| BackgroundAcute lung injury(ALI)and its more serious form--acute respiratory distress syndrome(ARDS)are a group of serious life-threatening clinical critical diseases,mainly showed as hypoxemia,pulmonary interstitial edema,formation of alveolar hyaline membrane and infiltration of inflammatory cells,which seriously interfere with the ventilation and exchange function of alveoli.There are many causes of ALI/ARDS,such as sepsis,mechanical stretch,pulmonary inflammation and gastric reflux.Sepsis and pneumonia are the most common infectious causes.Although protective mechanical ventilation,anti-infection and anti-shock therapy have achieved certain results in the treatment of ALI/ARDS,the mortality rate is still as high as 40%.Therefore,the basic research on the pathogenesis of ALI/ARDS is still very important.The mechanisms of ALI/ARDS are very complex,and the exact mechanism is not clear.The injury of pulmonary microvascular endothelial cells is considered to be the key step in the occurrence and development of ALI/ARDS.Pulmonary vascular endothelial cells form a vascular barrier through tight intercellular junctions,which is laid on the inner side of the vascular wall to prevent plasma and cell components from entering the tissue through circulation.When a variety of pathogenic factors act on endothelial cells,resulting in increased intercellular space and even endothelial cell necrosis,vascular endothelial barrier function is damaged,a large number of plasma components and blood cells enter the lung interstitium and alveolar cavity,leading to edema,alveolar respiratory membrane thickening,exudate forming a transparent membrane on the surface of alveolar cavity,alveolar exchange dysfunction,patients with uncorrectable hypoxemia.Therefore,maintaining the stability of vascular endothelial cell morphology and barrier function is an important measure and research idea for the treatment of ALI/ARDS.According to the current literature,the mechanisms of endothelial permeability increase caused by pulmonary vascular endothelial cell injury mainly focus on three aspects:the destruction of cell junctions between endothelial cells;cell contraction caused by cytoskeleton remodeling;cell necrosis caused by various strong pathogenic factors.Previous research results of our group found that mechanical stretch can lead to the abnormal number and function of p120 in alveolar epithelial cells,and then lead to the abnormal cell adhesion junction,and the increase of intercellular space.In animal experiments,the degree of interstitial and alveolar edema is positively correlated with the expression level of p120.However,the pathophysiological changes of pulmonary microvascular endothelial cells in LPS induced sepsis are still lack of in-depth study.Lipopolysaccharide(LPS)is a component of the outer cell wall of Gram-negative bacteria,which is often used as an important inflammatory inducer.LPS can bind to toll-like receptor 4(TLR4)and activate the downstream nuclear factor-?B(NF-?B)pathway to induce cell inflammation.In the early stage,our team established ARDS model by stimulating human pulmonary microvascular endothelial cells(HPMECs)with LPS for 4 hours.By using high-throughput cell sequencing and bioinformatics analysis,we found nearly 183 kinds of high expression mRNA.Among them,phospholipid Scramblase 4(PLSCR4)attracted our attention because of its significantly increased expression and its central position in the expression network.PLSCR4 belongs to the PLSCRs family.It is a group of single transmembrane proteins.It can transport phospholipids(PS)such as phosphatidylserine across the membrane in a Ca2+-dependent manner and increase the exposure of PS.The increased exposure of PS to the outside of cell membrane is considered to be the characteristic pathophysiological change of apoptosis and pyroptosis.As a newly discovered programmed cell death mode after cell necrosis and apoptosis,cell pyroptosis has the same characteristics as cell apoptosis,such as complete cell membrane structure,cell swelling and nuclear morphological changes.Different from apoptosis,pyroptosis is also called inflammatory necrosis because it is accompanied by the release of a large number of inflammatory factors.It has been reported that when HPMEC is stimulated by LPS,TNF-?,the pyroptosis occurred,and detect a large number of inflammatory cytokines IL-1 ? and IL-18 secretion.Therefore,it is inferred that the possibility of pyroptosis is greater in infectious pulmonary vascular endothelial injury.Pyroptosis is a kind of programmed cell death,also known as caspase dependent inflammatory necrosis.Different species and cell types activate different caspase subtypes during pyroptosis,mainly involving the activation of Caspase-1/4/5/11,downstream pyroptosis executive protein gasdermin D(GSDMD)and the release of inflammatory factors.It has been reported that the N-terminal activated fragment of GSDMD(GSDMD-NT)needs to be anchored on the cell membrane after binding with PS in order to perform pyroptosis.Therefore,combined with our previous experimental results,we put forward scientific questions:1.Whether the expression of PLSCR4 protein in hpmecs stimulated by LPS is consistent with the previous sequencing results,showing a significant increase?2.What role does plscr4 play in LPS induced HPMECs injury?Including the degree of inflammation and changes of endothelial permeability.3.Is the pathophysiological role of plscr4 related to pyroptosis?4.What are