Tubastatin A Ameliorates The Organ Injury Of CLP-Induced Sepsis By Decreasing The Formation Of NETs

Objective: Sepsis is a serious clinical disease,which lead to multiple organ failure and death.Studies have found that Tubastatin A(Tub A),an inhibitor of histone deacetylase 6(HDAC6),could significantly improves the survival rate of septic mice induced by lethal cecal ligation and puncture(CLP),alleviate liver and lung injury.However,the mechanism has not been elucidated.Neutrophil extracellular traps(NETs)can directly capture and kill pathogens,prevent them spreading in the blood circulation.Nevertheless,when triggers such as inflammation persist,excessive release of NETs instead cause tissue injury.The latest researches present that NETs are closely related to sepsis microcirculatory disorders.Therefore,this study observed the effect of Tub A on the injury of vital organs(liver,kidney,lung)and the formation of NETs through constructing the model of CLP-induced intraperitoneal infection in mice.Meanwhile,the effect of Tub A on microcirculation thrombosis of kidney was also evaluated.Methods: Male C57BL/6J mice were randomly divided into 3 groups,5 mice per group,Grouping was as follows: ⑴Sham group: open the abdomen,omitted CLP,DMSO intraperitoneal injection;⑵CLP group: CLP,DMSO intraperitoneal injection;⑶CLP+Tub A group: CLP,Tub A intraperitoneal injection(Tub A dissolved in DMSO),Treatment was administered 1 h following the CLP.At 24 hours after CLP,the whole blood was collected by extracting the eyeball,and the supernatant was separated to detect organ injury markers ALT and BUN.Pico Green? dye was used to detected quantitatively the level of cf-DNA.HE staining was used to observe the histopathological changes in vital organs(liver,kidney,lung).The formation of NETs in kidney was assessed by immunofluorescence.The formation of NETs and thrombin in the renal microcirculation was assessed by immunohistochemistry.Results: Compared with the sham group,mice in the CLP group showed symptoms of systemic inflammatory reactions following CLP,such as listlessness,Piloerection,and retardation.Compared with the CLP group,the activity of mice in the CLP + Tub A group was reduced,but the survival status was significantly improved.Under the light microscope,the morphological structures of liver,lung and kidney in the sham group were intact.Compared with the sham group,the mice in the CLP group showed multi-organ injury,hepatocyte bleeding,necrosis inflammatory cell infiltration;pulmonary interstitial congestion,consolidation and renal glomeruli and tubules injury and atrophy were visible.Compared with the CLP group,the liver,lung and kidney tissue injury and inflammatory cell infiltration of the mice in the CLP +Tub A group were alleviated,and the pathological injury score decreased(4.58±0.19 vs 3.30±0.22,4.58±0.29 vs 3.50±0.21,3.50±0.22 vs 2.06±0.21,p <0.05).Compared with the sham group,the levels of organ injury markers ALT and BUN in the CLP group were significantly increased(55.801±20.078 vs 196.027±22.822,p<0.05;15.252±3.312 vs58.993±10.123,p<0.05).Compared with the CLP group,the levels of ALT and BUN in the CLP+Tub A group were decreased(196.027±22.822 vs 143.666±12.532,p<0.05;58.993±10.123 vs 32.158±5.266,p < 0.05).Under the fluorescence microscope,No NETs-like structures were found in the sham group.Typical NETs structures were observed in the kidney of CLP group,Massive and diffuse distribution.Compared with the CLP group,NETs in the kidney of CLP+Tub A group was reduce(1.373±0.137 vs0.780±0.114,p<0.05).Compared with the sham group,the level of plasma cf-DNA in the CLP group was markedly elevated(0.44±0.003 vs 2.06±0.406,p<0.05).Compared with the CLP group,plasma cf-DNA level in the CLP + Tub A group was decreased(2.06±0.406 vs 1.12±0.146,p<0.05).In the sham group,there was almost no expression of NETs and thrombin in the renal microcirculation.In the CLP group,NETs and thrombin-positive regions were widely and diffusely distributed,coinciding with the CD31-positive regions.Compared with the CLP group,in the CLP+Tub A group,the formation of NETs in renal microcirculation decreased(3.712±0.099 vs 3.043±0.058,p<0.05),thrombin production decreased(2.866±0.206 vs 2.236±0.157,p<0.05).Conclusion: Tub A may alleviate organ injury induced by sepsis by means of decreasing the formation of NETs in the septic microcirculation,reduce microthrombosis,improve microcirculatory disorders.