Pulsatilla decoction is a famous traditional Chinese herbal formula for clearing heat and treating dysentery of animals or human. To elucidate its mechanism, many active components have been studied, however, the role...Pulsatilla decoction is a famous traditional Chinese herbal formula for clearing heat and treating dysentery of animals or human. To elucidate its mechanism, many active components have been studied, however, the roles of its polysaccharides still remain unclear. This study aimed to explore effects of polysaccharides from Pulsatilla decoction （PPD） on the microvascular endothelial glycocalyx （eGC）. The polysaccharides were extracted from PPD by water extraction and alcohol precipitation method. Mice were administered with PPD for 4 wk, and were then anesthetized with ether inhalation and were ifxed by cardiac perfusion with gradient concentration alcian blue solution. The jejunum was sampled and jejunal mucosa was prepared for ultrathin sections by routine method and was analyzed by transmission electron microscope. The results indicated that the eGC was observed as a strong electron-dense smooth linear margin or nonuniform conglomerates coating cell membranes, and PPD signiifcantly increased its thickness from （21.85±1.87） to （28.71±3.61） nm and improved its integrity. This study suggested that PPD may express their biological activities and protect against pathogenic factor damages by inlfuencing the eGC.展开更多
Objective: This study demonstrated that dexamethasone(DEX) protects the endothelial glycocalyx from damage induced by the inflammatory stimulus tumor necrosis factor-α(TNF-α) during severe acute pancreatitis(SAP), a...Objective: This study demonstrated that dexamethasone(DEX) protects the endothelial glycocalyx from damage induced by the inflammatory stimulus tumor necrosis factor-α(TNF-α) during severe acute pancreatitis(SAP), and improves the renal microcirculation. Methods: Ninety mice were evenly divided into 3 groups(Sham, SAP, and SAP+DEX). The SAP mice model was established by ligature of pancreatic duct and intraperitoneal injection of cerulein. Renal perfusion and function, and morphological changes of the glycocalyx were evaluated by laser Doppler velocimetry, electron microscopy, and histopathology(hematoxylin and eosin(H&E) staining), respectively. Serum levels of syndecan-1 and TNF-α were assessed by enzyme-linked immunosorbent assay(ELISA). The proàtectiveì effects of dexamethasone on the glycocalyx and renal microcirculation were evaluated. Results: Significantly high levels of serum TNF-α were detected 3 h after the onset of SAP. These levels might induce degradation of the glycocalyx and kidney hypoperfusion, resulting in kidney microcirculation dysfunction. The application of dexamethasone reduced the degradation of the glycocalyx and improved perfusion of kidney. Conclusions: Dexamethasone protects the endothelial glycocalyx from inflammatory degradation possibly initiated by TNF-α during SAP. This is might be a significant discovery that helps to prevent tissue edema and hypoperfusion in the future.展开更多
The incidence of diabetic kidney disease(DKD)is sharply increasing worldwide.Microalbuminuria is the primary clinical marker used to identify DKD,and its initiating step in diabetes is glomerular endothelial cell dysf...The incidence of diabetic kidney disease(DKD)is sharply increasing worldwide.Microalbuminuria is the primary clinical marker used to identify DKD,and its initiating step in diabetes is glomerular endothelial cell dysfunction,particularly glycocalyx impairment.The glycocalyx found on the surface of glomerular endothelial cells,is a dynamic hydrated layer structure composed of proteoglycans,glycoproteins,and some adsorbed soluble components.It reinforces the negative charge barrier,transduces the shear stress,and mediates the interaction of blood corpuscles and podocytes with endothelial cells.In the highglucose environment of diabetes,excessive reactive oxygen species and proinflammatory cytokines can damage the endothelial glycocalyx(EG)both directly and indirectly,which induces the production of microalbuminuria.Further research is required to elucidate the role of the podocyte glycocalyx,which may,together with endothelial cells,form a line of defense against albumin filtration.Interestingly,recent research has confirmed that the negative charge barrier function of the glycocalyx found in the glomerular basement membrane and its repulsion effect on albumin is limited.Therefore,to improve the early diagnosis and treatment of DKD,the potential mechanisms of EG degradation must be analyzed and more responsive and controllable targets must be explored.The content of this review will provide insights for future research.展开更多
Introduction The endothelial cells(ECs)lining every blood vessel wall constantly expose to the mechanical forces generated by the blood flow.The EC responses to these hemodynamic forces play a critical role in the hom...Introduction The endothelial cells(ECs)lining every blood vessel wall constantly expose to the mechanical forces generated by the blood flow.The EC responses to these hemodynamic forces play a critical role in the homeostasis of the circulatory system.In addition to forming a transport barrier between the blood and vessel wall,vascular ECs play important roles in regulating circulation functions.Besides biochemical stimuli,blood flow induced(hemodynamic)mechanical stimuli,such as shear stress,pressure and circumferential stretch,modulate EC morphology and functions by activating mechanosensors,signaling pathways,and gene and protein expressions.The EC responses to the hemodynamic forces(mechano-sensing and transduction)are critical to maintaining normal vascular functions.Failure in the mechano-sensing and transduction leads to serious vascular diseases including hypertension,atherosclerosis,aneurysms and thrombosis,to name a few[1].On the luminal surface of our blood vessels,there is a thin layer called