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Abstract: The present study was conducted to investigate the effects of eicosapentaenoic acid on glucocorticoid-induced bone changes in rats, and to compare them with those of alendronate. Thirty six male Wistar rats, 2.5 months of age, were divided into six groups (n = 6 each) and treated with 0.9% NaCl (control), methylprednisolone 7 mg/kg, once a week subcutaneously, methylprednisolone + alendronate 20 µg/kg, twice a week subcutaneously and methylprednisolone + 80 or 160 or 320 mg/kg eicosapentaenoic acid, per day orally, for 6 weeks. At the end of the experiment, serum and urinary parameters of bone metabolism determined and bone histomorphometric analyses performed on cancellous bone of femoral epiphysis and metaphysis and cortical bone of tibial diaphysis. There were no significant differences in serum and urinary parameters among groups. Decrease of epiphyseal and metaphyseal trabecular width, epiphyseal bone area/tissue area and increase of epiphyseal trabecular separation observed in the methylprednisolone group compared to control. Alendronate restored all of these parameters except metaphyseal trabecular width, which increased significantly by eicosapentaenoic acid at the doses of 80 and 160 mg/kg. Effects of alendronate and 160 mg/kg eicosapentaenoic acid on bone area/tissue area, alendronate and eicosapentaenoic acid at the doses of 80 and 160 mg/kg on trabecular separation and alendronate and eicosapentaenoic acid at doses of 160 and 320 mg/kg on epiphyseal trabecular width were statistically similar. Methylprednisolone did not significantly change cortical bone parameters including cortical width and marrow area/cortical area. Eicosapentaenoic acid, especially, at the dose of 160 mg/kg exerts beneficial effects on methylprednisolone-induced bone changes in rats; these effects are similar or sometimes even better than alendronate.
Osteoporosis is probably one of the main limitations to long-term glucocorticoid therapy . It has been demonstrated that prolonged exposure to glucocorticoids at supraphysiological doses induces osteoporosis associated with an increased risk of bone fracture . Bisphosphonates, such as alendronate, can counteract the negative effects of glucocorticoids on bone ; but diet therapy and life style changes that minimize bone loss would be very helpful to decrease the necessity for drug therapy to prevent osteoporosis .
It is known that dietary sources of long chain n-3 fatty acids are essential for maintaining optimum health, since mammals can not synthesize fatty acids with double bonds post the Δ position . Eicosapentaenoic acid is the major long chain n-3 essential fatty acid present in fish oil . Although best known for their cardioprotective role, long chain polyunsaturated fatty acids and their metabolites also regulate bone metabolism and consequently may have a potential in the prevention and/or treatment of osteoporosis . Fish oil or n-3 essential fatty acid consumption has previously shown to protect against ovariectomy-induced bone loss in rats and mice [7–9]. Eicosapentaenoic acid-enriched diet has prevented the loss of bone weight and strength in ovariectomized rats fed with low calcium diet .
The purpose of the present study was to investigate the putative positive effects of eicosapentaenoic acid on bone, in the presence of detrimental bone changes due to methylprednisolone administration in rats and to compare these effects with those of common anti-osteoporosis drug, alendronate.
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Epidemiological and longitudinal studies have reported a positive relationship between intake of n-3 family of long chain poly-unsaturated fatty acids and bone mineral density in postmenopausal women . In rats and mice, dietary supplementation with n-3 long chain polyunsaturated fatty acid-rich oils has improved maintenance of bone mass post-ovariectomy [7–9]. In addition, eicosapentaenoic acid-enriched diet has inhibited bone loss in ovariectomized rats fed with low calcium diet . However, there is no study conducted to demonstrate the effect of eicosapentaenoic acid on bone changes due to glucocorticoid administration. Glucocorticoid-induced osteopaenia and ovariectomy-induced osteopaenia are two contrasting types of osteopaenia. Histomorphometric studies performed by Nitta et al. demonstrated that the most characteristic consequence of methylprednisolone treatment in rats is a significant loss of trabecular bone resulting from thinning of trabecular bone with unchanged connectivity. Losses in the volume and thickness of trabecular bone in glucocorticoid-induced osteopaenia are due to acceleration of bone resorption (increase in Eroded Surface/Bone Surface) and depression of bone formation (Decrease in Osteoid Suface/Bone Surface). These characteristics are in a good contrast to those of ovariectomy-induced osteopaenia in which accelerated bone turnover in favour of bone resorption results in cut-but-non-thinned structure of the cancellous bone .
Regarding mechanism of action of glucocorticoids on bone, it is now apparent that their most important effect is decreasing bone formation. Glucocorticoids induce a 30% increase in osteoblast and osteocyte apoptosis and affect transcription of many of the genes responsible for synthesis of matrix constituents by osteoblasts such as type I collagen. Glucocorticoids also induce osteoclastogenesis and consequently increase bone resorption through enhancement of receptor activator of NF-κB (RANK) ligand (RANKL) production and inhibition of osteoprotegrin production. Because osteoprotegrin acts as a decoy receptor for RANKL, the final effect would be the overactivation of RANK which leads to an increase in bone resorption [17,18].
