| BackgroundMechanical ventilation can maintain airway patency,improve ventilation and oxygenation,prevent hypoxia and carbon dioxide accumulation,and ameliorate respiratory failure caused by underlying diseases with the ventilator.Mechanical ventilation is a way of ventilation which could replace,control or change the movement of spontaneous breathing with the mechanical devices.With the development of social economy and population ageing,respiratory failure,as a result of various reasons,the proportion of patients needing breathing machine treatment is increasing,and mechanical ventilation is the main method for the treatment of these diseases.Ventilation-induced lung injury(VILI)is the systemic hyperinflammatory reaction caused by the parameter regulation of ventilator or anesthetic machine and the primary lung diseases in patients undergoing mechanical ventilation with ventilator or anesthetic machine.Acute lung injury(ALI)can lead to acute respiratory distress syndrome(ARDS),and even mutiple organs disorder syndrome(MODS).Mechanical stretch can destroy the integrity of alveolar cell membrane,affect the permeability of alveolar membrane,cause pulmonary edema,at the same time,cause the imbalance of systemic inflammatory response,disrupt the balance of anti-inflammatory and pro-inflammatory factors,and then cause acute lung injury.The occurrence of VILI has a serious impact on the rehabilitation of patients.Meanwhile,the hospitalization time of patients prolongs obviously,the cost of treatment obviously increases,which brings the burden to patients and society.But the pathogenesis of VILI is still unclear.At present,the mechanism of VILI mainly focused on the biological injury caused by mechanical ventilation,including the imbalance of inflammatory reaction and the decrease of permeability barrier function.This study mainly focuses on the mechanism and treatment of VILI in these two aspects,and provides a new method for the treatment of VILI,which has important social value and scientific research value.E-cadherin is an important adherin junction in epithelial cells which plays an important role in maintaining cell polarity and integrity.p120-catenin is a kind of catenin belonging to the glycoprotein family,which regulates the signal transduction and cell adhesion.E-cadherin can form adherin junction through E-cadherin-mediated intercellular interaction and other protein combinations,which may increase the permeability of the barrier,suggesting that E-cadherin plays a key role in maintaining the barrier function.E-cadherin and several types of catenins(α,β,γ,and p120 catenin)can form complexus.Catenin is the target of cell signal transduction during inflammation,so it plays a role in regulating permeability.Cytokines include anti-inflammatory factors and pro-inflammatory factors,and mechanical stretch can lead to imbalance of inflammatory response.Tyrosine kinase is an enzyme coupled receptor distributed on the cytoplasmic membrane,which can be divided into three types:receptor tyrosine kinase,cytoplasmic tyrosine kinase,and nuclear tyrosine kinase.c-Src kinase is one of the kinases in cytoplasmic tyrosine kinase family,which plays a key role in many important physiological processes,such as cell growth,differentiation,transcription and so on.Our study demonstrated that c-Src kinase was involved in ventilation-induced lung injury and regulated cell barrier function.Nuclear transcription factor kappa B(NF-B)is an important transcriptional regulatory factor in cells,which participates in the inflammatory response of the body through a variety of cytokines.The activation of NF-kB is stimulated by a variety of substances,including inflammatory factors,integrins,chemicals that damaged DNA,mechanical forces,and so on.They bind to receptors on the surface of cells or within cells,causing signal transduction and activating NF-kB signaling pathways.Glutamine is the amide of glutamate,a non-essential amino acid in mammals,which can be transformed from glucose,is an important energy source for cell proliferation,and can be derived from skeletal muscles,lungs,liver,brain,and so on.Some