Effect of Bushen Zhuangdu Fang serum on mitochondrial apoptotic pathway of nucleus pulposus cells

doi:10.3969/j.issn.2095-4344.2644

Abstract

Abstract:

BACKGROUND: The mitochondrial apoptotic pathway is an important pathway in cell apoptosis. Previous studies have found that Bushen Zhuangdu Fang can improve intervertebral disc degeneration by inhibiting the mitochondrial apoptotic pathway in animal experiments. However, its mechanism of action is to be clarified.

OBJECTIVE: To observe the effect of serum containing Bushen Zhuangdu Fang on mitochondrial apoptotic pathway key proteins of human nucleus pulposus cells, and to explore the mechanism by which this drug-containing serum improves intervertebral disc degeneration.

METHODS: Thirty-seven male Sprague-Dawley rats were randomly divided into blank group, low-dose Chinese medicine group (0.506 g/kg per day), medium-dose Chinese medicine group (1.012 g/kg per day) and high-dose Chinese medicine group (2.024 g/kg per day). After 2 weeks of continuous administration, drug-containing serum was prepared. Human nucleus pulposus cells were randomly divided into normal group, cell model group, low-dose drug-containing serum group, medium-dose drug-containing serum group, and high-dose drug-containing serum group. The cell model group was treated with 200 μmol/L H₂O₂ for 6 hours, and the normal group received no treatment. The three drug-containing serum groups were treated with corresponding treatments for 48 hours. The pathological changes of nucleus pulposus cells were observed by transmission electron microscopy. Apoptotic rate of nucleus pulposus cells was detected by flow cytometry and mitochondrial membrane potential was detected by flow cytometry. Apaf1, Bcl-2, Bax and Cytc expressions were detected by real-time quantitative PCR and western blot assay.

RESULTS AND CONCLUSION: Compared with the normal group, the apoptotic rate of nucleus pulposus cells with obvious apoptotic morphology was significantly increased (P < 0.05), the expression of Apaf1, Cytc, and Bax were significantly increased at mRNA and protein levels (P < 0.05), and the mitochondrial membrane potential and expression of Bcl-2 mRNA and protein were significantly decreased in the cell model group (P < 0.05). After treatment with drug-containing serum, the apoptotic rate of nucleus pulposus cells decreased significantly (P < 0.05), the expression of Apaf1, Cytc, Bax and their proteins decreased significantly (P < 0.05), and the mitochondrial membrane potential, Bcl-2 and their proteins increased significantly (P < 0.05). Therefore, the serum containing Bushen Zhuangdu Fang can effectively inhibit apoptosis of nucleus pulposus cells in a dose-dependent manner. The drug-containing serum may alleviate intervertebral disc degeneration by reducing the expression of Apaf1, Cytc and Bax and increasing the expression of Bcl-2 at protein and gene levels, and inhibiting the mitochondrial apoptotic pathway.

Key words: intervertebral disc degeneration, nucleus pulposus cell apoptosis, mitochondrial pathway, drug-containing serum, Bushen Zhuangdu Fang

CLC Number:

Zhou Wenming, Lin Yifeng, Zhang Zhen, Chi Liye. Effect of Bushen Zhuangdu Fang serum on mitochondrial apoptotic pathway of nucleus pulposus cells[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(23): 3643-3648.

Figures/Tables 9

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