endothelial surface glycocalyx(ESG)which consists of proteoglycans,glycosaminoglycans(GAGs)and glycoproteins.The GAGs in the ESG are heparan sulfate(HS),hyaluronic acid(HA),chondroitin sulfate(CS),and sialic acid(SA)[2].In order to play important roles in vascular functions,such as being a mechanosensor and transducer for the endothelial cells(ECs)to sense the blood flow,a molecular sieve to maintain normal microvessel permeability and a barrier between the circulating cells and endothelial cells forming the vessel wall,the ESG should have an organized structure at the molecular level.Due to the limitations of optical and electron microscopy,the ultra-structure and organization of ESG has not been revealed until recent development of a super high resolution fluorescence optical microscope,STORM(Stochastic Optical Reconstruction Microscopy).The diffraction of a single fluorescence molecule can be described as the point spread function(PSF).When the light of wavelengthλexcites the fluorophore(emitter),the intensity profile of the spot is defined as the PSF with the width^0.6λ/NA,NA is the numerical aperture of the objective.The diffraction-limited image resolution,for a high numerical aperture objective lens,is^200 nm in the lateral direction and^500 nm in the axial direction,for a conventional fluorescence microscope.The key idea of the single-molecule localization microscopy is to light the molecule,in turn,to achieve the nanometer-level accuracy of their position and reconstruction into a super-resolution image,such as STORM.STORM employs photo-switching mechanisms to stochastically activate individual molecules(photo-switchable or photoactivatable fluorophores)within the diffraction-limited region at different times.Then images with sub-diffraction limit resolution are reconstructed from the measured positions of individual fluorophores[3].To trade the super spatial resolution(accuracy),STORM sacrifices its temporal resolution(efficiency)by switching the state and sequentially exciting the emitters at a high density.Rust et al[3]employed organic dyes and fluorescent proteins as photo-switchable emitters to trade temporal resolution for a super spatial resolution(~20 nm lateral and^50 nm axial at present,can go down to a couple of nanometers if using smaller peptides or antibody fragments instead of currently used whole anti-bodies),which is an order of magnitude higher than conventional confocal microscopy.In the current study,we employed STORM to reveal the major ultra-structural components of the ESG,HS and HA,and their organization at the surface of the cultured EC monolayer[4].Materials and methods We used newly acquired Nikon-STORM system to observe the ESG on in vitro EC(bEnd3,mouse brain microvascular endothelial cells)monolayers.After confluency,the bEnd3 cells were immunolabeled with anti-HS,fol-lowed by an ATT0488 conjugated goat anti-mouse IgG,and with biotinylated HA binding protein,followed by an AF647 conjugated anti-biotin.The ESG was then imaged by the STORM with a 100x/1.49 oil immersed lens.Multiple Reporters of ATT0488 and AF647 with alternating illumination were used to acquire the 3D images of HS and HA.The field of 256×256(40×40μm2)of HS and HA at the surface of ECs was obtained based on totally 40,000 of EM-CCD captured images for each reporter at a capturing speed of 19 ms/frame.Results HA is a long molecule weaving into a network which covers the endothelial luminal surface.The diameter of the HA segments is 185.3±44.7 nm,155.5±57.2 nm,and 156.9±56.1 nm,respectively,at the top,middle and bottom regions of the cell luminal surface.In contrast,HS is a shorter molecule,perpendicular to the cell surface.HA and HS are partially overlapped with each other at the endothelial luminal surface.We quantified the length,diameter,orientation,and density of HS at the top,middle and bottom regions of the endothelial surface.The diameter of the observed HS is 191.0±46.0 nm,284.3±71.1 nm,and 184.2±59.6 nm,and the length of the HS is 621.0±75.7 nm,651.0±118.0 nm,and 575.2±105.6 nm,respectively,at the top,middle and bottom regions of the cell luminal surface.For the HS orientation,its angle with the cell surface is 92.9±1.9,88.7±8.2,and 96.2±10.9 degree,respectively,at the top,middle and bottom regions.The angle of 90 degree is perfectly perpendicular to the cell surface.For the HS distribution,the average density is0.398 elements/μm2,0.345 elements/μm2 and 0.665 elements/μm2,respectively,and the distance between the adjacent HS is 1 694.4±628.1 nm,1 844.8±758.5 nm,and 1 221.9±450.7 nm,respectively,at the top,middle and bottom regions.Conclusions Our results suggest that HS plays a major role in mechanosensing and HA plays a major role in the molecular sieve,due to their organization,ultra-structure and distribution.展开更多
Covalent modification of bovine testicular hyaluronidase with chondroitin sulphate led to changes in the pattern of glycation of native and modified enzyme in its reaction with neutral saccharides and N-acetylhexosami...Covalent modification of bovine testicular hyaluronidase with chondroitin sulphate led to changes in the pattern of glycation of native and modified enzyme in its reaction with neutral saccharides and N-acetylhexosamines. Thus, mono- and di-saccharides inactivated the native hyaluronidase to a greater extent than the chondroitin sulfate-modified enzyme. N-acetylhexosamine, on the opposite, inactivated the modified hyaluronidase to a greater extent than the native one. These properties made it possible to use native and modified hyaluronidase as an informative research system for in vivo measurement of the predominant type of saccharide agents in the circulation. The proposed approach was experimentally substantiated by obtained results of the study on these interactions of hyaluronidase derivatives with hyaluronan fragments and their mixture. In a model of post-ischemic perfusion of the rat limb, the effect of hyaluronidase derivatives and their components on restoration of the microcirculation were tracked using laser Doppler flowmetry. Native hyaluronidase accelerated the restoration of initial level of microcirculation, but modified enzyme was markedly inhibited by glycocalyx degradation products. N-acetylhexosamine was positioned at the reducing terminal of these products as a natural label for these glycocalyx fragments. These and other data obtained under various experimental conditions supported the participation of endothelial glycocalyx in microcirculation disturbances.展开更多
