Effects Of Preconditioning Of Ulinastatin On GES-1 Cells Injury Induced By Oxygen And Glucose Deprivation

BackgroundAcute gastric mucosal lesion(AGML)is a common perioperative complication in clinical practice,but its pathogenesis is still not completely clear.It is generally believed that gastric mucosal ischemia is the key factor in the progress of AGML.In our previous work,the pathogenesis of AGML was explored from the aspects of invasive stress and non-invasive stress(psychological stress)by water-immersion restraint stress(WIRS)model in rats.However,its potential mechanism has not been explored,and the possible protective measures for AGML from the perspective of ischemia are still very limited.Ulinastatin(UTI),as a broad spectrum urinary trypsin inhibitor,has the functions of reducing reactive oxygen species(ROS),stabilizing lysosomal membrane,reducing inflammation and improving circulation.Previous studies showed that UTI has a protective effect on intestinal mucosa in critically ill patients,but whether it has a protective effect on gastric mucosa has not been reported.ObjectiveIn our study,the human gastric mucosal epithelial cell line(GES-1)was used to construct the "ischemic" model by oxygen and glucose deprivation(OGD).Our work are prepared to research the mechanism of AGML and the effects of ulinastatin preconditioning on GES-1 cells injury induced by oxygen and glucose deprivation from the perspective of gastric mucosal ischemia.Contents and Methods1.To build the OGD model for GES-1 cellsGES-1 cells were given different OGD time(0,2,4,6,8h)through glucose-free medium and three-gas incubater.Cell viability and apoptosis induced after oxygen and glucose deprivation were assessed by CCK-8,Hoechst staining and flow cytometry analysis.Western Blot was used to examine the protein expressions of Bcl-2 and Bax.2.To investigate the appropriate concentration of UTIThe GES-1 cells were treated with UTI at concentrations of 0,100,200,400,800,and 1000 U/mL respectively,the CCK-8 assay was performed 12 h later.The results showed that the cell viability of the group of 1000 U/ml was significantly lower than that in the group of OU/ml(P<0.01).Next,the cells were divided into normal control group,oxygen glucose deprivation group,different concentrations of UTI preconditioning group(UTI concentrations were 100,200,400,800 U/mL).The CCK-8 assay was performed to examine the cell viability.3.To investigate the mechanism of UTI to attenuate the OGD injury of GES-1 cellsGES-1 cells were randomly divided into normal control group(Group N),oxygen and glucose deprivation group(Group O)and ulinastatin preconditioning group(Group U).The cells in Group O and Group U were treated with OGD for 6 h,and Group U cells were treated with UTI(800 U/ml)for 12 h before OGD.The cell viability and apoptosis were determined by CCK-8 and flow cytometry analysis respectively.Western Blot was used to examine the protein expression of Caspase-3 and Cleaved Caspase-3.The TRPV1 mRNA expression was measured by quantitative real-time PCR.Results1.The results showed that as the OGD time was prolonged,the cells viability of GES-1 cells was significantly decreased,apoptotic cells and the apoptosis rate were significantly increased,and the ratio of apoptosis-related protein Bax/Bcl-2 increased markedly(P<0.01 or P<0.05).2.Compared with the oxygen glucose deprivation group,the cell viability increased significantly at UTI concentration of 400U/ml and 800U/ml,and the cell viability was the highest at the concentration of 800 U/ml(P<0.01).3.Compared with Group N,the viability of GES-1 were decreased,the apoptosis rate and the expression of Caspase-3 and Cleaved Caspase-3 were increased,and the TRPV1 mRNA expression decreased greatly in Group O(P<0.05).Compared with Group O,the mentioned changes above were significantly inhibited in Group U.ConclusionsThe OGD model of GES-1 cells was successfully built using glucose-free medium and three-gas incubater;Ulinastatin preconditioning could effectively inhibit the cells injury of GES-1 induced by OGD,which may be related to the reduced cell apoptosis and the upregulation of the TRPV1 mRNA expression.