Skip to main content

Advertisement

SpringerLink
Log in

Deletion of Mixed Lineage Kinase Domain Like Pseudokinase Aggravates Chronic Alcohol-Induced Liver Injury via Increasing Apoptosis

  • Original Article
  • Published:
Digestive Diseases and Sciences Aims and scope Submit manuscript

Abstract

Background and Aim

he mixed lineage kinase domain like pseudokinase (MLKL) is known to play a protective role in non-alcoholic fatty liver disease (NAFLD) via inhibition of necroptosis pathway. However, the role of MLKL in alcoholic liver disease (ALD) is not yet clear.

Method

C57BL/6N wild-type (WT) and MLKL-knockout (KO) mice (8–10 weeks old) were randomly divided into eight groups. To establish ALD model of different durations, ethanol (EtOH) was fed to WT and MLKL KO for 10 days, 4 weeks, and 8 weeks. The control group was fed with Lieber-DeCarli control diet for 8 weeks. Mortality, degree of hepatic inflammation, and steatosis were compared among the groups. Bulk mRNA transcriptome analysis was performed. Abundance of transcript and gene expressions were calculated based on read count or Transcript by Million (TPM) value.

Results

Survival rate of MLKL KO mice compared to WT was similar until 4 weeks, but the survival of MLKL KO mice significantly decreased after 8 weeks in ALD model. There was no difference in degree of inflammation, steatosis, and NAS scores between EtOH-fed MLKL KO and EtOH-fed WT mice at 10 days. However, at 4 weeks and 8 weeks, the degree of hepatic steatosis, NAS, and inflammation were increased in MLKL KO mice. RNA transcriptome data showed that fatty acid synthesis, and lipogenesis, mitochondria, and apoptosis-related pathways were upregulated in EtOH-fed MLKL KO mice compared to EtOH-fed WT mice. Although hepatocyte apoptosis (BAX/BCL2 ratio, caspase-3, and TUNEL staining) increased after EtOH intake; however, apoptosis was more significantly increased in EtOH-fed MLKL KO mice compared to the WT group. At the same time, hepatic cFLIP was decreased in EtOH-fed MLKL KO mice compared to the WT group.

Conclusion

MLKL deletion did not prevent chronic alcohol-induced liver damage independently of necroptosis and exacerbated hepatic steatosis by increasing hepatocyte apoptosis.

Graphical Abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data availability

Data available on request due to privacy/ethical restrictions.

Abbreviations

ALD:

Alcoholic Liver Disease

AST:

Aspartate Aminotransferase

ALT:

Alanine Aminotransferase

BAX:

Bcl-2-associated X protein

BCL2:

B-Cell Leukemia/Lymphoma Protein-2

CXCL1:

CXC Motif Chemokine Ligand-1

CXCL2:

CXC Motif Chemokine Ligand-2

c-FLIP:

Cellular FLICE Inhibitory Protein

EtOH:

Ethanol/Alcohol

FAS:

Fatty Acid Synthase

GAPDH:

Glyceraldehyde 3-phosphate Dehydrogenase

JNK:

C-Jun N-Terminal Kinase

p-JNK:

Phosphorylated JNK

RIPK1:

Receptor-interacting Protein Kinase 1

RIPK3:

Receptor-interacting Protein Kinase 3

MLKL:

Mixed-Lineage Kinase Domain Like Pseudokinase

NAFLD:

Non-alcoholic Liver Disease

NAS:

Non-Alcoholic Fatty Liver Disease Activity Score

Ppar-γ:

Peroxisome Proliferator Activated Receptor Gamma

SCD-1:

Stearoyl-Coenzyme A Desaturase-1

SerpinA12:

Serpin Peptidase Inhibitor, Clade A Member 12

SREBP1c:

Sterol Regulatory Element-binding Transcription Factor 1

TG:

Triglyceride

TLR4:

Toll-Like Receptor-4

TLR9:

Toll-Like Receptor-9

TUNEL:

Terminal deoxynucleotidyl Transferase dUTP Nick End Labeling

WT:

Wild Type

References

  1. Zhan C, Huang M, Yang X, Hou J. MLKL: functions beyond serving as the executioner of necroptosis. Theranostics 2021;11:4759–4769.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Oh JH, Saeed WK, Kim HY et al. Hepatic stellate cells activate and avoid death under necroptosis stimuli: hepatic fibrosis during necroptosis. J Gastroenterol Hepatol 2023;38:2206–2214.

