N-Desulfated/Acetylated Heparin Ameliorates The Shedding Of Glycocalyx In Septic Mice

Sepsis is a disorder of host response caused by infection,which leads to life-threatening organ dysfunction syndrome,which has the characteristics of high morbidity and high mortality.Clinical diagnosis and treatment is very difficult.According to reports,the number of patients who develop from infection to sepsis every year in the world reaches millions,and the mortality rate is 22%to 55%.Glycocalyx is a polysaccharide-protein complex structure on the cell surface.It is mainly composed of proteoglycans and glycosaminoglycans.It has many important physiological functions,such as maintaining vascular permeability,mechanical force conduction and regulation of vascular microenvironment.Function and so on.The study found that glycocalyx was firstly damaged in sepsis,followed by increased endothelial permeability,leakage of plasma proteins into the interstitial space,triggering tissue edema,and at the same time damaging the oxygen supply and nutrient supply of the tissue.Therefore,the development of drugs that can effectively protect glycocalyx is of great significance in reducing the mortality of patients with sepsis.The intestine is also known as the "starting organ" of the development of sepsis,and its infection is an important factor leading to the rapid spread of sepsis inside the body.In the intestinal tract,there is a complex intestinal environment,which releases various toxins such as endotoxins and pyrogens.The above toxins must break through the intestinal mucosal barrier before invading the blood circulation system,and glycocalyx is an important component of the intestinal mucosal barrier,which is seriously damaged in intestinal infection of sepsis.Therefore,the protection of intestinal glycosides is of great significance.Heparin is often used as a prophylactic hypercoagulable state in the treatment of sepsis,and heparin has also been found to have a significant inhibitory effect on heparanase which can cause glycocalyx destruction.However,heparin has a potential anti-coagulant activity and has a potential risk of bleeding in the treatment of sepsis.Therefore,the development of heparin derivatives with low anticoagulant activity and good glycocalyx protection has important clinical application value.The study found that N-desulfated acetylated heparin derivative(NDSAH)has lower anticoagulant activity,but better preserves the skeletal structure of heparin.In this study,we investigated the factors related to glycocalyx injury in NDSAH at the cellular level and sepsis mouse model,including Heparanase(HPA),Matrix metalloproteinase-9(MMP-9)and inflammatory factors.The effect was to investigate the value of NDSAH in inhibiting glycocalyx detachment and its protective effect on septic mice.The main results and conclusions of this research have the following aspects1.Preparation of heparin derivative NDSAH and its anticoagulant activity in vitro1.1 Preparation and structural confirmation of heparin derivative NDSAHThe heparin sodium is converted into heparinic acid by a strong acidic cationic resin,and then heparin pyridinium salt is prepared by neutralizing with pyridine.Subsequently,the N-sulfate on the heparin sugar chain was removed by heating in a methanol-DMSO-containing solution,and the obtained product was further acetylated with acetic anhydride to obtain a target product.The structure was examined by NMR and confirmed to be a heparin derivative of heparin dextran aldehyde acid N-position acetylated,which was recorded as NDSAH.1.2 Determination of anticoagulant titer of heparin derivative NDSAH by sheep plasma methodThe anti-coagulation titer of NDSAH was measured by the method of recalcification sheep plasma with heparin sodium as the standard,which was 32.1 IU/mg,indicating that the risk of NDSAH bleeding in the clinical day was significantly lower than that of heparin.2.Effect of heparin derivative NDSAH on survival rate of FIP sepsis mouse modelA mouse model of sepsis was prepared by intraperitoneal injection of Fecal pellets suspended in PBS(FIP).Heparin was used as a control to investigate the effect of NDASHA on the survival rate of mice.The results showed that both NDSAH and heparin could improve the survival rate of septic mice(p<0.05),with an increase of 62.5%and 50%,respectively.3.Protective effect of heparin derivative NDSAH on FIP sepsis mouse model3.1 Using Evans Blue as an indicator to investigate the effect of NDSAH on peritoneal permeability in mice with FIP sepsisIt was found that NDSAH can reduce peritoneal permeability of the mouse FIP sepsis model.3.2 Using 40kDa dextran-Texas red and 500kDa dextran-FITC as indicators,the effect of NDSAH on the total volume of vascular endothelial glycocalyx in FIP sepsis mice was examined.Using FIP sep's mouse model,500kDa dextran-FITC and 40kDa dextran-Texas red fluorescence detectors were injected intravenously to investigate the changes of total vascular endothelial glycocalyx in mice,normal group,FIP sepsis and NDSAH the total volume of vascular endothelium in mice mice was 105.9 ?L,37 ?L and 64?L,respectively.The results showed that NDSAH can effectively inhibit the detachment of whole body vascular endothelium during sepsis.4.Intestinal protective effect of heparin derivative NDSAH on FIP sepsis mouse model and its effect on inflammatory factor levels4.1 HE method to detect the protective effect of NDSAH on small intestine tissue of mice with FIP sepsis after 3h and 8h of modelingObservations were made at 3h and 8h after modeling.The results showed that after 3 h and 8 h of modeling,NDSAH showed significant protective effect on small intestinal tissue injury and necrosis in mice with FIP sepsis,and the protective effect of NDSAH was slightly stronger than that of heparin.4.2 Immunohistochemical method was used