Effects And Mechanisms Of Mechanical Sensing Protein Piezo1 On ARDS-associated Lung Fibrosis Promoted By Mechanical Ventilation

PartⅠPiezo1 is required for epithelial mesenchymal transition（EMT）promoted by mechanical stretch in bronchial epithelial cellsObjective Investigate whether mechanical stretch could promote the EMT in bronchial epithelial cells,as well as explore the role of Piezo1 in the process of EMT.Method Human bronchial epithelial cell line（BEAS-2B cells）was selected as the research object.BEAS-2B cells were stimulated with 40 m M HCl（culture medium p H =4.0）for 30 min to establish the vitro model of ARDS.BEAS-2B cells were subjected to stretch for 48 hours（20% amplitude and 0.33Hz）with FX-4000 cell stretch loading system to simulate mechanical ventilation in clinical patients The experiment was divided into A,B and C three parts.The section A was to investigate whether stretch could promote the EMT in bronchial epithelial cells.Four groups were divided,including PBS+Static group,HCL+Static group,PBS+Stretch group,HCL+Stretch group.The morphologic changes of BEAS-2B cells was observed by phase contrast microscopy.The levels of hydroxyproline and TGF-β1 in cell supernatant were determinate using ELISA.The changes of EMT proteins were gauged by western blot and immunofluorescent staining.The section B was to investigate the expression of Piezo1 in BEAS-2B cells.The expression of Piezo1 m RNA,Piezo2 m RNA,TRPV4 m RNA,TRPV1 m RNA,KCNK m RNA and stoml3 m RNA was measured by Q-PCR.The expression and distribution of Piezo1 in BEAS-2B cells were observed by immunofluorescence.The section C was to investigate whether Piezo1 knockout could affect mechanical stretch promoted EMT in bronchial epithelial cells.Two groups were divided,including WT+Stretch group,KO+Stretch group.The morphologic changes of BEAS-2B cells was observed by phase contrast microscopy.The levels of hydroxyproline and TGF-β1 in cell supernatant were determinate using ELISA.The changes of EMT proteins were gauged by western blot and immunofluorescent staining.Result Section A.Compared with PBS+Static group,the expression of E-cadherin,Cytokeratin-8 decreased,the expression of α-SMA,vimentin,hydroxyproline and TGF-β1 increased after HCl or stretch treatment alone.Compared with HCL+Static group,the expression of E-cadherin,Cytokeratin-8 decreased,the expression of α-SMA,Vimentin, hydroxyproline and TGF-β1 increased in HCl+Stretch group.（P<0.05）Section B.The expression of mechanical stretch sensitive protein Piezo1 m RNA was significantly higher than that of Piezo2 m RNA（P<0.01）,TRPV4 m RNA（P<0.05）,TRPA1 m RNA（P<0.01）,TRPC3 m RNA（P<0.01）,TRPC6 m RNA（P<0.01）,KCNK4 m RNA（P<0.01）and stoml3 m RNA（P<0.01）in BEAS-2B cells.Piezo1 was mainly expressed in the cell membrane,and a small part was expressed in the cytoplasm,but not in the nucleus.Section C.Compared with WT+Stretch group,the expressions of E-cadherin（P<0.01）and Cytokeratin-8（P<0.01）were significantly increased,while the expressions of hydroxyproline（P<0.001）,TGF-β1（P<0.01）,α-SMA（P<0.05）and Vimentin（P<0.01）were significantly decreased.Conclusion Mechanical stretch can promote the EMT of HCl-stimulated BEAS-2B cells.Mechanical stretch sensitive protein Piezo1 plays a key role in mechanical stretch promoted EMT in BEAS-2B cells.Part II Piezo1-mediated Ca²⁺ influx and ATP release accelerate EMT promoted by mechanical stretch in bronchial epithelial cellsObjective Investigate the role of Piezo1-mediated Ca²⁺ influx and ATP release in mechanical stretch promoted EMT in bronchial epithelial cells.Method The experiment was divided into A,B,C,D and E five parts.The section A was to investigate the effects of Piezo1 agonist Yoda1 on the intracellular Ca²⁺ concentration and ATP release in bronchial epithelial cells.The experiment was divided into 6 groups,including DMSO group,Yoda1 group,Gsmtx4+Yoda1 group,EGTA+ Yoda1 group,KO+DMSO group,KO+Yoda1 group.Fluo-3-AM fluorescence staining was used to detect intracellular Ca²⁺ flow and intracellular Ca²⁺ level.The level of ATP in cell supernatant was detected at 0min,5min,15 min,30min and 45 min,respectively.The section B was to investigate the effect of mechanical stretch on the intracellular Ca²⁺ concentration and ATP release in bronchial epithelial cells.The experiment