| Sepsis is a systemic inflammatory response syndrome caused by infection.As one of the serious complications of clinically critical patients such as trauma,burns and infection,sepsis is a precursor to multiple organ dysfunction syndrome(MODS).It is also a major problem facing today's critical care medicine.It is generally believed that the pathophysiological changes in patients with sepsis are closely related to Gram-negative bacteria and its lysate,endotoxin,also known as lipopolysaccharide(LPS).When LPS enters the body,it activates damaged endothelial cells,stimulates mononuclear-macrophages,and releases a variety of oxygen free radicals and inflammatory mediators,causing sepsis.Sepsis is essentially an uncontrolled inflammatory response and coagulopathy.The inflammatory cells and release of various inflammatory mediators during sepsis activates the coagulation system.The inflammatory response and coagulation activation promote and interact with each other,which further deteriorates the coagulation function,which is manifested by microvascular thrombosis in microvasculature,leading to microcirculatory disorders,which further develop into severe sepsis and septic shock.Sepsis coagulopathy refers to extensive systemic coagulation activation with high risk of bleeding and organ failure,the main feature of which is the imbalance of intravascular fibrin formation and clearance.It is characterized by enhanced coagulation reaction,severely reduced anticoagulant ability,and a significant decrease in fibrinolytic capacity,accompanied by a large consumption of coagulation factors and anticoagulant substances.Coagulation dysfunction plays an important role in the whole pathological process of sepsis,and is one of the key links in the development and prognosis of sepsis.Coagulation dysfunction caused by sepsis is mainly caused by blood coagulation activation,down-regulation of physiological anticoagulation mechanism and inhibition of fibrinolysis pathway.It is currently believed that the exogenous coagulation pathway triggered by tissue factor(TF)is the main pathway for clotting activation in sepsis.In sepsis,TF is mainly derived from vascular endothelial cells or monocytes.TF activates TF factor in the cycle and binds to form TF-VIIa complex,which can then activate a large number of prothrombin into thrombin through a cascade reaction.At the same time,a variety of pathways lead to down-regulation of physiological anticoagulant mechanisms,which is another important factor leading to blood clotting.There are three physiological anticoagulant pathways in the normal body:antithrombin ?(AT?),protein C(PC)system,and tissue factor pathway inhibitor(TFPI).The above anticoagulant pathways were significantly inhibited in sepsis.After the coagulation is activated,the fibrinolytic system is also activated.In sepsis,the fibrinolytic system is inhibited by the inactivation of the plasminogen activator inhibitor-1,which is produced in large amounts by the plasminogen activator.Endothelial cells line the inner surface of blood vessels and are capable of expressing a variety of anticoagulant-related components.Their structural and functional integrity contributes to maintaining the stability of the intravascular environment and is important for regulating vascular tone,maintaining the balance of coagulation/anticoagulation and immune function.Glycocalyx is a villus-like polysaccharide-protein complex structure covered by the inner side of vascular endothelial cells.As the skeleton structure of the surface layer of endothelial cells,it strongly affects the function of endothelial cells.Glycocalyx plays an important role in the formation of blood-tissue permeability barriers.It can regulate the interaction between blood cells and endothelial cells,and is necessary to maintain the normal structural function of vascular endothelial cells.At the same time,glycocalyx plays an important role in regulating vascular tone and inhibiting intravascular coagulation.In physiological conditions,vascular endothelial glycocalyx combines with anti-coagulation factors such as AT?,HC?,TM and TFPI,which gives endothelial cells anticoagulant properties under physiological conditions.Sepsis is essentially an excessive inflammatory response and coagulopathy.It is currently believed that vascular endothelial cells are one of the main target cells of inflammatory response,and can also serve as an effector cell,which plays an important role in microcirculatory disorders,septic shock and multiple organ dysfunction.In sepsis,due to LPS and the release of a large amount of inflammatory factors such as TNF-a and IL-6 released by inflammatory cells,endothelial cells change in morphology and function under their stimulation,a large amount of synthetic release of TF activates the exogenous coagulation reaction,while the expression of anticoagulant is reduced.In this inflammatory process,vascular endothelial glycocalyx is the first to be affected and destroyed before the endothelial cells themselves.For septic coagulopathy,endothelial cell damage caused by inflammatory factors and LPS is the initiation of coagulation activation.As the protective layer of endothelial cells,glycocalyx is destroyed before endothelial cells,which has become a portal link for the subsequent uncontrolled inflammatory reaction and