S1PR1 serves as a viable drug target against pulmonary fibrosis by increasing the integrity of the endothelial barrier of the lung

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with unclear etiology and limited treatment options. The median survival time for IPF patients is approximately 2-3 years and there is no effective intervention to treat IPF other than lung transplantation. As important components of lung tissue, endothelial cells (ECs) are associated with pulmonary diseases. However, the role of endothelial dysfunction in pulmonary fibrosis (PF) is incompletely understood. Sphingosine-1-phosphate receptor 1 (S1PR1) is a G protein-coupled receptor highly expressed in lung ECs. Its expression is markedly reduced in patients with IPF. Herein, we generated an endothelial-conditional S1pr1 knockout mouse model which exhibited inflammation and fibrosis with or without bleomycin (BLM) challenge. Selective activation of S1PR1 with an S1PR1 agonist, IMMH002, exerted a potent therapeutic effect in mice with bleomycin-induced fibrosis by protecting the integrity of the endothelial barrier. These results suggest that S1PR1 might be a promising drug target for IPF therapy.

Porphyromonas gingivalis infection causes umbilical vein endothelial barrier dysfunction in vitro by down-regulating ZO-1, occludin and VE-cadherin expression / 南方医科大学学报

OBJECTIVE@#To explore the molecular mechanisms of Porphyromonas gingivalis infection-induced umbilical vein endothelial barrier dysfunction in vitro.@*METHODS@#Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and after the formation of the endothelial barrier, the cells were infected with P. gingivals at a multiplicity of infection (MOI). The transepithelial electrical resistance (TEER) of the cell barrier was measured, and FITC-dextran trans-endothelial permeability assay and bacterial translocation assay were performed to assess the endothelial barrier function. The expression levels of cell junction proteins including ZO-1, occludin and VE-cadherin in the cells were examined by qRT-PCR and Western blotting.@*RESULTS@#In freshly seeded HUVECs, TEER increased until reaching the maximum on Day 5 (94 Ωcm2), suggesting the formation of the endothelial barrier. P. gingivals infection caused an increase of the permeability of the endothelial barrier as early as 0.5 h after bacterial inoculation, and the barrier function further exacerbated with time, as shown by significantly lowered TEER, increased permeability of FITC-dextran (40 000/70 000), and increased translocation of SYTO9-E. coli cross the barrier. MTT assay suggested that P. gingivals infection did not significantly affect the proliferation of HUVECs (P>0.05), but in P. gingivalsinfected cells, the expressions of ZO-1, occludin and VE-cadherin increased significantly at 24 and 48 h after bacterial inoculation (P < 0.05).@*CONCLUSION@#P. gingivals may disrupt the endothelial barrier function by down-regulating the expressions of the cell junction proteins (ZO-1, occludin, VE-cadherin) and increasing the permeability of the endothelial barrier.

Ruscogenin alleviates LPS-triggered pulmonary endothelial barrier dysfunction through targeting NMMHC IIA to modulate TLR4 signaling

Pulmonary endothelial barrier dysfunction is a hallmark of clinical pulmonary edema and contributes to the development of acute lung injury (ALI). Here we reported that ruscogenin (RUS), an effective steroidal sapogenin of Radix Ophiopogon japonicus, attenuated lipopolysaccharides (LPS)-induced pulmonary endothelial barrier disruption through mediating non-muscle myosin heavy chain IIA (NMMHC IIA)‒Toll-like receptor 4 (TLR4) interactions. By in vivo and in vitro experiments, we observed that RUS administration significantly ameliorated LPS-triggered pulmonary endothelial barrier dysfunction and ALI. Moreover, we identified that RUS directly targeted NMMHC IIA on its N-terminal and head domain by serial affinity chromatography, molecular docking, biolayer interferometry, and microscale thermophoresis analyses. Downregulation of endothelial NMMHC IIA expression in vivo and in vitro abolished the protective effect of RUS. It was also observed that NMMHC IIA was dissociated from TLR4 and then activating TLR4 downstream Src/vascular endothelial cadherin (VE-cadherin) signaling in pulmonary vascular endothelial cells after LPS treatment, which could be restored by RUS. Collectively, these findings provide pharmacological evidence showing that RUS attenuates LPS-induced pulmonary endothelial barrier dysfunction by inhibiting TLR4/Src/VE-cadherin pathway through targeting NMMHC IIA and mediating NMMHC IIA‒TLR4 interactions.

