Syndecan-1 May Affect Endothelial Cell Tight Junction Protein ZO-1 And Occludin In Sepsis By Regulating The Level Of Phospho-Smad2/3

Background and aims:Sepsis is a life-threatening organ dysfunction caused by the host’s dysfunctional response to infection,and is associated with significant morbidity and mortality.Because endothelial cells are both the target and source of inflammation,they play an important role in the local and systemic immune response.Highly selective endothelial barrier is essential for maintaining tissue fluid homeostasis and supporting normal organ function.The transport of vascular endothelium can occur not only through endothelial cells（cross cells）,but also between adjacent cells through the connection between endothelial cells.Tight junctions,such as Occludin and ZO-1,play an important role in maintaining endothelial barrier function.At present,a large number of studies have reported that when sepsis occurs,the endothelial tight junctions protein is destroyed,and the destruction of endothelial tight junctions may be an important mechanism leading to systemic injury in sepsis.Endothelial glycocalyx is a dense reticular structure,which is synthesized and secreted by endothelial cells,evenly distributed on the surface of vascular endothelial cells,and participates in a variety of physiological functions of vascular endothelium.At present,it has also been considered as a key regulator of the integrity of endothelial barrier.Proteoglycan constitutes the main scaffold of glycocalyx,and syndecan-1（SDC-1）,as an important component of glycocalyx,plays a vital role in maintaining the integrity of glycocalyx structure.Studies have shown that the degree of SDC-1 shedding from the glycocalyx may be significantly associated to the severity of sepsis,and the repair of the glycocalyx component SDC-1 may be a potential therapeutic target for sepsis.A large amount of evidence shows that the assembly,maintenance and disassembly of tight junctions can be regulated through signal pathways,thus affecting the barrier function of endothelium and epithelium.Transforming growth factor（TGF）-β/Smad signaling pathway has been proved to be closely related to tight junctions protein and cell permeability for many times.SDC-1 is considered to be a kind of heparin substance because of its unique heparin-sulfate chain,while TGF-βis a protein with heparin affinity.It has been confirmed in literature that SDC-1 can combine with TGF-β.Therefore,we speculate that SDC-1 may affect the expression of tight junctions of endothelial cells in sepsis by regulating the phosphorylation of Smad2/3,thus affecting the permeability of vascular endothelium.At present,the research based on sepsis tells us that when sepsis occurs,the endothelial glycocalyx structure is destroyed,resulting in the decrease of SDC-1expression in endothelial cells,and the significant destruction of tight junctions and the increase of endothelial permeability.These two manifestations often occur at the same time.Whether there is a certain relationship between them has not been confirmed.Whether SDC-1 can regulate Smad2/3 phosphorylation in endothelial cells and sepsis models and how to regulate Smad2/3 phosphorylation have not been confirmed.Based on these questions,we performed this study.Methods:1.Animal feeding and model establishmentUse male C57BL/6 mice of 6-8 weeks,weight 20-30g,specific pathogen free（SPF）as experimental animals.All mice were raised in an independent ventilated cage system before being used in the experiment.They were raised for 1 week according to the law of light and dark cycle every 12 hours to adapt to the environment.SDC-1^+/-genotype C57BL/6 male and female mice were bred together to produce offspring.Genotype identification of the toes of scissor mice after 1-2 weeks of breastfeeding.SDC-1^-/-genotype mice were used for the experiment.The mice were fasted for 12 hours before operation,and could be fed with water.Before anesthesia,the mice were weighed,and 0.3%pentobarbital sodium solution0.2m L/10g was used for anesthesia by intraperitoneal injection.After satisfactory anesthesia,prepare skin,disinfect,open the abdomen,locate the cecum and lift it out of the body.The sham-operation（Sham）group directly returns the cecum after exposing it.The cecal ligation and puncture（CLP）group ligates the cecum with silk thread at the middle point from the lower part of the ileocecal valve to the distal part of the cecum,gently push the contents of the cecum to the distal part of the cecum,at the middle of the ligation point and the distal part of the cecum,use 18G needle to penetrate once from the mesentery to the reverse mesentery.After pulling out the needle,squeeze out