coagulation disorder,which should be taken seriously.As a classic anticoagulant,heparin is well known and widely used.Heparin has also been found to protect against glycocalyx damage in sepsis in recent years.Heparin has also been used for anticoagulant therapy in sepsis,but there is a risk of bleeding.HP-sp is a partial uronic acid ring-opening derivative prepared by heparan oxidized by periodic acid and reduced by sodium borohydride.Previous research by our group has proved that HP-sp can effectively reduce the glycocalyx damage caused by sepsis.It can be speculated that this protective effect may help to alleviate coagulopathy in sepsis and exert anticoagulant effect,which is of positive significance for the treatment of sepsis.Therefore,this project intends to use heparin as a control to explore the role of HP-sp in the hypercoagulable state of sepsis and possible mechanisms.The main results of this project are:1.Preparation and in vitro anticoagulant activity of heparin derivative HP-sp1.1 Preparation and structural confirmation of heparin derivative HP-spHP-sp was prepared by subjecting heparin to sodium periodate oxidation and sodium borohydride reduction,and subjected to 1H-NMR measurement.The results showed that the heparin 1H-NMR spectrum had the C2-C3H2 and H3 signals of Glc's ortho-dihydroxy group at about ?3.40,and the signal disappeared in the 1H-NMR spectrum of HP-sp,which was consistent with the reaction.In addition,there is no significant difference in the structure of HP-sp compared with heparin.1.2 Determination of anticoagulant titer of heparin derivative HP-sp by sheep plasma methodThe inhibitory effect of HP-sp on plasma coagulation of recalcified sheep was investigated by sheep plasma method,and its anticoagulant activity was evaluated in vitro.As a result,the anticoagulant titer of HP-sp was determined to be 18.4 U/mg,which was less than 10%of heparin.2.Therapeutic effect of heparin derivative HP-sp on hypercoagulable state in sepsis mice2.1 The effect of HP-sp on tail bleeding time in sepsis miceThe intraperitoneal injection of LPS was used to replicate the model of sepsis mice with hypercoagulability.Heparin was used as a control to investigate the effect of HP-sp on clotting time in sepsis mice.The results showed that compared with the normal control group,the tail bleeding time of the LPS model group was significantly shortened after 5 hours of modeling(P<0.05);Compared with the LPS model group,the tail bleeding time of the heparin group and the HP-sp group was significantly prolonged(P<0.01);There was no significant difference in tail bleeding time between the heparin group and the HP-sp group(P>0.05).The tail bleeding time of heparin group and HP-sp group was significantly longer than that of normal group(P<0.05).This indicates that the sepsis mice model of LPS replication is hypercoagulable;Heparin can play a significant role in correcting blood hypercoagulability in mice with sepsis;The HP-sp with weak anticoagulant activity prepared in this study can also significantly improve the hypercoagulable state of mice with sepsis,and there was no significant difference from the heparin group.2.2 Effect of HP-sp on plasma D-dimer content in sepsis miceIntraperitoneal injection of LPS replicates sepsis mice with a hypercoagulable state model.Heparin was used as a control to investigate the effect of HP-sp on the content of D-dimer(early sensitive index of coagulation disorder)in peripheral blood of sepsis mice.The results showed that compared with the normal control group,the D-dimer content in the plasma of the LPS model group was significantly increased after 5h of modeling(P<0.05);Compared with the LPS model group,the plasma D-dimer content in the heparin group and the HP-sp group was significantly lower(P<0.05);There was no significant difference in plasma D-dimer content between the heparin group and the HP-sp group(P>0.05).Compared with the normal control group,the D-dimer content in the heparin group and the HP-sp group was slightly increased,but there was no significant difference(P>0.05).This indicates that the D-dimer content in the plasma of LPS-replicated sepsis mice is significantly increased.HP-sp can significantly reduce the D-dimer content in the plasma of septic mice,and its intensity is similar to that of heparin.Both can reduce the D-dimer content in the plasma of septic mice to a normal level.3.Inhibitory effect of heparin derivative HP-sp on thrombin3.1 HP-sp inhibits ?a by AT?Heparin was used as a control to examine the inhibitory effect of HP-sp on thrombin activity by AT? at the molecular level using the chromogenic substrate method.The results indicate that HP-sp does not have the ability to activate AT? and inactivate ?a.3.2 HP-sp inhibits thrombin by HC?Heparin and dermatan sulfate(DS)were used as controls to investigate the inhibitory effect of HP-sp on thrombin activity by HC? using the chromogenic substrate method.The results indicate that HP-sp can activate HC?,thereby antagonizing thrombin.4.Endothelial protective effect of heparin derivative HP-sp on sepsis miceTF,TFPI,sTM,sEPCR are all related to the structural integrity and functional status of endothelial cells.The mouse model of sepsis was injected by intraperitoneal injection of LPS.The content of these four components in peripheral blood