to detect the expression of TNF-?,IL-6,HPA,MMP-9,HS and Syndecan-1 in the small intestine of FIP sepsis mice after 3h and 8h modeling.4.2.1 TNF-?,IL-6 immunohistochemistry resultsThe results showed that the expression levels of TNF-a and IL-6 in the FIP sepsis group were significantly higher than those in the normal group(p<0.01),but compared with the model group,NDSAH significantly decreased the expression level of TNF-a and IL-6(p<0.01).Heparin also had an inhibitory effect on TNF-a expression(p<0.01)compared with the model group,but no Inhibitory effect on IL-6(p>0.05).After 8 hours of modeling,the expression of two inflammatory factors in the FIP model group decreased compared with that at 3 hour.Compared with the model group,NDSAH still showed a significant inhibition of TNF-a expression level(p<0.01),while IL-6 expression level decreased(p>0.05).Compared with the model group,the level of TNF-a in the heparin group was significantly decreased(p<0.01),and the expression level of IL-6 in the heparin group was decreased(p>0.05).4.2.2 HPA,MMP-9 immunohistochemistry resultsThe results showed that the expression levels of HPA and MMP-9 in the FIP sepsis group were significantly higher than those in the normal group(p<0.01).NDSAH can reduce the expression of HPA(p<0.05),but has no significant effect on MMP-9(p>0.05);heparin has no significant inhibitory effect on HPA and MMP-9(p>0.05).The expression of HPA and MMP-9 in the FIP sepsis model group was lower than that at 3h after modeling for 8 hours.At this time point,NDSAH significantly inhibited the expression of HPA(p<0.05),but did not significantly inhibit the inhibition of MMP-9(p>0.05);compared with the model group heparin presents the inhibition trend of HPA,MMP-9 expression levels,but no statistically significant(p>0.05)4.2.3 HS,Syndecan-1 immunohistochemistry resultsThe results showed that the expression levels of HS and Syndecan-1 in the FIP sepsis group were significantly lower than those in the normal group(p<0.01),after 3h of modeling and NDSAH significantly inhibited the detachment of HS and Syndecan-1 in the intestinal tissue of FIP sepsis mice.The content of important components HS and Syndecan-1 decreased(p<0.05).Compared with the model group,heparin significantly inhibited the decrease of Syndecan-1 content in sepsis(p<0.01),but had no significant effect on the decrease of HS content in sepsis(p>0.05).The content of HS and Syndecan-1 in the 8h model group increased after 3h model group.At this time point,the expression levels of HS and Syndecan-1 in the NDSAH group were higher than those in the FIP model group(p<0.05);while the expression level of Syncdean-1 in the heparin group was significantly different from that in the FIP group(p<0.01),no significant effect on HS expression level(p>0.05).4.3 ELISA method to detect the effect of NDSAH on the levels of MPO from mouse small intestine tissue with TNF-a and IL-6 in the mouse plasma4.3.1 ELISA method for detection of MPO content in small intestineThe results showed that the MPO level in the intestinal tissue of the FIP model group was significantly higher than that in the normal group(p<0.01).The NDSAH could significantly reduce the MPO level in the FIP sepsis mice(p<0.01).The heparin group was compared with the model group.There is a tendency to reduce the expression of MPO(p>0.05).4.3.2 Detection of plasma TNF-a and IL-6 by ELISAThe results showed that compared with the normal group,the plasma levels of TNF-a and IL-6 in the model group were significantly increased(p<0.01).Compared with the model group,NDSAH significantly reduced TNF-a and IL-6 level in plasma(p<0.01);heparin had a tendency to inhibit the up-regulation of TNF-a and IL-6 expression compared with the model group,but there was no significant difference(p>0.05).5.Effect of heparin derivative NDSAH on cell glycocalyx damage induced by LPS5.1 WGA-FITC fluorescence detectionThe cell glycocalyx injury model was prepared by LPS incubation method,and the fluorescence intensity of FITC was used as a quantitative index.It was found that compared with the normal group,LPS caused significant decrease in fluorescence intensity(p<0.05)in the three cells observed,HIEC,RAW264.7 macrophages and HUVEC,indicating that cell surface glycocalyx was damaged.Compared with the model group,NDSAH can significantly increase the fluorescence intensity(p<0.05)in all three cells,indicating that NDSAH has the effect of inhibiting the loss of glycocalyx in three cells induced by LPS;while heparin only causes HIEC cells glycocalyx injury protective effects induced by LPS,has significant protective effect(p<0.05).5.2 Effect of NDSAH on the effect of LPS-induced cell glycocalyx thickness and hardness by atomic force microscopyA cell glycocalyx damage model was prepared by LPS incubation.It was found that LPS acting on HIEC and RAW264.7 macrophages caused a decrease in the hardness and thickness of the surface of these two cells;NDSAH and heparin acted on both cells to attenuate the above changes caused by LPS.5.3 Laser confocal fluorescence microscopy to detect the effect of NDSAH on cell surface glycocalyx injury induced by LPSThe cell glycocalyx injury model was prepared by LPS incubation,and the cell membrane glycocalyx was stained by FITC-WAG It was found that LPS acts on HIEC,RAW264.7 macrophages and HUVEC cells,which leads to a decrease in cell surface glycocalyx integrity,while NDSAH and heparin can inhibit cell glycocalyx damage induced by LPS.6.ConclusionAs a non-anticoagulant heparin derivative,NDSAH can significantly improve the survival rate of septic mice,and inhibit the inflammatory factors TNF-a,IL-6,HPA,MMP-9 and MPO up-regulation,then inhibiting glycocalyx shedding from vascular endothelium and intestine.That makes the further development of NDSAH as a glycocalyx protectant for the treatment of sepsis.