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. 2021 Dec 8;4(4):e1181.
doi: 10.1002/jsp2.1181. eCollection 2021 Dec.

Hydrogen sulfide regulates autophagy in nucleus pulposus cells under hypoxia

Lei Yue  1 , Yongkai Hu  2 , Haoyong Fu  1 , Longtao Qi  1 , Haolin Sun  1
Affiliations

Hydrogen sulfide regulates autophagy in nucleus pulposus cells under hypoxia

Lei Yue et al. JOR Spine. .

Abstract

Objective: Hydrogen sulfide (H2S) has been found to act as an important gasotransmitter to regulate cell activities. This study aimed to investigate the effect of H2S on autophagy of nucleus pulposus (NP) cells under hypoxia and possible mechanism.

Materials and methods: NP cells were isolated from rat caudal discs. Cobalt chloride was used to mimic hypoxia, sodium hydrosulfide was used to emulate exogenous H2S and 3-methyladenine was used to block cell autophagy. Cell viability was assessed by phase contrast microscope and Cell Counting Kit-8 method. Moreover, expression of key autophagic proteins was analyzed via western blotting, and transmission electron microscopy was performed to detect autophagosomes.

Results: Hypoxia markedly impaired NP cell proliferation compared with control. Whereas H2S provided pro-proliferation and pro-autophagy effects on hypoxic NP cells. However, these beneficial impact of H2S on hypoxic NP cells were reversed by autophagy inhibitor.

Conclusions: Our results showed that H2S played a cytoprotective role in NP cells exposed to hypoxia in an autophagy-dependent manner.

Keywords: autophagy; hydrogen sulfide; nucleus pulposus.

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Conflict of interest statement

The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Cell grouping and determination of optimal concentration. (A) Isolation of rat nucleus pulposus cells from caudal disc and cell groupings according to different treatments of the third‐passage NP cells; (B‐D) The optimal concentration of different treatments was determined after 2 hours of different treatments by CCK‐8 assay, n = 3 replicates per group. ****P < .0001 by one‐way ANOVA test followed by Tukey's post hoc. 3‐MA, 3‐methyladenine; CoCl2, cobalt chloride; NaSH, sodium hydrosulfide; NP, nucleus pulposus; OD, optical density. Appendix S2 provided the numeric values of all the bar graphs
FIGURE 2
FIGURE 2
Effect of hypoxia, NaHS, and autophagy inhibitor on viability, proliferation and apoptosis of nucleus pulposus cells. (A) Cell proliferation of 4 days in different groups observed under phase‐contrast microscope, necrosis was defined as collapse of the plasma membrane and lysis of the cells; (B) CCK‐8 assay of NP cells was counted after 24 hours of different treatments. *P < .05 vs the control group, n = 3 replicates per group. **P < .01 by one‐way ANOVA test followed by Tukey's post hoc; ***P < .001 by one‐way ANOVA test followed by Tukey's post hoc; ****P < .0001 by one‐way ANOVA test followed by Tukey's post hoc. 3‐MA, 3‐methyladenine; CoCl2, cobalt chloride; NaSH, sodium hydrosulfide. Appendix S2 provided the numeric values of the bar graph
FIGURE 3
FIGURE 3
Expression of key autophagy markers in nucleus pulposus cells. Western blot analysis and density measurements. The expression level of (C) Bcl‐2 (n = 3 replicates per group), (D) Beclin‐1 (n = 3 replicates per group.), (E) ratio of LC3B II/GAPDH (n = 3 replicates per group.) and (F) ratio of LC3B II/LC3B I (n = 3 replicates per group) in hypoxia group was insignificantly lower than control group and significantly lower than NaHS + hypoxia group. The ratio was lowest in 3‐MA + NaHS + hypoxia group. *P < .05 by one‐way ANOVA test followed by Tukey's post hoc; **P < .01 by one‐way ANOVA test followed by Tukey's post hoc; ***P < .001 by one‐way ANOVA test followed by Tukey's post hoc. 3‐MA, 3‐methyladenine; CoCl2, cobalt chloride; NaHS, sodium hydrosulfide. Appendix S2 provided the numeric values of all the bar graphs
FIGURE 4
FIGURE 4
Autophagosome detection under TEM. (A) The number of autophagosomes (double‐membrane sequestering vesicles, marked by arrows) of nucleus pulposus cells in hypoxia group was attenuated compared with that of control group. The number of autophagosomes in NP cells of NaHS + hypoxia group was higher than in hypoxia group. The number of autophagosomes of NP cells in 3‐MA + NaHS + hypoxia group was the lowest among all groups (Scale bar: 2 μm in A1, B1, C1, D1, E1 and 0.5 μm in A2, B2, C2, D2, E2); (B) Bar chart of semi‐quantification of autophagosomes of different groups of cells of random visual fields under TEM. 3‐MA, 3‐methyladenine; CoCl2, cobalt chloride; NaSH, sodium hydrosulfide; TEM, transmission electron microscope
FIGURE 5
FIGURE 5
Schematic diagram of the cytoprotective effect of H2S on nucleus pulposus cells under hypoxia. H2S upregulates autophagy and downregulate apoptosis, resulting in beneficial effects on NP cells under hypoxia. The dotted line represents uncertain mechanism. H2S, hydrogen sulfide; NP, nucleus pulposus
See this image and copyright information in PMC

References

    1. Lan T, Shiyu H, Shen Z, Yan B, Chen J. New insights into the interplay between miRNAs and autophagy in the aging of intervertebral discs. Ageing Res Rev. 2021;65:101227. - PubMed
    1. Dowdell J, Erwin M, Choma T, Vaccaro A, Iatridis J, Cho SK. Intervertebral disk degeneration and repair. Neurosurgery. 2017;80(3S):S46‐S54. - PMC - PubMed
    1. Setton LA, Chen J. Cell mechanics and mechanobiology in the intervertebral disc. Spine (Phila Pa 1976). 2004;29(23):2710‐2723. - PubMed
    1. Li Z, Chen X, Xu D, Li S, Chan MTV, Wu WKK. Circular RNAs in nucleus pulposus cell function and intervertebral disc degeneration. Cell Prolif. 2019;52(6):e12704. - PMC - PubMed
    1. Chen JW, Ni BB, Zheng XF, Li B, Jiang SD, Jiang LS. Hypoxia facilitates the survival of nucleus pulposus cells in serum deprivation by down‐regulating excessive autophagy through restricting ROS generation. Int J Biochem Cell Biol. 2015;59:1‐10. - PubMed