| Objective: osteoporosis(OP)is a common systemic bone disease,which can easily lead to increased osteoporosis and fracture.The causes of osteoporosis are divided into primary and secondary.Secondary osteoporosis is a metabolic bone disease caused by diseases or drugs.The most common drug is the excessive use of glucocorticoids,which lead to the loss of bone mass in patients,and cause hormonal osteoporosis in severe cases,and then lead to osteoporotic fractures.Dexamethasone(DEX)is a common glucocorticoid drug in clinical practice.One of the most serious side effects of long-term large-scale use of dexamethasone is that it can affect the bone structure of patients of all ages,directly or indirectly affecting the endocrine system,resulting in osteoporosis and even fracture.Melatonin(MT)is a hormone secreted by the pineal gland of the brain.It is considered an antioxidant,which has a powerful antioxidant ability to eliminate free radicals,while being an effective scavenger of reactive oxygen species.Studies have shown that melatonin can affect bone metabolism by regulating calcium ion pathways,TRP pathways and so on to improve cell energy metabolism.There are few clinical studies on osteoporosis,and there is direction for basic research and clinical conversion,which is conducive to expanding the clinical indication of melatonin.The Akt/Nrf 2/HO-1 signaling pathway is an important intracellular signal transduction pathway involved in regulating biological processes such as oxidative stress and cell survival.Studies have shown that the application of glucocorticoids inhibits the activation of the Akt/Nrf 2 signaling pathway,resulting in increased oxidative stress and cell damage.Other studies have shown that melatonin significantly improve the anti-apoptosis ability of mouse osteoblasts under high glucose environment by activating the Nrf 2/HO-1 pathway.However,whether melatonin reduce oxidative stress by activating Akt/Nrf 2/HO-1 signaling pathway and reduce the damage of glucocorticoid to osteoblasts is worth research.In order to provide a new idea for finding a new treatment method for glucocorticoid-induced osteoporosis,we conducted this experiment to explore whether melatonin can inhibit the apoptosis of osteoblasts induced by glucocorticoids,so as to prevent and cure osteoporosis,and to explore the mechanism of melatonin in the Akt/Nrf 2/HO-1 signaling pathway in inhibiting glucocorticoid-induced apoptosis of osteoblasts.Methods:In the first part,the effects of melatonin on glucocorticoid-induced osteoporosis in vivo were studied through animal experiments.The experimental groups were:(1)blank control group(NC group);(2)Osteoporosis model group(DEX group);(3)Treatment group(DEX+MT group);(4)Melatonin control group(MT group).Bone tissue was observed morphologically and cytologically by HE staining,bone density and bone trabecular structure were quantitatively analyzed by Micro CT,bone mechanical properties were evaluated by biomechanical parameters of isolated femur,apoptotic protein expression was detected by Western blot,and m RNA expression was detected by PCR.In the second part of this paper,melatonin inhibits apoptosis and programmed necrosis of osteoblasts induced by glucocorticoid.(1)MC3T3-E1 osteoblast cell line was treated with DEX to make osteoporosis cell model,and osteoblasts were treated with different concentrations of DEX(0,400,800,1600μM)for 24 h and 48 h,respectively.The activity of osteoblasts was detected by CCK-8 assay,apoptotic protein expression was detected by Western blot,and apoptosis of osteoblasts was detected by Annexin V/PI staining flow cytometry.(2)Different dosing methods of MT and DEX were used for experimental grouping.Experimental groups:(1)NC group;(2)DEX group;(3)MT group;(4)DEX+MT group.CCK-8,flow cytometry and Western blot were used to detect cell activity and apoptosis.In the third part of this paper,the mechanism of inhibition of glucocorticoid damage to osteoblasts by melatonin activation of Akt/Nrf 2/HO-1 signaling pathway was studied.(1)Different groups of osteoblast cell lines were treated with Akt/Nrf2/HO-1 pathway inhibitor ML385.Experimental groups:(1)NC group;(2)DEX group;(3)MT group;(4)DEX+MT group;(5)DEX+MT+ML385 group.The expression of pathway-related proteins and cell activity were detected.(2)Transfection cell lines were established and the experimental groups were:(1)NC group;(2)DEX group;(3)DEX+MT group;(4)DEX+MT+si-NC group;(5)DEX+MT+si-Nrf-2 gene knockout group.The activity of osteoblasts was detected by CCK-8 assay.Apoptotic protein expression was detected by Western blot and apoptosis of osteoblasts was detected by Annexin V/PI staining flow cytometry.Results: 1.MT can increase bone mineral density and the formation of bone trabeculae,reduce the number of fat cells in the bone marrow cavity,and increase bone mineral density and bone strength in mice with hormone osteoporosis.MT can promote the expression of Nrf 2,Bcl-2 and osteogenic markers.2.When the appropriate concentration of DEX was 800μM and the appropriate treatment time was 48 hours,the treatment of DEX resulted in the most significant decrease in osteoblast activity and the most significant expression of apoptosis-related proteins.3.MT alleviates De X-induced osteoblast apoptosis.The combined action of MT and DEX can increase the activity of osteoblasts,decrease the apoptosis rate of osteoblasts and decrease the expression of osteoblast-related apoptotic proteins.4.Osteoblasts pretreated with DEX and MT were treated with Akt/Nrf 2/HO-1pathway inhibitor ML385,and the activity of osteoblasts was decreased and apoptosis was enhanced.These results indicated that MT alleviates the apoptosis of DEX on osteoblasts through Akt/Nrf 2/HO-1 pathway.5.MT alleviates the apoptotic effect of DEX on osteoblasts by activating Akt/Nrf 2/HO-1 pathway.DEX inhibits the expression of Nrf 2 and HO-1 proteins in this pathway,and MT can increase the expression levels of both.6.In established transfection cell lines,compared with DEX+MT group,Nrf 2knockout group showed decreased cell activity and increased apoptosis.Conclusions:1.Glucocorticoid-induced drugs can induce osteoporosis and osteoblast apoptosis in mice.2.Melatonin can alleviate osteoblast apoptosis induced by glucocorticoid.3.Melatonin can increase bone density and bone strength in mice with hormone type osteoporosis by promoting bone formation,thus playing a preventive and therapeutic role in bone loss caused by glucocorticoids.4.Melatonin can alleviate hormone-induced apoptosis of osteoblasts by activating Akt/Nrf 2/HO-1signaling pathway. |
