Study The Immunoregulation Effect Of Forsythia Suspense Extract On Sepsis Mouse Induced By CLP And Its Mechanism

Aim:Our objectives to investigate the immunological effect of Forsythia suspense extract(FS) in models of sepsis by cecal ligation and puncture(CLP) and in vitro,and elucidate the protective effect of FS on models of CLP and study its related mechanism,in order to supply new theory to clinical therapy.Methods:1.To investigate the effect of FS extract on the behaves of the mouse lymphocytes in vitro, lymphocytes were prepared from lymph nodes of BalB/c mice.Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the expression of CD69,CD25 and CD71 by T cells in response to Concanavalin(C onA).After the staining with CFDA-SE, mouse lymphocytes were stimulated with polyclonal activators C onA.The proliferation related index of mouse lymphocytes were analyzed with flow cytometry.The distribution of the cell-cycle was analyzed by PI staining together with Flow cytometry analysis.2.In order to elucidate the effect of FS extract on models of CLP and primarily study the protect mechanism on T lymphocytes of sepsis model induced by CLP,the experimental sepsis model was established by cecal ligation and puncture(CLP).Observed the mouse mortality rate of sepsis model in 7days and calculated the index about thymus and spleen.The plasma ALT/AST levels were detected by ALT/AST assay kit,respectively.After the staining with CFDA-SE,mouse lymphocytes were stimulated with polyclonal activator C onA.And the proliferation related index of mouse lymphocytes was analyzed with flow cytometry as well as MTT.3.Primarily study the protect mechanism on native immunity of sepsis model induced by CLP. Changes of reactive oxygen species(ROS) in neutrophils were analyzed by H₂DCFDA staining with flow cytometry only at 6h after CLP.Concentration of H₂O₂ and NO in serum were measured by H₂O₂ assay kit and NO assay kit at 6h after CLP,respectively.The effect of FS extract on cyto-phagocytesis in vitro was analyzed by flow cytometry and NO production of the peritoneal macrophages in vitro was measured by NO assay kit.Results:1.The studies in vitro showed that,as the concentration increased,FS extract significantly inhibited the activation and proliferation of the mouse lymphocytes induced by C onA.Flow cytometry analysis of the cell-cycle distribution revealed that FS extract block the progression of mouse lymphocytes into S phases.And it can also protect the apoptosis of thymus lymphcytes induced by dexamethasone.2.The studies in vivo demonstrated that FS extract can protect mice against lethality after CLP. It can also lead to splenic atrophy characterized by a reduction in organ size.However,compared with CLP-treated mice,FS extract can significantly inhibit sepsis induced atrophy in spleen and enhance the index of spleen and thymus.The plasma ALT/AST levels detected by ALT/AST assay kit showed that FS decrease the production of ALT/AST.The proliferation related index of mouse lymphocytes stimulated by C onA show that FS extract can also inhibit the proliferation of T lymphocytes in early state of inflammation of sepsis.3.After operation of CLP,the levels of ROS(6 h),H₂O₂(9 h) and NO in serum of CLP group were higher than Sham and Nature group.FS can decrease the all productions of them.The studies in vitro showed that FS may promote phagocytosis of peritoneal macrophages of normal mouse and inhibit NO production stimulated by LPS in vitro.Conlusion:1.Study in vitro shows that FS can significantly inhibit the activation and proliferation of the mouse lymphocytes,and blocks the progression of mouse lymphocytes into S phase.Moreover, it can also decrease the apotosis of mouse lymphocytes.2.The effects of FS have been studies in modles of CLP and in vivo.FS can prctect mouse thymus and spleen from destruction by sepsis.It can decrease the level of ALT in serum significantly and inhibit the proliferation of T lymphocytes in early state of inflamenation.3.In the early state of sepsis induced by CLP,FS may eliminate the toxic effect of free radical (ROS,H₂O₂ and NO) and increase the expression of the native immunity which contributes to the protective mechanisim from sepsis mouse.