| Background: Doxorubicin hydrochloride is one kind of the anthracycline anti-tumor drugs, which was used in clinical most commonly and it has a broader antitumor spectrum. However, the long-term use of doxorubicin can cause severe myocardial cell injury eventually led to the death of myocardial cells and manifested as an irreversible cardiomyopathy and congestive heart failure in clinical. Thereby, its clinical application was limited. Now, the doxorubicin cardio toxicity mechanism has not yet been fully elucidated, Previous research suggests that doxorubicin could cause the formation of a large number of oxygen radicals and lipid peroxidation in vivo and then cause myocardial energy metabolism disorder, eventually leading to impaired cardiac function. In recent years, the role of autophagy in cardiovascular disease has been concerned widespread. Macroautophagy, hereafter referred to as autophagy, is an intracellular process in which cytoplasmic materials are transported by autophagosomes to lysosomes for degradation. In different myocardial pathologies, the changes in autophagy may be a compensatory response of myocardial cells and may also lead to autophagic cell death. The purpose of this project is to study whether autophagy is involved in this process of adriamycin induced myocardial injury, and to study its mechanism of action.Objective: To study the effect and mechanism of autophagy in adriamycin induced myocardial injury model in mice and to provide a new strategy for the treatment of doxorubicin cardiomyopathy.Methods:Male C57mice,10-12weeks old, weighted24to28g were randomly divided into experimental and control groups. The mice of Experimental group were injected with doxorubicin hydrochloride by the dose of25mg/kg/d intraperitoneally one time resulted the acute myocardial injury model and the control group was given an equal dose of saline. The animals were sacrificed and cardiac tissue was harvested at different time points (Oh, Id,2d,4d) after the injection of dox and saline. The mice injected with DOX HCL and0.9%NaCl were injected with Evan’s blue dye (EBD,100mg/kg, i.p.)18hrs before the mice were anesthetized. For left ventricular cardiomyocyte cross-sectional area, coronal sections were deparaffinized and stained for membranes with Alexa488conjugated wheat germ agglutinin (WGA) and these sections were imaged for EBD (red) and Alexa-488(green) fluorescence observed under the fluorescence microscope. We will detect the expression of nrf2and its downstream genes as well as the autophagy related genes by Real time PCR and western blot. For the autophagy flux in vivo,10to12week-old male C57BL/6mice were randomly assigned to the four groups including saline+rap, saline+rap+cq, dox+rap,dox+rap+cq. Doxorubicin hydrochloride was administered by intraperitoneal (ip) injection at a dose of25mg/kg/d. Control mice received injections of saline at a comparable volume.The administration of rapamycin (2mg/kg), chloroquine (10mg/kg) was done via i.p. injections. Four hours after injection, animals were sacrificed and cardiac tissue was harvested immediately and for assessment of LC3and p62protein levels.Results:The body weight and heart weight of the experiment group mice injected with doxorubicin were gradually reduced with a statistically significant difference (p<0.01) compared with the control mice. The HE staining of ventricular sections demonstrated normal tissues in control mice whereas vacuolization of cardiomyocyte existed in the DOX treated mice. WGA staining showed that a large area of myocardial necrosis occurred in dox-treated mice campaired with the control group mice. The expression of Nrf2and its downstream gene NQO1, HO-1were gradually increased after the injection of doxorubicin. The expression of autophagy related gene such as Beclin1, ATG14L, ATG5, ATG7and LC3increased compared with the control mice, but the expression of Rubicon decreased compared with the control mice with a statistically significant difference (p<0.01). Western blot showed that the protein level of NQO1, LC3and P62are higher than the control mice,but there is no significant difference between the dox-treated mice and the control mice about the expression of ATG5,ATG7,LAMP1and Cathepthin D (p>0.05).There is a increased trend of Ubiquitin in the experimental group mice compared with control group.The autophagy flux in vivo showed that in the saline control group, the porteion level of LC3and P62are significantly higher in mice treated with rap and cq than the mice treated with rap only. But in the dox group, there is no significant difference in the expression of LC3and P62between the rap+cq treated mice and the rap treated mice.Conclusion:(1) Adriamycin activates the oxidative stress pathway.(2) Adriamycin activates the autophagy pathway.(3) The balance of autophagy flow was destroyed in the dox treated mice..(4) The protein degradation by the ubiquitin proteasome system was enhanced in the dox treated mice.. |
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