| Objective:The mechanism of ischemia-reperfusion injury is still not fully understood. In this study, we investigated the changes of cell viability, the endogenous HO-1 expression and its viriation after hypoxia-reoxygenation injury.Methods:The H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time (2,6,12,18, and 24 h). Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R. The effect of different durations of hypoxia/reoxygenation on myocardial cytotoxicity was measured.Results:As shown in Fig.1 A, H9c2 cells were subjected to different durations of reoxygenation (2,6,12,18, and 24 h). At 2 hours of reoxygenation, cell viability was at a minimum, and cytoxicity damage was greatest; however, by 18 hours of hypoxia-reoxygenation, cell viability had gradually stabilized. Significant differences occurred between the 12-,18-and 24-hour reoxygenation groups and the 2-hour reoxygenation group (***p< 0.001, vs. the normoxia group). RT-PCR (Fig. 1B) and Western Blotting (Fig.1C) were used to detect endogenous HO-1 expression in un-transduced H9c2 cells; HO-1 expression levels were highest at 2 hours of reoxygenation and were significantly lower at 12,18 and 24 hours of reoxygenation (**p< 0.01, vs. the normoxia group). The HO-1 protein expression level decreased over time after reoxygenation and remained at a stable, low level at 12 hours of reoxygenation.Conclusions:Hypoxia-reoxygenation would induced H9C2 cells injury. In this study, we found the extent of damage was approximately 40% after 6 hours of reoxygenation, and which we choose is as the model of myocardial hypoxia-reoxygenation injury.Objective:Mitochondria are the important energy conversion organelles in eukaryotic cells. In hypoxic environment, the mitochondria are the most sensitive organelles, it would be adjustmented by itself in order to adapt to the environment. Autophagy is another complex protective mechanism to cell damage in hypoxia. In this study, we investigated the changes of mitochondria functions and the level of autophagy after hypoxia-reoxygenation injury.Methods:The H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time (2h,6h,12h,18h, and 24h). Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R. Mitochondrial dysfunction (Flow cytometry quantified the ROS generation and JC-1 staining) and autophagy (The Confocal microscopy measured the autophagy). RFP-GFP-LC3 double-labeled adenovirus was used for testing. Flow cytometry quantified the apoptosis rate.Results:Cell viability was compared between the four groups. Significant differences were found between the normoxic group and the H/R group and between the H/R group and the Rapamycin/TSA group. Thus, pre-treatment with the autophagy inducer rapamycin or TSA inhibited H/R-induced damage, as demonstrated by the results obtained at 6 hours of reoxygenation. HO-1 expression was higher and p62 and LC3-? expression was lower in the normoxic group. In the H/R group, HO-1 expression was significantly lower, p62 expression was significantly higher, and LC3-II expression was also higher. After rapamycin or TSA induction, the use of autophagic markers, LC3II expression was significantly increased, p62 expression was significantly decreased, and autophagy was relatively constant. Moreover, rapamycin or TSA increased levels of HO-1. Fig.2C shows changes in autophagy in the four groups. In the H/R group, there was significant increase in yellow fluorescence, indicating impeded autophagy. In the rapamycin and TSA group, there was significant increase in red fluorescence, indicating unimpeded autophagy. During hypoxia-reoxygenation, autophagy occurred with impeded autophagy, and the expression levels of p62 and LC3-? were significantly increased. After myocardial hypoxia-reoxygenation, the mitochondrial membrane potential was significantly reduced, and mitochondrial ROS levels were significantly higher. After the use of autophagy markers, the level of autophagy was increased, the mitochondrial membrane potential was significantly increased, and mitochondrial ROS levels were significantly decreased. These results show that autophagy contributed to mitochondrial membrane stability and the generation of mitochondrial ROS. In H/R group, the apoptosis rate increased significantly, while in the Rapamycin and TSA group, apoptosis rate compared with the H/R group, there was significantly reduced.Conclusions:Autophagy had beed found in cadiac cells undergoing an I/R process, and a significant increasing in p62 and LC3-? levels was found as well. Rapamycin and TSA, as the autophagy-inducing pretreatment agents, could inhibit H /R injury and promote HO-1 protein levels. As the autophagy volumn increasing, the membrane potential of mitochondria was significantly increased, and ROS was significantly decreased.Objective:Mitochondrial dysfunction would ultimately lead to myocardial cell apoptosis and death during ischemia-reperfusion injuries. Autophagy could ameliorate mitochondrial dysfunction by autophagosome forming, which is a catabolic process to preserve the mitochondrial's structural and functional integrity. HO-1 induction and expression are important protective mechanisms. This study was focused on role of HO-1 during mitochondrial damage and its mechanism.Methods:The H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time (2h,6h,12h,18h, and 24h). Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R. Mitochondrial dysfunction (Flow cytometry quantified the ROS generation and JC-1 staining) and autophagy (The Confocal microscopy measured the autophagy). RFP-GFP-LC3 double-labeled adenovirus was used for testing. Flow cytometry quantified the apoptosis rate.Results:An Lv-HO-1-H9c2 cell line encoding lentivirus-mediated HO-1 overexpression was constructed. HO-1 overexpression in H9C2 cells significantly inhibited H/R-induced cell injury (**p<0.01,#p<0.05). Significant HO-1 overexpression was observed in the Lv-HO1-H9c2 cell line (5-fold,**p<0.01). After reoxygenation, the expression level of HO-1 mRNA was significantly reduced but remained significantly higher than that in the normoxic group. Under normoxic conditions, HO-1 expression was higher and LC-3 and p62 expression was lower in the Lv-HO1 group than in the Lv-scramble group (**p<0.01). Under H/R conditions, with HO-1 over-expression, HO-1 expression was increased, LC-3 expression was increased, and p62 expression was decreased, suggesting that autophagy was unimpeded (##p<0.01). For the Lv-scramble cell line, yellow fluorescence was significantly increased and red fluorescence was significantly decreased in the H/R group compared with the normoxic group (*p<0.05), indicating impeded autophagy. Under H/R conditions, red fluorescence was significantly increased in the Lv-HO1 group compared with the Lv-scramble group (##p<0.01), indicating unimpeded autophagy. During hypoxia-reoxygenation, autophagy was impeded, and the expression of p62 and LC3-? was significantly increased. These results suggest that HO-1 overexpression contributed to the level of autophagy and inhibited H/R-induced cell injury. After H/R treatment, the mitochondrial membrane potential was significantly increased (#p<0.05) and the mitochondrial ROS level was significantly decreased (**p<0.01,#p<0.05). In Lv-scramble-H/R group, the apoptosis rate increased significantly (** p<0.01) when compared with Lv-scramble-Normoxia group, while in the Lv-HO1-H/R group, apoptosis rate compared with the Lv-scramble-H/R group, there was significantly reduced (##p<0.01). after HO-1 was inhibited by ZnPP, the mitochondrial membrane potential was significantly decreased (** p<0.01), and the mitochondrial ROS level was significantly increased (** p <0.01).Conclusions:During myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect. |
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