The Role And Mechanism Of Cmpk2 In Glucocorticoid-induced Preosteoblasts Senescence

Objective: Glucocorticoid-induced osteoporosis(GIO)is a side effect of long-term glucocorticoid(GC)therapy,and the mechanisms of GIO remain unclear.Recent studies have found that GC may promote stem cell senescence in bone.Cellular senescence is manifested by growth arrest and loss of normal function but by active production of senescence-associated secretory phenotypes(SASP).Bone marrow mesenchymal stem cells(BMSC)senescence is associated with decreased proliferative capacity and osteogenic differentiation,and the production of pro-inflammatory and bone-resorbing factors.It was found that blocking GC receptors prevented premature aging of bone marrow stromal cells;high-dose GC promoted telomere shortening,which may be related to GC promoting cellular oxidative stress and inhibiting mitochondrial function.the molecular mechanism of how GC affects BMSC senescence via mitochondria remains unknown,and whether GC also promotes preosteoblasts senescence and whether it is related to mitochondria remains unclear.The objective of this study was to investigate the potential of DEX to promote cellular senescence in BMSCs and preosteoblasts.Additionally,we aimed to explore the impact of DEX on the expression of the mitochondria-related gene Cmpk2 during cellular senescence and assess the influence of Cmpk2 on mitochondrial function,cell senescence,and osteogenic differentiation in preosteoblasts treated with DEX.By shedding light on the molecular mechanisms underlying GIO,our study sought to uncover novel therapeutic strategies for this condition.Methods: 1.The GIO mouse model was constructed and divided into GIO and Con groups.After the Micro-CT test showed successful osteoporosis modeling,femoral sections of mice in each group were taken for staining of senescence-associatedβ-galactosidase(SA-β-Gal);the femur and tibia of mice in each group were used to extract primary BMSC.q RT-PCR and Western blot were used to detect senescence genes p21,p53,p16,PAI-1 in primary BMSC of mice.2.Preosteoblasts MC3T3-E1 were cultured in vitro and treated with different doses of DEX.Cell viability was measured by the CCK-8 test to determine the optimal dose and treatment time.The morphological changes were observed microscopically during DEX treatment.After DEX treatment in preosteoblasts,SA-β-Gal staining,q RT-PCR,and Western blot were performed to detect the levels of senescent cells and senescence genes p21,p53 and SASP genes PAI-1,IL-1β,and Mmp2 in the DEX and Con groups.Osteogenic differentiation marker genes ALP,OC,and Runx2 m RNA expression were measured by q RT-PCR after DEX treatment,and osteogenic differentiation ability and osteomineralization ability were measured by alkaline phosphatase(ALP)staining and alizarin red s staining.3.q RT-PCR and Western blot were performed to detect alterations of Cmpk2 in m RNA and protein levels in BMSC of GIO mice and preosteoblasts with DEX treatment compared to control.4.Cmpk2 si RNA and Con si RNA were transfected in preosteoblasts to verify transfection efficiency and interference effects.Preosteoblasts were treated in groups,divided into Con,DEX,and DEX+Cmpk2 si RNA group,and cellular reactive oxygen species and mitochondrial membrane potential were detected to clarify the effect of each treatment on mitochondrial function.5.Preosteoblasts were grouped and treated into the Con,DEX,DEX+Con si RNA,and DEX+Cmpk2 si RNA group,and SA-β-Gal staining was used to detect senescent cells.q RT-PCR and western blot was performed to detect the m RNA and protein levels of senescence genes and SASP genes in each group.6.Preosteoblasts were grouped and treated into the Con,DEX,and DEX+Cmpk2si RNA group.q RT-PCR was performed to detect the m RNA expression of osteogenic marker genes.Osteogenic differentiation ability and osteomineralization ability were measured by ALP staining and alizarin red s staining.Results: 1.Mice in the GIO group had significant osteoporotic features compared with the Con group,and the differences in bone parameters were statistically significant.Femoral sections in the GIO group had significant cell senescence shown by SA-β-Gal staining,and the expression of senescence genes in primary BMSC was significantly higher than that of control mice.2.High-dose DEX treatment significantly decreased the viability of preosteoblasts.The DEX dosing concentration was determined to be 50 μM and the treatment time was5 days.Compared with the control,DEX-treated preosteoblasts showed senescence microscopically,a significant increase in senescence gene expression,and a significant decrease in osteogenic marker gene expression,ALP activity,and mineralization capacity.3.Cmpk2 m RNA and protein levels were significantly higher in both BMSCs of GIO mice and preosteoblasts by DEX treatment than in controls.4.Cmpk2 expression was significantly decreased in preosteoblasts transfected with Cmpk2 si RNA,and Cmpk2 si RNA had good transfection efficiency and interference effect.The cellular ROS production in the DEX+Cmpk2 si RNA group was significantly lower than that in the DEX group;the mitochondrial membrane potential in the DEX+Cmpk2 si RNA group was significantly higher than that in the DEX group.5.The number of senescent cells,the expression of senescence genes and SASP gene were significantly lower in the DEX+Cmpk2 si RNA group than in the DEX group in preosteoblasts.6.The expression of osteogenic marker genes was significantly higher in the DEX+Cmpk2 si RNA group than in the DEX group in preosteoblasts;ALP activity and mineralized nodules were significantly higher in the DEX+Cmpk2 si RNA group than in the DEX group.Conclusion: 1.Glucocorticoids promote osteoporosis and cellular senescence in mice.2.Glucocorticoids promote MC3T3-E1 cell senescence and inhibit osteogenic differentiation.3.Cmpk2 is upregulated in BMSCs of GIO mice and DEX-treated MC3T3-E1 cells.4.Inhibition of Cmpk2 ameliorates DEX-induced mitochondrial dysfunction in MC3T3-E1.5.Inhibition of Cmpk2 reduces DEX-induced senescence of MC3T3-E1.6.Inhibition of Cmpk2 ameliorates DEX-induced osteogenic inhibition in MC3T3-E1.