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. 2021 Feb 20:164:315-328.
doi: 10.1016/j.freeradbiomed.2020.12.449. Epub 2021 Jan 9.

Role of necroptosis in chronic hepatic inflammation and fibrosis in a mouse model of increased oxidative stress

Sabira Mohammed  1 , Evan H Nicklas  2 , Nidheesh Thadathil  2 , Ramasamy Selvarani  2 , Gordon H Royce  2 , Michael Kinter  3 , Arlan Richardson  4 , Sathyaseelan S Deepa  5
Affiliations

Role of necroptosis in chronic hepatic inflammation and fibrosis in a mouse model of increased oxidative stress

Sabira Mohammed et al. Free Radic Biol Med. .

Abstract

Mice deficient in the antioxidant enzyme Cu/Zn-superoxide dismutase (Sod1-/- or Sod1KO mice) have increased oxidative stress, show accelerated aging and develop spontaneous hepatocellular carcinoma (HCC) with age. Similar to humans, HCC development in Sod1KO mice progresses from non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH) with fibrosis, which eventually progresses to HCC. Oxidative stress plays a role in NAFLD to NASH progression, and liver inflammation is the main mechanism that drives the disease progression from NASH to fibrosis. Because necroptosis is a major source of inflammation, we tested the hypothesis that increased necroptosis in the liver plays a role in increased inflammation and fibrosis in Sod1KO mice. Phosphorylation of MLKL (P-MLKL), a well-accepted marker of necroptosis, and expression of MLKL protein were significantly increased in the livers of Sod1KO mice compared to wild type (WT) mice indicating increased necroptosis. Similarly, phosphorylation of RIPK3 and RIPK3 protein levels were also significantly increased. Markers of pro-inflammatory M1 macrophages, NLRP3 inflammasome, and transcript levels of pro-inflammatory cytokines and chemokines, e.g., TNFα, IL-6, IL-1β, and Ccl2 that are associated with human NASH, were significantly increased. Expression of antioxidant enzymes and heat shock proteins, and markers of fibrosis and oncogenic transcription factor STAT3 were also upregulated and autophagy was downregulated in the livers of Sod1KO mice. Short term treatment of Sod1KO mice with necrostatin-1s (Nec-1s), a necroptosis inhibitor, reversed these conditions. Our data show for the first time that necroptosis-mediated inflammation contributes to fibrosis in a mouse model of increased oxidative stress and accelerated aging, that also exhibits progressive HCC development.

Keywords: Autophagy; Cu/Zn superoxide dismutase; Fibrosis; Hepatocellular carcinoma; Inflammation; Necroptosis; Necrostatin-1s; Non-alcoholic fatty liver disease; Non-alcoholic hepatosteatosis; Oxidative stress.

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Conflict of interest statement

