Effects of 6-gingerol on apoptosis of rat nucleus pulposus cells |
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Authors: | NAN Li-ping ZHANG Wen-jie FENG Xin-min WANG Feng ZHOU Shi-feng LIU Yang WANG Shu-guang CHEN Dong CAI Tong-chuan ZHANG Liang |
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Affiliation: | 1. Graduate School, Dalian Medical University, Dalian 116000, China;
2. Department of Orthopedics, Clinical Medical College of Yangzhou University, Yangzhou 225001, China;
3. International Zhuang Hospital, Guangxi University of Traditional Chinese Medicine, Nanning 530001, China |
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Abstract: | AIM: To study the effect of 6-gingerol on the apoptosis of rat nucleus pulposus cells and its possible mechanism. METHODS: Rat nucleus pulposus cells were isolated and cultured. The effects of 6-gingerol and hydrogen peroxide (H2O2) at different concentrations on the viability of nucleus pulposus cells were measured by CCK-8 assay. After 6-gingerol treatment, the protein level of p-Akt was determined by Western blot. The cells were divided into 4 groups:control group, H2O2 group, 6-gingerol group (6-gingerol + H2O2) and LY294002 group (6-gingerol + H2O2 + LY294002). The apoptotic rate and the levels of reactive oxygen species (ROS) were analyzed by flow cytometry. TUNEL fluorescence staining was used to observe the number of apoptotic cells. The morphological changes of mitochondria were observed under transmission electron microscope, and Western blot was used to determine the protein levels of caspase-3, Bcl-2, Bax, p-Akt, Akt and p53. The mRNA expression of aggrecan and type II collagen was measured by RT-qPCR. RESULTS: The results of CCK-8 assay showed that the optimal concentration of 6-gingerol for promoting the viability of rat nucleus pulposus cells was 24 mg/L, and the exposure condition of H2O2 at 80 μmol/L for 6 h was appropriate for establi-shing the cell damage model. 6-Gingerol increased the protein level of p-Akt in a time-dependent manner. The apoptotic rate, ROS level and TUNEL positive cells in H2O2 group were significantly increased compared with control group. The mitochondrial edema was obvious in H2O2 group compared with control group. The protein levels of pro-apoptotic molecules caspase-3, Bax and p53 were significantly increased, while anti-apoptotic protein Bcl-2, and mRNA expression of aggrecan and type II collagen were significantly decreased compared with control group (P<0.05). 6-Gingerol exerted a protective effect against H2O2-induced apoptosis and promoted the expression of anti-apoptotic proteins. However, this effect was weakened after treatment with PI3K/Akt signaling pathway inhibitor LY294002. CONCLUSION: H2O2 induces damage and dysfunction of rat nucleus pulposus cells, and 6-gingerol may inhibit H2O2-induced apoptosis of nucleus pulposus cells by activation of PI3K/Akt signaling pathway. |
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Keywords: | 6-Gingerol Nucleus pulposus cells Apoptosis Intervertebral disc degeneration PI3K/Akt signaling pathway |
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