Background:Ischemia reperfusion injury(IRI)is a major contributing factor to organ damage in liver transplantation(LT)impacting donor organ quality and patient survival.IRI-inflicted graft injury can be reduced by usi...Background:Ischemia reperfusion injury(IRI)is a major contributing factor to organ damage in liver transplantation(LT)impacting donor organ quality and patient survival.IRI-inflicted graft injury can be reduced by using hypothermic oxygenated machine perfusion(HOPE)as a preservation strategy instead of static cold storage(SCS).The endothelial glycocalyx is highly sensitive to IRI and its degradation during graft preservation and reperfusion was previously associated with inferior postoperative outcome after LT.Here,we aimed to measure glycocalyx degradation during and after HOPE in order to evaluate its potential for viability-assessment during machine perfusion and outcome prediction in patients undergoing LT.Methods:Glycocalyx degradation was quantified via enzyme-linked immunoassay(ELISA)for its main component syndecan-1(Sdc-1)in serum of 40 patients undergoing LT after HOPE.In addition,Sdc-1 was evaluated at multiple time points during HOPE.Patients were followed up for 3.5 years to assess postoperative complications including morbidity,the development of early allograft dysfunction(EAD)and graft survival.Results:Liver grafts which later developed EAD showed significantly higher Sdc-1 concentrations after 60 min of HOPE compared to grafts exhibiting normal postoperative function(P=0.02).Receiver operating characteristic analysis revealed a strong predictive potential with an area under the curve of 0.73.A cut-off at 808 ng/mL Sdc-1 at 60 min of HOPE allowed identification of a high-risk group with an incidence of EAD of 66.7%.Sdc-1 concentrations increased during all types of HOPE but were significantly higher in HOPE versus dual HOPE(D-HOPE)after 120 min of perfusion(P=0.02).Conclusions:Sdc-1 evaluated at 60 min during HOPE allows prediction of EAD after LT.Accordingly,Sdc-1 should be considered a potential additional biomarker for viability assessment during HOPE.展开更多
Objective:The endothelial glycocalyx(eGC)is a dynamic and multicomponent layer of macromolecules found at the surface of vascular endothelium,which is largely underappreciated.It has recently been recognized that eGC ...Objective:The endothelial glycocalyx(eGC)is a dynamic and multicomponent layer of macromolecules found at the surface of vascular endothelium,which is largely underappreciated.It has recently been recognized that eGC is a major regulator of endothelial function and may have therapeutic value in organ injuries.This study aimed to explore the role of the eGC in various pathologic and physiologic conditions,by reviewing the basic research findings pertaining to the detection of the eGC and its clinical significance.We also explored different pharmacologic agents used to protect and rebuild the eGC.Data sources:An in-depth search was performed in the PubMed database,focusing on research published after 2003 with keywords including eGC,permeability,glycocalyx and injuries,and glycocalyx protection.Study selection:Several authoritative reviews and original studies were identified and reviewed to summarize the characteristics of the eGC under physiologic and pathologic conditions as well as the detection and protection of the eGC.Results:The eGC degradation is closely associated with pathophysiologic changes such as vascular permeability,edema formation,mechanotransduction,and clotting cascade,together with neutrophil and platelet adhesion in diverse injury and disease states including inflammation(sepsis and trauma),ischemia-reperfusion injury,shock,hypervolemia,hypertension,hyperglycemia,and high Na+as well as diabetes and atherosclerosis.Therapeutic strategies for protecting and rebuilding the eGC should be explored through experimental test and clinical verifications.Conclusions:Disturbance of the eGC usually occurs at early stages of various clinical pathophysiologies which can be partly prevented and reversed by protecting and restoring the eGC.The eGC seems to be a promising diagnostic biomarker and therapeutic target in clinical settings.展开更多
Fluid resuscitation is an essential intervention in critically ill patients,and its ultimate goal is to restore tissue perfusion.Critical illnesses are often accompanied by glycocalyx degradation caused by inflammator...Fluid resuscitation is an essential intervention in critically ill patients,and its ultimate goal is to restore tissue perfusion.Critical illnesses are often accompanied by glycocalyx degradation caused by inflammatory reactions,hypoperfusion,shock,and so forth,leading to disturbed microcirculatory perfusion and organ dysfunction.Therefore,maintaining or even restoring the glycocalyx integrity may be of high priority in the therapeutic strategy.Like drugs,however,different resuscitation fluids may have beneficial or harmful effects on the integrity of the glycocalyx.The purpose of this article is to review the effects of different resuscitation fluids on the glycocalyx.Many animal studies have shown that normal saline might be associated with glycocalyx degradation,but clinical studies have not confirmed this finding.Hydroxyethyl starch(HES),rather than other synthetic colloids,may restore the glycocalyx.However,the use of HES also leads to serious adverse events such as acute kidney injury and bleeding tendencies.Some studies have suggested that albumin may restore the glycocalyx,whereas others have suggested that balanced crystalloids might aggravate glycocalyx degradation.Notably,most studies did not correct the effects of the infusion rate or fluid volume;therefore,the results of using balanced crystalloids remain unclear.Moreover,mainly animal studies have suggested that plasma may protect and restore glycocalyx integrity,and this still requires confirmation by high-quality clinical studies.展开更多
