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Ameliorative effects of bilirubin on cell culture model of non-alcoholic fatty liver disease

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Abstract

Background

Non-alcoholic fatty liver disease (NAFLD) is defined as the most prevalent hepatic disorder that affects a significant population worldwide. There are several genes/proteins, involving in the modulation of NAFLD pathogenesis; sirtuin1 (SIRT1), TP53-inducible regulator gene (TIGAR), and autophagy-related gene 5 (Atg5) are considered a chief group of these modulators that principally act by regulating the hepatic lipid metabolism, as well as preventing the lipid accumulation. Surprisingly, bilirubin, especially in its unconjugated form, might be able to alleviate NAFLD progression by decreasing lipid accumulation and regulating the expression levels of the above-stated genes.

Methods and results

Herein, the interactions between bilirubin and the corresponding genes’ products were first analyzed by docking assessments. Afterwards, HepG2 cells were cultured under the optimum conditions, and then were incubated with high concentrations of glucose to induce NAFLD. After treating normal and fatty liver cells with particular bilirubin concentrations for 24- and 48-hour periods, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay, colorimetric method, and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) were employed to assess cell viability status, intracellular triglycerides content, and mRNA expression levels of the genes, respectively. Intracellular lipid accumulation of HepG2 cells was significantly decreased after treating with bilirubin. Bilirubin also increased SIRT1 and Atg5 gene expression levels in fatty liver cells. TIGAR gene expression levels were variable upon the conditions and the cell type, suggesting a dual role for TIGAR during the NAFLD pathogenesis.

Conclusion

Our findings indicate the potential of bilirubin in the prevention from or amelioration of NAFLD through influencing SIRT1-related deacetylation and the process of lipophagy, as well as decreasing the intrahepatic lipid content.

Graphical abstract

In vitro model of NAFLD was treated with unconjugated bilirubin under the optimal conditions.Desirably, bilirubin moderated the accumulation of triglycerides within the cells possibly through modulation of the expression of SIRT1, Atg5, and TIGAR genes. In the context, bilirubin was shown to increase the expression levels of SIRT1 and Atg5, while the expression of TIGAR was demonstrated to be either increased or decreased, depending on the treatment conditions. Created with BioRender.com.

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Data availability

All datasets analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

Atg5/ATG5:

Autophagy-related gene/protein 5

BLVRA:

Biliverdin reductase

CLOCK:

Circadian Locomotor Output Cycles Kaput

CREB:

cAMP response element-binding protein

FOXO:

Fork head box class O

GSH:

Reduced form of glutathione

HCC:

Hepatocellular carcinoma

HMOX:

Heme oxygenase

IRS-2:

Insulin receptor substrate 2

LC3:

Light chain 3

LXR:

Liver X-receptor

3-MA:

3-methyladenine

NAD+ :

Nicotinamide adenine dinucleotide

NAFLD:

Non-alcoholic fatty liver disease

NF-κB:

Nuclear Factor-κB

PDB:

Protein Data Bank

PER2:

Period circadian regulator 2

PGC-1a:

PPAR gamma co-activator 1α

PPAR:

Peroxisome proliferator-activated receptor

SIRT1:

Silent information regulation homology 1

SPPARM:

Selective PPAR modulator

SREBP1c:

Sterol regulatory element binding protein 1c

TIGAR:

TP53 inducible glycolysis and apoptosis regulator

TG:

Triglycerides

TORC2:

Transducer of regulated CREB protein 2

UCB:

Unconjugated bilirubin

UGT1A1:

UDP-glucuronosyltransferase

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Acknowledgements

This research has been extracted from the M.Sc. thesis of Omid Vakili and was supported by Grant Number 97-01-01-17291 from Vice-chancellor for Research Affairs of Shiraz University of Medical Sciences, Shiraz, Iran.

Funding

This work was supported by Vice-chancellor for Research Affairs of Shiraz University of Medical Sciences (Grant number 97-01-01-17291).

Author information

Authors and Affiliations

  1. Autophagy Research Center, Department of Clinical Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

    Omid Vakili & Sayed Mohammad Shafiee

  2. Department of Clinical Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

    Omid Vakili

  3. Department of Clinical Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

    Mohammad Borji

  4. Clinical Biochemistry Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran

    Javad Saffari-Chaleshtori

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  1. Omid Vakili
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection, and analyses were performed by [Omid Vakili], [Javad Saffari-Chaleshtori], [Mohammad Borji], and [Sayed Mohammad Shafiee]. The first draft of the manuscript was written by [Omid Vakili] and all authors commented on previous versions of the manuscript. Supervision and project administration were conducted by [Sayed Mohammad Shafiee]. All authors read and approved the final manuscript.

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Correspondence to Sayed Mohammad Shafiee.

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The authors do not have any financial or non-financial interests to declare.

Ethical approval

This is an observational study. The Research Ethics Committee of Shiraz University of Medical Sciences has confirmed that no ethical approval is required.

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Vakili, O., Borji, M., Saffari-Chaleshtori, J. et al. Ameliorative effects of bilirubin on cell culture model of non-alcoholic fatty liver disease. Mol Biol Rep 50, 4411–4422 (2023). https://doi.org/10.1007/s11033-023-08339-y

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