| Sepsis is a life-threatening organ dysfunction caused by dysregulated host-to-infection immune response[1-3,[15]].Vascular barrier dysfunction and reduced microcirculation blood volume following sepsis are important causes of organ dysfunction[4].Although the treatment of sepsis has developed rapidly in recent years,there is still a lack of effective treatment for the vascular leakage.Therefore,it is of great significance to find effective measures for vascular barrier dysfunction caused by sepsis to reduce the mortality of sepsis.Pericytes are vascular wall cells and derived from mesenchyme,located in the basement membrane of lung and systemic capillaries.Pericytes are radial shape and connected with multiple endothelial cells to provide structural support while pericytes also control vascular tone,produce extracellular matrix components,and promote vascular homeostatic and angiogenic cytokines[5,6].Lung injury occurs early and is fatal following sepsis while the coverage rate of pericytes on pulmonary vascular endothelial cells plays a decisive role in pulmonary vascular barrier function[7,8].Our previous experiments showed that a large number of pericytes lose following sepsis,but the mechanism of pericytes injury remains unclear.Ferroptosis is a new type of programmed cell death characterized by iron-dependent lipid peroxide accumulation.Studies have found that ferroptosis is associated with many diseases,including sepsis[9].Ferroptosis cells release damage-related molecular patterns and lipid peroxides,further aggravating the inflammatory reaction following sepsis[10].Iron chelator can inhibit the progression of sepsis,improve the survival rate and prolong the survival time of septic mice[11].Ferroptosis inhibitors can improve organ function and relieve inflammatory reaction following sepsis[12].Whether pericytes undergo ferroptosis and pulmonary vascular barrier function could improve via inhibiting pericytes ferroptosis following sepsis remains unclear.This study established septic rat model by cecal ligation and perforation and established septic in vitro model by LPS treating pericytes.Proteomics,metabolomics,construction of pericytes targeting liposomes,adenovirus interference,flow cytometry,immunofluorescence,transmission electron microscopy,WB,detection of TER and FITC-BSA transmembrane permeability of endothelial cell,detection of FITC and EB of permeability of pulmonary to investigate the role and mechanism of pericytes ferroptosis in pulmonary vascular barrier dysfunction following sepsis both in vivo and in vitro.Research contents:PartⅠ:The link between pericytes loss and vascular leakage following sepsisSepsis rat model was established by cecal ligation and perforation,and the relationship between pericytes loss in pulmonary vessels and pulmonary vascular leakage following sepsis rats was studied.The number of pulmonary pericytes following sepsis was observed by immunofluorescence and WB.The changes of pulmonary vascular barrier function following sepsis were observed by EB and FITC-BSA permeability of pulmonary vessels.The changes of tight junction structure between endothelial cells were observed by TEM.PartⅡ:Pericytes ferroptosis plays a vital importance in pulmonary vascular leakage following sepsis1.In vitro,pericyte ferroptosis following LPS administration and the protective effect of Fer-1 on pericytesPericytes were extracted according to the literature[13],and the primary pericytes were identified by immunofluorescence.Pericytes were pretreated with Fer-1 first and then challenged with LPS to observe pericyte ferroptosis following LPS challenge and the protective effect of Fer-1 on pericytes.The content of reduced glutathione,the ratio of reduced glutathione to oxidized glutathione,and the expression of ferroptosis related proteins COX2 and GPX4 in pericytes are measured.The protective effect of Fer-1 on permeability of vascular endothelial cells was observed by TER and FITC-BSA.2.In vivo,ferroptosis of pericytes in pulmonary vessels of rats following sepsis and the protective effects of Fer-1 on pericyte ferroptosisFer-1 was injected intraperitoneally before cecal ligation and perforation.Flow cytometry was used to observe the effect of Fer-1 on the content of Lipid ROS in lung pericyte