Chinese Journal of Tissue Engineering Research ›› 2020, Vol. 24 ›› Issue (8): 1200-1206.doi: 10.3969/j.issn.2095-4344.2499

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Proliferative activity of nucleus pulposus cells in human intervertebral disc and intervention with Yishen Huoxue Tongluo Recipe

Chen Jiang1, Xiao Huideng2, Sun Qi1, Zhang Fan1, Zhu Yonggang2, Liu Zhichao2, Guo Feiyu2, Liu Genzhe2   

  1. 1Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing 100700, China; 2Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing 100010, China
  • Received:2019-07-16 Revised:2019-07-19 Accepted:2019-08-20 Online:2020-03-18 Published:2020-01-21
  • Contact: Liu Genzhe, MD, Chief physician, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing 100010, China
  • About author:Chen Jiang, MD, Associate chief physician, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing 100700, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81603638; 2017 Fundamental Research Fund of Beijing University of Chinese Medicine, No. 2017-JYB-JS-085; the Fundamental Research Funds for the Central Universities, No. 2018-JYB-XJQ010; 2019 Fundamental Research Fund of Beijing University of Chinese Medicine, No. 2019-JYB-JS-042; Young Talents Project of Dongzhimen Hospital of Beijing University of Chinese Medicine, No. DZMYS-201702

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Abstract:

BACKGROUND: The relationship between nuclear factor (NF)-κB signaling pathway and intervertebral disc degeneration is a hotspot in the field of orthopedics. In-depth investigation on various signaling pathways in the intervertebral disc contributes to understanding the mechanism of intervertebral disc degeneration.

OBJECTIVE: To investigate the regulation of the serum containing Yishen Huoxue Tongluo Recipe on the NF-κB signal pathway of human intervertebral disc nucleus pulposus cells under different hydrostatic pressures, attempting to explore the possible therapeutic mechanism and target of Yishen Huoxue Tongluo Recipe in the treatment of degenerative diseases of the intervertebral disc from the perspective of molecular biology.

METHODS: Passage 3 Human intervertebral disc nucleus pulposus cells were divided into eight groups and were cultured in the drug-containing serum of Yishen Huoxue Tongluo Recipe. The cells were intervened for 2, 4, and 6 hours under different hydrostatic loading conditions (0.3, 1, and 3 MPa). The morphology and growth of nucleus pulposus cells were observed by an inverted phase contrast microscope. Ultrastructural changes of intervertebral disc nucleus pulposus cells were observed by a transmission electron microscopy. Cell counting kit-8 method was used to detect the proliferation activity of nucleus pulposus cells. Annexin V-FITC/Propidium Iodide apoptotic kit was used to double-stain nucleus pulposus cells to detect the cell apoptotic rate. Western blot method was used to detect the changes of NF-κB p65, Collagen II, ADAMTS-4, MMP-13 and Caspase-3 in nucleus pulposus cells.

RESULTS AND CONCLUSION: (1) Under the same pressure and time, the morphology and growth of nucleus pulposus cells in the pressure+drug-containing serum groups were better than those in the normal pressure group and the simple pressure groups. Among them, the 0.3 and 1 MPa pressure+drug-containing serum groups had more intact cell morphology and better cell growth than the 3 MPa pressure+ drug-containing serum group. (2) The proliferative activity of nucleus pulposus cells was higher in the pressure+drug-containing serum group, and there was a significant difference between the 0.3 MPa pressure+drug-containing serum group and the 0.3 MPa simple pressure group (P < 0.05). (3) The apoptotic rate of nucleus pulposus cells in the pressure+drug-containing serum group was lower than that in the normal pressure group and simple pressure intervention group (P < 0.05). (4) The expression of Collagen II and Caspase-3 increased in the pressure+drug-containing serum group, while the expression of NF-kappa B p65, ADAMTS-4 and MMP-13 decreased in the pressure+drug-containing serum group (P < 0.05). To conclude, Yishen Huoxue Tongluo Recipe can increase cell activity, reduce cell apoptosis and effectively delay the degeneration of nucleus pulposus cells. Its mechanism is likely to promote the expression of Collagen II and Caspase-3 through the NF-κB signaling pathway of nucleus pulposus cells of intervertebral disc, and inhibit the expression of NF-κB p65, ADAMTS-4 and MMP-13.

Key words: hydrostatic pressure, NF-κB signaling pathway, nucleus pulposus cells, Yishen Huoxue Tongluo Recipe, intervertebral disc degeneration

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