Mechanism Of FAK Inhibitors In Alleviating Venous Inflammatory Injury Induced By Central Venous Catheter Administration

Objective:Central venous catheter administration can still cause venous thrombosis,catheter-related bloodstream infection and other complications.Catheter-related bloodstream infection is one of the main causes of nosocomial infection,which is significantly related to high hospitalization fee and the morbidity,mortality of nosocomial acquired sepsis.At present,a large number of studies have shown that prevention of infection,alleviation of venous inflammatory injury and reduction of bacterial adhesion during CVC administration can effectively prevent venous thrombosis and reduce the incidence of catheter-related bloodstream infection and related disease mortality.The aim of this study was to investigate the mechanism by which FAK inhibitor Y15 inhibits FAK/Akt phosphorylation and alleviates venous inflammatory injury induced by CVC.Methods:Part 1,1.The cells were randomly divided into normal control group,FAK inhibitor group(Y15 group),CVC+scratch+5-FU group and CVC+scratch+5-FU+Y15 group.Simulate the model of vascular endothelial cell injury induced by CVC retention by using Scratch method and CVCs and EA.hy926 cells co-culture,adding 5-FU(CVC+scratch+5-FU).2.The activity of EA.hy926 cells was determined by MTT colorimetric assay.The apoptosis rate of EA.HY926 cells was determined by flow cytometry.The levels of reactive oxygen species in each group were detected by DCF.Nitric oxide in the supernatant of cell culture in each group was determined by nitrate reductase method.The relationship between the treatment time of FAK inhibitor and FAK inhibitor and the changes of cell activity,apoptosis rate and oxidative stress in cell injury model induced by CVC+scratch+5-FU were analyzed.Part 2,1.The healing rate of each group of cells after scratch injury was detected by using the model of vascular endothelial cell injury induced by CVC+scratch+5-FU and the principle of cell scratching.To analyze the time-effect relationship between FAK inhibitor Y15 and EA.HY926 cell migration ability in cell injury model.To analyze the time-effect relationship between FAK inhibitor Y15 and EA.HY926 cell migration ability in cell injury model.Monocyte--EA.hy926 cell adhesion assay was used to determine the adhesion of monocyte in each group,and the influence of Y15 on the adhesion of monocyte in vitro injury model was analyzed.2.Western Blotting assay was used to detect the protein expression levels of FAK,Akt,p-FAK Tyr 397 and p-Akt Ser 473 in EA.hy926 cells,and to analyze the time-effect relationship between CVC and 5-FU-induced vascular endothelial cell injury,FAK inhibitor and FAK phosphorylation,Akt phosphorylation.Elisa experimental principle and method were used to detect the levels of cytokines CRP,IL-6,TNF-?,FIB,adhesion molecules VCAM-1 and ICAM-1 in the cell culture supernatants of different treatment groups at different time points.Meanwhile,RT-qPCR experimental principles and methods were used to verify the changes of the cytokines CRP,IL-6,TNF-?,eNOS,adhesion molecules VCAM-1 and ICAM-1 in mRNA levels in different groups at different time points.To analyze the time-effect relationship between the phosphorylation levels of FAK and Akt and the expression levels of related inflammatory factors,FIB,adhesion molecules VCAM-1 and ICAM-1 in vitro injury models.Part 3,1.An animal model of rabbit external jugular phlebitis induced by CVC input of 5-fu was established,and the body temperature of the rabbits at different time points was measured with a rectl thermometer.The number of colonies was observed after CVCs culture.Pathological sections were used to observe the pathological changes of external jugular vein,such as inflammatory cell infiltration,venous thrombosis,and granulation tissue.The relationship between Y15 inhibition of FAK,Akt phosphorylation and CVC combined with 5-FU induced pathological changes of external jugular vein was analyzed.2.The changes of inflammatory factors CRP,IL-6,TNF-? and fibrin FIB,adhesion molecules VCAM-1 and ICAM-1 mRNA were measured by RT-qPCR.Western Blotting was used to detect the differences in the expression levels of FAK,Akt,p-FAK Tyr 397 and p-Akt Ser 473 in the external jugular vein tissues of each group at different time of intervention.Results:Part 1,The differences of cell activity among groups and at different time point were statistically significant(F=145.400,P<0.001;F=42.980,P<0.001).At T1(intervention 24h),T2(intervention 48h),T3(intervention 72h),CVC+scratch+5-FU group and CVC+scratch+5-FU+Y15 group were significantly lower than the normal control group(P<0.001),and the cell activity of the CVC+scratch+5-FU+Y15 group was significantly lower than the CVC+scratch+5-FU group(P<0.001).The apoptosis rate was detected by flow cytometry.It was found that Y15 did not significantly change the apoptosis of EA.hy926 cells(P>0.05).Compared with the control group,the levels of reactive oxygen species in the CVC+scratch+5-FU group and the CVC+scratch+5-FU+Y15 group were significantly increased at each time point(P<0.001),and Y15 significantly reduced CVC+scratch+5-FU induced high ROS levels(P<0.001).In addition,the levels of NO in the CVC+scratch+5-FU group at T1,T2,and T3 and the CVC+scratch+5-FU+Y15 group at T2 and T3 were significantly lower than those in the