Arbutin Attenuates Glucocorticoid Induced Osteoporosis By Activating Osteoblast Autophagy

Objective: Osteoporosis is a systemic bone disease secondary to a variety of predisposing factors,characterized by osteopenia and deterioration of bone microstructure,accompanied by clinical symptoms such as bone fragility and increased risk of fracture.With the increase of the incidence of osteoporotic fractures,the morbidity and mortality also increase.Osteoporotic fracture brings great burden to society and family.Glucocorticoids are commonly used to treat inflammatory and immune-mediated diseases,such as allergies,rheumatoid arthritis and leukemia.Glucocorticoid related osteoporosis is one of the most common secondary osteoporosis.Long term use of glucocorticoid can lead to the imbalance of bone homeostasis in human body.When the bone loss and the formation of new bone decrease,it will eventually lead to osteoporosis.Therefore,there is an urgent need for effective treatment of glucocorticoid induced osteoporosis.Autophagy is a highly conserved behavior in eukaryotic cells.It has been confirmed that autophagy is involved in the regulation of a variety of cell behaviors,such as cell proliferation,differentiation and apoptosis.In the field of bone metabolism,autophagy can maintain bone homeostasis,and the imbalance of this pathway will lead to osteoporosis.Moreover,autophagy can maintain the activity of osteoblasts under stress,which is considered to be an important regulator of osteoblast differentiation and mineralization.In addition,there is evidence that glucocorticoid can inhibit the autophagy of osteoblasts,which is related to the pathogenesis of glucocorticoid induced osteoporosis.Arbutin is a natural hydroquinone glycoside existing in Vaccinium,Compositae,rhododendraceae and other plants,which has many biological activities such as skin whitening,hypoglycemic,neurotrophic,anti-inflammatory,tumor growth inhibition and so on.At present,it has been proved that arbutin can regulate the balance between osteoclasts and osteoblasts to maintain bone homeostasis.In view of the relationship between autophagy and glucocorticoid induced osteoporosis,we believe that it is an interesting and meaningful study whether arbutin affects glucocorticoid induced osteoporosis by regulating autophagy.Methods:In vivo experiment,according to the method of literature review,we selected 6-week-old healthy male mice,fed for a week,and then established the model.The mice were randomly divided into 4 groups,6 in each group,namely: A:control group,B: dexamethasone group,C: Dexamethasone + arbutin(low dose)group,D: Dexamethasone + arbutin(high dose)group.The mice were intraperitoneally injected with related drugs once a day for 5 weeks.Bone mineral density(BMD)and trabecular thickness were measured by ? CT(TB.TH)Trabecular number(TB.N),trabecular spacing(TB.SP)And bone volume / tissue volume(BV / TV).In each group,the eyeballs were taken and the serum was separated.The levels of ctx-1 and osteocalcin in serum were determined by ELISA kit.The expression levels of osteogenic markers and autophagy markers were detected by Western blot.In vitro,mouse MC3T3-E1 osteoblast precursor cells were cultured as the research object.The cells were intervened by the osteogenic differentiation medium containing 1 ? m dexamethasone and 100 ? m arbutin.After 3 days,Western blot was used to detect the autophagy activation of each group.After 7 days,Western blot was used to detect the osteogenic markers and alkaline phosphatase kit was used to detect the ALP activity expression of each group.After 18 days,alizarin red staining was used to evaluate the effect of arbutin on matrix mineralization of MC3T3-E1 cells.The formation of autophagy in MC3T3-E1 cells was detected by GFP-LC3 fluorescence.In order to estimate the effect of arbutin on autophagy,the expression changes of osteogenic and autophagy related indexes were further evaluated after 3-m A autophagy inhibitor was given.Results: 1.In animal experiments,?CT results showed that dexamethasone injection could cause bone loss and bone degeneration in mice.When different concentrations of arbutin were given for intervention,the bone loss of mice could be inhibited,which was more obvious in the high concentration of arbutin group.2.In animal experiments,he staining was used to observe the pathological morphology of mice femur.The results showed that arbutin could alleviate the effect of dexamethasone on the thinning and reducing of bone trabeculae in mice.3.In animal experiments,ELISA Kit analyzed the eyeball blood of mice in different groups.After DEX modeling,ctx-1 level and trap activity increased,while bone formation marker qsteocalcin level and ALP activity decreased,which was reversed after arbutin intervention.4.In animal experiments,Western blot detection showed that the expression levels of osteogenic markers and autophagy markers in the femur of mice after DEX intervention were significantly decreased,while the expression levels were significantly increased after arbutin administration compared with DEX group.5.In cell experiment,the expression of Runx2 and BMP2 in arbutin group was significantly higher than that in dex group.6.In cell experiment,ALP activity and alizarin red staining showed that the differentiation and mineralization ability of cells in arbutin group was significantly higher than that in dex group.7.In cell experiment,Western blot showed that arbutin group had more autophagy than DEX group.GFP-LC3 fluorescence tracing showed that the number of autophagosomes in arbutin group was more than that in dexamethasone group.8.In cell experiment,after 3-m A autophagy inhibitor was given,Western blot,ALP activity detection and alizarin red staining results confirmed that the ability of arbutin to enhance osteogenic differentiation and autophagy level could be inhibited by 3-m A.Conclusion: 1.Arbutin can reduce the bone loss caused by dexamethasone,so as to prevent osteoporosis caused by dexamethasone.2.Arbutin can improve the osteogenic differentiation ability and autophagy level of osteoblasts under the influence of dexamethasone.3.Autophagy pathway plays an important role in the prevention of dexamethasone induced bone loss by arbutin.