The Protective Effect And Mechanism Of Unfractionated Heparin On Rats Acute Lung Injury And Tight Junctions In Polymicrobial Sepsis

Sepsis is systemic inflammatory reaction caused by infection,which can lead to organ dysfunction.The pathophysiological mechanisms of acute lung injury（ALI）or acute respiratory distress syndrome（ARDS）include the destruction of alveolar epithelial barrier function and the pulmonary capillary endothelial cell injury,which leads to non-cardiogenic pulmonary edema.Sepsis-induced acute lung injury shows elevated plasma and lung tissue cytokines,fibrin deposition in the blood vessels and alveoli,and pulmonary vascular leakage.Tight junction proteins are important components for enhanced paracellular permeability.In vitro experiments showed that the expression of tight junction proteins including claudins,occludin and ZO-1 might be regulated by cytokines,growth factors or thrombins.Tight junction proteins downregulation or translocation can lead to tissue barrier dysfunction.Heparins can not only play a role in anticoagulation,but also interact with heparin-binding proteins,such as inflammatory factors,chemokines,and complements.As a result,heparins may participate in the immune response as their anti-inflammatory effects.Heparins in patients of sepsis,septic shock,sepsis and DIC can reduce their mortality.Treatment of endotoxemia mice or rats with unfractionated heparin or low molecular weight heparin can attenuate inflammation and organ injury,and improve survival rate.Up to this date,studies about tight junctions mainly focus on the brain tumors and meningitis,gastrointestinal cancers and inflammatory bowel diseases,inflammatory lung diseases,diabetic retinopathy.The relationship between sepsis induced-acute lung injury and tight junction proteins,including claudin-5,occludin and ZO-1,is rarely investigated.Futhermore,we have no idea that whether unfractionated heparin’s therapeutic effects on acute lung injury triggered by sepsis are associated with tight junctions.In this study,a rat model of sepsis-induced acute lung injury was established by cecal ligation and puncture,and the protective effects of unfractionated heparin on ALI and the expressions of claudin-5,occludin and ZO-1 were observed.We use human lung microvascular endothelial cells in vitro（HLMVECs）as a model of monolayer endothelial barrier and hypothesized that unfractionated heparin might protect HLMVECs tight junctions and attenuate microvascular leakage induced by lipopolysaccharide via ERK1/2 MAPK signaling pathway.Materials and methods Animals45 male Wistar rats were randomly divided into 3 groups:control group（n=15）,sepsis group（n=15）,heparin treatment sepsis group（n=15）.Control group were treated with sham operation,sepsis group and heparin treatment sepsis group were induced sepsis following the method of cecal ligation and puncture.In the heparin treatment sepsis group,rats were injected subcutaneously with unfractionated heparin 1000U/kg 30minutes before the operation and 12 hours after the operation respectively,while control group and sepsis group were injected subcutaneously with the same volume of saline at the same time.33 male Wistar rats were also used to evaluate the mortality of CLP model within 96 hr of the CLP procedure.Sample collectionThe rats were anesthetized,and then given unilateral bronchoalveolar lavage.The bronchoalveloar lavage fluid was collected,centrifuged and divided the supernatants,kept as the aliquot at-20℃ available for enzyme-linked immunosorbent assay.Take the left lung for the measurement of the lung wet to dry weight ratio.A small portion of the right lower lobe of the lung tissue was taken and fixed with 2.5%glutaraldehyde.The remaining right lower lobe of lung was fixed with 4%paraformaldehyde and embedded in paraffin for section,hematoxylin and eosin staining and immunohistochemistry.The middle lobe of right lung was taken and kept at-80℃ for Western blot detection.For the evaluation of lung microvascular permeability,rats were injected 1%Evans blue intravenously and sacrificed after 1 hour,then the left lung vessels were perfused with saline,the left lung was cut and sonicated finally.Cell cultureHuman lung microvascular endothelial cells（HLMVECs）purchased from Shanghai bioleaf biotech Co.were cultured in DMEM high glucose media supplemented with 10%fetal bovine serum 1%and penicillin/streptomycin.Cells were grown at 37℃,in a humidified 5%CO₂ incubator.Cell media were changed every 2 days.After 3-4 days culture,80-90%cell fusion achieved,and cells in an exponential phase which were ready for the experiment performance.Cells were grouped and treated with PBS,LPS,unfractionated heparin,and inhibitor of ERK（U0126）respectively.Experimental procedures1 Rats tissue detection（1）HE staining for histologic examination of lung injury.