Effect And Mechanism Of IGF-1 C.258A>G Synonymous Mutation On Senile Osteoporosis

With a worldwide aging population,senile osteoporosis（SOP）has become the most common age-related skeletal disease,characterized by reduced bone mass and degradation of bone tissue microarchitecture,increasing the risk of bone fragility and fracture,and seriously affecting human health.SOP disrupts the coordinated action of osteoclast bone resorption and osteoblast bone formation,increases osteoclast activity,and decreases osteoblast activity,resulting in a greater rate of bone resorption than bone formation,leading to the loss of bone structure.Insulin-like growth factor-1（IGF-1）plays an important role in SOP through its direct and indirect effects on osteoblast activity.Our group previously identified the presence and only one synonymous mutation c.258G>A（rs322131043）in the highly conserved IGF-1 gene coding region among species and found that this synonymous mutation altered IGF-1 gene expression and affected IGF-1 levels in the liver and blood of adult mice,but no growth and developmental changes were observed.Based on the above,this study aimed to clarify whether the IGF-1 c.258A>G synonymous mutation has an ameliorative effect on SOP in aged mice and its mechanism of action,expecting to explore the significance and function of this synonymous mutation,as well as to provide new ideas for the study of SOP mechanism.Firstly,the SOP model was constructed using IGF-1 c.258A>G synonymous mutation single base edited mice and wild-type mice（Ho,WT）by the D-galactose induction method.SOD,CAT,GSH-PX,and MDA were used to detect aging-related indexes in mice,and the results showed that the activities of SOD,CAT,and GSH-PX were significantly decreased and the MDA content was significantly increased after the induction of aging,and SOP model was successfully constructed.Meanwhile,we monitored the body weight,calculated organ index,tibia length,and tibia weight of WT and Ho SOP mice,and found that there was no significant difference in growth and development.To investigate whether the IGF-1 c.258A>G synonymous mutation affects IGF-1 expression in SOP mice,ELISA was performed to detect IGF-1 levels in the serum of WT and Ho mice at different growth and developmental periods.The results revealed that Ho SOP mice were significantly resistant to the reduced IGF-1secretion caused by aging（p<0.01）.Micro-CT and histological staining were used to observe the morphological structure of tibial trabeculae,and the results showed that WT SOP mice had significantly decreased bone mineral density and severe destruction of trabecular microstructure,while Ho SOP mice significantly increased bone mineral density,bone volume/tissue volume and trabecular number,and significantly decreased trabecular separation and structure model index.Ho SOP mice resisted bone loss and improved bone microarchitecture by increasing the number of osteoblasts,decreasing the number of osteoclasts,and increasing the collagen volume fraction.Further,the m RNA and protein expression of bone formation and bone resorption-related genes were detected by q RT-PCR and ELISA,and the results showed that the synonymous mutation could significantly promote the m RNA and protein expression of bone-formation-related genes and inhibit bone resorption genes.These results suggest that the synonymous mutation can significantly resist the decrease of IGF-1 secretion caused by aging,resist the bone loss caused by SOP,and improve SOP.Based on the fact that IGF-1 c.258A>G synonymous mutation can resist SOP-induced bone loss,this study was conducted to further explore the effect of IGF-1c.258A>G synonymous mutation on the proliferation and differentiation of senescent osteoblastic osteoclasts by in vitro experiments.Pre-osteoclasts（CNCCs）and pre-osteoblasts（OCLs）from WT and Ho mice were isolated and identified using ALP,ARS,and TRAP staining,and the results showed that two genotypes of pre-osteoclasts（CNCC-IGF-1-A/G）and pre-osteoblasts（OCL-IGF-1-A/G）were obtained.Meanwhile,an IGF-1 c.258A>G synonymous mutant MC3T3-E1 cell model was constructed using ABEmax base editor,and after the identification of genotypes,MC3T3-IGF-1-G was finally obtained.A senescence model of osteoblasts was constructed using H₂O₂ induction and identified byβ-galactosidase staining and senescence-associated gene q RT-PCR assay.Osteoblasts from two different sources were used to jointly explore the role of this synonymous mutation on their proliferation and differentiation,and subsequent experiments were performed based on the cellular senescence model.The expression of IGF-1 and its receptor IGF-1R protein in CNCC-IGF-1-A（group A）and CNCC-IGF-1-G（group G）were detected using Western Blot,and the results showed that the expression of IGF-1 and IGF-1R in group G was significantly higher than that in group A（P<0.05）.The effects of this synonymous mutation on osteoblast proliferation were investigated using CCK-8,Ed U,cell scratch assay,and q RT-PCR assay of osteoblast proliferation-related genes.The results showed that the proliferation ability,positive cells of Ed U staining,cell migration rate,and expression level of osteoblast proliferation-related genes were significantly higher in group G than in group A.Further detection of osteoblast differentiation indexes by ALP activity and staining,alizarin red staining,q RT-PCR,and ELISA showed that the synonymous mutation promoted osteoblast differentiation and the expression of m RNA and protein of genes for osteogenic differentiation.The experimental results of MC3T3-IGF-1-A and MC3T3-IGF-1-G were consistent with the above results.The effect of this synonymous mutation on osteoclast proliferation and differentiation was investigated using CCK-8,assay of osteoclast bone resorption ability,q RT-PCR of cell proliferation and bone resorption-related genes,and the results showed that the synonymous mutation had no significant effect on cell proliferation and proliferation-related gene expression,and affected the expression of bone resorption-related genes but had no significant effect on osteoclast bone resorption function.The above results indicated that the IGF-1 c.258A>G synonymous mutation affected the expression of IGF-1 protein in senescent osteoblasts and promoted the proliferation and differentiation of senescent osteoblasts,but did not affect the proliferation and differentiation of senescent osteoclasts.IGF-1 c.258A>G synonymous mutation on osteoblasts and osteoclasts alone may not reflect the real situation of bone homeostasis in animals.In this experiment,the osteoblast-osteoclast co-culture system was constructed using Transwell chambers to investigate the effect of this synonymous mutation on osteoblast activity and osteoclast differentiation in the co-culture system.Subsequently,the osteoblast and osteoclast activities in the co-culture system were identified by ALP staining,ARS staining,TRAP staining,and osteoclast bone resorption ability assay,and the results showed that the osteoblast-osteoclast co-culture system was successfully constructed,and the osteoblasts had osteogenic activity and osteoclasts had bone resorption activity.The results showed that the synonymous mutation promoted the expression of OPG and RANKL m RNA and protein in osteoblasts and significantly increased the ratio of OPG/RANKL.The above results resolved the effect and mechanism of IGF-1 c.258A>G synonymous mutation on SOP improvement at animal and cellular levels,respectively,and provided new ideas for the study of the SOP mechanism.