Study The Effect Of Ginsenoside Rg1on Mechanism Of Sepsis Mouse Induced By Cecal Ligation And Puncture

Objective1. To investigate the effects of Ginsenoside Rg1on the macrophages and T lymphocytesbehaviors.2. Study the effect of Rg1on the sepsis mouse induced by cecal ligation and puncture to discussthe related mechanism.Methods1. Influence of ginsenoside Rg1on murine peritoneal macrophages in vitroThe single cell suspension of murine peritoneal macrophages was prepared under sterilecondition. After pre-incubated by Rg1for4hours, the cells were stimulated with LPS at a finalconcentration of10μg/mL,24hours later, adding1μm diameter of the Yellow-Green Beads（1×10¹0/L）, using flow cytometry （FCM） to detect the phagocytosis ability of macrophages; theamount of NO produced by macrophages was detected by Griess kit. H2DCFDA staining wasused to detect the ROS prodcution by macrophages; Fluo-4/AM staining was used to detect theintracellular calcium concentration in macrophages; Sytox Green plus Fluorescence MicroplateRader were used to detect the cells survive condition.2. Effects of ginsenoside Rg1on murine lymphocytes in vitroAfter the3,3’-Dihexyloxacarbocyanine iodide （DIOC₆3） staining, the activity of thelymphocytes was analyzed with flow cytometry. Murine lymphocytes were stimulated by Con Aand treated with different concentration of Rg1. Cell proliferation was measured by carboxylfluorescin diacetate succinmidyl ester staining. The expression of CD69, CD25and CD71,whichwas the marker of early, middle, later activation of CD3⁺T lymphocyte, was measured by flowcytometry（FCM） combined with two-colour immunofluorescent staining of cell surface antigen.Through PI staining, the distribution of the cell cycle was analyzed with flow cytometry. We alsodetected the influence of Rg1on mitochondrial membrane potential by flow cytometry（FCM）combined with JC-1staining.3. The effects of Ginsenoside Rg1on the sepsis mouse induced by cecal ligation andpunctureThe sepsis model was established by cecal ligation and puncture （CLP）, in order toelucidate the effect of Rg1on models of CLP. Then, observing the surial rate of sepsis model in a week and calculating the index of thymus and spleen. The amount of NO produced by bloodserum was detected by Griess kit. The proliferation related index by Con A stimulating of mouselymphocytes was analyzed with flow cytometry after the staining with CFDA-SE.Results1. The studies in vitro showed that, Rg1could inhibit the phagocytosis ability of macrophagestimulated with or without LPS. It also indicated that Rg1could inhibit the production of NO andROS both in the presence and absence of LPS. In addition, Rg1could reduce the apoptosis ofperitoneal macrophages induced by ION. Furthermore, by CHX and CTX inducing, Rg1couldinhibit the cell apoptosis.2. The studies in vitro showed that, Rg1could enhance the activity of lymphocytes.CFDA-SE staining shows that Rg1efficiently enhanced the Con A-induced proliferation oflymphocytes in a time-and dose-dependent manner. Rg1showed modestly increasedproportions of CD69and CD71and CD25in Con A-stimulated CD3+T cells. FCM analysis of PIstaining implied that Rg1imposed a total cell cycle increased cells entering S phase and G2/Mphase. Furthermore, The Rg1could obviously inhibited the reduction of mitochondrialmembrane potential stimulated by Con A.3. The studies in vivo showed that, Rg1can protect mice against lethality after CLP and andenhance the index of spleen and thymus by sepsis induced. Rg1could inhibit the production ofNO in blood serum.Through the Con A stimulating, Rg1can also enhance the proliferation oflymphocytes in sepsis induced by CLP.ConclusionGinsenoside Rg1can regulate the behaviors of murine lymphocyte andperitonealmacrophages s in certain concentration range, moreover, it can protect the sepsis mice inducedby CLP.