Molecular dynamics investigation of the substrate binding mechanism in carboxylesterase.

A recombinant carboxylesterase, cloned from Pseudomonas putida and designated as rPPE, is capable of catalyzing the bioresolution of racemic 2-acetoxy-2-(2'-chlorophenyl)acetate (rac-AcO-CPA) with excellent (S)-enantioselectivity. Semirational design of the enzyme showed that the W187H variant could increase the activity by ∼100-fold compared to the wild type (WT) enzyme. In this study, we performed all-atom molecular dynamics (MD) simulations of both apo-rPPE and rPPE in complex with (S)-AcO-CPA to gain insights into the origin of the increased catalysis in the W187H mutant. Our results show differential binding of (S)-AcO-CPA in the WT and W187H enzymes, especially the interactions of the substrate with the two active site residues Ser159 and His286. The replacement of Trp187 by His leads to considerable structural rearrangement in the active site of W187H. Unlike in the WT rPPE, the cap domain in the W187 mutant shows an open conformation in the simulations of both apo and substrate-bound enzymes. This open conformation exposes the catalytic triad to the solvent through a water accessible channel, which may facilitate the entry of the substrate and/or the exit of the product. Binding free energy calculations confirmed that the substrate binds more strongly in W187H than in WT. On the basis of these computational results, we further predicted that the mutations W187Y and D287G might also be able to increase the substrate binding and thus improve the enzyme's catalytic efficiency. Experimental binding and kinetic assays on W187Y and D287G show improved catalytic efficiency over WT, but not W187H. Contrary to our prediction, W187Y shows slightly decreased substrate binding coupled with a 100-fold increase in turnover rate, while in D287G the substrate binding is 8 times stronger but with a slightly reduced turnover rate. Our work provides important molecular-level insights into the binding of the (S)-AcO-CPA substrate to carboxylesterase rPPEs, which will help guide future development of more efficient rPPE variants.

Celastrol induces cell cycle arrest by MicroRNA-21-mTOR-mediated inhibition p27 protein degradation in gastric cancer.

OBJECTIVE: Celastrol has anti-cancer effects by increase of apoptosis of gastric cancer cells. However, its role in gastric cancer cell cycle is still unclear. The aim of this study was to investigate the effect and mechanism of celastrol on gastric cancer cell cycle. METHODS: The effects of celastrol on cell cycle in BGC-823 and MGC-803 cells were assayed via flow cytometry analysis. The expression of p27 and mTOR was detected by real-time PCR and western blot. The activity of mTOR and mTORC2 was measured by mTOR and mTORC2 kinase assays. miR-21 mimic was used to up-regulate miR-21 expression and mTOR expression plasmid was used to increase mTOR level in gastric cancer cells. RESULTS: Celastrol caused G2/M cell-cycle arrest that was accompanied by the down-regulation of miR-21 expression. In particular, miR-21 overexpression reversed cell cycle arrest effects of celastrol. Further study showed that celastrol increased levels of the p27 protein by inhibiting its degradation. miR-21 and mTOR signaling pathway was involved in the increase of p27 protein expression in BGC-823 and MGC-803 cells treated with celastrol. Significantly, miR-21 overexpression restored the decrease of mTOR activity in cells exposed celastrol. CONCLUSIONS: The effect of celastrol on cell cycle arrest of gastric cancer cells was due to an increase of p27 protein level via inhibiting miR-21-mTOR signaling pathway.

Unusually broad substrate profile of self-sufficient cytochrome P450 monooxygenase CYP116B4 from Labrenzia aggregata.

A new member of the CYP116B subfamily-P450LaMO-was discovered in Labrenzia aggregata by genomic data mining. It was successfully overexpressed in Escherichia coli, purified, and subsequently characterized spectroscopically, and its catalytic properties were assessed. Substrate profiling of the P450LaMO revealed that it was a versatile catalyst, exhibiting hydroxylation and epoxidation activities as well as O-dealkylation and asymmetric sulfoxidation activities. Diverse compounds, including alkylbenzenes, aromatic bicyclic molecules, and terpenoids, were shown to be hydroxylated by P450LaMO. Such diverse catalytic activities are uncommon for the bacterial P450s, and the P450LaMO-mediated stereoselective hydroxylation of inactivated C-H bonds-ubiquitous and relatively unreactive in organic molecules-is particularly unusual. The self-sufficient nature of P450LaMO, coupled with its broad substrate range, highlights it as an ideal template for directed evolution towards various applications.

Cloning and Characterization of a Novel Esterase from Rhodococcus sp. for Highly Enantioselective Synthesis of a Chiral Cilastatin Precursor.

