Ellagic Acid (EA) Promotes Osteoblastic Differentiation of Dental Pulp-Derived Stem Cells (DPSCs) by Upregulating KLF2 and by Inducing Autophagy/Mitophagy

Abstract ID 100163 Poster Board 365 Objective: Examine the ability of EA to promote osteoblastic differentiation in dental pulp-derived stem cells, elucidate the in-depth regulatory mechanisms underlying EA-induced osteogenic differentiation, and investigate the specific role of KLF2 in this osteogenesis process. Background: Osteoporosis is a systemic metabolic bone disorder that causes bone deterioration. Current treatments help only to slow the loss of bone density but do not facilitate any bone regeneration,underscoring the need to understand the underlying mechanisms for novel therapies. Several cellular functions and molecular signaling pathways regulate the coupling of osteoblasts and osteoclasts. Recently, autophagy has emerged as a mechanism in bone remodeling and regeneration (2). On the other hand, polyphenolic compounds are grabbing the attention of researchers due to their considerable anti-inflammatory and antioxidant effects. Numerous fruits, such as pomegranates, pecans, and raspberries, contain a significant amount of ellagic acid (EA), which is a flavonoid polyphenol. However, there were no reported studies regarding its role in modulating autophagy and mitophagy during osteogenic differentiation of DPSCs. Various KLF (Krüppel-like factor) proteins impact bone health and disease, such as KLF2. It is expressed in primary osteoblasts, which is upregulated during osteoblast differentiation. Previously studies showed that KLF2 promotes osteoblast differentiation and mineralization. Therefore, KLF2 might be a promising therapeutic target for bone disease (1). Methods: Herein, we studied the role of EA in regulating osteogenesis in dental pulp-derived stem cells which are self-renewing multipotent cells that are capable of mediating tissue regeneration via autophagy and mitophagy and elucidated the effects of EA on structural and functional molecules related to osteogenesis. To check the efficacy of EA to induce osteogenic differentiation, ellagic acid was added to the cultured DPSCs to induce osteogenic differentiation for 7 and 14 days. Then to evaluate the osteogenic potential of EA, first, we performed the Alizarin Red staining to observe the calcium depositions, which is used as a hallmark of osteogenic differentiation. Later, we assessed the expression of osteoblastic-specific markers using RT-PCR, western blot, and Immunocytochemistry. Results: Our results have shown that the polyphenol EA upregulated the osteoblastic-specific markers and demonstrated that the EA promotes the expression of Runx2, SPP1, and SPARC along with BMP2/SMAD1/5/8 pathway molecules and upregulated the expression of KLF2 at the mRNA and protein levels. These findings indicate that EA promotes osteogenesis in DPSCs via BMP2 signaling. Apart from these, we noticed that autophagic markers such as BECN1, ATG3, ATG5, ATG7, and Mitophagy markers, such as PARKIN, DRP1, and FIS1 were significantly upregulated in the presence of EA. Conclusion: These data indicate that EA facilitates osteogenic differentiation of DPSC through modulating autophagy and mitophagy by inducing KLF2 and upregulating the BMP2 signaling. As studies have indicated BMP-2 is known to enhance osteogenic differentiation, and because of the relationship between KLF2 and autophagy, we intend to develop a potential stem cell therapy for osteoporosis using human DPSCs.