Effects And Mechanisms Of Piezo1 On The Regulation Of Intestinal Epithelial Barrier Function

Part 1 The role of Piezo1 in regulating intestinal barrier functionAim To explore the role of Piezo1 in the regulation of intestinal barrier function and TJsMethods Cells were infected with lentiviral vector to knockdown and overexpress Piezo1.Caco-2 cells were divided into Piezo1-knockdown（Piezo1-KD）,Piezo1-overexpression（Piezo1-OV）group,Control-KD（CON-KD）and Control-OV（CON-OV）groups.The expression of Piezo1,Claudin-1,Occludin,ZO-1 were evaluated using PCR,Western Blot,and Immunostaining.The Caco-2 monolayer and colon epithelium were divided into control,6μg/ml Ruthenium Red（Ru R）,2μg/ml Gs MTx4 and 1μg/ml Yoda1 groups.An in vitro epithelial barrier model was established.The barrier function were evaluated by measuring trans-epithelial resistance（TEER）and FITC-FD4 flux rate of both the Caco-2 monolayer and colon epithelium.Results1.Piezo1 was abundantly expressed in the mouse intestinal epithelium and cellular membrane of Caco-2 cell monolayers2.The Piezo1-KD group showed higher TEER than the control group（492.68±23.39 vs.431.85±27.56Ω×cm²,n=6,P=0.003）,while no significant difference was found in FD4 influx(0.45±0.02 vs.0.44±0.02 ng×m L^-1cm^-2min^-1,n=6,P=0.085).The Piezo1-OV group showed a sharp decrease in TEER（224.68±22.24 vs 393.05±44.00Ω×cm²,n=6,P<0.001）and an increase in FD4 influx(1.01±0.14 vs 0.42±0.07 ng×m L^-1cm^-2min^-1,n=6,P<0.001).In the Piezo1-KD group,the expression of Claudin-1 was higher（n=6,P=0.024 in PCR,P=0.0006 in western-blot）.In the Piezo1-OV group,the expression of Claudin-1 was significantly decreased（n=6,P=0.016 in PCR,P<0.001 in western-blot）.Occludin was also decreased in the Piezo1-OV group（n=6,P=0.002 in PCR and western-blot）,while no significant difference was found in the Piezo1-KD group（n=5,P=0.431 in PCR,P=0.474 in western-blot）3.In Caco-2 monolayer,TEER in the Ru R and Gs MTX4 group were higher than the control（CON）group（426.0±6.19 and 428.27±6.74 vs 432.0±5.42Ω×cm²,n=6,P=0.027 and 0.006,respectively）,FD4 influx in the Gs MTX4 group was lower than the CON group(0.44±0.03 vs 0.53±0.02 ng×m L^-1cm^-2min^-1,n=6,P=0.03).TEER in the Yoda1 group was significantly lower（354.82±8.33Ω×cm²,n=6,P<0.001）,FD4 influx in the Yoda1 group was higher(0.64±0.03 ng×ml^-1cm^-2min^-1,n=6,P<0.001)than the CON group.In a mouse model,activation of Piezo1 by Yoda1 significantly decreased TEER（78.90±2.88 vs 54.00±3.57Ω×cm²,n=5,P=0.006）and increased FD4permeability(1.59±0.08 vs 1.15±0.05 ng×m L^-1cm^-2min^-1,n=5,P=0.001)4.Immunostaining showed colocalization of Piezo1 and Claudin-1.ConclusionChanges of Piezo1 activity and expression can alter barrier function and permeability.Piezo1 can regulate barrier function by affecting the expression of Claudin-1 and Occludin.Part 2 Piezo1 regulates intestinal barrier function via ROCK1/2 signaling pathwaysAim To explore the potential pathways including ROCK and Erk in regulating intestinal barrier functionMethods Cells were infected with lentiviral vector to knockdown and overexpress Piezo1.ROCK inhibitor（Y-27632）and activator（U46619）were used with Caco-2 cells.Caco-2 cells are divided into eight groups:CON-KD,PIEZO-KD,CON-KD+Y-27632,Piezo1-KD+Y-27632,CON-OV,Piezo1-OV,CON-OV+U46619,Piezo1-OV+U46619 groups.The expression of Claudin-1,ZO-1,Occludin were evaluated by immunostaining,RT-PCR,and western blot.The expression of ROCK1/2,Erk1/2 were evaluated by RT-PCR and western blot.Results1 The expression of ROCK1 and ROCK2 in the Piezo1-KD group is higher than the CON-OV group（n=5,6;P<0.001,P<0.001）.Thr455/Ser456 ROCK1and Ser1366 ROCK2 were also higher expressed in the Piezo1-KD group（n=6;P=0.034,P<0.001）,and lower expressed in the Piezo1-OV group（n=6;P=0.035,P=0.003）.No significant changes were observed in Erk1/2expression.（n=6;P=0.952,P=0.923）2 Inhibition of ROCK1/2 decreased Claudin-1 expression in Piezo1-KD group（n=6,P<0.001）.The CON-OV+U46619 group showed no difference in Claudin-1expression compared with that in the CON-OV group（n=6,P=0.074）.In the Piezo1-OV+U46619 group,U46619 increased Claudin-1 expression compared to that in Piezo1-OV group（n=6,P<0.001）.No changes were found in the expression of Occludin and ZO-1 after inhibiting ROCK1/2（P>0.05）。ConclusionManipulation of Piezo1 changed phosphorylated ROCK1/2 expressionInterference on ROCK1/2 reversed the effect of Piezo1 on Claudin-1...