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. 2017;2017:8640284.
doi: 10.1155/2017/8640284. Epub 2017 Jun 8.

Hydrogen Sulfide Inhibits Autophagic Neuronal Cell Death by Reducing Oxidative Stress in Spinal Cord Ischemia Reperfusion Injury

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Free PMC article

Hydrogen Sulfide Inhibits Autophagic Neuronal Cell Death by Reducing Oxidative Stress in Spinal Cord Ischemia Reperfusion Injury

Lei Xie et al. Oxid Med Cell Longev. .
Free PMC article

Abstract

Autophagy is upregulated in spinal cord ischemia reperfusion (SCIR) injury; however, its expression mechanism is largely unknown; moreover, whether autophagy plays a neuroprotective or neurodegenerative role in SCIR injury remains controversial. To explore these issues, we created an SCIR injury rat model via aortic arch occlusion. Compared with normal controls, autophagic cell death was upregulated in neurons after SCIR injury. We found that autophagy promoted neuronal cell death during SCIR, shown by a significant number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling- (TUNEL-) positive cells colabeled with the autophagy marker microtubule-associated protein 1 light chain 3, while the autophagy inhibitor 3-methyladenine reduced the number of TUNEL-positive cells and restored neurological and motor function. Additionally, we showed that oxidative stress was the main trigger of autophagic neuronal cell death after SCIR injury and N-acetylcysteine inhibited autophagic cell death and restored neurological and motor function in SCIR injury. Finally, we found that hydrogen sulfide (H2S) inhibited autophagic cell death significantly by reducing oxidative stress in SCIR injury via the AKT-the mammalian target of rapamycin (mTOR) pathway. These findings reveal that oxidative stress induces autophagic cell death and that H2S plays a neuroprotective role by reducing oxidative stress in SCIR.

