Effectsof IRhom2 On Ventilator-induced Lung Injury By Notch Signaling Pathway

BackgroundMechanical ventilation is a supportive therapy to maintain respiratory function and to minimize the patients’ burden of breathing in a variety of clinical and surgical situation.However,it may cause lung damage,resulting in ventilator-induced lung injury(VILI).Lung protective ventilation strategy is a standard treatment for patients with acute respiratory distress syndrome(ARDS)and it attracts more and more attention to the clinical significance of VILI.For the ARDS and other high-risk patients who need mechanical ventilation supportive therapy,preventing and relieving the VILI is still a hotspot and difficulty in perioperative anesthesia medicine and critical care medicine research.The mechanism of VILI includes barotrauma,volutrauma,atelectrauma and biotrauma.High volume or high pressure mechanical ventilation can cause the release of pro-inflammatory medium by many different clinically relevant mechanism.The barotrauma,volutrauma,atelectrauma and biotrauma are not mutually exclusive because by causing stress rupture,the barotrauma,volutrauma and atelectrauma can all lead to biotrauma,making it a focus of current researches.Alveolar macrophages(AM)play a vital role in immune homeostasis and the maintenance of the host defense and they are main producers of proinflammatory cytokines after lung’s exposure to harmful stimulus.Macrophages can be quickly activated by mechanical ventilation,which suggests that they may play an important role in the pathogenesis of VILI.But the mechanism about alveolar macrophages’ activation and regulation by mechanical ventilation has not been fully elucidated.The “three-step proteolytic model” of Notch signaling pathway:Firstly,the Notch receptors are cut by Furin and assembled into mature heterodimer receptors in golgi apparatus;then the heterodimer receptors are transported to the cell surface to bind the ligands and are cut by ADAM 10/Kuz or ADAM 17/TACE;finally they are cut byγ-secretase and cause the release of NICD.NICD is transferred to the nucleus and combined with CSL to active the target genes such as Hes and ultimately affects the cell differentiation,proliferation and apoptosis.Notch signal can increase the activation of nuclear factor-kappa B(NF-κB),which is confirmed can promote the occurrence of VILI,therefore,Notch signaling pathway might play a regulatory role in VILI.In addition,there are experiments show that TACE plays a significant role in the activation of Notch signaling pathway.Inactive rhomboid-like protein2(iRhom2)belongs to Rhomboid protein family and more than 10 kinds of Rhomboid protein family members have been find by far,they are involved in the various kinds of signal transduction inside and outside of cells.In macrophages,iRhom2 is combined with TACE and promotes its release from endoplasmic reticulum,however,absence of iRhom2 leads to the failure of TACE’s releasing from endoplasmic reticulum and transferring to the cell surface and further affects the function of TACE.Thus,iRhom2 may affect the VILI by iRhom2/TACE/Notch pathway.By studying the role of Notch signaling pathway in VILI and then regulating the iRhom2 in alveolar macrophages to observe the extent of the activation of Notch signaling pathway,it is helpful to clarify the mechanism of iRhom2 affecting VILI.Further more,it provides a new clue and target for clinical intervention of VILI and theoretical basis and clinical strategies for prophylaxis and treatment of VILI.Part1.The function of mechanical ventilation on Notch signaling pathway in alveolar macrophagesObjective:To build the VILI model in healthy mice and study the function of mechanical ventilation on Notch signaling pathway in alveolar macrophages.Methods:C57BL/6 mice were randomly divided into 3 groups(n=3)including Control(Con),low tidal volume(LVT,VT=7ml/kg)and high tidal volume(HVT,VT=20ml/kg)group.The LVT and HVT mice were mechanically ventilated for 4 hours while the control mice underwent the tracheotomy without ventilation.Lung injury was evaluated by histology,lung injury score,wet/dry ratio(W/D ratio),alveolar capillary permeability to Evans bluealbumin(EBA)and the protein level in bronchoalveolar lavage fluid(BALF)and pulmonary inflammation was evaluated by cell counting and measurements of inflammatory cytokine.Alveolar macrophages were extracted and used to evaluate the NICD protein expression of Notch signaling pathway by Western Blot,while Hes-5m RNA expression by RT-PCR.Results:Compared with control and LVT group,HVT mechanical ventilation could cause lung injury and increase W/D ratio,EBA,protein level in BALF and inflammato- rycytokine.NICD protein and Hes-5 m RNA expression also increased significantly in HVT group.Conclusion:HVT mechanical ventilation could cause lung injury and activate Notch signaling pathway in alveolar macrophages.Part2.The function of Notch signaling pathway in VILIObjective: To study the function of Notch signaling pathway in VILI.Methods:C57BL/6 mice were randomly divided into 3 groups(n=3)including Control(Con),high tidal volume(HVT)and DAPT group.DAPT group received Notch inhibitor DAPT 100mg/kg before high tidal volume mechanical ventilation and other groups received equal DMSO.Lung injury was evaluated by histology,lung injury score,W/D ratio,EBA and the protein level in BALF and pulmonary inflammation was evaluated by cell counting and measurements of inflammatory cytokine.Alveolar macrophages were extracted and used to evaluate the NICD protein expression by Western Blot.Results:Compared with HVT group,lung injury was alleviated significantly in DAPT group and W/D ratio,EBA,protein level in BALF and inflammatory cytokine were also decreased in DAPT group.The result of Western Blot showed that NICD protein expression also decreased significantly in DAPT group.Conclusion:Notch signaling pathway was involved in the formation of VILI and inhibiting this pathway could significantly reduce the degree of lung injury.Part3.The effect of iRhom2 on Notch signaling pathway in alveolar macrophagesObjective: To study the effect of iRhom2 on Notch signaling pathway in macrophages.Methods:RAW264.7 cells were randomly divided into 2 groups(n=3)including Control(R-Con)and iRhom2 down-regulation group(R-iRhom2-/-).RAW264.7 cells were collected and used to evaluate the NICD protein expression by Western Blot.Results:Compared with control group,NICD protein expression decreased significantly in iRhom2 down-regulation group.Conclusion:iRhom2 down-regulation could reduce the expression of NICD in macrophages.