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6-gingerol protects nucleus pulposus-derived mesenchymal stem cells from oxidative injury by activating autophagy
Li-Ping Nan1, Feng Wang2, Yang Liu3
1Department of Orthopedic, Tongji University School of Medicine, Shanghai Tenth People's Hospital, Tenth People's Hospital of Tongji University, Shanghai 200072, China.
Background:
To date, there has been no effective treatment for intervertebral disc degeneration (IDD). Nucleus pulposus-derived mesenchymal stem cells (NPMSCs) showed encouraging results in IDD treatment, but the overexpression of reactive oxygen species (ROS) impaired the endogenous repair abilities of NPMSCs. 6-gingerol (6-GIN) is an antioxidant and anti-inflammatory reagent that might protect NPMSCs from injury.
Aim:
To investigate the effect of 6-GIN on NPMSCs under oxidative conditions and the potential mechanism.
Methods:
The cholecystokinin-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of 6-GIN. ROS levels were measured by 2´7´-dichlorofluorescin diacetate analysis. Matrix metalloproteinase (MMP) was detected by the tetraethylbenzimidazolylcarbocyanine iodide assay. TUNEL assay and Annexin V/PI double-staining were used to determine the apoptosis rate. Additionally, autophagy-related proteins (Beclin-1, LC-3, and p62), apoptosis-associated proteins (Bcl-2, Bax, and caspase-3), and PI3K/Akt signaling pathway-related proteins (PI3K and Akt) were evaluated by Western blot analysis. Autophagosomes were detected by transmission electron microscopy in NPMSCs. LC-3 was also detected by immunofluorescence. The mRNA expression of collagen II and aggrecan was evaluated by real-time polymerase chain reaction (RT-PCR), and the changes in collagen II and MMP-13 expression were verified through an immunofluorescence assay.
Results:
6-GIN exhibited protective effects against hydrogen peroxide-induced injury in NPMSCs, decreased hydrogen peroxide-induced intracellular ROS levels, and inhibited cell apoptosis. 6-GIN could increase Bcl-2 expression and decrease Bax and caspase-3 expression. The MMP, Annexin V-FITC/PI flow cytometry and TUNEL assay results further confirmed that 6-GIN treatment significantly inhibited NPMSC apoptosis induced by hydrogen peroxide. 6-GIN treatment promoted extracellular matrix (ECM) expression by reducing the oxidative stress injury-induced increase in MMP-13 expression. 6-GIN activated autophagy by increasing the expression of autophagy-related markers (Beclin-1 and LC-3) and decreasing the expression of p62. Autophagosomes were visualized by transmission electron microscopy. Pretreatment with 3-MA and BAF further confirmed that 6-GIN-mediated stimulation of autophagy did not reduce autophagosome turnover but increased autophagic flux. The PI3K/Akt pathway was also found to be activated by 6-GIN. 6-GIN inhibited NPMSC apoptosis and ECM degeneration, in which autophagy and the PI3K/Akt pathway were involved.
Conclusion:
6-GIN efficiently decreases ROS levels, attenuates hydrogen peroxide-induced NPMSCs apoptosis, and protects the ECM from degeneration. 6-GIN is a promising candidate for treating IDD.
Insights
6-gingerol (6-GIN) protects nucleus pulposus-derived mesenchymal stem cells (NPMSCs) from oxidative damage by reducing reactive oxygen species (ROS) and apoptosis. This antioxidant may offer a novel treatment for intervertebral disc degeneration (IDD).
Area of Science:
- Biomedical Engineering
- Regenerative Medicine
- Cell Biology
Background:
- Intervertebral disc degeneration (IDD) lacks effective treatments.
- Nucleus pulposus-derived mesenchymal stem cells (NPMSCs) show potential for IDD therapy.
- Oxidative stress impairs NPMSC function, limiting their therapeutic efficacy.
Purpose of the Study:
- To investigate the protective effects of 6-gingerol (6-GIN) on NPMSCs under oxidative stress.
- To elucidate the underlying mechanisms, including autophagy and the PI3K/Akt pathway.
Main Methods:
- NPMSCs were exposed to hydrogen peroxide (H2O2) with or without 6-GIN.
- Cell viability, ROS levels, apoptosis, and extracellular matrix (ECM) markers were assessed.
- Autophagy markers, apoptosis-related proteins, and PI3K/Akt pathway proteins were analyzed via Western blot.
- Autophagosomes were visualized using transmission electron microscopy and immunofluorescence.
Main Results:
- 6-GIN protected NPMSCs against H2O2-induced injury, reducing ROS and apoptosis.
- 6-GIN treatment increased autophagy (Beclin-1, LC-3) and activated the PI3K/Akt pathway.
- Autophagy flux was enhanced, and NPMSC apoptosis and ECM degeneration were inhibited.
- 6-GIN promoted ECM expression by reducing MMP-13 levels.
Conclusions:
- 6-GIN effectively mitigates oxidative stress and apoptosis in NPMSCs.
- 6-GIN activates autophagy and the PI3K/Akt pathway, protecting the ECM.
- 6-GIN represents a promising therapeutic candidate for intervertebral disc degeneration.
