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A20 attenuates pyroptosis and apoptosis in nucleus pulposus cells via promoting mitophagy and stabilizing mitochondrial dynamics

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Abstract

Background

A20 is an anti-inflammatory molecule in nucleus pulposus (NP) cells. The anti-inflammatory properties of A20 are mainly attributed to its ability to suppress the NF-κB pathway. However, A20 can protect cells from death independently of NF-κB regulation. This study aimed to investigate the effects of A20 on pyroptosis and apoptosis of NP cells induced by lipopolysaccharide (LPS).

Methods

NP cells induced by LPS were used as an in vitro model of the inflammatory environment of the intervertebral disc. Pyroptosis, apoptosis, and mitophagy marker proteins were detected. Then, NP cells were transfected with A20 overexpressed lentivirus or A20-siRNA. Annexin V FITC/PI, Western blotting, and immunofluorescence assays were used to detect the apoptosis, pyroptosis, and mitophagy of NP cells. Furthermore, the expressions of A20, related proteins, and related inflammatory cytokines were detected by western blotting, and ELISA.

Results

Apoptosis and pyroptosis of NP cells increased gradually treated with LPS for 12 h, 24 h, and 48 h. Differently, the level of mitophagy increased first and then decreased, and was the highest at LPS treatment for 12 h. Overexpression or knockdown of A20 in NP cells revealed that A20 attenuated the pyroptosis, apoptosis, and production of inflammatory cytokines of NP cells induced by LPS, while A20 sponsored mitophagy, reduced ROS production and collapse of mitochondrial membrane potential (ΔΨm). Moreover, A20 also promoted mitochondrial dynamic homeostasis and attenuated LPS-induced excessive mitochondrial fission. Excitingly, inhibition of mitophagy attenuated the effect of A20 on the negative regulation of pyroptosis of NP cells induced by LPS. Pyroptosis was accompanied by a large release of inflammatory cytokines. Inhibition of pyroptosis also significantly reduced apoptosis of NP cells. Finally, The mitochondria-targeted active peptide SS-31 inhibited LPS-induced pyroptosis and ROS production in NP cells.

Conclusions

To sum up, A20 attenuates pyroptosis and apoptosis of NP cells via promoting mitophagy and stabilizing mitochondrial dynamics. Besides, A20 reduces LPS-induced NP cell apoptosis by inhibiting NLRP3 inflammasome-mediated pyroptosis. It provides theoretical support for the reduction of functional NP cell loss in the intervertebral disc through the gene-targeted intervention of A20.

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Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We are very grateful for the experimental technical support provided by Southeast University School of Medicine.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 81871810, 81702201).

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Author notes

  1. Xin Peng, Cong Zhang and Zhi-Min Zhou: contributed equally to this work.

Authors and Affiliations

  1. Medical School of Southeast University, Nanjing, China

    Xin Peng, Jia-Wei Gao, Zhan-Yang Qian, Jun-Ping Bao, V. L. F. Cabral & Xiao-Tao Wu

  2. Department of Orthopedics, Zhongda Hospital, Southeast University, Nanjing, China

    Cong Zhang, Zhi-Min Zhou, Kun Wang, Hang-Yu Ji & Xiao-Tao Wu

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  1. Xin Peng
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  2. Cong Zhang
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Peng, X., Zhang, C., Zhou, ZM. et al. A20 attenuates pyroptosis and apoptosis in nucleus pulposus cells via promoting mitophagy and stabilizing mitochondrial dynamics. Inflamm. Res. 71, 695–710 (2022). https://doi.org/10.1007/s00011-022-01570-6

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  • DOI: https://doi.org/10.1007/s00011-022-01570-6

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