Protection Of HES/HBOC On Vascular Permeability And Its Relationship To Endothelial Glycocalyx

Hemorrhagic shock is a common clinical critical disease,and an important cause of early death in wartime and peacetime tramua.Fluid resuscitation is an important measure to restore tissue perfusion and hemodynamics in hemorrhagic shock patients.Vascular leakage is a common complication of hemorrhagic shock and fluid resuscitation,which is the leading cause of organ dysfunction.Crystalloids and colloids are the commonly used resuscitation fluids.Recent years hemoglobin based oxygen carrier(HBOC)is widely paid attension as a oxygencarrying fluid,profoundly study of the protective effect of different resuscitation fluids on vascular leakage has important clinical significance.Hydroxyethyl starch(HES)and HBOC are important colloid resuscitation fluid and oxygen carrier fluids,which played important role in hemorrhagic shock resuscitation.Whether HES and HBOC have protective effect on vascular permeability and the mechanism are not determinal,it need to be elucidated.Endothelial glycocalyx is a network dynamic structure covering the surface of vascular endothelial cells.Pathological stimulation such as hemorrhagic shock and sepsis could result in the destruction of the structure and function of endothelial glycocalyx,and lead to vascular leakage.It is unclear whether endothelial glycocalyx participates in the protection of vascular permeability by HES/HBOC after hemorrhagic shock.Intercellular junction also plays an important role in the function of endothelial barrier.Hemorrhagic shock and hypoxia resulted in decreased expression of intercellular junction protein,increased intercellular space,and increased transmembrane transport of proteins and other macromolecules.Whether the protective effect of HES/HBOC on pulmonary vascular leakage in hemorrhagic shock is related to intercellular junction and endothelial glycocalyx has not been reported.Therefore,hemorrhagic shock rats and hypoxic vascular endothelial cells were used to observed the role of HES/HBOC on the protection of pulmonary vascular permeability in hemorrhagic shock,and the relationship to endothelial glycocalyx.Main research contents and methods:Part I: The protective effect of HES on vascular permeability of hemorrhagic shock rats and its relationship to endothelial glycocalyx1.The protective effect of HES on pulmonary vascular permeability in hemorrhagic shock rats: hemorrhagic shock SD rat model and hypoxic treated vascular endothelial cells(VECs)were used to observe the changes of pulmonary vascular permeability after hemorrhagic shock and HES infusion;the changes of transmembrane resistance(TER)and FITC-BSA transmittance of vascular endothelial cells after hypoxia and HES incubation were also measured.2.The protective effect of HES on pulmonary vascular endothelial glycocalyx in hemorrhagic shock: observed the changes in the structure of endothelial glycocalyx and the expression of hyaluronic acid,heparan sulfate,and chondroitin sulfate in pulmonary veins after hemorrhagic shock and HES infusion;the effect of HES on the expression of hyaluronic acid,heparan sulfate,and chondroitin sulfate in hypoxic VECs was also observed in vitro.3.The role and mechanism of endothelial glycocalyx in the protection of HES on pulmonary vascular permeability after hemorrhagic shock: the effects of HES on the expression of endothelial glycocalyx in hypoxic VECs and the permeability of VECs after inhibition of the expression of each component of endothelial glycocalyx by the specific endothelial glycocalyx inhibitors were observed.The effects of HES on the expression of heparinase,hyaluronidase,and neuraminidase in hypoxic VECs were also observed.4.The effect of HES on intercellular junction and its relationship with endothelial glycocalyx in hemorrhagic shock: The effects of HES on the expression of intercellular junction proteins ZO-1,VE-cadherin,and occludin were observed in hemorrhagic shock rat model and hypoxic VECs model.The effects of inhibiting endothelial glycocalyx on the protection of intercellular junction in HES were also observed.Part II: Effect and mechanism of HBOC on pulmonary vascular leakage and pulmonary function in hemorrhagic shock1.Protective effect of HBOC on pulmonary vascular permeability in hemorrhagic shock: the effects of HBOC infusion on the permeability of FITC-BSA and Evans Blue in lung tissue were observed by using SD rat model of hemorrhagic shock and hypoxic VECs model;the effects of HBOC on TER and the transmittance of FITC-BSA in VECs were also observed by using hypoxic VECs model.2.Protective effect of HBOC on endothelial glycocalyx and intercellular junction of pulmonary vein in hemorrhagic shock: the effects of HBOC on the structure of endothelial glycocalyx and the expression of its components were observed by using hemorrhagic shock rat model and hypoxic VECs model in vivo and in vitro,and the expression of intercellular junction protein was also observed.3.Effect of HBOC on mitochondrial function and structure of endothelial cells: the effects of HBOC on oxygen delivery and consumption in rats with hemorrhagic shock were observed in vivo.The effects of HBOC on mitochondrial function including membrane potential,ATP level,and oxygen consumption rate of VECs were measured by using hypoxic VECs model,and the changes of mitochondrial structure were also observed.4.Protective effect of HBOC on lung injury in hemorrhagic shock rats: the effect of HBOC infusion on the blood levels of TNF-? and IL-6 in hemorrhagic shock rats was observed;at the same time,the effects of HBOC on the number of leukocytes,concentration of protein in alveolar lavage fluid and the recruitment and infiltration of neutrophils in lung tissue were observed.Results:Part I: The protective effect of HES on vascular permeability of hemorrhagic shock rats and its relationship to endothelial glycocalyx1.Protective effect of HES on pulmonary vascular permeability in hemorrhagic shockPulmonary vascular permeability was significantly increased after hemorrhagic shock,which was manifested by increased pulmonary vascular permeability to FITC-BSA and Evans Blue;the barrier