Expression Of ZIPK In Endothelial Cell Adhesion Molecules And Its Function In ALI Of Septic Mice

Endothelial cells(EC)are distributed in almost all tissues and organs of the body and have essential physiological functions.They form a semi-permeable barrier between blood and tissues through adhesion junctions and tight junctions between adjacent ECs,allowing only small molecules to pass through,while large molecules and cells cannot pass through to maintain the organism's homeostasis.In pathological states such as inflammation,EC is activated,and endothelial permeability is increased,while the expression of adhesion molecules on its surface is elevated,and leukocytes and macromolecules from the blood enter tissues and organs through the endothelial layer,triggering severe inflammation and causing tissue and organ damage.In our previous study,we found that Zipper-interacting Protein Kinase(ZIPK)is a critical molecule in the regulation of endothelial permeability,which mediates the disruption of blood-brain barrier function in vivo and Thrombin and Tumor Necrosis Factor-alpha(TNF)in vitro.Necrosis factor alpha(TNF-?)and other induced contraction of microvascular EC and elevated endothelial permeability;the expression of ZIPK was elevated in TNF-?-stimulated human umbilical vein endothelial cells(HUVEC),and ZIPK also mediated the expression of adhesion molecules;we also found that Tamoxifen injection of UBC-Cre^ERT2+/+-Zipk^flox/flox-induced knockdown(KO)of Zipk gene expression was protective against acute lung injury induced by endotoxin lipopolysaccharide(LPS)lung exposure in mice.In this paper,we further investigated the role of ZIPK on LPS-mediated expression of EC adhesion molecules and used KO mice to initially observe the effect of ZIPK deletion on the pathological process of cecal ligation and puncture(CLP)-induced sepsis in a mouse model of sepsis.The role of ZIPK in the expression of adhesion molecules in EC was first investigated.Analysis by Q-RT PCR and Western Blotting revealed that only very low levels of VCAM-1,ICAM-1,and E-selectin were expressed in untreated HUVEC,and LPS(50 ng/m L)treatment significantly induced their protein and RNA expression;the protein levels of VCAM-1 and ICAM-1 The protein levels of both VCAM-1 and ICAM-1 were elevated approximately 20-fold,and the mRNA levels of VCAM-1,ICAM-1 and E-selectin were also significantly elevated.After treatment with the specific inhibitor TC-DAPK6(0.1 and 0.5?M),the protein expression of VCAM-1 and ICAM-1 was significantly inhibited,with the inhibition rate reaching about 80%;the expression level of adhesion molecules in HUVEC was also significantly inhibited by small interfering RNA(si RNA)knocking down the expression of ZIPK,with the inhibition rate reaching about60%.The above findings suggest that ZIPK is involved in the regulation of adhesion molecule expression in LPS-induced HUVEC.Further study revealed that inhibition of ZIPK significantly inhibited the activation of JNK and AKT in LPS-induced HUVEC,suggesting that ZIPK may regulate the expression of adhesion molecules through JNK and AKT signaling pathways.In this paper,we also found that inhibition of ZIPK significantly decreased the expression levels of inflammatory factors such as interleukin-6(IL-6),interleukin-8(IL-8),and TNF-?in LPS-induced HUVEC.Sepsis is a severe threat to human health due to systemic tissue and organ damage caused by an uncontrolled host response to infection or injury.Acute lung injury(ALI)is an essential pathological process of sepsis.Currently,CLP is a common surgical model for triggering sepsis in mice.In the CLP-induced sepsis model,intestinal bacteria enter the blood circulation from the abdominal cavity,triggering a systemic inflammatory response and tissue damage,and even organ failure.Since EC activation is an essential cause of sepsis,preliminary in vivo studies were conducted based on the above findings that ZIPK regulates LPS-induced expression of adhesion molecules and inflammatory factors.Moderate and severe mouse sepsis models were established by 25%and 50%cecum ligation puncture,respectively,and correlation analysis was performed at 8 h and 24 h postoperatively using histochemical staining,Q-RT PCR,and ELISA.The results showed that the lung tissues of 25%of the mice with moderate sepsis model of cecum ligation were significantly edematous at 8 h and 24 h postoperatively,with more inflammatory cell infiltration in the alveoli and thickened alveolar walls;other organs such as liver,kidney,and heart all had different degrees of inflammatory cell infiltration.The above results indicated that the CLP sepsis mouse model was successfully established in this paper.The lung pathology score of KO mice decreased at 8 h and 24 h after surgery compared with that of wild-type(WT)mice.The mRNA expression levels of Vcam-1 and Icam-1 in the lung tissues of KO mice were lower than those of WT mice at 8 h postoperatively;the lung tissues of KO mice showed a decreasing trend of pro-inflammatory factor Il-1?mRNA expression at 24 h and Tnf-?mRNA expression at 8 h,but there was no statistical difference.Inflammatory factors including TNF-?,IL-6,and IL-10 in plasma and alveolar lavage fluid were not significantly altered.It is suggested that knocking down ZIPK has a tendency to improve ALI and other factors in moderate septic mice,but the effect is not significant.In this paper,we also found that the decrease in body temperature was significantly inhibited(to 120 h)in cecum ligation 50%of severe sepsis KO mice compared with WT mice;the level of body weight loss in KO mice was also lower than that in WT mice;suggesting that inhibition of ZIPK may improve the pathological process of severe sepsis.In conclusion,the results of this paper suggest that ZIPK is involved in the regulation of LPS-induced EC adhesion molecules and some inflammatory factors expression,and its role may be mainly through promoting the activation of PI3K/AKT and JNK signaling pathways;ZIPK knockdown tends to improve pathological lung injury,bacterial leakage,weight loss,and inflammatory factors expression in the acute phase(8 h and 24 h after surgery)of moderate sepsis mice There was a trend of improvement,but it was not significant.In addition,ZIPK knockdown effectively prevented the decrease of body temperature in mice with severe sepsis and improved the decrease of body weight to some extent.This paper provides new evidence for the involvement of ZIPK in the regulation of EC activation and microvascular endothelial barrier disruption during the inflammatory response and tentatively finds that ZIPK knockdown tends to improve the early pathological process of moderate sepsis in mice and may improve the early symptoms of severe sepsis.