The 70% ethanol extract of the whole plant of Souliea vaginata was purified by multi-chromatographic methods including macroporous resin,silica gel,Sephadex LH-20,and C18-reversed-phase column chromatography. A new spirocyclic cycloartane triterpenoid was isolated and identified as( 16 R*,20 R*,23 S*,24 R*,25 S*)-16,23: 23,26-diepoxy-15α,24,25-trihydroxy-9,19-cycloart-3β-O-β-D-xylopyranoside( 1),and named as soulieoside S. Its planar structure and relative configuration were determined by spectroscopic techniques including 2 D NMR and HRESI-MS. As one of the main components of S. vaginata,compound 1 was evaluated for its anti-inflammatory activity by a lipopolysaccharide( LPS)-stimulated NO production model in RAW264. 7 macrophages,but it didn't show NO production inhibitory effect.
Actaea/metabolism* ; Glycosides ; Lipopolysaccharides ; Molecular Structure ; Triterpenes/metabolism*

OBJECTIVEEndotoxemia is a serious syndrome resulting in multi-organ failure. Once it happens, the penetration of small intestine epithelium increases, body liquid losses, then effective circulating blood decreases and serious metabolic acidosis, serious hypotension, systolic failure, and even shock may occur. In this pathological process, endotoxin, tumor necrosis alpha and systolic dysfunction play important roles. Nowadays, many studies have been done to resolve the systolic dysfunction, but too much attention had been paid to the followings: the depressions of myocardium caused by tumor necrosis alpha, other inflammatory factors, endotoxin and metabolic acidosis; the disturbance of blood vessel-nerve regulations; nitric oxide (NO)/inducible nitric oxide synthase (iNOS) over-synthesis and the decreased density of beta-receptors in the myocardium and/or their activities. Little attention has been paid to the relationship between alpha sarcmeric actin (alpha-SA) and systolic dysfunction during endotoxemia. Glutamine (Gln) can be metabolized into glutathione, an eliminator of free radical. It has been used in preventing myocardial damage from reperfusion. This study aimed to observe the dynamic changes of alpha-SA and mRNA expressions in rats with endotoxemia and examine the effects of Gln on them.

METHODSClassical rat model of endotoxemia was established by intraperitoneal injection of LPS (4 mg/kg, Escherichia coli O55:B5, Sigma). 121 Wistar 18-day-rats were divided into three groups randomly, (1) 0 h control group (normal saline: 1 ml/kg, n = 11). (2) LPS group (LPS: 4 mg/kg, n = 55). (3) Gln group (LPS: 4 mg/kg and immediately 13.64%; Gln: 1 ml/kg, Fresenus, n = 55), Furthermore, LPS and Gln groups were divided into 2, 4, 6, 24 and 72 h time points (n = 11). Each time point of LPS and Gln as well as control rats were anaesthetized at each time point with 1% chloral hydrate injected intraperitoneally at the dosage of 1 ml/kg. Then rats were sacrificed at appoint time, and the hearts were isolated. Eight of them were put in 76 degrees C liquid nitrogen and then frozen in minute 80 degrees C icebox in order to measure the expression of alpha-SA mRNA by RT-PCR. Three of them were fixed in 4% formaldehydum polymerisatum for 12 to 16 h, then the expression of alpha-SA was detected by immunohistochemistry.

RESULTS(1) Compared to 0 h, the expressions of alpha-SA and mRNA in LPS group were significantly depressed (P < 0.01). In LPS group, the lowest was at 6 - 24 h, while in Gln group, it was postponed to 24 h. At 72 h, there was no difference in expressions of alpha-SA between Gln and 0 h group (P > 0.05). (2) Comparing at same time point, the expressions of alpha-SA were significant higher in Gln group than those in LPS group, while the expressions of alpha-SA mRNA in Gln group were high at 4-72 h. There was, however, no significant difference at early phase (P > 0.05).

CONCLUSIONAlpha-SA and its mRNA expression were depressed in LPS-induced endotoxemia, especially from 6 to 24 h. It could damage the systolic function. alpha-SA decrease in endotoxemia was due to the inhibited synthesis other than the promoted degradation. Glutamine could inhibit the effects of LPS on both alpha-SA and its mRNA expressions.