function of VECs was damaged,the TER value of VECs was decreased,and the FITC-BSA permeability was increased after hypoxia;HES infusion ameliorated the leakage of FITC-BSA and Evans Blue in pulmonary vessels after hemorrhagic shock,and also had the protective effect on the TER value and the transmission rate of FITC-BSA after hypoxic VECs.It was suggested that HES had protective effect on pulmonary vascular permeability after hemorrhagic shock.2.Role of endothelial glycocalyx in the protective effect of HES on pulmonary vascular permeability in hemorrhagic shockThe structure of pulmonary vascular endothelial glycocalyx was destroyed after hemorrhagic shock,which showed that the thickness of pulmonary vascular endothelial glycocalyx was decreased and the expression of heparan sulfate,hyaluronic acid,and chondroitin sulfate in pulmonary vascular endothelial glycocalyx was significantly decreased;the levels of heparan sulfate,hyaluronic acid,and chondroitin sulfate in plasma were significantly increased after hemorrhagic shock.HES infusion had a significant protective effect on pulmonary vascular endothelial glycocalyx,the expression of components of endothelial glycocalyx was increased.Inhibiting endothelial glycocalyx could attenuate the protective effect of HES on barrier function of VECs,the protective effect of HES on vascular permeability was antagonized by endothelial glycocalyx inhibitors.It was found that HES protected endothelial glycocalyx mainly by down-regulating the expression of heparinase,hyaluronidase,and neuraminidase.3.Protective effect of HES on intercellular junction in hemorrhagic shock and its relationship with endothelial glycocalyxThe expressions of intercellular junction proteins ZO-1,VE-cadherin,and occludin were significantly decreased after hemorrhagic shock and hypoxia.HES could improve the expression of ZO-1,VE-cadherin,and occludin.Inhibition of endothelial glycocalyx did not affect the expression of intercellular junction proteins in VECs,and it did not affect the protective effect of HES on intercellular junction proteins after hemorrhagic shock or hypoxia.The results suggested that HES could protect intercellular junction after hemorrhagic shock,but it has nothing to do with endothelial glycocalyx.Part II: The protective effect and mechanism of HBOC on pulmonary vascular leakage and pulmonary function in hemorrhagic shock1.Protective effect of HBOC on pulmonary vascular permeability in hemorrhagic shockPulmonary vascular permeability was significantly increased after hemorrhagic shock,and the pulmonary vascular permeability to FITC-BSA and Evans blue was significantly increased;the barrier function of hypoxic VECs was also destroyed,the TER value of endothelial cells was significantly decreased,and the transmittance of FITC-BSA was significantly increased.HBOC had protective effect on pulmonary vascular permeability,it could improve the pulmonary vascular permeability of FITC-BSA and Evans Blue induced by hemorrhagic shock,and improved the decrease of TER value of VECs and the increase of transmittance of FITC-BSA induced by hypoxia.It was suggested that HBOC could protect pulmonary vascular permeability after hemorrhagic shock.2.Protective effect of HBOC on pulmonary vascular endothelial glycocalyx and intercellular junction in hemorrhagic shockAfter hemorrhagic shock,the endothelial glycocalyx in pulmonary vessels was destroyed,the structure was damaged,the expression of components was significantly decreased;the expression of intercellular junction proteins ZO-1,VE-cadherin,and occludin was also significantly decreased.HBOC infusion significantly improved the expression of heparan sulfate,hyaluronic acid,and chondroitin sulfate;and it also increased the expression of ZO-1,VE-cadherin,and occludin.Further studies have shown that inhibition of endothelial glycocalyx could attenuate the protective effect of HBOC on FITC-BSA permeability of hypoxic VECs.These results suggested that endothelial glycocalyx and intercellular junction played important role in the protection of pulmonary vascular permeability in hemorrhagic shock by HBOC.3.Effect of HBOC on mitochondrial function and structure of endothelial cells in hemorrhagic shockOxygen delivery and consumption of rats were significantly reduced after hemorrhagic shock.HBOC could significantly improve the oxygen delivery and consumption of rats after hemorrhagic shock.There was mitochondrial dysfunction in VECs after hypoxia,which were manifested in the decrease of mitochondrial membrane potential,ATP level,oxygen consumption rate in VECs;the morphology of mitochondria was changed and showed obvious fragmentation.HBOC could significantly improve ATP production,mitochondrial membrane potential,oxygen consumption rate,and the morphology of mitochondria.The fragmentation of mitochondria was significantly reduced after HBOC incubation in hypoxic VECs.4.Protective effect of HBOC on pulmonary function in hemorrhagic shockThere were obvious lung function damage after hemorrhagic shock,which manifested in obvious pathological changes in lung tissue,the number of leukocyte in alveolar lavage fluid and the concentration of protein were significantly increased,and the levels of inflammatory cytokines including TNF-? and IL-6 were also significantly increased;HBOC could obviously improve them.It was suggested that HBOC could improve the lung dysfunction induced by hemorrhagic shock.Conclusion1.HES had protective effect on pulmonary vascular permeability by protecting endothelial glycocalyx and intercellular junction proteins after hemorrhagic shock;the protective effect of HES on endothelial glycocalyx was mainly achieved by down-regulating the expression of heparinase,hyaluronidase,and neuraminidase.2.HBOC had protective effects on pulmonary vascular leakage and lung function while exerting volume resuscitation;the protective effect of HBOC on endothelial permeability was mainly achieved by protecting the endothelial glycocalyx and intercellular junction.HBOC protect the endothelial glycocalyx and intercellular junction possibly by improving the tissue oxygen delivery,and therefore improving the mitochondrial structure and function.