BACKGROUND:The present study aims to investigate whether mannitol facilitates central nervous system(CNS) entry of vancomycin and alleviates methicillin-resistant Staphylococcus aureus(MRSA)intracranial infection.METH...BACKGROUND:The present study aims to investigate whether mannitol facilitates central nervous system(CNS) entry of vancomycin and alleviates methicillin-resistant Staphylococcus aureus(MRSA)intracranial infection.METHODS:Blood-brain barrier(BBB) permeability was assessed by measuring the concentration of sodium fl uorescein(NaF) in the brain tissues of rats and fl uorescein isothiocyanate-dextran(FITC-dextran)in a single-cell layer model.Neutrophil infiltration in the brain tissue,inflammatory cytokine levels in the serum,neurological function,and 7-day survival rates were used to evaluate therapeutic eff ects of mannitol and vancomycin in MRSA-infected rats.Syndecan-1 and fi lamentous actin(F-actin) levels were measured,and the relationship between F-actin and the endothelial glycocalyx layer(EGL) was explored via the depolymerization agent cytochalasin D and the polymerization agent jasplakinolide.RESULTS:Following mannitol administration,the NaF and vancomycin concentrations in the brain tissue increased rapidly within 5 min and remained stable for 30 min,indicating that mannitol increased BBB permeability for 30 min.In vitro,mannitol treatment led to significantly greater FITC-dextran permeation through a single-cell layer compared to controls.In the MRSA intracranial infection model,rats treated with mannitol and vancomycin simultaneously presented less infl ammation,improved neurological function,and increased 7-day survival rate compared to rats treated with vancomycin and mannitol at 10-hour intervals.Further experiments revealed that mannitol decreased the expression of syndecan-1 in brain tissues,which was confi rmed by in vitro experiments showing that mannitol signifi cantly decreased syndecan-1 via F-actin depolymerization.CONCLUSION:Mannitol may enhance the therapeutic effi cacy of vancomycin against intracranial MRSA infection by decreasing the endothelial glycocalyx of the BBB via F-actin depolymerization.展开更多
BACKGROUND Injury to the glomerular filtration barrier causes diabetic kidney disease(DKD),and glomerular endothelial-mesenchymal transition damages the filtration barrier of glomerular endothelial cells.Shenfushu gra...BACKGROUND Injury to the glomerular filtration barrier causes diabetic kidney disease(DKD),and glomerular endothelial-mesenchymal transition damages the filtration barrier of glomerular endothelial cells.Shenfushu granules(SFSGs)can treat chronic renal failure;however,their role and mechanism in DKD remain unclear.AIM To investigate the role of SFSGs in delaying DKD progression and their underlying mechanism in a streptozotocin-induced DKD mouse model.METHODS The microalbumin content in the urine and the blood glucose,creatinine,and blood urea nitrogen levels in the serum were measured.The expression and distribution ofα-smooth muscle actin(α-SMA),heparan sulfate(HS)and cluster of differentiation(CD)31 were observed through immunofluorescence or immunohistochemistry.Western blotting was conducted to measure the expression of CD31,α-SMA,PIK3R1,protein kinase B(AKT),phospho-PIK3R1,phospho-AKT,and heparanase-1.Network pharmacology was conducted to screen and identify the core components and targets of SFSGs.Molecular docking and dynamic simulations were performed to evaluate the binding ability of the core components of SFSGs to their core targets.RESULTS Compared with those in the model group,the 24-hour microalbuminuria(188.2±20.1 and 140.4±24.7 vs 323.2±44.4),serum creatinine(79.4±2.6 and 68.7±6.0 vs 110.2±4.8),blood urea nitrogen(14.4±1.1 and 13.1±0.5 vs 19.5±1.1),and renal index(20.3±1.0 and 19.6±0.8 vs 25.3±1.7)were significantly lower in the SFSGs(2.08 and 4.16 g/kg/day extract)-treated DKD mice.SFSGs inhibited the down regulation of CD31 and the upregulation ofα-SMA in the glomerular endothelial cells of DKD mice.Additionally,SFSGs suppressed the decrease in glycocalyx thickness and the expression of its component HS.Network pharmacology revealed that PIK3R1 was the core target of SFSGs.SFSGs markedly downregulate the expression of phospho-PIK3R1,phospho-AKT,and heparanase-1.However,the PIK3R1 agonist abolished the regulatory effect of SFSGs on the expression of CD31,α-SMA,and heparanase-1.CONCLUSION Collectively,these results suggest that SFSGs can significantly delay DKD progression and inhibit injury to the glycocalyx and the endothelial-mesenchymal transition of glomerular endothelial cells.This mechanism is related to PIK3R1/AKT/heparanase-1 signaling pathway regulation.展开更多