Actins ; metabolism ; Animals ; Endotoxemia ; metabolism ; Glutamine ; pharmacology ; Lipopolysaccharides ; Myocardium ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar

Objective To investigate the effects of lipopolysaccharide (LPS) on human pulmonary vascular endothelial cells (HPVECs) cytoskeleton and perform biological analysis of the microRNA (miRNA) spectrum. Methods The morphology of HPVECs was observed by microscope, the cytoskeleton by FITC-phalloidin staining, and the expression of VE-cadherin was detected by immunofluorescence cytochemical staining; the tube formation assay was conducted to examine the angiogenesis, along with cell migration test to detect the migration, and JC-1 mitochondrial membrane potential to detect the apoptosis. Illumina small-RNA sequencing was used to identify differentially expressed miRNAs in NC and LPS group. The target genes of differentially expressed miRNAs were predicted by miRanda and TargetScan, and the functional and pathway enrichment analysis was performed on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Further biological analysis of related miRNAs was carried out. Results After the LPS got induced, the cells became round and the integrity of cytoskeleton was destroyed. The decreased expression of VE-cadherin was also observed, along with the decreased ability of angiogenesis and migration, and increased apoptosis. Sequencing results showed a total of 229 differential miRNAs, of which 84 miRNA were up-regulated and 145 miRNA were down-regulated. The target gene prediction and functional enrichment analysis of these differential miRNA showed that they were mainly concentrated in pathways related to cell connection and cytoskeleton regulation, cell adhesion process and inflammation. Conclusion In vitro model of lung injury, multiple miRNAs are involved in the process of HPVECs cytoskeleton remodeling, the reduction of barrier function, angiogenesis, migration and apoptosis.
Humans ; Lipopolysaccharides/pharmacology* ; Endothelial Cells/metabolism* ; MicroRNAs/metabolism* ; Lung/metabolism* ; Cytoskeleton ; Gene Expression Profiling

This study aimed to examine the number of circulating Toll-like receptor 4 (TLR4) + CD14+ monocytes in patients with different stages of chronic kidney disease (CKD), their responses to lipopolysaccharide (LPS), and to explore the potential association of the number of TLR4+CD14+ monocytes with clinical laboratory measures. The numbers of TLR4+CD14+, LPS-stimulated TNF-α+CD14+ and interleukin (IL)-6+CD14+ monocytes were determined by flow cytometry in 9 patients with stage 3 CKD, 11 with stage 4 CKD, 16 with stage 5 CKD, and 19 healthy controls (HCs). Their laboratory tests were performed by routine methods and the potential association among these measures was analyzed by Pearson's correlation analysis. The numbers of CD14+, CD14+TLR4+, LPSstimulated TNF-α+CD14+ and IL-6+CD14+ monocytes in patients with CKD were significantly less than those of HCs (all P<0.05), and were negatively associated with patient disease severity. The number of CD14+TLR4+ monocytes was positively correlated with estimated glomerular filtration rate (eGFR, P<0.001) and the levels of hematocrit (P<0.01), but negatively correlated with the levels of blood urine nitrogen, serum creatinine, and C-reactive protein (P<0.001 for all), in the CKD patients. Our data indicate that significant reduction in the number of TLR4+ monocytes and their impaired responses to LPS may be associated with the progression of CKD in Chinese patients.
Cytokines ; metabolism ; Humans ; Kidney Failure, Chronic ; metabolism ; Lipopolysaccharide Receptors ; metabolism ; Lipopolysaccharides ; pharmacology ; Monocytes ; metabolism ; Toll-Like Receptor 4 ; metabolism

OBJECTIVE: To detect cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release in human nasal epithelia (HNE) induced by lipopolysaccharide (LPS) in different concentration gradient and time gradient, and to investigate their roles in nasal inflammatory pathogenesis. METHOD: Western Blot and fluorescent real time quantitative PCR were performed to detect the expression of COX-2 in HNE induced by LPS and blocked by selective inhibitor of COX-2. The concentrations of PGE2 were determined by enzyme immunoassay. RESULT: Low expressions of COX-2 and PGE2 were detected in normal HNE. COX-2 expression and PGE2 release increased in HNE induced by LPS in time-dependent or dose-dependent manner. The increased release of PGE2 was later than that of COX-2 expression. COX-2 expression and PGE2 release were dose-dependently attenuated by selective inhibitor of COX-2. CONCLUSION LPS effectively induces COX-2 expression and PGE2 release in HNE. And COX-2 is responsible for the synthesis of PGE2. These results indicate that the increased expression of COX-2 and PGE2 is involved in the inflammation of HNE induced by LPS in vitro.
Cells, Cultured ; metabolism ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; biosynthesis ; metabolism ; Epithelial Cells ; metabolism ; Humans ; Lipopolysaccharides ; Nasal Mucosa ; cytology ; metabolism