（2）ELISA for measurement of IL-6 content in bronchoalveolar lavage fluid.（3）Wet to dry weight ratio was calculated to assess pulmonary edema.（4）Evans blue dyes content in lung tissue was detected for assessment of lung microvascular endothelial permeability.（5）Observed ultrastructure of lung blood gas barrier by transmission electron microscope.（6）Western blot analysis of the expression of tight junction protein claudin-5,occludin,ZO-1 in lung tissue.（7）Immunohistochemical analysis of the expression and localization of tight junction protein claudin-5,occludin,ZO-1 in lung tissue.2 Cellular detection（1）Transendothelial electrical resistance（TEER）measurement of HLMVECs（2）FITC-dextran permeability assay of HLMVECs（3）Ultrastructure of HLMVECs tight junctions observed by transmission electron microscopy.（4）ELISA for measurement of IL-6 content in HLMVEC supernatants.（5）Western blot analysis of claudin-5,occludin,ZO-1,p-ERK1/2 and ERK1/2expressions in HLMVECs.Statistical analysisData were analyzed with SPSS18.0 software.Data from counting materials were expressed as a mean value±standard deviation.Significance of differences among groups was tested by one-way analysis of variance（ANOVA）,followed by comparing least significant difference between two groups.P values<0.05 were considered significant.ResultsPart 1 Effects of unfractionated heparin on acute lung injury and tight junctions in septic rats1 Observation of animal modelIn the control group,all rats can eat and move as usual,without abnormal magnifestations.However,septic rats showed generalized weakness,malaise,chills,piloerection,reduced appetite and food intake,diarrhea and decreased motor activity,besides,they breathed more deeply and frequently.Septic rats treated with heparin showed a little malaise and weakness,ate and moved less than control group but better than septic rats without heparin treatment.All control rats remained alive,whereas the sepsis group（CLP-only）exhibited a lower survival rate compared with the sepsis+UFH group at 96 h.2 Lung histopathologyIn the control group,the alveoli were opened uniformly,alveolar wall and septa were thin and clear.In the sepsis group,the alveolar collapsed,the alveolar wall and septa widened significantly,with congestion,swellen and significant inflammatory cells infiltration in septa.In the heparin treatment group,the alveolar septa swelling and inflammatory cells infiltration were attenuated compared with sepsis group.3 IL-6 in bronchoalveolar lavage fluidIn sepsis group,IL-6 level increased significantly compared with the control group（27.76±4.10 vs.2.25±0.24,p<0.0001）.In heparin treatment sepsis group,IL-6 level was lower than that of sepsis group（12.50±1.65 vs.27.76±4.10,p<0.0001）,but still higher than the control group（12.50±1.65 vs.2.25±0.24,p<0.0001）.4 Wet to dry weight ratioIn sepsis group,lung wet weight increased significantly than the control group（8.906±1.598 vs.4.319±0.100,p<0.0001）.In heparin treatment sepsis group,wet weight was lower than that of sepsis group（5.389±0.444 vs.8.906±1.598,p<0.0001）,but still higher than the control group（5.389±0.444 vs.4.319±0.100,p<0.0001）.5 Lung vascular leakage of Evans blue dyeEvans blue content sepsis group in lung tissue was significantly higher than the control group（44.81±5.10 vs.11.29±2.82,p<0.0001）.In heparin treatment sepsis group,EBD was lower than that of sepsis group（24.94±5.90 vs.44.81±5.10,p<0.0001）,but still higher than the control group（24.94±5.90 vs.11.29±2.82,p<0.0001）.6 Ultrastructure of lung blood gas barrierIn the control group,the alveolar epithelial and microvascular endothelial cells had no swelling or dissolution;furthermore,there were no large gap between cells.In the sepsis group,the alveolar epithelial membranes were dissolved,cell boundary was not clear,as well as basal membrane disrupted and endothelial cells swellen.The alveolar epithelial injury and vascular endothelial cell swelling were attenuated in the heparin treatment sepsis group.7 Western blot analysis of tight junction protein claudin-5,occludin,ZO-1 expression in lung tissueThe expression of claudin-5,occludin and ZO-1 in septic rats lung,were significantly downregulated compared with the control group.However,heparin treatment