The Influence Of Recombinant Human Thrombopoietin On Platelet Apoptosis Induced By Lipopolysaccharide In Vitro

Objective:Lipopolysaccharide(LPS)is the main pathogenic factor of G~-,and it is the key factor to the pathogenesis of sepsis associated thrombocytopenia.Firstly,in this study,we tried to establish an in vitro model of sepsis through using specific concentration of LPS to effect the healthy human washed platelets.Secondly,we used different concentrations of recombinant thrombopoietin(rhTPO)to intervene this model.Our study was intended to investigate the influence of LPS on platelet apoptosis in vitro and the influence of rhTPO on platelet apoptosis induced by LPS.Content and Mehtods:PartⅠ:In vitro,we prepared washed platelets from healthy human and observed platelets under microscope.Then we maked the washed platelets incubate with different concentration of LPS(0μg/mL,5μg/mL,10μg/mL,20μg/mL,40μg/mL)to establish the model of sepsis in vitro.Moreover,we detected the expression levels of Bcl-xl,Bax and Bak in platelet lysis solution by Weston Blot,and estimated the effect of platelet apoptosis induced by LPS and chosed the optimal concentration.PartⅡ:After the in vitro model of sepsis established,we used different concentrations of rhTPO(0mg/mL,25mg/mL,50mg/mL,100mg/mL)to intervene.Similarly,we tested the expression of Bcl-xl,Bax and Bak in platelet lysis solution by Weston Blot,Enzyme linked immunosorbent assay(Elisa)to detect the level of CD40L in platelet reaction and lysis solution,respectively.Microplate reader was used to determinate the activation of Caspase-3,Caspase-8 and Caspase-9.Results:PartⅠ:Under microscope,the platelets were in the shape of circular double disc,without nucleus,single scattered.The platelet count was about 5×10~8/mL,no red blood cells,and white blood cell count was<1/μL.Western blot indicated that the expression of apoptotic protein Bax and Bak was increased and anti apoptotic protein Bcl-xL was also shown increasing trend after different concentration of LPS induced.The increasing trend manifested as a dose-dependent manner and the optimum LPS concentration was 10μg/mL.The semi quantitative analysis of protein bands showed that the expression of Bak and Bax were increased compared with the control group(p<0.05),and the expression of Bcl-xl was higher than the control group(p<0.05).PartⅡ:Western Blot showed that the expression of apoptotic protein Bax,Bak and anti apoptotic protein Bcl-xl were increased in the LPS group compared with the control group,which indicate that the sepsis model was established successfully.The expression of Bax and Bak was reduced in LPS and rhTPO group compared with LPS group,and in a concentration dependent manner,while Bcl-xl expression was increased.The semi quantitative analysis of each protein bands showed that the differences of each group were statistically significant(p<0.05).The result of ELISA suggested that the CD40L expression of platelets reaction liquid was significantly increased in LPS group compared with the control group(71.28±12.30 vs 30.34±5.25,p<0.05),and the CD40L expression of platelets lysis buffer was also elevated(873.9±60.0 vs 793.7±55.2,p<0.05).LPS combined with different concentration of rhTPO groups compared with the single use of LPS group,CD40L concentration of the reaction solution was increased(81.75±9.44,91.20±10.16,112.39±9.30 vs71.28±12.30,p<0.05),as well as the CD40L concentration of platelets lysis buffer(1046.9±124.5,1195.2±146.1,1349.2±142.2 vs 873.9±60.0,p<0.05).Microplate reader indicated that the levels of Caspase-3,Caspase-8 and Caspase-9 was not distinctly increased in LPS group compared with the control group(p>0.05),and there was not significant elevated in LPS combined with low,medium and high concentration of rhTPO group compared with the single use of LPS group.Conclusion:1.LPS could induce the apoptosis of platelets in vitro,which showed that the expression of Bak,Bax and Bcl-xl was increased,and the effect was dose-dependent.2.rhTPO could inhibit the LPS-induced apoptosis of platelet in vitro,which manifest that the expression of apoptotic protein Bak,Bax decreased and the expression of anti-apoptotic protein Bcl-xl increased,and this effect was concentration dependent.3.rhTPO can improve platelet function and promote the activation againest LPS,manifested as the expression of CD40L was increased.