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.
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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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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).
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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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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)
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)
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)
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)
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)
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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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DOI: https://doi.org/10.1007/s10565-021-09635-8