Application Research Of Z-DEVD-fmk To Reduce Red Blood Cell Storage Damage

Objective:It observes cysteine-containing aspartate-specific protease,Caspase-3’s inhibitor Z-DEVD-fmk’s function of inhibiting the activity of Caspase-3 in red blood cells of storage(sRBC)to reduce storage damages of sRBC.Methods:1.Extract fresh sRBC and divide it into six groups:the group of normal saline(NS)(Group A),the group of 10μmol/L Z-DEVD-fmk inhibitor(Group B),the group of 30μmol/L Z-DEVD-fmk inhibitor(Group C),the group of 50μmol/L Z-DEVD-fmk inhibitor(Group D),the group of 100μmol/L Z-DEVD-fmk inhibitor(Group E)and the DMSO group(Group F).Add normal saline of 0.9%100μl into Group A;add Z-DEVD-fmk 100μl of different concentrations respectively into Group B,Group C,Group D and Group E and gently shake each to make them well mixed and the medicine evenly distributed and their final concentrations are respectively 10μmol/L,30μmol/L,50μmol/L,100μmol/L;add equivalent dimethyl sulfoxid(DMSO)100μl into Group F.Store them in a refrigerator within the inside temperature(4 ± 2)℃.2.Record and measurement index2.1 Extra-erythrocyte osmotic pressure:Measure the extra-erythrocyte osmotic pressure on the 1st and 21st days after drug addition2.2 MDA content:the thiobarbituric acid method detect the content of MDA in red blood cells of storge on the 1,7,14 and 21 days after the addition of the drug.2.3 Caspase-3 activity and ATP content:The Caspase-3 activity and ATP contentin in red blood cells of storge were detected by ELISA method on the 1,7,14 and 21 days after the addition of the drug.2.4 PS exposure rate on the stored red blood cell membrane was detected by a universal cytometer on the 1,7,14,and 21 days after the addition of the drug.Results:1.Extra-erythrocyte osmotic pressure:The extra-erythrocyte osmotic pressures of each group were in the normal osmotic pressure range on the 1st and 21st days after adding the medicine.2.The thiobarbituric acid method detects MDA content in stored red blood cell:On the 1,7,14,21 days after administration of stored red blood cell,as the stored time prolonged,the MDA content in the stored red blood cell of each group gradually increased,and there are statistically significant differences in MDA content at different time points within each group(P<0.05).On the 7,14,and 21 days after administration of stored red blood cell,compared with group A,the MDA content in the stored red blood cell of groups B,C,D,and E were lower than that of groups A(P<0.05),but there was no statistical difference in group F(P>0.05);Compared with group E,the increase of MDA content in stored red blood cell of group B and C was below that of group E.(P<0.05).3.ELISA method detects Caspase-3 activity in stored red blood cell:On the 1,7,14 and 21 days after administration of the stored red blood cell,as the stored time prolonged,the Caspase-3 activity in the stored red blood cell of each group gradually increased,and There are statistically significant differences in Caspase-3 activity at different time points within each group(P<0.05).On the 7,14,and 21 days after administration of stored red blood cell,compared with group A,the activity level of Caspase-3 in stored red blood cell of groups B,C,D,and E were lower than that of group A(P<0.05),and group F had no difference(P>0.05).Compared with the group E,the Caspase-3 activity in the stored red blood cell of the groups B and C was below that of the group E(P<0.05).4.ELISA method detects ATP content in the stored red blood cell:On the 1,7,14 and 21 days after administration of the stored red blood cell,with the prolonged storage time,the ATP content in the stored red blood cell of each group gradually decreased,and there are statistically significant differences in ATP content at different time points within each group(P<0.05).On the 7,14,and 21 days after administration of stored red blood cell,compared with group A,the ATP levels in the stored red blood cell of groups B,C,D,and E were higher than those in group A(P<0.05),there was no difference in group F(P>0.05).Compared with group E,the ATP concenntration in the stored red blood cell of group B and C was higher than that of group E(P<0.05).5.PS exposure rate on membrane of the stored red blood cell:On the 1,7,14 and 21 days after administration of the stored red blood cell,as the storage time increased,the exposure rate of PS on the red blood cell membrane of each group gradually increased,and there are statistically significant differences in PS exposure rate of stored red blood cell membrane of each group at different time points(P<0.05).On the 7,14,and 21 days administration of the stored red blood cell,compared with group A,the PS exposure rate on the stored red blood cell membrane of groups B,C,D,and E were lower than that of the grouop A(P<0.05),but there was no difference in group F(P>0.05)Compared with the E group,the PS exposure rate on the stored red blood cell membrane of the B and C groups was lower than that of the E group(P<0.05).Conclusion:1.As the storage time increases,the ability of stored red blood cells to resist oxidative stress decreases,and the activity of Caspase-3 increases,which further damages stored red blood cells.2.Using Z-DEVD-fmk to inhibit the activity of Caspase-3 can effectively reduce the storage damage of the stored red blood cell.In this experiment,the appropriate concentration range is 10-30μmol/L.3.The damage mechanism of stored red blood cells may be related to the increase in apoptosis mediated by Caspase-3,Z-DEVD-fmk has a certain protective effect.