following sepsis.The effect of Fer-1 on pericyte loss following sepsis in rats was observed.The pericytes coverage of pulmonary vascular endothelial cells was observed by immunofluorescence,and the expression of pericyte markers PDGFR-βand NG2 in pulmonary vessels was detected.To observe the effect of Fer-1 on ferroptosis in pulmonary vessels,the expression of ferroptosis related proteins GPX4 and COX2 and the content of lipid peroxide in pulmonary vessels were measured.3.The protective effects of Fer-1 on pulmonary vascular barrier and lung function in rats following sepsisFer-1 was injected intraperitoneally before cecal ligation and perforation.The effect of Fer-1 on pulmonary vascular barrier in septic rats,including measuring the EB and FITC-BSA permeability and the ultrastructure of pulmonary vascular endothelial cells.The effects of Fer-1 on lung function were observed,including the changes of neutrophil infiltration,lung wet-dry mass ratio and arterial blood gas indexes.PartⅢ:The mechanism of pericytes ferroptosis following sepsis1.A combination of proteomics and metabolomics analysis to identify the key molecule mediating pericytes ferroptosis following sepsisA combination of proteomics and metabolomics analysis showed septic pericytes underwent metabolic reprogramming and lipid metabolism was seriously disturbed.Proteomics suggests that ACSL4 is significantly upregulated in septic pericytes while ACSL4is not only a key executive molecule of ferroptosis,but also a regulator of lipid metabolism.Therefore,this study investigated the mechanism of ACSL4 mediating pericytes ferroptosis following sepsis.2.The effect of regulation of ACSL4 in pericytes on ferroptosis following sepsisThis study established a pericytes ACSL4 knockdown model via adenovirus interference to observe the effect of regulation of ACSL4 in pericytes on ferroptosis following sepsis and the contents of Lipid ROS and LPO in pericytes,and the expression of ferroptosis related proteins COX2 and GPX4 in pericytes were measured.3.The mechanism of ACSL4 mediating pericytes ferroptosis following sepsisThis study established a pericytes ACSL4 knockdown model via adenovirus interference to observe the effect of regulation of ACSL4 in pericytes on mitochondrial function following sepsis.Mitochondrial membrane potential,mitochondrial respiratory function and ROS content in mitochondrial were detected.PGC-1α（PPAR gamma-Coactivator-1）is an important protein that promotes mitochondrial proliferation and increases mitochondrial oxidation function.WB was used to detect the effect of regulating ACSL4 in pericytes on the expression of PGC-1αin septic pericytes.4.The mechanism of YAP1 regulating the expression of ACSL4 in pericytesHippo pathway can sense a variety of external stimuli and phosphorylate Yes-associatedprotein1（YAP1）through protein kinase chains,leading to cytoplasmic retention and degradation.The non-phosphorylated form of YAP1 can enter the nucleus and bind to transcription factors to activate target genes for transcriptional regulation.Proteomics suggested that Hippo pathway was significantly down-regulated in septic pericytes.This study investigated the relationship between YAP1 and ACSL4 expression in septic pericytes.The changes of YAP1 expression level and nuclear translocation in septic pericytes were detected.This study established a pericytes YAP1 knockdown model via adenovirus interference to observe the effect of regulation of YAP1 in pericytes on the cell viability and the expression of ACSL4 in septic pericytes.PartⅣ:The effect of targeted-regulation of ACSL4 in pericytes on pulmonary vascular barrier and organ function in septic rats1.The effect of regulation of ACSL4 in pericytes on permeability of vascular endothelial cells following sepsisThis study established a pericytes ACSL4 knockdown model via adenovirus interference and then co-incubated pericytes with endothelial cells to determine the effect of regulation of ACSL4 in pericytes on permeability of vascular endothelial cells following sepsis.The TER and FITC-BSA permeability were measured.2.Construction and physical and chemical evaluation of liposomes carrying Rosi and targeting pericytes（Rosi@LNP-aminopeptidase A/NG2）Thin layer chromatography was used to encapsulate Rosi