Background:Low cardiac output syndrome(LCOS)is a frequent and serious complication after pediatric cardiac surgery.Endothelial glycocalyx(EG)degradation,indicated by elevated syndecan-1,contributes to microvascular dy...Background:Low cardiac output syndrome(LCOS)is a frequent and serious complication after pediatric cardiac surgery.Endothelial glycocalyx(EG)degradation,indicated by elevated syndecan-1,contributes to microvascular dysfunction and postoperative instability.The relationship between syndecan-1 dynamics and surgical risk categories remains unclear.Objective:To examine the association between perioperative syndecan-1 levels and clinical outcomes across Risk Adjustment for Congenital Heart Surgery(RACHS-1)categories.Methods:We analyzed 106 children(RACHS-1 categories 2–4)undergoing elective cardiac surgery with cardiopulmonary bypass(CPB).Syndecan-1 was measured at baseline(T0),4 h(T4),and 72 h(T72).Outcomes included LCOS,vasoactive inotropic score(VIS),Pediatric Logistic Organ Dysfunction(PELOD-2),pediatric intensive care unit(PICU)stay,and mortality.Analyses used Kruskal–Wallis,Bonferroni post hoc tests,Spearman correlation,and multivariable regression adjusted for CPB duration,cross-clamp time,and pre-PICU status.Results:Syndecan-1 differed significantly across RACHS groups at T0(p=0.044)and T72(p=0.015).RACHS score was weakly correlated but significant with syndecan-1 at T72(r=0.238,p=0.019)and decline from T4–T72(r=0.249,p=0.013),indicating delayed recovery at higher risk.RACHS-4 patients had the highest VIS and PELOD-2 scores and longer PICU stay.In adjusted models,RACHS-3 was associated with higher syndecan-1 at T72(β=+51.9,p=0.016),higher VIS 0–4 h(β=+4.9,p=0.008),and increased LCOS risk(OR 5.99,95%CI 1.61–25.70,p=0.010).RACHS-4 showed greater organ dysfunction but LCOS risk was attenuated(OR 0.19 vs.RACHS-3,p=0.035).Mortality was highest in RACHS-4(17.6%)but not statistically significant(p=0.368).Conclusion:Higher RACHS categories are linked with delayed EG recovery,greater vasoactive support,and more severe organ dysfunction.Syndecan-1 kinetics at 72 h,alongside VIS and LCOS,may serve as adjunctive markers for postoperative risk stratification in pediatric cardiac surgery.展开更多
基金supported by grants from the National Natural Science Foundation of China (31001083)the Scientific Research Program of Beijing Municipal Commission of Education of China (KM201110020014, KZ201110020021)the Beijing Natural Science Foundation, China (6142004)
文摘Pulsatilla decoction is a famous traditional Chinese herbal formula for clearing heat and treating dysentery of animals or human. To elucidate its mechanism, many active components have been studied, however, the roles of its polysaccharides still remain unclear. This study aimed to explore effects of polysaccharides from Pulsatilla decoction （PPD） on the microvascular endothelial glycocalyx （eGC）. The polysaccharides were extracted from PPD by water extraction and alcohol precipitation method. Mice were administered with PPD for 4 wk, and were then anesthetized with ether inhalation and were ifxed by cardiac perfusion with gradient concentration alcian blue solution. The jejunum was sampled and jejunal mucosa was prepared for ultrathin sections by routine method and was analyzed by transmission electron microscope. The results indicated that the eGC was observed as a strong electron-dense smooth linear margin or nonuniform conglomerates coating cell membranes, and PPD signiifcantly increased its thickness from （21.85±1.87） to （28.71±3.61） nm and improved its integrity. This study suggested that PPD may express their biological activities and protect against pathogenic factor damages by inlfuencing the eGC.
基金Project supported by the National Natural Science Foundation of China(No.81501644)
文摘Objective: This study demonstrated that dexamethasone(DEX) protects the endothelial glycocalyx from damage induced by the inflammatory stimulus tumor necrosis factor-α(TNF-α) during severe acute pancreatitis(SAP), and improves the renal microcirculation. Methods: Ninety mice were evenly divided into 3 groups(Sham, SAP, and SAP+DEX). The SAP mice model was established by ligature of pancreatic duct and intraperitoneal injection of cerulein. Renal perfusion and function, and morphological changes of the glycocalyx were evaluated by laser Doppler velocimetry, electron microscopy, and histopathology(hematoxylin and eosin(H&E) staining), respectively. Serum levels of syndecan-1 and TNF-α were assessed by enzyme-linked immunosorbent assay(ELISA). The proàtectiveì effects of dexamethasone on the glycocalyx and renal microcirculation were evaluated. Results: Significantly high levels of serum TNF-α were detected 3 h after the onset of SAP. These levels might induce degradation of the glycocalyx and kidney hypoperfusion, resulting in kidney microcirculation dysfunction. The application of dexamethasone reduced the degradation of the glycocalyx and improved perfusion of kidney. Conclusions: Dexamethasone protects the endothelial glycocalyx from inflammatory degradation possibly initiated by TNF-α during SAP. This is might be a significant discovery that helps to prevent tissue edema and hypoperfusion in the future.
基金Supported by the Natural Science Foundation of Shandong Province of China,No.ZR2019MH072.
文摘The incidence of diabetic kidney disease(DKD)is sharply increasing worldwide.Microalbuminuria is the primary clinical marker used to identify DKD,and its initiating step in diabetes is glomerular endothelial cell dysfunction,particularly glycocalyx impairment.The glycocalyx found on the surface of glomerular endothelial cells,is a dynamic hydrated layer structure composed of proteoglycans,glycoproteins,and some adsorbed soluble components.It reinforces the negative charge barrier,transduces the shear stress,and mediates the interaction of blood corpuscles and podocytes with endothelial cells.In the highglucose environment of diabetes,excessive reactive oxygen species and proinflammatory cytokines can damage the endothelial glycocalyx(EG)both directly and indirectly,which induces the production of microalbuminuria.Further research is required to elucidate the role of the podocyte glycocalyx,which may,together with endothelial cells,form a line of defense against albumin filtration.Interestingly,recent research has confirmed that the negative charge barrier function of the glycocalyx found in the glomerular basement membrane and its repulsion effect on albumin is limited.Therefore,to improve the early diagnosis and treatment of DKD,the potential mechanisms of EG degradation must be analyzed and more responsive and controllable targets must be explored.The content of this review will provide insights for future research.
