Roles of RAGE/ROCK1 Pathway in HMGB1-Induced Early Changes in Barrier Permeability of Human Pulmonary Microvascular Endothelial Cell.

Background: High mobility group box 1 (HMGB1) causes microvascular endothelial cell barrier dysfunction during acute lung injury (ALI) in sepsis, but the mechanisms have not been well understood. We studied the roles of RAGE and Rho kinase 1 (ROCK1) in HMGB1-induced human pulmonary endothelial barrier disruption. Methods: In the present study, the recombinant human high mobility group box 1 (rhHMGB1) was used to stimulate human pulmonary microvascular endothelial cells (HPMECs). The endothelial cell (EC) barrier permeability was examined by detecting FITC-dextran flux. CCK-8 assay was used to detect cell viability under rhHMGB1 treatments. The expression of related molecules involved in RhoA/ROCK1 pathway, phosphorylation of myosin light chain (MLC), F-actin, VE-cadherin and ZO-1 of different treated groups were measured by pull-down assay, western blot and immunofluorescence. Furthermore, we studied the effects of Rho kinase inhibitor (Y-27632), ROCK1/2 siRNA, RAGE-specific blocker (FPS-ZM1) and RAGE siRNA on endothelial barrier properties to elucidate the related mechanisms. Results: In the present study, we demonstrated that rhHMGB1 induced EC barrier hyperpermeability in a dose-dependent and time-dependent manner by measuring FITC-dextran flux, a reflection of the loss of EC barrier integrity. Moreover, rhHMGB1 induced a dose-dependent and time-dependent increases in paracellular gap formation accompanied by the development of stress fiber rearrangement and disruption of VE-cadherin and ZO-1, a phenotypic change related to increased endothelial contractility and endothelial barrier permeability. Using inhibitors and siRNAs directed against RAGE and ROCK1/2, we systematically determined that RAGE mediated the rhHMGB1-induced stress fiber reorganization via RhoA/ROCK1 signaling activation and the subsequent MLC phosphorylation in ECs. Conclusion: HMGB1 is capable of disrupting the endothelial barrier integrity. This study demonstrates that HMGB1 activates RhoA/ROCK1 pathway via RAGE, which phosphorylates MLC inducing stress fiber formation at short time, and HMGB1/RAGE reduces AJ/TJ expression at long term independently of RhoA/ROCK1 signaling pathway.

Treatment of low molecular weight heparin inhibits systemic inflammation and prevents endotoxin-induced acute lung injury in rats.

To determine whether low molecular weight heparin (LMWH) is able to reduce pulmonary inflammation and improve the survival in rats with endotoxin-induced acute lung injury (ALI). Rat ALI model was reproduced by injection of lipopolysaccharide (LPS) into tail vein. Rats were divided randomly into three groups: control group, ALI group, LMWH-treated group. Blood was collected and lung tissue was harvested at the designated time points for analysis. The lung specimens were harvested for morphological studies, streptavidin-peroxidase immunohistochemistry examination. Lung tissue edema was evaluated by tissue water content. The levels of lung tissue myeloperoxidase (MPO) were determined. Meanwhile, the nuclear factor-kappa B (NF-κB) activation, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) levels and high mobility group box 1 (HMGB1) and intercellular adhesion molecule-1 (ICAM-1) protein levels in the lung were studied. In survival studies, a separate group of rats were treated with LMWH or sterile saline after LPS administration. Then, the mortality was recorded. Treatment with LMWH after ALI was associated with a reduction in the severity of LPS-induced lung injury. Treatment with LMWH significantly decreased the expression of TNF-α, IL-1ß, HMGB1 and ICAM-1 in the lung of ALI rats. Similarly, treatment with LMWH dramatically diminished LPS-induced neutrophil sequestration and markedly reduced the enhanced lung permeability. In the present study, LMWH administration inhibited the nuclear translocation of NF-κB in the lung. Survival was significantly higher among the LMWH-treated group compared with the ALI group. These data suggest that LMWH attenuates inflammation and prevents lethality in endotoxemic rats.

