Bioinformatics Analysis Of Human Zo-1 Protein And The Role Of 20(s)-protopanaxadiol In Lipopolysaccharideinduced Endothelial Barrier Injury

Sepsis means that the body produces a variety of physiological disorders in the state of infection,resulting in a variety of organ damage and dysfunction,seriously endangering the lives of patients.The pathogenesis of sepsis involves a highly complex comprehensive response,the central link of which is the change of endothelial barrier function.Tight junction is not only an important part of the paracellular pathway of endothelial cell barrier,but also regulates the function of endothelial cells.ZO-1 is an important component of tight junction protein.At present,there are many studies on human ZO-1,but the bioinformatics analysis of human ZO-1 has not been reported.Therefore,the bioinformatics analysis of human ZO-1 can provide a theoretical basis for the study of human ZO-1 in the treatment of septic cell barrier injury.20（S）-protopanaxadiol（20（S）-PPD）belongs to protopanaxadiol ginsenoside,which has a good effect on anti-inflammation and protection of blood vessels.However,it has not been reported whether20（S）-PPD can reduce the damage of endothelial barrier caused by inflammation.Therefore,whether 20（S）-PPD can improve the damage of endothelial barrier induced by LPS is a question worthy of study.In the first part of the experiment,bioinformatics methods were used to reveal various physical and chemical properties and biological functions of human tight junction protein ZO-1,so as to provide ideas and theoretical methods for the treatment of cell barrier damage in human sepsis.In this part,bioinformatics tools were used to predict and analyze the physical and chemical properties,structure,function and homologous relationship among species of human ZO-1 protein.The results show that human ZO-1 contains27191 atoms and 1748 amino acids,the theoretical isoelectric point is 6.24,most of the negatively charged residues are negatively charged acidic proteins,the instability index is 59.28,the average hydrophilic total number is-0.911,which belongs to unstable hydrophilic proteins,and the subcells are located in the cytoplasm and do not contain signal peptides and transmembrane helix structures.The secondary structure consists ofα-helix,β-folding,β-rotation and random crimp,which are coiled together to form a higher tertiary structure,which contains 144 serine sites,54 threonine sites and 34 tyrosine sites,the N-terminal has three PDZ domains and SH3domains,and the C-terminal has an actin binding domainαdomain and an unknown functional region ZU5 domain.Human ZO-1 has high homology with rat.KEGG signal pathway analysis shows that human ZO-1 interacts with a variety of tight junction proteins and participates in a variety of biological reactions.Therefore,as an important part of tight junction,ZO-1participates in a variety of regulation of paracellular pathways and can be used as a potential target for the treatment of sepsis-related endothelial barrier injury.In the second part of the experiment,human umbilical vein endothelial cell（HUVECs）was used as the study cell to establish an in vitro septic model induced by LPS to study whether 20（S）-PPD can reduce the injury of endothelial barrier induced by LPS.First of all,the cell viability was detected by CCK-8 method to determine the safe concentration range of 20（S）-PPD on HUVECs.Then,combined with the previous research results in the laboratory,CCK-8 method was used to detect the effect of LPS on the viability of HUVECs cells at different times,and the model of HUVECs damage induced by LPS was established.Secondly,HUVECs in logarithmic growth phase were divided into control group,LPS group and 20（S）-PPD+LPS group（P+L）.The effects of 20（S）-PPD on LPS-induced HUVECs cell viability and endothelial barrier damage were detected by CCK-8method and Transwell chamber.Finally,the effects of 20（S）-PPD on ZO-1and cytoskeleton protein F-actin induced by LPS in HUVECs cells were observed by immunofluorescence.The results showed that 20（S）-PPD significantly increased the cell viability of HUVECs at 2.5μmol·L^-1.When HUVECs was injured by 10μg/m L LPS,the cell viability decreased with the prolongation of LPS treatment time,and the difference was significant from3h.The cell viability and TEER value of 20（S）-PPD pretreatment group were significantly higher than those of LPS group,while Pa value was significantly lower than that of LPS group（P<0.05）.Therefore,2.5μmol·L^-120（S）-PPD and 10μg/m L LPS were selected for follow-up experiment.The immunofluorescence results showed that compared with the control group,there was obvious depolymerization and ectopic phenomenon of F-actin and no obvious microfilament in LPS group,and there was also depolymerization and ectopic phenomenon in F-actin of P+L group,but there was still obvious microfilament expression compared with LPS group.Compared with the control group,the fluorescence of ZO-1 in LPS group decreased and there were obvious deletion and ectopic phenomena.The expression of ZO-1 in P+L group was also decreased and deleted,but the phenomenon of ZO-1 deletion was less than that in LPS group.These results suggest that human ZO-1,as an important part of tight junction,participates in a variety of regulation of paracellular pathways and has the possibility of becoming a therapeutic target for sepsis.20（S）-PPD can attenuate the decrease of viability and high permeability of HUVECs cells induced by LPS.The molecular mechanism may be related to the increased expression of cytoskeleton protein F-actin and connexin ZO-1.