was divided into 6 groups,including DMSO group,stretch group,Gsmtx4+Stretch group,EGTTA+ Stretch group,KO+DMSO group,KO+Stretch group.BEAS-2B cells were stained by Fluo3-AM fluorescence staining and the intracellular Ca²⁺ concentration was determined by flow cytometry.The concentration of ATP in cell supernatant was detected at 0min,5min,15 min,30min and 45 min,respectively.The section C was to investigate the effects of Piezo1-mediated Ca²⁺ influx on ATP release in bronchial epithelial cells stimulated by mechanical stretch.The experiment was divided into six groups,including DMSO group,Stretch group,BAPTAM-AM+Stretch group,KO + DMSO group,KO+Stretch group,KO+ BAPTAM-AM+Stretch group.The ATP level in cell supernatant was detected at 45 min.The section D was to investigate the role of exogenous ATP in mechanical stretch promoted EMT in bronchial epithelial cells.The experiment was divided into 4 groups,including PBS group,ATP group,HCL group,HCL+ATP group.After the experiment,the morphological changes of BEAS-2B cells were observed with inverted light microscope.The levels of hydroxyproline and TGF-β1 in cultured supernatant were determined by ELISA.The expression of cytokeratin-8 and E-cadherin in epithelial cells and α-SMA and Vimentin in interstitial cells were detected by Western Blot and immunofluorescence.The section E part was to investigate the role of Piezo1-mediated ATP release in the EMT of bronchial epithelial cells induced by mechanical stretch.The experiment was divided into 3 groups,including HCl+Static group,HCl +Stretch group,HCl+Stretch+Apyrase group.After the experiment,the morphological changes of BEAS-2B cells were observed with inverted light microscope.The levels of hydroxyproline and TGF-β1 in cultured supernatant were determined by ELISA.The expression of cytokeratin-8 and E-cadherin in epithelial cells and α-SMA and Vimentin in interstitial cells were detected by Western Blot and immunofluorescence.Result Section A.Yoda1 increased the intracellular Ca²⁺ level in BEAS-2B cells.Gsmtx4 or Piezo1 knockout significantly inhibited the increase of intracellular Ca²⁺ induced by Yoda1.EGTA can significantly inhibit the increase of intracellular Ca²⁺ induced by Yoda1.Yoda1 induced the increase of extracellular ATP.（5）The increase of extracellular ATP induced by Yoda1 was significantly inhibited by Gsmtx4 or Piezo1 knockout.Section B.Stretch stimulation increased the intracellular Ca²⁺ level in BEAS-2B cells.Gsmtx4 or Piezo1 knockout significantly inhibited the increase of intracellular Ca²⁺ induced by stretch stimulation.EGTA can significantly inhibit the increase of intracellular Ca²⁺ induced by stretch stimulation.Stretch stimulation induced the increase of extracellular ATP.The increase of extracellular ATP induced by Stretch stimulation was significantly inhibited by Gsmtx4 or Piezo1 knockout.The increase of extracellular ATP induced by stretch stimulation was significantly inhibited by Gsmtx4,Piezo1 knockout or EGTA.Section C.BAPTA-AM inhibited extracellular ATP release induced by mechanical stimulation（P<0.001）,but had no significant effect on ATP release in Piezo1 knockouted BEAS-2B cells.Section D.Compared with PBS group,the level of hydroxyproline and TGF-β1 in ATP group was not significantly changed,but the level of hydroxyproline（P<0.05）and TGF-β1（P<0.01）in HCl group was slightly increased.Compared with HCl group,the level of hydroxyproline（P<0.001）and TGF-β1（P<0.001）in ATP+ HCl group was significantly increased.Compared with PBS group,the expressions of epithelial markers and interstitial markers in ATP group were not significantly changed,but the expressions of E-cadherin（P<0.05）and Cytokeratin-8（P<0.05）in BEAS-2B cells were slightly decreased in HCl group,and α-SMA（P<0.05）and vimentin（P<0.05）were slightly increased in HCl group.Compared with HCl group,the expression of E-cadherin（P<0.05）and Cytokeratin-8（P<0.05）in ATP+ HCl group was significantly decreased,the expression of α-SMA（P<0.05）and vimentin（P<0.05）were significantly increased.Section E.Compared with Static group,the levels of hydroxyproline（P<0.0001）and TGF-β1（P<0.001）in stretch group were significantly increased.Compared with Stretch group,the levels of hydroxyproline（P<0.001）and TGF-β1（P<0.01）in the Stretch+Apyrase group were