Department of Orthopedic Surgery, The Fourth Affiliated Hospital, International Institutes of Medicine, Zhejiang University School of Medicine, No. N1 Shangcheng Road, Yiwu, 322000, People's Republic of China.
Department of Orthopedic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, No. 88, Jiefang Road, Hangzhou, 310009, People's Republic of China.
Stem Cell Res Ther. 2022 Aug 19;13(1):424. doi: 10.1186/s13287-022-03125-2.
Aucubin (AU), an iridoid glucoside isolated from many traditional herbal medicines, has anti-osteoporosis and anti-apoptosis bioactivities. However, the effect of AU on the treatment of bone-fracture remains unknown. In the present study, the aims were to investigate the roles and mechanisms of AU not only on osteoblastogenesis of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) and anti-oxidative stress injury in vitro, but also on bone-fracture regeneration by a rat tibial fracture model in vivo.
CCK-8 assay was used to assess the effect of AU on the viability and proliferation of hBM-MSCs. The expression of specific genes and proteins on osteogenesis, apoptosis and signaling pathways was measured by qRT-PCR, western blotting and immunofluorescence analysis. ALP staining and quantitative analysis were performed to evaluate ALP activity. ARS and quantitative analysis were performed to evaluate calcium deposition. DCFH-DA staining was used to assess the level of reactive oxygen species (ROS). A rat tibial fracture model was established to validate the therapeutic effect of AU in vivo. Micro-CT with quantitative analysis and histological evaluation were used to assess the therapeutic effect of AU locally injection at the fracture site.
Our results revealed that AU did not affect the viability and proliferation of hBM-MSCs. Compared with control group, western blotting, PCR, ALP activity and calcium deposition proved that AU-treated groups promoted osteogenesis of hBM-MSCs. The ratio of phospho-Smad1/5/9 to total Smad also significantly increased after treatment of AU. AU-induced expression of BMP2 signaling target genes BMP2 and p-Smad1/5/9 as well as of osteogenic markers COL1A1 and RUNX2 was downregulated after treating with noggin and LDN193189. Furthermore, AU promoted the translocation of Nrf2 from cytoplasm to nucleus and the expression level of HO1 and NQO1 after oxidative damage. In a rat tibial fracture model, local injection of AU promoted bone regeneration.
Our study demonstrates the dual effects of AU in not only promoting bone-fracture healing by regulating osteogenesis of hBM-MSCs partly via canonical BMP2/Smads signaling pathway but also suppressing oxidative stress damage partly via Nrf2/HO1 signaling pathway.
桃叶珊瑚苷（AU）是从多种传统草药中分离得到的环烯醚萜苷，具有抗骨质疏松和抗细胞凋亡的生物活性。然而，AU 对骨折治疗的作用尚不清楚。本研究旨在探讨 AU 不仅在体外对人骨髓间充质基质细胞（hBM-MSCs）成骨作用和抗氧化应激损伤的作用，而且在体内大鼠胫骨骨折模型中对骨折再生的作用和机制。
CCK-8 法检测 AU 对 hBM-MSCs 活力和增殖的影响。采用 qRT-PCR、western blot 和免疫荧光分析检测成骨、凋亡和信号通路相关基因和蛋白的表达。通过 ALP 染色和定量分析评估 ALP 活性。通过 ARS 和定量分析评估钙沉积。采用 DCFH-DA 染色评估活性氧（ROS）水平。建立大鼠胫骨骨折模型验证 AU 在体内的治疗效果。采用微 CT 结合定量分析和组织学评价评估 AU 局部注射在骨折部位的治疗效果。
结果表明，AU 不影响 hBM-MSCs 的活力和增殖。与对照组相比，western blot、PCR、ALP 活性和钙沉积证实 AU 处理组促进了 hBM-MSCs 的成骨作用。AU 处理后，磷酸化 Smad1/5/9 与总 Smad 的比值也显著增加。用 noggin 和 LDN193189 处理后，AU 诱导的 BMP2 信号靶基因 BMP2 和 p-Smad1/5/9 以及成骨标志物 COL1A1 和 RUNX2 的表达下调。此外，AU 促进了 Nrf2 从细胞质向细胞核的易位以及 HO1 和 NQO1 的表达水平在氧化损伤后。在大鼠胫骨骨折模型中，AU 局部注射促进了骨再生。
本研究表明，AU 具有双重作用，不仅通过调节 hBM-MSCs 的成骨作用部分通过经典的 BMP2/Smads 信号通路促进骨折愈合，而且还通过 Nrf2/HO1 信号通路抑制氧化应激损伤。