Protective effect of protein kinase C inhibitor on rat renal vascular endothelial injury induced by lipopolysaccharide / 中华危重病急救医学

Objective To investigate the protective effect of protein kinase C (PKC) inhibitor rottlerin on rat renal vascular endothelial injury induced by lipopolysaccharide (LPS). Methods Rat renal microvascular endothelial cells cultured for 3-6 generations were divided into three groups according to random number table: blank control group in which cells were not challenged, LPS group in which cells were only stimulated by LPS 10 mg/L for 24 hours, and PKC inhibitor group in which cells were treated with PKC inhibitor rottlerin 2 μmol/L 30 minutes before LPS stimulation. The levels of tumor necrosis factor-α (TNF-α) and interleukins (IL-1β, IL-8) were determined by enzyme-linked immunosorbent assay (ELISA). Monolayer permeability was determined by Transwell assay. The expressions of PKC, RhoA and vascular endothelial-cadherin (VE-cadherin) were detected by Western Blot. The morphological characteristic and distribution of F-actin was measured by laser confocal fluorescence microscope. Results Compared with blank control group, the levels of inflammatory cytokines at 24 hours after 10 mg/L LPS stimulation were significantly increased in LPS group [TNF-α (ng/L): 397.3±25.4 vs. 46.8±8.9, IL-1β(ng/L): 76.7±11.2 vs. 12.6±3.2, IL-8 (ng/L): 574.5±31.4 vs. 73.2±9.6, all P < 0.05], the permeability of endothelial cells was significantly increased (A value: 1.32±0.03 vs. 0.36±0.02, P < 0.05), while the expressions of PKC and RhoA were significantly up-regulated (PKC/β-actin: 0.88±0.02 vs. 0.61±0.03, RhoA/β-actin: 0.96±0.01 vs. 0.49±0.03, both P < 0.05), VE-cadherin expression was significantly down-regulated (VE-cadherin/β-actin: 0.51±0.01 vs. 0.72±0.04, P < 0.05), and the F-actin distribution disorder had obvious stress fiber formation. Compared with LPS group, the levels of inflammatory cytokines were significantly lowered in PKC inhibitor group [TNF-α (ng/L): 127.4±14.6 vs. 397.3±25.4, IL-1β(ng/L): 43.2±7.8 vs. 76.7±11.2, IL-8 (ng/L): 212.7±18.2 vs. 574.5±31.4, all P < 0.05], the permeability of endothelial cells was significantly decreased (A value: 0.81±0.02 vs. 1.32±0.03, P < 0.05), the expressions of PKC and RhoA were significantly down-regulated (PKC/β-actin: 0.44±0.03 vs. 0.88±0.02, RhoA/β-actin: 0.63±0.05 vs. 0.96±0.01, both P < 0.05), the VE-cadherin expression was significantly up-regulated (VE-cadherin/β-actin: 0.69±0.03 vs. 0.51±0.01, P < 0.05), and the F-actin remodeling and stress fiber formation were significantly reduced. Conclusion PKC inhibitor could significantly attenuate the damage of vascular endothelial barrier induced by LPS, and plays an important role in endothelial cell barrier.

Sphingosine 1-phosphate signaling pathway:a novel target for regulation of epithelial and endothelial barrier function / 中国药科大学学报

@#Sphingosine 1-phosphate(S1P)is a pleiotropic sphingolipid metabolite that has been shown to regulate important physiological function by activation of its G-protein-coupled receptors S1PRs. S1P has been identified as an important signaling molecule in maintaining epithelial and endothelial barrier function. S1P signaling pathway is involved in epithelial and endothelial barrier function by regulation of adherens junction and tight junction assembly, cytoskeletal reorganization, and focal adhesion formation. Thus, S1P signaling pathway may become a novel therapeutic target for cell barrier dysfunction during some illnesses such as acute lung injury, inflammatory bowel disease and sepsis. In this review, the research progress of S1P signaling pathway in regulating epithelial and endothelial barrier function and the application of S1P in barrier dysfunction-related diseases were summarized, so as to provide references for future research.

