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Activation of HSP70 impedes tert-butyl hydroperoxide (t-BHP)-induced apoptosis and senescence of human nucleus pulposus stem cells via inhibiting the JNK/c-Jun pathway

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Abstract

The endogenous repair failure of degenerated intervertebral disk (IVD) is highly related to the exhaustion of nucleus pulposus stem cells (NPSCs). Excessive oxidative stress could induce apoptosis and senescence of NPSCs, thus, declining the quantity and quality of NPSCs. Heat shock protein 70 (HSP70) is a family of cytoprotective and antioxidative proteins. However, there is no report on the protective effects of HSP70 on oxidative stress-induced NPSC impairments and underlying mechanisms. In the present study, we treated NPSCs with tert-butyl hydroperoxide (t-BHP) in vitro to simulate an oxidative stress condition. HSP70 inducer TRC051384 was used to evaluate the cytoprotective effects of HSP70. The results suggested that HSP70 impeded t-BHP-mediated cell viability loss and protected the ultrastructure of NPSCs. Moreover, t-BHP could induce mitochondrial apoptosis and p53/p21-mediated senescence of NPSCs, both of which were significantly inhibited in HSP70 activation groups. Excessive oxidative stress and mitochondrial dysfunction reinforced each other and contributed to the cellular damage processes. HSP70 decreased reactive oxygen species (ROS) production, rescued mitochondrial membrane potential (MMP) collapse, and blocked ATP depletion. Finally, our data showed that HSP70 downregulated the JNK/c-Jun pathway. Taken together, activation of HSP70 could protect against t-BHP-induced NPSC apoptosis and senescence, thus, improving the quantity and quality of NPSCs. Therefore, HSP70 may be a promising therapeutic target for IVD degeneration.

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All data generated or analyzed during this study are included in the manuscript.

Abbreviations

LBP:

Low back pain

IVD:

Intervertebral disk

IVDD:

Intervertebral disk degeneration

NP:

Nucleus pulposus

NPSC:

Nucleus pulposus stem cell

HSP70:

Heat shock protein 70

JNK:

C-Jun N-terminal kinase

CCK-8:

Cell-counting kit-8

PI:

Propidium iodide

TEM:

Transmission electron microscopy

MDA:

Malondialdehyde

ROS:

Reactive oxygen species

mtROS:

Mitochondrial ROS

MMP:

Mitochondrial membrane potential

SASP:

Senescence-associated secretory phenotype

TRC:

TRC051384

t-BHP:

Tert-Butyl hydroperoxide

NS:

No statistically significant difference

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Acknowledgements

We would like to thank the researchers and study participants for their contributions.

Funding

This study was funded by the Major Research Plan of National Natural Science Foundation of China (Grants 91649204), the National Natural Science Foundation of China (No. 81974352), and the National Key Research and Development Program of China (Grants 2016YFC1100100).

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Author notes

  1. Shuo Zhang, Weijian Liu and Peng Wang have contributed equally to this work.

Authors and Affiliations

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

    Shuo Zhang, Weijian Liu, Peng Wang, Binwu Hu, Xiao Lv, Baichuan Wang & Zengwu Shao

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

    Songfeng Chen

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  1. Shuo Zhang
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Contributions

ZS and BW contributed to the study conception and design. SZ, WL, and PW performed the experiments and analyzed the data. BH wrote the first draft of the manuscript. XL and SC substantially edited the manuscript. All authors reviewed and approved the final manuscript.

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Correspondence to Baichuan Wang or Zengwu Shao.

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Experimental protocols of the present study were approved by the medical ethics committee of Tongji Medical College, Huazhong University of Science and Technology.

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Supplementary Fig. 1 The transfection efficacy of siRNA for

HSPA1A. (a) The mRNA levels of HSPA1A in human NPSCs. Data were normalized to GAPDH. (b) Representative western blot graphs and statistical analysis of HSP70 expression in human NPSCs. (TIF 383 kb)

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Zhang, S., Liu, W., Wang, P. et al. Activation of HSP70 impedes tert-butyl hydroperoxide (t-BHP)-induced apoptosis and senescence of human nucleus pulposus stem cells via inhibiting the JNK/c-Jun pathway. Mol Cell Biochem 476, 1979–1994 (2021). https://doi.org/10.1007/s11010-021-04052-1

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