Chinese Journal of Tissue Engineering Research ›› 2025, Vol. 29 ›› Issue (1): 8-15.doi: 10.12307/2024.734

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Effect of Wenshen Tongluo Zhitong formula on mouse H-type bone microvascular endothelial cell/bone marrow mesenchymal stem cell co-culture system

Zhou Shijie1, Li Muzhe1, Yun Li1, Zhang Tianchi1, Niu Yuanyuan1, Zhu Yihua1, Zhou Qinfeng1, Guo Yang1, 2, Ma Yong1, 2, 3, Wang Lining1, 3   

  1. 1New Technology Laboratory of Bone Injury Repair and Reconstruction, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China; 2 Wuxi Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Clinical Medical Innovation Center for Degenerative Osteoarthropathy of Traditional Chinese Medicine, Wuxi 214071, Jiangsu Province, China; 3 School of Chinese Medicine & School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China
  • Received:2023-10-17 Accepted:2023-12-11 Online:2025-01-08 Published:2024-05-18
  • Contact: Wang Lining, Lecturer, New Technology Laboratory of Bone Injury Repair and Reconstruction, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China; School of Chinese Medicine & School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China
  • About author:Zhou Shijie, Doctoral candidate, New Technology Laboratory of Bone Injury Repair and Reconstruction, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 82074458 (to GY); Natural Science Foundation Project of Jiangsu Province, No. BK20221351 (to GY); Youth Project of Natural Science Foundation of Jiangsu Province, No. BK20220470 (to WLN); Jiangsu Provincial Colleges and Universities Natural Science Research Project, No. 22KJB360012 (to WLN); NATCM's Project of High-level Construction of Key TCM Disciplines (NATCM’s Human Education Letter[2023]No. 85) (to MY); Funded Project of Jiangsu Provincial Clinical Medicine Innovation Center for Degenerative Osteoarthrosis of Traditional Chinese Medicine (JSTCM’s Science and Education[2021]No. 4) (to MY) 

Abstract:

BACKGROUND: Bone relies on the close connection between blood vessels and bone cells to maintain its integrity. Bones are in a physiologically hypoxic environment. Therefore, the study of angiogenesis and osteogenesis in hypoxic environment is closer to the microenvironment in vivo. 

OBJECTIVE: To explore the influence of Wenshen Tongluo Zhitong (WSTLZT) formula on H-type bone microvascular endothelial cell/bone marrow mesenchymal stem cell co-culture system in hypoxia environment and its related mechanism. 

METHODS: Enzyme digestion method and flow sorting technique were used to isolate and identify H-type bone microvascular endothelial cells. Mouse bone marrow mesenchymal stem cells were isolated and obtained by bone marrow adhesion method. H-type bone microvascular endothelial cell/bone marrow mesenchymal stem cell hypoxic co-culture system was established using Transwell chamber and anoxic culture workstation. WSTLZT formula powder was used to intervene in each group at a mass concentration of 50 and 100 μg/mL. The angiogenic function of H-type bone microvascular endothelial cells in the co-culture system was evaluated by scratch migration test and tube formation test. The osteogenic differentiation ability of bone marrow mesenchymal stem cells in the cocultured system was evaluated by alkaline phosphatase staining and alizarin red staining. The protein and mRNA expression changes of PDGF/PI3K/AKT signal axis related molecules in H-type bone microvascular endothelial cells in the co-cultured system were detected by Western Blotting and q-PCR, respectively. 

RESULTS AND CONCLUSION: (1) Compared with the normal oxygen group, the scratch mobility and new blood vessel length of H-type bone microvascular endothelial cells were significantly higher (P < 0.05); the osteogenic differentiation capacity of bone marrow mesenchymal stem cells was higher (P < 0.05); the expression of PDGF/PI3K/AKT axis-related molecular protein and mRNA increased (P < 0.05) in the hypoxia group. (2) Compared with the hypoxia group, scratch mobility and new blood vessel length were significantly increased in the H-type bone microvascular endothelial cells (P < 0.05); bone marrow mesenchymal stem cells had stronger osteogenic function (P < 0.05); the expression of PDGF/PI3K/AKT axis-related molecular proteins and mRNA further increased (P < 0.05) after treatment with different dose concentrations of WSTLZT formula. These findings conclude that H-type angiogenesis and osteogenesis under hypoxia may be related to the PDGF/PI3K/AKT signaling axis, and WSTLZT formula may promote H-type vasculo-dependent bone formation by activating the PDGF/PI3K/AKT signaling axis, thereby preventing and treating osteoporosis.

Key words: Wenshen Tongluo Zhitong formula, H-type bone microvascular endothelial cell, bone marrow mesenchymal stem cell, hypoxia, angiogenesis, osteogenesis

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