A correlation study of blood-brain barrier permeability changes and EBA, VEGF expression in rats following radiation-induced brain injury / 天津医药

Objective To explore the dynamic changes of endothelial barrier antigen (EBA) and vascular endothelial growth factor (VEGF) expressions in cerebral cortex under the condition of blood-brain barrier damage in rats following radi?ation-induced brain injury, which provided clinical references. Methods Forty-eight clean grade male SD rats were divid?ed into the control group and 7 d, 14 d, 28 d after brain irradiation group (n=12 for each group) by using stochastic indicator method. The radiation-induced brain injury model was established by using electronic computer X-ray tomography tech?nique. The 3%Evans blue (EB) was injected into rats according to the dose of 3 mL/kg via the tail vein, then the blood ves?sels of cerebral cortex were exposed after having a craniotomy. EB extravasation was detected by microcirculation micro?scope. The permeability of blood-brain barrier was evaluated by using microscope vascular camera device. The expressions of EBA and VEGF in the cerebral cortex were measured by immunohistochemistry staining in each group. Results Both of EB extravasation and VEGF expression in rat cerebral cortex were significantly increased in injury group at day 7, 14 and 28 after brain irradiation compared with those of control group (P<0.05), and which were gradually decreased from day 7 to day 28 after brain irradiation. There were significant differences in EB extravasation and VEGF expression between the injury subgroups (P<0.05). There was a positive correlation between EB extravasation and VEGF expression (r=0.898, P<0.001). The expression levels of EBA were decreased at different time points in injury groups compared with those of control group (P<0.05), and gradually increased from day 7 to 28 after injury. There were significant differences in expression levels of EBA between injury subgroups (P<0.05). The expression of EBA was negatively correlated with EB extravasation (r=-0.866, P<0.001). Conclusion The increases of blood-brain barrier permeability have important relation to the decreases of EBA expression and the increases of VEGF expression after radiation-induced brain injury.

Inhibition of multidrug resistance-associated protein 4 protects against pulmonary vascular endothelial barrier dysfunction in septic rats / 中华急诊医学杂志

Objective To explore the effects of multidrug resistance-associated protein 4 (MRP4) inhibition on pulmonary vascular endothelial barrier dysfunction in septic rats.Methods Sixty Sprague Dawley rats were randomly (random number) divided into three groups:sham-operated group,sepsis group,and sepsis plus MRP4 inhibitor treatment group,with 20 rats in each group.Sepsis was induced by cecal ligation and puncture.MRP4 inhibitor MK571 (20 mg/kg) was administrated by intraperitoneal injection 30 minutes before induction of sepsis.Twenty-four later,serum interlukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels were measured by enzyme-linked immunosorbent assay.Lung injury was assessed by histopathological examination.Lung vascular permeability was evaluated by quantitation of Evans blue dye extravasation from vascular space to lung parenchyma.Results Compared with sham group,serum IL-6 and TNF-α levels were significantly higher in sepsis group.In addition,lung injury and lung vascular permeability were elevated in sepsis group compared to sham group.Importantly,MRP4 inhibitor treatment significantly decreased serum IL-6 and TNF-α levels,improved lung injury and reduced lung vascular permeability in septic rats.Conclusions Inhibition of MRP4 protects against pulmonary vascular endothelial barrier dysfunction in septic rats.

In vitro treatment with Treponema pallidum membrane protein Tpp17 increased the permeability of vascular endothelial barrier / 中华微生物学和免疫学杂志

Objective To investigate the in vitro effects of Treponema pallidum membrane protein Tpp17 on the permeability of endothelial barrier for further investigation on the immunopathogenesis of syphi-lis.Methods A cellular model of in vitro monolayer was established by using human umbilical vein endo-thelial cells ( HUVECs) .Cell-ELISA and a TMB kit were respectively used to measure the expression of VE-cadherin and the flux of horseradish peroxidase ( HRP) by monolayer HUVECs after stimulation with the re-combinant Tpp17 (rTpp17) protein.THP-1 cells stained with Calcein AM were added to the top of HUVEC monolayer in Transwell culture.Then, the numbers of THP-1 cells in the upper wells and beneath the HUVEC monolayer were counted by using a fluorescence microscope.The rTpp17 protein-treated HUVECs were fixed in 4%buffered paraformaldehyde and stained with rhodamine-phalloidin for observing the distri-bution of F-actin under a confocal laser scanning microscope.Results Compared with the control group, the expression of VE-cadherin in HUVECs was decreased, while the permeability of HUVEC monolayer was increased upon the stimulation with rTpp17 protein (P<0.05).Moreover, rTpp17 protein-induced F-actin redistribution and increased transendothelial migration of THP-1 cells were observed in rTpp17 protein-trea-ted HUVECs as compared with those of the control group (P<0.05).Conclusion Treponema pallidum membrane protein Tpp17 could suppress the expression of VE-cadherin and enhance the redistribution of F-actin, resulting in an enhanced transendothelial migration of THP-1 cells and an increased permeability of HUVEC monolayer.The Tpp17 protein might play an important role in the immunopathogenesis of syphilis.

