Activated Platelets Autocrine 5-Hydroxytryptophan Aggravates Sepsis-induced Acute Lung Injury By Mediated Neutrophils Extracellular Traps Formation

【Objective】To investigate the effect of peripheral 5-hydroxytryptamine on the neutrophils extracellular traps formation and its effect on lung injury in sepsis mice,and to explore its mechanism preliminarily.【Methods】（1）The mice genotype was identified by PCR amplification and agarose electrophoresis,and the target gene mice were screened;Wild-type（WT）and Tph1knockout(Tph1^ko)C57 mice（6-8 weeks）were selected according to the random number method.Mice were divided into WT mice sham group,WT mice sepsis group,Tph1^komice sham group and Tph1^komice sepsis group,The mice in the sham group received sham surgery（only the abdominal cavity was opened and the cecum was gently turned without ligation and puncture,and then the abdomen was closed）;the mice in the sepsis group received cecal ligation and puncture（CLP）to establish a sepsis model;the survival rate of the mice in each group within 72 hours was observed;At the same time,the lung tissue of each group of mice was collected 12hours after surgery,and the pathological damage of the lung tissue of the mice in each group was observed under the light microscope by hematoxylin and eosin（HE）staining;The pulmonary edema of mice in each group was observed by calculating the dry/wet weight ratio of lung tissue.Then the differences in the contents of tumor necrosis factor-α（TNF-α）and interleukin-6（IL-6）in the bronchoalveolar lavage fluid of mice between each group were detected by enzyme-linked immunosorbent assay（ELISA）.（2）The differences in the formation rate of neutrophils extracellular traps in lung tissues of mice in each group and the correlation between platelet index CD41 and neutrophils extracellular traps were detected by tissue immunofluorescence method.（3）Combined with in vivo experiments,the effect of exogenous5-hydroxytryptamine on neutrophils extracellular traps in the absence of platelets was detected by cellular immunofluorescence.At the same time,the effects of WT mice-derived platelets and Tph1^komice-derived platelets on the neutrophils extracellular traps were detected.Flow cytometry was used to detect the effects of WT mice-derived platelets and Tph1^komice-derived platelets on neutrophils extracellular traps.【Results】（1）The survival rate results showed that the survival rates of the WT mice sepsis group and the Tph1^komice sepsis group were significantly lower than those of the sham group of the respective strains of mice.The survival rate of Tph1^komice sepsis group was significantly higher than that of WT mice sepsis group（P<0.05）;HE staining results of mice lung tissue showed that the lung tissue structure of WT mice and Tph1^komice sham group was intact,no obvious exudation,and no obvious thickening of alveolar walls.However,the alveolar structure of the mice in the sepsis group was damaged,a large number of inflammatory cells were infiltrated,and the alveolar wall was thickened,and the degree of lung injury in the sepsis group of Tph1^komice was less than that of the WT mice.The results of lung dry/wet weight ratio of mice showed that the lung dry/wet weight ratio of sham mice group was higher than that of sepsis mice group,while the lung dry/wet weight ratio of WT sepsis mice group was lower than that of Tph1^kosepsis mice group（P<0.05）.ELISA results showed that there was no significant difference in the contents of TNF-αand IL-6 in bronchoalveolar lavage fluid between the WT mice sham group and the Tph1^komice sham group;The contents of TNF-αand IL-6 in the bronchoalveolar lavage fluid of the mice in the sepsis group were significantly higher than those in the sham operation group,and the levels of the above indexes in the WT mice sepsis group were significantly higher than those in the Tph1^komice sepsis group（P<0.05）.（2）The results of tissue immunofluorescence showed that no obvious neutrophils extracellular traps were formed in the lung tissue of the WT mice sham group and the Tph1^komice sham group.However,a large number of neutrophils extracellular traps were detected in the lung tissue of the mice in the sepsis group,which was significantly higher than that in the sham operation group,and the amount of neutrophils extracellular traps generated in Tph1^komice sepsis group was significantly lower than that in WT mice sepsis group（P<0.05）.（3）The results of tissue immunofluorescence showed that the lungs from sham mice did not show neutrophils extracellular traps formation,while the lungs from WT mice sepsis group showed a mass of the accumulation of platelets and neutrophils extracellular traps.However,the lungs from Tph1^komice sepsis group showed a significant decrease of the accumulation of platelets and neutrophils extracellular traps.（4）The results of cellular immunofluorescence showed that exogenous5-hydroxytryptamine could not directly induce neutrophils extracellular traps formation,nor could it increase neutrophils extracellular traps formation induced by phorbol 12-myristate 13-acetate（PMA）;At the same time,regardless of whether the platelets derived from WT mice or Tph1^komice were intervened with exogenous serotonin,the formation of neutrophils extracellular traps could not be induced without PMA activation;PMA-activated WT mice-derived platelets significantly increased PMA-induced neutrophils extracellular trap formation,but Tph1^komice-derived platelets did not.Flow cytometry results in the co-culture experiment of murine platelets from various strains and neutrophils from WT mice showed that the expression level of neutrophils citrulline histones H3（Cit H3）was lower in Tph1^komice-derived platelets under PMA activation compared with WT mice-derived platelets.【Conclusions】Activated platelets promote neutrophils extracellular traps formation in lung tissue of septic mice through autocrine 5-hydroxytryptamine signaling.Peripheral 5-hydroxytryptamine deficiency protects the lung from sepsis-induced injury and improves survival in septic mice by inhibiting neutrophils extracellular traps formation in lung tissue,reducing inflammatory cytokine production.