the upstream effectors of plscr4?To further improve the mechanism of plscr4 pathway.ObjectiveIn this study,we established cell and animal models of sepsis by LPS stimulation to explore the role of PLSCR4 in HPMECs injury,as well as the changes of cell inflammation and permeability.Through the study of the mechanism of PLSCR4,we hope to provide a new idea for the clinical treatment of ARDS;Through DNA pull-down experiment and protein mass spectrometry experiment,to improve the research of PLSCR4 pathway,which may provide reference for the basic experimental research of PLSCR4.MethodsIn this study,HPMECs and mice were used as research objects to establish sepsis ARDS model by LPS stimulation and siRNA interference,WB,enzyme-linked immunosorbent assay(ELISA),fluorescence assay and tracer flux assay(TFA),hematoxylin eosin(he)staining,DNA pull-down and protein detection were used to study the role and mechanism of plscr in hpmecs injury,as well as the upstream regulatory factors,so as to provide a new target for the clinical and basic research of ARDS.Part 1 Effect and mechanisms of PLSCR4 in LPS induced HPMECs injuryHuman pulmonary microvascular endothelial cells(HPMECs)were purchased from sciencecell research laboratories.The cells were resuscitated,cultured,subcultured and cryopreserved according to the standard operating instructions provided by ScienCell research laboratories.The cells were cultured in a 5%CO2 cell incubator at 37?.The cell culture medium,serum as well as growth factor and antibody used in the experiment were recommended by ScienCell company.Experimental grouping:HPMECs were divided into four groups:Sc siRNA group,Sc siRNA+LPS group,PLSCR4 siRNA+LPS group,and PLSCR4 siRNA group.HPMECs were transfected with SC siRNA,PLSCR4 siRNA with polyplus transfection respectively,and then cultured in the incubator for 48 hours.When the confluence reached to 80%-90%,LPS working solution was added to the experimental group with the final concentration of 1 ? g/ml,and then put into the cell incubator to continue to culture for 4 hours.After treatment:1.The protein of HPMECs was extracted using RIPA lysate,and the expression of plscr4 was detected by western blot;2.After collecting cell culture medium,the expression of IL-1 ? and IL-18 in cell culture medium of each group was detected by ELISA;3.After HPMECs were cultured in Transwell chamber to 90%confluence,the permeability of HPMECs was detected by tracer flux assay;4.After modeling sepsis,the medium was removed and washed with sterile DPBS for two times.After that,Annexin V-FITC and propidium iodide(PI)were added and incubated in the dark room for 15 minutes.Then,the degree of PS exposure on cell membrane and the degree of nuclear staining were observed by fluorescence microscope to determine the degree of pyroptosis and the distribution of PS;5.Western blot was used to detect the expression of pyroptosis related proteins,such as caspase-1,caspase-1,P20,GSDMD,GSDMD-NT,and to observe whether the expression of PLSCR4 has direct effect on the pyroptosis pathway;6.The protein of cell membrane and cytoplasm were extracted,and the distribution of GSDMD-NT in cell membrane and cytoplasm was detected by western blot;7.C57BL/6 mice were raised to 8-10 weeks old and weighed 20-25g.They were randomly divided into four groups.The grouping was the same as the cell experiment.siRNA was injected into the retinal vein plexus.Western blot was used to detect the knockout efficiency.Then LPS solution was injected into the lung through tracheotomy under anesthesia,then the mice were placed upright and rotated to ensure the even distribution of LPS solution in the alveoli.After 4 hours,the mice were killed and the lung tissue was taken.The pathological changes of lung tissue were observed by lung injury score,wet/dry weight ratio(W/D)and HE staining,and the effect of PLSCR4 was verified by animal experiments.Part 2 Study on upstream transcription factors of PLSCR4Through the first part of the experimental study,we have a preliminary understanding of the role and mechanism of plscr4 in LPS induced HPMECs.In order to improve the study upstream pathway of PLSCR4,we used DNA pull-down technology and protein mass spectrometry analysis to find the possible upstream transcription factors or regulatory factors of PLSCR4.HPMECs were divided into control group and LPS group.When the confluence reached to 80-90%in T-75 culture bottle in the LPS group,LPS working solution was added with the final concentration of 1 ? g/ml,then after 4 hours:1.Using DNA pull-down technique,DNA sequence probe of PLSCR4 promoter was designed.After PCR amplification and labeling,DNA-magnetic bead complex was prepared.Then protein samples were mixed with the complex.Protein-DNA-magnetic bead complex was obtained at 4? overnight.After elution,the supernatant of the complex was obtained.Finally,western blot was used to analyze the protein for the differential expression bands between the control group and LPS group by silver staining,to determin the molecular weight range of the differential expression bands;2.After obtaining the differentially expressed band,the information of total protein samples in DNA pull-down test was obtained by protein mass spectrometry experiment,and combined with silver staining results to further determine the possible promoter protein information;3.After obtaining the protein information of promoter through the two experimental techniques above,the next step for cell experiment