Objective To investigate the effect of long intergenic non-coding RNA COX2 (lincRNA-COX2) on apoptosis and polarization of Listeria monocytogenes (Lm)-infected RAW264.7 cells. Methods RAW264.7 cells were cultured and divided into control group (uninfected cells), Lm infection group, negative control of small interfering RNA (si-NC) group, si-NC and Lm infection group, small interfering RNA of lincRNA-COX2 (si-lincRNA-COX2) group, si-lincRNA-COX2 and Lm infection group. RAW264.7 cells were infected with MOI=10 Lm for 6 hours, and then the inhibition efficiency of siRNA transfection was detected by fluorescence microscope and quantitative real-time PCR (qRT-PCR). The expression levels of cleaved-caspase-3(c-caspase-3), caspase-3, B-cell lymphoma-2 (Bcl2), Bcl2 associated X protein (BAX), arginase 1 (Arg1), inducible nitric oxide synthase (iNOS) were detected by Western blot analysis. Results c-caspase-3/caspase-3, BAX/Bcl2 and iNOS were significantly up-regulated, while the level of Arg1 was down-regulated in Lm-infected RAW264.7 cells compared with control group. LincRNA-COX2 knockdown inhibited the increase of protein levels for BAX/Bcl2, c-caspase-3/caspase-3 and iNOS in Lm-infected RAW264.7 cells, while the level of Arg1 in Lm-infected RAW264.7 cells was up-regulated. Conclusion Knockdown of lincRNA-COX2 can inhibit cell apoptosis and suppress the macrophage polarization into M1 type in Lm-infected RAW264.7 cells.
Apoptosis/genetics* ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/metabolism* ; Cyclooxygenase 2/metabolism* ; Listeria monocytogenes/pathogenicity* ; Macrophages/microbiology* ; RNA, Long Noncoding/metabolism* ; RNA, Small Interfering/genetics* ; Animals ; Mice

This paper was mainly to discuss the potential role and mechanism of Lianhua Qingwen Capsules(LHQW) in inhibiting pathological inflammation in the model of acute lung injury caused by bacterial infection. For in vitro study, the mRNA expression of MCP-1 in RAW264.7 cells and THP-1 cells, the content of MCP-1 in cell supernatant, as well as the effect of LHQW on chemotaxis of macrophages were detected. For in vivo study, mice were randomly divided into 7 groups, including normal group, model group(LPS 5 mg·kg~(-1)), LHQW 300, 600 and 1 200 mg·kg~(-1)(low, middle and high dose) groups, dexamethasone 5 mg·kg~(-1) group and penicillin-streptomycin group. Then, the anal temperature was detected two hours later. Dry weight and wet weight of lung tissues in mice were determined; TNF-α and MCP-1 levels in alveolar lavage fluid and MCP-1 in serum were detected. In addition, the infiltration of alveolar macrophages was also observed and the infiltration count of alveolar macrophages was measured by CCK-8 method. HE staining was also used to observe the inflammatory infiltration of lung tissues in mice. Both of the in vitro and in vivo data consistently have confirmed that: by down-regulating the expression of MCP-1, LHWQ could efficiently decrease the chemotaxis of monocytes toward the pulmonary infection foci, thus blocking the disease development in ALI animal model.
Acute Lung Injury ; microbiology ; Animals ; Bacterial Infections ; drug therapy ; Bronchoalveolar Lavage Fluid ; Capsules ; Chemokine CCL2 ; metabolism ; Chemotaxis ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Lipopolysaccharides ; Lung ; Macrophages ; drug effects ; Mice ; RAW 264.7 Cells ; Random Allocation ; THP-1 Cells ; Tumor Necrosis Factor-alpha ; metabolism

