Expression of HMGB1 in septic serum induces vascular endothelial hyperpermeability.

The aim of the present study was to investigate the effects of high mobility group protein B1 (HMGB1), which is expressed in the serum of patients with sepsis, on vascular endothelial permeability. Sera from patients with sepsis were used to treat endothelial cells (ECs), and the effect on endothelial permeability was evaluated using immunofluorescence. The morphologies of endothelial cytoskeletal actin and vascular endothelial (VE)­cadherin were assessed using laser scanning confocal microscopy. The protein expression levels of HMGB1, B­cell lymphoma 2 (BCL­2) and BCL­2­associated X protein (BAX) were detected using western blotting. EC apoptosis was measured using flow cytometry. The results demonstrated that HMGB1 was significantly expressed in the serum 24 h following the onset of sepsis, and the expression levels peaked at 48 h, which were sustained until 96 h post­onset. Compared with the control group, treatment of the ECs with 20% septic serum in vitro significantly increased endothelial monolayer permeability (P<0.01), markedly induced transcellular filamentous (F)­actin rearrangement with stress fiber formation, and resulted in the localization of VE­cadherin fragmentations at the cell borders with increased gaps between ECs. Furthermore, flow cytometry showed that the apoptotic rate of ECs was significantly increased following treatment with septic serum. In addition, the expression levels of BAX were significantly increased, whereas the expression levels of BCL­2 were significantly decreased. Pretreatment with an HMGBI inhibitor (ethyl pyruvate; 5 µM) 24 h prior to treatment with the septic serum attenuated the effects of septic serum treatment. Together, these findings suggested that treatment of ECs with sera from patients with sepsis may induce the loss of vascular endothelial monolayer integrity, elicit the formation of endothelial F­actin stress fibers and initiate VE­cadherin redistribution, which may be attributed to high levels of HMGB1 in the serum. This mechanism also appears to involve changes in the activation of BAX and BCL­2, resulting in EC apoptosis.

Proinflammatory effects and molecular mechanisms of interleukin-17 in intestinal epithelial cell line HT-29.

AIM: To evaluate the proinflammatory effects and molecular mechanisms of interleukin (IL)-17 in intestinal epithelial cell line HT-29. METHODS: HT-29 cells were cultured with IL-17, tumor necrosis factor (TNF)-α, or the combination of both IL-17 and TNF-α. Real-time PCR and Western blot were used to measure the gene expression levels of neutrophil chemokines CXCL1, CXCL2, CXCL5, CXCL6, IL-8 and TH-17 cell chemokine CCL20, the phosphorylation levels of p38 and TNF-α, and the expression level of IL-8, after using the p38 inhibitor in HT-29 cells. The stable Act1 knockdown HT-29 cell line was established to further test the phosphorylation changes of p38, after using IL-17 and TNF-α. RESULTS: After HT-29 cells were cultured with IL-17 and TNF-α, the expression levels of neutrophil chemokines (CXCL1, CXCL2, CXCL5, CXCL6, IL-8) and Th17 chemokine (CCL20) significantly improved (24.96 ± 2.53, 28.47 ± 2.87, 38.08 ± 2.72, 33.47 ± 2.41, 31.7 ± 2.38, 44.37 ± 2.73, respectively), and the differences were all statistically significant (P < 0.01). Western blot results showed that IL-17 obviously enhanced the phosphorylation level of p38, which was induced by TNF-α. Compared with the control group, the expression level of IL-8 significantly declined (9.47 ± 1.36 vs 3.06 ± 0.67, P < 0.01) when TH-29 cells were cultured with IL-17 and TNF-α. p38 inhibition assay showed that the p38 pathway played an essential role in the inflammatory response induced by IL-17. p38 phosphorylation levels could not be changed after using IL-17 and TNF-α in the stable Act1 knockdown HT-29 cell line. CONCLUSION: IL-17 significantly promoted the gene expression levels of TNF-α-induced neutrophil chemokines and Th17 cell chemokine. It is obvious that IL-17 and TNF-α have synergistic effects on p38.

Effect of serum from patients with severe acute pancreatitis on vascular endothelial permeability.

