The Molecular Mechanism Of Endothelial Cell KLK8 Involved In Sepsis Lung Injury

Sepsis is an inflammatory response syndrome caused by dysregulation of the body’s immune response to infection,which can lead to multiple organ failure and life-threatening disease.Lung is the most commonly affected organ,and the exact mechanism of acute lung injury is unclear,endothelial cell injury plays a key role.Kallikrein-related peptidases-8（KLK8）is a tissue kinase-releasing enzyme and highly associated with inflammation.Our previous study demonstrated that KLK8is involved in hyperglycemia-induced endothelial cell injury of renal fibrosis,but its significance in sepsis is still limited.Objective:To investigate whether KLK8 is involved in sepsis and the possible mechanism,hopefully providing a new intervention target for sepsis.Methods:1.To investigate the expression and localization of KLK8 in lipopolysaccharide（LPS）-induced sepsis with endothelial cell injury.（1）The C57BL/6J mice were used to construct an LPS-induced sepsis model and evaluate the KLK8 protein levels in lung tissue.（2）Immunofluorescence was performed to study KLK8 expression in lung tissues of LPS-induced sepsis mice.（3）KLK8 and cytokine interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）were detected by real-time transcription-quantitative polymerase chain reaction（RT-q PCR）.2.To confirm knockout effects of KLK8 in the endothelial cells during sepsis.（1）We used the endothelial KLK8 knockout mice to construct the LPS-induced sepsis model,and the seven-day survival rate after LPS was observed in mice;（2）The levels of blood oxygen saturation,total cell count of bronchoalveolar lavage exudate,and cytokines interleukin-1 beta（IL-1β）,IL-6,and monocyte chemoattractant protein-1（MCP-1）were measured in LPS model mice to assess lung injury.3.To examine overexpression effects of KLK8 in endothelial cells and possible mechanisms during sepsis.（1）The lentiviral vector which overexpresses KLK8 was designed to infect the endothelial cells（Human umbilical vein endothelial cells）.（2）We investigated the proliferation and migration ability via cell proliferation and scratch assay in the infected endothelial cells.Furthermore,we detected the cytokine Interleukin-1 beta（IL-1β）,Interleukin-6（IL-6）,and Tumor necrosis factor-α（TNF-α）.（3）Fluorescein isothiocyanate（FITC-dextran）assay was performed to detect the permeability of infected endothelial cells.The human acute monocytic leukemia cells（THP-1）was used to detect inflammatory cells’adherent and chemotactic capacity to endothelial cells.（4）The interaction of KLK8 with protein kinase receptor（PAR）was proved by immunoprecipitation.（5）Adminstreated PAR2 inhibitor to interfere with the infected endothelial cells assessing whether the function of endothelial cells was altered.Results:1.High expression of KLK8 was observed in the LPS-induced sepsis lung tissue,which was located in the endothelial cells.We also found that LPS-induced endothelial cell injury promoted cytokines IL-6,TNF-α,and KLK8 expression.2.The endothelial cell KLK8 knockout(KLK8^tie2cre)mice decreased the total number of leakage cells in the alveolar,eased the related cytokine IL-6 expression in mice,improved blood oxygenation,and increased the7-days survival rate post-modeling.3.Overexpression of KLK8 in endothelial cells enhanced cytokine expression but also increased permeability of endothelial cells,and the adherent and chemotactic capacity of inflammatory cells to endothelial cells.4.Inhibiting PAR2 attenuated the inflammatory response,improved endothelial cells’permeability,and weakened the adherent and chemotaxis ability of inflammatory cells to endothelial cells.Conclusions:High expression of KLK8 in sepsis can promote endothelial cell injury and acute lung injury,and KLK8 acts on endothelial cells probably through cleavage PAR2 to activate the inflammation signaling pathway.