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SCIENCE CHINA Life Sciences, https://doi.org/10.1007/s11427-019-1761-1

Hydrogen selenide, a vital metabolite of sodium selenite, uncouples the sulfilimine bond and promotes the reversal of liver fibrosis

Dongrui Luan 1, Zengteng Zhao 1, Dandan Xia 2, Qiuling Zheng 2, Xiaonan Gao 1, Kehua Xu 1,*, Bo Tang 1
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  • ReceivedMar 23, 2020
  • AcceptedJun 20, 2020
  • PublishedSep 1, 2020
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Abstract

Sodium selenite has alleviating effects on liver fibrosis; however, its therapeutic molecular mechanism remains unclear. Herein, hydrogen selenide, a major metabolite of Na2SeO3, was tested to uncouple the sulfilimine bond in collagen IV, the biomarker of liver fibrosis. A mouse model of liver fibrosis was constructed via a CCl4-induced method, followed by the administration of0.2 mg kg−1 Na2SeO3 via gavage three times per week for 4 weeks. Changes in H2Se, NADPH, and H2O2 levels were monitored in real time by using NIR-H2Se, DCI-MQ-NADPH, and H2O2 probes in vivo, respectively. H2Se continuously accumulated in the liver throughout the Na2SeO3 treatment period, but the levels of NADPH and H2O2 decreased. The expression of collagen IV was analyzed through Western blot and liquid chromatography-mass spectrometry. Results confirmed that the sulfilimine bond of collagen IV in the fibrotic mouse livers could be broken by H2Se with the Na2SeO3 treatment. Therefore, the therapeutic effect of Na2SeO3 on liver fibrosis could be mainly attributed to H2Se that uncoupled the sulfilimine bond to induce collagen IV degradation. This study provided a reasonable explanation for the molecular mechanism of the in vivo function of Na2SeO3 and the prevention of liver fibrosis by administering inorganic selenium.


Funded by

the National Natural Science Foundation of China(21575081,21775091,21535004,91753111)

the Key Research and Development Program of Shandong Province(2018YFJH0502)


Acknowledgment

This work was supported by the National Natural Science Foundation of China (21575081, 21775091, 21535004 and 91753111) and the Key Research and Development Program of Shandong Province (2018YFJH0502). We thank Professor K.W. Michael Siu, University of Windsor, for valuable discussion and constructive suggestions.


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

The supporting information is available online at https://doi.org/10.1007/s11427-019-1761-1. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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    Figure 1

    Therapeutic effect of sodium selenite on mice with liver fibrosis during the early and middle stages of the disease. A, Schematic of the experimental design. (i) The control group (mice sacrificed on the 8th week); (ii) the fibrosis group (CCl4 induced for 8 weeks and mice sacrificed on the 8th week); (iii) the Na2SeO3 treatment group (CCl4 induced for 8 weeks, Na2SeO3 treated for 4 weeks, and mice sacrificed on the 12th week); (iv) another control group (mice sacrificed on the 12th week); and (v) another fibrosis group (CCl4 induced for 8 weeks and mice sacrificed on the 12th week). B, H&E staining of liver sections in groups (i) to (v). Black arrows indicate collagen proliferation, collagen bridge formation, and fibrosis. Red arrow shows liver cell degeneration and cytoplasmic vacuolation. C and D, Serum levels of ALT and AST. *, P<0.05 vs. the CCl4 group. Data are the mean±standard error of the mean (SEM), n=5.

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    Figure 2

    Sodium selenite downregulates the overexpression of collagen IV. A, IF staining of collagen IV in the liver of the control and fibrosis groups. B, WB assay of collagen IV expression in the control and fibrosis groups. C, WB assay of collagen IV expression in the control, fibrosis (CCl4), and Na2SeO3 treatment groups (CCl4+Na2SeO3). The relative levels of collagen IV were normalized with β-actin as the loading control. Data were shown as mean±SEM. *, P<0.05 vs. the control group and #, P<0.05 vs. the CCl4 group. All experiments were performed in triplicates.

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    Figure 3

    Hydrogen selenide accumulation in the fibrotic mouse liver. A, Representative in vivo near-infrared fluorescent imaging on the specific days when the fibrotic mice were treated with Na2SeO3 by gavage. After Na2SeO3 was administered by gavage for 2 h, the mice were orthotopically injected with the NIR-H2Se probe (10 μmol L−1) in the liver. Then, the fluorescence images were collected every 4 h (2, 6, 10, and 14 h). B, Graphs showing the normalized fluorescence intensities in (A) correspondingly. Data were mean±SEM; n=5.

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    Figure 4

    The accumulation of H2Se, NADPH and H2O2 in the mouse livers. A, In vivo fluorescence images of the mouse livers treated with Na2SeO3 on different days. The fluorescence intensities of H2Se, NADPH, and H2O2 in fibrotic mouse livers treated with Na2SeO3 were collected on specific days. The mice were orthotopically injected with the NIR-H2Se probe (10 μmol L−1), DCI-MQ-NADPH (10 μmol L−1), and the H2O2 probe (10 μmol L−1) 7 h after Na2SeO3 gavage. Then 1 h later the fluorescent signals were acquired, respectively. Mice were treated in the same conditions on all specific days. B, Normalized fluorescence intensities corresponding to (A). Data were mean±SEM; n=5.

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    Figure 5

    Western blot and mass spectrometric analysis of H2Se uncoupling the sulfilimine bond. A and B, WB analysis of NC1 proteins in different groups. Lane (a): 15 μg of pure NC1 protein; Lane (b): NC1 protein isolated from the control group (15 μg); Lane (c and e): NC1 protein isolated from the CCl4-induced group (15 μg for c, 20 μg for e); Lane (d and f): NC1 protein isolated from the Na2SeO3 treatment group (15 μg for d, 20 μg for f); Lane (g): NC1 protein isolated from the CCl4-induced group treated with H2Se (5 mmol L−1) at room temperature for 30–40 min (20 μg). C and D, Full-scan MS1 of α2-Met93- and α6-Hyl211-containing peptides. The m/z 998.38 (+3) ion, m/z 998.74 (+3) ion, m/z 999.05 (+3) ion, and m/z 999.40 (+3) ion correspond to the Met93-containing peptide derived from the α2 NC1 domain; the m/z 1,151.01 (+2) ion, m/z 1,151.52 (+2) ion, m/z 1,152.00 (+2) ion, m/z 1,152.51 (+2) ion, m/z 1,153.01 (+2) ion, and m/z 1,160.48 (+2) ion correspond to the α6-Hyl211-containing peptide derived from the a6 NC1 domain. The α2-Met93- and α6-Hyl211-containing peptides deviated from their expected theoretical peptide masses by −48 and +46 atomic mass units, respectively. The modifications referred to the olefin fragment derived from Met93 and a methylsulfenamide fragment derived from Hyl211 that resulted from the Cope elimination. E, Schematic of the uncoupling progression of NC1 hexamers by H2Se.