studies showed that glutamine could decrease immunomodulatory,reduce the release of TNF-α，IL-1 β and IL-6,and alleviate lung injury and improve the prognosis of acute respiratory distress syndrome(ARDS).But the protective mechanism of glutamine on VILI is still unclear.In the study,we studied the mechanism of VILI treated with glutamine by regulating cell barrier function through c-Src kinase and cellular inflammatory response through NF-κB signaling pathway.Part 1 The effect of mechanical stretch on inflammatory factor,E-cadherin,and p120-cateninObjectiveIn this study,we simulated VILI in vivo and in vitro,and then measured the changes of interleukin 6(IL-6),Tumor necrosis factor-a(TNF-a),interleukin 10(IL-10),the expression of E-cadherin and p120-catenin.MethodsIn vivo,18 healthy C57BL/6 mice weighing 25-30 g were randomly divided into three groups with random number table(n= 6 in each group):control(Group C);low tidal volume(Group L);high tidal volume(Group H).Mice in Group C were not treated with anything.Mice in the other two groups were treated with mechanical ventilation.Mice were treated with a tidal volume of 7 mL/kg,a respiratory rate of 120 times/min,and positive end-expiratory pressure(PEEP)of 5 cm H20 in Group L,and a tidal volume of 20 mL/kg,a respiratory rate of 40 times/min,and PEEP of 0 cm H20 in Group H.The common ventilation parameters were set as follows:I/E ratio of 1:2,and a fraction of inspired oxygen of 21%,mechanical ventilation time of 4 h.After 4 h of mechanical ventilation,mice were sacrificed,and the lung injury score was recorded.The lungs were removed;the right lung upper lobe was quickly frozen in liquid nitrogen,which was used for the western blotting,and the remnant right lung tissue was fixed in 4%paraformaldehyde for 48-72 h for hematoxylin and eosin(H and E)staining.The left lung was lavaged to collect the bronchoalveolar lavage(BAL)fluid for the detection of total cells,the percentage of neutrophils,IL-6,TNF-a,and IL-10 with ELISA,and then used to calculate the pulmonary wet-to-dry(W/D)ratio to quantify the magnitude of pulmonary edema.After measuring the wet lung weight,tissues were incubated in a 70℃ incubator for 72 h to gain the wet-to-dry.In vitro,MLE-12 cells were divided into three groups with random number table:control group(Group sham),8%mechanical stretch group(Group 8%CS),20%mechanical stretch group(Group 20%CS).Cell monolayers were mounted onto the Flexercell system with a cyclic stretching pattern of a frequency 0.5 Hz for 30 cycles/min and a stretch-to-relaxation relation of 1:1 in Group 8%CS and Group 20%CS.The cyclic stretch time is 4h in Group 8%CS and Group 20%CS.After MLE-12 cells were treated with cyclic stretch,the expressions of E-cadherin,p120-catenin were measured with Western blotting,the expressions of IL-6,IL-10,and TNF-a were measured with ELISA and real time PCR.ResultsIn vivo1.From HE staining,the alveolar congestion,infiltration or aggregation of neutrophils in the airspace or the vessel wall,and thickening of the alveolar wall were caused in Group H,and HE staining in Group L was not significantly changed.2.From lung injury scores,compared with Group C,the lung injury scores increased in Group H;compared with Group L,the lung injury scores increased in Group H(P<0.05)3.From wet-to-dry(W/D)ratio,compared with Group C,it increased in Group H(P<0.05);compared with Group L,it increased in Group H(P<0.05).4.From bronchoalveolar lavage(BAL)fluid,compared with Group C,total cell counts the percentage of neutrophils,IL-6,and TNF-a in BAL fluid were higher in Group H(P<0.05),IL-10 was lower in Group H(P<0.05);compared with Group L,total cell counts the percentage of neutrophils,IL-6,and TNF-a in BAL fluid were higher in Group H(P<0.05),IL-]0 was lower in Group H(P<0.05).5.From western blotting,compared with Group C,the expressions of E-cadherin and p120-catenin were lower in Group H(P<0.05);compared with Group L,the expressions of E-cadherin and p120-catenin were lower in Group H(P<0.05).In vitro1.From ELISA and Real time PCR,compared with Group sham,the expressions of IL-6 and TNF-α increased in Group 20%CS(P<0.05);compared