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

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  • ReceivedMar 23, 2020
  • AcceptedJun 20, 2020
  • PublishedSep 1, 2020


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)


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.



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.


[1] Ahmed Z., Ahmed U., Walayat S., Ren J., Martin D.K., Moole H., Koppe S., Yong S., Dhillon S.. Liver function tests in identifying patients with liver disease. Clin Exp Gastroenterol, 2018, 11: 301-307 CrossRef PubMed Google Scholar

[2] Andueza A., Garde N., García-Garzón A., Ansorena E., López-Zabalza M.J., Iraburu M.J., Zalba G., Martínez-Irujo J.J.. NADPH oxidase 5 promotes proliferation and fibrosis in human hepatic stellate cells. Free Radic Biol Med, 2018, 126: 15-26 CrossRef PubMed Google Scholar

[3] Bataller R., Brenner D.A.. Liver fibrosis. J Clin Invest, 2005, 115: 209-218 CrossRef PubMed Google Scholar

[4] Bhave G., Cummings C.F., Vanacore R.M., Kumagai-Cresse C., Ero-Tolliver I.A., Rafi M., Kang J.S., Pedchenko V., Fessler L.I., Fessler J.H., et al. Peroxidasin forms sulfilimine chemical bonds using hypohalous acids in tissue genesis. Nat Chem Biol, 2012, 8: 784-790 CrossRef PubMed Google Scholar

[5] Brewer A.C., Mustafi S.B., Murray T.V.A., Rajasekaran N.S., Benjamin I.J.. Reductive stress linked to small HSPs, G6PD, and Nrf2 pathways in heart disease. Antioxid Redox Signal, 2013, 18: 1114-1127 CrossRef PubMed Google Scholar

[6] Brigelius-Flohé R.. Selenium compounds and selenoproteins in cancer. Chem Biodivers, 2008, 5: 389-395 CrossRef PubMed Google Scholar

[7] Casino P., Gozalbo-Rovira R., Rodríguez-Díaz J., Banerjee S., Boutaud A., Rubio V., Hudson B.G., Saus J., Cervera J., Marina A.. Structures of collagen IV globular domains: insight into associated pathologies, folding and network assembly. IUCrJ, 2018, 5: 765-779 CrossRef PubMed Google Scholar

[8] Chen W., Rock J.B., Yearsley M.M., Ferrell L.D., Frankel W.L.. Different collagen types show distinct rates of increase from early to late stages of hepatitis C-related liver fibrosis. Hum Pathol, 2014, 45: 160-165 CrossRef PubMed Google Scholar

[9] Clement B., Grimaud J.A., Campion J.P., Deugnier Y., Guillouzo A.. Cell types involved in collagen and fibronectin production in normal and fibrotic human liver. Hepatology, 1986, 6: 225-234 CrossRef PubMed Google Scholar

[10] Cosgrove D., Liu S.. Collagen IV diseases: a focus on the glomerular basement membrane in Alport syndrome. Matrix Biol, 2017, 57-58: 45-54 CrossRef PubMed Google Scholar

[11] Crosas-Molist E., Fabregat I.. Role of NADPH oxidases in the redox biology of liver fibrosis. Redox Biol, 2015, 6: 106-111 CrossRef PubMed Google Scholar

[12] Dimmeler S., Zeiher A.M.. A “reductionist” view of cardiomyopathy. Cell, 2007, 130: 401-402 CrossRef PubMed Google Scholar

[13] Duarte S., Baber J., Fujii T., Coito A.J.. Matrix metalloproteinases in liver injury, repair and fibrosis. Matrix Biol, 2015, 44: 147-156 CrossRef PubMed Google Scholar

[14] Fidler A.L., Darris C.E., Chetyrkin S.V., Pedchenko V.K., Boudko S.P., Brown K.L., Gray Jerome W., Hudson J.K., Rokas A., Hudson B.G.. Collagen IV and basement membrane at the evolutionary dawn of metazoan tissues. eLife, 2017, 6: e24176 CrossRef PubMed Google Scholar

[15] Hahn E., Wick G., Pencev D., Timpl R.. Distribution of basement membrane proteins in normal and fibrotic human liver: collagen type IV, laminin, and fibronectin. Gut, 1980, 21: 63-71 CrossRef PubMed Google Scholar

