The Role Of Cellular Senescence In The Long Bone Growth Retardation Of Young Mice Offspring Induced By Prenatal Dexamethasone Exposure

Objective:Prenatal dexamethasone exposure(PDE)can effectively promote fetal lung maturity and reduce the incidence of respiratory diseases in infants.Therefore,dexamethasone is widely used in clinical to prevent neonatal respiratory diseases.However,studies have shown that prenatal application of dexamethasone(PDE)has toxic effects on the development of long bone in offspring mice,but the mechanism is not clear.The aim of this study was to explore the cellular mechanism of PDE on the early bone development of offspring mice.Methods:Based on the clinical treatment and dosage of dexamethasone for pregnant women with risk of premature delivery,the pregnant mice(C57BL/6)were injected subcutaneously with dexamethasone(1.2mg/kg/D)on the gestational day 12-14,and the pregnant mice in the control group were injected with the same amount of normal saline.The tibias of 12 weeks old mice offspring were collected and the microstructure of cancellous bone was analyzed by micro CT.The tibia/femur of 2,4 and 6 weeks old mice offspring were collected,fixed and decalcified,dehydrated with ethanol and embedded in paraffin.H&E staining was used to analyze the morphology of bone tissue,Goldner trichrom staining was used to staining mineralized bone,TRAP kit was used to detect the number of osteoclasts,senescence-associated ?-galactosidase was used to detect the cellular senescence,Immunofluorescence staining was performed to detect Ki-67,a marker for cell proliferation,Nestin,a marker for osteoprogenitors,and endomucin and CD31,markers for H-type blood vessels in bone tissue;flow cytometry was used to analyze the number of CD45-Sca+CD105+CD29+bone marrow mesenchymal stem cells.PDE pregnant mice were treated with vehicle(200 ?l 1%methyl cellulose)or D+Q during GD 12-14 by oral gavage at dosages of 5 mg/kg/day and 50 mg/kg/day,respectively,in 200 ?l 1%methyl cellulose.Femurs of 2-week-old mice offspring were collected for HE staining,?-galactosidase staining,Goldner trichrome staining and nestin immunofluorescence detection.For in vitro experiments,bone marrow stromal cells extracted from 10 week old mice were cultured in vitro,and CFU-F staining was used to detect the clonal formation and proliferation ability of BMSCs;RNA was extracted two weeks after osteogenic differentiation in vitro,the expression of Runx2,OCN,BSP,ALP and osterix were detected by real-time quantitative PCR,and the osteogenic differentiation ability of BMSCs was detected by alizarin red and von Kossa staining three weeks after osteogenic differentiation in vitro.Results:Micro CT analysis showed that the bone volume fraction(BV/TV),trabeculae bone number(Tb.N)decreased and the trabecular bone separation(Tb.Sp)increased in 3-month-old PDE offspring mice.H&E staining showed that PDE could reduce length of bone trabeculae in 2,4 and 6-week-old mice.It is suggested that PDE may retard postnatal bone growth in young offspring mice.Quantitative results of Goldner's trichrome staining and TRAP staining showed that the number of osteoblasts and osteoclasts in PDE offspring mice decreased.Senescence-associated ?-galactosidase staining and Ki-67 immunofluorescence staining showed that PDE resulted in the increase of cellular senescence and the decrease of proliferation of epiphyseal cells.Endomucin and CD31 fluorescence co-immunostaining showed that the number of H-type blood vessels in the bone of the offspring mice was significantly reduced compared with the control group.The results of immunofluorescence and flow cytometry showed that PDE reduced the number of Nestin+cells and CD45-Sca+CD105+CD29+ stem cells in the offspring mice.Further study demonstrated that senolytics D+Q can significantly rescue postnatal bone growth retardation induced by PDE.In vitro experiments showed that PDE decreased the proliferation and osteogenic differentiation of BMSCs.Conclusion:PDE may induce the aging of the local bone cells of the offspring mice,cause the change of the local bone microenvironment,reduce the number of H-type blood vessels,the proliferation of bone marrow mesenchymal stem cells and the directional differentiation of osteoblasts,so as to reduce the long bone mass of the offspring mice.Inhibition of cellular senescence may be proposed for treating retardation of bone growth caused by PDE.