    Article  CAS  PubMed  Google Scholar 

  3. Oh JH, Park S, Hong E et al. Novel inhibitor of mixed-lineage kinase domain-like protein the antifibrotic effects of a necroptosis antagonist. ACS Pharmacol Transl Sci 2023;6:1471–1479.

    Article  CAS  PubMed  Google Scholar 

  4. Zhou Y, Wu R, Wang X, Bao X, Lu C. Roles of necroptosis in alcoholic liver disease and hepatic pathogenesis. Cell Prolif 2022;55:e13193.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Afonso MB, Rodrigues PM, Mateus-Pinheiro M et al. RIPK3 acts as a lipid metabolism regulator contributing to inflammation and carcinogenesis in non-alcoholic fatty liver disease. Gut 2021;70:2359–2372.

    Article  CAS  PubMed  Google Scholar 

  6. Saeed WK, Jun DW, Jang K et al. Decrease in fat de novo synthesis and chemokine ligand expression in non-alcoholic fatty liver disease caused by inhibition of mixed lineage kinase domain-like pseudokinase. J Gastroenterol Hepatol 2019;34:2206–2218.

    Article  CAS  PubMed  Google Scholar 

  7. Wu X, Poulsen KL, Sanz-Garcia C et al. MLKL-dependent signaling regulates autophagic flux in a murine model of non-alcohol-associated fatty liver and steatohepatitis. J Hepatol 2020;73:616–627.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Tonon M, Piano S. Alcohol-related cirrhosis: the most challenging etiology of cirrhosis is more burdensome than ever. Clin Mol Hepatol 2021;27:94–96.

    Article  PubMed  Google Scholar 

  9. Saeed WK, Jun DW, Jang K, Koh DH. Necroptosis signaling in liver diseases: an update. Pharmacol Res 2019;148:104439.

    Article  CAS  PubMed  Google Scholar 

  10. Saeed WK, Jun DW, Jang K et al. Mismatched effects of receptor interacting protein kinase-3 on hepatic steatosis and inflammation in non-alcoholic fatty liver disease. World J Gastroenterol 2018;24:5477–5490.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhang Z, Xie G, Liang L et al. RIPK3-mediated necroptosis and neutrophil infiltration are associated with poor prognosis in patients with alcoholic cirrhosis. J Immunol Res 2018;2018:1509851.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Miyata T, Wu X, Fan X et al. Differential role of MLKL in alcohol-associated and non-alcohol-associated fatty liver diseases in mice and humans. JCI Insight. 2021. https://doi.org/10.1172/jci.insight.140180.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Roychowdhury S, McMullen MR, Pisano SG, Liu X, Nagy LE. Absence of receptor interacting protein kinase 3 prevents ethanol-induced liver injury. Hepatology 2013;57:1773–1783.

    Article  CAS  PubMed  Google Scholar 

  14. Chen H, McKeen T, Chao X et al. The role of MLKL in Hepatic ischemia-reperfusion injury of alcoholic steatotic livers. Int J Biol Sci 2022;18:1096–1106.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wu X, Fan X, McMullen MR et al. Macrophage-derived MLKL in alcohol-associated liver disease: regulation of phagocytosis. Hepatology. 2022. https://doi.org/10.1016/S0168-8278(22)00478-0.

