Relationship between endothelial damage and p120-catenin in paraquat intoxication and the protective effect of mangiferin / 中华危重病急救医学

ABSTRACT

Objective To investigate the relationship between endothelial damage and p120-catenin (p120-ctn) in a model of paraquat intoxication,and the modulatory effect of mangiferin on p120-ctn.Methods Human umbilical vein endothelial cells (HUVECs) were cultured in two compartment spreading apparatus in vitro.The endothelial cells were divided into three groups:control group (cultured in DMEM with 10％ fetal bovine serum),paraquat group (paraquat was added to the medium with final concentration of 0.05 μmol/L) and mangiferin group (cultured in medium with addition of paraquat for 30 minutes,then mangiferin was added in a final concentration of 20 μmol/L).The cellular permeability at 6,12,24,48,72 hours after culture in the three groups was measured.The expressions of p120-ctn 1A,p120-ctn 3A mRNA and p120-ctn protein were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot analysis.The distribution of p120-ctn protein was observed by immunofluorescence.Results Compared with control group,cellular permeability in paraquat and mangiferin groups were increased with prolongation of time,and peaked at 72 hours [(29.86 ± 3.98)％,(24.39 ± 2.79)％ vs.(11.71 ± 1.67)％,both P＜0.05].The cellular permeability was significantly lower in mangiferin group than that in paraquat group at different time points (all P＜0.05).At 6 hours after intoxication,the expressions of p120-ctn 1A,p 120-ctn 3A mRNA (gray value) and p 120-ctn protein (gray value) were significantly lower in paraquat group than those in control group (p120-ctn 1A mRNA:0.150 ± 0.024 vs.0.433 ± 0.024,p120-ctn 3A mRNA:0.316 ± 0.043 vs.0.701 ±0.020,p120-ctn protein:0.485 ±0.031 vs.0.763 ±0.038,all P＜0.01).The expressions of p120-ctn 1A,p120-ctn 3A mRNA and p120-ctn protein were significantly higher in mangiferin group than those in paraquat group from 6 hours on (p120-ctn 1A mRNA:0.281 ± 0.021 vs.0.150 ± 0.024,p120-ctn 3A mRNA:0.602 ± 0.042 vs.0.316 ± 0.043,p120-ctn protein:0.675 ± 0.031 vs.0.485 ± 0.031,all P＜0.01),and they were gradually increased with prolongation of time,and peaked at 72 hours (p120-ctn 1A mRNA:1.376 ±0.128 vs.0.150 ± 0.024,p120-ctn 3A mRNA:1.251 ± 0.059 vs.0.316 ± 0.043,p120-ctn protein:0.844 ± 0.050 vs.0.485 ± 0.031,all P＜ 0.01).Under upright fluorescence microscope,p120-ctn was mainly distributed in the cell membrane in control group,with a slight expression in cytoplasm,and no expression in the nuclei.With prolongation of time,p120-ctn expression in the cell membrane was gradually decreased in paraquat group,while it was increased in the cytoplasm and nuclei,with blurring of cell membrane and widening of cellular gap.p120-ctn expression was improved on the cell membrane in mangiferin group at corresponding time points,with decreased in expression in nuclei and cytoplasm.Conclusion The p120-ctn protein plays an important role in the enhancement of endothelial permeability in paraquat intoxication,and mangiferin may attenuate endothelial injury in paraquat intoxication possibly through modulation of p 120-ctn protein.