Downregulation of HMGB1 protects against the development of acute lung injury after severe acute pancreatitis.

OBJECTIVE: To examine the effect of downregulation of high mobility group box 1 (HMGB1) on severe acute pancreatitis (SAP) associated with acute lung injury (ALI), and its subsequent effect on disease severity. METHODS: Wistar rats were given an IV injection of pRNA-U6.1/Neo-HMGB1, pRNA-U6.1/Neo-vector or saline before induction of SAP. Then, SAP was induced in rats by the retrograde injection of 5% sodium taurocholate into the pancreatic duct. The control group received only a sham operation. Lung and pancreas samples were harvested after induction of SAP. The protein levels of HMGB1, matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1) in lung tissue were investigated. The severity of pancreatic injury was determined by a histological score of pancreatic injury, serum amylase, and pancreatic water content. The lung injury was evaluated by measurement of pulmonary microvascular permeability, lung myeloperoxidase activity and malondialdehyde levels. RESULTS: The results found that in pRNA-U6.1/Neo-HMGB1 treated rats, serum tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) levels were decreased and the severity of pancreatic tissue injury was less compared with either untreated SAP or pRNA-U6.1/Neo-vector treated rats (P<0.05). The administration of pRNA-U6.1/Neo-HMGB1 in SAP-induced rats downregulated the DNA binding activity of the nuclear factor-kappa B (NF-κB) and the expressions of MMP-9 and ICAM-1 in lung. Thus, compared with the untreated SAP rats, the inflammatory response and the severity of ALI decreased (P<0.05). CONCLUSIONS: These results demonstrate that HMGB1 could augment Inflammation by inducing nuclear translocation of NF-κB, thus aggratating the severity of SAP associated with ALI.

shRNAs targeting high-mobility group box-1 inhibit E-selectin expression via homeobox A9 in human umbilical vein endothelial cells.

The nucleus of the endothelial cell contains large amounts of high-mobility group box protein 1 (HMGB1), a cytokine mediator of inflammation, and the endothelium may be a crucial source of HMGB1 during the inflammatory response. Therefore, the downregulation of HMGB1 expression by RNA interference (RNAi) may decrease inflammatory activity. The aim of this study was to investigate the possible mechanism of action and the effect of HMGB1 on homeobox A9 (HOXA9) and E-selectin expression. Recombinant human full-length HMGB1 was cloned by PCR amplification from human umbilical vein endothelial cells (HUVECs) and then subcloned into a pcDNA­3.1-myc-his-B vector. Specific short hairpin RNAs (shRNAs) for the HMGB1 target sequence and a scrambled sequence were designed, synthesized and cloned into a pRNA­U6.1/Neo vector. Specific small interfering RNAs (siRNAs) for the HOXA9 target sequence were commercially prepared and the expression levels of HMGB1, HOXA9, intracellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1) and E-selectin were detected using real-time PCR and western blot analysis. The expression of the full-length HMGB1 gene and protein was verified in HUVECs. The shRNA for HMGB1 and siRNA for HOXA9 successfully decreased the expression levels of HMGB1 and HOXA9, respectively. ICAM1, VCAM1 and E-selectin were downregulated through HMGB1 interference in HUVECs, and HMGB1 shRNA decreased E-selectin expression by HOXA9. These results demonstrated the potential use of specific siRNA targeting HMGB1 expression for the development of novel therapeutic agents for inflammatory disorders.

Protective effect of ethyl pyruvate on pancreas injury in rats with severe acute pancreatitis.

