Study On Prevention Effect Of Intestinal Injury In Sepsis/Severe Sepsis Rats Preconditioning With PA-MSHA

Objective: To investigate the possible mechanisms of PA-MSHA on the prevention effect on abdominal sepsis by detecting the concentration of cytokine(IL-1, IL-6,TNF-α, IL-10) in intestinal tissues and ascites, the expression of ICAM-1 and VCAM-1 on intestinal tissues, the expression of TLR4 on neutrophils in the peripheral blood and the effect on the in vitro chemotaxis of blood neutrophils through establishing models of sepsis and severe sepsis.Methods:(1)40 healthy SPF SD rats(230±10g), male, were randomly divided into 5 groups: control group, sepsis group, PA- sepsis group, severe sepsis group, PA- severe sepsis group.(2) Rats in the control group, sepsis group and severe sepsis group were preadministered subcutaneously with saline at a does of 0.3ml/day for 7 consecutive days. While rats in PA-sepsis group and PA-severe sepsis group receied the same dose of PA-MSHA instead of saline.(3) after 7 days, the control group were intraperitoneally injected with 3ml 0.9% sodium chloride; to establish a sepsis model, 1.6m L/kg body weight human stool suspension, diluted in saline to 3 ml, were injected into the abdomen of rats in sepsis group and PA-sepsis sepsis group.(as described by Gonnert FA). to establish the severe sepsis model, 3m L/kg body weight human stool suspension, diluted in saline to 3 ml, were injected into the abdomen of rats in severe sepsis group and PA-severe sepsis group(4) when the model was successfully established, rats were anesthetized with 3% pentobarbital sodium.(1)Peritoneal lavage and the abdominal cavity lavage was recovered.(recovery rate≥80%).Enzyme-linked immunosorbentassay(ELISA) was adopted to detect the concentration of cytokines(IL-1, IL-6, TNF-a, IL-10) in the abdominal cavity lavage.(2)blood from abdominal aorta was used for blood cultures. Neutrophils from peripheral blood were isolated and the expression of TLR4 on the surface of neutrophils was detected by flow cytometry. The number of neutrophil chemotactic test in vitro Transwell.(3)When the animals were sacrificed, pathological changes were observed by HE staining of intestinal tissue.(4)The expression of cytokines(IL-1, IL-6, TNF-, IL-10) and adhesion molecules(ICAM-1, VCAM-1) in the intestinal homogenate were measured by ELISA.Results: 1、Invasive mean arterial pressure(MAP) and blood culture: Compared with the control group, the MAP of the other four groups was significantly decreased. There was no bacterial growth in the blood cultures of the control group. The results of the other four groups of blood culture were all E. coli(+).2、Pathological changes: rats were dissected immediately after the experiment and gross sample was observed. The intestine tissue in the control groups was pink, which means had a good blood supply; while the intestine tissues in the other four groups were dark, congested, edema, accumulated of gas or adhered to the surrounding tissues to some extent. Paraffin section and HE staining: the intestinal epithelial cells in the control group were intact, and the epithelium and lamina were closely connected, without the infiltration of inflammatory cell. The intestinal glands were normal. The other 4 groups have different degrees of epithelial cell damage, inflammatory cell infiltration, the inherent layer edema and capillary dilation..3、Concentrations of cytokines in peritoneal lavage fluid:(1)Pro-inflammatory cytokine concentration(IL-1, IL-6, TNF-α): Compared with the control group, promoting inflammatory cytokine concentrations in the other four groups were increased(P < 0.05); The severe sepsis group is higher than the sepsis group(P < 0.05); Sepsis group was higher than the PA-sepsis sepsis group(P < 0.05); Severe sepsis group was higher than that the PA-severe sepsis group(P < 0.05).(2)Anti-inflammatory cytokine concentration(IL-10): Compared with the control group, the other four groups were increased(P < 0.05); sepsis group was higher than the PA-sepsis group(P < 0.05); severe sepsis group was higher than PA-severe sepsis group(P < 0.05).4、Intestinal cytokines and adhesion molecular concentration:(1)Pro-inflammatory cytokine concentration(IL-1, IL-6, TNF-α): Compared with the control group, promoting inflammatory cytokine concentrations in the other four groups were increased(P < 0.05); The severe sepsis group is higher than the sepsis group(P < 0.05); Sepsis group was higher than the PA-sepsis sepsis group(P < 0.05); Severe sepsis group was higher than that the PA-severe sepsis group(P < 0.05).(2)Anti-inflammatory cytokine concentration(IL-10): Compared with the control group, the other four groups were increased(P < 0.05); sepsis group was lower than the PA-sepsis group(P < 0.05); severe sepsis group was lower than PA-severe sepsis group(P < 0.05).(3)The adhesion molecules concentration(ICAM-1 and VCAM-1): The average intestinal expression of ICAM-1 and VCAM-1 in the sepsis group, the PA-sepsis group, the severe sepsis group and the PA-severe sepsis group was elevated, as compared with the that in the control group(P <0.05); The sepsis group was higher expression than the PA-sepsis group in both ICAM-1 and VCAM-1(P <0.05);However, the expression level of ICAM-1 and VCAM-1 in severe sepsis group was lower than PA-severe sepsis group(P < 0.05);While there was no significant change in severe sepsis group compared with PA-severe sepsis group(P=0.262).5、TLR4 expression on neutrophils was detected by Flow cytometry : Compared with control group, the expression of TLR4 in the other four groups were increased(P < 0.05); Sepsis group were lower than that of severe sepsis, sepsis group; Sepsis group is higher than the PA- sepsis group(P < 0.05); Severe sepsis group is higher than the PA- severe sepsis group(P < 0.05).6、The number of neutrophil chemotactic test in vitro Transwell:Compared with control group, the expression of TLR4 in the other four groups were increased(P < 0.05); Severe sepsis group were less than that of sepsis group; Sepsis group was less than the PA- sepsis group(P < 0.05); Severe sepsis group was less than the PA- severe sepsis group(P < 0.05).Conclusion: 1、Sepsis and severe sepsis caused by abdominal infection can lead to the increase of local(ascites and intestinal) cytokine factors in rats. PA-MSHA pretreatment can reduce the intestinal inflammatory damage and prevent sepsis in rats by reducing the local cytokine concentrations2Abdominal infection-induced sepsis and severe sepsis can elevate the expression of intestinal ICAM-1 and VCAM-1 in rats; PA-MSHA pretreatment can inhibit the expression of ICAM-1 and VCAM-1 in the sepsis group. In which way the injury of intestinal maybe reduced.3、PA-MSHA preconditioning could inhibit the expression of TLR4 on the surface of the neutrophil, improve neutrophil immune paralysis, in which way reducing the level of systemic inflammation and reducing tissue damage.4、PA-MSHA pretreatment can reduce systemic and local(abdominal and intestinal) inflammatory response, in which way the intestinal injury induced by sepsis could be prevent. so as to improve the effect of the neutrophil paralysis in sepsis rats.