Investigation Of The Pathogenesis And Drug Treatment Of Acute Ethanol-induced Myocardial Apoptosis

Objective:Acute alcoholism is a state of arousal followed by inhibition which is caused by excessive consumption of alcohol or alcoholic beverages at one time.The incidence of acute alcohol induced-myocardial injury is increasing for years,but there is still a lack of effective treatment and drugs at present,which has attracted the extensive attention of clinicians.It has been reported that myocardial apoptosis is one of the main pathogenesis of acute alcoholic myocardial injury.Nucleolar protein 66(NO66,also known as ribosomal oxygenase 1)plays a key role in the regulation of apoptosis and response to acute ethanol treatment.However,its effect and mechanism on ethanol-induced myocardial apoptosis are unclear.It remains to be further explored that whether and how NO66 is involved in myocardial apoptosis caused by acute ethanol.The current clinical treatment for acute alcoholic myocardial injury only includes temperance and symptomatic treatment of clinical manifestations such as heart failure and arrhythmia.In recent years,effective inhibition of myocardial apoptosis has gradually become a new direction to treatment of acute alcohol induced-myocardial injury.Traditional Chinese medicine extraction plays an important role in drug research and development.Tanshinone IIA is an active ingredient with anti-apoptotic,anti-inflammatory and anti-oxidation effects extracted from Salvia miltiorrhiza.However,its role in acute alcoholic myocardial apoptosis has not been studied.In summary,the purpose of this study is to reveal the molecular mechanism of NO66 in ethanol-induced myocardial cell apoptosis,and to clarify the protective effect and mechanism of Tanshinone IIA on acute ethanol-induced myocardial apoptosis.Our study may provide a new target and a new strategy for clinical protection against ethanol-induced myocardial injury.Methods:The first part of the study:The AC16 cell line was selected for our study.After the cells were treated with different concentrations of ethanol,CCK-8 assay was used to detect cell viability,LDH assay was used to detect the level of cell damage,flow cytometry was used to detect cell apoptotic rate,Western blot was used to detect apoptotic-related protein level.The appropriate ethanol concentration was selected to establish ethanol induced AC16 myocardial cell apoptosis model.The levels of NO66and PI3K/Akt pathway-related m RNA and protein were detected by Western blot and RT-q PCR.The overexpressed NO66 AC16 cell was constructed,and then the cells were treated with LY294002 and ethanol.The apoptotic level was detected by flow cytometry.The expression of PI3K/Akt pathway-related protein was detected by Western blot.The overexpressed PTEN and NO66 AC16 cell was constructed,and then the cells were treated with ethanol.The apoptotic level was detected by flow cytometry.The NO66 and PTEN m RNA level was detected by RT-q PCR.The NO66?PTEN and pAkt/Akt protein level was detected by Wetern blot.The second part of the study:The acute ethanol-induced myocardial injury model was established in C57BL/6 mice.Mice were randomized into four groups:control group,ethanol group,ethanol+Tan IIA(5 mg·kg^-1·d^-1)group,ethanol+Tan IIA(10mg·kg^-1·d^-1)group.Heart structure and function of mice were evaluated using an echocardiographic system.The body weight and heart weight of the mice were measured.The pathological scores were made after HE staining of the myocardium to evaluate the different level of histopathological changes between different groups.The TUNEL staining was performed on the myocardium of mice to calculate the apoptosis rate of myocardium.Western blot was used to detect the protein level in heart tissue.The third part of the study:The H9c2 cell line was selected for our study.The appropriate ethanol concentration was selected to establish ethanol induced H9c2 myocardial cell apoptosis model.After treated with different concentrations of Tan IIA,the cell viability was detected by CCK-8 assay to evaluate the toxicity of Tan IIA.In ethanol-induced H9c2 cells apoptosis model,Tan IIA was administered,and cell viability was detected by CCK-8 assay to determine the optimal concentration of Tan IIA and the optimal action time of Tan IIA at the concentration.H9c2 cells were cultured with ethanol and different concentrations of Tan IIA for 24 hours.The apoptotic rate of cell was detected by flow cytometry.The levels of PI3K/Akt pathway-related protein were detected by Western blot.The PI3K specific inhibitor(LY294002)was applied,and then H9c2 cells were treated with ethanol and Tan IIA.The apoptotic rate of cell was