Rutin promotes osteogenic differentiation of MC3T3-E1 cells:regulating the formation of neutrophil extracellular traps

Li Jie; Liu Yang; Wang Dayu; Wan Qiang; Zhu Jiayi; Feng Wenjun; Chen Jinlun; Jie Ke; Huang Yiwei; Xin Pengfei; Zeng Jianchun; Zeng Yirong; Zhang Haitao

doi:10.12307/2026.793

Abstract：BACKGROUND:Rutin can effectively prevent osteoporosis,but its mechanism of action remains unclear. OBJECTIVE:To investigate the effect of rutin on osteogenesis of MC3T3-E1 cells under the action of neutrophil extracellular traps. METHODS:(1)Human myeloid leukemia dHL60 cells were stimulated with phorbol 12-myristate 13-acetate to induce neutrophil extracellular trap formation in vitro.dHL60 cells were divided into four groups and cultured.The control group received Hank's balanced salt solution,while the other three groups received 50 nmol/L phorbol esters.The latter two groups were then treated with either 250 μmol/L rutin or 250 μmol/L rutin and 5 U/mL DNase I.dHL60 cell apoptosis was assessed by flow cytometry,and the expression of marker genes and proteins associated with neutrophil extracellular trap formation was determined by RT-qPCR and western blot assay.(2)MC3T3-E1 cells were divided into six groups for culture:the control group was treated with Hank's balanced salt solution,and the other five groups were treated with 50 nmol/L phorbol esters;dHL60 cells and 50 nmol/L phorbol esters;100 μmol/L rutin;dHL60 cells,50 nmol/L phorbol esters,and 250 μmol/L rutin;and dHL60 cells,50 nmol/L phorbol esters,250 μmol/L rutin,and 5 U/mL DNase I.Flow cytometry was used to assess apoptosis in MC3T3-E1 cells treated with neutrophil extracellular traps.Alkaline phosphatase staining and Alizarin red staining were used to determine the osteogenic and mineralization abilities of MC3T3-E1 cells treated with neutrophil extracellular traps.RT-qPCR and western blot assay were used to examine the expression of osteogenesis-related genes and proteins in MC3T3-E1 cells treated with neutrophil extracellular traps. RESULTS AND CONCLUSION:(1)Compared with the blank control group,rutin significantly inhibited the mRNA and protein expressions of protein arginine deiminase 4,myeloperoxidase,and neutrophil elastase in dHL60 cells(P<0.000 1).Compared with the rutin group alone,the combined intervention of rutin and DNase I had a more significant downregulation effect on protein arginine deiminase 4,myeloperoxidase,and neutrophil elastase(P<0.05),indicating that rutin could significantly inhibit the formation of neutrophil extracellular traps.(2)After dHL60 cells induced neutrophil extracellular traps and then co-cultured with MC3T3-E1 cells,the mRNA and protein expressions of Runt-related transcription factor 2,β-catenin,and bone morphogenetic protein 2 in MC3T3-E1 cells were significantly downregulated(P<0.000 1),and the apoptosis rate was significantly increased(P<0.000 1),which indicate that neutrophil extracellular traps significantly inhibit the osteogenic capacity of MC3T3-E1 cells in vitro and promote their apoptosis.Rutin alone or combined with DNase I significantly improved the apoptosis and osteogenic capacity of MC3T3-E1 cells exposed to neutrophil extracellular traps,and the combined effect of rutin and DNase I was more pronounced than that of rutin alone,suggesting that rutin may inhibit the formation of neutrophil extracellular traps,thereby improving the osteogenic capacity of MC3T3-E1 cells.(3)Molecular docking and molecular dynamics simulations revealed that rutin binds well to the target proteins of protein arginine deiminase 4,myeloperoxidase,and neutrophil elastase,indicating that rutin can specifically inhibit the formation of neutrophil extracellular traps.