The Impacts Of Ulinastatin On Mitochondrial Membrane Potential, BCL-2 And MCU In Rat Hippocampal Neurons Following Oxidative Stress Injury

Objective:To observe the changes of mitochondrial membrane potential,energy metabolism,BCL-2 and MCU in hippocampal neuron following oxidative stress damage.The potential protective effects of ulinastatin on oxidative stress injury in hippocampal neurons and the possible mechanism were also studied.Methods:Primarily cultured neurons with serum-free medium on the ninth day were randomly divided into 4 groups:control group(N),H2O2 group(H),1000u/ml ulinastatin+H2O2 group(UTI1+H)and 2000 u/ml ulinastatin+H2O2 group(UTI2+H),which were isolated from the fetal rats hippocampus of SD pregnant rats and the oxidative stress injury model was established by using H2O2.Next,to investigate the morphological changes of neurons with inverted phase contrast microscope,evaluating the activity of Na+-K+-ATPase and Ca2+-Mg2+-ATPase with spectrophotometer,detecting the mitochondrial membrane potential with fluorescence microscope and apoptosis rate with Annexin-V-Fluorescein/PI and Hoechst33342/PI,and quantifying the assays of BCL-2 and MCU with RT-PCR and Western bloting.Results:1.The primary hippocampal neurons on the 8 to 14 days were the best cultured with serum-free medium,which had stereoscopic nucleus and reticular axons.The purity of neurons was up to 92.05% which was meeting the experimental requirements.2.In the H group,the neurons were characterd by cytoplasmic concentration,marginal nucleus,dilated cell gap,axonal disruption,and a few suspending cells,while the axons of neurons in UTI1+H and UTI2+H group were still connected into a network and the cell body had a halo.3.The activity of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in H group was lower than that in N group,UTI1+H and UTI2+H groups(P<0.05).The activity between UTI1+H group and UTI2+H group has significant difference(P<0.05).4.The mitochondrial membrane potential was lower and apoptosis rate was higher in H group than those in N group,UTI1+H and UTI2+H group,and the difference between UTI1+H group and UTI2+H group was statistical significance(P<0.05).5.The expression of BCL-2 was lower and MCU was higher in H group than those in N group,UTI1+H and UTI2+H group,and the difference of BCL-2 between UTI1+H and UTI2+H group was statistical significance(P<0.05),while there were no significant difference of MCU between the two group(P>0.05).Conclusion:1.Oxidative stress injury can trigger energy metabolism disorders,mitochondrial membrane potential decreased,cell apoptosis of neurons.2.The concentration of 1000u/ml,2000 u/ml ulinastatin can improve the oxidative stress injury to hippocampus neuron cell,which may be related to reducing cell apoptosis by maintaining cell energy metabolism,stabilizing mitochondrial membrane potential,increasing the BCL-2 expression and decreasing the expression of MCU.