verification was carried out:HPMECs were cultured in six well plate and divided into four groups.The changes of plscr4 expression after possible promoter protein silencing were detected by blot;IL-1 ? and IL-18 were detected by ELISA to determine the degree of inflammation of HPMECs;the permeability of monolayer endothelial cells was detected by TFA;the changes of pyroptosis of HPMECs after promoter protein silencing were observed by fluorescence microscope.ResultsPart 1 Effect and mechanisms of PLSCR4 in LPS induced HPMECs injury1.The protein expression of plscr4 was detected by western blot.After LPS stimulated HPMECs for 4 hours,western blot showed that the expression of PLSCR4 in sc siRNA+LPS group was significantly increased compared with the sc siRNA group,which indicated that the experimental results were consistent with the previous sequencing results;after adding PLSCR4 siRNA,the expression of PLSCR4 was significantly decreased,which meant the interference efficiency was statistically significant(P<0.05);2.The inflammatory factors were detected by ELISA.The concentrations of IL-1? and IL-18 in the sc siRNA+LPS group were significantly higher than those in the sc siRNA group,which indicated that the establishment of inflammation model of sepsis induced by LPS was established.And the concentrations of IL-1 ? and IL-18 in PLSCR4 siRNA+LPS group were higher than those in sc siRNA+LPS group(P<0.05),suggesting that PLSCR4 plays a protective role in the inflammatory process of HPMECs;3.Tracer flux assays were used to detect the permeability of monolayer endothelial cells.HPMECs were inoculated into Transwell chamber to simulate human pulmonary microvascular endothelium,and then stimulated by the LPS.It was found that after silencing the expression of PLSCR4,the amount of FITC-dextran in the lower chamber of PLSCR siRNA+LPS group increased significantly(P<0.05),which meant the monolayer endothelial permeability increased significantly(P<0.05);It is suggested that the normal expression of PLSCR4 could not only reduce the inflammatory degree of HPMECs,but also reduce the damage of vascular endothelial permeability;4.The distribution of PS on the surface of HPMECs was observed by fluorescence microscope to reflect the function level of PLSCR4.And observe the fluorescence intensity of nuclear PI to reflect the integrity of cell membrane,it was found that when PLSCR4 was silenced,the distribution of PS on the outer side of cell membrane decreased,while the nuclear staining deepened,suggesting that when PLSCR4 was silenced,the distribution of PS on the outer side of cell membrane decreased,With the decrease of PLSCR4 function,the damage of cell membrane integrity increased,and that the protective effect of PLSCR4 may be related to the maintenance of cell membrane integrity during pyroptosis;5.Western blot was used to detect the expression of key executive proteins in pyrolytic pathway.In order to study the protective mechanism of PLSCR4,western blot was used to detect the key proteins of pyroptosis pathway.It was found that there was no significant difference in the expression of caspase-1,caspase-1 P20,GSDMD and GSDMD-NT between sc siRNA+LPS group and PLSCR4 siRNA+LPS group,suggesting that the protective effect of PLSCR4 was not directly mediated by interfering with pyroptosis signaling pathway;6.After silencing plscr4,the expression of GSDMD-NT in the cytoplasm of PLSCR4 siRNA+LPS group was significantly higher than that of sc siRNA+LPS group(P<0.05),but the expression of gsdmd-nt in the membrane was significantly lower than that of SC siRNA+LPS group(P<0.05);7.By establishing a mouse model of lung infection,measuring the W/D,lung injury score and he staining of lung tissue,it was found that PLSCR4 played a protective role consistent with the cell experiment.Part 2 Study on upstream transcription factors of PLSCR41.in the DNA-pull down experiment,cell samples were processed by pull down,elution and gel electrophoresis to obtain silver staining bands.It was found that at 18.4 KDa,there was significant difference between LPS group and control group.2.The protein samples obtained from DNA pull-down experiment were identified by protein mass spectrometry,and the total protein information of control group and LPS group was obtained.The protein was p62280 in 18.4 kDa interval;3.In the verification experiment of P62280,when P62280 was silenced and hpmecs were stimulated with LPS for 4 hours,the change of plscr4 protein expression was basically consistent with that of p62280,and the degree of inflammation and monolayer endothelial permeability in P62280 siRNA+LPS group were also increased(P<0.05);fluorescence microscope also observed The increase of nuclear staining in siRNA P62280+LPS group suggested that the degree of pyroptosis was more serious than that in sc siRNA+LPS group.ConclusionsIn this study,we found that PLSCR4 plays a protective role in LPS induced hpmecs injury,which can reduce the secretion of inflammatory factors and protect the permeability of monolayer endothelial cells.When PLSCR4 was silenced by siRNA,the inflammatory process and damage of permeability increased correspondingly.In the experiment of improving the mechanism of PLSCR4,P62280 was found that it could act as an upstream transcription factor to regulate the expression of PLSCR4 in inflammation.Further study of this mechanism pathway can provide new therapeutic ideas and targets for the clinical treatment of ARDS. |
PDF Full Text Request