The Role Of AMPK Activation In Effect Of Hydrogen Sulfide Against Doxorubicin-induced H9c2Cardiomyocytes Apoptosis

Background and ObjectiveDoxorubicin is commonly used for the treatment of malignant tumor, but it canelicit dilated cardiomyopathy (DCM) and heart failure for tumor patients.Doxorubicin-induced cardiomyocytes apoptosis plays an important role in itsmyocardial toxicity. Exploring the side effect of doxorubicin on the heart is ofimportant theoretical and application value for preventing and treating DCM and heartfailure. Hydrogen sulfide is considered to be the third endogenous gasotransmitterafter nitric oxide and carbon monoxide. It has a protective effect on cardiomyocytes.Adenosine monophosphate activated protein kinase (AMPK) is not only a cellularenergy sensor and regulator, but also a pressure sensor. It can regulate reactive oxygenspecies/redox balance, cell apoptosis and proliferation to maintain cellularhomeostasis. The goal of the present study is to explore the new mechanism ofhydrogen sulfide involving in activation of AMPK against doxorubicin-induced H9c2cardiomyocytes apoptosis.MethodsThe viability of H9c2cardiomyocytes was detected by MTT assay; the cellapoptosis tested by Hoechst33258staining was observed under fluorescencemicroscope; fluorescence microscope was used to detect the cellular ROS level afterincubation with DCFH-DA fluorescent probe, and the average of green fluorescenceintensity values was semi-quantitatively analyzed by the Image Pro Plus software; theexpressions of p-AMPK, AMPK and p53protein in H9c2cardiomyocytes wereassayed by Western Blot. Results1. H9c2cardiomyocytes were treated with different doxorubicin （0、0.25、0.5、1、2、5μmol/L）. The results from MTT assay and Hoechst33258staining revealed thatdoxorubicin reduced cell viability and promoted cell apoptosis in aconcentration-dependent manner. It was observed that5μmol/L doxorubicin couldmarkedly induce H9c2cardiomyocytes apoptosis.2. H9c2cardiomyocytes were pretreated with different NaHS（0、25、50、100、200μmol/L）for30min followed by doxorubicin. Our results showed that NaHS couldprotect against doxorubicin-induced H9c2cardiomyocytes apoptosis in aconcentration-dependent manner. It was found that the effect of doxorubicin on H9c2cardiomyocytes could be obviously antagonized by100μmol/L NaHS.3. Cells were randomly divided into4groups: control group，5μmol/L DOXgroup，100μmol/L NaHS＋5μmol/L DOX group，5mmol/L PPG＋5μmol/L DOXgroup. Compared NaHS+DOX group with DOX group, the ratio of p-AMPK/AMPKwas1.4-fold highe（rp＜0.05)，and the expression of p53protein and the level of ROSwere decreased by51.4%（p＜0.05）and40%（p＜0.01）respectively, while theopposite effects were in PPG+DOX group. These results suggested that H2S againstdoxorubicin-induced H9c2cardiomyocytes apoptosis could be associated with theactivation of AMPK, the reduction of p53expression and ROS level.4. H9c2cardiomyocytes were pretreated with AMPK inhibitor compound C.Cells were randomly divided into5groups: control group，5μmol/L DOX group，100μmol/L NaHS＋5μmol/L DOX group，10μmol/L compound C＋100μmol/LNaHS＋5μmol/L DOX group，10μmol/L compound C＋5μmol/L DOX group.Compared compound C+NaHS＋DOX group with NaHS＋DOX group, it wasobserved that the ratio of p-AMPK/AMPK was reduced by25.8%（p＜0.05）, and theexpression of p53protein, ROS level and cell apoptosis rate were1.9-fold（p＜0.05）,1.4-fold（p＜0.05）and1.6-fold（p＜0.05）higher respectively. ConclusionsHydrogen sulfide protects against doxorubicin-induced H9c2cardiomyocytesapoptosis via activating AMPK, and reducing the level of reactive oxygen species andthe expression of p53protein.