| Sepsis is a life-threatening organ dysfunction caused by the imbalance of the body’s response to infection.It is a common disease in ICU.The pathophysiological process of sepsis is very complex.Despite the remarkable curative effect of anti-infection therapy and organ function support therapy,the fatality rate of sepsis is still as high as 30%-50%.According to statistics,about 5.3 million people worldwide die of sepsis every year.There are two peaks of death in sepsis patients,one is systemic inflammation and the other is immunosuppression.At the current level of treatment,most sepsis patients can pass through the stage of severe systemic inflammation and enter the stage of immunosuppression,while those in the stage of immunosuppression often die from uncontrollable secondary infections and multiple organ dysfunction syndrome(MODS).Studies have shown that lymphocytes distributed in lymph nodes,spleens,lungs,small intestines and other organs of dead sepsis patients have extensive apoptosis.As the largest gland in human body,liver plays an important role in metabolism and immune homeostasis,and is also one of the most common organs damaged by sepsis.It also plays a key role in the development and occurrence of sepsis.Immunosuppression caused by apoptosis of immune cells and imbalance of inflammatory factors has become an important cause of death in sepsis patients.The mortality rate is as high as 40%-80%.In recent years,a large number of studies have shown that as a newly discovered damage associated molecular pattern(DAMP)molecule,extracellular histones in sepsis are mainly released by neutrophil extracellular traps(NETs)and apoptotic or necrotic cells.Once it released outside the cell,cytotoxicity can be produced through a variety of signaling pathways,causing apoptosis of endothelial cells,lymphocytes,macrophages,innate immune cells,and releasing a large number of cytokines,which can induce immune response disorder,resulting in organ damage and even body death.Clinical studies found that the content of histone-DNA mixture in peripheral serum of patients with severe sepsis and septic shock increased significantly.Therefore,extracellular histone has become a potential target for sepsis treatment.Heparin belongs to glycosaminoglycan,and its main disaccharide unit is disaccharide trisulfate(IdoA2S03-GlcNS036S03),which has strong electronegativity.In 2014,Wildhagen et al confirmed by surface plasmon resonance(SPR)that heparin can bind histone directly and effectively reduce the cytotoxicity of histone to achieve therapeutic effect on sepsis mice.It is well known that heparin has a strong anticoagulant effect,in addition,it also has anti-inflammatory,immunoregulatory properties,calyx protection and other functions.However,the potential risk of hemorrhage of heparin limits its use in sepsis.Heparin derivatives,which can effectively replace heparin and have strong affinity with extracellular histones and reduce anticoagulant activity,have become the direction of research and development through derivatization of heparin in various ways.In our previous studies,we found that C2-C3 fractured heparin derivatives(HP-SP)prepared by periodate oxidation and sodium borohydride reduction did not destroy the main chain structure of heparin,while retaining the high degree of sulfation of heparin.Our team also found that HP-SP could significantly improve the hypercoagulability of sepsis mice,although its anticoagulant activity was significantly reduced in vitro;HP-SP could protect the calyx of brain,small intestine and heart of sepsis mice by inhibiting the expression of heparanase and matrix metalloproteinase-9;HP-SP also had anti-tumor activity.Heparin derivatization methods mainly include degradation to low molecular fragments,desulfurization and destruction of its anticoagulant pentose structure.In this study,different sizes of heparin fragments dp6,dp12 and dp24,low sulfated heparin sulfate and HP-SP were selected as research objects,and HP-SP,as an extracellular histone antagonist,was selected from molecular,cellular and animal levels.The protective effects of HP-SP on lymphocyte apoptosis and macrophage phagocytosis induced by extracellular histone were investigated.Septic immunosuppressive mice were duplicated by double-hit method,and the protective effects of HP-SP on lymphocyte(CD4,CD8)and the regulatory effects of inflammatory factors(IL-6,TNF-alpha,IL-lbeta and IL-10)in the model mice were investigated.To protect the immune related organs(spleen,thymus,lymph node,liver,small intestine)and kidney,and to improve the survival rate of sepsis immunosuppressed mice.The results and conclusion