| Mechanical stretch caused by ventilator and anesthesia machine damage the lung tissue in clinical anesthesia and intensive care units.Mechanical factors increase the transpulmonary pressure and shear stress,damage the integrity of alveolar cell membrane,impaired barrier function,increase permeability,and then cause lung edema,and even acute lung injury,namely ventilator induced lung injury(VILI).At present,the mechanisms of VILI have not been fully studied,and its pathogenesis research mainly focuses on four aspects:inflammatory response,lung surfactant,lung surface tension and alveolar permeability.This study mainly explores the role of alveolar permeability in the pathogenesis of VILI,the upstream genes and the expressions of tight junction and adherin junction.Part 1 Expressions of E-cadherin,Occludin,Elk1,and MMP-9 in VILIObjectiveTo explore the expressions of E-cadherin,Occludin,Elk1,and MMP-9 treated with high tidal volume mechanical ventilation and 20%cyclic stretch.MethodsIn vivo,thirty C57BL/6 mice were randomly divided into 3 groups(n=10):control group(MV 0h group),high tidal volume 2h group(MV 2h group),high tidal volume 4h group(MV 4h group).The mice which in MV 0h group did not treat with mechanical ventilation.The mice treated with high tidal volume mechanical ventilation in MV 2h group and MV 4h group.The parameters in MV 2h group and MV 4h group as follows:tidal volume for 20mL/kg,respiratory frequency for 40/min,positive end-expiratory pressure(PEEP)of 0cm H2O,respiratory ratio(I:E)of 1:2,the ventilation time for 2h and 4h.After high tidal volume mechanical ventilation,lung injury score,wet/dry weight ratio(W/D),Evans blue dye,bronchoalveolar lavage fluid(BALF)and HE staining were evaluated the lung injury.The distribution of E-cadherin and Occludin was observed by immunofluorescence.The expressions of E-cadherin,Occludin,Elk1,and MMP-9 were measured by Western Blot.In vitro,MEL-12 cells were randomly divided into 3 groups:CS 0h group,CS 2h group,CS 4h group.MLE-12 cells in CS 0h group were not treated with cyclic stretch.MLE-12 cells were treated with cyclic stretch in CS 2h group and CS 4h group.The stretch time is 2h and 4h.After cyclic stretch,MLE-12 cells were lysed to pick up the total protein to measure the expressions of E-cadherin,Occludin,Elk1,and MMP-9 by Western Blot.MLE-12 cells were observed the distribution of E-cadherin and Occludin by immunofluorescence.ResultsIn vivo,HE staining found that the lung tissue of mice had pulmonary congestion,alveolar wall thickening slightly,alveolar transparent membrane formation in MV 2h group;the lung tissue of mice had pulmonary congestion severely,alveolar hemorrhage,alveolar wall thickening and alveolar transparent membrane formation in MV 4h group.Compared with MV 0h group,lung injury score,wet/dry weight ratio(W/D),evans blue dye,total cell counts of BALF increased in MV 2h group and MV 4h group(P<0.05).Compared with MV 0h group,the expressions of E-cadherin and Occludin decreased(P<0.05),the expressions of Elkl and MMP-9 increased in MV 2h group and MV 4h group by Western Blot(P<0.05).Immunofluorescence found that fluorescence expressions of E-cadherin and Occludin attenuated in MV 2h group and MV 4h group.In vitro,immunofluorescence found that fluorescence expressions of E-cadherin and Occludin attenuated in CS 2h group and CS 4h group.Compared with CS 0h group,the expressions of E-cadherin and Occludin decreased,the expressions of Elk1 and MMP-9 increased in the CS 2h group and the CS 4h group by Western Blot(P<0.05).ConclusionsHigh tidal volume mechanical ventilation and pathological cyclic stretch reduced the expressions of E-cadherin and Occludin,damaged alveolar permeability,caused lung edema