with liposomes.By constructing peptides targeting NG2 and aminopeptidase A and loading them on the surface of liposomes,Rosi@LNP-aminopeptidase A/NG2 was synthesized successfully.The morphology of liposomes was characterized by TEM and DLS,including particle size,potential,drug loading and encapsulation rate of liposomes.The release curve of liposome in pH7.4 PBS was detected and immunofluorescence was used to determine the pericytes targeting property of liposomes.3.The protective effect of Rosi@LNP-aminopeptidase A/NG2 on lung pericytes following sepsisLiposomes（Rosi 0.2mg/kg）were injected through caudal vein before cecal ligation and perforation.Flow cytometry was used to observe the effect of Rosi@LNP-aminopeptidase A/NG2 on the content of Lipid ROS in lung pericytes in rats following sepsis.The effects of Rosi@LNP-aminopeptidase A/NG2 on the number of lung pericytes in rats were observed,including the changes of pericytes coverage of pulmonary vascular endothelial cells in rats detected by immunofluorescence,and the changes of PDGFR-βand NG2 expression detected.To observe the effects of Rosi@LNP-aminopeptidase A/NG2 on pulmonary vascular ferroptosis in rats,the expression of ferroptosis related proteins GPX4 and COX and the content of LPO in pulmonary vessels were measured.4.The protective effect of Rosi@LNP-aminopeptidase A/NG2 on pulmonary vascular barrier and lung function following sepsisLiposomes（Rosi 0.2mg/kg）were injected through caudal vein before cecal ligation and perforation.The effect of Rosi@LNP-aminopeptidase A/NG2 on pulmonary vascular barrier in septic rats,including measuring the EB and FITC-BSA permeability and the ultrastructure of pulmonary vascular endothelial cells.The effects of Rosi@LNP-aminopeptidase A/NG2 on lung function were observed,including the changes of neutrophil infiltration,lung wet-dry mass ratio and arterial blood gas indexes.5.The protective effect of Rosi@LNP-aminopeptidase A/NG2 on animal survival and organ functionLiposomes（Rosi 0.2mg/kg）were injected through caudal vein before cecal ligation and perforation.The effect of Rosi@LNP-aminopeptidase A/NG2 on organ function was detected by measuring AST、ALT、Crea、Urea and c Tn T,and blood flow of intestine、liver and kidney.The survival rate and survival time in the 72h-window were recorded.Results:PartⅠ:The link between pericytes loss and vascular leakage following sepsisThe expression levels of PDGFR-βand NG2 in pulmonary vessels were significantly decreased,and the cell coverage of pericytes on endothelial cells in pulmonary vessels was significantly decreased following sepsis.Pulmonary vascular barrier function was severely damaged,which was manifested by significantly increased pulmonary vascular permeability to EB and FITC-BSA and significantly increased opening degree of pulmonary vascular endothelial cell tight junction.Pulmonary structure was destroyed and a large number of neutrophils infiltrated,and pulmonary edema was severe.Arterial blood gas results showed that oxygen partial pressure,oxygen saturation and pH were significantly decreased,and carbon dioxide partial pressure was significantly increased.The results suggest that following sepsis,a large number of pericytes lose,pulmonary vascular barrier function and pulmonary function are seriously damaged,and the decrease of pericytes is closely related to vascular leakage following sepsis.PartⅡ:Pericytes ferroptosis plays a vital importance in pulmonary vascular leakage following sepsis1.LPS treatment caused severe pericytes ferroptosis,and Fer-1,an ferroptosis inhibitor,reduced pericytes ferroptosis and improved vascular endothelial cells barrier functionFer-1 improved cell viability and reduces ferroptosis in pericytes by significantly reducing the contents of Lipid peroxides and ferrous ions,and significantly increasing the contents of reduced glutathione and the ratio of reduced glutathione to oxidized glutathione in pericytes.The expression of ferroptosis related protein GPX4 was significantly increased,while the expression of COX was significantly decreased.Fer-1 pretreated pericytes were co-cultured with vascular endothelial cells,and Fer-1 pretreatment significantly increased TER and decreased FITC-BSA permeability.These results suggested that serious