The Effects Of RhTPO On Mature Platelets In Lipopolysaccharide Induced Thrombocytopenia

Objective:To investigate the effect of rh TPO on mature platelets in mice with thrombocytopenia(TCP)induced by lipopolysaccharide(LPS),and to provide theoretical basis for the clinical application of rh TPO in patients with thrombocytopenia.Content:Male SPF C57BL/6 mice were randomly divided into 5 groups:(1)sham-operated group(Sham group);(2)experimental control group(LPS group);(3)low dose group:LPS+rh TPO 1.35×10~3 U/kg·d group(L Group);(4)medium dose group:LPS+rh TPO 2.7×10~3 U/kg·d group(M group);(5)high dose group:LPS+rh TPO5.4×10~3 U/kg·d group(H Group).In the model group,single intraperitoneal injection of LPS 30 mg/kg was used to make TCP model,while in the Sham group,intraperitoneal injection of the same volume of normal saline was used.The drug intervention was started immediately after modeling,and the corresponding dose of rh TPO was injected subcutaneously every 24 h.All groups were observed for 72 h.The first part:To study the therapeutic effect of different doses of rh TPO on LPS induced TCP mice.The changes of platelet count in peripheral blood and 72 h survival rate were observed.The second part:To study the effect of rh TPO on LPS induced mature platelets in TCP mice.The proportion of CD61~+CD62P~+cells in platelet rich plasma was detected by flow cytometry to reflect the platelet activation;The expression of CD41 in lung and spleen was detected by immunohistochemistry,which reflected the condition of platelet seizure in organs.The levels of IL-1βand IL-18 in platelet rich plasma were detected by ELISA,the expression of Caspase-1 and gasdermin D(GSDMD)in platelet rich plasma was detected by flow cytometry,which reflected the condition of platelet scorch.Results:Part 1.After LPS modeling,the platelet count of mice in each group began to decrease,and the platelet of mice in L group increased significantly at 48-72h;the survival rate of mice in LPS group was significantly lower than Sham group,and the survival rate of mice in L group was slightly higher than LPS group,but the difference was not statistically significant;combined with the results of platelet count and mortality,the establishment of mouse TCP model was successful.Part 2.After LPS,the proportion of CD61~+CD62P~+cells in platelet rich plasma of mice in each group was significantly increased.The proportion of CD61~+CD62P~+cells in group L was significantly lower than LPS group.The change of CD61~+CD62P~+cells in group M was not significant compared with LPS group.The proportion of CD61~+CD62P~+cells in group H was slightly higher than LPS group.The positive expression of CD41 in the lung and spleen of LPS model group was significantly increased.After LPS model making,the positive expression of CD41 in the lung of rh TPO group(L,M group)was significantly decreased compared with LPS group,while the positive expression of CD41 in the lung of H group was not significantly changed compared with LPS group;after LPS model making,the positive expression of CD41 in the spleen of rh TPO group(L,M,H Group)was significantly reduced;the positive expression of CD41 was significantly lower than LPS group.The levels of IL-1βand IL-18 in the platelet rich plasma of mice in LPS group were significantly higher than Sham group,while those in L group were significantly lower than LPS group.Compared with Sham group,GSDMD protein in platelet rich plasma of LPS model mice was significantly higher,L group was significantly lower than LPS group,and M,H group was not significantly changed compared with LPS group.Compared with Sham group,Caspase-1 of platelet rich plasma in LPS group was significantly higher,L and M group was significantly lower than LPS group,while H group was not significantly changed compared with LPS group.Conclusion:Intraperitoneal injection of LPS induced TCP in mice.Low dose rh TPO has certain protective effect on LPS induced TCP mice,and its mechanism may be related to inhibiting the activation of mature platelets,reducing the internal attachment of platelets and inhibiting the pyroptosis of platelets.The higher dose of rh TPO had less protective effect on LPS induced TCP mice,suggesting that there might be threshold effect in the application of rh TPO in LPS induced TCP.