Quercetin promotes osteogenic differentiation of senescent jaw bone marrow mesenchymal stem cells

Abstract

BACKGROUND: Age-related degeneration is closely associated with bone metabolic imbalance. In the jaw, this manifests as alveolar bone resorption, tooth loosening, and even loss. Impaired osteogenic differentiation potential of senescent jaw bone marrow mesenchymal stem cells is a critical factor hindering jaw bone regeneration. Quercetin, a natural flavonoid compound, exhibits antioxidant, anti-inflammatory, and cell differentiation-regulating properties, yet effect and mechanism of quercetin in osteogenic differentiation of senescent jaw bone marrow mesenchymal stem cells remain unclear. OBJECTIVE: To investigate the effects of quercetin on the proliferation, migration, osteogenic differentiation, and senescence of aged jaw bone marrow mesenchymal stem cells. METHODS: Jaw bone marrow mesenchymal stem cells were isolated from the mandibles of 10 8-week-old SD rats and cultured using a combination of bone marrow flushing and bone slice digestion. Jaw bone marrow mesenchymal stem cells were subcultured to the third and seventh passages, serving as the young and senescent groups, respectively. The quercetin group was treated with quercetin based on the senescent group. CCK-8 assay was used to detect the effects of 0.01, 0.1, 1, 10, 100 μmol/L quercetin on proliferation of senescent jaw bone marrow mesenchymal stem cells, and the optimal concentration was selected. Cell scratch assay was used to observe cell migration ability. RT-qPCR and western blot were used to detect the expression of senescence markers. β-galactosidase staining was used to observe the proportion of positive cells. After 7 days of osteogenic induction, RT-qPCR and western blot were used to detect the expression of osteogenic markers. After 14 days of osteogenic induction, alkaline phosphatase staining was performed. After 21 days of osteogenic induction, alizarin red staining was performed. Western blot was used to detect the expression of phosphorylated protein kinase B, protein kinase B, phosphorylated mammalian target of rapamycin, and mammalian target of rapamycin. RESULTS AND CONCLUSION: Compared with the young group, the proliferation ability of the senescent group decreased. Compared with the senescent group, 1 μmol/L quercetin significantly promoted the proliferation of senescent jaw bone marrow mesenchymal stem cells (P < 0.01). Compared with the senescent group, the migration ability of senescent jaw bone marrow mesenchymal stem cells in the quercetin group was improved, the proportion of β-galactosidase positive cells was significantly reduced, and the mRNA and protein expression of senescence-related P16, P53, and P21 were decreased (P < 0.05). After osteogenic induction, compared with the senescent group, the quercetin group showed increased calcium nodule formation, alkaline phosphatase staining area, and mRNA and protein expression of alkaline phosphatase, osteopontin, and Runt-related transcription factor 2 (P < 0.05). Compared with the senescent group, the phosphorylation levels of protein kinase B and mammalian target of rapamycin in the quercetin group were significantly reduced (P < 0.05). These results indicate that quercetin can inhibit senescence of jaw bone marrow mesenchymal stem cells caused by multiple passages and promote osteogenic differentiation by regulating the protein kinase B/mammalian target of rapamycin signaling pathway.

1. Introduction

With the acceleration of global population aging, the incidence of oral and maxillofacial diseases such as jaw bone resorption and alveolar bone defects in the elderly has increased significantly [1-2]. Jaw bone marrow mesenchymal stem cells (JBMMSCs) are mesenchymal stem cells present in the jaw bone with multilineage differentiation potential. As important seed cells for bone tissue engineering, their osteogenic differentiation ability directly affects the process of bone defect repair. However, bone marrow mesenchymal stem cells undergo functional decline during aging, manifested as decreased proliferation, migration, and osteogenic differentiation abilities [3-5]. Cellular senescence not only affects cell function but also synthesizes senescence-associated secretory phenotype (SASP) into the bone marrow microenvironment. This senescent microenvironment leads to decreased proliferative activity and osteogenic differentiation potential of JBMMSCs, and through secretion of a large number of pro-inflammatory factors, causes chronic inflammation and spreads the senescent state to adjacent cells [6-8]. This has become a key bottleneck restricting the therapeutic effect of bone regeneration in elderly patients [9]. Traditional drug interventions often have problems such as low bioavailability and significant adverse reactions. Therefore, exploring safe and effective strategies to regulate the function of senescent JBMMSCs has important clinical significance.

Quercetin is a natural flavonoid compound widely present in vegetables and fruits. As a dietary polyphenol, it is easily ingested through daily diet or supplements. In recent years, it has been found to have significant antioxidant, anti-inflammatory, and pro-differentiation activities [10-13]. The combination of quercetin and the protein kinase inhibitor dasatinib has shown bone protective effects in animal models of osteoporosis, and its efficacy in treating other aging-related diseases is also significant [14-16]. Of particular note, quercetin has been shown to reduce the expression of cellular senescence markers P16 and senescence-associated β-galactosidase (SA-β-gal) in various cell types, suggesting its potential as a senolytic agent. However, its specific effects on senescent JBMMSCs and the underlying mechanisms remain largely unexplored.