Effects Of The Different Time Of Hypoxia Reoxygenation And Scutellarin On Autophagy Of Myocardial Cells

[Objective] The purpose of this study is to investigate the effect of different time of hypoxia reoxygenation (HR) on cell autophagy and cell viability, and further study the characteristics of Yunnan Medicinal Plant Erigeron breviscapus main active ingredient-scutellarin (SCU)’s cell damage effect through inhibiting autophagy antagonized cells induced by HR effect. In vitro HR model in this subject was established on rat myocardial cell(H9C2) and the level of changes of microtubule-associated protein 1 light chain 3 beta and microtubule-associated protein 1 light chain 3 alpha included in autophagy related gene were detected. Meanwhile, the different reoxygenation time and the effect of SCU on the expression of autophagy and cell injury were described.[Methods]1 H9C2 cells were as the subjects in this project, the HR model was hypoxia for 2h and then reoxygenation. The myocardial cell viability was detected by MTT.2 According to the different reoxygenation time, it was divided into normal control group (group A), hypoxia group (group B), reoxygen 2h group (group C), reoxygen 12h group (group D) and reoxygen 24h group (group E).On the basis of different concentration of SCU after HR, it was divided into normal control group (group A), reoxygenation group (group B), 1uM SCU group (Group C), lOuM SCU group (Group D),30uM SCU group (Group E) and 100um SCU group (Group F).The expression of LC3 II/LC3 I in each groups was detected by Western Blot, and the activity of myocardial cells were detected by MTT. 3 Data were analyzed by SPSS statistical software, and independent samples t test was conducted between the two groups. Single factor variance analysis (one-way ANOVA) was used in differences between groups, and LSD test after variance analysis was used in comparison between the two groups. Excel graphics software was used to mapping and with P<0.05 to determine the difference was statistically significant.[Results]1 Validation of the model of hypoxia reoxygenation injury:MTT results show that the viability of the myocardial cells after hypoxia 2h/reoxygenation 2h was significantly lower than that in the normal control group(P<0.05), which showed that the method of establishing hypoxia reoxygenation model was successful.2 The effect of different reoxygenation time on the expression of LC3 protein after hypoxia of myocardial cells:The ratio of LC3 II/LC3 I of the cells in group B (hypoxia group), group C (reoxygen 2h group), group D (reoxygen 12h group), group E (reoxygen 24h group) was significantly higher than that of group A (normal control group) (P<0.05). Among them, the ratio of LC3 II/LC3 I of the cells in group C and D were significantly higher than that of group B(P<0.05), paticlularly the group D was the highest (P<0.05), and there was no significant difference between group B and E.3 The effect of different reoxygenation time on myocardial cell viability after hypoxia of myocardial cells:MTT results showed that the cell viability in the group B (hypoxia group), group C (reoxygen 2h group), group D (reoxygen 12h group) and group E (reoxygen 24h group) was significantly lower than group A (normal control group) (P<0.05). Among them, the cell viability in the group C and D was significantly lower than that in the group B (P<0.05), particularly the cell activity in the group D was the lowest (P<0.05), and the cell activity in the group E was higher than that in the group B (P<0.05).4 Effect of SCU on the expression of LC3 in myocardial cells treated with HR:The ratio of LC3 II/LC3 I of the cells in group C (1uM SCU group), group D (10uM SCU group), group E (30uM SCU group) and group F (100uM SCU group) was lower than that in group B (reoxygenation group) (P<0.05). Among them, the ratio of LC3 II/LC3 I in group E and F was the lowest (P<0.05), and there was no significant difference between the two groups. Meanwhile, there was also no significant difference between the group C and the D.5 Effect of SCU on the viability of myocardial cells treated with HR:The cell viability in group C (luM SCU group), group D (lOuM SCU group) and group E (30uM SCU group) was higher than that in group B (reoxygenation group) (P<0.05). Among them, the cell viability of group E was the highest (P<0.05). There was no significant difference between group C and D. The cell viability in group F (100uM SCU group) was higher than that in group B (reoxygenation group) (P<0.05), but lower than that in group C (1uM SCU group).[Conclusion]1 H9C2 cultured in the three gas incubator to hypoxia culture for 2h and aerobic culture for 2h given 2% of oxygen can obviously decreased the viability of the myocardial cells, and can effectively simulate the myocardial hypoxia reoxygenation injury in vitro;2 Hypoxia can activate autophagy, and autophagy increased significantly after reoxygenation for 2h. Autophagy was activated most obviously at 12 hours and decreased to the level of hypoxia group at 24 hours;3 Cells were damaged by hypoxia, and further increase of cellular damage was occured after oxygen inhalation. At 12 hours, the cell viability was the lowest, and the cell viability was improved at 24h, but it was still unable to return to normal levels.4 At 2h of hypoxia and reoxygenation and 12h of reoxygenation, the viability of myocardial cell decreased with the increase of cell autophagy.5 SCU can reduce the autophagy in myocardial cells after HR, and the effect is significant in 30uM and 100uM.6 SCU can improve the viability of myocardial cell after HR, and the effect was significant at 30uM.7 SCU can improve the myocardial viability by inhibiting autophagy, and the effect is the best when the concentration is 30uM.