detected by flow cytometry,and the levels of PI3K/Akt pathway-related protein were detected by Western blot.Results:Part I:The study on the mechanism of NO66 in acute ethanol-induced apoptosis of AC16 cardiomyocytesAC16 cells were directly exposed to different doses(0 to 800 m M)of ethanol for 24 h.We selected 400 m M ethanol treatment for 24 h as the acute ethanol-induced AC16cardiomyocytes apoptosis model.NO66 m RNA and protein levels in the 400 m M ethanol group were lower than those in the control group.Our results showed that ethanol administration(400 m M)reduced the ratio of pAkt/Akt and Bcl-2/Bax,while PTEN,p53,and the cleaved caspase-3 expression were increased.NO66 overexpression reduced ethanol-induced AC16 cell apoptosis.Our results showed that acute ethanol exposure could induce the reduction of the pAkt/Akt and Bcl-2/Bax and increasing of PTEN,p53,and cleaved caspase-3 levels in AC16 cells,which was significantly reversed by NO66overexpression.LY294002 significantly declined the ratio of pAkt/Akt and Bcl-2/Bax,while increased the expression of p53 and caspase-3 activity in NO66-overexpressing AC16 cells.In PTEN-overexpressing AC16 cells,the level of pAkt/Akt was decreased,while NO66 expression was not affected.Overexpression of NO66 inhibited the increase of PTEN expression induced by ethanol,and increased the reduction of pAkt/Akt ratio induced by ethanol.PTEN overexpression suppressed the anti-apoptotic effect of NO66on AC16 cells with ethanol administration and greatly weakened the effect of NO66 on activating ethanol-induced pAkt/Akt reduction.Part ?:To observe the effect of tanshinone IIA on acute ethanol-induced myocardial injury in miceThere was no significant difference in body or heart weight between the four groups.After the treatment with ethanol,the left ventricular shortening-fraction(FS)and ejection fraction(EF)decreased,and the left ventricular end-systolic diameter(LVESD)of mice increased.But FS and EF were gradually increased and LVESD was gradually decreased after the mice were treatment the increased concentration of Tan IIA.Acute ethanol exposure caused myocardial injury in mice,but the myocardial injury gradually improved after treating with increased concentration of Tan IIA.Acute ethanol exposure caused myocardial apoptosis in mice,but the level of apoptosis gradually decreased after treating with increased concentration of Tan IIA.Compared with the heart tissue in control group,cleaved caspase-3 and p53 levels were increased in the ethanol group,and the ratio of pAkt/Akt and Bcl-2/Bax was decreased.But the levels of cleaved caspase-3and p53 were gradually decreased,and the ratio of pAkt/Akt and Bcl-2/Bax was gradually increased after treatment with increased concentration of Tan IIA.Part ?:The investigation of the mechanism of tanshinone IIA on acute ethanol-induced myocardial apoptosis at cellular and molecular levelsH9c2 cardiac cells were treated with different doses(0 to 400 m M)of ethanol for 24 h.We selected 200 m M ethanol treatment for 24 h as the acute ethanol-induced H9c2myocardial cell apoptosis model.Tan IIA(0.3?M?1?M?3?M?10?M and 30?M)showed no cytotoxicity to H9c2 cells.Tan IIA(10?M)had the strongest protective effect on acute ethanol induced cell damage,and its effect increased with time.Therefore,the10?M Tan IIA was used as the working concentration of the subsequent experiments,the3?M Tam IIA was used as the low concentration control.The working time of Tan IIA is24 h.Flow cytometry results showed that acute ethanol exposure led to the increasing of apoptotic rate,but the apoptotic rate of H9c2 cells was gradually decreased after treatment with an increasing concentration of Tan IIA.Compared with the control group,cleaved caspase-3 and p53 levels of cells were increased in the ethanol group,and the ratio of pAkt/Akt and Bcl-2/Bax was decreased.But the levels of cleaved caspase-3 and p53 were gradually decreased,and the ratio of pAkt/Akt and Bcl-2/Bax was gradually increased after treatment with an increasing concentration of Tan IIA.The apoptotic rate of cells in LY294002+Tan IIA group was higher than that in Tan IIA group.Compared with Tan IIA group,levels of Bcl-2/Bax were reduced as well as p53 and caspase-3expression increased in Tan IIA+LY294002 group.Conclusion:1.Overexpression of NO66 actives Akt phosphorylation by suppressing PTEN expression,thereby protecting ethanol-induced cell apoptosis in human AC16cardiomyocytes.2.Tanshinone IIA attenuates acute ethanol ingestion-induced heart damage in mice by exerting anti-apoptotic effects.3.Tanshinone IIA alleviates acute ethanol ingestion-induced H9c2 cardiomyocytes apoptosis mainly through activating the PI3K/Akt pathway.