of septic mice was determined by ELISA.The protective effect of HP-sp on endothelial injury in septic mice was investigated.4.1 Effect of HP-sp on plasma TF content in sepsis miceTF is a trigger for coagulation activation in sepsis.The results showed that compared with the normal group,the plasma TF content in the LPS group was significantly increased,and there was almost no significant change during the 3?11 h period after modeling,and it was always maintained at a high level.Compared with the LPS group,the TF content in the heparin group and the HP-sp group was similar to that in the LPS group at 3h after model establishment,but then showed a significant downward trend.The TF content of the two groups was lower than that of the LPS group at 7h,and at 11h,the TF content in the heparin group was close to the normal group.This indicates that both heparin and HP-sp can reduce the plasma TF content in sepsis mice.4.2 Effect of HP-sp on plasma TFPI content in sepsis miceTFPI is a natural inhibitor of TF and is one of the main components of the body's physiological anticoagulation.The results showed that compared with the normal group,the TFPI content in the LPS group was significantly increased 3h after modeling,while the TFPI content in the LPS group decreased linearly during the 3?11h period.Compared with the LPS group,the TFPI content in the heparin group was similar to that in the LPS group during the 3?7h period,but during the 7?11 h period,the TFPI content was significantly higher than that of the LPS group,and the decline trend was relatively flat;Compared with the LPS group,the TFPI content in the HP-sp group was lower during the 3?7h period,but it showed a significant increase trend.After 7h,the content change was not obvious,but significantly higher than the LPS group.At 11 h,the HP-sp group content was still higher than the HP group.This indicates that both HP and HP-sp can increase the plasma TFPI content in sepsis mice.The change of TFPI/TF ratio showed that the TFPI/TF ratio of LPS group increased slightly at 3h compared with the normal group,but remained unchanged within 3?7h,but decreased significantly at 7?11h.Compared with LPS group,the ratio of HP group and HP-sp group showed an upward trend within 3?7h,and HP-sp group was significantly higher than LPS group at 7h,then the ratio was basically unchanged,while HP group always showed an upward trend,and at 11h,it was significantly higher than the LPS group.This indicates that both HP and HP-sp can significantly increase the ratio of TFPI/TF in the plasma of sepsis mice.4.3 Effect of HP-sp on plasma sTM content in sepsis micesTM is formed by TM shedding into the blood and serves as a marker of endothelial damage.The results showed that compared with the normal group,the plasma sTM content in the LPS group was significantly increased,which decreased slightly within 3?7h after modeling,and there was almost no significant change during 7?11h,which was always maintained at a high level.Compared with the LPS group,the content of HP group and HP-sp group was similar to that of LPS group at 3h after model establishment,but then showed a downward trend.At 7 h,the content of HP-sp group was lower than that of LPS group,and at 11h,both were lower than the LPS group,and the HP-sp group was lower than the HP group.This indicates that HP-sp can significantly reduce the plasma sTM content in sepsis mice,and the intensity of heparin is low,mainly concentrated after 7h.4.4 Effect of HP-sp on plasma sEPCR content in sepsis miceEPCR is a high-affinity endothelial cell surface receptor of PC/APC,which is shed into blood into sEPCR.sEPCR has the effect of inhibiting APC,and can also reflect the condition of endothelial injury.The results showed that compared with the normal group,the sEPCR content of LPS group increased significantly,showing a trend of increasing first and then decreasing.Compared with LPS group,heparin group and HP-sp group showed a downward trend.At 3h,HP-sp group was higher than LPS group,and the content of both groups was lower than LPS group at 7h.The content of HP group still decreased significantly during 7?11h,while the content of HP-sp group decreased slightly.At 11h,the content of heparin group was similar to that of normal group,while the content of HP-sp group was close to that of LPS group,so HP-sp was not as effective as heparin.This indicates that heparin can significantly reduce the plasma sEPCR content in septic mice,and HP-sp can significantly reduce its content within 3?7h.5.ConclusionHP-sp is a derivative of heparin partial uronic acid ring opening.HP-sp has weak anticoagulant activity due to its structural heparin anticoagulant pentose structure.However,it is still possible to significantly improve the hypercoagulable state of mice caused by sepsis.This study has found that HP-sp anticoagulation mainly depends on its activation of HCII to inactivate thrombin.At the same time,HP-sp can also inhibit the initiation of coagulation activation in sepsis and the inhibition of physiological anticoagulant pathways in sepsis(TFPI and PC system)by protecting endothelial cells,thereby exerting anticoagulant effect.This study laid the foundation for the development of HP-sp as a new mechanism for anticoagulant therapy in sepsis. |
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