a small amount of feces to ensure that the puncture point is unobstructed.Return the caecum and close the abdomen layer by layer.After the operation,the mice were resuscitated by subcutaneous injection of normal saline and intraperitoneal injection of ceftriaxone for anti-infection.2.Cell cultureHuman umbilical vein endothelial cells（HUVEC）were used as experimental cells,and DMEM complete medium（DMEM basic medium plus 1%penicillin mixture,10%fetal bovine serum,1%endothelial cell growth additive）was used for culture,and cultured in a cell incubator at 37℃and 5%CO2.Change the medium every 2 days.Cell passage can be carried out when the growth reaches 80-90%density.Use of lipopolysaccharide（LPS）10μg/m L as irritant.HUVEC with good growth status was selected to be infected with CRISPR/CAS9single vector lentivirus.When the cells were digested and passaged to 30%density,a suitable amount of purinomycin resistant virus solution was added.After screening,the fluorescence expression was observed by fluorescence microscope and western blot was used to identify the infection efficiency.SDC-1 knockout（KO）and SDC-1over-expression（OE）stable cell lines were prepared.3.Experimental groupingThe animals were randomly divided into WT Sham group,WT CLP group,SDC-1^-/-Sham group and SDC-1^-/-CLP group;There are 20 mice in each group for survival rate experiment,and 6 mice in each group for other experiment.Cell experiment was divided into vector NC group,vector LPS group,SDC-1 KO NC group,SDC-1 KO LPS group,SDC-1 OE NC group and SDC-1 OE LPS group.4.Experimental method（1）Draw the survival curve of mice:observe the animal status every 6 hours after the establishment of the animal model,record the number of deaths every 24 hours until96 hours,and draw the survival curve.（2）The wet to dry weight ratio of lung tissue was measured to evaluate the pulmonary edema in each group.（3）The content of Evans blue in lung tissue was measured to evaluate the pulmonary microvascular leakage.（4）Western blot was used to detect the protein expression level of SDC-1 protein,tight junctions protein ZO-1,Occludin and signal pathway Smad2/3,phospho-Smad2/3in lung tissue or HUVEC cells.（5）The fluorescence intensity of tight junctions protein ZO-1 and Occludin in lung tissue or HUVEC cells was detected by immunofluorescence.（6）HUVEC performed FITC-dextran permeability test to evaluate the permeability of cell monolayer.5.Statistical analysisUse Image J 1.53 software to count the gray value of each western blot band and the fluorescence intensity of the immunofluorescence image.Use Graph Pad Prism 9.5.0software to statistically analyze the data.After verifying that the data conforms to the normal distribution,use t test for comparison between the two groups,and use analysis of variance（ANOVA）for comparison between multiple groups.P<0.05 indicates that there is statistical difference.Results:1.The simultaneous down-regulation of SDC-1 and tight junctions proteins in sepsis resulted in increased vascular endothelial permeability.At the animal level,western blot showed that the content of SDC-1 in the lung tissue of septic mice began to decrease at 6 hours after CLP（compared with Sham group,P<0.01）,and the decrease was the most obvious at 24 hours（compared with Sham group,P<0.001）.ZO-1 protein and Occludin protein also decreased significantly 24 hours after CLP（compared with Sham group,P<0.0001 and P<0.01,respectively）.The contents of SDC-1,ZO-1 and Occludin in the lung tissue of mice were down-regulated at 24 hours after CLP.In the follow-up animal experiment,we used CLP stimulation for 24 hours as the stimulation condition of sepsis mice.The lung wet-dry ratio of mice in 24 hours after CLP was significantly higher than that in Sham group（6.666±0.556 vs.4.719±0.189,P<0.0001）,and the Evans blue content per gram of lung tissue was also significantly higher than that in Sham group（143.983±17.162μg vs.96.446±4.017μg,P<0.001）。At the cell level,the western blot results showed that the content of SDC-1 protein in HUVEC decreased significantly after 12 hours of LPS stimulation（compared with NC group,P<0.001）,and the decrease was more obvious at 24 hours（compared with NC group,P<0.0001）.The content of ZO-1 and Occludin protein in HUVEC also decreased significantly at 24 hours after LPS stimulation（compared with NC group,P<0.0001,respectively）.The contents of SDC-1,ZO-1 and Occludin in HUVEC were down-regulated synchronously after 24 hours of LPS stimulation.We used LPS 10μg/m L in the subsequent cell experiment stimulation for 24 hours was used as the stimulation condition for the sepsis injury of HUVEC cell monolayer.At the same time,we observed that after 24 hours of LPS stimulation,the leakage of FITC-dextran by HUVEC was significantly higher than that of NC group.The concentration of FITC-dextran in the lower chamber of Transwell in the two groups was 0.518±0.034mg/m L and 0.29±0.015 mg/m L（P<0.001）.2.Effect of SDC-1 deficiency on tight junction protein expression and vascular endothelial permeability in mice lung tissue.