The Role Of Proteinase 3 In The Inflammatory Response Of Early Sepsis And Its Regulatory Mechanism

Background:Hyperinflammatory response that induced by enormous amounts of actived proinflammatory cytokines is the leading cause of death in early sepsis.Researches have shown that blocking the function of IL-1? and IL-18 simultaneously could remarkablely increase the survival rate of early sepsis model mice,thus finding a single target that could block the activation of various kinds of proinflammatory cytokine at early stage of sepsis would make sense.The expression of membrane proteinase 3(m PR3)on neutrophils has a positive regulatory effect on various proinflammatory factors.Epigenetic regulation of gene function has been identified as an important mechanism regulating myeloid cell function in patients with sepsis.The regulation mechanism of m PR3 on neutrophil in sepsis has not been reported.In our research,we aimed to clarify the relationship between PR3 expression and the severity and proinflammatory factors in septic patients.Then,we explored the mechanism underlying the regulation of PR3 expression to provide a target to the treatment of early sepsis.Methods: Patients satisfying the definition of sepsis were included and peripheral blood was collected within 24 h after diagnosis.Healthy volunteers matched with sex and age were enrolled as control group.We isolated the neutrophils and measured the expression of PR3 on neutrophils by flow cytometry and compared the level of them between the septic patients and healthy volunteers.The levels of plasma cytokines IL-1? and TNF-? were measured by ELISA.The differences in cytokines between septic patients and healthy volunteers were compared.We measured the expression of JMJD3 by real-time PCR and compared the level of JMJD3 between the septic patients and healthy volunteers.Intervention measures were implemented and experiment was divided into four groups: blank control group,LPS-stimulated group,LPS-stimulated+GSK-J4 group and GSK-J4 group.The above four groups of neutrophils were cultured with monocyte respectively.We measured the m RNA levels of PR3 by real-time PCR and the expression of PR3 on neutrophils by flow cytometry,and differences among the groups were compared.The levels of IL-1? in the culture supernatant were measued by ELISA and compared among groups.Western blot was used to detect Histone H3 lysine 27 trimethylation(H3K27me3)expression changes in each group.Results: Compared with healthy volunteers,the level of PR3 expression on neutrophils in septic patients were significantly higher;Compared with the m PR3 low expression group,the levels of TNF-a and IL-1? were significantly increased in the m PR3 high expression group in patients with sepsis.Compared with the m PR3 low expression group,the levels of APACHEII score were significantly increased in the m PR3 high expression group in patients with sepsis.Compared with healthy volunteers,the m RNA levels of JMJD3 in septic patients were significantly higher.In vitro experiments showed that,under the condition of single neutrophil culture: compared with blank control group,the levels of m PR3 and IL-1? in LPS-stimulated group were significantly higher;The levels of m PR3 and IL-1? were significantly lower in the group treated with GSK-J4 when compared with non-treatment group.The protein level of H3K27me3 was significantly decreased after LPS stimulated neutrophils in vitro.Compared with LPS-stimulated group without GSK-J4 intervention,the protein level of H3K27me3 was significantly increased after GSK-J4 intervention.Under the co-culture conditions: IL-1? levels in the supernatant of LPS-stimulated group were significantly higher than those in the blank control group;the levels of IL-1? in the supernatant of GSK-J4-treated co-cultured cells were significantly lower than those without GSK-J4 intervention.The level of IL-1? in LPS-stimulated co-culture supernatant was significantly higher than that of the corresponding individual cultured cells.Conclusions: The expression of PR3 on neutrophils was elevated in early sepsis,which associated with the increase of inflammatory factors and severity of disease.JMJD3 was activated in patients at early stage of sepsis.JMJD3 regulated the expression of neutrophil m PR3 during sepsis-induced excessive inflammation.JMJD3 may regulate the expression of m PR3 on neutrophils by modifying H3K27me3.Expression of m PR3 on neutrophils may increase the release of proinflammatory cytokine IL-1? in the early sepsis,which may have an amplification effect on the inflammatory response.