[16] Hernandez-Gea V., Friedman S.L.. Pathogenesis of liver fibrosis. Annu Rev Pathol, 2011, 6: 425-456 CrossRef PubMed Google Scholar

[17] Kieliszek M., Lipinski B., Błażejak S.. Application of sodium selenite in the prevention and treatment of cancers. Cells, 2017, 6: 39 CrossRef PubMed Google Scholar

[18] Kisseleva T.. The origin of fibrogenic myofibroblasts in fibrotic liver. Hepatology, 2017, 65: 1039-1043 CrossRef PubMed Google Scholar

[19] Kong F., Ge L., Pan X., Xu K., Liu X., Tang B.. A highly selective near-infrared fluorescent probe for imaging H2Se in living cells and in vivo. Chem Sci, 2016, 7: 1051-1056 CrossRef PubMed Google Scholar

[20] Liu S.B., Ikenaga N., Peng Z.W., Sverdlov D.Y., Greenstein A., Smith V., Schuppan D., Popov Y.. Lysyl oxidase activity contributes to collagen stabilization during liver fibrosis progression and limits spontaneous fibrosis reversal in mice. FASEB J, 2015, 30: 1599-1609 CrossRef PubMed Google Scholar

[21] Liu W., Hou T., Shi W., Guo D., He H.. Hepatoprotective effects of selenium-biofortified soybean peptides on liver fibrosis induced by tetrachloromethane. J Funct Foods, 2018a, 50: 183-191 CrossRef Google Scholar

[22] Liu Y., Liu Q., Hesketh J., Huang D., Gan F., Hao S., Tang S., Guo Y., Huang K.. Protective effects of selenium-glutathione-enriched probiotics on CCl4-induced liver fibrosis. J Nutr Biochem, 2018b, 58: 138-149 CrossRef PubMed Google Scholar

[23] Liu Y., Liu Q., Ye G., Khan A., Liu J., Gan F., Zhang X., Kumbhar S., Huang K.. Protective effects of selenium-enriched probiotics on carbon tetrachloride-induced liver fibrosis in rats. J Agric Food Chem, 2014, 63: 242-249 CrossRef PubMed Google Scholar

[24] Lopez-Sanchez I., Dunkel Y., Roh Y.S., Mittal Y., De Minicis S., Muranyi A., Singh S., Shanmugam K., Aroonsakool N., Murray F., et al. GIV/Girdin is a central hub for profibrogenic signalling networks during liver fibrosis. Nat Commun, 2014, 5: 4451 CrossRef PubMed ADS Google Scholar

[25] Luan D., Gao X., Kong F., Song X., Zheng A., Liu X., Xu K., Tang B.. Cyclic regulation of the sulfilimine bond in peptides and NC1 hexamers via the HOBr/H2Se conjugated system. Anal Chem, 2018, 90: 9523-9528 CrossRef PubMed Google Scholar

[26] Mak K.M., Chen L.L., Lee T.F.. Codistribution of collagen type IV and laminin in liver fibrosis of elderly cadavers: immunohistochemical marker of perisinusoidal basement membrane formation. Anat Rec, 2013, 296: 953-964 CrossRef PubMed Google Scholar

[27] Mak K.M., Mei R.. Basement membrane type IV collagen and laminin: An overview of their biology and value as fibrosis biomarkers of liver disease. Anat Rec, 2017, 300: 1371-1390 CrossRef PubMed Google Scholar

[28] Martin K., Pritchett J., Llewellyn J., Mullan A.F., Athwal V.S., Dobie R., Harvey E., Zeef L., Farrow S., Streuli C., et al. PAK proteins and YAP-1 signalling downstream of integrin beta-1 in myofibroblasts promote liver fibrosis. Nat Commun, 2016, 7: 12502 CrossRef PubMed ADS Google Scholar

[29] Mas M.R., Comert B., Oncu K., Vural S.A., Akay C., Tasci I., Ozkomur E., Serdar M., Mas N., Alcigir G., et al. The effect of taurine treatment on oxidative stress in experimental liver fibrosis. Hepatol Res, 2004, 28: 207-215 CrossRef PubMed Google Scholar

[30] McCall A.S., Cummings C.F., Bhave G., Vanacore R., Page-McCaw A., Hudson B.G.. Bromine is an essential trace element for assembly of collagen IV scaffolds in tissue development and architecture. Cell, 2014, 157: 1380-1392 CrossRef PubMed Google Scholar