（1）The mortality of SDC-1^-/-septic mice is higher than that of WT septic mice.At 96 hours after operation of Sham and CLP,all mice in WT Sham group and SDC-1^-/-Sham group survived,and the 96-hour mortality of mice in SDC-1^-/-CLP group was significantly higher than that in WT CLP group[17（85%）vs.11（55%）],with significant statistical difference（P<0.05）.In addition,we observed early death in SDC^-/-CLP group mice.At 24 hours after the induction of sepsis model,more than half of SDC-1^-/-CLP group mice died（n=11,55%）,and the number of deaths was significantly higher than WT CLP group mice（n=6,30%）.（2）The expression of tight junctions protein ZO-1 and Occludin and vascular endothelial permeability in lung tissue of SDC-1^-/-mice was down-regulated.Western blot results showed that the expression of ZO-1 and Occludin in lung tissue of mice in SDC-1^-/-Sham group was significantly lower than that in WT Sham group（P<0.01 and<0.0001,respectively）.The immunofluorescence results showed that the fluorescence intensity of ZO-1 and Occludin in lung tissue of mice in SDC-1^-/-Sham group was significantly lower than that in WT Sham group（P<0.0001,respectively）.Compared with the WT Sham group,the SDC-1^-/-Sham group showed an increase in lung wet-dry ratio（5.587±0.124 vs.4.719±0.189,P<0.001）,and an increase in Evans blue content per gram of lung tissue（123.307±11.095μg vs.96.449±4.017μg,P<0.001）.（3）SDC-1 deficiency further aggravated the damage of tight junctions and vascular endothelial permeability in CLP mice.Western blot results showed that the expression of ZO-1 and Occludin in lung tissue of SDC-1^-/-CLP mice was further reduced compared with WT CLP group（P<0.01 and<0.0001,respectively）.The immunofluorescence results showed that the fluorescence intensity of ZO-1 and Occludin in the lung tissue of mice in SDC-1^-/-CLP group was also further reduced compared with WT CLP group（P<0.01 and<0.0001,respectively）.The lung wet-dry ratio of mice in the SDC-1^-/-CLP group was further increased than that in the WT CLP group（7.932±0.244 vs.6.666±0.556,P<0.0001）,and the Evans blue content per gram of lung tissue in the SDC-1^-/-CLP group was also further increased than that in the WT CLP group（184.495±4.268μg vs.143.983±17.162μg,P<0.0001）.3.The effect of overexpression and knockout of SDC-1 on tight junction protein expression and vascular endothelial permeability in HUVEC.（1）Overexpression and knockout of SDC-1 can regulate the expression of HUVEC monolayer tight junctions protein.Western blot results showed that the expression of ZO-1 and Occludin in HUVEC of SDC-1 KO NC group was significantly lower than that of vector NC group after knocking out SDC-1 without LPS stimulation（P<0.0001,respectively）.The overexpression of SDC-1 showed that the expression of ZO-1 and Occludin in HUVEC of SDC-1 OE NC group was significantly higher than that of vector NC group（P<0.0001and<0.01,respectively）.The immunofluorescence results showed that the fluorescence intensity of ZO-1 and Occludin in HUVEC of SDC-1 KO NC group was significantly lower than that of vector NC group（P<0.01 and<0.001,respectively）after knocking out SDC-1 without LPS stimulation.The overexpression of SDC-1 showed that the fluorescence intensity of ZO-1 and Occludin in HUVEC of SDC-1 OE NC group was significantly higher than that of vector NC group（P<0.001 and<0.0001,respectively）.（2）SDC-1 deficiency further aggravated LPS-induced HUVEC monolayer tight junctions damage and vascular endothelial permeability damage.Western blot results showed that the expression of ZO-1 and Occludin in HUVEC of SDC-1 KO LPS group was further decreased than that of vector LPS group（P<0.0001,respectively）.The results of immunofluorescence showed that the fluorescence intensity of ZO-1 and Occludin in HUVEC of SDC-1 KO LPS group was further lower than that of vector LPS group（P<0.05 and<0.0001,respectively）.At the same time,we observed the leakage of FITC-dextran from the monolayer of HUVEC knocked out by SDC-1when stimulated by LPS.The results showed that the leakage of FITC-dextran in SDC-1KO LPS group was the most serious in each group,with a significant statistical difference compared with the vector LPS group（0.721±0.04 mg/m L vs.0.485±0.015mg/m L,P<0.0001）.