control group(P<0.001),but after Y15 treatment with CVC+scratch+5-fu group,the level of NO at each time point increased significantly.Part 2,1.At T2 and T3,compared with the control group,Y15 significantly increased the scratch healing rate(P<0.001),but the scratch healing rate was significantly lower in the CVC+scratch+5-FU group(P<0.001).With the intervention of Y15,the scratch healing rate of cells in the CVC+scratch+5-fu group was significantly increased.Mononuclear cell-EA.hy926 cell adhesion assay showed that after 72h treatment,the adhesion of monocytes in CVC+scratch+5-fu+Y15 group was significantly lower than that in CVC+scratch+5-fu group(P<0.001),and there was no difference with the normal control group(P>0.05).2.Western Blotting results showed that the expression levels of p-FAK Tyr 397 and p-Akt Ser 473 in CVC+scratch+5-FU group were significantly higher than those in the normal control group(P<0.001),and were time-dependent.Mean value of normal control group and CVC+scratch+5-FU group:relative expression level of p-FAK Tyr 397(treatment 24h:0.36+0.032 VS 0.62+0.038;treatment 48h:0.36±0.032 VS 0.97±0.049;treatment 72h:0.36±0.032 VS 1.27±0.044),;relative expression level of p-Akt Ser 473(treatment 24h:0.36±0.073 VS 0.62±0.050);treatment 48h:0.36±0.073 VS 0.97±0.075;treatment 72h:0.36±0.073 VS 1.27±0.036).Compared with CVC+scratch+5-fu+Y15 group,p-FAKTyr 397 at T1 and T3 and p-Akt Ser 473 protein expression levels at T2 and T3 were significantly down-regulated(P<0.05)in a time-dependent manner.Mean of CVC+scratch+5-FU group vs CVC+scratch+5-FU+Y15 group:relative expression level of p-FAK Tyr 397(treatment 24h:0.62±0.038 VS 0.39±0.05;treatment 48h:0.97±0.049 VS 0.55±0.014;treatment 72h:1.27±0.044 VS 0.88±0.014);relative expression level of p-Akt Ser 473(treatment 24h:0.62±0.050 VS 0.49±0.029;treatment 48h:0.97±0.075 VS 0.78±0.030;treatment 72h:1.27±0.036 VS 1.05±0.061)?3.Elisa and RT-qPCR results showed cytokine CRP,IL-6,TNF-?,FIB,eNOS,ICAM-1,VCAM-1 and integrin ?1 levels differences in groups and in different time points were statistically significant(P<0.001 or P<0.01),and there was an interaction between time and groups(P<0.001).Part 3,1.The main changes of rabbit body temperature were as follows:The body temperature of the CVC+5-fu group at 2 weeks,the 4-week group and the 6-week group was significantly higher than that of the normal control group at the CVCs catheterization day and the first day,the second day,and the third day after CVCs catheterization and the day of execution(P<0.001).However,compared with the normal control group,the body temperature of CVC+5-fu+Y15 group at 2 weeks and 4 weeks was significantly increased at 0,the first day and the second day after CVCs catheterization(P<0.001),and there was no difference in rabbit body temperature between the third day after CVCs catheterization and the day of execution.Compared with the normal control group,CVC+5-fu+Y15 6 weeks group showed significantly higher body temperature(P<0.05)at 0 days,1 day after CVCs catheterization and the day of execution,and no difference at other time points(P>0.05).2.Histopathological examination results showed that in the CVC+5-fu group,vitreous changes and/or myxoid changes occurred in the vascular endothelium at 2 weeks of intervention,and the vascular intima was shed,and the blood was bleeding under the internal model;Four weeks after intervention,thrombosis,granulation tissue and infiltration of neutrophils and monocytes were observed in the lumen;At the 6th week of intervention,scar tissue was formed under the intima.CVC+5-fu+Y15 group:the degree of vascular endothelial injury was significantly reduced;No obvious pathological changes were observed in the vascular endothelium at 2 or 4 weeks after intervention,and hyaline and/or myxoid changes occurred in the vascular endothelium at 6 weeks;No thrombosis was observed in the 5 rabbits.3.CVC+5-FU group:Compared with the normal control group,the levels of cytokines VC AM-1,ICAM-1,CRP,IL-6,TNF-? and integrin?1 mRNA were significantly up-regulated(P<0.001),while eNOS mRNA levels were significantly down-regulated(P<0.001),and p-FAK Tyr 397 and p-Akt Ser 473 protein expression levels were significantly up-regulated(P<0.001).CVC+5-FU+Y15 group:Compared with CVC+5-FU group,the levels of cytokines VCAM-1,ICAM-1,CRP,IL-6 and integrin?1 mRNA were significantly down-regulated(P<0.001).The level of eNOS mRNA was significantly up-regulated(P<0.001),and the phosphorylation levels of FAK Tyr 397 and Akt Ser 473 were significantly decreased(P<0.001).Conclusions:1.After Y15 treatment of CVC+scratch+5-FU-induced damaged cells,the cell apoptosis rate did not change significantly,and the ROS level was significantly down-regulated,but it was still higher than the normal control group,and the NO level was significantly increased.2.In vitro,Y15 treated the model group,inhibited the phosphorylation of FAK Tyr 397 and Akt Ser 473,enhanced the migration ability of EA.hy926 cells,decreased the number of adhesion to THP-1 cells,and related cytokine VCAM-1.ICAM-1,CRP,IL-6,TNF-?,FIB and integrin?1 levels were down-regulated to varying degrees,while eNOS levels were up-regulated.It was suggested that Y15 alleviated the degree of damage of EA.hy926 cells 3.Through the rabbit model,this study further verified the role of Y15 in slowing down CVC+5-fu induced venous inflammatory injury.