and lung injury.Part 2 Effects of E-cadherin,Occludin and permeability treated with Elk1 or MMP-9 knockdown in VILIObjectiveTo explore the effects of E-cadherin,Occludin and permeability treated with Elk1 or MMP-9 knockdown in VILI.MethodsIn vitro,MEL-12 cells were randomly divided into 8 groups:CS 0h group,si-Nc group,si-Elk1 group,si-MMP-9 group,CS 4h group,CS 4h+si-Nc group,CS 4h+si-Elk1 group,CS 4h+si-MMP-9 group.Negative control siRNA,Elk1 siRNA,or MMP-9 siRNA was transfected into MLE-12 cells for 48h.MLE-12 cells were lysed to pick up the total protein to measure the expressions of Elkl and MMP-9 by Western Blot.MLE-12 cells were treated with cyclic stretch in CS 4h group,CS 4h+si-Nc group,CS 4h+si-Elk1 group,and CS 4h+si-MMP-9 group.The parameters in CS 4h group,CS 4h+si-Nc group,CS 4h+si-Elk1 group,and CS 4h+si-MMP-9 group:amplitude 20%,frequency 0.5HZ,stretch and relax ratio of 1:1,stretch time 4h.After cyclic stretch,MLE-12 cells were lysed to pick up the total protein to measure the expressions of E-cadherin,Occludin,Elk1,and MMP-9 by Western Blot.MLE-12 cells were observed the distribution of E-cadherin and Occludin by immunofluorescence.In vivo,Forty C57BL/6 mice were randomly divided into 4 groups(n=10):MV 0h group,MV 4h group,MV 4h+si-Elk1 group,MV 4h+si-MMP-9 group.The mice did not treat with mechanical ventilation in MV 0h group.mice were injected by caudal vein with Elk1 siRNA(2.5 ?g/g)or MMP-9 siRNA(2.5 ?g/g),10%glucose solution,DEPC water,and the EntransterTM In Vivo Transfection reagent at the dosage of 200 ?L according to the manufacturer's instructions.After 72 h of transfection,the mice were treated with mechanical ventilation for 4 h in MV 4h+si-Elk1 group and MV 4h+si-MMP-9 group.The parameters in MV 4h group,MV 4h+si-Elk1 group and MV 4h+si-MMP-9 group as follows:tidal volume for 20mL/kg,respiratory frequency for 40/min,positive end-expiratory pressure(PEEP)of 0cm H2O,respiratory ratio(I:E)of 1:2,the ventilation time for 4h.After high tidal volume mechanical ventilation,lung injury score,wet/dry weight ratio(W/D),evans blue dye,bronchoalveolar lavage fluid(BALF)and HE staining were evaluated the lung injury.The distribution of E-cadherin and Occludin was observed by immunofluorescence.The expressions of E-cadherin,Occludin,Elk1,and MMP-9 were measured by Western Blot.ResultsIn vitro,immunofluorescence found that fluorescence expressions of E-cadherin and Occludin attenuated in CS 4h group,fluorescence expressions of E-cadherin and Occludin enhanced in CS 4h+si-Elk1 group and CS 4h+si-MMP-9 group.Compared with si-Nc group,the Elk1 level decreased 80%in si-Elk1 group(P<0.05),the MMP-9 level decreased 70%in si-MMP-9 group(P<0.05).Compared with CS 4h group,the expressions of E-cadherin,Occludin,Elk1,and MMP-9 were not statistical significance in CS 4h+si-Nc group(P>0.05).Compared with CS 4h+si-Nc group,the expressions of E-cadherin and Occluidn increased(P<0.05),Elk1 decreased in CS 4h+si-Elk1 group(P<0.05);the expressions of E-cadherin and Occluidn increased(P<0.05),MMP-9 decreased in CS 4h+si-MMP-9 group(P<0.05).In vivo,HE staining found that the lung tissue of mice had pulmonary congestion severely,alveolar hemorrhage,alveolar wall thickening and alveolar transparent membrane formation in MV 4h group;pulmonary congestion,alveolar hemorrhage,alveolar wall thickening decreased in MV 4h+si-Elk1 group and MV 4h+si-MMP-9 group.Compared with MV 4h group,lung injury score,wet/dry weight ratio(W/D),evans blue dye,total cell counts of BALF decreased in MV 4h+si-Elk1 group and MV 4h+si-MMP-9 group(P<0.05).Compared with MV 4h group,the expressions of E-cadherin and Occludin increased in MV 4h+si-Elk1 group and MV 4h+si-MMP-9 group by Western Blot(P<0.05).Immunofluorescence