Unfractionated Heparin Improves Coagulation By Inhibiting Heparinase To Protect Glycocalyx Of Endothelial Cells In Sepsis

Objective: Severe inflammation and activated coagulation in sepsis always lead to injury and dysfunction of endothelial cells,which is the main cause of microcirculation failure and organ failure.The glycocalyx is negatively charged and its thickness varies according to diffrent vessels in the organs.The glycocalyx and endothelial cells are the first barrier to isolate pathogens and organs,which play a key role in the process of sepsis.In physiological state,the glycocalyx has many functions such as anti-thrombosis,inhibition of cell adhesion and prevention of tissue edema.In the early stage of sepsis,the glycocalyx is damaged,resulting in leukocyte adhesion and coagulation activation.Heparanase(HPA)degrades the glycocalyx through various pathways,leading to endothelial cell injury with increased inflammation,coagulation activation and tissue edema,microcirculation disorder and even multiple organ failure.In addition to the traditional anticogulation effect,unfractionated heparin(UFH)also plays important roles in immune regulations.A large number of basic and clinical studies have confirmed that UFH can protect the glycocalyx in sepsis.But the exact mechanisms are not clear.To our knowledge,whether UFH can protect the glycocalyx by reducing the expression of heparinase and thus play a protective role in sepsis has not been reported yet.Therefore,animal and cell studies are necessary to confirme in order to provide theoretical basis for the roles and mechanisms of UFH in sepsis.The purpose of this study was to investigate the effects of UFH on the glycocalyx of endothelical cells and the coagulation function in sepsis.We also explored the mechanism involved.Methods:1.Tissue samples :50 male C57BL/6 mice aged 8-10 weeks were collected from the Laboratory Animal Center of China Medical University.20 mice were treated with cecal ligation and puncture(CLP).Conforming to the criteria of sepsis caused by CLP and the fatality rate of 50% indicated successful modeling.30 mice were randomly divided into 5 groups: control group,equal dose of 0.9% Na Cl was injected through tail vein.CLP group,the sepsis model was made by CLP.CLP+ UFH group,UFH 8 U/20g(dose selected according to our research group’s previous studies)was injected through tail vein 1 hour after CLP.CLP+Heparinase(HPA)inhibitor group,HPA inhibitor20mg/kg(dose selected according to our research group’s previous studies)was injected intraperitoneally.CLP+UFH+HPA inhibitor group,injected HPA inhibitor 20 mg/kg was injected intraperitoneally before CLP modeling,and UFH 8 U/20 g was injected through tail vein 1 hour later.Lungs were collected 4 hours(time point selected according to our research group’s previous studies)after CLP modeling or 0.9%Na Cl injection.2.Hematoxylin and Eosin(HE)staining was performed to evaluate lung histopathological score3.The degree of pulmonary edema was quantified by measuring the wet/dry of lung and the percentage of lung water content.4.Cell culture: Human pulmonary microvascular endothelial cells(HPMECs)were purchased from Shanghai Baili Biotechnology Co,LTD.The cells were divided into 5groups: control group,equal volume of PBS was added..Lipopolysaccharide(LPS)group,LPS 10 μg/ m L(dose selected according to our research group’s previous studies)was added.LPS+UFH group,UFH 10 U/ m L was added after 1 hour of LPS 10 μg/ m L.LPS+HPA inhibitor group,both LPS 10μg/ m L and HPA inhibitor 150 μg/ m L(dose selected according to our research group’s previous studies)were added.LPS+HPA inhibitor +UFH group,HPA inhibitor 150 μg/ m L and LPS 10μg/ m L were added,UFH10 U/ m L was given 1h later.Cells were collected 4h after LPS or PBS.5.m RNA levels of tissue factor(TF),fibrinogen(Fib),interleukin-1 β(IL-1β),syndecan-1 and HPA in lung tissues were detected by real-time quantitative polymerase chain reaction(q RT-PCR).The m RNA levels of TF,IL-1β,syndecan-1 and HPA in HPMECs were detected by q RT-PCR..6.Immunofluorescence: syndecan-1 and HPA were labeled with immunofluorescence.The fluorescence intensity was analyzed by Image J software.7.Western Blot(WB)were used to evaluate the expressions of syndecan-1 and HPA proteins in all groups.8.Statistical analysis: SPSS 26.0 software was used for statistical analysis and Graph-Pad Prism 8.0 software was used for plotting.All values were expressed as mean± standard deviation(mean ± SD).Multiple group comparisons were performed by one-way analysis of variance(ANOVA)and Tukey’s post hoc test.Least significant heparinase,differences(LSD-T test)was used for comparison between groups.Brown-Forsythe and Welch ANOVE tests were used when the variances were unequal.P<0.05 was considered statistically signifcant diference.Results: CLP leads to lung injury and pulmonary edema,increased exprssions of HPA,decreased m RNA and protein expressions of syndecan-1 in lungs,increased expressions of TF,Fib and IL-1β m RNA,suggesting activation of coagulation and inflammation.HPA inhibitors reduced the severity of lung injury and pulmonary edema induced by CLP,inhibited the degradation of syndecan-1 in lung,and reduced the expression of TF and Fib in lung tissues,suggesting that HPA promoted the degradation of theglycocalyx and leaded to coagulation activation.UFH reduced the lung injury and pulmonary edema caused by CLP,inhibited the expressions of HPA,increased the expressions of syndecan-1,reduced the m RNA expressions of TF,Fib and IL-1β in lung tissues.These results suggested that UFH might protect CLP-induced lung glycocalyx injury through HPA pathway.In cellexperimens,UFH inhibited LPS-induced expression of HPA,increased the m RNA and protein expression of syndecan-1,decreased the expressions of TF and IL-1β,suggesting that UFH proteced the glycocalyx by inhibiting HPA expression,thereby inhibiting the activation of coagulation and inflammation.These results suggested that UFH might protect against LPS-induced gylcocalyx injury through HPA pathway.Conclusion: HPA promoted the degradation of endothelial glycocalyx in sepsis,leading to the activation of coagulation and inflammation.UFH might inhibit the degradation of glycocalyx by inhibiting the expression of HPA.