Figures

Figure 1
Figure 1
SCIR injury induces neuronal cell apoptosis. Immunofluorescence analysis of TUNEL (a) and caspase-3 (b) in the spinal cord after I/R. This image shows results obtained from six rats with I/R. Scale bars represent 10 μm. (c) Western blot of cleaved caspase-3 in the spinal cord extracts from normal and I/R rats. (d) Densitometric analysis of the immunoblot reported in Figure 1(c). Samples from six normal and six I/R rats were pooled together. ∗∗∗P < 0.001. Data were analyzed using t-test and represent three independent experiments. SCIR: spinal cord ischemia reperfusion; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; I/R: ischemia reperfusion.
Figure 2
Figure 2
SCIR injury induces neuronal cell autophagy. (a) Western blots of p62, Atg12-Atg5, and LC3 in the spinal cord extracts from normal and I/R rats. Samples from six normal and six I/R rats were pooled together. (b) Densitometric analysis of the immunoblot reported in Figure 2(a). (c) Immunofluorescence analysis of LC3 in the spinal cord after I/R. This image represents six rats with I/R. Scale bar represent 10 μm. Arrows designate regions of 400x magnification shown in insets. ∗∗∗P < 0.001. Data were analyzed using one-way ANOVA. Data represent three independent experiments. SCIR: spinal cord ischemia reperfusion; I/R: ischemia reperfusion; LC3: microtubule-associated protein 1 light chain 3.
Figure 3
Figure 3
Autophagy promotes neuronal cell apoptosis in SCIR injury. (a) Immunofluorescence analysis of TUNEL and LC3 in the spinal cord after I/R treated with or without 3-MA. (b) Immunofluorescence analysis of caspase-3 in the spinal cord after I/R treated with or without 3-MA. (c) Western blot analysis of p62, Atg12-5, LC3-II, and cleaved caspase-3 after I/R injury treated with or without 3-MA. (d) Densitometric analysis of the immunoblot reported in Figure 3(c). (e) BBB scores of animals after SCIR treated with or without 3-MA. Images represent six rats with I/R treated with or without 3-MA. Scale bars represent 10 μm. ∗∗∗P < 0.001. Data were analyzed using one-way ANOVA in (d) and two-way ANOVA in (e) and represent three independent experiments. SCIR: spinal cord ischemia reperfusion; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; LC3: microtubule-associated protein 1 light chain 3; I/R: ischemia reperfusion; 3-MA: 3-methyladenine; BBB: Basso, Beattie, and Bresnahan; ANOVA: analysis of variance.
Figure 4
Figure 4
Oxidative stress induces neuronal autophagic cell death in SCIR injury. (a) Immunofluorescence analysis of ROS in the spinal cord after SCIR treated with or without NAC. (b) MDA concentration and (c) SOD activity in the spinal cord after I/R treated with or without NAC. (d) Immunofluorescence analysis of TUNEL and LC3 in the spinal cord after I/R treated with or without NAC. (e) Immunofluorescence analysis of caspase-3 in the spinal cord after I/R treated with or without NAC. (f) Western blot analysis of p62, Atg12-5, LC3-II, and cleaved caspase-3 after I/R injury treated with or without NAC. (g) Densitometric analysis of the immunoblot reported in Figure 4(f). (h) BBB scores of animals after SCIR treated with or without NAC. Images represent six rats with I/R treated with or without NAC. Scale bars represent 10 μm. ∗∗∗P < 0.001. Data were analyzed using one-way ANOVA in (b), (c), and (g) and two-way ANOVA in (h). Data represent three independent experiments. SCIR: spinal cord ischemia reperfusion; ROS: reactive oxygen species; NAC: N-acetyl-L-cysteine; MDA: malondialdehyde; SOD: superoxide dismutase; I/R: ischemia reperfusion; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; BBB: Basso, Beattie, and Bresnahan; ANOVA: analysis of variance.
Figure 5
Figure 5
H2S inhibits neuronal autophagic cell death after SCIR injury. (a) Immunofluorescence analysis of ROS in the spinal cord after I/R treated with or without H2S. (b) MDA concentration and (c) SOD activity in the spinal cord after I/R treated with or without H2S. (d) Immunofluorescence analysis of TUNEL and LC3 in the spinal cord after I/R treated with or without H2S. (e) Immunofluorescence analysis of caspase-3 in the spinal cord after I/R treated with or without H2S. (f) Western blot analysis of cleaved caspase-3, Bax, BLC2, LC3, Atg12-Atg5, p62, p-AKT, and p-mTOR in the spinal cord extracts from normal and I/R rats treated with or without H2S. (g) Densitometric analysis of the immunoblot reported in (f). (h) BBB scores of animals after SCIR treated with or without H2S. Images represent six rats with I/R treated with or without H2S. Scale bars represent 10 μm. ∗∗∗P < 0.001. Data were analyzed using one-way ANOVA in (b), (c), and (g) and two-way ANOVA in (h) and represent three independent experiments. H2S: hydrogen sulfide; SCIR: spinal cord ischemia reperfusion; ROS: reactive oxygen species; I/R: ischemia reperfusion; MDA: malondialdehyde; SOD: superoxide dismutase; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; LC3: microtubule-associated protein 1 light chain 3; BBB: Basso, Beattie, and Bresnahan; ANOVA: analysis of variance.
Figure 6
Figure 6
H2S inhibits neuronal autophagic cell death via the AKT-mTOR pathway. (a) Immunofluorescence analysis of TUNEL and LC3 in the spinal cord after I/R treated with or without Ly294002 or rapamycin. (b) Immunohistochemistry staining of Bax in the spinal cord after I/R treated with or without Ly294002 or rapamycin. (c) BBB scores of animals after SCIR treated with or without Ly284002 or rapamycin. (d) Western blots of cleaved caspase-3, Bax, BCL2, LC3, Atg12-Atg5, p62, P-AKT, and P-mTOR in spinal cord extracts from normal and I/R rats treated with or without H2S. (e) Densitometric analysis of the immunoblot reported in (d). Samples from six normal and six I/R rats were pooled together. Images represent six rats per group with different treatments. Scale bar represent 10 μm. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Data were analyzed using one-way ANOVA in (e) and two-way ANOVA in (c). H2S: hydrogen sulfide; LY: Ly294002; RA: rapamycin; mTOR: the mammalian target of rapamycin; LC3: microtubule-associated protein 1 light chain 3; I/R: ischemia reperfusion; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; BBB: Basso, Beattie, and Bresnahan; SCIR: spinal cord ischemia reperfusion; ANOVA: analysis of variance.

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References

    1. Zhang Q., Huang C., Meng B., Tang T., Shi Q., Yang H. Acute effect of ghrelin on ischemia/reperfusion injury in the rat spinal cord. International Journal of Molecular Sciences. 2012;13(8):9864–9876. doi: 10.3390/ijms13089864. - DOI - PMC - PubMed
    1. Fang B., Li X. Q., Bao N. R., et al. Role of autophagy in the bimodal stage after spinal cord ischemia reperfusion injury in rats. Neuroscience. 2016;328:107–116. doi: 10.1016/j.neuroscience.2016.04.019. - DOI - PubMed
    1. Bischoff M. S., Di Luozzo G., Griepp E. B., Griepp R. B. Spinal cord preservation in thoracoabdominal aneurysm repair. Perspectives in Vascular Surgery and Endovascular Therapy. 2011;23(3):214–222. doi: 10.1177/1531003511400622. - DOI - PubMed
    1. Jiang H., Xiao J., Kang B., Zhu X., Xin N., Wang Z. PI3K/SGK1/GSK3beta signaling pathway is involved in inhibition of autophagy in neonatal rat cardiomyocytes exposed to hypoxia/reoxygenation by hydrogen sulfide. Experimental Cell Research. 2016;345(2):134–140. doi: 10.1016/j.yexcr.2015.07.005. - DOI - PubMed
    1. Temiz C., Temiz C., Solmaz I., et al. The effects of splenectomy on lipid peroxidation and neuronal loss in experimental spinal cord ischemia/reperfusion injury. Turkish Neurosurgery. 2013;23(1):67–74. doi: 10.5137/1019-5149.JTN.6825-12.1. - DOI - PubMed

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