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GSK-3β inhibition protects human nucleus pulposus cell against oxidative stress-inducing apoptosis through mitochondrial pathway

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Abstract

Background

Oxidative stress in the intervertebral disc leads to nucleus pulposus (NP) degeneration by inducing cell apoptosis. However, the molecular mechanisms underlying this process remain unclear. Increasing evidence indicates that GSK-3β is related to cell apoptosis induced by oxidative stress. In this study, we explored whether GSK-3β inhibition protects human NP cell against apoptosis under oxidative stress.

Methods and results

Immunofluorescence staining was used to show the expression of GSK-3β in human NP cells (NPCs). Flow cytometry, mitochondrial staining and western blot (WB) were used to detect apoptosis of treated NPCs, changes of mitochondrial membrane potential and the expression of mitochondrial apoptosis-related proteins using GSK-3β specific inhibitor SB216763. Co-Immunoprecipitation (Co-IP) was used to demonstrate the interaction between GSK-3β and Bcl-2. We delineated the protective effect of GSK-3β specific inhibitor SB216763 on human NPCs apoptosis induced by oxidative stress in vitro. Further, we showed SB216763 exert the protective effect by preservation of the mitochondrial membrane potential and inhibition of caspase 3/7 activity during oxidative injury. The detailed mechanism underlying the antiapoptotic effect of GSK-3β inhibition was also studied by analyzing mitochondrial apoptosis pathway in vitro.

Conclusions

We concluded that the GSK-3β inhibitor SB216763 protected mitochondrial membrane potential to delay nucleus pulposus cell apoptosis by inhibiting the interaction between GSK-3β and Bcl-2 and subsequently reducing cytochrome c(Cyto-C) release and caspase-3 activation. Together, inhibition of GSK-3β using SB216763 in NPCs may be a favorable therapeutic strategy to slow intervertebral disc degeneration.

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Data availability

All data generated or analyzed during this study are included in this published article and available from the corresponding author upon reasonable request.

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Funding

This study was partially supported by grants from the National Key Research and Development Program of China (No. 2016YFA0101300).

Author information

Author notes

  1. Kai Zhu and Song Guo contributed equally to this work.

Authors and Affiliations

  1. Department of Orthopaedics, Shanghai East Hospital, Tongji University School of Medicine, No.150 Jimo Road, Shanghai, 200120, People’s Republic of China

    Kai Zhu & Jun Tan

  2. Department of Orthopaedics II, Qingdao No.8 People’s Hospital, No.84 Fengshan Road, Qingdao, 266121, People’s Republic of China

    Kai Zhu, Guoyi Han & Qinglei Xu

  3. Research Center for Translational Medicine, Translational Medical Center for Stem Cell Therapy and Institute for Regenerative Medicine, Shanghai East Hospital, Tongji University School of Medicine, No.150 Jimo Road, Shanghai, 200120, People’s Republic of China

    Kai Zhu, Mengmeng Ma & Wenjie Tang

  4. Department of Orthopaedics, Shanghai General Hospital, Shanghai Jiaotong University, No.100 Haining Road, Shanghai, 200080, People’s Republic of China

    Song Guo

  5. Department of Anesthesiology, Qingdao No.8 People’s Hospital, No. 84 Fengshan Road, Qingdao, 266121, People’s Republic of China

    Xiancheng Qiang

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Contributions

KZ: Acquisition of data, Validation and Methodology, Drafting the manuscript. SG: Drafting the manuscript, Validation and Methodology. GH: Analysis and/or interpretation of data. XQ: Analysis and/or interpretation of data. MM: Acquisition of data. QX: Analysis and/or interpretation of data. WT: Conception and design of study. JT: Conception and design of study. All authors approved to submit this version to this publication.

Corresponding authors

Correspondence to Wenjie Tang or Jun Tan.

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Competing interests

The authors declare no conflict of interest.

Ethical approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Shanghai East Hospital, Tongji University School of Medicine.

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Informed consent was obtained from all individual participants included in the study.

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Supplementary Information

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11033_2022_7218_MOESM1_ESM.tif

Supplementary file1 (TIF 1806 kb) Fig. S1 Co-expression of Aggrecan and Collagen II in human nucleus pulposus cells (NPCs). The expression of Aggrecan and Collagen II were mainly in the cytoplasm of NPCs (100×). Scale bars, 100 μm

11033_2022_7218_MOESM2_ESM.tif

Supplementary file2 (TIF 314 kb) Fig. S2 Expression of GSK-3β in human nucleus pulposus cells (NPCs). The expression of GSK-3β was mainly in the cytoplasm of NPCs (100×) Scale bars, 100 μm

Supplementary file3 (TIF 394 kb) Fig. S3 TBHP cytotoxicity analysis using CCK-8

11033_2022_7218_MOESM4_ESM.tif

Supplementary file4 (TIF 76 kb) Fig. S4 TBHP caused oxidative stress damage of NPCs. 8-OHdG positive rates at different treating time points as described above. Data are expressed as the mean ± SD. 8-OHdG: 8-hydroxydeoxyguanosine*p < 0.05

11033_2022_7218_MOESM5_ESM.tif

Supplementary file5 (TIF 508 kb) Fig. S5 TBHP caused dysfunction of mitochondrial membrane potential using TMRM and MTG staining. TMRM: Tetramethylrhodamine methylester Scale bars, 50 μm

11033_2022_7218_MOESM6_ESM.tif

Supplementary file6 (TIF 399 kb) Fig. S6 shRNA2 effectively induced the down-regulation of GSK-3β in NPCs. a Western blot analysis of GSK-3β in each treatment group as described above. b The mRNA level of GSK-3β in each treatment group as described above. Data are expressed as the mean ± SD *p < 0.05

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Zhu, K., Guo, S., Han, G. et al. GSK-3β inhibition protects human nucleus pulposus cell against oxidative stress-inducing apoptosis through mitochondrial pathway. Mol Biol Rep 49, 3783–3792 (2022). https://doi.org/10.1007/s11033-022-07218-2

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  • DOI: https://doi.org/10.1007/s11033-022-07218-2

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