with Group 8%CS,the expressions of IL-6 and TNF-α increased in Group 20%CS(P<0.05).Compared with Group sham,the expression of IL-10 decreased in Group 20%CS(P<0.05);compared with Group 8%CS,the expression of IL-10 decreased in Group 20%CS(P<0.05).2.From western blotting,compared with Group sham,the expressions of E-cadherin and p120-catenin decreased in Group 20%CS(P<0.05);compared with Group 8%CS,the expressions of E-cadherin and p120-catenin decreased in Group 20%CS(P<0.05).Conclusions1.High tidal volume ventilation and 20%cyclic stretch could cause VILI,the expressions of IL-6 and TNF-α increased and IL-10 decreased.2.High tidal volume ventilation and 20%cyclic stretch could decrease the expressions of E-cadherin and p120-catenin.Part 2 Glutamine regulates inflammatory response by regulating NF-κB signaling pathway in VILIObjectiveIn this study,we simulated VILI in vivo and in vitro,and then investigated the effect of glutamine on the expressions of IL-6,TNF-a,and IL-10 in VILI,glutamine could decrease the expressions of IL-6,TNF-a,and improve that of IL-10 by inhibiting the activation of NF-κB signaling pathway.MethodsIn vivo,30 healthy C57BL/6 mice weighing 25-30 g were randomly divided into five groups with random number table(n= 6 in each group):control(Group C);low tidal volume(Group L);low tidal volume + glutamine(Group L+G);high tidal volume(Group H);and high tidal volume + glutamine(Group H + G).Mice in Group C were not treated with anything.Mice in the other four groups were treated with mechanical ventilation.Mice were treated with a tidal volume of 7 mL/kg,a respiratory rate of 120 times/min,and positive end-expiratory pressure(PEEP)of 5 cm H20 in Group L and Group L + G,and a tidal volume of 20 mL/kg,a respiratory rate of 40 times/min,and PEEP of 0 cm H20 in Group H and Group H+G.The common ventilation parameters were set as follows:I/E ratio of 1:2,and a fraction of inspired oxygen of 21%,mechanical ventilation time of 4 h.Mice in Group L+G and Group H+G were pretreated with glutamine(0.75 g/kg at an intravenous(I.V.)bolus)30 min before anesthesia.After 4 h of mechanical ventilation,mice were sacrificed.The left lung was lavaged to collect the bronchoalveolar lavage(BAL)fluid for the detection of total cells,the percentage of neutrophils,IL-6,TNF-a,and IL-10 with ELISA and TNF-κB p65 with real time PCR.In vitro,MLE-12 cells were divided into three groups with random number table:control group(Group sham),mechanical stretch group(Group CS),and mechanical stretch pretreated with glutamine group(Group CS+G).Glutamine(4 mM)was added to the plate of confluent MLE-12 cells 60 min before cyclic stretch in Group CS+G.Cell monolayers were mounted onto the Flexercell system with a cyclic stretching pattern of a frequency 0.5 Hz for 30 cycles/min and a stretch-to-relaxation relation of 1:1 in Group CS and Group CS+G The cyclic stretch time is 4h in Group CS and Group CS+G.After MLE-12 cells were treated with cyclic stretch,apoptosis was measured with flow cytometry,the expressions of IL-6,IL-10,and TNF-a were measured with ELISA and real time PCR,the expression of NF-kB p65 was measured with real time PCR.ResultsIn vivo1.From bronchoalveolar lavage(BAL)fluid,compared with Group C,total cell counts the percentage of neutrophils were higher in Group H(P<0.05);compared with Group H,total cell counts and the percentage of neutrophils were lower in Group H+G(P<0,05).2.From bronchoalveolar lavage(BAL)fluid,compared with Group C,the expressios of IL-6 and TNF-a were higher in Group H(P<0.05),the expression of IL-10 was lower in Group H(P<0.05);compared with Group H,the expressions of IL-6 and TNF-a were lower in Group H+G(P<0.05),the expression of IL-10 was higher in Group H+G(P<0.05).3.From bronchoalveolar lavage(BAL)fluid,compared with Group C,the expression of NF-κB p65 was higher in Group H(P<0.05);compared with Group H,the expression of NF-κB p65 was lower in Group H+G(P<0.05).In vitro1.From apoptosis,compared with Group sham,it increased in Group CS(P<0.05);compared with Group CS,it decreased in Group CS+G(P<0.05).2.From ELISA and real time PCR,compared with Group