ferroptosis occurred in LPS treated pericytes,and Fer-1 could significantly reduce pericytes ferroptosis and improve endothelial cell barrier function in vitro.2.Lung pericytes ferroptosis in pulmonary vascular vessels in rats is closely related to pulmonary vascular leakage,and Fer-1 could alleviate pulmonary vascular leakage in rats following sepsisFer-1 alleviated lung pericytes ferroptosis following sepsis by significantly reducing the content of Lipid ROS in lung pericytes detected by flow cytometry.Fer-1 reduced pulmonary vessels ferroptosis in rats,which was characterized by significantly decreased lipid peroxide contents in pulmonary vessels and the increased expression of ferroptosis related protein GPX4 and decreased expression of COX2.Fer-1 significantly reduced lung pericytes loss following sepsis,which was embodied that the pericytes coverage on pulmonary vascular endothelial cells increased significantly,and the expression of pericytes markers NG2 and PDGFR-βincreased significantly.Fer-1 significantly reduced the pulmonary vascular leakage following sepsis,which was manifested as a significant decrease in the pulmonary vascular permeability to EB and FITC-BSA and improved endothelial cell tight junction.Fer-1significantly improved lung function,including reduced pulmonary neutrophil infiltration,and reduced pulmonary edema.Fer-1 significantly alleviated the inflammatory response of sepsis rats,improved the 72-hour survival rate and prolonged survival time.These results suggested that ferroptosis was closely related to pulmonary vascular leakage following sepsis,and Fer-1 could reduce pericytes ferroptosis and loss,reduce pulmonary vascular leakage and improve lung function following sepsis,and then reduce inflammatory response,improve animal survival rate and prolong survival time.PartⅢ:The mechanism of pericytes ferroptosis following sepsis1.The changes of proteomics in septic pericytesPrincipal component analysis and sample correlation heat map indicated that there was a large degree of dispersion and low similarity between the septic pericytes and the normal pericytes,which could be used for subsequent analysis.Among them,203 proteins were significantly up-regulated and 93 proteins were significantly down-regulated.KEGG enrichment analysis of different proteins showed that ferroptosis pathway,lipid metabolism pathway including glycerol phospholipid metabolism pathway and fatty acid synthesis pathway were significantly up-regulated,while Hippo pathway was significantly down-regulated.2.The changes of metabolomics in septic pericytesPrincipal component analysis and sample correlation heat map indicated that there was a large degree of dispersion and low similarity between the septic pericytes and the normal pericytes,which could be used for subsequent analysis.A total of 111 metabolites were significantly different between the two groups,among which reduced glutathione,cysteine and cystine were significantly down-regulated,while oxidized glutathione was significantly up-regulated.KEGG enrichment analysis of the different metabolites showed that lipid metabolism-related pathways were significantly up-regulated.3.Regulation of ACSL4 in pericytes improved mitochondrial function and reduced ferroptosis in septic pericytesThe results indicated that metabolic septic pericytes underwent metabolism reprogramming and lipid metabolism was seriously disturbed.Proteomics indicated that ACSL4 was significantly up-regulated in septic pericytes,and knockdown ACSL in pericytes significantly improved mitochondrial function,including decreased ROS production in mitochondria,increased mitochondrial membrane potential,improved respiratory function,and increased PGC-1αexpression.These results suggested that the expression of PGC1-αwas inhibited by ACSL4 in septic pericytes,which destroyed mitochondrial function,promoted ROS generation in mitochondria,caused severe oxidative stress,and then induced ferroptosis in septic pericytes.Reducing the expression of ACSL4 could increase the expression of PGC-1α,improve mitochondrial function,reduce ferroptosis,and then restore pericytes viability.4.Regulation of YPA1 in pericytes reduced the expression of ACSL4 and improved pericytes viability following LPS treatmentThe expression