Shandong University of Traditional Chinese Medicine, Jinan, China.
The First Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China.
Phytomedicine. 2024 Dec;135:155572. doi: 10.1016/j.phymed.2024.155572. Epub 2024 Apr 3.
Our previous study demonstrated that Du-Zhong-Wan (DZW) promoted osteoporotic fracture (OPF) healing by enhancing osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and angiogenesis of endothelial cells (ECs). However, the heterogeneity of BMSCs and ECs, as well as the specific molecular mechanism underlying these effects, still require further evaluation.
The primary objective of this study was to elucidate the heterogeneity of BMSCs and ECs, as well as the cellular-level mechanism of DZW against OPF through single-cell RNA sequencing.
In this study, we presented a single-cell atlas of mouse femoral callus, comparing samples with and without DZW treatment, utilizing single-cell RNA sequencing. Variable genes were identified using the FindVariableGenes (FVG) and principal component analysis (PCA) analysis. Additionally, uniform manifold approximation and projection (U-MAP) was employed to reduce and visualize the distinct subclusters. The CellPhoneDB2 method was employed to analyze intercellular communication and quantify the interaction between ligands and receptors within distinct cell clusters. The osteogenic differentiation capacity of BMSCs was assessed by micro-CT, alkaline phosphatase (ALP), and alizarin red S (ARS) assay. The scratch wound assay and tube formation assay were utilized to assess the angiogenic capabilities of ECs in vitro. Additionally, western blot and immunofluorescence experiments were utilized to elucidate the related protein expression.
Consistent with our previous studies, DZW obviously promoted osteoporotic fracture healing. Moreover, this study discovered 14 cell clusters at the femoral fracture callus, where the BMSCs most actively interacted with ECs, through single-cell sequencing. Notably, DZW significantly elevated the proportion of Lepr BMSCs and Podxl ECs subgroup, which were respectively considered essential cells for osteoblastogenesis and angiogenesis of arteriolar vessels. The increased proportion of Podxl ECs was partially attributed to vascular endothelial growth factor (VEGF), secreted by BMSCs, which were able to be reversed by YAP pharmacological inhibitor verteporfin. Furthermore, the western blot assay revealed elevated expression levels of YAP/β-catenin, VEGF, RUNX2, and OCN in BMSCs treated with DZW, which were counteracted by verteporfin.
The data above indicates that DZW elevates the proportion of LEPR BMSCs and Podxl ECs, therefore contributing for the osteogenic ability of BMSCs and BMSCs-mediated angiogenesis via activation of the YAP/β-catenin/VEGF axis, which provides novel potential targets and mechanism for DZW in treating OPF in sub-clusters and molecular level.
我们之前的研究表明，杜仲丸（DZW）通过增强骨髓间充质干细胞（BMSCs）的成骨分化和内皮细胞（ECs）的血管生成来促进骨质疏松性骨折（OPF）的愈合。然而，BMSCs 和 ECs 的异质性，以及这些作用的具体分子机制，仍需要进一步评估。
本研究的主要目的是通过单细胞 RNA 测序阐明 BMSCs 和 ECs 的异质性以及 DZW 对抗 OPF 的细胞水平机制。
在这项研究中，我们利用单细胞 RNA 测序，展示了小鼠股骨骨痂的单细胞图谱，比较了有和没有 DZW 治疗的样本。使用 FindVariableGenes（FVG）和主成分分析（PCA）分析鉴定可变基因。此外，采用均匀流形逼近和投影（U-MAP）来减少和可视化不同的亚群。使用 CellPhoneDB2 方法分析细胞间通讯，并量化不同细胞群中配体和受体之间的相互作用。通过微 CT、碱性磷酸酶（ALP）和茜素红 S（ARS）测定评估 BMSCs 的成骨分化能力。利用划痕实验和管形成实验评估 ECs 的体外血管生成能力。此外，还进行了 Western blot 和免疫荧光实验以阐明相关蛋白表达。
与我们之前的研究一致，DZW 明显促进了骨质疏松性骨折的愈合。此外，通过单细胞测序，这项研究在股骨骨折骨痂中发现了 14 个细胞簇，其中 BMSCs 与 ECs 最活跃地相互作用。值得注意的是，DZW 显著增加了 Lepr BMSCs 和 Podxl ECs 亚群的比例，这两个亚群分别被认为是成骨细胞发生和小动脉血管生成所必需的细胞。Podxl ECs 比例的增加部分归因于由 BMSCs 分泌的血管内皮生长因子（VEGF），而 YAP 药理学抑制剂维替泊芬可以逆转这一现象。此外，Western blot 检测显示，用 DZW 处理的 BMSCs 中 YAP/β-catenin、VEGF、RUNX2 和 OCN 的表达水平升高，而维替泊芬则拮抗了这一作用。
上述数据表明，DZW 增加了 LEPR BMSCs 和 Podxl ECs 的比例，从而通过激活 YAP/β-catenin/VEGF 轴来提高 BMSCs 的成骨能力和 BMSCs 介导的血管生成，这为 DZW 在亚群和分子水平治疗 OPF 提供了新的潜在靶点和机制。