A novel nonheme chloroperoxidase (RhEst1), with promiscuous esterase activity for enantioselective hydrolysis of ethyl (S)-2,2-dimethylcyclopropanecarboxylate, was identified from a shotgun library of Rhodococcus sp. strain ECU1013. RhEst1 was overexpressed in Escherichia coli BL21(DE3), purified to homogeneity, and functionally characterized. Fingerprinting analysis revealed that RhEst1 prefers para-nitrophenyl (pNP) esters of short-chain acyl groups. pNP esters with a cyclic acyl moiety, especially that with a cyclobutanyl group, were also substrates for RhEst1. The Km values for methyl 2,2-dimethylcyclopropanecarboxylate (DmCpCm) and ethyl 2,2-dimethylcyclopropane carboxylate (DmCpCe) were 0.25 and 0.43 mM, respectively. RhEst1 could serve as an efficient hydrolase for the bioproduction of optically pure (S)-2,2-dimethyl cyclopropane carboxylic acid (DmCpCa), which is an important chiral building block for cilastatin. As much as 0.5 M DmCpCe was enantioselectively hydrolyzed into (S)-DmCpCa, with a molar yield of 47.8% and an enantiomeric excess (ee) of 97.5%, indicating an extremely high enantioselectivity (E = 240) of this novel and unique biocatalyst for green manufacturing of highly valuable chiral chemicals.

Artesunate exerts an anti-immunosuppressive effect on cervical cancer by inhibiting PGE2 production and Foxp3 expression.

Artesunate (ART), derived from a common traditional Chinese medicine, has beeen used an antimalarial for several years. In this study, the effect and mechanism of ART on anti-human cervical cancer cells was examined. The level of prostaglandin E2 (PGE2 ) and the population of CD4+CD25+Foxp3 regulatory T cells (Treg) in peripheral blood were detected by flow cytometry. In vivo antitumor activity was investigated in mice with cervical cancer by the subcutaneous injection of various concentrations of ART. The concentrations of PGE2 in the supernatants of CaSki cells were measured using an ELISA kit. Cyclooxygenase-2 (COX-2) and Foxp3 expression were determined using quantitative polymerase chain reaction (qPCR) and western blot analysis. The effect of ART on the viability of CaSki and Hela cells was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. It was identified that the level of PGE2 and the population of CD4+CD25+Foxp3 Treg cells in the peripheral blood were significantly higher in cervical cancer patients and mice with cervical cancer. ART was capable of inhibiting orthotopic tumor growth, which correlated with a decrease in the level of PGE2 and the percentage of Treg cells in mice with cervical cancer. Furthermore, ART decreased COX-2 expression and the production of PGE2 in CaSki and Hela cells. Notably, the supernatants of CaSki cells treated with ART lowered the expression of Foxp3 in Jurkat T cells, which was capable of being reversed by exogenous PGE2 . Our data revealed that ART may elicit an anti-tumor effect against cervical cancer by inhibition of PGE2 production in CaSki and Hela cells, which resulted in the decrease of Foxp3 expression in T cells. Therefore, ART may be an effective drug for immunotherapy of cervical cancer.

Celastrol induces apoptosis of gastric cancer cells by miR-21 inhibiting PI3K/Akt-NF-κB signaling pathway.

OBJECTIVE: Celastrol, a plant triterpene, has anticancer effects by increase of apoptosis. In the present study, the mechanism of celastrol on gastric cancer cell apoptosis was examined. METHODS: The effect of celastrol on PI3K/Akt and the NF-κB signaling pathway was evaluated with Western blot and luciferase reporter assay. miR-21 expression was determined using real-time PCR. miR-21 inhibitor and miR-21 mimic were used to downregulate and upregulate miR-21 expression, respectively. RESULTS: It was identified that celastrol was capable of inducing apoptosis of gastric cancer cells, which was mediated via inhibiting the activation of PI3K/Akt and NF-κB. A strong activator of Akt, IGF-1 restored NF-κB activity in cells treated with celastrol. Celastrol could also significantly suppress miR-21 expression. Furthermore, miR-21 inhibitor could decrease phospho-Akt expression and NF-κB activity. Notably, upregulation of miR-21 expression can increase PI3K/Akt and NF-κB activity and decrease apoptosis of gastric cancer cells treated with celastrol, which could be reversed by PI3K inhibitor. CONCLUSIONS: Our data revealed that the effect of celastrol on apoptosis was due to miR-21 inhibiting the PI3K/Akt-dependent NF-κB pathway.

Celastrol induces apoptosis of gastric cancer cells by miR-146a inhibition of NF-κB activity.

BACKGROUND: Celastrol, a plant triterpene, is known to play important role in inhibiting proliferation and inducing apoptosis of gastric cancer cells. In the present study, the mechanism of celastrol on gastric cancer cells apoptosis was examined. METHODS: We assessed effect of celastrol on NF-κB signaling pathway in gastric cancer cells using western blot and luciferase reporter assay. The real-time PCR was used to evaluate the effect of celastrol on miR-146a expression, and miR-146a mimic to evaluate whether over-expression of miR-146a can affect NF-κB activity. Finally, the effect of miR-146a on celastrol-induced anti-tumor activity was assessed using miR-146a inhibitor. RESULTS: Celastrol decreased gastric cancer cells viability in a dose-dependent. Celastrol also reduced IκB phosphorylation, nuclear P65 protein levels and NF-κB activity. Furthermore, Celastrol could increase miR-146a expression and up-regulation of miR-146a expression could suppress NF-κB activity. More important, down-regulation of miR-146a expression can reverse the effect of celastrol on NF-κB activity and apoptosis in gastric cancer cells. CONCLUSIONS: In this study, we demonstrated that the effect of celastrol on apoptosis is due to miR-146a inhibition of NF-κB activity.

