| Objective: Glucocorticoids(GCs)are widely applied to treat inflammatory and immunemediated clinical complications.However,long-term use and over-doses of GCs result in frequent bone loss.Glucocorticoid-induced osteoporosis(GIO)is a metabolic bone disease and has been universally acknowledged as the most common secondary and iatrogenic form of osteoporosis.The decline of bone mass per unit volume is due to reduced newborn bone and more bone resorption.It is accompanied with the decrease of the bone quality and the occurrence of osteoporotic fracture.Although in clinical practice,medical staff has raised their precaution consciousness of the GIO,the awareness of side effects and catastrophic damage brought by glucocorticoids are still lacked.elucidation of the pathogenic mechanism and prevention of GIO is insufficient.Therefore,it is necessary to develop new drug for prevention and treatment.In recent years,the natural bioactive extracts have received a high degree of recognition,among of which,the medical value of gastrodin has been favored by people for long.We focused on exploring the full potential of gastrodin and whether it can targetedly reverse glucocorticoid-induced osteoporosis.Therefore,this research discussed about the effects of gastrodin and dexamethasone on the biological behavior of osteoblasts by deeply investigating the possible regulatory mechanism in changes of Nrf2 signaling pathway.On this basis,the effects of gastrodin on microstructure and biomechanical properties of bone tissue were verified in a glucocorticoid-induced osteoporotic rats model.Methods: 1.MC3T3-E1 was selected as pre-osteogenic cell line and primary osteoblasts were isolated from Sprague-Dawley newborn mice cranial bone.By using the method of CCK-8 assay,alkaline phosphatase activity assay,Annexin V-FITC/PI apoptosis test and Hoechst33258 dyeing,the protective effect of gastrodin on DEX induced biological behavior of osteoblast was detected.2.RNA interference(si RNA)technique,mito SOX? Red,DCFH-DA fluorescent probes and JC–1 mitochondrial membrane potential detection were used to investigate the specific mechanism of Nrf2 pathway.3.Alizarin Red staining was used to detect gastrodin's effect on dexathamasone-induced inhibition of osteogenic differentiation and mineralization.4.Real-time quantitative PCR and Western blotting analysis were used to detect the expression level of osteogenic differentiation,apoptosis, mitochondria and endoplasmic reticulum stress related transcription factors and proteins.5.Micro-computed tomography scanning technique was used to observe threedimensional microstructure of femur bone.6.The change of bone density was detected by double energy X-ray absorption spectrometry.7.The morphological changes of bone trabecula were compared by H&E staining.8.Three-point bending experiments were performed to test the biomechanical properties of femurs.Results: 1.The primary osteoblasts were isolated from cranial bone of Sprague-Dawley newborn mice for further analysis.2.CCK-8 assay,alkaline phosphatase activity assay indicated that GSTD significantly reduced high-dosed dexamethasone toxicity of osteoblasts as well as ALP activity in a dose-dependent manner.3.Annexin V-FITC/PI apoptosis assay and Hoechst33258 staining results showed pretreatment of GSTD protected primary osteoblasts from dexamethasone-induced apoptosis and enhanced cell aurvivability.4.Alizarin Red staining result showed GSTD modulated dexamethasoneinduced matrix mineralization by increasing calcium deposits formation and promoting osteogenic differentiation.5.By RNA interference of Nrf2,transfection efficiency results suggested that the cell transfection was successfully accomplished.Compared with the control group,expression of Nrf2 in the si-Nrf2 group was significantly decreased than transfection group.Mito SOX? Red probe assay and JC-1 mitochondrial membrane potential detection showed that high-dosed dexamethasone treatment significantly increased mitochondrial-targeted reactive oxygen species level and resulted in membrane potential disorder,pretreatment of gastrodin maintained homeostasis by decreasing mitochondrial stress,mitochondrial membrane potential and Nrf2 silent group weakened gastrodin's protective effect on osteoblasts.ATP activity measurement and DCFH-DA fluorescence probe detection showed that gastrodin reversed dexamethasone-induced increase total reactive oxygen species in cells,and significantly improved ATP generation.The above results suggested that gastrodin was able to protect cell mitochondrial function by regulating the Nrf2 signaling pathway.6.Real-time PCR detection results showed that gastrodin relieved the suppression of dexamethasone on osteogenetic differentiation markers expression level in MC3T3-E1,such as Runx2,Osterix,BMP-2,and OCN.Under the same conditions,western blot results indicated that the expression of Runx2 and OCN was up-regulated and the expression level of PPAR?iso2 was decreased by gastrodin,the expression level of Nrf2,HO-1 and NQO-1 were increased as well.In primary osteoblasts under the same condition,endoplasmic reticulum stress related marker GRP78,phosphoelf2? and CHOP were decreased,mitochondrial apoptosis indicators AIF,cleaved caspase3,bax,cytochrome C were decreased while anti-apoptosis bcl-2 upregulated.The expression level of HO-1 and NQO-1 induced by dexamethasone were regulated through Nrf2 signaling pathway.7.H&E staining on decalcified bone section illustrated that dexamethasone induced osteoporotic rat model was built.The density of bone trabecula in the dexamethasone group decreased and its width decreased.The gastrodin treatment group showed a improvement on density with a concentration dependence.8.Double energy X-ray absorption spectrometry suggested gastrodin led to evident improvement of bone mineral density against erosion of bone microstructure by dexamethasone.9.Microcomputed tomography(micro-CT)provided a three-dimensional image of the microstructure of the femur.Data derived from the structural trabecular bone parameters revealed that compared with the dexamethasone model group,GSTD upregulated trabecular number(Tb.N),bone volume/tissue volume(BV/TV),trabecular thickness(Tb.Th),and connectivity density(Conn.D),but clearly downregulated trabecular separation(Tb.Sp)and the structural model index(SMI)in femurs.Collectively,gastrodin improved the three-dimensional structure of proximal femoral under the dexamethasone induction.These data suggested that GSTD improved bone microstructure in a GIO model.10.By three bending test,it was indicated that the maximum bending load and stiffness of the femur could be increased under the condition of dexamethasone.Conclusion: Gastrodin can significantly alleviate the inhibitive effects of dexamethasone on osteoblast proliferation and alkaline phosphatase activity.At the same time,by activation of Nrf2 and its downstream signaling molecules NQO-1 and HO-1,gastrodin was able to reduce mitochondria-targeted and cellular oxidative stress induced by dexamethasone,maintaining the mitochondria and endoplasmic reticulum homeostasis,and prevent osteoblast apoptosis.Gastrodin can also relieve the dexamethasone-inhibition of calcium nodule formation,improving osteogenic differentiation.In glucocorticoidinduced osteoporotic rats,it can be indicated that gastrodin can prevent destruction of the bone trabecular microstructure,raise bone trabecular density,and improve bone microstructure parameters.Furthermore,gastrodin can increase bone mineral density,increase bone strength and strengthen its biomechanical properties. |
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