The Effects Of Celecoxib On Human Brain Microvascular Endothelial Cells Release 6-keto-PGF_1?,TXB₂ And Apoptosis After Radiation

Part1:The effects of Celecoxib on human brain microvascular endothelial cells apoptosis after radiationObjective:To observe the effects of Celecoxib on human brain microvascular endothelial cells release 6-keto-PGF1?,TXB2 and apoptosis after radiation.Methods:The logarithmic growth phase cells were divided into control groups?Con?,irradiation groups?IR?and combination groups?IR+C?,and the further experiments underwent after HBMECs incubated with celecoxib for 24h.CCK-8 assay was used to determine the half inhibitory concentration of HBMECs?IC50?and to determine the appropriate drug concentration.Clone formation assay was used to calculate the clone formation rate and take shape the survival curve.Annexin V-FITC/PI double staining method was used to measure the apoptosis rates of HBMECs at different time points in different groups.Western blot was used to measure the protein expression of Cleaved caspase-3,P-JNK and Cox-2.Results:1.CCK-8 assay showed that the inhibition rates of the groups with different drugconcentration were 4.52±0.13%,6.31±0.07%,9.01±0.10%,15.26±0.70%,70.65±089%.The IC50 was 81.50±0.75?mol/L which obtained by the statistic software of SPSS19.0,and 30?mol/L drug concentration?sublethal concentration?was used in the further experiments.2.The single-hit multi-target model?y=1-?1-exp?-k^*x??^{^}N?was uesed to evaluated the radiosensitivity of celecoxib on HBMECs.The values of D0,Dq,SF2 and SER were calculated.Results suggested that there was no radiosensitizing effects when HBMECs cultured with 30?mol/L of celecoxib.?SER=0.96?.3.Compared with Con groups,the apoptosis rates of IR or IR+C groups increased over time?p<0.05?;Compared with IR groups,the apoptosis rates of IR+C groups decreased over time?p<0.05?.4.Western bloting showed that the expressions of Cleaved caspase-3,P-JNK and Cox-2 were rare in normal HBMECs,but increased significantly in IR groups,and the expressions of Cleaved caspase-3 and Cox-2 increased over time.Compared with the IR groups,the expressions decreased in IR+C groups.Conclusions:The apoptosis rates of HBMECs increased after irradiation,while the30?mol/L concentration of celecoxib cuold reduce the apoptosis rates after radiation.The mechanisms may be related to the inhibition of Cox-2 expression,which could influence the expression of Cleaved caspase-3 and P-JNK in HBMECs.Part2:The effects of Celecoxib on human brain microvascular endothelial cells release 6-keto-PGF_1?and TXB₂ after radiation.Objective:To observe the changes of celecoxib release 6-keto-PGF_1?,TXB₂ and the ratio of TXB₂/6-keto-PGF_1?on HBMECs after radiation.Methods:The IR groups and IR+C groups were incubated with 30?mol/L celecoxib for 24h befere radiation.The culture medium was centrifugated for 10 minutes at approximately 3000 r/min.The following experiment was according to the procedures of specification.Read the Optical Density?O.D.?at 450nm using a Microplate Reader.Sample concentrations were calculated after compared with the standard curves.Results:Compared with Con groups,the TXB₂ in IR groups and IR+C groups?24,48h?were increased?p<0.05?,and the 6-keto-PGF_1?in IR and IR+C groups were decreased after6h?p<0.05?;Compared with the IR groups,the TXB₂ in IR+C groups were decreased?p<0.05?,while the 6-keto-PGF_1?were not?p>0.05?;Compared with Con groups,the ratios of TXB₂/6-keto-PGF_1?were increased in IR and IR+C groups?p<0.05?,but decreased in IR+C groups compared with IR groups?p<0.05?.Conclusions:Celecoxib?30?mol/L?could inhibite the TXB₂ releasing and decrease the ratio of TXB₂/6-keto-PGF1?,after ridiation on HBMECs,but not the releasing of6-keto-PGF_1?.Showed that celecoxib could inhibite the cells from releasing the adverse factors of thrombosise after radiation,this provided the cytological evidences of celecoxib treated radiation-induced brain injury by inhibiting the formation of thrombosis.