sham,the expressions of IL-6 and TNF-a increased in Group CS(P<0.05);compared with Group CS,the expressions of IL-6 and TNF-a decreased in Group CS+G(P<0.05).Compared with Group sham,the expression of IL-10 decreased in Group CS(P<0.05);compared with Group CS,the expression of IL-10 increased in Group CS+G(P<0.05).3.From real time PCR,compared with Group sham,the expression of NF-κB p65 increased in Group CS(P<0.05);compared with Group CS,the expression of NF-κB p65 decreased in Group CS+G(P<0.05).Conclusions1.High tidal mechanical ventilation and 20%cyclic stretch can increase the cell apoptosis and activate NF-κ2B signaling pathway.2.Glutamine can inhibit the activation of NF-λB signaling pathway,decrease the cell apoptosis induced by mechanical stretch,and stabilize the balance of inflammatory response.Part 3 Glutamine can enhance the barrier function by inhibiting the activation of c-Src kinase in VILIObjectiveIn this study,we simulated VILI in vivo and in vitro,and then investigated the effect of glutamine on the expressions of E-cadherin and p120-catenin in VILI,glutamine could improve the expressions of E-cadherin and p 120-catenin by inhibiting the activation of c-Src.MethodsIn vivo,the groups and experimental preconditioning were in accordance with the experiment in vivo in part 2.After 4 h of mechanical ventilation,mice were sacrificed,and the]ung injury score was recorded.The lungs were removed;the right lung upper lobe was quickly frozen in liquid nitrogen,which was used for the western blotting,and the remnant right lung tissue was fixed in 4%paraformaldehyde for 48-72 h for hematoxylin and eosin(H and E)staining.The left lung was used to calculate the pulmonary wet-to-dry(W/D)ratio to quantify the magnitude of pulmonary edema.In vitro,the groups and experimental preconditioning were in accordance with the experiment in vitro in part 2.After MLE-12 cells were treated with cyclic stretch,the expressions of E-cadherin,p120-catenin,c-Src were measured by Western blotting,the distribution of E-cadherin were measured with immunofluorescence.ResultsIn vivo1.From lung injury scores,compared with Group C,the lung injury scores increased in Group H;compared with Group H(P<0.05),the lung injury scores decreased in Group H+G(P<0.05).2.From wet-to-dry(W/D)ratio,compared with Group C,it increased in Group H;compared with Group H(P<0.05),it decreased in Group H+G(P<0.05).3.From HE staining,the alveolar congestion,infiltration or aggregation of neutrophils in the airspace or the vessel wall,and thickening of the alveolar wall were caused in Group H..In the H+G group,the infiltration of the alveolar cavity and the wall neutrophils was significantly decreased,the pulmonary congestion and pulmonary hemorrhage decreased,and the thickening of the alveolar wall decreased.4.From western blotting,compared with Group C,the expressions of E-cadherin and p120-catenin were lower in Group H(P<0.05);compared with Group H,the expressions of E-cadherin and p120-catenin were higher in Group H+G(P<0.01).Compared with Group C,the expression of c-Src phosphorylation was higher in Group H(P<0.05);compared with Group H,the expression of c-Src phosphorylation was lower in Group H+G(P<0.05).In vitro1.From western blotting,compared with Group sham,the expressions of E-cadherin and p120-catenin decreased in Group CS(P<0.01);compared with Group CS,the expressions of E-cadherin and p120-catenin increased in Group CS+G(P<0.05).Compared with Group sham,the expression of c-Src phosphorylation increased in Group CS(P<0.05);compared with Group CS,the expression of c-Src phosphorylation decreased in Group CS+G(P<0.05).2.From immunofluorescence,the distribution of E-cadherin was more limited under microscope in the stretching groups than in the non-stretching group,and the distribution of E-cadherin was better in the glutamine group than in the stretching group.Conclusions1.High tidal mechanical ventilation and 20%cyclic stretch can phosphorylate the c-Src.2.Glutamine can inhibit the phosphorylation of c-Src,increase the expressions of E-cadherin and p120-catenin and improve the barrier function caused by adhreins. |