（3）Overexpression of SDC-1 can improve HUVEC monolayer tight junctions protein and vascular endothelial permeability damage induced by LPS.Western blot results showed that the expression of ZO-1 and Occludin in HUVEC of SDC-1 OE LPS group was significantly higher than that of vector LPS group（P<0.0001and<0.001,respectively）.The immunofluorescence results showed that the fluorescence intensity of ZO-1 and Occludin in HUVEC of SDC-1 OE LPS group was significantly higher than that of vector LPS group（P<0.05 and<0.01,respectively）.At the same time,we observed the leakage of FITC-dextran from the monolayer of HUVEC overexpressed by SDC-1 when stimulated by LPS.The results showed that the leakage of FITC-dextran in the SDC-1 OE LPS group was significantly improved compared with that in the vector LPS group（0.404±0.037 mg/m L vs.0.485±0.015 mg/m L,P<0.01）.4.Both SDC-1 deficiency and sepsis stimulation resulted in an increase in Smad2/3phosphorylation level.（1）SDC-1 deficiency can induce the increase of Smad2/3 phosphorylation.At the animal level,western blot results showed that there was no statistical difference in the expression of Smad2 and Smad3 protein in the lung tissue of mice in WT Sham group,WT CLP group,SDC-1^-/-Sham group and SDC-1^-/-CLP group.After knocking out SDC-1 without CLP stimulation,it was found that the phosphorylation levels of Smad2 and Smad3 in lung tissue of mice in SDC-1^-/-Sham group were significantly higher than those in WT Sham group（P<0.01,respectively）.At the cellular level,the results of western blot showed that there was no statistical difference in the expression of Smad2 and Smad3 protein in HUVEC in the vector NC group,the vector LPS group,the SDC-1 KO NC group,and the SDC-1 KO LPS group.After SDC-1 knockout without LPS stimulation,it was observed that the phosphorylation levels of Smad2 and Smad3 in HUVEC of SDC-1 KO NC group were also significantly higher than those of vector NC group（P<0.0001 and<0.001,respectively）.（2）Increased Smad2/3 phosphorylation induced by sepsis stimulation.At the animal level,western blot showed that the phosphorylation levels of Smad2and Smad3 in the lung tissue of WT CLP group were significantly higher than those of WT Sham group（P<0.01,respectively）.In the lung tissue of mice in SDC-1^-/-CLP group,the phosphorylation level of Smad2 and Smad3 reached the highest level,which was significantly different from that in SDC-1^-/-Sham group（P<0.05,respectively）and WT CLP group（P<0.05,respectively）.At the cellular level,the results of western blot showed that the phosphorylation levels of Smad2 and Smad3 in HUVEC in the vector LPS group were significantly higher than those in the vector NC group（P<0.0001 and<0.01,respectively）,while the phosphorylation levels of Smad2 and Smad3 in HUVEC in the SDC-1 KO LPS group were the highest in each group,with a significant statistical difference compared with the SDC-1 KO NC group（P<0.0001,respectively）,and there was also a significant statistical difference compared with the vector LPS group（P<0.0001,respectively）.5.SDC-1 overexpression can significantly inhibit Smad2/3 phosphorylation.Western blot results showed that there was no statistical difference in the expression of Smad2 and Smad3 protein in HUVEC in the vector NC group,the vector LPS group,the SDC-1 OE NC group,and the SDC-1 OE LPS group.After overexpression of SDC-1without LPS stimulation,it was observed that the phosphorylation level of Smad2 and Smad3 in HUVEC of SDC-1 OE NC group was significantly lower than that of vector NC group（P<0.05 and<0.0001,respectively）.The overexpression of SDC-1significantly inhibited the increase of Smad2/3 phosphorylation of HUVEC caused by LPS.The phosphorylation level of Smad2 and Smad3 in HUVEC of SDC-1 OE LPS group was significantly lower than that of vector LPS group（P<0.001 and<0.0001,respectively）.Conclusion:1.When sepsis occurs,SDC-1 and tight junctions proteins of endothelial cells are down-regulated synchronously,resulting in increased vascular endothelial permeability.2.SDC-1 can regulate the expression of tight junctions protein ZO-1 and Occludin.3.SDC-1 deficiency will further aggravate the sepsis injury,which is manifested by a significant increase in mortality,a significant down-regulation of tight junctions protein,and a significant increase in endothelial cell permeability.Overexpression of SDC-1 can reverse the above injury,suggesting that SDC-1 may become a new therapeutic target for sepsis.4.SDC-1 can regulate the phosphorylation level of Smad2/3.Down-regulation of SDC-1leads to an increase in the phosphorylation level of Smad2/3,while up-regulation of SDC-1 can inhibit the phosphorylation of Smad2/3.5.SDC-1 may affect the expression of tight junctions protein in sepsis by regulating the phosphorylation of Smad2/3,thus affecting the permeability of endothelial cells.