BACKGROUND: Systemic inflammatory mediators have an important role in the development of acute pancreatitis. In this study, we investigated the effect of ethyl pyruvate (EP) on pancreas injury in rats with severe acute pancreatitis (SAP) and its possible mechanism. METHODS: We randomly allocated rats into the following three experimental groups: control and SAP- and EP-treated. Then, we recorded the mortality rate. We harvested tissue specimens for morphological studies, streptavidin-peroxidase immunohistochemistry examination, and Western blot analysis. We tested the levels of pancreatic tissue malondialdehyde and the activity of serum amylase, myeloperoxidase in the pancreas. In addition, we studied nuclear factor-κB (NF-κB) activation, tumor necrosis factor-α levels, and high mobility group box 1 protein expression levels in the pancreas. RESULTS: Treatment with EP after SAP was associated with a reduction in the severity of SAP and pancreas injury. Treatment with EP significantly decreased the expression of tumor necrosis factor-α and high mobility group box 1, and ameliorated malondialdehyde concentration and myeloperoxidase activity in the pancreas in SAP rats. Compared with the SAP group, treatment with EP significantly decreased the number of inflammatory cell infiltration, markedly inhibited pancreatic NF-κB DNA binding, and increased the survival rates. CONCLUSIONS: This study demonstrates that preventing the activation of NF-κB by EP ameliorates tissue injury associated with experimental murine acute pancreatitis. This result provides an important insight into the molecular biology of acute pancreatitis.

[Effect of down-regulation of high mobility group box-1 expression on proliferation and migration abilities of human umbilical vein endothelial cells].

OBJECTIVE: To investigate the effect of down-regulation of high mobility group box-1 (HMGB1) expression on the proliferation and migration abilities of human umbilical vein endothelial cell ( HUVEC) in vitro. METHODS: The method of stable transfection was used to transfect the pRNA-u6.1/Neo-control and pRNA-u6.1/ Neo-HMGB1 short hairpin RNA (shRNA) plasmid to HUVEC cells, and control and HMGB1 shRNA cell lines were reproduced, and the cells were identified by Western blotting test and real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR). Then methyl thiazolyl tetrazolium (MTT) was used for the determination of the proliferative ability, flow cytometry was used for determining the changes in cell cycle distribution ability of HUVEC, and the migratory ability was detected by scratch test. RESULTS: Western blotting and RT -PCR detection proved the production of stable transfection cell lines was successful, and the content of HMGB1 protein and mRNA expression in cells were significantly lower after cultivation for 72 hours than those of control cells (protein: 0.436 ± 0.027 vs. 1.017 ± 0.038, I= 12.180, P=0.000; mRNA: 0.436 ± 0.031 vs. 1.020 ± 0.051, T=9.660, P= 0.001 ). The results of MTT showed that the proliferation ability of HMGB1 shRNA cell lines was lower obviously 2, 3, 4, 5 days after transfection than that of control cells ( 2 days: 0.210 ± 0.023 vs. 0.240 ± 0.011, T= 1.050, P=0.351; 3 days: 0.240 ± 0.022 vs. 0.361 ± 0.030, T=3.203, P=0.033; 4 days: 0.373 ± 0.031 vs. 0.531 ± 0.033, T=3.530, P=0.022; 5 days: 0.441 ± 0.031 vs. 0.602 ± 0.030, T=4.180, P=0.106). Flow cytometry results showed that the number of HMGB 1 shRNA cells in S phase was significantly lower than that of the control cell line ( ( 13.10 ± 1.10 )% vs. (21.12 ± 1.20)%, T=4.950, P=0.001). Scratch test showed that the healing ability of HMGB1 shRNA cell line was lowered significantly at 12 hours as compared with that of control ( (20.17 ± 3.33 )% vs. ( 88.53 ± 3.15 )% , T= 14.142, P=0.000). CONCLUSION: HMGB 1 shRNA can significantly inhibit HUVEC cell proliferation and migration.

[Construction of stable human umbilical vein endothelial cells line expressing short hairpin RNA (shRNA) targeting high mobility group box-1 gene].

