Effects Of Flos Lonicerae On Mouse T Lymphocytes Behavior And Immunoregulation In Sepsis

Aim:To investigate the effect of Flos lonicerae on mouse T lymphocytes behavior and sepsis,and elucidate the protective mechanism,in order to supply theory to clinical therapy.Methods:1.Lymphocytes isolated from lymphoid nodes of BalB/c mouse were stained with fluorescence conjugated monoclonal antibodies and flow cytometry analysis was employed to detect the effects of FL on the activation of mouse lymphocytes treated with Con A.The effects of FL on the proliferation rate of mouse lymphocytes were also analyzed by CFDA-SE staining.The distribution of the cell-cycle was analyzed by PI staining together with flow cytometry analysis.2.For experiment sepsis,mouse was intraperitoneal injection(i.p.) of a bacterial suspension containing 10⁸ live escherichia coli(E.coli) cells.We monitored the effect of FL on the mouse mortality rate in the experimental sepsis.The spleens and thymi were removed and cell yield was counted,and we compared spleen index and thymi index of different groups.Apoptosis of thymocytes DEX were detected by AnnexinV-FITC/PI staining together with flow cytometry.E.coli stained by CFDA-SE and flow cytometry was utilized to observe the effect of FL on macrophage phagocytosis.3.For LPS-induced experimental sepsis,mice were given intraperitoneal injections of a mixture of LPS and D-galactosamine.The proliferation related index of mouse lymphocytes was analyzed with flow cytometry and MTT.Changes of reactive oxygen species(ROS) in blood were analyzed by H₂DCFDA staining together with flow cytometry.Serum ALT,AST levels and the pathological changes were obtained to study the protective of FL on liver function of septic mouse.Concentration of H₂O₂ in serum was measured by H₂O₂ kit.The effect of FL on NO production of the peritoneal macrophage in vitro was measured by Griess Reagent assay kit.Results:1.The results indicated that FL ihibited the activation of mouse lymphocytes(P＜0.05).CFDA-SE analysis also showed the proliferation of mouse lymphocytes were obviously inhibited by different concentrations of FL.Flow cytometry analysis of the cell-cycle distribution revealed that the cell number in G0/G1 was increased,while the cell number in S and G2/M was decreased as the concentration of FL increased.2.The results demonstrated that septic mouse induced by the intraperitoneal injection of E.coli led to splenic atrophy.However,compared with septic mouse,FL could significantly increase thymus index and spleen index.FL could inhibit the apoptosis of thymus cells induced by Dex(P＜0.01).The survival rate of FL group was much higher than the sepsis group.FL promoted the phagocytosis.3.Establishing sepsis model by intraperitoneal injections of LPS was to analysis the protective mechanism of FL on septic mouse.Compared with the control group,the lymphocytes proliferation reponses of septic mouse were reduced upon stimulation with Con A,and the levels of ALT,AST and H₂O₂ in serum and ROS in blood were increased significantly compared with the normal group,Meanwhile,the levels of inflammatory mediators were increased and morphological structures were damaged..But in the interference group FL could enhance proliferation of lymphocytes,and inhibit the levels of ALT,AST and H₂O₂ in serum and ROS in blood.Moreover,the levels of inflammatory mediators were significantly lower than septic group.FL could inhibit NO production of peritoneal macrophage induced by LPS with Griess Reagent assay kit.Conclusion:1.FL could significantly inhibit the activation and proliferation of mouse lymphocytes in vitro.Flow cytometry analysis of the cell-cycle distribution revealed that FL can inhibit the progression of mouse lymphocytes growing into S and G2/M phase. That indicated FL inhibited specific cellular immune response.These results supply theory to clinical therapy.2.FL could inhibit atrophy in spleen and thymus induced by sepsis.FL could inhibit the apoptosis of thymus cells in vitro and promote the phagocytosis of peritoneal macrophage significantly in sepsis,which contribute to the protective effect on the sepsis mouse in the experimental sepsis induced by the intraperitoneal injection of E.coli.3.In the experimental sepsis induced by LPS,FL may eliminate the toxic effect of free radical and enhance the proliferative response of lymphocytes,which contribute to the protective effect on the sepsis mouse.