Department of Orthopaedics, Kunming Municipal Hospital of Traditional Chinese Medicine, The Third Affiliated Hospital of Yunnan University of Chinese Medicine, Kunming, Yunnan 650599, P.R. China.
The Fifth Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510405, P.R. China.
Int J Mol Med. 2022 Sep;50(3). doi: 10.3892/ijmm.2022.5167. Epub 2022 Jul 7.
Total flavonoids of (TFRD), extracted from the kidney‑tonifying Traditional Chinese medicine , can be effective in treating osteoporosis, bone fractures and defects. However, the pharmacological effects of TFRD on the specific vessel subtype CD31Emcn during distraction osteogenesis (DO) remains unclear. The present study aimed to investigate the effects of TFRD on CD31Emcn vessels in a rat model of DO. In the present study, tibial DO models were established using 60 rats with a distraction rate of 0.2 mm per day for 20 days. Co‑immunofluorescence staining of CD31 and endomucin (Emcn) was conducted to determine CD31Emcn vessels. Radiographic, angiographic and histological analyses were performed to assess bone and vessel formation. Tube formation, alkaline phosphatase (ALP) and Von Kossa staining assays were performed to test angiogenesis of endothelial precursor cells (EPCs) and osteogenesis of bone marrow‑derived mesenchymal stem cells (BMSCs). Additionally, expression levels of platelet‑derived growth factor (PDGF)‑BB, VEGF, runt‑related transcription factor 2 (RUNX2) and Osterix (OSX) were determined by western blotting and reverse transcription‑quantitative PCR. The assays demonstrated that TFRD markedly promoted CD31Emcn vessel formation during DO, whereas PDGF‑BB neutralizing antibody suppressed vessel formation. Furthermore, the ALP, Von Kossa staining and tube formation assays indicated that TFRD notably elevated the angiogenic capacity of EPCs and osteogenic capacity of BMSCs under stress conditions, which was significantly suppressed by blocking PDGF‑BB. The protein and mRNA levels of PDGF‑BB, VEGF, RUNX2 and OSX were upregulated by TFRD, but downregulated by blocking PDGF‑BB. Thus, TFRD could facilitate CD31Emcn vessel formation and subsequently enhance angiogenic‑osteogenic coupling to regenerate bone defects during DO via the PDGF‑BB/VEGF/RUNX2/OSX signaling axis, which indicated that CD31Emcn vessels could be a potential novel therapeutic target for DO, and TFRD may represent a promising drug for promoting bone regeneration in DO by increasing CD31Emcn vessels.
从补肾中药中提取的总黄酮（TFRD）可有效治疗骨质疏松症、骨折和骨缺损。然而，TFRD 对牵张成骨（DO）过程中特定血管亚型 CD31Emcn 的药理作用尚不清楚。本研究旨在探讨 TFRD 对 DO 大鼠模型中 CD31Emcn 血管的影响。本研究采用 60 只大鼠建立胫骨 DO 模型，每天牵引 0.2mm，共 20 天。采用 CD31 和内粘蛋白（Emcn）共免疫荧光染色确定 CD31Emcn 血管。进行影像学、血管造影和组织学分析以评估骨和血管形成。进行管形成、碱性磷酸酶（ALP）和 Von Kossa 染色测定以检测内皮祖细胞（EPC）的血管生成和骨髓间充质干细胞（BMSC）的成骨作用。此外，通过 Western blot 和逆转录-定量 PCR 测定血小板衍生生长因子（PDGF）-BB、血管内皮生长因子（VEGF）、成骨特异性转录因子 2（RUNX2）和骨形成蛋白 2（OSX）的表达水平。结果表明，TFRD 可显著促进 DO 过程中 CD31Emcn 血管形成，而 PDGF-BB 中和抗体则抑制血管形成。此外，ALP、Von Kossa 染色和管形成测定表明，TFRD 可显著提高应激条件下 EPC 的血管生成能力和 BMSC 的成骨能力，而阻断 PDGF-BB 则显著抑制其作用。TFRD 可上调 PDGF-BB、VEGF、RUNX2 和 OSX 的蛋白和 mRNA 水平，但阻断 PDGF-BB 则下调其水平。因此，TFRD 可通过 PDGF-BB/VEGF/RUNX2/OSX 信号通路促进 CD31Emcn 血管形成，从而增强血管生成-成骨偶联，促进 DO 过程中的骨缺损再生，表明 CD31Emcn 血管可能成为 DO 的潜在新治疗靶点，TFRD 可能通过增加 CD31Emcn 血管来促进 DO 中的骨再生，是一种有前途的药物。