    Article  PubMed  Google Scholar 

  16. Miyata T, Nagy LE. Programmed cell death in alcohol-associated liver disease. Clin Mol Hepatol 2020;26:618–625.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Wu J, Huang Z, Ren J, Zhang Z, He P, Li Y, Ma J et al. Mlkl knockout mice demonstrate the indispensable role of Mlkl in necroptosis. Cell Res 2013;23:994–1006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhu Q, Han X, Peng J, Qin H, Wang Y. The role of CXC chemokines and their receptors in the progression and treatment of tumors. J Mol Histol 2012;43:699–713.

    Article  CAS  PubMed  Google Scholar 

  19. Wang K. Molecular mechanisms of hepatic apoptosis regulated by nuclear factors. Cell Signal 2015;27:729–738.

    Article  CAS  PubMed  Google Scholar 

  20. Suda J, Dara L, Yang L, Aghajan M, Song Y, Kaplowitz N, Liu ZX. Knockdown of RIPK1 markedly exacerbates murine immune-mediated liver injury through massive apoptosis of hepatocytes, independent of necroptosis and inhibition of NF-kappaB. J Immunol 2016;197:3120–3129.

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This research was supported by Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (RS-2021-DD121073, Republic of Korea). And This study was supported by The Research Supporting Program of The Korean Association for the Study of the Liver (KASL2019-04).

Author information

Author notes

  1. Keon Hwi Im and Waqar Khalid Saeed are equally contributed as co-first authors.

  2. Dae Won Jun and Eileen L. Yoon have equally contributed to this work.

Authors and Affiliations

  1. Department of Translational Medicine, Graduate School of Biomedical Science and Engineering, Hanyang University, 222 Wangsimni-Ro, Seongdong-Gu, Seoul, Republic of Korea

    Keon Hwi Im, A. Hyeon Lee, Ji Eun Kim, Seung Min Lee & Dae Won Jun

  2. Department of Biomedical Sciences, Pak-Austria Fachhochschule: Institute of Applied Sciences and Technology, Mang, Haripur, Pakistan

    Waqar Khalid Saeed

  3. Faculty of Science, Bachelor of Medical Sciences, University of Western Ontario (ON), London, Canada

    Eun Bin Kim

  4. Department of Internal Medicine, Hanyang University College of Medicine, 222 Wangsimni-Ro, Seongdong-Gu, Seoul, 133-791, Republic of Korea

    Xuanyuan hanning, Dae Won Jun & Eileen L. Yoon

  5. Department of Pathology, Hanyang University School of Medicine, 222 Wangsimni-Ro, Seongdong-Gu, Seoul, Republic of Korea

    Hyun Sung Kim

  6. Hanyang Institute of Bioscience and Biotechnology, Hanyang University, 222 Wangsimni-Ro, Seongdong-Gu, Seoul, Republic of Korea

    Dae Won Jun & Eileen L. Yoon

Authors
  1. Keon Hwi Im
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Waqar Khalid Saeed
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eun Bin Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Hyeon Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ji Eun Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Seung Min Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xuanyuan hanning
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hyun Sung Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Dae Won Jun
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Eileen L. Yoon
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Guarantor of the article: Dae Won Jun, Concept and design: Eileen L. Yoon, Data collection and management: Keon Hwi Im, Xuanyuan hanning, Eun Bin Kim, A Hyeon Lee, Ji Eun Kim, Histology interpretation: Hyun Sung Kim, Interpretation of data: Seung Min Lee, Writing of the manuscript: Waqar Khalid Saeed, and Keon Hwi Im, All authors have approved the final version of the manuscript.

Corresponding author

Correspondence to Dae Won Jun.

Ethics declarations

Conflict of interest

None of the authors has any potential financial conflicts to declare.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Im, K.H., Saeed, W.K., Kim, E.B. et al. Deletion of Mixed Lineage Kinase Domain Like Pseudokinase Aggravates Chronic Alcohol-Induced Liver Injury via Increasing Apoptosis. Dig Dis Sci (2024). https://doi.org/10.1007/s10620-024-08310-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s10620-024-08310-2

Search

Navigation