Inhibition Of Histone Acetylation By Curcumin Reduces Alcohol-induced Fetal Cardiac Apoptosis

ObjectivePrenatal alcohol exposure may cause cardiac development defects.It is known that alcohol can induce cardiac apoptosis and myocardium dysplasia,however the underlying mechanisms are not yet clear.Although,our previous studies have suggested that histone modification plays a vital role in alcohol induced fetal cardiac development abnormalities.This study investigates the phenomenon further,particularly by examining the effect of histone acetylation regulation mechanisms on alcohol induced cardiac apoptosis.Methods(1)Between E7.5 and E15.5,the pregnant mice were given ethanol(56% v/v in saline)using oral gavage at dosage of 10?l/g every day.Controls group were given isovolumetric glucose saline solution simultaneously.At E 17.5,all pregnant mice were euthanized using carbon dioxide asphyxia and the embryonic hearts were promptly retrieved from the mouse pups.1)Western blot were used to detect the acetylation of histone H3K9;2)Q-PCR assay were used to detect the mRNA of caspse-3,caspase-8 and bcl-2,and the expression of caspase-3,active-caspase-3,caspase-8,active-caspase-8 and bcl-2 were detected by western blot;3)We evaluated the acetylation levels of histone H3K9 in the promoter of apoptosis genes using Ch IP assay kit;4)TUNEL assay was used to assess the mode of apoptosis of alcohol treatment.(2)The cardiac progenitor cells were divided into five groups: alcohol;curcumin;alcohol + curcumin;DMSO;and control.We used 200 mM alcohol and 25?M curcumin to intervene our cells and the cells were collected after a 24 h treatment.1)Western blot were used to detect the acetylation of histone H3K9;2)Western blot were used to detect the expression of caspase-3,active-caspase-3,caspase-8,active-caspase-8 and bcl-2;3)Flow cytometry(FCM)assay was used to detect the apoptosis levels.ResultsTUNEL assay revealed that positively stained cells were significantly higher in the alcohol group.Western blotting revealed that alcohol increases active-caspase-3 and active-caspase-8,whereas it reduces caspase-3,caspase-8 and bcl-2.Furthermore,we observed that alcohol exposure enhanced acetylation of histone H3K9 in embryonic hearts.Ch IP assay showed that alcohol significantly increased the acetylation of histone H3K9 in the promoter of caspase-3 and caspase-8,and decreased the acetylation of histone H3K9 in the promoter of bcl-2.Through in vitro experiments,we found that alcohol treatment increased the expression of active-caspase-3,active-caspase-8 and the acetylation of histone H3K9,and decreased the expression of caspase-3,caspase-8 and bcl-2 in cardiac cells.Surprisingly,when we intervened cardiac cells with curcumin the up-regulation of active-caspase-3,active-caspase-8 and acetylation of histone H3K9,and the down-regulation of caspase-3,caspase-8 and bcl-2 were all reversed.Moreover,flow cytometry assay demonstrated that the high apoptosis level that was induced by alcohol in cardiac cells were down-regulated after curcumin treatment.ConclusionThese findings indicate that histone modification may play an important role in mediating alcohol induced fetal cardiac apoptosis,possibly through the up-regulation of acetylation of H3K9 in the promoters of apoptosis genes.This study also shows that curcumin may reverse alcohol-induced fetal cardiac apoptosis,which provide further implications that curcumin is protective againstalcohol abuse during pregnancy.