Human intestinal bacteria play an important role in the metabolism of herbal medicines, leading to the variations in their pharmacological profile. The present study aimed to investigate the metabolism of Xiao-Cheng-Qi decoction (XCQD) by human intestinal bacteria and to discover active component combination (ACC) contributing to the anti-inflammatory activity of XCQD. The water extract of XCQD was anaerobically incubated with human intestinal bacteria suspensions for 48 h at 37 °C. A liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) method was performed for identification of the metabolites. In addition, the anti-inflammatory effects of XCQD and biotransformed XCQD (XCQD-BT) were evaluated in vitro with cytokines in RAW264.7 cells induced by lipopolysaccharide (LPS). A total of 51 compounds were identified in XCQD and XCQD-BT. Among them, 20 metabolites were proven to be transformed by human intestinal bacteria. Significantly, a combination of 14 compounds was identified as ACC from XCQD-BT, which was as effective as XCQD in cell models of inflammation. In conclusion, this study provided an applicable method, based on intestinal bacterial metabolism, for identifying combinatory compounds responsible for a certain pharmacological activity of herbal medicines.
Animals ; Anti-Inflammatory Agents ; isolation & purification ; pharmacology ; therapeutic use ; Bacteria ; metabolism ; Biotransformation ; Cytokines ; metabolism ; Drugs, Chinese Herbal ; chemistry ; metabolism ; Feces ; microbiology ; Gastrointestinal Microbiome ; drug effects ; Humans ; Inflammation ; chemically induced ; drug therapy ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; metabolism ; Mice ; Models, Biological ; Molecular Structure ; RAW 264.7 Cells

OBJECTIVE: Soluble triggering receptors expressed by myeloid cells-1 (sTREM-1) and inflammatory cytokine tumor necrosis factor-α (TNF-α) in macrophage cells were stimulated by Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) to investigate the expression of triggering receptors expressed by myeloid cells-1 (TREM-1) and further explore the correlation between TREM-1 and the pathogenesis of periodontitis. METHODS: THP-1 cells (a human monocytic cell line derived from an acute monocytic leukemia patient) were induced to differentiate THP-1 macrophages by phorbol-12-myristate-13-acetate and were injected with 0 (blank control), 0.5, or 1.0 μg·mL⁻¹ Pg-LPS. The THP-1 cells were then grouped in accordance with incubation time, and each group was incubated for 4, 6, 12, or 24 h. The expression of the TREM-1 mRNA in macrophages was detected by real-time quantitative polymerase chain reaction, while the expression of TREM-1 protein was detected by Western blot; the site where TREM-1 protein expression was observed in macrophages was detected by immunofluorescence staining, and the expression of soluble sTREM-1 and TNF-α in cell culture medium was detected by enzyme-linked immunosorbent assay. RESULTS: Compared with the blank control group, the expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in Pg-LPS-stimulated macrophages was significantly upregulated (P<0.05). The expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in the supernatant of cell culture was higher in the 1.0 μg·mL⁻¹ Pg-LPS group than in the 0.5 μg·mL⁻¹ group; this expression was statistically significant since the 6, 4, and 4 h time point (P<0.05). Cell immunofluorescence staining showed that TREM-1 protein was positive when the THP-1 macrophages was stimulated by Pg-LPS (1.0 μg·mL⁻¹) for 24 h, and the staining sites of TREM-1 were mainly located in the cell membrane of the macrophages (P<0.05). The expression level of TNF-α increased in groups stimulated by Pg-LPS, and the expression level of TNF-α was significantly higher in 1.0 μg·mL⁻¹ Pg-LPS stimulated groups than in 0.5 μg·mL⁻¹ Pg-LPS-stimulated groups since the 6 h time point (P<0.05). The expressions of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in 0.5 μg·mL⁻¹ Pg-LPS-stimulated macrophages were positively correlated with one another (r=1, P<0.05), but no statistically significant correlation was found in the expression of TNF-α. The positive correlation between sTREM-1 and TNF-α expressions was detected when macrophages were stimulated by 1.0 μg·mL⁻¹ Pg-LPS (r=1, P<0.05). CONCLUSIONS The expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in the culture supernatant in Pg-LPS-stimulated macrophages was significantly upregulated on the basis of the concentration of Pg-LPS; moreover, their upregulation was positively correlated with one another. The expression of TNF-α in the supernatant of cell culture was also upregulated and was positively correlated with the expression of sTREM-1 at the group of high Pg-LPS concentration (1.0 μg·mL⁻¹). Results reveal that TREM-1, which has been realized as a proinflammatory receptor protein, can promote the development of periodontitis by regulating the expression of TNF-α in macrophages.
Adult ; Humans ; Lipopolysaccharides ; Macrophages ; metabolism ; Myeloid Cells ; Periodontitis ; metabolism ; microbiology ; Porphyromonas gingivalis ; pathogenicity ; Triggering Receptor Expressed on Myeloid Cells-1 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

