Title: Mechanism of reduction of protection induced by hypoxic postconditioning in diabetic myocardiocytes subjected to hypoxia-reoxygenation: the relationship with DJ-1 expression
Abstract: Objective
To evaluate the mechanism of reduction of protection induced by hypoxic postconditioning (HPO) in diabetic myocardiocytes subjected to hypoxia-reoxygenation (H/R), and the relationship with DJ-1 expression.
Methods
H9c2 cells cultured in normal culture atmosphere were randomly divided into 6 groups (n=36 each) using a random number table: control group (group C), group H/R, group HPO, plasmid carrying DJ-1 gene transfection group (group D), plasmid carrying DJ-1 gene transfection+ H/R group (group DH), and plasmid carrying DJ-1 gene transfection+ H/R+ HPO group (group DHH). H/R was induced by 4 h of hypoxia followed by 2 h of reoxygenation.H9c2 cells were cultured in high glucose culture medium (30 mmol/L) for 48 h in C, H/R and HPO groups.H/R model was established in H/R and HPO groups.HPO was induced by 3 cycles of 5 min reoxygenation and 5 min hypoxia before the onset of reoxygenation in group HPO.In D, DH and DHH groups, the plasmid carrying DJ-1 gene (pEX-2-EGFP-DJ-1) was transfected into the cells, and then the cells were cultured in high glucose culture medium and subjected to H/R and HPO, respectively.At 2 h of reoxygenation, the cell survival rate was measured using the cell counting kit-8 assay, and the level of lactate dehydrogenase (LDH) in the supernatant was detected by enzyme-linked immunosorbent assay, the autophagosomes were examined with a transmission electron microscope, and the expression of DJ-1 and p62 and ratio of microtubule-associated protein 1 light chain 3 Ⅱ/Ⅰ (LC3 Ⅱ/Ⅰ) in cells were detected by Western blot.
Results
Compared with group C, the cell survival rate and DJ-1 expression were significantly decreased, and the LDH activity was significantly increased in H/R and HPO groups (P 0.05). Compared with group D, the cell survival rate was significantly decreased, and the LDH activity was significantly increased in group DH (P 0.05).
Conclusion
The mechanism of reduction of protection induced by HPO in diabetic myocardiocytes subjected to H/R is related to high glucose-induced inhibition of DJ-1 expression and decrease in autophagy.
Key words:
Anoxia; Ischemic postconditioning; Diabetes mellitus; Myocytes, cardiac; Reperfusion injury; Intracellular signaling petides and proteins
Publication Year: 2016
Publication Date: 2016-04-20
Language: en
Type: article
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