The Mechanism Of Dendritic Cell On Inflammation In Multiple Organs Dysfunction Syndrome

Objective: (1) To explore the effect of DC on inflammatory in MODS animal model. (2) To explore the effect mechanism of Carbachol on immunological function through MODS animal model. (3) To observe protective effect of Flt3L on immunological function and organs in MODS.Method: The MODS model of mice was reproduced by zymosan injection in the peritoneal cavity of the C57BL/6 mice. The mice were randomly divided into four groups as follows: normal control group (n=30), MODS group(n=150), carbachol treatment group(n=30)and Flt3L treatment group(n=30). MODS group was further divided into 6 hour, 24 hour, 48 hour, 6 day and 10-12 day post trauma. Carbachol was given pre. Flt3L 5μg/kg was given via the peritoneal cavity on the fifth day after trauma in Flt3L treatment group. Animals of all groups were sacrificed at designated time points, and blood and spleen samples were harvested. Separated blood serum was stored in -20℃. Spleen sample was divided into two parts as following: one part was used to store in LIN , and the other was used to procure DC by MACS microbeads by using column of nylon wool. DC phenotypes , peripheral blood T cell subpopulation and I-A~b of PBMCs were analyzed by flow cytometry and the cytokine released into supernatants were also determined. Cytokines in supernatant and tissue homogenate were determined by ELISA kits for mice. CD86, IL-1β, IL-10 and HMGB1 were detected by immunohistochemical method. NF-KBp65 mRNA expression was measured by by real-time quantitative PCR.Result: (1) The expression of IL-1β, IL-10 and HMGB1 post the zymosan trauma. The significant increase in splenic IL-1βpositive cell number was detected at 6 and 48 hour. After 24 hours it began to decrease to the normal levels. The significantly elevated expression of splenic HMGB1 positive cell number was detected at 6-48 hour group. On the 6th day it maintained at the normal level. On 10-12 days the level began to increase. The elevated expression of splenic IL-10 positive cell number was detected at 48 hour and reached the peak on 10-12 day. (2) After zymosan trauma the mouse spleen DC quantity obviously increased in 6 and 24 hour group. After 48 hour there was a drop. On the 6th day the DC numbers maintained at the normal level. On 10~12th the DC numbers raised obviously. The significantly elevated expression of MHC- II and CD86 was detected at 6 and 24 hour and suggested DC mature. At 48 hour and on the 6th day the expression of MHC-II and CD86 were at the normal level. On 10-12 day MHC-II and CD86 expression decreased obviously. The level of IL-12p70 in supernatant of cultured DC obviously increased at 6 and 24 hour and restored to the normal level after 48 hour. On the 10~12th day the IL-12p70 quantity reduced obviously. After the trauma NF-κB p65mRNA expression of cultured DC at 6 and 24 hour group remarkably increased and restored to the normal level on the 48 hour and 6th day group. In 10-12 day group, NF-κB p65mRNA dropped obviously. (3) In trauma 6 hour group, the ALT in the experimental mouse internal organs functional indicator increased, other changes are not obvious. The expression of I-A~b by the mononuclear cells, the CD4~+T cell number and the CD4/CD8 ratio declined remarkably in peripheral blood. Pathological changed mainly for internal organs extravasated blood, dropsy; the trauma latter on the 6th day group, the experimental mouse functional indicator had the change for the better, the pathological changes were not obvious.The expression of I-A~b by the mononuclear cells, the CD4~+T cell number and the CD4/CD8 ratio increased properly in peripheral blood. The trauma latter on the 10~12th day group, each biochemical indicator rised, The expression of I-A~b by the mononuclear cells in peripheral blood dropped to the perigee, the CD4~+T cell also obviously reduced, the CD4/CD8 ratio dropped obviously. Many main internal organs pathological changes were serious. (4) In the carbachol treatment group the expression of I-A~b by DC and the level of IL-12 p70 decreased. The numbers of IL-1βand HMGB1 positive cells also decreased in the spleen. The ALT in the experimental mouse internal organs function target decreased. to normal level. The pathological changes of organs were relieved. Carbachol (1μmol/ml, 10μmol/ml and 100μmol/ml) could significantly inhibit the increase of MHC-II expression and IL-12p70 secretion produced by DC responsed to LPS, especially the concentration of 100μmol/ml. (5) The expression of I-A~b by DC , the levels of IL-12 p70 in the spleen, the expression of I-A~b by the mononuclear cells, the CD4~+T cell number, the CD4/CD8 ratio, ALT and BUN increased remarkably in Flt3L treatment group, whereas, the level of IL-10 decreased. The pathological changes of organs were relieved.Conclusion: (1) Splenic DCs have close correlation and regularity with the proinflammatory and anti-inflammatory factors in the course of MODS. DCs have the ability to regulate the inflammatory. NF-κB/IL-12 rountine is a important link in regulating the immunity and inflammatory. (3) Carbachol may interrupt the development of SIRS through suppressing the maturation and activation of DCs and attenuating inflammatory reaction in the early stage of MODS. (4) Flt3L may improve immunological suppression and prevent the formation of MODS through promoting the maturation and activation of tolerance DCs in the advanced stage.