Orofacial Reconstruction and Regeneration Laboratory, The Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, China; Department of Oral and Maxillofacial Surgery, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, China.
Orofacial Reconstruction and Regeneration Laboratory, The Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, China; Department of Oral Implantology, The Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, China.
Cell Signal. 2021 Nov;87:110138. doi: 10.1016/j.cellsig.2021.110138. Epub 2021 Aug 28.
The osteogenic differentiation ability of adipose-derived stem cells (ASCs) is attenuated in type 2 diabetic osteoporosis (Dop) mice. Several studies suggest autophagy and Notch signaling pathway play vital roles in cell proliferation, differentiation, and osteogenesis. However, the mechanisms of autophagy and Notch signaling in the osteogenic differentiation of Dop ASCs were unclear. Thus, it is meaningful to reveal potential correlations between autophagy, Notch signaling, and osteogenesis, and explore involved molecular mechanisms in Dop ASCs.
The diabetic osteoporosis C57BL/6 mouse model, which was confirmed by micro-CT and HE & Masson staining, was established through high-sugar and high-fat diet and streptozotocin injection. ASCs were obtained from the inguinal subcutaneous fat of Dop mice. The multi-differentiation potential of ASCs was evaluated by staining with Alizarin Red (osteogenesis), Oil Red O (adipogenesis), and Alcian blue (chondrogenesis). Cell viability was assessed by Cell Counting Kit-8 assay. Torin1, an inhibitor of mTOR, was used to stimulate the autophagy signaling pathway. DAPT, a γ-secretase inhibitor, was used to suppress Notch signaling pathway activity. Gene and protein expression of autophagy, Notch signaling pathway, and osteogenic factors were detected by real-time quantitative PCR, western blot, and immunofluorescence microscopy.
Our findings showed autophagy and osteogenic differentiation ability of Dop ASCs exhibited downward trends that were both rescued by Torin1. Notch signaling was suppressed in Dop ASCs, but upregulated when autophagy was activated. After activation of autophagy, DAPT treatment led to decreased Notch signaling pathway activation and attenuated osteogenic differentiation ability in Dop ASCs.
Downregulated autophagy suppressed Notch signaling, leading to a reduced osteogenic differentiation capacity of Dop ASCs, and Torin1 can rescue this process by activating autophagy. Our findings contribute to understanding the mechanism underlying impairment of the osteogenic differentiation ability of Dop ASCs.
2 型糖尿病骨质疏松症（Dop）小鼠脂肪来源干细胞（ASCs）的成骨分化能力减弱。有几项研究表明，自噬和 Notch 信号通路在细胞增殖、分化和成骨中起着至关重要的作用。然而，Dop ASCs 成骨分化过程中自噬和 Notch 信号通路的机制尚不清楚。因此，揭示自噬、Notch 信号通路与成骨之间的潜在相关性，探讨 Dop ASCs 中涉及的分子机制具有重要意义。
通过高糖和高脂肪饮食联合链脲佐菌素注射，建立了经 micro-CT 和 HE&Masson 染色证实的糖尿病骨质疏松 C57BL/6 小鼠模型。从 Dop 小鼠腹股沟皮下脂肪中获得 ASCs。通过茜素红（成骨）、油红 O（脂肪生成）和阿利新蓝（软骨生成）染色评估 ASCs 的多向分化潜能。通过细胞计数试剂盒-8 测定细胞活力。使用 mTOR 抑制剂 Torin1 刺激自噬信号通路。使用 γ-分泌酶抑制剂 DAPT 抑制 Notch 信号通路活性。通过实时定量 PCR、western blot 和免疫荧光显微镜检测自噬、Notch 信号通路和成骨因子的基因和蛋白表达。
我们的研究结果表明，Dop ASCs 的自噬和成骨分化能力呈下降趋势，而 Torin1 可挽救这一下降趋势。Dop ASCs 中的 Notch 信号受到抑制，但自噬激活时上调。自噬激活后，DAPT 处理导致 Dop ASCs 中 Notch 信号通路激活减弱，成骨分化能力降低。
下调的自噬抑制了 Notch 信号，导致 Dop ASCs 的成骨分化能力降低，而 Torin1 通过激活自噬可以挽救这一过程。我们的研究结果有助于理解 Dop ASCs 成骨分化能力受损的机制。