OBJECTIVE: To construct the short hairpin RNA (shRNA) targeting high mobility group box-1 (HMGB1) and culture the stable human umbilical vein endothelial cell (HUVEC) line expressing this shRNA. METHODS: Based on the HMGB1 gene sequence, shRNA was designed, synthesized and subcloned into the pRNA-u6.1/Neo vector, while negative controls were also established. Then the recombinant vector was transfected into HUVEC cell line and the cell was screened with G418 and assayed by using real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. RESULTS: Restriction endonuclease digestion test and sequencing verification showed that the recombinant pRNA-u6.1/Neo vector expressing this shRNA targeting HMGB1 was successfully constructed and the stable HUVEC cell line expressing this shRNA was developed. The real time RT-PCR and Western blotting was used to detect that recombinant plasmid in HUVEC cell effect on expression of HMGB1 was reduced. (mRNA: 0.4635 ± 0.0342 vs. 1.0340 ± 0.0352, protein: 0.4510 ± 0.0200 vs. 1.0210 ± 0.0110, both P<0.05). CONCLUSION: The recombinant pRNA-u6.1/Neo vector expressing shRNA targeting HMGB1 was successfully constructed and the stable HUVEC cell line expressing this shRNA was developed, and therefore allowed further investigation regarding the function of HMGB1 gene in the HUVEC cell line.

Therapeutic treatment with ethyl pyruvate attenuates the severity of liver injury in rats with severe acute pancreatitis.

OBJECTIVE: The objective of the study was to evaluate the effect of ethyl pyruvate (EP) in ameliorating liver injury in rats with severe acute pancreatitis (SAP) and its possible mechanism. METHODS: Rats were randomly divided into control group, SAP group, and EP-treated group. Then, the tissue specimens were harvested for morphological studies, immunohistochemistry examination, reverse transcriptase-polymerase chain reaction, and Western blot analysis. The DNA-binding activity of nuclear factor κB was measured by electrophoretic mobility shift assay. The concentrations of serum amylase, alanine aminotransferase, and pancreatic tissue malondialdehyde and the activity of myeloperoxidase in the liver were determined. RESULTS: Treatment with EP after SAP was associated with a reduction in the severity of SAP and liver injury. Treatment with EP significantly decreased the hepatic mRNA expression of tumor necrosis factor α and interleukin 1ß and ameliorated the activity of myeloperoxidase in the liver in SAP rats. Compared with the SAP group, treatment with EP significantly decreased the infiltration of inflammatory cells and markedly inhibited hepatic nuclear factor κB DNA binding; EP therapy dramatically inhibited high-mobility group box 1 expression from inflamed hepatic tissue. CONCLUSIONS: Our results demonstrate that EP might play a therapeutic role in liver inflammation in this SAP model, and these beneficial effects of EP are because of the modulation of high-mobility group box 1 and other inflammatory cytokine responses.

HMGB1 activates nuclear factor-κB signaling by RAGE and increases the production of TNF-α in human umbilical vein endothelial cells.

OBJECTIVE: High mobility group box chromosomal protein 1 (HMGB1) is a lately discovered candidate molecule identified as an important extracellular mediator in systemic inflammation. Systemic inflammation results in endothelial cell activation and microvascular injury. In the present study, we investigated the effects of HMGB1 on the activation of human umbilical vein endothelial cells (HUVECs) and defined pathways activated by HMGB1. METHODS: HUVECs obtained by collagenase treatment of umbilical cord veins were stimulated in vitro with HMGB1. The activation of HUVECs was studied regarding (i) the kinetics of tumor necrosis factor-α (TNF-α) production in HUVECs, (ii) HMGB1-induced up-regulation of receptor for advanced glycation end products (RAGE), (iii) HMGB1-induced nuclear translocation of nuclear factor kappa B (NF-κB) in HUVECs, (iv) the activation of signalling transduction pathways. RESULTS: HUVECs activation was stimulated by HMGB1 partially in a RAGE-dependent manner. Additionally, the HMGB1-induced activation of HUVECs was significantly inhibited by anti-RAGE monoclonal antibody and Ethyl pyruvate (EP) that had been shown to be an effective anti-inflammatory agent. Short-term prestimulation of HUVECs with HMGB1 caused a time-dependent increase in the secretion of TNF-α and expression of RAGE. Furthermore, HMGB1 stimulation resulted in nuclear translocation of transcription factor NF-κB. Most importantly, pretreatment with anti-RAGE monoclonal antibody significantly decreased the amounts of TNF-α and inhibited the nuclear translocation of NF-κB. Additionally in HUVECs cultures, EP specifically inhibited activation of NF-κB signaling pathway that are critical for TNF-α release. CONCLUSIONS: In conclusion, Our data present a link between HMGB1and RAGE function of endothelial cells and demonstrate the pathway activated by HMGB1. These findings may provide a novel therapeutic strategy to improve the endothelial cells function.

