The Effect And Mechanism Of ATRA On Glucocorticoid-induced Osteoporosis

Objective:Glucocorticoid-induced osteoporosis(GIOP)is the most common secondary osteoporosis.GIOP is mainly characterized as decreased bone formation.Patients with GIOP are vulnerable to fractures,and the subsequent delayed union or nonunion.All-trans retinoic acid(ATRA)can promote the osteogenic differentiation in mesenchymal stem cells(MSCs).However,the effect of ATRA on osteogenic inhibition remains unclear.This study aims to clarify whether ATRA can reverse glucocorticoid-inhibited osteogenic differentiation in vitro and GIOP in vivo,and the possible underlying mechanism.Methods:1.Bone morphogenetic protein 9(BMP9)-transfected MSCs were recruited as an in vitro osteogenic model.The model was combined with different concentrations of ATRA or dexamethasone(DEX).RTq PCR,Western blot and immunofluorescent staining were used to detect the changes of osteogenic markers,such as RUNX2,ALP,and OPN.ALP staining and Alizarin red S staining were recruited to evaluate the changes of ALP activity and matrix mineralization.2.The rat GIOP model was constructed by intraperitoneal injection of1 mg/kg DEX,twice a week for 8 weeks.Meanwhile,intragastric administration of ATRA was recruited for GIOP prevention and treatment.The reversal effect of ATRA on GIOP were assessed by ELISA detection of serum osteoblastic marker osteocalcin(OCN)and osteoclastic marker tartrate-resistant acid phosphatase 5b(TRACP-5b),histological staining(H&E and Masson’s trichrome staining)and μCT analysis.3.RTq PCR and Western blot were recruited to analyze the receptor subtypes(RARα,RARβ and RARγ)activated by ATRA.Meanwhile,the corresponding receptor inhibitor was recruited to investigate its effect on the reversal process of ATRA.4.RNA-sequencing(RNA-seq)was used to detect and analyze the differential genes involved in the reversal process of ATRA.5.Adenovirus-mediated overexpression and knockdown of Serpina3 n were constructed and combined with ATRA or DEX to detect its effect on DEX-inhibited osteogenic differentiation and the reversal process of ATRA.6.Chromatin immunoprecipitation(Ch IP)was recruited to explore the relationship between RARβ and the differential gene Serpina3 n.Results:1.ATRA enhanced BMP9-induced ALP activity in mouse embryonic fibroblasts(MEFs),C3H10T1/2 and C2C12.ATRA promoted the protein level of ALP,RUNX2 and matrix mineralization induced by BMP9 in MEFs.High concentrations of DEX reduced BMP9-induced ALP activity and expression of osteogenic markers.2.When DEX is combined with ATRA,ATRA increased the ALP activity and the expression of osteogenic markers inhibited by DEX.3.In rat GIOP model,the serum OCN,type I collagen,bone volume,trabecular number and trabecular thickness in ATRA prevention and treatment groups were higher than those in GIOP group,and the difference is statistically significant.4.During the reversal of ATRA,the expression of RARβ was increased,moreover,the RARβ inhibitor Le135 partially blocked the reversal effect of ATRA on DEX-inhibited osteogenic differentiation.RNA-seq showed that Serpina3 n was significantly up-regulated by DEX and down-regulated by ATRA.5.Overexpression of Serpina3 n attenuated ATRA-induced ALP activity,knockdown of Serpina3 n attenuated the inhibitory effect of DEX on BMP9-induced ALP activity.Ch IP rusult showed that RARβ can negatively regulate the expression of Serpina3 n.Conclusion:ATRA can reverse DEX-inhibited osteogenic differentiation both in vitro and in vivo,which may be closely related to the inhibition of ATRA/RARβ on DEX-promoted Serpina3n;ATRA has a positive therapeutic effect on rat GIOP model;Serpina3n may become a new target of GIOP.