The Effect Of Thalidomide On LPS Induced Endothelial Cell Injury And Its Preliminary Mechanism

Objective:To explore the role of thalidomide in oxidative stress injury and its possible mechanism.Methods:Lipopolysaccharides?Lipopolysaccharides,LPS?is used to construct the oxidative stress damage model in vitro.0.25?g/ml,0.5?g/ml,1?g/ml,2?g/ml LPS are used to treat HUVEC for 6 h,12 h,24 h respectively.The best building time and concentration are determined by flow cytometry.MTT assay is used to determine the cytotoxic concentration of thalidomide.This study is divided into six groups:Control group,model group,thalidomide alone treatment group,1?g/ml thalidomide+1?g/ml LPS,10?g/ml thalidomide+1?g/ml LPS,100?g/ml thalidomide+1?g/mlLPS.The cells are pretreat with Thalidomide for 6h,12h,24h and then treated with LPS for 24h.TUNEL and flow cytometry are used to determine the optimal pretreatment time of thalidomide and the protection of different concentrations of thalidomide.The expression of NF-?B downstream P65 and P-P65 is detected by Western Blot.And Flow cytometry is used to detect the production of ROS and mitochondrial membrane potential.The expression of Bax,Bcl2 and Hif-1?are mearsured by Q-pcr.Results:MTT results show that the concentration of thalidomide is harmless in the range of0¹25?g/m L,so the maximum concentration of thalidomide is selected as the concentration of subsequent tests.Flow cytometry results show that low concentration of LPS can lead to apoptosis,.Compared with the treatment for 6h and 12h,0.25?g/ml and 0.5?g/ml LPS treat the cells for 24h respectively,the apoptosis rate increase significantly?P<0.05?.And after1?g/ml LPS process HUVEC for 24h,the apoptosis rate is up to 92%,the difference was statistically significant when compared with the apoptosis rate after treatment for 6 h and 12h?P<0.05?..2?g/ml LPS treat HUVEC has no difference in the apoptosis rate after treatment with 1?g/ml LPS at each time point.The apoptosis rate of 100?g/ml Thalidomide pretreatment for 6 h is obviously lower than the model group,and the difference is statistically significant?P<0.05?.But when compared with pretreatment of 12 h,24 h,the apoptosis rate is basically no change,and there is no statistically significant difference?P>0.05?.In addition,the apoptosis rate?31.5%?of 100?g/ml thalidomide pretreatment is compared with that of 10?g/ml?52.6%?and 1?g/ml?68.6%?.The apoptosis rate decrease significantly,and the difference is statistically significant?P<0.05?.Results of Western Blot show that the maximum phosphorylation levels expression of p-P65 is at 15 min.At the same time,the expression of total P65 has not changed and the expression of p-P65 is gradually reduce with the increase of concentration of Thalidomide,the difference is statistically significant.?P<0.05?.The production of ROS is gradually decreased and the mitochondrial membrane potential rise to different extent alone with the increase of pretreatment concentration of Thalidomide and when compared with model group,the difference was statistically significant?P<0.05?.Meanwhile,by detecting the expression of Bax,Bcl₂ and Hif-1?,we find that the expression of Bax gradually decreased with the increase of thalidomide concentration,and the expression of Bcl₂ gradually increase.The difference was statistically significant?P<0.05?.The expression of Hif-1?decrease with the increase of thalidomide concentration.Conclusion:LPS can successfully build oxidative stress damage model in vitro.And pretreatment with Thalidomide can protect mitochondria function to alleviate oxidative stress damage.This study provides a better understanding of clinical research of Thalidomide.