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

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. dHL60 cells were divided into 4 groups: control group received Hank's balanced salt solution; the other three groups received 50 nmol/L phorbol 12-myristate 13-acetate; the latter two groups additionally received 250 μmol/L rutin or 250 μmol/L rutin plus 5 U/mL DNase I. Apoptosis of dHL60 cells was detected by flow cytometry; mRNA and protein expression of neutrophil extracellular trap marker genes were detected by RT-qPCR and western blot. (2) MC3T3-E1 cells were divided into 6 groups: control group received Hank's balanced salt solution; the other five groups received 50 nmol/L phorbol 12-myristate 13-acetate; dHL60 cells and 50 nmol/L phorbol 12-myristate 13-acetate; 100 μmol/L rutin; dHL60 cells, 50 nmol/L phorbol 12-myristate 13-acetate, and 250 μmol/L rutin; dHL60 cells, 50 nmol/L phorbol 12-myristate 13-acetate, 250 μmol/L rutin, and 5 U/mL DNase I. Apoptosis of MC3T3-E1 cells under neutrophil extracellular traps was detected by flow cytometry; alkaline phosphatase staining and alizarin red staining were used to determine osteogenic and mineralization abilities; RT-qPCR and western blot were used to detect osteogenic-related gene and protein expression. RESULTS AND CONCLUSION: (1) Compared with the blank control group, rutin significantly inhibited the mRNA and protein expression of protein arginine deiminase 4, myeloperoxidase, and neutrophil elastase in dHL60 cells (P < 0.000 1); compared with the rutin group, the combination of rutin and DNase I had a more significant downregulation effect (P < 0.05), indicating that rutin can significantly inhibit neutrophil extracellular trap formation. (2) After inducing neutrophil extracellular traps from dHL60 and co-culturing with MC3T3-E1, the mRNA and protein expression 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 significantly increased (P < 0.000 1), indicating that neutrophil extracellular traps can significantly inhibit osteogenic ability and promote apoptosis of MC3T3-E1 cells in vitro. After intervention with rutin alone or rutin combined with DNase I, the apoptosis and osteogenic ability of MC3T3-E1 cells under neutrophil extracellular traps were significantly improved, and the effect of rutin combined with DNase I was more significant than rutin alone, indicating that rutin may inhibit neutrophil extracellular trap formation, thereby improving the osteogenic ability of MC3T3-E1 cells. (3) Molecular docking and molecular dynamics simulations showed that rutin binds well to protein arginine deiminase 4, myeloperoxidase, and neutrophil elastase target proteins, indicating that rutin can target and inhibit neutrophil extracellular trap formation.

1. Introduction

Osteoporosis is characterized by reduced bone mass and deterioration of bone microarchitecture, particularly trabecular bone in vertebrae and long bones, leading to increased risk of hip and vertebral fractures, with high disability and mortality rates [1]. Epidemiological data indicate that in Europe, the proportion of people aged over 65 has reached 21.2%, while in the United States, more than 10 million people over 50 suffer from osteoporosis [2]. In China, the prevalence of osteoporosis is approximately 19.2% among those over 50 and 32.0% among those over 60 [3]. Current prevention and treatment strategies for osteoporosis remain limited, necessitating further exploration of more effective interventions.

The pathogenesis of osteoporosis primarily stems from an imbalance in bone metabolic homeostasis, where the dynamic equilibrium between bone formation mediated by osteoblasts and bone resorption dominated by osteoclasts is disrupted, ultimately leading to progressive bone loss [4]. The bone marrow is a complex microenvironment containing various immune cells, including neutrophils, T cells, and B cells. Neutrophils are produced from myeloid precursor cells in the bone marrow and are regulated by multiple cytokines. Recent evidence suggests that neutrophils and their extracellular traps (NETs) play a role in bone metabolism and inflammatory bone diseases. However, the specific impact of NETs on osteoblast function and the potential regulatory effects of natural compounds like rutin remain largely unexplored.