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Induction of Sestrin2 by pterostilbene suppresses ethanol-triggered hepatocyte senescence by degrading CCN1 via p62-dependent selective autophagy

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Abstract

Hepatocyte senescence is a key event participating in the progression of alcoholic liver disease. Autophagy is a critical biological process that controls cell fates by affecting cell behaviors like senescence. Pterostilbene is a natural compound with hepatoprotective potential; however, its implication for alcoholic liver disease was not understood. This study was aimed to investigate the therapeutic effect of pterostilbene on alcoholic liver disease and elucidate the potential mechanism. Our results showed that pterostilbene alleviated ethanol-triggered hepatocyte damage and senescence. Intriguingly, pterostilbene decreased the protein abundance of cellular communication network factor 1 (CCN1) in ethanol-exposed hepatocytes, which was essential for pterostilbene to execute its anti-senescent function. In vivo studies verified the anti-senescent effect of pterostilbene on hepatocytes of alcohol-intoxicated mice. Pterostilbene also relieved senescence-associated secretory phenotype (SASP), redox imbalance, and steatosis by suppressing hepatic CCN1 expression. Mechanistically, pterostilbene-forced CCN1 reduction was dependent on posttranscriptional regulation via autophagy machinery but not transcriptional regulation. To be specific, pterostilbene restored autophagic flux in damaged hepatocytes and activated p62-mediated selective autophagy to recognize and lead CCN1 to autolysosomes for degradation. The protein abundance of Sestrin2 (SESN2), a core upstream modulator of autophagy pathway, was decreased in ethanol-administrated hepatocytes but rescued by co-treatment with pterostilbene. Induction of SESN2 protein by pterostilbene rescued ethanol-triggered autophagic dysfunction in hepatocytes, which then reduced senescence-associated markers, postponed hepatocyte senescence, and relieved alcohol-caused liver injury and inflammation. In conclusion, this work discovered a novel compound pterostilbene with therapeutic implications for alcoholic liver disease and uncover its underlying mechanism.

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Data availability

All data included in this study are available upon request by contact with the corresponding author.

Code availability

Not applicable.

Abbreviations

ALT:

alanine aminotransferase

AO:

acridine orange

AST:

aspartate aminotransferase

ATG7:

autophagy-related protein 7

BafA1:

bafilomycin A1

BECN1:

Beclin1

CCK-8:

cell count kit-8

CCN1:

cellular communication network factor 1

CHX:

cycloheximide

Co-IP:

co-immunoprecipitation

CQ:

chloroquine

DMSO:

dimethylsulfoxide

ELISA:

enzyme-linked immunosorbent assay

FBS:

fetal bovine serum

GSH:

hlutathione

GSSG:

oxidized glutathione

H&E:

hematoxylin and eosin

HMGA1:

high mobility group A1

IFNγ:

interferon gamma

LAMP:

lysosome-associated membrane protein 1

LC3:

microtubule-associated protein 1 light chain 3

3-MA:

3-methyladenine

MDC:

monodansylcadaverine

mTOR:

mechanistic target of rapamycin

NBR1:

neighbor of BRCA1 gene 1

NC:

negative control

NDP52:

nuclear dot protein 52

OPTN:

optineurin

PBS:

phosphate-buffered saline

PTS:

pterostilbene

ROS:

reactive oxygen species

SA-β-gal:

senescence-associated β-galactosidase

SASP:

senescence-associated secretory phenotype

SD:

standard deviation

SESN2:

Sestrin2

TC:

total cholesterol

TEM:

transmission electron microscopy

TERT:

telomerase reverse transcriptase

TG:

triglyceride

TNFα:

tumor necrosis factor alpha

TRF:

telomeric repeat binding factor

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Funding

This work was supported by the National Natural Science Foundation of China (No. 81803606 and No. 81900531, China), The Science and Technology Project of Jiangsu Provincial Administration of Traditional Chinese Medicine (No. YB201847, China), Nantong Science and Technology Project (No. MS12018051, China), Innovation and Entrepreneurship Training Program for Undergraduates of Nantong University (No. 2020207, China), and Scientific Research Startup Foundation for Talents of Nantong University (China).