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.. Effect of Sod1 deficiency on necroptosis.
(A) Left panel: Immunoblots of liver extracts prepared from WT (blue bars), Sod1KO untreated (red bars) and Sod1KO mice treated with Nec-1s (green bars) for P-MLKL, MLKL and β-tubulin. Right panel: Graphical representation of quantified blots normalized to β-tubulin. (B) Left panel: Immunoblots of liver extracts for P-RIPK3, RIPK3, and β-tubulin. Right panel: Graphical representation of quantified blots normalized to β-tubulin. (C) Transcript levels for MLKL and RIPK3. (D) Left panel: Immunoblots of liver extracts for cleaved caspase-3 and total caspase-3. Right panel: Graphical representation of quantified blots normalized to total caspase-3. (E) Left panel: Immunoblots of liver extracts for LC3-I, LC3-II, p62 and β-actin. Right panel: Graphical representation of quantified blots normalized to β-actin. Data were obtained from 5 to 7 mice per group and are expressed as the mean ± SEM. (ANOVA, *WT-Veh vs Sod1KO-Veh; #WT-Veh vs Sod1KO-Nec-1s; ^ Sod1KO-Veh vs Sod1KO-Nec-1s; */#/^ P ≤ 0.05).
Figure 2.
Figure 2.. Effect of Sod1 deficiency and necroptosis on inflammation.
Transcript levels of F4/80 (A), and proinflammatory M1 macrophage markers CD68,CD86, and TLR4 (B) in the livers of WT (blue bars), Sod1KO untreated (red bars) and Sod1KO mice treated with Nec-1s (green bars). (C) Left panel: Immunoblots of liver extracts for phospho-NF-κB (p65), NF-κB and β-actin. Right panel: Graphical representation of quantified blots normalized to β-actin. (D) Left panel: Immunoblots of liver extracts for NLRP3 and β-tubulin. Right panel: Graphical representation of quantified blots normalized to β-tubulin. (E) Transcript levels of NLRP3. Data were obtained from 5 to 7 mice per group and are expressed as the mean ± SEM. (ANOVA, *WT-Veh vs Sod1KO-Veh; #WT-Veh vs Sod1KO-Nec-1s; ^ Sod1KO-Veh vs Sod1KO-Nec-1s; */#/^ P ≤ 0.05).
Figure 3.
Figure 3.. Effect of Sod1 deficiency and necroptosis on proinflammatory cytokines and chemokines.
Transcript levels of proinflammatory cytokines and chemokines (A), interleukins (B), and members of TNFα family (C) in the livers of WT (blue bars), Sod1KO untreated (red bars) and Sod1KO mice treated with Nec-1s (green bars). Data were obtained from 5 to 7 mice per group and are expressed as the mean ± SEM. (ANOVA, *WT-Veh vs Sod1KO-Veh;#WT-Veh vs Sod1KO-Nec-1s; ^ Sod1KO-Veh vs Sod1KO-Nec-1s; */#/^ P ≤ 0.05).
Figure 4.
Figure 4.. Effect of Sod1 deficiency and necroptosis on glycolytic pathway.
The relative expression of glycolytic enzymes in the livers Sod1KO mice (red bars) and Sod1KO mice treated with Nec-1s (green bars), compared to WT mice, as assessed by targeted quantitative proteomics. Expression of proteins in WT mice is taken as 100%. The values are normalized to internal standards and housekeeping proteins. (ANOVA, *WT-Veh vs Sod1KO-Veh; #WT-Veh vs Sod1KO-Nec-1s; ^ Sod1KO-Veh vs Sod1KO-Nec-1s; */#/^ P ≤ 0.05).
Figure 5.
Figure 5.. Effect of Sod1 deficiency and necroptosis on lipid metabolism.
Transcript levels of proteins involved lipid metabolism and transport (A) and cholesterol metabolism and transport (B) in the livers Sod1KO mice (red bars) and Sod1KO mice treated with Nec-1s (green bars), compared to WT mice. (C) Protein expression of as assessed by targeted quantitative proteomics. (D) Left panel: Immunoblots of liver extracts for SREBP1, SREBP2, P-ACC, ACC, and β-tubulin. Right panel: Graphical representation of SREBP1 and SREBP2 blots normalized to β-tubulin and P-ACC normalized to ACC. (ANOVA, *WT-Veh vs Sod1KO-Veh; #WT-Veh vs Sod1KO-Nec-1s; ^ Sod1KO-Veh vs Sod1KO-Nec-1s; */#/^ P ≤ 0.05).
Figure 6.
Figure 6.. Effect of Sod1 deficiency and necroptosis on antioxidant proteins and stress response.
The relative expression of antioxidant enzymes (A), Lon protease 1 (B), and stress-related proteins (C) in the livers of Sod1KO mice (red bars) and Sod1KO mice treated with Nec-1s (green bars), compared to WT mice, as assessed by targeted quantitative proteomics. Expression of these proteins in WT mice is taken as 100%. The values are normalized to internal standards and housekeeping proteins. (D) Left panel: Immunoblots of liver extracts for 4-HNE. Right panel: Graphical representation of quantified blots normalized to total protein in Coomassie stained gel. Data were obtained from 5 to 6 mice per group and are expressed as the mean ± SEM. (ANOVA, *WT-Veh vs Sod1KO-Veh; #WT-Veh vs Sod1KO-Veh; ^ Sod1KO-Veh vs Sod1KO-Nec-1s; */#/^ P ≤ 0.05).
Figure 7.
Figure 7.. Effect of Sod1 deficiency and necroptosis on markers of fibrosis.
(A) Transcript levels of pro-fibrotic cytokines TGFβ, CTGF, PDGFα, and PDGFβ in the livers of WT (blue bars), Sod1KO untreated (red bars) and Sod1KO mice treated with Nec-1s (green bars). (B) Top panel: Immunoblots of liver extracts for TGFβ and β-tubulin. Bottom panel: Graphical representation of quantified blots normalized to β-tubulin. (C) Left panel: Immunoblots of liver extracts for desmin and β-tubulin. Right panel: Graphical representation of quantified blots normalized to β-tubulin. (D) Transcript levels of Col1α2 and Col3α1. (E) Transcript levels of MMPs and TIMPs. (F) ALT activity as measured in the serum of WT (blue bars), Sod1KO (red bars) and Sod1KO mice treated with Nec-1s (green bars). (G) Left panel: Immunoblots of liver extracts prepared from WT (blue bars), Sod1KO untreated (red bars) and Sod1KO mice treated with Nec-1s (green bars) for P-STAT3 and STAT3. Right panel: Graphical representation of quantified phospho-blots normalized to total protein. Data were obtained from 5 to 7 mice per group and are expressed as the mean ± SEM. (ANOVA, *WT-Veh vs Sod1KO-Veh; #WT-Veh vs Sod1KO- Nec-1s; ^ Sod1KO-Veh vs Sod1KO-Nec-1s; */#/^ P ≤ 0.05).
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References

    1. Marcellin P, Kutala BK, Liver diseases: A major, neglected global public health problem requiring urgent actions and large-scale screening., Liver Int. Off. J. Int. Assoc. Study Liver. 38 Suppl 1 (2018) 2–6. 10.1111/liv.13682. - DOI - PubMed
    1. Byass P, The global burden of liver disease: a challenge for methods and for public health., BMC Med. 12 (2014) 159. 10.1186/s12916-014-0159-5. - DOI - PMC - PubMed
    1. Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M, Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes., Hepatology. 64 (2016) 73–84. 10.1002/hep.28431. - DOI - PubMed
    1. Marengo A, Rosso C, Bugianesi E, Liver Cancer: Connections with Obesity, Fatty Liver, and Cirrhosis., Annu. Rev. Med. 67 (2016) 103–117. 10.1146/annurev-med-090514-013832. - DOI - PubMed
    1. Goh GB-B, McCullough AJ, Natural History of Nonalcoholic Fatty Liver Disease., Dig. Dis. Sci. 61 (2016) 1226–1233. 10.1007/s10620-016-4095-4. - DOI - PMC - PubMed

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