Experimental Study On IGF-1C And P24 Peptide Synergistically Promote Bone Marrow Stem Cell Osteogenesis Through JNK/p38 Signaling Pathway In Vitro

Background:Bone defect caused by trauma,tumor resection,infection or congenital malformation is a common clinical disease.Bone tissue engineering is considered to be an important means to reconstruct bone defects;growth factor is one of the important elements of bone tissue engineering.In recent years,Insulin-like growth factors-1（IGF-1）and bone morphogenetic protein 2（BMP-2）have both been proven to promote bone marrow mesenchymal stem cells（BMSCs）are osteogenic,and their osteogenic ability has a synergistic effect,but the growth factors are expensive,difficult to obtain,and easy to inactivate.The C domain of IGF-1 and BMP-2 derived peptides P24,both of which have similar biological activities as growth factors,are readily available,and have fewer side effects.Can these two peptides synergistically promote bone formation in BMSCs in vitro?What is the mechanism?This study is to explore the osteogenic effects and mechanisms of these two peptides,and will provide a research basis for the application of peptides in tissue engineering to repair bone defects.Objective:This study aimed to investigate the effect of IGF-1C and P24 peptides on the osteogenic diferentiation of BMSCs and its mechanism.Method:1.In order to determine the optimal concentration of IGF-1C and P24 peptides to promote the proliferation and osteogenic differentiation of BMSCs in vitro,Incubated BMSCs with IGF-1C and P24,and determined the promotion of BMSCs proliferation and osteogenesis by cell counting kit-8（cck-8）and detection of alkaline phosphatase（ALP）activity.2.In order to study the effects of IGF-1C and P24 peptides alone and in combination on the proliferation and osteogenic differentiation of BMSCs,the two peptides were cultured with BMSCs alone or in combination,and the proliferation was measured by cck-8 and flow cytometry.ALP staining,Alizarin red S staining（ARS）and Quantitative Real-time Polymerase Chain Reaction（qRT-PCR）,Western Blot（WB）to detect the effects of single and combined culture on the osteogenic differentiation of BMSCs.3.To explore the mechanism of IGF-1C and P24 peptides synergistically promoting the osteogenic differentiation of BMSCs.Detected the phosphorylation level of JNK and p38,the expression of RUNX2 protein,the formation of calcified nodules and ALP staining by Westen Blot and ARS staining with or without specific inhibitors.Result:1.When BMSCs were cocultured in vitro with IGF-1C and P24,the optimal concentration for promoting proliferation and osteogenic differentiation was 50μg/mL within the concentration range of this study.2.IGF-1C and P24 can synergistically promote the proliferation of BMSCs,promoted ALP activity and the formation of calcified nodules,and up-regulated the expression of Runx2,OCN and OSX genes and proteins.3.IGF-1C and P24 can synergistically activate the JNK and p38 signaling pathways and increase the levels of p-JNK and p-p3 8 in BMSCs;adding JNK inhibitor（SP600125）or p38（SB203580）can inhibit p-JNK,p-p38 level,which can block Runx2 expression and inhibit the formation of calcified nodules.Conclusion:IGF-1C and P24 can synergistically activate the JNK and p38 signaling pathways of BMSCs,thereby increasing ALP activity,increasing Runx2,OCN and OSX gene and protein expression,and promoting the formation of calcified nodules,thereby inducing the formation of new bone.