基金supported by NIH-1SC1CA153325-01,NSF-MRI CBET 1337746 and 1UG3TR002151-01
文摘Introduction The endothelial cells(ECs)lining every blood vessel wall constantly expose to the mechanical forces generated by the blood flow.The EC responses to these hemodynamic forces play a critical role in the homeostasis of the circulatory system.In addition to forming a transport barrier between the blood and vessel wall,vascular ECs play important roles in regulating circulation functions.Besides biochemical stimuli,blood flow induced(hemodynamic)mechanical stimuli,such as shear stress,pressure and circumferential stretch,modulate EC morphology and functions by activating mechanosensors,signaling pathways,and gene and protein expressions.The EC responses to the hemodynamic forces(mechano-sensing and transduction)are critical to maintaining normal vascular functions.Failure in the mechano-sensing and transduction leads to serious vascular diseases including hypertension,atherosclerosis,aneurysms and thrombosis,to name a few[1].On the luminal surface of our blood vessels,there is a thin layer called endothelial surface glycocalyx(ESG)which consists of proteoglycans,glycosaminoglycans(GAGs)and glycoproteins.The GAGs in the ESG are heparan sulfate(HS),hyaluronic acid(HA),chondroitin sulfate(CS),and sialic acid(SA)[2].In order to play important roles in vascular functions,such as being a mechanosensor and transducer for the endothelial cells(ECs)to sense the blood flow,a molecular sieve to maintain normal microvessel permeability and a barrier between the circulating cells and endothelial cells forming the vessel wall,the ESG should have an organized structure at the molecular level.Due to the limitations of optical and electron microscopy,the ultra-structure and organization of ESG has not been revealed until recent development of a super high resolution fluorescence optical microscope,STORM(Stochastic Optical Reconstruction Microscopy).The diffraction of a single fluorescence molecule can be described as the point spread function(PSF).When the light of wavelengthλexcites the fluorophore(emitter),the intensity profile of the spot is defined as the PSF with the width^0.6λ/NA,NA is the numerical aperture of the objective.The diffraction-limited image resolution,for a high numerical aperture objective lens,is^200 nm in the lateral direction and^500 nm in the axial direction,for a conventional fluorescence microscope.The key idea of the single-molecule localization microscopy is to light the molecule,in turn,to achieve the nanometer-level accuracy of their position and reconstruction into a super-resolution image,such as STORM.STORM employs photo-switching mechanisms to stochastically activate individual molecules(photo-switchable or photoactivatable fluorophores)within the diffraction-limited region at different times.Then images with sub-diffraction limit resolution are reconstructed from the measured positions of individual fluorophores[3].To trade the super spatial resolution(accuracy),STORM sacrifices its temporal resolution(efficiency)by switching the state and sequentially exciting the emitters at a high density.Rust et al[3]employed organic dyes and fluorescent proteins as photo-switchable emitters to trade temporal resolution for a super spatial resolution(~20 nm lateral and^50 nm axial at present,can go down to a couple of nanometers if using smaller peptides or antibody fragments instead of currently used whole anti-bodies),which is an order of magnitude higher than conventional confocal microscopy.In the current study,we employed STORM to reveal the major ultra-structural components of the ESG,HS and HA,and their organization at the surface of the cultured EC monolayer[4].Materials and methods We used newly acquired Nikon-STORM system to observe the ESG on in vitro EC(bEnd3,mouse brain microvascular endothelial cells)monolayers.After confluency,the bEnd3 cells were immunolabeled with anti-HS,fol-lowed by an ATT0488 conjugated goat anti-mouse IgG,and with biotinylated HA binding protein,followed by an AF647 conjugated anti-biotin.The ESG was then imaged by the STORM with a 100x/1.49 oil immersed lens.Multiple Reporters of ATT0488 and AF647 with alternating illumination were used to acquire the 3D images of HS and HA.The field of 256×256(40×40μm2)of HS and HA at the surface of ECs was obtained based on totally 40,000 of EM-CCD captured images for each reporter at a capturing speed of 19 ms/frame.Results HA is a long molecule weaving into a network which covers the endothelial luminal surface.The diameter of the HA segments is 185.3±44.7 nm,155.5±57.2 nm,and 156.9±56.1 nm,respectively,at the top,middle and bottom regions of the cell luminal surface.In contrast,HS is a shorter molecule,perpendicular to the cell surface.HA and HS are partially overlapped with each other at the endothelial luminal surface.We quantified the length,diameter,orientation,and density of HS at the top,middle and bottom regions of the endothelial surface.The diameter of the observed HS is 191.0±46.0 nm,284.3±71.1 nm,and 184.2±59.6 nm,and the length of the HS is 621.0±75.7 nm,651.0±118.0 nm,and 575.2±105.6 nm,respectively,at the top,middle and bottom regions of the cell luminal surface.For the HS orientation,its angle with the cell surface is 92.9±1.9,88.7±8.2,and 96.2±10.9 degree,respectively,at the top,middle and bottom regions.The angle of 90 degree is perfectly perpendicular to the cell surface.For the HS distribution,the average density is0.398 elements/μm2,0.345 elements/μm2 and 0.665 elements/μm2,respectively,and the distance between the adjacent HS is 1 694.4±628.1 nm,1 844.8±758.5 nm,and 1 221.9±450.7 nm,respectively,at the top,middle and bottom regions.Conclusions Our results suggest that HS plays a major role in mechanosensing and HA plays a major role in the molecular sieve,due to their organization,ultra-structure and distribution.
文摘Covalent modification of bovine testicular hyaluronidase with chondroitin sulphate led to changes in the pattern of glycation of native and modified enzyme in its reaction with neutral saccharides and N-acetylhexosamines. Thus, mono- and di-saccharides inactivated the native hyaluronidase to a greater extent than the chondroitin sulfate-modified enzyme. N-acetylhexosamine, on the opposite, inactivated the modified hyaluronidase to a greater extent than the native one. These properties made it possible to use native and modified hyaluronidase as an informative research system for in vivo measurement of the predominant type of saccharide agents in the circulation. The proposed approach was experimentally substantiated by obtained results of the study on these interactions of hyaluronidase derivatives with hyaluronan fragments and their mixture. In a model of post-ischemic perfusion of the rat limb, the effect of hyaluronidase derivatives and their components on restoration of the microcirculation were tracked using laser Doppler flowmetry. Native hyaluronidase accelerated the restoration of initial level of microcirculation, but modified enzyme was markedly inhibited by glycocalyx degradation products. N-acetylhexosamine was positioned at the reducing terminal of these products as a natural label for these glycocalyx fragments. These and other data obtained under various experimental conditions supported the participation of endothelial glycocalyx in microcirculation disturbances.
