The resveratrol attenuates ethanol-induced hepatocyte apoptosis via inhibiting ER-related caspase-12 activation and PDE activity in vitro.
BACKGROUND
Endoplasmic reticulum (ER) stress plays a key role in cell apoptosis pathways. Caspase-12, a proapoptotic gene induced by ER stress, is also the key molecule in ER-related apoptosis. The purpose of this study is to evaluate the protective activity and possible mechanism of resveratrol (ResV) against ethanol (EtOH)-induced apoptosis in human hepatocyte Chang cell line.


METHODS
The human hepatocyte Chang cell line was used to test the hypothesis that ResV may alleviate the liver cell apoptosis induced by EtOH. ER stress-inducible proteins and silent mating type information regulation 2 homolog 1 (SIRT1) were assayed by Western blot. Cell viability was studied by MTT assay and apoptosis was measured by Annexin-V and propidium iodide assay. Caspase-12 activation was examined by immunofluorescence staining. Alcohol dehydrogenase-2 (ADH-2) and aldehyde dehydrogenase-2 (ALDH-2) were measured by polymerase chain reaction amplified product length polymorphism. Phosphodiesterase (PDE) activity was assayed in cell lysates using a cyclic nucleotide PDE assay.


RESULTS
EtOH exposure significantly increased the expression of ER stress markers and activated signaling pathways associated with ER stress. These include GRP78, p-IRE1α, p-eIF2α, p-PERK, ATF4 as well as cleaved caspase-3/12, CHOP/GADD153, and Bax in human hepatocyte Chang cell line. The expression of these proteins were significantly down-regulated by ResV (10 μM) in a SIRT1-dependent manner. ResV can inhibit EtOH-, tunicamycin-, thapsigargin-induced caspase-12 activation. ADH-2 and ALDH-2 activities are lower in this cell line. PDE activity increased by EtOH was inhibited by ResV (10 μM).


CONCLUSIONS
The results indicate that (i) EtOH-induced activation of caspase-12 could be one of the underlying mechanisms of hepatocyte apoptosis; (ii) EtOH-induced cell apoptosis was alleviated via ResV (10 μM) by inhibiting ER stress and caspase-12 activation in a SIRT1-dependent manner; and (iii) SIRT1 activated indirectly by ResV (10 μM) attenuates EtOH-induced hepatocyte apoptosis partly through inhibiting PDE activity.
Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
L Q
LQ
Liu
Z Q
ZQ
Fan
Y F
YF
Tang
Z J
ZJ
Ke
D000426:Alcohol Dehydrogenase / Q000235:genetics; D000444:Aldehyde Dehydrogenase / Q000235:genetics; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D017209:Apoptosis / Q000187:drug effects*; D053454:Caspase 12 / Q000378:metabolism*; D061945:Caspase Inhibitors / Q000494:pharmacology; D002460:Cell Line; D002492:Central Nervous System Depressants / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D000077185:Resveratrol; D056564:Sirtuin 1 / Q000378:metabolism; D013267:Stilbenes / Q000494:pharmacology*
D061945:Caspase Inhibitors; D002492:Central Nervous System Depressants; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D010726:Phosphodiesterase Inhibitors; D013267:Stilbenes; D000431:Ethanol; C489332:ADH1B protein, human; D000426:Alcohol Dehydrogenase; C096127:ALDH2 protein, human; D000444:Aldehyde Dehydrogenase; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D010727:Phosphoric Diester Hydrolases; D053454:Caspase 12; C447939:SIRT1 protein, human; D056564:Sirtuin 1; D000077185:Resveratrol
Apoptosis; Caspase-12 Activation; Endoplasmic Reticulum Stress; Ethanol; Resveratrol