Study The Effect Of Morin On Murine T Lymphocytes Behavior And Immunoregulation In DTH And Sepsis

Aim:To investigate the effect of morin on murine T lymphocytes behaviors, DNFB-inducedDTH and E. coli-induced sepsis, in order to explore the mechanism of immunoregulation effectof morin.Methods:Murine lymph node-derived T lymphocytes were separated and stimulated withconcanavalinA (ConA) or phorbol 12,13-dibutyrate (PDB) plus ionomycin (Ion), establishing thedexamethasone (Dex)-induced T lymphocyte apoptosis model. To investigate the effect of morinon T lymphocyte behaviors and Dex-induced thymocyte apoptosis. Using fluorescent-labeledantibody to detect the molecule expressed on the surface of T cells, carboxylfluorescein diacetate,succinimide ester (CFDA-SE) staining to detect T cell proliferation, propidium iodide (PI)staining to detect the cell-cycle of T cell and sub-diploid ratio of Dex-induced thymocyte.Establishing the Dinitrofluorbenzene (DNFB)-induced delayed type hypersensitivity (DTH)model to evaluate the immunoregulation effect of morin on DTH, the effect was observed bythymus index, spleen index and the impact of morin on the inflammatory cell infiltration andtissue edema of of murine ear. Using the light microscopy to observe the histological changes ofmouse ear, the monotetrazolium (MTT) colorimetry to observe the proliferation of lymph nodelymphocytes and spleen lymphocytes respectively; establishing sepsis model by injectingintraperitoneal injection (i.p.) with a bacterial suspension containing 10~8 live escherichia coli (E.coli) cells, monitored the effect of morin on the mouse mortality rate in the sepsis model,using Annexin-V/PI staining to mesure apoptosis of lymphocyte, Griess reaction to detect NOsecretion of peritoneal macrophage, and flow cytometry and fluorescence microscope to observephagocytosis of peritoneal macrophage.Results:1. The optical working concentration of Morin was 25, 50 and 100μmol/L, and the mosteffective concentration of morin was 100μmol/L. Morin could down-regulate the ratio of CD69~+T cells and decrease the proliferation indexes (PI) of T cells stimulated by ConA or PDB inCFSE staining experiment.. FCM analysis of PI staining implied that morin could increase theratio of S phase cells in Con A or PDB plus Ion group; The assay of sub-diploid ratio of thymusby PI staining revealed that morin could increase the sub-diploid ratio of thymus induced byDex.2. The results of ear weighting showed that morin significantly inhibited DTH reaction(P＜0.05). Histological observation indicated 70 mg/ml morin markedly reduced the infiltrationof lymphocytes. The proliferation of lymphocytes derived from lymph node or spleen was alsonotably decreased by morin treatment (P＜0.05). Morin could decrease the thymus index andspleen index.3. The survival rate ofmorin (35 mg/ml) group was much higher than sepsis group; Griessreaction analysis revealed that the morin could significantly suppressed the NO production frommacrophages stimulated with LPS. We also found that phagocytosis of peritoneal macrophagewas enhanced in mice after injection subcutaneously (s.c.) with 10 mg/ml morin(P＜0.05), theimpact ofmorin on the apoptosis of lymphocyte in sepsis group was slight (P＞0.05).Conclusion:1. Morin can significantly inhibit ConA stimulated activation and ConA or PDB plus Ionstimulated proliferation of murine T lymphocytes, in which the S phase lagging induced bymorin may serve as one of the major mechanisms, morin can also enhance the apoptosis of thymus cells induce by Dex.2. Morin can significantly inhibit inflammatory cells infiltration and tissue edema of ear inthe DTH mode mice. Suppression of DTH by morin may be ascribed to the reduced proliferationof lymph node cells and spleen lymphocytes.3. Morin can protect mice against lethality after i.p. of E. coli. Treatment with morinmarkedly enhanced phagocytosis of phagocytes. Morin can also inhibit the production of NOfrom peritoneal macrophages induced by LPS. These results suggest that morin can effectivelyprevent and treat sepsis and microbial infection.