of this study are as follows:1.Study on the affinity between histone and heparin derivatives of glucuronic acid with different molecular weight,degree of sulfation and open-loop at molecular and cellular levels1.1 Molecular level study on the interaction of different heparin derivatives with histonesThe affinity of different molecular weight,sulfated heparin derivatives and HP-SP to histone was studied by SPR technique.The results showed that the order of affinity between different molecular weight heparin derivatives and histone was:heparin sodium≈enoxaparin sodium≈dp24≈dp 12>dp6;heparin glucuronic acid ring-opening derivatives and histone affinity was:heparin≈HP-SP;Dalteparin>M402;different sulfated heparin derivatives and histone affinity.The order of size was heparin>heparan sulfate.1.2 Protective effects of different heparin derivatives on histone-induced apoptosis of endothelial cells at cellular levelThe apoptotic protective effect of extracellular histone on EA.hy 926 endothelial cells was studied by flow cytometry.The results showed that the effects of 100 μg/mL heparin on EA.hy 926 cell apoptosis induced by 100 μg/mL histone were observed.The survival rates of endothelial cells in each group(blank control group,histone group,heparin group,dp6 group,dp 12 group,dp24 group,enoxaparin group,daheparin,M402 group,HP-SP group,heparin sulfate group)were as follows:(92.53±0.47)%、(31.83±2.48)%、(90.26±1.80)%、(49.77±0.47)%、(76.03± 1.08)%、(81.50±0.79)%、(84.7±1.87)%、(87.87±1.02)%、(84.4±1.93)%、(89.13 ± 1.87)%、(85.77±0.76)%.The protective effects of heparin derivatives with different molecular weights on endothelial cells were as follows:dp6<dp12<dp24<heparin;the protective effects of heparin derivatives with open-loop glucuronic acid on endothelial cells.The survival rate of endothelial cells protected by different sulfated heparin derivatives was:heparin>acetylheparin sulfateThe above-mentioned studies at the molecular and cellular levels showed that the affinity of heparin and its derivatives to histone and the degree of antagonistic cytotoxicity to histone were related to their molecular weight and sulfation degree.The opening of glucuronic acid had no significant effect on them HP-SP was selected as the most antagonistic histone at the molecular and cellular levels.Excellent heparin derivatives.2.Determination of anticoagulant activity of HP-SP in vitro and its effect on survival rate of mice induced by histoneThe anticoagulant titer of HP-SP w as 17.08 IU/mg by sheep plasma anticoagulant assay.After injecting 100 mg/kg histone into tail vein of mice and injecting 30 mg/kg heparin sodium and HP-SP solution into tail vein 2 minutes earlier,the 24h survival rate of mice was investigated.The 24h survival rate of mice in histone group was 0%Both HP-SP and heparin could rapidly antagonize the protective effect of histone And the 24h survival rate of mice in histone group was 100%The results showed that the anticoagulant activity of HP-SP prepared by heparin glucuronic acid ring-opening reaction decreased significantly,and it could rapidly antagonize histone in vivo to achieve the protection of the body3.HP-SP antagonizes the protective effect of extracellular histone on lymphocytes and macrophages3.1Protective effect of HP-SP on lymphocyteThe protective effects of HP-SP and heparin on lymphocyte apoptosis induced by histone(50 μg/mL)for 1 h were determined by flow cytometry.The results showed that the survival rate of lymphocyte in blank group was(87.20+2.22)%,histone group was(29.89+2.57)%,heparin group was(84.77+1.64)%,and HP-SP group was(84.91+2.55)%.The results showed that histone had strong cytotoxicity to lymphocyte.HP-SP and heparin could antagonize histone to protect lymphocyte(P<0.05)3.2 Protective effect of HP-SP on macrophage phagocytosisThe effect of histone on macrophage phagocytosis was investigated by neutral red assay at different concentration and time.The results showed that the effect of histone on macrophage phagocytosis was time-dependent(6h,12h,24h)and concentration-dependent(25,50,100,200,300,400,500 μg/mL).When the concentration of histone was 300 μg/mL,the phagocytosis was inhibited at 6h and 12h respectively(P>0.05,compared with blank).The best condition was the concentration of histone 300 μg/mL for 12 h to study the protective effect of HP-SP on macrophagesThe protective effects of HP-SP and heparin at different concentrations(50,100,200,400 μg/mL)on macrophage phagocytosis were further investigated.The results showed that HP-SP and heparin at low dose(50 μg/mL)could protect macrophages by antagonizing histone.When the concentration was 100 μg/mL,the