Role of PI3K/Akt signal pathway in anoxia-reoxygenation injury attenuated by emulsified isoflurane pretreatment in neonatal rat cardiomyocytes

Abstract

Objective To evaluate the role of PI3K/Akt signal pathway in anoxia-reoxygenation (A/R) injury attenuated by emulsified isoflurane pretreatment in neonatal rat cardiomyocytes. Methods The cardiomyocytes of 60 neonatal Wistar rats (1-3 days old) were incubated in 24 well culture plates and randomly assigned into 6 groups (n=8 each): control group (group C);A/R group ;EI group;EI + wortmannin (specific inhibitor of PI3K) group (group EIW);wortmannin group (group W);fat emulsion group (group F). In group C, the cardiomyocytes were cultured for 4 h. In group A/R, the cardiomyocytes were exposed to 95% N2-5% CO2 saturated ischemic solution for 2 h followed by 2 h reoxygenation with 95 % O2-5% CO2 saturated reperfusion solution. In group EI, emulsified isoflurane was added to the culture medium immediately before cardiomyocyte hypoxia with the final concentration of 1.68 mmol/L. In group EIW, emulsified isoflurane and wortmannin were added to the culture medium in combination immediately before cardiomyocyte hypoxia with the final concentrations of 1.68 mmol/L and 100 nmol/L respectively. In group W and F, wortmannin and 30% fat emulsion were added to the culture medium immediately before cardiomyocyte hypoxia with the final concentration of 100 nmol/L and 0.05% respectively. The supernatant was taken from culture medium to measure LDH activity and cTnI concentration, and myocardial cell homogenate was prepared to measure SOD activity and MDA content and to detect phosphorylation-Akt (p-Akt) expression by Western blot at the end of 2 h reoxygenation. Results The supernatant LDH activity, cTnI concentration and cardiomyocyte MDA content were significantly higher, while SOD activity lower in the other 5 groups than in group C (P 0.05). The supernatant LDH activity, cTnI concentration and cardiomyocyte MDA content were significantly higher, while cardiomyocyte SOD lower in group EIW, W, and F than in group EI (P<0.05). The cardiomyocyte p-Akt expression was significantly increased in ascending order from group C, EIW, A/R, F to group EI (P<0.05). Conclusion Emulsified isoflurane pretreatment can attenuate A/R injury in neonatal rat cardiomyocytes via activation of PI3K/Akt signal pathway. Key words: Isoflurane; Fat emulsions, intravenous; 1-Phosphatidylinositol 3-kinase; Myocytes, cardiac; Cell hypoxia; Oxygen