level of YAP1 in septic pericytes was significantly increased,and the nuclear translocation of YAP1 in septic pericytes was increased.Knockdown YAP1 in septic pericytes could significantly reduce the expression of ACSL4 and improve pericytes viability.These results suggested that YAP1 promoted the expression of ACSL4 in septic pericytes,thus promoting ferroptosis,and silence of YAP1 could reduce the expression of ACSL4,thus improving pericytes viability.PartⅣ:The effect of targeted-regulation of ACSL4 in pericytes on pulmonary vascular barrier and organ function in septic rats1.The effect of regulation of ACSL4 in pericytes on permeability of vascular endothelial cells following sepsisIn vitro,knockdown ACSL4 in pericytes and then co-cultured vascular endothelial cells with pericytes.TER was significantly increased and FITC-BSA permeability was reduced.2.Construction and physical and chemical evaluation of liposomes carrying Rosi and targeting pecicytes（Rosi@LNP-aminopeptidase A/NG2）In this experiment,Rosi@LNP-AA/NG2 was successfully prepared by thin film dispersion,and its morphology and particle size were investigated by TEM and DLS.The particle size was about 100nm,and the shape was spherical.Immunofluorescence was used to observe the property of pericyte targeting and the results showed that Rosi@LNP-AA/NG2had good pericyte targeting.The cumulative release of Rosi@LNP-AA/NG2 in pH7.4 PBS solution was determined by HPLC.In addition,the drug loading and encapsulation rate of Rosi@LNP-AA/NG2 were 8.6%and 85%,respectively,which provided the possibility for further in vivo experiments.3.The protective effect of Rosi@LNP-aminopeptidase A/NG2 on lung pericytes following sepsisCompared with Rosi@LNP,Rosi@LNP-AA/NG2 significantly reduced the lung pericytes ferroptosis in septic rats,as shown by the decreased contents of Lipid Ros.Following sepsis,ferroptosis in pulmonary vessels was significantly reduced,including decreased lipid peroxide content,increased expression of ferroptosis protein GPX4 and decreased expression of COX2.Pericytes loss was significantly reduced,which was manifested by increased expression of PDGFR-βand NG2,and significantly increased pericytes coverage on pulmonary vascular endothelial cells.4.The protective effect of Rosi@LNP-aminopeptidase A/NG2 on pulmonary vascular barrier and lung function following sepsisCompared with Rosi@LNP,Rosi@LNP-AA/NG2 significantly improved the pulmonary vascular barrier,which showed that the pulmonary vascular permeability to EB and FITC-BSA was significantly decreased,and the tight junction of pulmonary vascular endothelial cells was improved.Rosi@LNP-AA/NG2 significantly improved lung pathological structure,reduced neutrophil infiltration,reduced pulmonary edema,and improved lung function.5.The protective effect of Rosi@LNP-aminopeptidase A/NG2 on animal survival and organ functionCompared with Rosi@LNP,Rosi@LNP-AA/NG2 significantly reduced inflammation in sepsis rats,improved the pathological structure of heart,liver and kidney,improved the function of liver and kidney and alleviated myocardial injury and increased blood flow of liver,kidney and intestinal tract.The 72-hour survival rate and survival time of sepsis rats were significantly improved.Conclusion:1.Lung pericytes underwent ferroptosis in rats following sepsis,which plays an important role in pericytes loss.Fer-1,an inhibitor of ferroptosis,can significantly reduce lung pericytes ferroptosis,reduce pericytes loss,and improve the pulmonary vascular barrier following sepsis.2.The mechanism of pericytes ferroptosis is closely related to the downregulation of Hippo pathway,and increased nuclear translocation of YAP1 promotes ACSL4 expression in pericytes following sepsis.By inhibiting the expression of PGC-1α,ACSL4 leads to the impairment of mitochondrial function,the production of ROS in mitochondria,and the accumulation of Lipid peroxide and Lipid ROS in pericytes,leading to pericytes ferroptosis.3.Targeted-regulation of ACSL4 expression in pericytes can significantly reduce lung pericytes ferroptosis,reduce lung pericytes loss,improve pulmonary vascular barrier,and play a protective role in the function of vital organs in rats following sepsis. |
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