Study The Effects Of Andrographolide On Mechanism Of Sepsis Mouse Induced By Cecal Ligation And Puncture

AIM:To investigate the effect of Andrographolide on T Lymphocyte behaviors in vitro, and elucidate the immunoregulation effect of AN on sepsis models induced by cecal ligation and puncture, in order to explore the mechanism of AN.Methods:1. To investigate the effect of Andrographolide on the behaviors of the mouse lymphocytes in vitro, in order to elucidate the mechanism of the immunosuppressive effect of Andrographolide. Cell suspensions were prepared from mouse lymph nodes. T lymphocytes were treated with different concentrations of Andrographolide, and stimulated with polyclonal activators Con A. Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the expression of CD69. After the staining with CFDA-SE, T lymphocytes were stimulated with polyclonal activators Con A. Flow cytometry (FCM) was used to detect the proliferation of T lymphocytes. The cell-cycle distribution was analyzed by propidium iodide staining. DIOC6(3) staining was used to analyze the apoptosis of lymphocytes induced by dexa methason (DEX). Fluorescence conjugated microbeads and flow cytometry were used to detect phagocytosis. Griess kit was used to detect the production of NO induced by LPS+IFN-y from macrophage. 2. In order to elucidate the effect of Andrographolide 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 CLP. The proliferation related index of mouse lymphocytes was analyzed with MTT. Flow cytometry were used to detect the apoptosis of thymocytes. H2DCFDA staining was used to analyze the changes of reactive oxygen species (ROS) in peripheral blood together with flow cytometry.Results:1. The studies in vitro showed that, In a dose-dependent manner, Andrographolide (10μmol/L, 20μmol/L,30μmol/L,40μmol/L) can significantly inhibit T lymphocytes activation index (P<0.01) stimulated by Con A, and inhibit proliferation index (P<0.01) stimulated by Con A, as well as the progression of T lymphocytes in S phase. Andrographolide can significantly reduce the apoptosis of lymphocytes which were induced by DEX. Andrographolide could promote phagocytosis of macrophages, but inhibit the production of NO which was stimulated with LPS+IFN-y.2. The studies in vivo demonstrated that Andrographolide can significantly inhibit sepsis induced atrophy in spleen and enhance the index of spleen and thymus. The proliferation related index of mouse lymphocytes stimulated by Con A show that Andrographolide can also inhibit the proliferation of T lymphocytes in early state of inflammation of the sepsis, and Andrographolide can also inhibit the apoptosis of thymocytes. Andrographolide can inhibit the production of ROS from neutrophils.Conclusion:Andrographolide has significantly effect on behaviors of mouse T lymphocytes and macrophages, and can protect the early state of inflammation of the sepsis model induced by CLP.