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Author notes

  1. Yiming Jiang and Ying Zhou contributed equally to this work.

Authors and Affiliations

  1. School of Pharmacy, Nantong University, 19 Qixiu Road, Nantong, 226001, Jiangsu, China

    Yiming Jiang, Ying Zhou, Xinqi Wang, Xiaofeng Bao & Chunfeng Lu

  2. School of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, 210023, Jiangsu, China

    Wenxuan Xu

  3. Department of Pharmacology, School of Pharmacy, Wannan Medical College, 22 West Wenchang Road, Wuhu, 241002, Anhui, China

    Huanhuan Jin

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  1. Yiming Jiang
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  2. Ying Zhou
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Contributions

Yiming Jiang (data curation: equal; formal analysis: lead; investigation: equal); Ying Zhou (formal analysis: equal; investigation: equal); Wenxuan Xu (formal analysis: equal; validation: lead); Xinqi Wang (visualization: equal); Huanhuan Jin (visualization: equal); Xiaofeng Bao (methodology: equal); Chunfeng Lu (conceptualization: lead; formal analysis: equal; funding acquisition: lead; project administration: lead; resources: lead; software: lead; supervision: lead; writing – original draft: lead; writing – review & editing: lead).

Corresponding author

Correspondence to Chunfeng Lu.

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Ethics approval

The study was approved by the Institutional and Local Committee on the Care and Use of Animals of Nantong University (Nantong, Jiangsu, China).

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Supplementary information

Figure S1

Quantification of SA-β-gal staining. Human hepatocytes were treated with vehicle, 100-mM ethanol or plus pterostilbene (5, 10, and 20 μM) for 24 h. Data are expressed as mean ± SD, #P<0.05 versus Vehicle, *P<0.05 versus Ethanol. (PNG 49 kb)

High Resolution Image (TIF 262 kb)

Figure S2

Verification of interference efficiency on autophagy. (A) Hepatocytes were treated with CQ, 3-MA, or NH4Cl at indicated concentrations for 24 h. Western blot analysis of intracellular p62 protein abundance. Data are expressed as mean ± SD, ##P<0.01 and ###P<0.001 versus Vehicle. (B) Hepatocytes were transfected with 100-pmol siNC or siATG7 for 24 h. Western blot analysis of intracellular ATG7 and p62 protein abundance. (C) Hepatocytes were transfected with 100-pmol siNC or siBECN1 for 24 h. Western blot analysis of intracellular BECN1 and p62 protein abundance. Data are expressed as mean ± SD, ##P<0.01 and ###P<0.001 versus siNC. (PNG 257 kb)

High Resolution Image (TIF 1551 kb)

Figure S3

Verification of interference efficiency of BafA1 on autophagy. Hepatocytes were treated with 5-nM BafA1 for 24 h. Western blot analysis of intracellular p62 protein abundance. Data are expressed as mean ± SD, ##P<0.01 versus Vehicle. (PNG 55 kb)

High Resolution Image (TIF 301 kb)

Figure S4

Quantification of co-localization of CCN1 and p62 in hepatocytes. Hepatocytes were treated with vehicle, 100-mM ethanol or plus 20-μM pterostilbene for 24 h. Data are expressed as mean ± SD, #P<0.05 versus Vehicle, *P<0.05 versus Ethanol. (PNG 35 kb)

High Resolution Image (TIF 147 kb)

Figure S5

Verification of interference efficiency on SESN2. Hepatocytes were transfected with 100-pmol siNC or siSESN2 for 24 h. Western blot analysis of intracellular SESN2 protein abundance. Data are expressed as mean ± SD, ##P<0.01 versus siNC. (PNG 71 kb)

High Resolution Image (TIF 396 kb)

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Jiang, Y., Zhou, Y., Xu, W. et al. Induction of Sestrin2 by pterostilbene suppresses ethanol-triggered hepatocyte senescence by degrading CCN1 via p62-dependent selective autophagy. Cell Biol Toxicol 39, 729–749 (2023). https://doi.org/10.1007/s10565-021-09635-8

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