The Effects Of Farmesyltransferase Inhibitor,FTI-277, On Immune Function And Outcome Of Mice With Sepsis

Part I The relationship of sepsis and farneslated protein/farnesyltransferaseactivityObjective:To investigate therelationship of sepsis and farnesylation and explore the effect of farnesyltransferase inhibitor,FTI-277,on the farnesylation in septic mice.Methods:Create the sepsis model in mice with cecal ligation and puncture (CLP),using sham operation as a control.At2h after CLP or sham operation, inject FTI-277(25mg/kg BW, IP),or inject PBS. There are four groups: Sham+PBS; Sham+FTI-277; CLP+PBS; CLP+FTI, each group with4-6mice. At16h after CLP or sham operation, euthanize the mice with cervical dislocation. Isolate the spleen, then detect the farnesylated protein in the spleen of mice with immunohistochemistry and ELISA. Evaluate the farnesyltransferase activity with3H. The data are compared with one-way analysis of variance followed by Newman-Keuls comparison or student's t test.Results:Sepsis increased farnesylated protein and farnesyltransferase activity in mouse spleen and FTI-277reversed it.Conclusion:Fanesyltransferase activity and farnesylated protein were increased by sepsis in mouse spleen. FTI-277has the effect on reducing it. Part Ⅱ The relationship of farnesyltransferase inhibitor and phagocytosis of macrophagesObjective:To study the effect of farnesyltransferase inhibitor,FTI-277,on the phagocytotic activity of macrophages.Methods:Create the sepsis model in mice with cecal ligation and puncture (CLP),using sham operation as a control.At2h after CLP or sham operation, inject FTI-277(25mg/kg BW, IP),or inject PBS. There are four groups: Sham+PBS; Sham+FTI-277; CLP+PBS; CLP+FTI, each group with4-6mice. At16h after CLP or sham operation, euthanize the mice with cervical dislocation. Take the blood and peritoneal lavage from the mice. Evaluate the bacteria load in the blood and peritoneal lavage with bacterial culture.Analyze the phagocytosis of mavrophages with flowcytometry. The data are compared with one-way analysis of variance followed by Newman-Keuls comparison or student's t test.Results:The bacterial load were extremely high in the blood and peritoneal lavage of septic mice, and the treatment of FTI-277improved bacterial clearance in both blood and peritoneal lavage. Sepsis increased the total number of macrophages in the peritoneal cavity of septic mice, and impaired the phagocytotic activity, which could be reversed by FTI-277.Conclusion:Frnesyltransferase inhibitor played a critical role in improving the bacterial clearance and phagocytosis of macrophages. Part Ⅲ The study on the relationship of farnesyltransferaseinhibitor and immune function of septic miceObjective:To investigate the immunosuppression induced by sepsisand explore the effect of FTI-277on the immunosuppression.Methods:Create the sepsis model in mice with cecal ligation and puncture (CLP),using sham operation as a control.At2h after CLP or sham operation, inject FTI-277(25mg/kg BW, IP),or inject PBS. There are four groups: Sham+PBS; Sham+FTI-277; CLP+PBS; CLP+FTI, each group with4-6mice. At16h after CLP or sham operation, euthanize the mice with cervical dislocation. Isolate the splenocytes. Evaluate the expression of CD4+Foxp3+T cells and PD-L1/PD-1in the spleen with flowcytometry. Culture the splenocytes ex vivo together with anti-CD3+CD28antibody. Measure the concentration of IFN-γ and IL-4in the supernatant. Test the proliferation activity of T cells with resazurin dye solution. The data are compared with one-way analysis of variance followed by Newman-Keuls comparison or student's t test.Results:After CLP, the percentage of CD4+Foxp3+T cells were increased in the spleen, the secretion of IFN-y were inhibited, the proliferative response to anti-CD3+CD28antibodies of T cells were impaired and the expression of PD-L1and PD-1were enhanced. FTI-277has the effect on improving all the changes above.Conclusion:The results implied that sepsis induced the dysfunction of splenic T cells of mice, resulting in the immunosuppreion of splenocytes. FTI-277significantly improved the immunosuppression of splenocytes. Part IV The relationship of farnesyltransferase inhibitor and prognosis of septic miceObjective:To evaluate the prognosis of septic mice with the treatment of farnesyltransferase inhibitorMethods:Create the sepsis model in mice with cecal ligation and puncture (CLP),using sham operation as a control.At2h after CLP or sham operation, inject FTI-277(25mg/kg BW, IP),or inject PBS. There are four groups: Sham+PBS; Sham+FTI-277; CLP+PBS; CLP+FTI, each group with4-6mice. To evaluate the survival time based on observation for7days after CLP or sham operation. At16h after CLP or sham operation, euthanize the mice with cervical dislocation. Take the blood from mice. Measure the concentration of HMGB1by ELIS A. Evaluate the cardiac function with echocardiography and LV function test. The data are compared with one-way analysis of variance followed by Newman-Keuls comparison or student's t test.Results:FTI-277dramatically improved the survival rate of septic mice, inhibited the elevation of HMGB1and reduced the apoptotic cells in spleen and thymus.Conclusion:One of the mechanism for FTI-277improving the survival rate of septic mice may relate to its function on suppressing the secretion of HMGB1and reducing the apoptosis in spleen and thymus.