文摘Background:Ischemia reperfusion injury(IRI)is a major contributing factor to organ damage in liver transplantation(LT)impacting donor organ quality and patient survival.IRI-inflicted graft injury can be reduced by using hypothermic oxygenated machine perfusion(HOPE)as a preservation strategy instead of static cold storage(SCS).The endothelial glycocalyx is highly sensitive to IRI and its degradation during graft preservation and reperfusion was previously associated with inferior postoperative outcome after LT.Here,we aimed to measure glycocalyx degradation during and after HOPE in order to evaluate its potential for viability-assessment during machine perfusion and outcome prediction in patients undergoing LT.Methods:Glycocalyx degradation was quantified via enzyme-linked immunoassay(ELISA)for its main component syndecan-1(Sdc-1)in serum of 40 patients undergoing LT after HOPE.In addition,Sdc-1 was evaluated at multiple time points during HOPE.Patients were followed up for 3.5 years to assess postoperative complications including morbidity,the development of early allograft dysfunction(EAD)and graft survival.Results:Liver grafts which later developed EAD showed significantly higher Sdc-1 concentrations after 60 min of HOPE compared to grafts exhibiting normal postoperative function(P=0.02).Receiver operating characteristic analysis revealed a strong predictive potential with an area under the curve of 0.73.A cut-off at 808 ng/mL Sdc-1 at 60 min of HOPE allowed identification of a high-risk group with an incidence of EAD of 66.7%.Sdc-1 concentrations increased during all types of HOPE but were significantly higher in HOPE versus dual HOPE(D-HOPE)after 120 min of perfusion(P=0.02).Conclusions:Sdc-1 evaluated at 60 min during HOPE allows prediction of EAD after LT.Accordingly,Sdc-1 should be considered a potential additional biomarker for viability assessment during HOPE.
基金General Program of National Natural Science Foundation of China(No.WJ2018H205).
文摘Objective:The endothelial glycocalyx(eGC)is a dynamic and multicomponent layer of macromolecules found at the surface of vascular endothelium,which is largely underappreciated.It has recently been recognized that eGC is a major regulator of endothelial function and may have therapeutic value in organ injuries.This study aimed to explore the role of the eGC in various pathologic and physiologic conditions,by reviewing the basic research findings pertaining to the detection of the eGC and its clinical significance.We also explored different pharmacologic agents used to protect and rebuild the eGC.Data sources:An in-depth search was performed in the PubMed database,focusing on research published after 2003 with keywords including eGC,permeability,glycocalyx and injuries,and glycocalyx protection.Study selection:Several authoritative reviews and original studies were identified and reviewed to summarize the characteristics of the eGC under physiologic and pathologic conditions as well as the detection and protection of the eGC.Results:The eGC degradation is closely associated with pathophysiologic changes such as vascular permeability,edema formation,mechanotransduction,and clotting cascade,together with neutrophil and platelet adhesion in diverse injury and disease states including inflammation(sepsis and trauma),ischemia-reperfusion injury,shock,hypervolemia,hypertension,hyperglycemia,and high Na+as well as diabetes and atherosclerosis.Therapeutic strategies for protecting and rebuilding the eGC should be explored through experimental test and clinical verifications.Conclusions:Disturbance of the eGC usually occurs at early stages of various clinical pathophysiologies which can be partly prevented and reversed by protecting and restoring the eGC.The eGC seems to be a promising diagnostic biomarker and therapeutic target in clinical settings.
文摘Fluid resuscitation is an essential intervention in critically ill patients,and its ultimate goal is to restore tissue perfusion.Critical illnesses are often accompanied by glycocalyx degradation caused by inflammatory reactions,hypoperfusion,shock,and so forth,leading to disturbed microcirculatory perfusion and organ dysfunction.Therefore,maintaining or even restoring the glycocalyx integrity may be of high priority in the therapeutic strategy.Like drugs,however,different resuscitation fluids may have beneficial or harmful effects on the integrity of the glycocalyx.The purpose of this article is to review the effects of different resuscitation fluids on the glycocalyx.Many animal studies have shown that normal saline might be associated with glycocalyx degradation,but clinical studies have not confirmed this finding.Hydroxyethyl starch(HES),rather than other synthetic colloids,may restore the glycocalyx.However,the use of HES also leads to serious adverse events such as acute kidney injury and bleeding tendencies.Some studies have suggested that albumin may restore the glycocalyx,whereas others have suggested that balanced crystalloids might aggravate glycocalyx degradation.Notably,most studies did not correct the effects of the infusion rate or fluid volume;therefore,the results of using balanced crystalloids remain unclear.Moreover,mainly animal studies have suggested that plasma may protect and restore glycocalyx integrity,and this still requires confirmation by high-quality clinical studies.
基金supported by the National Natural Science Foundation for Young Scientists of China (grant no.2002074)the Natural Science Foundation of Guangdong Province(2023A1515010267, 2023A1515012665, 2024A1515010073)+1 种基金the China International Medical Foundation Cerebrovascular Disease Youth Innovation Fund (Z-2016-20-2201)the Medical Leading Talents Fund of Guangdong Province (KJ012019430)。
文摘BACKGROUND:The present study aims to investigate whether mannitol facilitates central nervous system(CNS) entry of vancomycin and alleviates methicillin-resistant Staphylococcus aureus(MRSA)intracranial infection.METHODS:Blood-brain barrier(BBB) permeability was assessed by measuring the concentration of sodium fl uorescein(NaF) in the brain tissues of rats and fl uorescein isothiocyanate-dextran(FITC-dextran)in a single-cell layer model.Neutrophil infiltration in the brain tissue,inflammatory cytokine levels in the serum,neurological function,and 7-day survival rates were used to evaluate therapeutic eff ects of mannitol and vancomycin in MRSA-infected rats.Syndecan-1 and fi lamentous actin(F-actin) levels were measured,and the relationship between F-actin and the endothelial glycocalyx layer(EGL) was explored via the depolymerization agent cytochalasin D and the polymerization agent jasplakinolide.RESULTS:Following mannitol administration,the NaF and vancomycin concentrations in the brain tissue increased rapidly within 5 min and remained stable for 30 min,indicating that mannitol increased BBB permeability for 30 min.In vitro,mannitol treatment led to significantly greater FITC-dextran permeation through a single-cell layer compared to controls.In the MRSA intracranial infection model,rats treated with mannitol and vancomycin simultaneously presented less infl ammation,improved neurological function,and increased 7-day survival rate compared to rats treated with vancomycin and mannitol at 10-hour intervals.Further experiments revealed that mannitol decreased the expression of syndecan-1 in brain tissues,which was confi rmed by in vitro experiments showing that mannitol signifi cantly decreased syndecan-1 via F-actin depolymerization.CONCLUSION:Mannitol may enhance the therapeutic effi cacy of vancomycin against intracranial MRSA infection by decreasing the endothelial glycocalyx of the BBB via F-actin depolymerization.
