Effect Of Propofol On Splenic Dendritic Cells Differentiation And The Level Of TNF-α、IL-10 In Septic Mice

Objectives We made sepsis mice model with the cecum ligation puncture(CLP) in our study. To comprehensively assesse the inflammation status on different phases, we take a comparative observation of the number, the maturity degree and the subtype of the dendritic cells between the endotoxin group and the propofol treatment group, besides, we detected the level of TNF-a and IL-10 at every phase point. In order to offer the basis for the reasonable application of sedative drugs in the ICU, this experiment explains that the propofol can affect sepsis immune balance from the perspective of dendritic cells turning into to regulatory dendritic cells in sepsis mice.Methods Sixty mice were randomly divided into three groups with 20 mice at each group: sham group,CLP model with propofol treatment group(P group),CLP model with 0.9% normal saline group(NS group).We made sepsis model in the P group and NS group according to the cecum ligation perforation method(Cecal ligation puncture, CLP),while the sham group only open flip the cecum.After the success of the sepsis model building,the mice in the NS group and the propofol group were respectively given equal 0.9% saline and propofol(15 mg/kg)through intraperitoneal injection,while the mice in sham group were without any further processing.DCs were isolated by DC Isolation Kit according to manufacturer’s instructions. The collected mice spleen DCs don’t contain antibodies and magnetic beads and were then used for follow-up study.CD11clowCD45 RBhigh DCs were purified through CD11 c and CD45 RB magnetic beads. After incubated 24 h,CD11clowCD45RBhigh DCs together with incubation buffer were added into flow tube to centrifuge.Then,absorbed the supernatant and saved under-20℃.After centrifugation,the sedimentary cells were re-suspended with PBS buffer and add CD11c-APC,CD45 RBPE,CD40-FITC,CD80-FITC,CD86-FITC,I-a/e FITC and avoid light incubation for 15 minutes.Soon afterwards,flow cytometry was used to detect and analyze the dendritic cell and its subsets in each group. The cytokine levels of TNF-α and IL-10 were measured by ELISA(Enzyme-Linked Immunosorbnent Assay).Results 1 The splenic dendritic cells were divided into three subsets according to the FACS analysis:CD11clowCD45RBlow DCs,CD11clowCD45 RBhigh DCs(namely regulatory dendritic cells) and CD11chighCD45 RBlow DCs(namely conventional dendritic cells).2 Afterwith isotype control,all the surface molecules in dendritic cells including CD40,CD80,CD86 and MHC-II are in a state of lower expression or weak expression.3 Compared with the results of the NS group,there was no significant difference in the proportion of CD11clowCD45 RBhighDCs and CD11chighCD45 RBlow DCs between these two groups at 0~6h,while the proportion of R2(regulatory DC cells) in propofol group significantly increased at 12 h. However, the proportion of CD11chighCD45 RBlowDCs in the propofol group was decreased.In contrast to the NS group, the proportion of R2(regulatory DC cells) in Propofol group was significantly increased at 24 and 36 hour point(P<0.05). 4 Compared with the results of the sham group,the levels of IL-10 and TNF-α in NS group and Propofol group both increased markedly(P<0.05).In addition,compared with the results of the NS group,the level of IL-10 increases largely while the level of TNF-α decreases significantly(P<0.05) as time goes by.5 Pearson linear correlation analysis showed that expression level of IL-10 in incubation buffer and the percentage of R2.Conclusions 1 Both TNF-α and IL-10 have a certain degree of rise in early sepsis stage.This result shows that the immune state is imbalance in early sepsis.2 The application of propofol in sepsis can affect the spleen dendritic cell differentiation to CD11clowCD45 RBhighDCs. Besides,propofol,to a certain extent,can increase the level of IL-10 and decrease the level of TNF-α at the same time.3 There is a positive correlation between the regulatory dendritic cells and the increasing of the level of IL-10.Therefore,we speculated that propofol play a role in the early sepsis through promoting dendritic cells to regulatory dendritic cells differentiation and increasing the negative immunity regulation(r=0.355,P<0.05).