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Critical contribution of RIPK1 mediated mitochondrial dysfunction and oxidative stress to compression-induced rat nucleus pulposus cells necroptosis and apoptosis

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Abstract

The aim of this study was to investigate whether RIPK1 mediated mitochondrial dysfunction and oxidative stress contributed to compression-induced nucleus pulposus (NP) cells necroptosis and apoptosis, together with the interplay relationship between necroptosis and apoptosis in vitro. Rat NP cells underwent various periods of 1.0 MPa compression. To determine whether compression affected mitochondrial function, we evaluated the mitochondrial membrane potential, mitochondrial permeability transition pore (mPTP), mitochondrial ultrastructure and ATP content. Oxidative stress-related indicators reactive oxygen species, superoxide dismutase and malondialdehyde were also assessed. To verify the relevance between oxidative stress and necroptosis together with apoptosis, RIPK1 inhibitor necrostatin-1(Nec-1), mPTP inhibitor cyclosporine A (CsA), antioxidants and small interfering RNA technology were utilized. The results established that compression elicited a time-dependent mitochondrial dysfunction and elevated oxidative stress. Nec-1 and CsA restored mitochondrial function and reduced oxidative stress, which corresponded to decreased necroptosis and apoptosis. CsA down-regulated mitochondrial cyclophilin D expression, but had little effects on RIPK1 expression and pRIPK1 activation. Additionally, we found that Nec-1 largely blocked apoptosis; whereas, the apoptosis inhibitor Z-VAD-FMK increased RIPK1 expression and pRIPK1 activation, and coordinated regulation of necroptosis and apoptosis enabled NP cells survival more efficiently. In contrast to Nec-1, SiRIPK1 exacerbated mitochondrial dysfunction and oxidative stress. In summary, RIPK1-mediated mitochondrial dysfunction and oxidative stress play a crucial role in NP cells necroptosis and apoptosis during compression injury. The synergistic regulation of necroptosis and apoptosis may exert more beneficial effects on NP cells survival, and ultimately delaying or even retarding intervertebral disc degeneration.

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Abbreviations

IVD:

Intervertebral disc

IVDD:

IVD degeneration

NP:

Nucleus pulposus

RIPK1:

Receptor-interacting protein kinase 1

RIPK3:

Receptor-interacting protein kinase 3

MLKL:

Mixed lineage kinase domain-like

PCD:

Programmed cell death

NF-κB:

Nuclear factor-κB

MDA:

Malondialdehyde

SOD:

Superoxide dismutase

mtROS:

Mitochondrial ROS

LDH:

Lactate dehydrogenase

CypD:

Cyclophilin D

MMP:

Mitochondrial membrane potential

mPTP:

Mitochondrial permeability transition pore

SiRNA:

Small interfering RNA

Nec-1:

Necrostatin-1

z-VAD:

Z-VAD-FMK

CsA:

Cyclosporine A

NAC:

N-acetyl-l-cysteine

BHA:

Butyl-4-hydroxyanisole

NS:

No significant statistical significance

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Acknowledgements

This study was supported by National Natural Science Foundation of China (Grant No. 81572203), the National Key Research and Development Program of China (Grant No. 2016YFC1100100) and the Youth Innovation Fund of The First Affiliated Hospital of Zhengzhou University (Grant No. YNQN 2017037).

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Author notes

  1. Songfeng Chen, Xiao Lv and Binwu Hu have contributed equally to this work.

Authors and Affiliations

  1. Department of Orthopaedics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China

    Songfeng Chen, Hongjian Liu & Jianzhong Xu

  2. Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China

    Xiao Lv, Binwu Hu, Lei Zhao, Shuai Li, Zhiliang Li, Xiangcheng Qing & Zengwu Shao

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  1. Songfeng Chen
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Contributions

SC, XL, BH and ZS designed the research. SC, XL, BH and LZ performed the experiments. SC, XL, and SL acquired and analyzed the data. SC, XL, BH, XQ, HL and JX contributed to writing of the manuscript. Finally, all authors have reviewed and approved the final submitted manuscript. The integrity of this work is guaranteed by Dr Songfeng Chen, Dr Xiao Lv, Dr Binwu Hu, and Dr Zengwu Shao.

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Correspondence to Songfeng Chen or Zengwu Shao.

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Chen, S., Lv, X., Hu, B. et al. Critical contribution of RIPK1 mediated mitochondrial dysfunction and oxidative stress to compression-induced rat nucleus pulposus cells necroptosis and apoptosis. Apoptosis 23, 299–313 (2018). https://doi.org/10.1007/s10495-018-1455-x

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