The Effect And Mechanism Of Astragalus Polysaccharides By Interfering With The DC Cells Of CLP Mice On Sepsis

Sepsis is a systemic inflammatory response when subjected to external causes, in fact, is an immune disorder process that mainly for early immune excessive, late immunosuppression. The function and secretion of DC is inhibited during sepsis, thus the activation or recovery of DC’s activity can greatly improve the symptoms of sepsis. So we can regard DC cell as a target for treatment of sepsis.Along this line of thought, If we just need to find out a way to activate DC cells to achieve the purpose of the treatment of sepsis?Pharmacopoeia requirements Astragalus, dried roots of Mongolia or Astragalus Astragalus films, mainly produce in the northeast and north of China, has been widely used in traditional medicine. Recent researchs find that Astragalus contains many biological active ingredients, such as polysaccharides, saponins, flavonoids, especially polysaccharides studied so much which is readily available, inexpensive, of clear effective ingredient and mature extraction technology. A large number of scientific experiments show APS has a broad influence on specific immune and non-specific immune. Our laboratory have preliminarily found that APS could active normal mice spleen of DC. So we try Astragalus polysaccharide(APS) for a drug to treat sepsis by target of DC cell to investigate mechanisms of action and provide a basis for clinical treatment for APS.In view of this, this study is divided into two parts:The first part: In vivo experiments, preliminarily study the changes of DC’s number、phenotype and inflammatory factors after APS intervented;The second part: in vitro experiments to further clarify how the APS have an effect on the DC, so as to have a protective effect on septic mice and provide the theory for APS’ applications to the treatment of sepsis as DC a target by cell reinfusion. The first part In vivo experiments, preliminarily study the influence of APS on the DC subsets of CLP mice Methods: 1. Model and Group: 60-C57BL/6-SPF grade healthy male mice, CLP sepsis model mice by the application of cecal ligation and puncture(CLP)were divided into four groups(CLP, APS-50mg/g, APS-100mg/g, APS-200mg/g). Another 15-C57BL/6, SPF healthy male mice, CLP sepsis model mice by the application of cecal ligation and puncture(CLP) were divided into four groups(CLP, DC-105, DC-5x105, DC-106). 2. Cells: Immune beads separation and purification of mouse spleen DC, Respectively get spleen DC cells after 50mg/g、 100mg/g 、200mg/g of APS intraperited for the first day、second day、third day、fifth day(the day CLP modeled recorded as 0 d); 3. Survival rate:at first dayof CLP, the mice in DC group were intraperitoneally injected into mice of 105/DC、5x105/DC、 106 /DC, CLP group injected NS, respectively observed mice at the first day、second day、third day、fifth day; 4. Enzyme-linked assay(ELISA): to detect the expression of cytokines such IL-10, IL-12, IL-6, TNF-α in blood form the supernatant;5. Fluorescence staining: Get two DC subsets by DC stained the CD11c-APC, CD45RB-PE antibody, DC labeled CD40, CD80, CD86, I-A\E to get their expression of surface molecules; 6. Flow cytometry: Flow cytometry Analysis after(5) antibody staining, as well as changes of number after DC being stimulated; 7. Statistical Methods: Statistical analysis by SPSS13.0 statistical software, measurement data that the groups were compared using ANOVA, P <0.05 statistically significant representative. Results: 1. In vivo experiments, CLP mice after being injected by the normal DC can improve its viability, indicating that DC have innate protective effect on the CLP mice; 2. After APS injected into CLP mice, the surface molecules on DC(CD40, CD80, CD86, I-A\E) comparation to untreated expression, was significantly increased, APS can intervene the expression of DC’s phenotype molecule. 3. Spleen DC of CLP mice after APS being injected, a significant increase in the number treated than the one untreated, and the impact on CD11 chighDCs in a dose dose-dependent way, while the CD45 chighDCs increase and lower; 4. Get IL-12, IL-10 from the supernatant of spleen DC after APS-CLP mice, found IL-12 of CD11 chighDCs secretion a dose-dependent relationship with APS, but the IL-10 of CD45 chighDCs secretion a negative correlation; 5. Get IL-6, TNF-γ from the supernatant of mice eye’s blood after APS-CLP mice, APS hardly have significant effect on CLP mice but its release significantly reduces in the post-inflammatory cytokines. Conclusion:In vivo experiments, preliminarily find that APS can affect the function and phenotype of DC, and mainly play a role in the latter part of immunosuppression in sepsis. The Second Part In vitro experiments to further clarify how APS have an effect on DC, so as to have a protective effect on septic mice Methods: 1. Model and Group: 20-C57BL/6, SPF healthy male mice, CLP sepsis model mice by the application of cecal ligation and puncture(CLP) were divided into four groups(CLP, APS-50ug/ml, APS-100ug/ml, APS-200ug/ml), the day CLP modeled recorded as 0 d; 2. Cell culture: CD11 chigh DCs, CD45 chigh DCs independently culture; CD11 chigh DCs、 CD45 chigh DCs and CD4+ T cells were mixed with cultured;the normal DCs after 50ug/ml、100ug/ml、200ug/ml APS intervented for 2h; 3. Enzyme-linked assay(ELISA):To detect the expression of cytokines(IL-2 IL-6、IL-10, IL-12,TNF-γ) in the blood form supernatant; 4. Flow cytometry: analysis of the number of CD11 chigh DCs, CD45 chigh DCs after APS stimulated. 5. Cell transfusion: Get 105 /DC by APS stimulated for 2h,then in vitro injected into the first day of CLP mice to observe and record the death of mice; 6. Statistical Methods: SPSS13.0 statistical software for statistical analysis, measurement data that the groups were compared using ANOVA, P <0.05 representative of the difference was statistically significant. Results: 1. After two DC subsets was stimulated respectively by the APS(50ug/ml, 100ug/ml, 200ug/ml), result found the total quantity of DC increased, CD11 chigh DCs intervented by APS obviously in a dose-dependent manner, but does not affect CD45 chigh DCs obviously, the initial description of the impact on the DC after APS stimulated mainly affect CD11 chighDCs.2. After APS(50ug/ml, 100ug/ml, 200ug/ml) stimulated, the secretion of the IL-10 and IL-12 found that there is a dose-dependent relationship on the secretion of IL-12 on CD11 chigh DCs after APS stimulated, and the secretion of IL-10 on CD45 chigh DCs after APS stimulated had close negative correlation with APS.3. CD11 chigh or CD45 RBhigh DCs after APS(50ug/ml, 100ug/ml, 200ug/ml) stimulated and CD4+ T cells were mixed culture system to detect CD4+ T cell-related cytokines(IL-4, TNF-α), indicating that APS can be further activated T lymphocyte, thereby enable the transfer of Th2 to Th1 response. 4. After the reinfusion of DCs stimulated by APS into CLP mice, the viability of the CLP mice significantly improved; Comparation to those ones untreated, reinfusion into mice also significantly increased. Preliminarily note the DC’s intervention function in vitro and reinfusion to the body has a protective effect on sepsis. Conclusion:In vitro experiments to further clarify how APS intervene to DC: inducted mouse spleen dendritic cells to differentiate into producing IL-12-CD11chighCD45 Rblow DC subsets, and further activated T lymphocyte, thereby enable the transfer of Th2 to Th1 response. In addition, the effect of APS on T cell differentiation to Th1 was not associated with the inhibition of IL-10 production in CD11clowCD45 RBhigh DCs. Through the APS intervention of DC in vitro and cell reinfusion,provides a basis for in sepsis clinical application.