[31] Metes-Kosik N., Luptak I., Dibello P.M., Handy D.E., Tang S.S., Zhi H., Qin F., Jacobsen D.W., Loscalzo J., Joseph J.. Both selenium deficiency and modest selenium supplementation lead to myocardial fibrosis in mice via effects on redox-methylation balance. Mol Nutr Food Res, 2012, 56: 1812-1824 CrossRef PubMed Google Scholar

[32] Miyaki E., Imamura M., Hiraga N., Murakami E., Kawaoka T., Tsuge M., Hiramatsu A., Kawakami Y., Aikata H., Hayes C.N., et al. Daclatasvir and asunaprevir treatment improves liver function parameters and reduces liver fibrosis markers in chronic hepatitis C patients. Hepatol Res, 2016, 46: 758-764 CrossRef PubMed Google Scholar

[33] Muriel P.. Role of free radicals in liver diseases. Hepatol Int, 2009, 3: 526-536 CrossRef PubMed Google Scholar

[34] Niemelä O., Risteli L., Sotaniemi E.A., Risteli J.. Type IV collagen and laminin-related antigens in human serum in alcoholic liver disease. Eur J Clin Invest, 1985, 15: 132-137 CrossRef PubMed Google Scholar

[35] Paik Y.H., Kim J., Aoyama T., De Minicis S., Bataller R., Brenner D.A.. Role of NADPH oxidases in liver fibrosis. Antioxid Redox Signal, 2014, 20: 2854-2872 CrossRef PubMed Google Scholar

[36] Pan X., Wang X., Wang L., Xu K., Kong F., Tang B.. Near-infrared fluorescence probe for monitoring the metabolic products of vitamin C in HepG2 cells under normoxia and hypoxia. Anal Chem, 2015, 87: 7092-7097 CrossRef PubMed Google Scholar

[37] Pan X., Zhao Y., Cheng T., Zheng A., Ge A., Zang L., Xu K., Tang B.. Monitoring NAD(P)H by an ultrasensitive fluorescent probe to reveal reductive stress induced by natural antioxidants in HepG2 cells under hypoxia. Chem Sci, 2019, 10: 8179-8186 CrossRef PubMed Google Scholar

[38] Parola M., Robino G.. Oxidative stress-related molecules and liver fibrosis. J Hepatol, 2001, 35: 297-306 CrossRef Google Scholar

[39] Pedchenko V., Kitching A.R., Hudson B.G.. Goodpasture’s autoimmune disease—A collagen IV disorder. Matrix Biol, 2018, 71: 240-249 CrossRef PubMed Google Scholar

[40] Popov Y., Sverdlov D.Y., Sharma A.K., Bhaskar K.R., Li S., Freitag T.L., Lee J., Dieterich W., Melino G., Schuppan D.. Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice. Gastroenterology, 2011, 140: 1642-1652 CrossRef PubMed Google Scholar

[41] Rajasekaran N.S., Connell P., Christians E.S., Yan L.J., Taylor R.P., Orosz A., Zhang X.Q., Stevenson T.J., Peshock R.M., Leopold J.A., et al. Human αB-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in mice. Cell, 2007, 130: 427-439 CrossRef PubMed Google Scholar

[42] Richter K., Konzack A., Pihlajaniemi T., Heljasvaara R., Kietzmann T.. Redox-fibrosis: Impact of TGFβ1 on ROS generators, mediators and functional consequences. Redox Biol, 2015, 6: 344-352 CrossRef PubMed Google Scholar

[43] Robertson W.E., Rose K.L., Hudson B.G., Vanacore R.M.. Supramolecular organization of the α121-α565 collagen IV network. J Biol Chem, 2014, 289: 25601-25610 CrossRef PubMed Google Scholar

[44] Rojkind M., Giambrone M.A., Biempica L.. Collagen types in normal and cirrhotic liver. Gastroenterology, 1979, 76: 710-719 CrossRef Google Scholar

[45] Schuppan D.. Structure of the extracellular matrix in normal and fibrotic liver: collagens and glycoproteins. Semin Liver Dis, 1990, 10: 1-10 CrossRef PubMed Google Scholar