significantly decreased.Compared with Static group,the expressions of Ecadherin（P<0.01）and Cytokeratin-8（P<0.01）in Stretch group were decreased,and the interstitial cell markers α-SMA（P<0.01）and vimentin（P<0.01）were increased.Compared with Stretch group,the expressions of E-cadherin（P<0.05）and Cytokeratin-8（P<0.05）in BEAS-2B cells in Stretch+Apyrase group were significantly increased,and the expressions of interstitium cell markers α-SMA（P<0.05）and vimentin（P<0.05）were significantly decreased.Conclusion Piezo1 regulates the mechanical stretch promoted EMT in BEAS-2B cell by mediating Ca²⁺ influx and ATP release.Part III Piezo1 accelerate ARDS-associated pulmonary fibrosis promoted by mechanical ventilationObjective To study the role of Piezo1 in mechanical ventilation promoted pulmonary fibrosis and EMT.Method The mouse ARDS model was induced by tracheal instillation of HCL（PH=1.2,2ml/kg）.After 48 hours,the mice were intubated through the oral and ventilated for 2 hours with peak inspiratory pressure（PIP）of 22 cm H2 O,PEEP of 2 cm H2 O,respiratory rate（RR）of 70 breaths per minute.The animals were then extubated observed for 14 days.The experiment is divided into three parts: A,B,and C.The section A was to study whether mechanical ventilation can promote the development of pulmonary fibrosis in mice.SPF-grade healthy male C57BL/6 mice were randomly divided into 4 groups,including Control group,HCL group,MV group,HCL+MV group.After 14 days of mechanical ventilation,the mice were sacrificed and lung tissues were collected.HE staining and Masson staining were used to evaluate the degree of lung injury and pulmonary fibrosis in mice.The levels of COL-1,hydroxyproline and TGF-β1 in lung tissues were detected.Western blot and immunofluorescence staining were used to detect the expression of epithelial cell markers E-cadherin and Cytokeratin-8,and the expression of mesenchymal cell markers α-SMA and vimentin.The section B was to studies the expression and distribution of Piezo1 in mouse lung tissue.The expression and distribution of Piezo1 in mouse lung tissue were detected by immunofluorescence.The section C was to study whether the Piezo1 protein knockdown can reduce ARDSrelated pulmonary fibrosis promoted by mechanical ventilation.Adeno-associated virus（AAV）transfection was used to knockdown the expression of Piezo1 in lung tissue.Then the experiment was divided into 4 groups,including Sc RNA transfection group,Sh RNA transfection group,Sc RNA transfection+MV group,Sh RNA transfection+ MV group.After 14 days,the mice were sacrificed and lung tissues were collected.HE staining and Masson staining were used to evaluate the degree of lung injury and pulmonary fibrosis in mice.The levels of COL-1,hydroxyproline and TGF-β1 in lung tissues were detected.Western blot and immunofluorescence staining were used to detect the expression of epithelial cell markers Ecadherin and Cytokeratin-8,and the expression of mesenchymal cell markers α-SMA and vimentin.Result Section A.Compared with the control group,the degree of lung injury,the deposition of blue collagen in lung tissue,the levels of COL-1,hydroxyproline and TGF-β1 in lung tissue were significantly increased in HCl group and MV group.（2）Compared with the control group,the expressions of E-cadherin and Cytokeratin-8 were significantly downregulated,while the expressions of vimentin and α-SMA were significantly increased.（3）The combined application of HCl and MV will further promote these changes.Section B.Piezo1 was widely expressed in lung epithelial cells.In lung epithelial cells,Piezo1 was mainly expressed in the cell membrane,with a small amount of expression in the cytoplasm and no expression in the nucleus.Section C.Compared with Sc RNA+MV group,the degree of lung injury,the collagen deposition in lung tissue,the levels of COL-1,hydroxyproline and TGF-β1 in lung tissue were significantly reduced in Sh RNA+MV group.Compared with Sc RNA+MV group,the expressions of E-cadherin and Cytokeratin-8 were significantly up-regulated,while the expressions of vimentin and α-SMA were significantly down-regulated in Sh RNA + MV group.Conclusion Mechanical ventilation can aggravate ARDS related pulmonary fibrosis.Piezo1 plays a key role in mechanical ventilation promoted ARDS related pulmonary fibrosis.