The Mechanism Of Morphine Induced Cardiaprotection Against Ischemia/reperfusion Injury Via Autophagy Regulation

Objectives Although previous studies have demonstrated that autophagy plays an important role in myocardial ischemia/reperfusion injury, it is not clear whether autophagy is involved in morphine induced cardioprotection. This study will explore whether morphine protected the myocardial cells against oxidative stress through inhibiting autophagy and its molecular mechanism.Methods 650 μM H2O2 were used to induce rat heart tissue-derived H9c2 cells’ mitochondrial damage. Cell activity was detected by MTT. Meanwhile, in order to detect the m PTP opening, cells were loaded with TMRE and imaged with confocal microscopy. Atg5 m RNA expressions were evaluated by quantitative RT-PCR. Meanwhile, western blot was used to test LC3, Beclin1 and m TO R phosphorylation.Results 1 MTT assay was used to detect cell activity. Compared to the H2O2 group, cells treated with morphine(0.1 μM) showed a significantly increase in the absorbance ratio(114.24±1.64%, P<0.05), indicating that morphine could increase cell activity. 2 The TMRE fluorescence intensity results showed that, compared to H2O2 control group, morphine can obviously inhibit the m PTP opening(88.00±2.74%, P<0.05), similarly to morphine, the effect of H2O2 on TMRE fluorescence was inhibited by 3-MA(88.67±4.50%, P<0.05), suggesting that morphine could prevent oxidative stress ind uced mitochondrial injury, which may be related to autophagy. 3 Real Time RT-PCR results showed that, compared with the blank control group, H2O2 can obviously increase Atg5 m RNA expression(183.70±10.87%, P<0.05) which was inhibited by morphine(127.20± 7.72%, P<0.05). Western blot results pointed that H2O2 increased LC3 phosphorylation which was reversed by morphine(125.91±5.12%, P<0.05). Morphine could also inhibite H2O2 induced m TOR inactivation(78.24±4.20%, 99.69±6.01%, P<0.05). But there is not a significant difference between different groups in Beclin1 expression.The data suggest that morphine protected the heart via negative regulating autophagy. 4 Western blot results showed, comparing to the H2O2 group, morphine increased m TOR phosphorylation which was inhibited by m TO R agonist Rapamycin(H2O2+Rap+Mor: 79.34±4.02%, P<0.05). Rapamycin could also inhibited morphine induced LC3 expression increasing(H2O2+Rap+Mor: 154.18±13.38%, P<0.05), suggesting that morphine inhibited autophagy by activating m TO R.Conclusions Morphine may inhibite oxidative stress injury induced m PTP opening by activating the negative regulation mechanisms of autophagy.