Study On The Mechanism Of IL-18 Enhancing The Osteogenic Differentiation Of HBMSC Through The SLC7A5/c-MYC Regulatory Axis

Objective: Splenic rupture combined with limb fracture is a common clinical high-energy multiple injury,and total splenectomy is the first emergency measure.However,there are very few reports about the impact of splenectomy on fractures.The preliminary clinical study of our research group found that the healing of the fracture was significantly delayed in patients undergoing splenectomy with combined traumatic fractures and splenic rupture,which is considered to be related to the body’s immune inflammation after splenectomy.Immune function changes lead to changes in the release of inflammatory factors related to fracture healing.The inflammatory factors are significantly reduced and thus affect the healing of fractures.IL-18 is a powerful pro-inflammatory factor that plays an important role in the repair of bone defects It has an effect on bone marrow mesenchymal stem cells,osteoblasts,and osteoclasts,but the specific mechanism is still unclear.Therefore,this article will discuss the specific mechanism of IL-18 on fracture healing.Methods: IL-18 was divided into 1ng/ml,10ng/ml,50ng/ml,100ng/ml concentration groups to induce osteogenic differentiation and find the optimal concentration group,using alkaline phosphatase(ALP)staining and alizarin red staining To detect the effect of IL-18 on the osteogenic differentiation of hBMSCs.Perform q RT-PCR and Western Blot to evaluate the expression of bone-specific markers ALP,RUNX2,BMP2 and pathway related proteins SLC7A5,c-MYC gene and protein,and immunofluorescence to detect the expression of SLC7A5,c-MYC protein.Use Si RNA to knock out SLC7A5,c-MYC or SLC7A5 specific blocker JPH203 and c-MYC specific blocker 10058-F4 for blocking,and detect the expression of pathway related proteins SLC7A5,c-MYC,and detect bone specificity markers ALP,RUNX2,BMP2 gene and protein expression levels.Results: IL-18 can increase calcium deposition and alkaline phosphatase activity in the osteogenic differentiation of hBMSCs.At the same time,IL-18 can enhance the expression of ALP,RUNX2,and BMP2 through the SLC7A5/c-MYC pathway,and silence SLC7A5,c-MYC can reduce the expression of ALP,RUNX2,BMP2 genes and proteins,and IL-18 intervention can significantly reduce the inhibitory effect of Si RNA.In addition,treatment of hBMSCs with SLC7A5 specific inhibitor JPH203 and c-MYC specific inhibitor 10058-F4 gave similar results,which can significantly reduce the osteogenic effect of IL-18 on bone marrow mesenchymal stem cells.Conclusion:1.IL-18 can significantly promote the differentiation of hBMSCs into osteoblasts,showing a concentration-dose dependence,and the effect is most obvious at100ng/ml.2.The SLC7A5/c-MYC signaling pathway plays a regulatory role in the osteogenic differentiation of hBMSCs.3.IL-18 promotes the osteogenic differentiation of hBMSCs through the SLC7A5/c-MYC pathway,which further proves that the IL-18 plays an important role in the fracture healing process,and provides a new theoretical basis for whether fractures with splenic rupture can protect the spleen.