[46] Schuppan D., Besser M., Schwarting R., Hahn E.G.. Radioimmunoassay for the carboxy-terminal cross-linking domain of type IV (basement membrane) procollagen in body fluids. Characterization and application to collagen type IV metabolism in fibrotic liver disease. J Clin Invest, 1986, 78: 241-248 CrossRef PubMed Google Scholar

[47] Seyer J.M., Hutcheson E.T., Kang A.H.. Collagen polymorphism in normal and cirrhotic human liver. J Clin Invest, 1977, 59: 241-248 CrossRef PubMed Google Scholar

[48] Su S., Zhao Q., He C., Huang D., Liu J., Chen F., Chen J., Liao J.Y., Cui X., Zeng Y., et al. miR-142-5p and miR-130a-3p are regulated by IL-4 and IL-13 and control profibrogenic macrophage program. Nat Commun, 2015, 6: 8523 CrossRef PubMed ADS Google Scholar

[49] Svegliati S., Spadoni T., Moroncini G., Gabrielli A.. NADPH oxidase, oxidative stress and fibrosis in systemic sclerosis. Free Radical Biol Med, 2018, 125: 90-97 CrossRef PubMed Google Scholar

[50] Tacke F., Trautwein C.. Mechanisms of liver fibrosis resolution. J Hepatol, 2015, 63: 1038-1039 CrossRef PubMed Google Scholar

[51] Than M.E., Henrich S., Huber R., Ries A., Mann K., Kühn K., Timpl R., Bourenkov G.P., Bartunik H.D., Bode W.. The 1.9-angstrom crystal structure of the noncollagenous (NC1) domain of human placenta collagen IV shows stabilization via a novel type of covalent Met-Lys cross-link. Proc Natl Acad Sci USA, 2002, 99: 6607-6612 CrossRef PubMed ADS Google Scholar

[52] Vanacore R., Ham A.J.L., Voehler M., Sanders C.R., Conrads T.P., Veenstra T.D., Sharpless K.B., Dawson P.E., Hudson B.G.. A sulfilimine bond identified in collagen IV. Science, 2009, 325: 1230-1234 CrossRef PubMed ADS Google Scholar

[53] Vanacore R.M., Ham A.J.L., Cartailler J.P., Sundaramoorthy M., Todd P., Pedchenko V., Sado Y., Borza D.B., Hudson B.G.. A role for collagen IV cross-links in conferring immune privilege to the goodpasture autoantigen. J Biol Chem, 2008, 283: 22737-22748 CrossRef PubMed Google Scholar

[54] Vanacore R.M., Shanmugasundararaj S., Friedman D.B., Bondar O., Hudson B.G., Sundaramoorthy M.. The α1.α2 network of collagen IV. J Biol Chem, 2004, 279: 44723-44730 CrossRef PubMed Google Scholar

[55] Veidal S.S., Karsdal M.A., Nawrocki A., Larsen M.R., Dai Y., Zheng Q., Hägglund P., Vainer B., Skjøt-Arkil H., Leeming D.J.. Assessment of proteolytic degradation of the basement membrane: a fragment of type IV collagen as a biochemical marker for liver fibrosis. Fibrog Tissue Repair, 2011, 4: 22 CrossRef PubMed Google Scholar

[56] Weekley C.M., Harris H.H.. Which form is that? The importance of selenium speciation and metabolism in the prevention and treatment of disease. Chem Soc Rev, 2013, 42: 8870-8894 CrossRef PubMed Google Scholar

[57] Wells R.G.. Cellular sources of extracellular matrix in hepatic fibrosis. Clin Liver Dis, 2008, 12: 759-768 CrossRef PubMed Google Scholar

[58] Wu D., Chen L., Kwon N., Yoon J.. Fluorescent probes containing selenium as a guest or host. Chem, 2016, 1: 674-698 CrossRef Google Scholar

[59] Zeisberg M., Kramer K., Sindhi N., Sarkar P., Upton M., Kalluri R.. De-differentiation of primary human hepatocytes depends on the composition of specialized liver basement membrane. Mol Cell Biochem, 2006, 283: 181-189 CrossRef PubMed Google Scholar

[60] Zhao Y., Wei K., Kong F., Gao X., Xu K., Tang B.. Dicyanoisophorone-based near-infrared-emission fluorescent probe for detecting NAD(P)H in living cells and in vivo. Anal Chem, 2018, 91: 1368-1374 CrossRef PubMed Google Scholar

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.