This study aimed to explore the potentiating effect and mechanism of the extract of Jingfang Granules(JFG) on the activation of macrophages. The RAW264.7 cells were treated with JFG extract and then stimulated by multiple agents. Subsequently, mRNA was extracted, and reverse transcription-polymerase chain reaction(RT-PCR) was used to measure the mRNA transcription of multiple cytokines in RAW264.7 cells. The levels of cytokines in the cell supernatant were detected by enzyme-linked immunosorbent assay(ELISA). In addition, the intracellular proteins were extracted and the activation of signaling pathways was determined by Western blot. The results showed that JFG extract alone could not promote or slightly promote the mRNA transcription of TNF-α, IL-6, IL-1β, MIP-1α, MCP-1, CCL5, IP-10, and IFN-β, and significantly enhance the mRNA transcription of these cytokines in RAW264.7 cells induced by R848 and CpG in a dose-dependent manner. Furthermore, JFG extract also potentiated the secretion of TNF-α, IL-6, MCP-1, and IFN-β by RAW264.7 cells stimulated with R848 and CpG. As revealed by mechanism analysis, JFG extract enhanced the phosphorylation of p38, ERK1/2, IRF3, STAT1, and STAT3 in RAW264.7 cells induced by CpG. The findings of this study indicate that JFG extract can selectively potentiate the activation of macrophages induced by R848 and CpG, which may be attributed to the promotion of the activation of MAPKs, IRF3, and STAT1/3 signaling pathways.
Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Plant Extracts/metabolism* ; Lipopolysaccharides/pharmacology* ; Macrophages ; Cytokines/metabolism* ; RNA, Messenger/metabolism*

During Toxoplasma gondii infection, macrophages, dendritic cells, and neutrophils are important sources of pro-inflammatory cytokines from the host. To counteract the pro-inflammatory activities, T. gondii is known to have several mechanisms inducing down-regulation of the host immunity. In the present study, we analyzed the production of proand anti-inflammatory cytokines from a human myelomonocytic cell line, THP-1 cells, in response to treatment with T. gondii lysate or lipopolysaccharide (LPS). Treatment of THP-1 cells with LPS induced production of IL-12, TNF-alpha, IL-8, and IL-10. Co-treatment of THP-1 cells with T. gondii lysate inhibited the LPS-induced IL-12, IL-8 and TNF-alpha expression, but increased the level of IL-10 synergistically. IL-12 and IL-10 production was down-regulated by anti-human toll-like receptor (TLR)-2 and TLR4 antibodies. T. gondii lysate triggered nuclear factor (NF)-kappaB-dependent IL-8 expression in HEK293 cells transfected with TLR2. It is suggested that immunosuppression induced by T. gondii lysate treatment might occur via TLR2-mediated NF-kappaB activation.
Animals ; Cell Line ; Cytokines/*biosynthesis ; Humans ; Inflammation/metabolism ; Lipopolysaccharides/*pharmacology ; Macrophages/*drug effects/*metabolism ; Toxoplasma

OBJECTIVETo compare the ability of the polysaccharide from various Porphyromonas gingivalis (Pg) type and clinical strains in inducing THP-1 cells to produce cytokines interleukin(IL)-1β, IL-8, and tumor necrosis factor(TNF)-α, in order to analyze the immunogenicity of Pg polysaccharide components and the virulence-associated factors of this periodontal pathogen.

METHODSThe bacterial polysaccharide was extracted from high virulent Pg strains, W83, SJD2, SJD12 and low virulent Pg, ATCC33277, SJD4, SJD5, and SJD11 by phenol-water extraction. The extracted polysaccharide was used to stimulate the THP-1 cells with different simulation periods and doses. The level of the cytokines, including IL-1β,IL-8 and TNF-α in the cell culture suspension was measured by enzyme-linked immunosorbent assay(ELISA).

RESULTSThe polysaccharide extraction of Pg strains was composed of lipopolysaccharide(LPS) and capsular polysaccharide. The secretion of IL-1β, IL-8 and TNF-α, produced by the THP-1 cells showed in a time- and dose-dependent manner in the medium containing 10% fetal bovine serum. The level of these cytokines of the high virulent strains was higher than that of the low virulent strains in medium containing 1% fetal bovine serum.Four hours after stimulation with polysaccharide extracted from high virulent strains, the levels of IL-1β,IL-8, and TNF-α in the cell suspension were (1 639 ± 497), (1 648 ± 513) and (140 ± 48) µg/L, respectively, whereas for low virulent strains, the levels of IL-1β, IL-8, and TNF-α were (773 ± 382), (892 ± 400) and (67 ± 33) µg/L, respectively.

CONCLUSIONSPolysaccharide extracted from Pg could induced the THP-1 cells to secrete the cytokines of IL-1β, IL-8 and TNF-α. The level of the cytokines produced by the THP-1 cells associates with the bacterial virulent properties.

Cytokines ; metabolism ; Interleukin-1beta ; Interleukin-8 ; Lipopolysaccharides ; physiology ; Porphyromonas gingivalis ; metabolism ; Tumor Necrosis Factor-alpha

Animals ; Cyclooxygenase 2 ; metabolism ; Lipopolysaccharides ; Liver Failure ; metabolism ; Male ; Mice ; Mice, Inbred BALB C

Cell Line ; Cyclin-Dependent Kinase 5 ; metabolism ; Humans ; Lipopolysaccharides ; Microglia ; cytology ; drug effects ; metabolism ; Stilbenes ; pharmacology