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.

VTSNN: a virtual temporal spiking neural network.

Spiking neural networks (SNNs) have recently demonstrated outstanding performance in a variety of high-level tasks, such as image classification. However, advancements in the field of low-level assignments, such as image reconstruction, are rare. This may be due to the lack of promising image encoding techniques and corresponding neuromorphic devices designed specifically for SNN-based low-level vision problems. This paper begins by proposing a simple yet effective undistorted weighted-encoding-decoding technique, which primarily consists of an Undistorted Weighted-Encoding (UWE) and an Undistorted Weighted-Decoding (UWD). The former aims to convert a gray image into spike sequences for effective SNN learning, while the latter converts spike sequences back into images. Then, we design a new SNN training strategy, known as Independent-Temporal Backpropagation (ITBP) to avoid complex loss propagation in spatial and temporal dimensions, and experiments show that ITBP is superior to Spatio-Temporal Backpropagation (STBP). Finally, a so-called Virtual Temporal SNN (VTSNN) is formulated by incorporating the above-mentioned approaches into U-net network architecture, fully utilizing the potent multiscale representation capability. Experimental results on several commonly used datasets such as MNIST, F-MNIST, and CIFAR10 demonstrate that the proposed method produces competitive noise-removal performance extremely which is superior to the existing work. Compared to ANN with the same architecture, VTSNN has a greater chance of achieving superiority while consuming ~1/274 of the energy. Specifically, using the given encoding-decoding strategy, a simple neuromorphic circuit could be easily constructed to maximize this low-carbon strategy.

[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.

Influence of Different Bifurcation Angles on the Flame Propagation of Gas Explosions in Three-Way Bifurcated Pipes.

Exploring the flame propagation law in the process of gas explosion under different bifurcation angles is of great significance to the design of coal mine roadway and the prevention of gas explosion accidents. To study the variation of flame propagation law with bifurcation angle, an in-house experimental system based on a small scale three-way bifurcated pipe was developed to perform gas explosion experiments using mixtures of premixed methane-air with a methane concentration of 9.5%. Numerical simulations were conducted to study the propagation of the explosion flame. The results show that, (i) during the flame propagation process, the flame morphology evolves in the following manner: hemispherical, concave entrainment-deformation-flattening; (ii) in the case of gas explosion of three-way bifurcated pipes, there are significant differences in damage at different positions, and the damage at the pipe connection is the most serious. (iii) Although the parameters of the explosion flame in the bifurcated pipe exhibit similar trends across four different bifurcation angles, the values of the flame parameters obtained by the experiments and numerical simulations were not completely consistent. (iv) When the bifurcation angle is between the 45 and 75° bifurcation range, the area of the turbulent vortex formed by the air flow increases as the angle of the pipe widens. The research results analyze the propagation law of gas deflagration flame in the bifurcated pipeline, providing reference for the propagation mechanism of gas deflagration in underground bifurcated roadway and the formulation of prevention measures, which is conducive to preventing the propagation of gas explosion, reducing the intensity, and reducing the loss caused by gas explosion. However, large-scale tests are needed to determine the applicability of small-scale tests and calculations in this paper to full-scale mine conditions.

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.

[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.

Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding.

A new carboxylic esterase RhEst1 which catalyzes the hydrolysis of (S)-(+)-2,2-dimethylcyclopropanecarboxylate (S-DmCpCe), the key chiral building block of cilastatin, was identified and subsequently crystallized in our previous work. Mutant RhEst1A147I/V148F/G254A was found to show a 5-fold increase in the catalytic activity. In this work, molecular dynamic simulations were performed to elucidate the molecular determinant of the enzyme activity. Our simulations show that the substrate binds much more strongly in the A147I/V148F/G254A mutant than in wild type, with more hydrogen bonds formed between the substrate and the catalytic triad and the oxyanion hole. The OH group of the catalytic residue Ser101 in the mutant is better positioned to initiate the nucleophilic attack on S-DmCpCe. Interestingly, the "170-179" loop which is involved in shaping the catalytic sites and facilitating the product release shows remarkable dynamic differences in the two systems. Based on the simulation results, six residues were identified as potential "hot-spots" for further experimental testing. Consequently, the G126S and R133L mutants show higher catalytic efficiency as compared with the wild type. This work provides molecular-level insights into the substrate binding mechanism of carboxylic esterase RhEst1, facilitating future experimental efforts toward developing more efficient RhEst1 variants for industrial applications.

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.

[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.

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.

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.

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.