Role of Akt/mTOR signaling pathway in ulinastatin-induced reduction of oxidative stress injury to cardiomyocytes of rats

Abstract

Objective To evaluate the role of protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway in ulinastatin-induced reduction of oxidative stress injury to cardiomyocytes of rats. Methods H9c2 cells were inoculated in 96-well plates at a density of 104 cells/well.The cells were divided into 5 groups using a random number table: control group(group C, n=12), H2O2 group(n=12), H2O2 plus ulinastatin group(group HU, n=12), H2O2 plus ulinastatin plus LY294002 group(group HUL, n=9)and H2O2 plus LY294002 group(group HL, n=9). In group H2O2, H2O2(final concentration 200 μmol/L)was added, and the cells were incubated for 12 h in the incubator.In HU, HL and HUL groups, ulinastatin(final concentration 50 U/ml), LY294002(final concentration 10 μmol/L), and ulinastatin(final concentration 50 U/ml)plus LY294002(final concentration 10 μmol/L)were added, respectively, the cells were incubated for 2 h, H2O2(final concentration 200 μmol/L)was then added, and the cells were incubated for 12 h in the incubator.The cell survival rate was measured by methyl thiazolyl tetrazolium assay.The levels of superoxide dismutase(SOD)and malondialdehyde(MDA)were determined by using water soluble tetrazolium-1 and thiobarbituric acid colorimetric assays.Cell apoptosis was measured by flow cytometry, and apoptosis rate(AR)was calculated.The expression of Akt, phosphorylated Akt(p-Akt), mTOR, phosphorylated mTOR(p-mTOR), activated caspase-3 and activated poly-ADP-ribose polymerase(PARP)was detected by Western blot.The p-Akt/AKT and p-mTOR/mTOR ratios were calculated. Results Compared with group C, the cell survival rate was significantly decreased, AR was increased, the SOD activity was decreased, the MDA content was increased, the expression of activated caspase-3 and PARP was up-regulated(P 0.05). Compared with group H2O2, the cell survival rate was significantly increased, AR was decreased, the SOD activity was increased, the MDA content was decreased, the p-Akt/AKT and p-mTOR/mTOR ratios were increased, and the expression of activated caspase-3 and PARP was down-regulated in group HU(P<0.05). Compared with group HU, the cell survival rate was significantly decreased, AR was increased, and the expression of activated caspase-3 and PARP was up-regulated in group HUL(P<0.05). Conclusion The mechanism by which ulinastatin reduces oxidative stress injury to cardiomyocytes is related to activation of Akt/mTOR signaling pathway in rats. Key words: Trypsin inhibitors; Protein-serine-threonine kinases; Oxidative stress; Myocytes; Cardiac