Bioactive Cyclic Peptide D7 Inhibits Adipogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells Induced By Osteonecrosis Of Femoral Head And Its Mechanism

Background and purpose:Steroid-induced osteonecrosis of the femoral head(SONFH)caused by glucocorticoids(GCs)is the most serious effect in long-term or over-dose steroid therapy,that is characterized by interruption of blood supply,necrosis of subchondral bone,and eventually bone collapse of the femoral head,resulting in severe hip joint pain and dysfunction.Once diagnosed,approximately 80%of patients typically progress to femoral head collapse within 1-5 years if it is not treated in a timely manner.In spite of total hip arthroplasty(THA)is the most effective treatment for patients with osteonecrosis of femoral head(ONFH)in the terminal stages,the longevity of prostheses after total hip arthroplasty in young adults or active populations are still been a challenge for orthopedist,which are related to postoperative prosthesis loosening,polyethylene liner wear,and periprosthetic infection.Bone marrow mesenchymal stem cells(BMSCs)play an essential function in the pathophysiology process of ONFH and represent a promising method for cell therapy and tissue regeneration medicine due to BMSCs own extraordinary capacity for self-renewal and multilineage differentiation.Although the pathophysiological mechanism of steroid-induced osteonecrosis of femoral head remains unknown,it is clear that long-term GCs use induced decreased osteogenic and chondrogenic differentiation,and increased adipogenic differentiation of bone marrow mesenchymal stem cells.Bioactive cyclic peptide D7 is a newly discovered cyclic peptide with specific affinity for BMSCs,but its function in regulating adipogenic differentiation remains unclear.In this study,dexamethasone(DEX)was used to stimulate mouse bone marrow mesenchymal stem cells to simulate the environment of SONFH in vitro,to observe whether cyclic peptide D7 could inhibit the adipogenic differentiation of mouse bone marrow mesenchymal stem cells,and to explore its possible regulatory mechanism.Materials and methods:1.BMSCs from mouse tibia and femur were extracted and cultured to passage 4-6 for research.The affinity of D7 peptide to mouse BMSCs was studied by fluorescent cytochemistry;the toxicity of D7 peptide and dexamethasone to mouse BMSCs was detected by CCK-8 cell proliferation assay,and the concentration of D7 and dexamethasone was determined.2.After 7 days of adipogenic differentiation of mouse BMSCs,oil red O staining(ORO staining)was performed to observe the size and number of intracellular lipid droplets.Expression of adipogenic key markers such as Peroxisome Proliferator-activated Receptors gamma(PPARg),enhancer-binding protein alpha(CEBPA),and adiponectin(ADIPOQ)at the protein and transcription levels was detected by western blot(WB)and reverse transcription-quantitative polymerase chain reaction(RT-qPCR).3.The overlapping targets of D7 peptide in ONFH were analyzed by network pharmacology analysis,and the activation of ERK1/2 signaling pathway in mouse BMSCs was detected by Western Blot.After blocking this signaling pathway,its effects on the adipogenic changes of mouse BMSCs stimulated by DEX and D7 peptides were observed through Oil Red O staining and RT-qPCR.Results:1.Fluorescence cytochemistry showed that D7 peptide had a good affinity for mouse BMSCs,and D7 peptide mainly existed in the cytoplasm of BMSCs.Appropriate concentrations of DEX(10uM)were obtained by CCK-8 cell proliferation assay to simulate ONFH conditions and a safe dose of D7(20uM)in mouse BMSCs.2.A certain concentration of DEX(10uM)can significantly increase the size and number of lipid droplets in mouse BMSCs,and can increase the expression of PPARg,CEBPA,ADIPOQ at the protein and transcription levels.However,D7 peptide could obviously inhibit DEX-induced adipogenic differentiation.3.The results of network pharmacology analysis showed that D7 peptide had an activating effect on the ERK1/2 MAPK signaling pathway in mouse BMSCs,which was further verified by WB results.the inhibitor of this signaling pathway could block the effect of D7 peptide on the adipogenic differentiation of mouse BMSCs.Conclusion:Cyclic peptide D7 can inhibit DEX-induced adipogenic differentiation of mouse BMSCs by activating the ERK1/2-MAPK signaling pathway.This study suggests that D7 may participate in the pathophysiological process of BMSCs after glucocorticoid-induced ONFH,and provides a potential approach for the treatment of early glucocorticoid-induced ONFH.