found that fluorescence expressions of E-cadherin and Occludin attenuated in CS 4h group,fluorescence expressions of E-cadherin and Occludin enhanced in CS 4h+si-Elk1 group and CS 4h+si-MMP-9 group.ConclusionsElkl and MMP-9 are the key factors to regulate E-cadherin and Occludin in VILI,Elk1 or MMP-9 knockdown reverses lung injury due to high tidal volume mechanical ventilation and pathological cyclic stretch.Part 3 Interaction between Elkl and MMP-9 and imbalance of MMP-9/TIMP-1 ratio in VILIObjectiveTo investigate the interaction between Elkl and MMP-9 and imbalance of MMP-9/TIMP-1 ratio in VILI.MethodsIn vitro,MEL-12 cells were randomly divided into 6 groups:CS 0h group,si-Elk1 group,si-MMP-9 group,CS 4h group,CS 4h+si-Nc group,CS 4h+si-Elk1 group,CS 4h+si-MMP-9 group.Elk1 siRNA,or MMP-9 siRNA was transfected into MLE-12 cells for 48h.MLE-12 cells were lysed to pick up the total protein to measure the expressions of Elkl and MMP-9 by Western Blot.MLE-12 cells were not treated with cyclic stretch in CS 0h group.The stretch time is 4h in CS 4h group,CS 4h+si-Elk1 group,and CS 4h+si-MMP-9 group.After cyclic stretch,MLE-12 cells were lysed to pick up the total protein to measure the expressions of Elkl and MMP-9 by Western Blot.In vivo,Forty C57BL/6 mice were randomly divided into 4 groups(n=10):MV 0h group,MV 4h group,MV 4h+si-Elk1 group,MV 4h+si-MMP-9 group.The mice did not treat with mechanical ventilation in MV 0h group.mice were injected by caudal vein with Elk1 siRNA(2.5 ?g/g)or MMP-9 siRNA(2.5?g/g),10%glucose solution,DEPC water,and the EntransterTM In Vivo Transfection reagent at the dosage of 200 ?L according to the manufacturer's instructions.After 72 h of transfection,the mice were treated with mechanical ventilation for 4 h in MV 4h+si-Elk1 group and MV 4h+si-MMP-9 group.The parameters in MV 4h group,MV 4h+si-Elk1 group and MV 4h+si-MMP-9 group as follows:tidal volume for 20mL/kg,respiratory frequency for 40/min,positive end-expiratory pressure(PEEP)of 0cm H2O,respiratory ratio(I:E)of 1:2,the ventilation time for 4h.After high tidal volume mechanical ventilation,lung injury score,wet/dry weight ratio(W/D),evans blue dye,bronchoalveolar lavage fluid(BALF)and HE staining were evaluated the lung injury.The expressions of Elk1 and MMP-9 were measured by Western Blot.ResultsIn vitro,immunoprecipitation found that there were the expressions of E-cadherin and Occludin in Input group,compared with CS 0h,the expressions of E-cadherin and Occludin increased in CS 4h group.Using MMP-9 for precipitation,it was revealed that MMP-9 and Elk1 were precipitated and the amount of protein precipitated increased with tensile time.Compared with CS 4h group,the expressions of Elk1 and MMP-9 decreased in CS 4h+si-Elk1 group(P<0.05).Compared with CS Oh group,the MMP-9 level decreased(P<0.05),the Elk1 level was not statistical significance in si-MMP-9 group(P>0.05).Compared with CS 4h group,the MMP-9 level decreased(P<0.05),the Elk1 level was not statistical significance in CS 4h+si-MMP-9 group(P>0.05).In vivo,compared with MV 4h group,the expressions of Elk1 and MMP-9 decreased in MV 4h+si-Elk1 group(P<0.05);the MMP-9 level decreased(P<0.05),the Elk1 level was not statistical significance in MV 4h+si-MMP-9 group(P>0.05).Compared with MV Oh,the TIMP-1 level and the MMP-9/TIMP-1 ratio increased in MV 4h group(P<0.05).Compared with MV 4h group,the TIMP-1 level and the MMP-9/TIMP-1 ratio decreased in MV 4h+si-Elk1 group and MV 4h+si-MMP-9 group(P<0.05).ConclusionsElk1 and MMP-9 interact in VILI,mechanical force activates Elk1,regulates MMP-9 activation,causes imbalance of MMP-9 and TIMP-1 and increases MMP-9/TIMP-1 ratio. |
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