Evaluation of the protective effects of resveratrol on hydrocortisone-induced growth inhibition in the peripubertal rat epiphyseal plate

Glucocorticoids are frequently used in the treatment of many inflammatory diseases according to their strong anti-inflammatory properties. They show their physiological and pharmacological effects by binding to the nuclear-type glucocorticoid receptor (GR) located inside the cell, like other steroid substances. Since this receptor is found in almost all cells in the human body, adverse effects can be observed even at therapeutic doses. Deterioration in bone mineralization and suppression of height growth are important adverse effects that can be seen with chronic steroid use in childhood. When glucocorticoids are used in chronically, they suppress the pituitary release of adrenocorticotropic hormone (ACTH), growth hormone (GH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH). They reduce the biological activity of insulin-like growth factor-1 (IGF-1), which controls enchondral ossification in epiphyseal cartilage, by decreasing its synthesis and release. As a result, they lead to osteoporotic changes and inhibition of height growth in long-term use. Resveratrol is an important antioxidant substance with phytoestrogenic properties. It has come up with its effect on reducing the incidence of cardiovascular disease, and subsequently, its positive effects on bone health in osteoporosis-like bone pathologies have been reported in clinical and preclinical studies. It has been shown to increase bone mineral density in postmenopausal women. It has been shown that it can increase osteoblastic bone formation and decrease osteoclastic resorption in experimental animal osteoporosis models and increase tibia length by delaying epiphyseal closure in rabbit studies. In our study, to investigate the protective effects of resveratrol on the growth inhibition of the epiphyseal plate with hydrocortisone, peripubertal Wistar Albino rats were randomized into 6 groups, one of which was the control group. After a standard AP tibia radiograph was taken, 10 mg/kg/day subcutaneous hydrocortisone, 10mg/kg/day subcutaneous hydrocortisone + 50 mg/kg/day resveratrol with orogastric gavage, and resveratrol alone at doses of 10-50-100 mg/kg/day has been applied for 10 days respectively. After the control radiograph was taken on the tenth day, the rats were sacrificed, blood was sampled, and the tibia bones were dissected under appropriate conditions and stored for histopathological evaluation. Bone biomarkers sclerostin and osteopontin were measured from the collected blood samples with the "Enzyme-Linked ImmunoSorbent Assay" (ELISA) method. Cartilage zones of the epiphyseal plate (resting, proliferative, hypertrophic and total zone) were measured histomorphometrically in pathological samples. Apoptosis marker caspase-3 and a transcription factor NF-kB p65 were evaluated semiquantitatively by immunohistochemical staining method. According to our study findings, hydrocortisone administered subcutaneously at a dose of 10mg/kg/day for 10 days significantly decreased the tibia elongation evaluated by radiography (23.4% vs 15.1%, p<0.001). In the group in which resveratrol was given by gavage at a dose of 50 mg/kg/day with 10 mg/kg/day sc hydrocortisone, inhibition of tibia elongation disappeared. In the comparisons made with the control group in the histomorphometric measurements, the total zone thickness was found to be significantly decreased in the hydrocortisone and resveratrol 50 mg/kg (RSV50) groups (554.95±17.14 μm, vs. 483.74±14.66 μm, 384.91±20.52 μm, p<0.05, p<0.001). The total zone measurement of the resveratrol 50mg/kg and hydrocortisone 10mg/kg group was similar to the control group. The resting zones were determined to be significantly thinner than the control in the groups in which resveratrol was administered alone (p<0.001). The osteogenic zone distal to the epiphyseal cartilage was immunohistochemically stained for caspase-3 and NF-kB p65 expression and evaluated semi quantitatively. The apoptosis rates in the osteogenic area were found to be higher in the groups in which hydrocortisone was administered 10 mg/kg/day and resveratrol 100 mg/kg/day (p<0.05). NF-kB p65 expression was similar in all groups. The blood level of the bone formation inhibitor sclerostin was significantly reduced in the high-dose resveratrol group (100 mg/kg/day) compared to the control group (1,342 pg/ml (897-1,617), p<0.001)). Osteopontin blood level, which is accepted as an indicator of bone mineralization, was found to be significantly increased in the high-dose resveratrol group compared to the control (24,491 pg/ml (16,345-72,408), p<0.05). In conclusion, inhibition of tibia bone elongation and growth plate total zone thickness with subcutaneous 10 mg/kg/day hydrocortisone was reversed with 50 mg/kg/day oral resveratrol administration in our study. While doses of 10-50-100 mg/kg/day of resveratrol alone did not affect the radiographic linear elongation of the tibia, a dose of 50 mg/kg/day caused a decrease in growth plate thickness. It has been thought that the phytoestrogenic property of resveratrol and its accelerating effect on chondrocyte cellular senescence against the inhibitory effect of hydrocortisone on gonadotropin secretion and decelerating effect on cellular senescence may explain this condition.