Establishment And Application Research Of A Sepsis Model In Wild Type Mice

Objective:Sepsis and it triggered septic shock and multiple organ failure(MOF) are common concerns of clinicians and researches. Now it’s believed that the fundamental pathological mechanism of sepsis is the imbalance of inflammatory response and anti-inflammatory responses. Adenosine and its A2 A receptor can mediate immune suppression signals to alleviate tissue injury. It has been revelled that adenosine A2 A receptor knockout mice had a higher survival rate post intestinal bacteria insults compared with WT. However, theeffects of endothelial A2 Areceptor on sepsis is not clear. The endothelial A2 Areceptorspecificallyknockout mice and antagonist of A2 A receptor were used to study the connection. Mthods:1.The established sepsis model in wild type mice In this research sepsis was induced by subjecting 9~12W old male mice to cecalligation and puncture(CLP). The ligated sites were choosed at 50% or 75% of the cecum. Survival rate was recorded after model established.Bacteria in blood and peritoneal fluidwere cultured on plates. The white blood cells, neutrophils, monocytes, lymphocytes and platelets were ccounted with routine blood instrument. The levels of ALT, AST and BUN were detected with blood biochemistry analyzer. Cytokines including GM-CSF, INF-γ, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-15, IL-17, MIP-1α, MIP-1β and MIP-2 were detected by Luminex. Endothelial dysfunction biomarkers including TM, E-selection, VCAM-1, s ICAM-1, VEGF and VEGFR-1 were detected by ELISA. The thrombosis and other pathological changes of lung, liver and kidney were detected by HE.2.The therapeutical effects of antagonisting A2 Areceptor on sepsisIn accordance with the operation of C57 mice, A2Afloxp/floxpcdh5cre/+ and A2Afloxp/floxpmice were subjected to CLP. Meanwhile, A2Afloxp/floxp mice were given A2 AR antagonist KW6002(10mg/kg) to evaluate the therapeutic mechanism. Survival rates was recorded andbacteria in blood and peritoneal fluidwere counted. Routine blood and blood biochemical indicator were detected. Exudation of tissues were detected by evans blue. The levels of cytokines IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-15, MIP-1α, MIP-1β and MIP-2. Endothelial dysfunction biomarkers including TM, E-selection, VCAM-1, s ICAM-1, VEGF and VEGFR-1 were detected. The thrombosis and other pathological changes of lung, liver and kidney. Taken all of the datas together to evaluate the correlation between A2 AR inhibitor and endothelial A2 A receptor knockout. Results:1. The established sepsis model in wild type miceThe survival rate of wild type mice was closely associated with the ligated sites. Mice died totally within 4d when ligated 75% of the cecum, while 40% of the mice can live to 12 d when ligated 50% of the cecum. The blood routine and blood biochemistry tests showed that total number of white blood cells, neutrophils, lymphocytes and monocytes droped sharply, while ALT, AST and BUN rised. The levals of proinflammatory factors IL-1α, IL-6, TNF-αand anti-inflammatory factors IL-10 and chemokine MIP-1α, MIP-1β, MIP-2 had significant difference. The endothelial dysfunction molecules, especially s-ICAM-1, E-slectin and VEGF rise a lot. Congestion and disseminated intravascular coagulation(DIC) was visible between mesenchyme of lung tissues in mice post CLP.2.The therapeutical effects of antagonisting A2 Areceptor on sepsis A2Afloxp/floxpcdh5cre/+mice are endothelial A2 AR conditionally knockout mice. Under normal conditions the penetration function was not affected, while under the CLP induced sepsis conditions, the lung, liver and kidney permeation were declined. A2Afloxp/floxpcdh5cre/+, A2Afloxp/floxpmice and A2 AR antagonist KW6002(10mg/kg) given and vehicle groups were subjected to CLP. The final results showed:(1)The 12 day mortality rate of A2Afloxp/floxp mice was 95% and A2Afloxp/floxp cdh5cre/+mice was 55%. Endothelial A2 AR conditionally knockout mice had a higher survival rate. The mortality rate of vehicle group was 85% and KW6002 group was 38%, which was remarkable lower.(2)The bacteria load in blood of A2Afloxp/floxp cdh5cre/+mice 16 h post CLP had a significant decline compared with A2Afloxp/floxp mice and at 48 h there was a similar decline. The bacteria load in peritoneal fluidof A2Afloxp/floxp cdh5cre/+mice 16 h post CLP had significant decline compared with A2Afloxp/floxp mice and at 48 h there was a similar decline. The bacteria load in blood and peritoneal fluid of KW6002 group all had a decline tendency compared with vehicle group.(3)The boood routine test showed WBC numbers were similar between A2Afloxp/floxp mice A2Afloxp/floxp cdh5cre/+mice 16 or 48 h post CLP. The number of neutrophiles had a decline tendency but other WBC were similar. WBC numbers were similar between KW6002 group and vehicle group and neutrophiles at 16 h post CLP had a decline tendency, other WBC were similar.(4)Cytokines detection showed that levels of IL1α, IL10, IL6, MIP1β and MIP2 of A2Afloxp/floxp cdh5cre/+mice had a significant decline compared with A2Afloxp/floxp mice. The levels of MIP1α, TNFα were similar decline. The levels of cytokines detected at 48 h were similar. The levels of IL1α, IL10, IL6, MIP1β, MIP2 of KW6002 group had a significant decline compared with vehicle group. Levels of MIP1α and TNFα were similar. The levels of cytokines detected at 48 h were similar.(5)Endothelial dysfunction biomarkers showed that, compared with A2Afloxp/floxp mice,levels of TM, TF of A2Afloxp/floxp cdh5cre/+mice haddecline tendency and VEGF was similar. At 48 h, TM matained decline tendency and VEGF declined, TF was similar. Compared with vehicle group, KW6002 group had a significant decline in TM, TF and VEGF levels at 16 h post CLP. TM matained decline tendency and VEGF, TF was similar.(6)Histopathological detection showed pulmonarycongestion was obvious of A2Afloxp/floxp mice and big thrombosis within the blood vessels.Visible thrombosis also emerged within lung tissue interval of A2Afloxp/floxp cdh5cre/+mice. Compared with vehicle group, KW6002 group alleviated the congestion and thrombosis. More severe thrombosis of lung was visible at every group 48 h post CLP. The hepatic detection showed visiblecongestion and thrombosis occasionally at each group. Congestion and thrombosis emerged within kidney of the A2Afloxp/floxp mice and vehicle group, Which was less in A2Afloxp/floxp cdh5cre/+mice and KW6002 group. Conclusions:1.The established sepsis model in C57 wild type mice had obvious septic characteristics, which including amount of bacteria emerged in the blood, liver and kidney function damaged, proinflammatory factors IL-6 and chemokines raised andendothelial dysfunction biomarkers like TM, VEGF changed.2.Antagonist of adenosine A2 AR could improve septic mice survival rate, reduce the bacteria load in blood, lower the levals of IL6, IL1α, IL10, MIP1α, MIP1β, and MIP2 and reduce VEGF, TM, E-slectin and TF levels.3.Selectively knockout of endothelial A2 Areceptor could also improve the survival rate of mice with sepsis, reduce the bacteria load in blood, maintain the liver and kidney function, lower the levals of IL6 and MIP2 and reduce TM, E-slectin, VEGFR1 and TF expression.4.The therapeutic effects of antagonist of A2 AR had close correlations with endothelial A2 A receptors inhibition.