Department of Orthopedics, Fuzhou Second Hospital, Fuzhou 350007, China.
Department of Orthopedics, Affiliated Jiangyin Hospital of Nantong University, Jiangyin 214400, China.
Exp Biol Med (Maywood). 2022 Oct;247(19):1764-1775. doi: 10.1177/15353702221116879. Epub 2022 Aug 11.
Morroniside is known to improve osteoporosis by promoting osteoblastogenesis. The activation of PI3K/Akt/mTOR signaling is a significant mechanism in morroniside-promoted osteoblastogenesis. It is well known that protective autophagy is an important factor in osteoblastogenesis. However, the activation of mTOR signaling can inhibit autophagy. This study aimed to investigate the relationship between mTOR signaling and autophagy in morroniside-regulated osteoblastogenesis. In this study, we investigated the effect of morroniside on the autophagic activity (LC3 conversion rate, LC3-puncta formation, and autophagosome number) of differentiated osteoblast precursors (MC3T3-E1 cells). Then, we identified the roles of mTOR knockdown in morroniside-regulated alterations of autophagy and osteogenic parameters in MC3T3-E1 cells. Next, mTOR knockdown and overexpression were used to observe the roles of mTOR in morroniside-regulated alterations of autophagic molecules (Atg7, Atg13, and Beclin1). Subsequently, the additional value of the above autophagic molecules on morroniside-regulated osteogenic parameters in MC3T3-E1 cells was analyzed based on lentiviral transduction. Finally, combined with morroniside and TAT-Beclin1, the roles of Beclin1 upregulation in the effects of morroniside was investigated. Our experimental data showed that morroniside promoted both the mTOR activity and autophagy in MC3T3-E1 cells. Morroniside-upregulated autophagic activity and Atg13 or Beclin1 protein level in MC3T3-E1 cells were enhanced by mTOR knockdown. Furthermore, Morroniside-upregulated Atg13 and Beclin1 expression was reversed by mTOR overexpression. Importantly, autophagy upregulation with overexpression of the autophagic gene, Atg13 or BECN1 (gene form of Beclin1), significantly promoted osteoblastogenesis regulated by morroniside. The promotional effect of morroniside on bone microarchitecture, bone mass, and bone parameters (including trabecular bone area and OCN expression in trabecular bone) in ovariectomized (OVX) mice was enhanced by TAT-Beclin1 administration. In conclusion, the autophagy-enhancing drugs related to Beclin1 or Atg13 may be an effective adjuvant therapy in the treatment of osteoporosis with morroniside.
莫诺苷通过促进成骨细胞生成来改善骨质疏松症。PI3K/Akt/mTOR 信号的激活是莫诺苷促进成骨细胞生成的重要机制。众所周知，保护性自噬是成骨细胞生成中的一个重要因素。然而，mTOR 信号的激活可以抑制自噬。本研究旨在探讨 mTOR 信号与莫诺苷调节的成骨细胞生成中的自噬之间的关系。在这项研究中，我们研究了莫诺苷对分化的成骨前体细胞（MC3T3-E1 细胞）中自噬活性（LC3 转化率、LC3 斑点形成和自噬体数量）的影响。然后，我们确定了 mTOR 敲低在莫诺苷调节的 MC3T3-E1 细胞中自噬和成骨参数改变中的作用。接下来，使用 mTOR 敲低和过表达来观察 mTOR 在莫诺苷调节的自噬分子（Atg7、Atg13 和 Beclin1）改变中的作用。随后，基于慢病毒转导分析上述自噬分子对 MC3T3-E1 细胞中莫诺苷调节的成骨参数的额外价值。最后，结合莫诺苷和 TAT-Beclin1，研究了上调 Beclin1 在莫诺苷作用中的作用。我们的实验数据表明，莫诺苷促进了 MC3T3-E1 细胞中的 mTOR 活性和自噬。在 MC3T3-E1 细胞中，莫诺苷上调自噬活性和 Atg13 或 Beclin1 蛋白水平被 mTOR 敲低增强。此外，mTOR 过表达逆转了莫诺苷上调的 Atg13 和 Beclin1 表达。重要的是，通过过表达自噬基因 Atg13 或 BECN1（Beclin1 的基因形式）上调自噬，显著促进了莫诺苷调节的成骨细胞生成。TAT-Beclin1 给药增强了莫诺苷对去卵巢（OVX）小鼠骨微结构、骨量和骨参数（包括小梁骨面积和小梁骨中 OCN 表达）的促进作用。总之，与 Beclin1 或 Atg13 相关的自噬增强药物可能是莫诺苷治疗骨质疏松症的有效辅助治疗方法。