caused the upregualtion of claudin-5,occludin and ZO-1（p<0.05）.8 Immunohistochemical analysis of the expression and localization of tight junction protein claudin-5,occludin,ZO-1 in lung tissueIn the control group,claudin-5 was mainly expressed in alveolar capillary endothelial cell membrane and cytoplasm edge,while occludin and ZO-1 expression exsisted in both alveolar epithelium and alveolar capillary endothelium.Occludin located in the cell membrane and cytoplasm edge,ZO-1 located in the cytoplasm near the cell membrane,all of the three junction proteins located as a wavy,linear distribution.The expressions of claudin-5,occludin and ZO-1 in lung tissue of septic rats were decreased,even disappeared or losed normal continuous localization in alveoli or capillaries,only a few scattered spots or patchy brown staining left.Treated with heparin improved the expression and localization of claudin-5,occludin and ZO-1,partly restored continuous and wavy,linear distribution.Part 2 Effects of unfractionated heparin and U0126 on HLMVECs permeability,tight junction and ERK1/2 MAPK signaling pathway1 LPS-induced transendothelial cell resistance decrease in HLMVECsLPS stimulation could lead to the decrease of HLMVECs TEER compared with baseline,which was dependent on the time of LPS exposure.LPS5μg/ml exposure for 24hours,HLMVECs TEER decreased a little than that without LPS exposure,however,the difference between the two group was not significant（p>0.05）.LPS incubation with 10,20,50,100μg/ml for 24hours,HLMVECs TEER decreased significantly compared with control group（p<0.05）.2 LPS induces downregulation of HLMVECs tight junction proteinLPS5μg/ml induced significantly decrease in the expression of occludin and ZO-1（p<0.05）,but not in claudin-5（p>0.05）.LPS10μg/ml,LPS 20μg/ml,LPS 50μg/ml caused significant downregulation of claudin-5,occludin,ZO-1 expression（p<0.05）.3 Unfractionated heparin,U0126 attenuates LPS-induced FITC-dextran leakage in HLMVECsLPS10μg/ml increased FITC-dextran leakage in HLMVECs（LPS10μg/ml vs.control,21.08±1.27 vs.10.89±1.87 cm/s,p<0.0001）.Pre-treatment with unfractionated heparin 10U/ml inhibited LPS-induced FITC-dextran leakage markbly（LPS10μg/ml+UFH10U/ml vs.LPS10μg/ml,14.22±2.24 vs.21.08±1.27 cm/s,p<0.005）.Similarly,pre-treatment with U0126 25μM inhibited LPS-induced FITC-dextran leakage effectively（LPS10μg/ml±U0126 25μM vs.LPS10μg/ml,12.84±0.57 vs.21.08±1.27 cm/s,p<0.005）.4 Unfractionated heparin attenuates LPS-induced ultrastructure disruption of HLMVECs tight junctionIn the control group,tight junction was a compact linear structure between the endothelial cells.LPS10μg/ml induced gap formation between adjecent cells and a large number of vacuoles presented at cell membrane,opened the tight junction.Pre-treatment with unfractionated heparin 10U/ml attenuated the disruption of endothelial tight junction structure.5 Unfractionated heparin inhibits LPS-induced IL-6 secretion in HLMVECs supernatantsLPS10μg/ml stimulation resulted in a significant increase in secreted IL-6 relative to that in control cells（p<0.0001）,whereas pre-treatment with unfractionated heparin10U/ml decreased the level of secreted IL-6（p<0.0001）.6 Unfractionated heparin prevents LPS-induced downregulation of tight junction proteins in HLMVECsLPS10μg/ml induced obvious downregulation of claudin-5,occludin,ZO-1 in HLMVECs（p<0.005）.Unfractionated heparin pre-treatment effectively upregulated claudin-5,occludin,ZO-1 protein level（p<0.05）.7 Unfractionated heparin inhibits LPS induced phosphorylation of ERK 1/2 MAPK in HLMVECsLPS10μg/ml stimulated the expression of p-ERK1/2 MAPK significantly（p<0.0001）,while pre-treatment with unfractionated heparin 10U/ml inhibited LPS-induced p-ERK1/2 MAPK expression（p<0.05）.8 U0126 prevents LPS-induced downregulation of tight junction proteins in HLMVECsThe expressions of claudin-5,occludin and ZO-1 in LPS10μg/ml group were significantly lower than that in control group（p<0.05）.The expressions of claudin-5,occludin and ZO-1 in LPS10μg/ml with U0126 25μM pre-treatment were higher than those in LPS group（p<0.05）.Conclusion1 Unfractionated heparin can attenuate acute lung injury of septic rats through inhibiting inflammation and upregulating claudin-5,occludin and ZO-1 protein expression.2 Unfractionated heparin prevents LPS-induced ultrastructural disruption of tight junctions and leakage of HLMVECs.3 Unfractionated heparin might protect HLMVECs permeability by regulating the expression of claudin-5,occludin and ZO-1 via ERK1/2 MAPK signaling pathway...