Role of high-mobility group box 1 protein in the pathogenesis of intestinal barrier injury in rats with severe acute pancreatitis.

OBJECTIVE: To investigate the role of high-mobility group box 1 (HMGB1) in the development of intestinal barrier injury of severe acute pancreatitis (SAP) and to examine the effect of ethyl pyruvate (EP) on intestinal inflammation in rats with SAP. METHODS: Rats were randomly divided into the following experimental groups: control, SAP, and EP treated. Then, the distal ileum was harvested for morphological studies, streptavidin-peroxidase immunohistochemistry examination, and Western blot analysis. The concentrations of plasma amylase, endotoxin, and diamine oxidase (DAO) and the activity of myeloperoxidase (MPO) in the intestine were determined. RESULTS: We found that the expression of HMGB1 was up-regulated in the ileal mucosa within 6 hours and then remained elevated for more than 48 hours after SAP. Meanwhile, the levels of plasma amylase, endotoxin, and DAO and the activity of MPO in the intestinal mucosa were rapidly increased after SAP. Whereas treatment with EP significantly decreased the expression of intestinal HMGB1, the levels of plasma amylase, endotoxin, and DAO ameliorated the activity of MPO in the intestine in SAP rats. CONCLUSIONS: Our results demonstrate that HMGB1 participates in intestinal barrier injury in SAP and EP might play a therapeutic role in intestinal inflammation in this SAP model.

[Therapeutic effects of low molecular weight heparin and aspirin on acute lung injury in rat].

OBJECTIVE: To investigate the possible effects of nuclear factor-KappaB (NF-KappaB) activation on the expression of intercellular adhesion molecular-1 (ICAM-1) and P-selectin in murine acute lung injury (ALI) and assess the potential beneficial effects and mechanism of low molecular weight heparin (LMWH) and aspirin (ASA). METHODS: Rat ALI model was reproduced by injection of lipopolysaccharide into tail vein. Sixty rats were divided randomly into four groups (n=15): normal control group, ALI group, LMWH group and ASA group. The change in NF-KappaB activity in the lung to show its relation with ICAM-1 and P-selectin was observed by immunohistochemistry. RESULTS: Compared with normal control group, the activity of NF-KappaB and the expression of ICAM-1 and P-selectin increased significantly in the lung tissue of ALI group (all P<0.05). Compared with ALI group, the level of NF-KappaB activity and the expression of ICAM-1 and P-selectin were obviously down regulated, and also the pathological lesion and inflammatory response of lung were improved in LMWH and ASA groups. However, the therapeutic effects of ASA were stronger than those of LMWH (all P<0.05). CONCLUSION: NF-KappaB activation plays an important role in ALI. NF-KappaB takes part in the activation of many kinds of inflammatory cells such as neutrophil, endothelial cells and so on, and it adjusts the genetic expression of ICAM-1 and P-selectin. LMWH and ASA show their beneficial effects on lung injury in both functional and morphological aspects. But the mechanism is different. LMWH can indirectly inhibit the activation of NF-KappaB, improve lung microcirculation and decrease the adhesion of neutrophil and platelet. ASA plays the role as an inhibitor of NF-KappaB activation.