Negative Effect And Mechanisms Of Uremic Toxins On The Intestinal Barrier Function

Part one: Effects of uremia serum on the structure and function of intestinal barrier functionObjective: To observe the effect of uremia serum on the structure and function of intestinal barrier function and provide theoretical basis to explore the direct evidence that uremic conditions lead to intestinal dysfunction.Methods: Fasting blood was collected from patients with uremia and healthy volunteers and detect creatinine,urea nitrogen,albumin and other important biochemical indicators.The proliferation of Caco-2 cells which were treated with different concentrations of serum(10%,20%,30%,40%)were determined by CCK-8 and the time gradient of 24,48,72 h were set.Caco-2 cells were divided into control group,heath group,uremia group.Immunofluorescence was used to detect the distribution and morphological changes of ZO-1,Occludin and Claudin-1 in Caco-2 cells.Western blot was used to detect the expression of the above proteins.Caco-2 cells were used to establish monolayers.The integrity of Caco-2 monolayers were measured in different ways(morphology,AKP activity and fluorescein sodium penetration experiment).The Caco-2 monolayers were divided into control group,heath group and uremia group,and the barrier function of monolayers were tested by sodium fluorescein permeation test.Results:(1)The serum levels of Scr,Urea,UA in patients with uremia were significantly higher than those in healthy controls(p <0.01).(2)Compared with the heath group,30% and 40% uremia serum could significantly inhibit the proliferation of Caco-2 cells after 72 hours of culture(P<0.01),the proliferation of Caco-2 cells cultured in 40% healthy serum for 72 h was lower than that in 30% healthy serum group.Therefore,serum concentration of 30%,72 hours of action time was used for subsequent experiments.(3)Immunofluorescence indicated that uremia serum could change the distribution of ZO-1,Occludin and Claudin-1 protein.Western blotting showed that uremia serum could significantly down-regulate the expressions of ZO-1,Occludin and Claudin-1(P <0.01).(4)Caco-2 cells formed a complete and dense monolayer after 21 days.Uremic serum could obviously increase the sodium fluorescein permeability in Caco-2 monolayer(P <0.01).Conclusions:(1)The serum levels of Scr,Urea,UA increased significantly.(2)The uremia serum could significantly down-regulate the expressions of ZO-1,Occludin and Claudin-1,and changed their distribution.(3)Uremia serum could obviously increase the permeability of Caco-2 monolayer.Part two: Negative effect and mechanisms of urea combined urease on the intestinal barrier functionObjective: To observe the effect of urea on the structure and function of intestinal barrier function.To study the effect of urea combined urease on the structure and function of intestinal epithelial barrier,and explore the role of NF-?B / MLCK pathway in it.Methods: The proliferation of Caco-2 cells which were treated with different concentrations of urea(0,10,20,40mmol/L)were determined by CCK-8 and the time gradient of 24,48,72 h were set.Western blotting was used to detect the expression of the ZO-1,Occludin and Claudin-1 proteins.Caco-2 cells were divided into control group,urea group,urease group,urea combined urease group.Immunofluorescence was used to detect the distribution and morphological changes of ZO-1,Occludin and Claudin-1 in Caco-2 cells and western blot was used to detect the expression of the above proteins.Meanwhile,the expression of NF-?B P65,p-NF-?B P65,I?B?,p-I?B?,MLCK,MLC,p-MLC in Caco-2 cellsin Caco-2 cells would be detected.The Caco-2 monolayers were divided into control group,urea group,urease group,urea combined urease group,and the barrier function of monolayers were tested by sodium fluorescein permeation test.Results:(1)Urea could not affect the proliferation and the expression of the tight junction proteins of Caco-2 cells,even in high concentrations of 40mmol/L.(2)Compared with control group,urea group and the urease group,urea combined urease could significantly inhibit the proliferation of Caco-2 cells(P <0.01).(3)Immunofluorescence indicated that urea combined urease could change the distribution of ZO-1,Occludin and Claudin-1 protein.Western blot showed that urea combined urease could significantly down-regulate the expressions of ZO-1,Occludin and Claudin-1,compared with control group,urea group and urea combined urease group,the difference was statistically significant(P<0.01).(4)Urea combined urease could obviously increase the sodium fluorescein permeability in Caco-2 monolayer,compared with control group,urea group and urea combined urease group,the difference was statistically significant(P <0.01).(5)Compared with control group,urea group and urea combined urease group,the ratio of p-NF-?B P65/NF-?B P65?p-I?B?/I?B??p-MLC/MLC and the expression increased significantly in urea combined urease group(P <0.01).Conclusions:(1)Urea could not inhibit the proliferation of Caco-2 cells in the concentration range of 0-40 mmol / L,and had no significant effect on the expression of tight junction proteins.(2)The urea combined urease could significantly down-regulate the expressions of ZO-1,Occludin and Claudin-1,and changed their distribution.(3)Urea combined ureasecould obviously increase the permeability of Caco-2 monolayer.(4)Urea combined with urease could activate NF-?B/MLCK pathway,which might be have damaging effects on the intestinal barrier function.