The Effect Of Neutrophil Extracellular Traps On Intestinal Barrier Function In Acute Intestinal Ischemia

Objective:Acute intestinal ischemia is uncommon abdominal vascular critical disease but carries a greater mortality.Severe sepsis,shock and trauma can result in inadequate blood flow of intestine which may contribute to intestinal dysfunction.With worsening hypoxemia,the intestinal histological alterations may result in destruction of intestinal epithelial barrier function and induce to systemic inflammation,which is generally believed to be the cause of MODS.Previous research has indicated that systemic inflammation may be a main cause of intestinal injury following ischemia and neutrophil plays an important role in disease progression.In 2004,Brinkmann,et al identified a novel mechanism in which neutrophils create extracellular traps,known as neutrophil extracellular traps(NETs),which comprise chromatin and granule proteins.The understanding of NETs has recently improved due to the increased intensity of NETs research over the last decade.NETs appear to play a protective role by trapping microorganisms to prevent them from spreading.However,damaged-associated molecular pattern molecules(DAMPs)released from NETs have also been shown to exert harmful effects by triggering coagulation,inflammation and cell death.A large number of intracellular and nuclear self-antigen are released during NETs formation,which can induce autoantibody formation,leading to the occurrence of various autoimmune diseases.And NETs-targeted therapy has substantially improved in a range of animal models.The aim of this study is to elucidate the hypothesized mechanism of NETs on intestinal barrier and may provide the new therapy target and theoretical basis how intestinal dysfunction developed during acute intestinal ischemia.Part 1Methods:The acute intestinal ischemia model rats were subjected to warm ischemia for 1 h by clamping the superior mesenteric artery.The DNase-1 treatment group received a single DNase-1 injection intravenously o h,2 h,4 h and 6 h following 1 h ischemia.The rats were sacrificed at 24 h.Blood samples and ileum tissues were collected.A capture enzyme-linked immunosorbent assay(ELISA)was used to quantify the NET levels in the serum and an immunofluorescence assay was used to identify NETs in vivo.The tissues were surgically excised and subjected to histological study by staining with hematoxylin and eosin.Western blotting analysis and mmunohistochemistry were performed to reflect the functional integrity of the intestinal mucosa barrier.The IL-6,IL-10 and tumor necrosis factor-alpha(TNF-?)levels in the serum,which reflect the severity of inflammation,were measured using corresponding ELISA kits.Results:NETs were observed in the serum and ileum tissue during acute intestinal ischemia injury,whereas fewer NETs were detected in the sham group.Notably,we detected lower NET levels following DNase-1 intervention.The rats subjected DNase-1 injection 0 h showed the most serious pathologic damage.And rats subjected DNase-1 injection 4 h showed relatively minor injuries compared with other groups.Part 2Methods:Proper selection of the best time point depends upon the results of the first part.We investigate the therapeutic effect of DNase-1 treatment and DNase-1 combined with enoxaparin treatment.We aimed to investigate the effect of combination therapies on intestinal barrier.Results:DNase-1 treatment and enoxaparin treatment can improve intestinal injury effectively.Combination therapies can inhibit histopathological changes significantly.And combination therapies are superior to a monotherapeutic regimen.Conclusion:(1)NETs was significantly associated with the intestinal injury during acute intestinal ischemia;(2)The time point of 4 h may be the best timing of intervention by DNase-1.At this point,DNase-1 treatment may reduce the early proinflammatory response and ameliorates the disruption of tight junction proteins after acute intestinal ischermia.The early deletion of NETs may exacerbate tissue injury.(3)Anticoagulant therapy may improve the outcome of intestine and decrease the NETs expression in a rat model of acute intestinal ischemia.(4)Combinations of anticoagulant therapy and NET-targeted interventions can significantly restore intestinal barrier structures and attenuate the systemic inflammation.