The impact of Interleukin-1β on the structure of human umbilical vein endothelial cells and aquaporin-1 expression / 中华肝胆外科杂志

Objective To explore the role of IL-1β in capillary leak syndrome by observing the alterations of AQP-1 expression,apoptosis,and ultrastructural of vascular endothelial cells under the action of IL-1β.Methods Umbilical vein endothelial cells (UVEC) in vitro were randomly allocated into 3 groups:time,concentration,and control.In the time group,UVECs were treated with culture medium containing 20 μg/L IL-1β for3 h(T1),8 h(T2),12 h(T3) and 24 h(T4).In the concentration group,UVECs were treated with culture medium containing 0.2 μg/L(C1),2 μg/L(C2) and 20 μg/L(C3) IL-1β for 24 h.In the control group,UVECs were treated with culture medium without IL-1β for 24 h.The changes of AQP-1 mRNA and protein expression were detected by real-time PCR and Western blot.Apoptosis was detected by flow cytometry,and cell ultrastructural changes were observed by electron microscopy.Results AQP-1 mRNA and protein expression of T1-T4 in the time group and C1-C3 in the concentration group were lower than those of the control group (P ＜ 0.05).The apoptotic rate was increased,and mitochondrial swelling,vacuolar degeneration,karyolysis and necrosis were observed under electron microscopy.These were more pronounced with time or concentration increases.Conclusions IL-1β can cause a decrease of AQP-1 mRNA and protein expression,increase in apoptotic rate and increase in damage to the cells'ultrastructure.This is an important reason for damage to the vascular endothelial barrier and may be associated with capillary leak syndrome.

Effect of Dachengqi decoction on pancreas aquaporin 1 in rats with acute necrotizing pancreatitis / 中华胰腺病杂志

Objective To detect the expression of aquaporin 1 in pancreas of rats with acute necrotizing pancreatitis (ANP) and to study the effect of Dachengqi decoction on it.MethodsOne hundred and sixty male SD rats were randomly divided into control group ( C group,n =32 ),ANP group ( n =32),Dexamethasone group (De group,n =32),Acetazolamide group (A group,n =32) and Dachengqi decoction group (DD group,n =32).ANP model was induced by retrograde injection of 5％ sodium taurocholate into the biliary and pancreatic duct.Rats in De group received dexamethasone (4 mg/kg) intravenously after ANP induction; while rats in A group received 1 ml acetazolamide via gastric lavage 2 h before ANP induction; rats in DD group received 2 ml Dachengqi decoction via gastric lavage 48,24,2h before ANP induction; rats in C group received laparotomy.Eight rats in each group were sacrificed at 3 h,6 h,12 h and 18h after induction of ANP models.Quantity of ascites and levels of serum amylases were measured.Pathological changes in pancreas tissue were detected by HE and electron microscope.Capillary permeability in pancreas tissue was detected by Evans Blue (EB) extravasations method.AQP1 expression in pancreas tissue was detected by real-time PCR and Western blotting.ResultsLevels of serum amylase in ANP group was significantly higher,and the pancreatic injuries were obvious ; the levels of serum amylase in De group and DD group was lower than that in ANP group,and the pancreatic injuries were attenuated.The levels of serum amylase in A group were higher than that in ANP group,and the pancreatic.injuries were more severe than that in ANP group.Six hours after ANP induction,the levels of EB in pancreas were (13.44 ±2.56),(126.35 ± 14.80),(86.31 ± 14.46),( 108.99 ± 15.07 ),(78.29 ± 16.85 ) mg/L In C group,ANP group,De group,A group and DD group,and the expression of AQP1 mRNA in pancreatic tissue was ( 170.07 ± 22.48 ) ％,( 83.93 ± 8.98 ) ％,( 117.09 ±10.70 ) ％,( 69.00 ± 8.98 ) ％,( 112.82 ± 11.79 ) ％ ; and the expression of AQP1 protein was 0.23 ± 0.06,0.10 ±0.02,0.32 ±0.03,0.13 ±0.02,0.45 ±0.04.The content of EB in ANP group was higher than that in C group,while the expression of AQP1 mRNA and protein in ANP group was significantly lower than that in C group (P ＜ 0.05 ).The content of EB in De group and DD group was significantly lower than that in ANP group,while the expression of AQP1 mRNA and protein was significantly higher than that in ANP group (P ＜ 0.05).ConclusionsAQP1 plays an important role in the pathogenesis of capillary endothelial barrier dysfunction in rats with ANP.Dachengqi Decoction can attenuate pancreatic injuries of rats by regulating the expression of AQP1.