protection of HP-SP and heparin on macrophage phagocytosis was the greatest(P<0.001,compared with histone group)The above studies on the protective effect of HP-SP on immune cells show that extracellular histones are highly cytotoxic to lymphocytes and macrophages.Low doses of HP-SP and heparin can rapidly antagonize the apoptosis of extracellular protective lymphocytes and the phagocytosis of macrophages 4.Regulation of HP-SP on inflammatory factor in immunosuppressive mice with sepsis and protection of CD4,CD8 lymphocytes and immune organsAfter 72 h of CLP,sepsis immunosuppressive mice were induced by intraperitoneal injection of 5 mg/kg LPS.After 4 h of CLP operation,HP-SP and heparin groups were injected with HP-SP(8 mg/kg)and heparin(3 mg/kg)respectively through caudal vein for 3 days.Both sham-operation group and CLP group were injected with the same dose of saline via caudal vein.4.1 Effects of HP-SP on plasma levels of IL-6,IL-10 and extracellular histone in septic immunosuppressed miceAfter 96 h of CLP modeling,the levels of IL-6,IL-10 and extracellular histone in plasma of mice were measured by ELISA.The results showed that the levels of IL-6 in CLP group(early stage of sepsis)increased significantly(P<0.05,compared with sham operation group),but the levels of IL-10 and extracellular histone did not increase significantly;the levels of IL-6 in CLP-LPS group(immunosuppressive stage)decreased significantly.IL-10 and extracellular histone levels increased significantly(P<0.05,compared with CLP group).When HP-SP and heparin were given in advance,both of them could significantly reduce IL-10 and extracellular histone levels,but there was no significant change in IL-6 levels(P>0.05,P<0.05,compared with CLP-LPS group).The results showed that after the second hit,the release of IL-6 and IL-10 in mice in the immunosuppressive phase of sepsis(CLP-LPS group)was significantly inhibited(P<0.05),and the release of IL-10 was significantly increased(P<0.05),which accorded with the characteristics of imbalance of inflammation level in the immunosuppressive phase,and the level of extracellular histone was not affected.Epidemic suppression period increased significantly.When heparin and HP-SP were given in advance,they could inhibit the release of IL-10 by reducing the level of extracellular histone to protect the apoptosis of immune cells,but had no significant effect on the level of proinflammatory factor IL-6.There was no significant difference between non-anticoagulant HP-SP and heparin(P>0.05).4.2 Immunohistochemical determination of Histone H4,CD4 and CD8 in spleen,thymus and lymph nodes of immunosuppressive mice with sepsisImmunohistochemistry was used to detect the expression of H4,CD4 and CD8 in spleen,lymph node and thymus of mice 96 h after CLP modeling.The results showed that CD4 and CD8 lymphocytes in spleen,lymph node and thymus of mice in CLP-LPS group were significantly decreased(P<0.05,which was not related to sham operation).CD8 lymphocyte was weaker than CD4 lymphocyte,and the expression of histone H4 in three immune organs was significantly increased(P<0.05).The proportion of CD4/CD8 was further investigated.At present,the proportion of CD4/CD8 in spleen,lymph node and thymus of mice in CLP-LPS group decreased significantly(P<0.05).When heparin and HP-SP were given in advance,both of them could effectively alleviate the apoptosis of CD4 and CD8 lymphocytes in immune organs by antagonizing the cytotoxicity of extracellular histone H4(P<0.05);HP-SP could effectively increase the ratio of CD4/CD8 in spleen and lymph nodes(P<0.05),but the ratio of CD4/CD8 in thymus was increased.The ratio of CD4/CD8 in lymph nodes was increased by heparin(P<0.05),but the ratio of CD4/CD8 in spleen and thymus was improved,but there was no significant difference.These results indicated that CD4 and CD8 lymphocyte apoptosis occurred in spleen,thymus and lymph nodes of mice in the immunosuppressive phase(CLP-LPS group),and the ratio of CD4/CD8 decreased significantly,which was closely related to the cytotoxicity of extracellular histones in the three organs.When heparin and HP-SP were given in advance,both of them could effectively alleviate the apoptosis of CD4 and CD8 lymphocytes in immune organs by antagonizing the cytotoxicity of extracellular histone H4(P<0.05),and the improvement of lymph nodes was stronger than that of spleen and thymus tissues.The observation of HE also supported the results.4.3 Effect of HP-SP on pathological changes of spleen,thymus and lymph nodes in sepsis immunosuppressed miceThe pathological changes of spleen,thymus and lymph nodes were evaluated by HE staining