Acanthopanax exosome-like nanovesicles promote osteogenic differentiation of human bone marrow mesenchymal stem cells

Abstract

BACKGROUND: Acanthopanax and its extracts exhibit osteogenic effects, but the osteogenic potential and mechanisms of acanthopanax exosome-like nanovesicles remain unclear. OBJECTIVE: To investigate the molecular mechanism by which acanthopanax exosome-like nanovesicles promote osteogenic differentiation of human bone marrow mesenchymal stem cells and their preventive role in osteoporosis. METHODS: (1) Human bone marrow mesenchymal stem cells were extracted by gradient density centrifugation. Exosome-like nanovesicles derived from acanthopanax were isolated by differential centrifugation and sucrose gradient density centrifugation. (2) Human bone marrow mesenchymal stem cells were treated with 0, 2.5, and 5 μg/mL acanthopanax exosome-like nanovesicles. Osteogenic differentiation was assessed by alkaline phosphatase staining, Alizarin red staining, qRT-PCR, and western blot assay. (3) Key pathways were identified by transcriptome sequencing and verified using a transforming growth factor β1 receptor inhibitor. (4) An ovariectomized rat model of osteoporosis was established, and acanthopanax exosome-like nanovesicles were administered intraperitoneally for 12 weeks. Bone microarchitecture was analyzed by Micro-CT, and osteogenic protein expression was detected by histological staining. RESULTS AND CONCLUSION: (1) Acanthopanax exosome-like nanovesicles exhibited a typical cup-shaped or disc-shaped morphology. (2) In vitro experiments confirmed that acanthopanax exosome-like nanovesicles dose-dependently promoted osteogenic differentiation of bone marrow mesenchymal stem cells, as evidenced by increased alkaline phosphatase activity, enhanced mineralization nodule formation, and upregulated osteogenic gene expression. (3) Transcriptome analysis revealed that acanthopanax exosome-like nanovesicles activated the transforming growth factor β1/Smad2/3 signaling pathway, upregulating transforming growth factor β1 and phosphorylated Smad2/3 protein expression, and the transforming growth factor β1 receptor inhibitor partially suppressed their osteogenic effect. (4) Animal experiments showed that after intervention with 5 mg/kg acanthopanax exosome-like nanovesicles, bone mineral density, bone volume fraction, and trabecular thickness were significantly increased in ovariectomized rats (P < 0.05), collagen fiber formation was evident, and the expression of Runt-related transcription factor 2, osteocalcin, and transforming growth factor β1 proteins in bone tissue was upregulated. No obvious toxicity was observed in major organs by histological examination. These results indicate that acanthopanax exosome-like nanovesicles promote osteogenic differentiation of human bone marrow mesenchymal stem cells by activating the transforming growth factor β1/Smad2/3 pathway and effectively improve osteoporosis.

1. Introduction

Osteoporosis is a widespread chronic disease in the aging global population [1]. With advancing age, bone mineral density gradually decreases, significantly increasing the risk of fractures [2]. Current therapeutic drugs include bisphosphonates [3], selective estrogen receptor modulators, and parathyroid hormone analogs [4-6]. Although these agents achieve certain efficacy, long-term use may lead to adverse reactions such as osteonecrosis of the jaw, atypical fractures, and thrombosis, along with issues of drug tolerance and poor compliance [5,7]. Therefore, developing novel therapeutic strategies with good efficacy and high safety has become an urgent priority in the field of osteoporosis prevention and treatment. Bone remodeling depends on the dynamic balance between osteoblasts and osteoclasts, and the decreased differentiation capacity of bone marrow mesenchymal stem cells into osteoblasts is a key mechanism in the pathogenesis of osteoporosis [8-9]. Enhancing osteogenic differentiation has become an effective strategy for treating osteoporosis.

Acanthopanax, the root bark of Acanthopanax senticosus, has the effects of nourishing the liver and kidney, and strengthening bones and tendons. Modern pharmacological studies indicate that Acanthopanax and its extracts have multiple activities in the prevention and treatment of osteoporosis. Eleutheroside B inhibits osteoclast formation by suppressing the expression of nuclear factor of activated T cells 1 and c-Fos induced by receptor activator of nuclear factor-κB ligand, thereby improving osteoporosis symptoms in ovariectomized mice [10]. Acanthopanax extract also upregulates serum osteocalcin levels [11], exerting osteogenic effects. Furthermore, eleutheroside B reduces the expression of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-23, exerting anti-inflammatory and immunomodulatory effects, thereby further alleviating osteoporosis symptoms [12-13]. Syringin, another major active component of Acanthopanax, has been shown to significantly increase bone mineral density, bone mineral content, and trabecular thickness, inhibit osteoclast enzyme activity, and improve trabecular microarchitecture [14]. In summary, the active components of Acanthopanax have good potential in promoting bone formation and inhibiting bone resorption.

Although the active components of traditional Chinese medicine have positive effects on the prevention and treatment of osteoporosis, their clinical application is still limited by low bioavailability, complex active components, and unclear mechanisms. In recent years, plant-derived exosome-like nanovesicles (ELNs), as novel natural nano-delivery systems, have attracted widespread attention due to their good biocompatibility, stable encapsulation of active components, and cross-species regulation of target cell functions [15-16]. Studies have shown that plant-derived exosome-like nanovesicles can be used as drug delivery vehicles and have therapeutic potential in various diseases. However, the osteogenic effects and mechanisms of Acanthopanax-derived exosome-like nanovesicles have not been fully elucidated. This study aims to investigate the molecular mechanisms by which Acanthopanax exosome-like nanovesicles promote osteogenic differentiation of human bone marrow mesenchymal stem cells and their preventive effects on osteoporosis.