Abstract: AIM: To construct eukaryotic plasmid pFLAG-AMPKα2, and explore the effect on cardiomyocytes subjected to anoxia/reoxygenation (A/R) injury. METHODS: Total mRNA was extracted from H9c2 cells, and cDNA was formed by reverse transcription. The AMPKα2 coding sequence was amplified by polymerase chain reaction (PCR) and cloned into pFLAG-CMV-4. The plasmid was transfected into H9c2 cells and AMPKα protein was detected by Western blot. A/R injury model was established, cell viability was detected by MTT assay, LDH activity was analyzed with an automatic biochemical analyzer. The percentage of apoptosis, and intracellular ROS were measured by flow cytometry. SOD and GSH-Px were measured by a colorimetric method.RESULTS: AMPKα2 was successfully constructed into pFLAG-CMV-4 the expression vector. The length of the fragment identified by restriction enzyme digestion was 1700 bp. AMPKα2 protein was significant increase after transfecting with pFLAG-AMPKα2. After anoxia/reoxygenation in H9c2 cells, LDH activity, ROS production, and apoptosis were remarkably increased, while cell viability, activities of SOD and GSH-Px were decreased compared with control group (P<0.01). However, the overexpression of AMPKα2 with transfecting pFLAG-AMPKα2 could reverse above index and then protect against A/R injury. CONCLUSION: Recombinant plasmid pFLAG-AMPKα2 could protect H9c2 cells against A/R injury and the mechanism involving improvement of oxidative stress.

Key words: AMPK, Anoxia/reoxygenation injury, Apoptosis, ROS, Oxidative stress