted by osteoblasts, is a crucial molecular in bone regeneration progress . Upon evolution of the different osteocalcin levels between the control and treatment groups, a significant decrease was observed in model group, however, osteocalcin indiosgenin-treated groups were gradually increased with dose up, indicating diosgenin could induce ossification, producing much bone matrix, and leading to the normal balance of bone metabolism. From a mechanistic point of view, the diosgenin treatment can either raises the amount of osteoblasts, or activates the activity of the existed cells . In fact, recent published data have already proved that, consumption diet with rich olive oil was associatied with the increase serum osteocalcin levels [29, 30]. It seems that the diosgenin-mediated increase of osteocalcin is likely to be a key factor associated with the inhibition of bone loss.
Estradiol is an estrogenic hormone with two hydroxyl groups in molecular structure. Absence of estradiol is a well known and probably the most important cause of osteoporosis in premature and menopause female . Our results indicate that the protective effect of estradiol on diosgenin-induced bone loss occurs, estradiol content in diosgenin-treated groups were increased in different degrees. Ferretti et al.  suggested that the estradiol can prevent osteoblast apoptosis by suppression of estrogen receptor β (ERβ). Our results suggest the possible mechanism that diosgenin improves the level of estradiol and inhibits the high bone turnover, while the inhibition principle was still completely blunted. The role of diosgenin influencing estradiol expression needs further study.
Recently, the possibility that naturally occurring phytochemicals from edible materials may reduce the risk of bone diseases has gained considerable interest. Zhang et al. demonstrated that higher does ethanol extract of Lepidium meyenii was effective in preventing bone loss . Rao et al. reported that lycopene in tomato inhibited osteoclastic mineral resorption by decreasing tartrate-resistant acid phosphatase formation . Citrus fruits are rich in micronutrients, limonoids, and polyphenolic compounds, which was important in improving the BMD and bone biomechanical properties and decreasing the bone resorption . A recent report showed that an extract of Prunus mume affected the proliferation and differentiation of pre-osteoblastic MC3T3-E1 cells . Almost all these research based on the mechanisms that antioxidant vitamins may exert favorable effects on BMD and osteoporotic risk by scavenging free radicals and thereby reducing oxidative stress . Although Shishodia and Aggarwal  suggest the diosgenin suppress osteoclastogenesis through restrain of NF-κB-regulated gene expression and reinforce of cytokines-induced apoptosis, the detailed mechanisms of inhibiting effect of diosgenin are still unknown, futher studies are needed to identify the bioactive components and the mechanisms of the action.
According to the experimental results mentioned above, we can make the following conclusions. First, diosgenin possesses a potential inhibitory effect against osteoporosis via promoting bone formation and inhibiting bone absorption, and regulating bone metabolism toward negative balance. Second, diosgenin has certain prevention and cure function for osteoporosis in rats induced by retinoic acid. Our study provides a theoretical base for the further development of diosgenin.
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