Bone loss due to estrogen deficiency is primarily as a result of increased osteoclast activity and accelerated bone resorption .
The aim of the present study was to determine whether eicosapentaenoic acid would exert beneficial effects on bone in glucocorticoid-treated rats and to compare the effects of eicosapentaenoic acid with those of the common anti-osteoporosis drug, alendronate.
It has been demonstrated that the key histological feature of glucocorticoid-induced cancellous bone loss is a reduction in the trabecular thickness, reflecting suppressed bone formation . In the present study, methylprednisolone induced cancellous bone loss that was manifested by a significant decrease in both epiphyseal and metaphyseal trabecular width and epiphyseal bone area/tissue area and an increase in epiphyseal trabecular separation. Alendronate restored all of these parameters except metaphyseal trabecular width, which increased significantly by eicosapentaenoic acid at the doses of 80 and 160 mg/kg. Effects of alendronate and 160 mg/kg eicosapentaenoic acid on bone area/tissue area, alendronate and eicosapentaenoic acid at the doses of 80 and 160 mg/kg on trabecular separation and alendronate and eicosapentaenoic acid at doses of 160 and 320 mg/kg on epiphyseal trabecular width were statistically similar. The effect of eicosapentaenoic acid on attenuating cancellous bone loss was found to be dose-dependent and the most effective dose was 160 mg/kg, except on increasing epiphyseal trabecular width that the effect of 320 mg/kg was only slightly better than 160 mg/kg.
It seems that glucocorticoid-induced cancellous osteopaenia in rats is a good responder to eicosapentaenoic acid. As noted previously, at the present study eicosapentaenoic acid with the dose of 160 mg/kg significantly attenuated cancellous bone loss due to glucocorticoid administration regarding histomorphometric parameters. In the study performed by Poulsen et al. (2007), diet supplemented with 0.5 g/kg body weight/day of eicosapentaenoic acid fed to ovariectomized rats for 16 weeks did not significantly increase bone mineral density compared to ovariectomized control rats . Considering different histomorphometric features and pathogenesis pathways of glucocorticoid-induced osteopaenia and ovariectomy-induced osteopaenia, as stated above, it is plausible that these two kinds of osteopaenia respond differently to the same agent. A fact that should be noted here is that these researchers did not perform bone histomorphometric study so the alterations in local bone formation and resorption and the modification of bone architecture remain uncertain.
Although glucocorticoid-induced osteoporosis is more evident in cancellous than in cortical bone , it has been also reported that glucocorticoid administration induces cortical osteopaenia in rats [21–23]. In the present study, methylprednisolone did not significantly change cortical bone parameters including cortical width and marrow area/cortical area of tibial diaphysis compared to control. Iwamoto et al. reported that treatment of rats with methylprednisolone for 4 weeks induced cancellous osteopaenia without significantly affecting cortical bones; however, continuation of the same regimen for 8 weeks resulted in cortical osteopaenia in addition to cancellous osteopaenia that was manifested by a decrease in the percent of cortical area and an increase in the percent of marrow area of tibial diaphysis . It seems that cortical osteopaenia due to glucocorticoid administration needs more time or total amount of drug than cancellous osteopaenia to be manifested.
Alendronate and eicosapentaenoic acid in three different doses did not exert any significant positive or negative effects on cortical bone parameters compared to control.
Serum bone resorption marker (CTX), bone formation markers (osteocalcin and alkaline phosphatase), calcium, phosphorus and creatinine concentrations were statistically similar among groups as well as urinary calcium/creatinine and phosphorus/creatinine ratios. In the study performed by Unoki, administration of prednisolone 2.5 mg/kg six times a week for 8 weeks to rats, although induced cancellous osteopaenia, did not significantly change urinary calcium and phosphorus compared to control group . In the study performed by Wang et al., administration of methylprednisolone at the doses of 0, 2.5, 5, 10 or 20 mg/kg to male rats daily for 4 weeks did not significantly change the values of serum calcium, phosphorus and resorption marker (Pyridinoline) among groups and osteocalcin and alkaline phosphatase decreased significantly only in 10 and 20 mg/kg methylprednisolone-treated groups .
This lack of significant difference in serum and urine biochemical markers in the present study may be due to a lack of statistical power to detect differences between groups or more probably due to relatively low cumulative dose of methylprednisolone used. Another explanation may be that by considering this fact that methylprednisol one sodium succinate is a short to medium acting glucocorticoid, there is a possibility that its effects on biochemical markers of bone metabolism have been vanished during the one week time course between the last dose of methylprednisol one and collecting of samples.
It seems that in the present study, histomorphometric parameters have been more appropriate indicators of detrimental changes of bone due to glucocorticoid administration in rats at least in cancellous bone.
In conclusion, eicosapentaenoic acid especially at the dose of 160 mg/kg exerts appreciable positive effects on detrimental bone changes due to glucocorticoid administration in rats. These effects are similar or even better (as seen with metaphyseal trabecular width) than a common anti-osteoporosis agent, alendronate.