after 96 h of CLP modeling.The results showed that lymphocytes in lymph nodes,thymus and spleen were decreased and arranged loosely in CLP-LPS group.There were a large number of neutrophils and macrophages in spleen.When heparin and HP-SP were given in advance,both of them could significantly improve the pathological changes of the three groups,and the improvement of lymph nodes was stronger than that of spleen and thymus.The above results showed that the number of lymphocytes in immune organs(spleen,thymus and lymph nodes)of mice in immunosuppressive phase decreased and arranged loosely.Immunohistochemical results have confirmed that there are high levels of histone monomers(H4)in the three organs.Therefore,the decrease of lymphocytes in these three organs is related to the toxicity of extracellular histone.When heparin and HP-SP were given in advance,both of them could inhibit the apoptosis of lymphocyte in immune organs by antagonizing the extracellular histone,and improve the histopathological damage,which was related to the antagonism of HP-SP and heparin against the extracellular histone,the protection of immune cells,the inhibition of the release of IL-10 and the improvement of tissue immunosuppression.The above HP-SP effects on spleen and thymus.lymph node protection studies have shown that early administration of HP-SP and heparin can regulate inflammation by antagonizing cytotoxicity of extracellular histone in vivo,inhibiting the release of anti-inflammatory factor IL-10 in immunosuppressive mice,and protecting CD4 and CD8 lymphocytes in spleen,lymph nodes and thymus from apoptosis.Inhibited immune disorders are regulated to improve the immune suppression of sepsis to achieve the protection of the body.5.Protective effect of HP-SP on liver injury in septic immunosuppressed miceAfter 72 h of CLP modeling,sepsis mice were induced by intraperitoneal injection of 5 mg/kg LPS.After 4 h of CLP modeling,8 mg/kg HP-SP and 3 mg/kg heparin sodium were injected into tail vein for intervention.5.1 Effects of HP-SP on extracellular histone,IL-6,TNF-a,IL-1p,IL-10 and extracellular histone levels in liver tissues of septic immunosuppressive miceAfter 24h and 96 hh of CLP modeling,the extracellular histones,IL-6,TNF-a,IL-1β and IL-10 in liver tissue of mice were determined by ELISA.The results showed that the contents of IL-6,TNF-α,IL-1β,IL-10 and extracellular histone in CLP-LPS group increased significantly at 24 h and 96 h(P<0.05),and at 96 h,the levels of extracellular histone and IL-6 were significantly lower than those at 24 h,but the levels of IL-10 were significantly higher.When heparin and HP-SP were given in advance,the levels of extracellular histone,IL-6,TNF-α,IL-1β,and IL-10 in liver tissue were significantly decreased(P<0.05).The results showed that in the early stage of sepsis(24h),hepatic tissue was dominated by pro-inflammatory factors(IL-6,TNF-a,IL-1β)due to the toxicity of extracellular histones,while in the immunosuppressive stage of sepsis(96h),anti-inflammatory factors(IL-10)were dominant.When heparin and HP-SP were given interventional therapy,heparin and HP-SP could pass through.Over-decreasing the level of extracellular histone in liver tissue,and then reducing the levels of IL-6,TNF-a,IL-1β and IL-10 inflammatory factors,to achieve the protection of liver tissue.The results were supported by the detection of HE in the liver and the enzymology of liver function.5.2 Effect of HP-SP on liver function indexes AST and ALT in septic immunosuppressive mice After 12 h,24 h and 96 h of CLP modeling,the levels of AST and ALT in serum of mice were determined by microporous method.The results showed that the levels of AST and ALT in CLP-LPS mice increased significantly at 12h,24h and 96 h(P<0.05),and continued to increase within 24 h after operation.When heparin and HP-SP were administered in advance,both of them could reduce liver injury by antagonizing extracellular histone to regulate inflammation level and reduce AST and ALT levels.These results suggest that liver injury occurs in the early stage of sepsis in mice,which is related to the release of inflammatory cytokines by extracellular histones in liver tissue,leading to hepatocyte apoptosis and necrosis.When heparin and HP-SP were given in advance,both of them could reduce the levels of TNF-a,IL-1β,IL-6 and IL-10 by antagonizing extracellular histones,and reduce the degree of liver injury,thereby reducing the levels of AST and ALT in serum.5.3 Effect of HP-SP on relative liver weight and liver tissue pathological damage in immunosuppressive mice with sepsisAfter 24 h and 96 h of CLP modeling,the results showed that