ABSTRACT
The aim of this paper was to investigate whether the mechanism of salvianolic acid B in protecting H9 c2 cardiomyocytes from hypoxia/reoxygenation injury is related to the regulation of mitochondrial autophagy mediated by NIX. H9 c2 cardiomyocytes were cultured in vitro and divided into normal group, model group and salvianolic acid B group(50 μmol·L~(-1)). Hypoxia/reoxygenation injury model was established by hypoxia for 4 h and reoxygenation for 2 h. In normal group, high glucose DMEM medium was used for culture. Those in model group were cultured with DMEM medium without glucose and oxygen, and no drugs for hypoxia and reoxyge-nation. In salvianolic acid B group, salvianolic acid B prepared by glucose-free DMEM medium was added during hypoxia, and the other process was as same as the model group. The cell viability was evaluated by CCK-8 assay. The leakage of lactate dehydrogenase(LDH) was detected by microplate method. The levels of intracellular reactive oxygen species(ROS) and mitochondrial membrane potential(ΔΨm) were measured by chemical fluorescence method. The level of intracellular adenosine triphosphate(ATP) was mea-sured by fluorescein enzyme method. The autophagy related proteins LC3-Ⅰ, LC3-Ⅱ, apoptosis related protein cleaved caspase-3 and mitochondrial autophagy receptor protein NIX were detected by Western blot. As compared with the normal group, the activity of H9 c2 cardiomyocytes and ATP level were decreased(P<0.05); LDH leakage and ROS production were increased(P<0.01); ΔΨm was decreased(P<0.01); LC3-Ⅱ/LC3-Ⅰ ratio, cleaved caspase-3 and NIX protein expression levels were increased(all P<0.05) in the model group. As compared with the model group, the activity of cells and ΔΨm were significantly increased(P<0.01); ATP level was increased(P<0.05); LDH leakage and ROS generation were decreased(P<0.01); LC3-Ⅱ/LC3-Ⅰ ratio was decreased(P<0.01); cleaved caspase-3 and NIX expression levels were decreased(P<0.05) in the salvianolic acid B group. The protective effect of salvianolic acid B on hypoxia/reoxygenation injury of H9 c2 cardiomyocytes may be associated with inhibiting mitochondrial auto-phagy. The specific mechanism may be related to inhibiting the activation of mitochondrial autophagy mediated by NIX, increasing ΔΨm, reducing ROS production, reducing the expression of cleaved caspase-3, LC3-Ⅱ, and increasing cell viability.
Subject(s)
Humans , Apoptosis , Autophagy , Benzofurans , Cell Hypoxia , Cell Survival , Hypoxia , Myocytes, CardiacABSTRACT
Objective To investigate the effect of prostaglandin E1 (PGE1)on alveolar cells apoptosis in rat lung impact in-jury model.Methods SD rats were divided into 3 groups (normal control group,lung injury control group and PGE1 treated group).PaO2 and pulmonary coefficient were detected after 24 h of impact.TUNEL labeling was used to evaluate apoptosis and Western blot was used to estimate protein expression levels of beclin-1,LC3 Ⅱ/LC3 Ⅰand NIX.Results After 24 h of impacting, there were obvious structural damage and pneumonedema in rat lung.Compared to normal control group,the PaO2 of lung injury control group decreased and the apoptosis of alveolar cells increased significantly(P <0.05).Furthermore,the expression levels of Beclin-1,LC3Ⅱ/LC3Ⅰ and NIX in the impacting control group were increased (P <0.05 ).In the PGE1 treated group,the PaO2 were decreased compared to normal control group(P <0.05),but these expression levels were higher significantly than lung injury control group (P <0.05).The expression levels of apoptosis,Beclin-1,LC3Ⅱ/LC3Ⅰ and NIX in the PGE1 treated group were in-creased compared to normal control group(P <0.05),but these expression levels were lower significantly than lung injury control group (P <0.05).Conclusion PGE1 could alleviate alveolar cells apoptosis after lung impacting injury,and which effect may as-cribe to PGE1 inhibiting NIX-mediated autophagy and autophagic apoptosis of alveolar cells.