基金Supported by the Changsha Science and Technology Major Project,No.kh2205035the Hunan Traditional Chinese Medicine Science and Technology Project,No.2021061the Natural Science Foundation of Hunan Province,No.2025JJ80080。
文摘BACKGROUND Injury to the glomerular filtration barrier causes diabetic kidney disease(DKD),and glomerular endothelial-mesenchymal transition damages the filtration barrier of glomerular endothelial cells.Shenfushu granules(SFSGs)can treat chronic renal failure;however,their role and mechanism in DKD remain unclear.AIM To investigate the role of SFSGs in delaying DKD progression and their underlying mechanism in a streptozotocin-induced DKD mouse model.METHODS The microalbumin content in the urine and the blood glucose,creatinine,and blood urea nitrogen levels in the serum were measured.The expression and distribution ofα-smooth muscle actin(α-SMA),heparan sulfate(HS)and cluster of differentiation(CD)31 were observed through immunofluorescence or immunohistochemistry.Western blotting was conducted to measure the expression of CD31,α-SMA,PIK3R1,protein kinase B(AKT),phospho-PIK3R1,phospho-AKT,and heparanase-1.Network pharmacology was conducted to screen and identify the core components and targets of SFSGs.Molecular docking and dynamic simulations were performed to evaluate the binding ability of the core components of SFSGs to their core targets.RESULTS Compared with those in the model group,the 24-hour microalbuminuria(188.2±20.1 and 140.4±24.7 vs 323.2±44.4),serum creatinine(79.4±2.6 and 68.7±6.0 vs 110.2±4.8),blood urea nitrogen(14.4±1.1 and 13.1±0.5 vs 19.5±1.1),and renal index(20.3±1.0 and 19.6±0.8 vs 25.3±1.7)were significantly lower in the SFSGs(2.08 and 4.16 g/kg/day extract)-treated DKD mice.SFSGs inhibited the down regulation of CD31 and the upregulation ofα-SMA in the glomerular endothelial cells of DKD mice.Additionally,SFSGs suppressed the decrease in glycocalyx thickness and the expression of its component HS.Network pharmacology revealed that PIK3R1 was the core target of SFSGs.SFSGs markedly downregulate the expression of phospho-PIK3R1,phospho-AKT,and heparanase-1.However,the PIK3R1 agonist abolished the regulatory effect of SFSGs on the expression of CD31,α-SMA,and heparanase-1.CONCLUSION Collectively,these results suggest that SFSGs can significantly delay DKD progression and inhibit injury to the glycocalyx and the endothelial-mesenchymal transition of glomerular endothelial cells.This mechanism is related to PIK3R1/AKT/heparanase-1 signaling pathway regulation.
文摘Background:Low cardiac output syndrome(LCOS)is a frequent and serious complication after pediatric cardiac surgery.Endothelial glycocalyx(EG)degradation,indicated by elevated syndecan-1,contributes to microvascular dysfunction and postoperative instability.The relationship between syndecan-1 dynamics and surgical risk categories remains unclear.Objective:To examine the association between perioperative syndecan-1 levels and clinical outcomes across Risk Adjustment for Congenital Heart Surgery(RACHS-1)categories.Methods:We analyzed 106 children(RACHS-1 categories 2–4)undergoing elective cardiac surgery with cardiopulmonary bypass(CPB).Syndecan-1 was measured at baseline(T0),4 h(T4),and 72 h(T72).Outcomes included LCOS,vasoactive inotropic score(VIS),Pediatric Logistic Organ Dysfunction(PELOD-2),pediatric intensive care unit(PICU)stay,and mortality.Analyses used Kruskal–Wallis,Bonferroni post hoc tests,Spearman correlation,and multivariable regression adjusted for CPB duration,cross-clamp time,and pre-PICU status.Results:Syndecan-1 differed significantly across RACHS groups at T0(p=0.044)and T72(p=0.015).RACHS score was weakly correlated but significant with syndecan-1 at T72(r=0.238,p=0.019)and decline from T4–T72(r=0.249,p=0.013),indicating delayed recovery at higher risk.RACHS-4 patients had the highest VIS and PELOD-2 scores and longer PICU stay.In adjusted models,RACHS-3 was associated with higher syndecan-1 at T72(β=+51.9,p=0.016),higher VIS 0–4 h(β=+4.9,p=0.008),and increased LCOS risk(OR 5.99,95%CI 1.61–25.70,p=0.010).RACHS-4 showed greater organ dysfunction but LCOS risk was attenuated(OR 0.19 vs.RACHS-3,p=0.035).Mortality was highest in RACHS-4(17.6%)but not statistically significant(p=0.368).Conclusion:Higher RACHS categories are linked with delayed EG recovery,greater vasoactive support,and more severe organ dysfunction.Syndecan-1 kinetics at 72 h,alongside VIS and LCOS,may serve as adjunctive markers for postoperative risk stratification in pediatric cardiac surgery.