there was no significant difference in relative liver weight among the groups at 24 h(P>0.05),but there was no significant difference in relative liver weight at 96 h(P>0.05,compared with sham operation group).HE staining showed that most of the hepatocytes in imunosuppressed mice showed moderate swelling,narrowing of hepatic sinuses and focal inflammatory necrosis at 96 h.When heparin and HP-SP were given in advance,liver pathological damage could be effectively improved.The above-mentioned studies on liver injury show that HP-SP and heparin can reduce the release of IL-6,TNF-a,IL-1β and IL-10 inflammatory factors in liver tissue by reducing the level of extracellular histone,thereby reducing liver injury.AST.The decrease of ALT level and HE staining both support this conclusion.6.Protective effect of HP-SP on small intestine and kidney of sepsis immunosuppressed mice and its effect on 7-day survival rate of sepsis immunosuppressed mice6.1 Observation of general conditions and weight change of mice in 7 days7 days continuity observed that sepsis mice after CLP appeared a variety of pathological manifestations,namely,mental malaise,tail sparse water-like feces,periocular secretions,some of the inner canthus bleeding symptoms,weight loss,and abdominal stench after dissection.The results showed that the model of sepsis mice was successful.Observing the weight change of mice in 7 days,we found that the weight of mice in CLP-LPS group decreased significantly within 7 days,and the intervention of heparin and HP-SP could significantly improve the weight loss of mice.6.2 HE staining of small intestine and kidney in mice and 7-day survival rateAfter 96 h of CLP modeling,HE staining of small intestine and kidney of mice showed that villi atrophied and epithelial cells fell off obviously in CLP-LPS group,and the renal capsule became thicker.When heparin and HP-SP were given in advance,the pathological changes of small intestine and liver could be effectively improved.The 7 day survival rate was 14.2%in CLP-LPS group.When heparin and HP-SP were given in advance,the survival rate of sepsis mice was significantly increased.The survival rate of heparin group was 42.8%,and that of HP-SP group was 35.7%.These phenotypic studies show that early administration of HP-SP and heparin can improve the immunosuppressive state by antagonizing the cytotoxicity of extracellular histone to regulate inflammation level and protect immune cells in vivo,thus achieving the protective effect on intestinal mucosal injury and kidney injury in sepsis mice,and at the same time improving the body of sepsis mice effectively.The 7 day survival rate of sepsis mice was significantly reduced and increased.8.ConclusionIn this study,we found that HP-SP with low anticoagulant activity can antagonize the cytotoxicity of extracellular histone in vitro and in vivo,thereby exerting a multifaceted protective effect on immune-related organs and immune cells of sepsis-immunosuppressed mice,improving the immunosuppressive state,suggesting that HP-SP may prevent sepsis from entering more thorny stage from SIRS.Hands and dangerous CARS stage are of great value in the treatment of sepsis.9.Innovation and potential application value of this topic(1)Heparin derivatives can interact with histones,but the mechanism of their action has not been systematically studied.The interaction between heparin and its derivatives and histone with molecular weight,sulfation degree and glucuronic acid ring opening was investigated at molecular and cellular levels.The results showed that the affinity of heparin and its derivatives to histone decreased with the decrease of molecular weight and sulfation degree of heparin,while the opening of heparin non-sulfated glucuronide had no significant effect on it.(2)The potential risk of hemorrhage of heparin limits its clinical application.It was found that HP-SP had little anticoagulant activity and retained the strong sulfation degree of heparin,which had almost the same affinity histone as heparin.(3)Extracellular histones,as DAMP molecules,are mostly focused on early studies of sepsis.In this study,we found that in the immunosuppressive phase of sepsis,extracellular histones still have a high level,which provides a new research direction for the intervention of immunosuppressive phase.10.Future research directions of this topicThe molecular mechanism of HP-SP antagonistic histone on immune cell protection and the regulatory effect and mechanism of HP-SP antagonistic extracellular histone on disordered immune level in sepsis need to be further studied. |
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