OBJECTIVE: This study aimed to investigate the effect of serum taken from patients with severe acute pancreatitis (SAP) on vascular endothelial permeability. METHODS: The monolayer permeability of endothelial cells (ECs) was assessed. Morphological changes in ECs, induced by serum from patients with SAP were assessed. Expressions of RhoA, myosin light chain (MLC) phosphorylation, and VE-cadherin protein were detected by Western blot. RESULTS: Compared with the control group, 20% SAP serum significantly increased endothelial monolayer permeability (P < 0.01), markedly induced transcellular F-actin redistribution with stress fiber formation and VE-cadherin derangement with fragmentations located at the cell borders, and increased gaps between ECs. Furthermore, Western blotting showed that SAP serum induced rapid activation of Rho protein, and markedly increased the level of phosphorylated MLC. However, pretreatment with Y-27632 (an inhibitor for Rho kinase) significantly inhibited endothelial hyperpermeability and the morphological changes of F-actin rearrangement and VE-cadherin redistribution. This was associated with a down-regulation of Rho protein expression and a reduction in the level of MLC phosphorylation. CONCLUSIONS: The SAP serum induces the loss of vascular endothelial monolayer integrity, with endothelial F-actin stress fiber formation and VE-cadherin redistribution. One of the mechanisms for this process involves the activation of the Rho/Rho kinase signaling pathway.

Study of Astragalus mongholicus polysaccharides on endothelial cells permeability induced by HMGB1.

Astragalus mongholicus polysaccharide (APS) shows various biological activities. Here, we explored the effect of APS on high mobility group protein 1 (HMGB1) -induced endothelial cell permeability. The results indicated APS pretreatment effectively inhibited HMGB1-induced increased permeability in endothelial cells (ECs). Signal transduction studies showed APS inhibited not only the activation of small guanylate Rho and its downstream effector Rho kinase (ROCK), but also the subsequent phosphorylation of myosin light chain (MLC) in ECs. In conclusion, our investigations suggested that APS inhibited HMGB1-induced increased permeability in ECs, regulated by Rho/ROCK signal pathways.

[A study on increased permeability and morphological changes in actin cytoskeleton and tight junction of vascular endothelial cells induced by tumor necrosis factor-alpha].

OBJECTIVE: To study the effect of human tumor necrosis factor-alpha (TNF-alpha) on permeability of human vascular endothelial cell (EA.hy926) monolayer and its mechanism. METHODS: 5, 10, 20 microg/L TNF-alpha was respectively added to the cultured endothelial cell monolayer for 24 hours, or 10 microg/L TNF-alpha for 6, 12, 24 hours. Human vascular endothelial cell (EA.hy926) monolayer permeability was measured by detecting fluorescence intensity of fluorescein isothiocyanate (FITC) labeled dextran. Immunofluorescence and laser confocal microscopy were used to assess vascular endothelial actin cytoskeleton (F-actin) and tight junction protein (ZO-1) distribution. Western blotting was used to assess ZO-1 expression. RESULTS: Compared with control group, TNF-alpha significantly increased endothelial permeability and induced F-actin redistribution and stress fiber formation with ZO-1 derangement. Gaps increased obviously between endothelial cells. Furthermore, Western blotting showed that TNF-alpha reduced ZO-1 expression in a dose- and time-dependent manner. CONCLUSION: TNF-alpha increased endothelial cell permeability by damaging integrity of endothelial barrier function.

Effects of gut barrier dysfunction and NF-kappaB activation on aggravating mechanism of severe acute pancreatitis.

OBJECTIVE: To study the effects of gut-derived endotoxin translocation and NF-kappaB activation on the aggravating mechanism of severe acute pancreatitis (SAP) and of treatment with pyrrolidine dithiocarbamate (PDTC) on rats with SAP. METHODS: SD rats were randomly divided into sham operation group (SO), SAP group, SAP + lipopolysaccharide(LPS) group, pyrrolidine dithiocarbamate (PDTC) treatment group and LPS group. Biochemical parameters and cytokines were examined in the serum. Multiple organs pathological slices were examined. Expression of NF-kappaB mRNA in the liver tissue was detected by RT-PCR. Activation of NF-kappaB by the method of streptomycin avidin-peroxidase (SP) and expression of NF-kappaB p65 protein and its binding activity were analyzed by Western blot and electrophoretic mobidity shift assay (EMSA). RESULTS: Compared with sham operation group, the concentration of TNF-alpha, alanine aminotransferase (ALT), and diamine oxidase (DAO) in serum significantly increased in SAP + LPS group (P < 0.05). Pathological changes were markedly observed in tissues and the expression of NF-kappaB mRNA in the liver significantly increased (P < 0.05) also, the activation of NF-kappaB and binding activity of NF-kappaB p65 protein in the liver markedly increased (P < 0.01) in SAP + LPS group. Treatment with PDTC markedly reduced concentration of ALT, DAO and TNF-alpha, and the expression of NF-kappaB, and the pathologic scores, as well as significantly decreased the expression of NF-kappaB p65 protein. CONCLUSION: The activation and overexpression of NF-kappaB may participate in the aggravating mechanism of SAP. Treatment with PDTC has a protective effect on multiple organs damage in SAP.