To investigate cytotoxic secondary metabolites of Micrococcus sp. R21, an actinomycete isolated from a deep-sea sediment (-6 310 m; 142 degrees 19. 9' E, 10 degrees 54. 6' N) of the Western Pacific Ocean, column chromatography was introduced over silica gel, ODS, and Sephadex LH-20. As a result, eight compounds were obtained. By mainly detailed analysis of the NMR data, their structures were elucidated as cyclo(4-hydroxy-L-Pro-L-leu) (1), cyclo(L-Pro-L-Gly) (2), cyclo( L-Pro-L-Ala) (3), cyclo( D-Pro-L-Leu) (4), N-β-acetyltryptamine (5), 2-hydroxybenzoic acid (6), and phenylacetic acid (7). Compound 1 exhibited weak cytotoxic activity against RAW264. 7 cells with IC50 value of 9.1 μmol x L(-1).
Animals ; Biological Factors ; chemistry ; isolation & purification ; metabolism ; pharmacology ; Cell Survival ; drug effects ; Macrophages ; cytology ; drug effects ; Magnetic Resonance Spectroscopy ; Mass Spectrometry ; Mice ; Micrococcus ; chemistry ; genetics ; isolation & purification ; metabolism ; Molecular Structure ; Phylogeny ; RAW 264.7 Cells ; Seawater ; microbiology ; Secondary Metabolism

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin-resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.
Actinobacillus pleuropneumoniae/genetics/*pathogenicity/*physiology ; Animals ; *Apoptosis ; Bacterial Proteins/genetics/metabolism ; Blotting, Southern ; Exotoxins/*genetics ; Hemolysin Proteins/genetics/metabolism ; *Hemolysis ; Macrophages, Alveolar/metabolism/*microbiology ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Swine ; Virulence

OBJECTIVETo determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism.

METHODSOmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays.

RESULTSThe bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01).

CONCLUSIONExpression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Antigens, Bacterial ; genetics ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; metabolism ; Cell Line ; Chaperonin 60 ; genetics ; metabolism ; Humans ; Leptospira interrogans ; genetics ; immunology ; pathogenicity ; Lipoproteins ; genetics ; metabolism ; Macrophages ; microbiology

The incidence of diabetes mellitus is increasing among companion animals. This disease has similar characteristics in both humans and animals. Diabetes is frequently identified as an independent risk factor for infections associated with increased mortality. In the present study, homozygous diabetic (db/db) mice were infected with Listeria (L.) monocytogenes and then treated with the anti-diabetic drug exendin-4, a glucagon-like peptide 1 analogue. In aged db/db mice, decreased CD11b+ macrophage populations with higher lipid content and lower phagocytic activity were observed. Exendin-4 lowered high lipid levels and enhanced phagocytosis in macrophages from db/db mice infected with L. monocytogenes. Exendin-4 also ameliorated obesity and hyperglycemia, and improved ex vivo bacteria clearance by macrophages in the animals. Liver histology examined during L. monocytogenes infection indicated that abscess formation was much milder in exendin-4-treated db/db mice than in the control animals. Moreover, mechanistic studies demonstrated that expression of ATP binding cassette transporter 1, a sterol transporter, was higher in macrophages isolated from the exendin-4-treated db/db mice. Overall, our results suggest that exendin-4 decreases the risk of infection in diabetic animals by modifying the interaction between intracellular lipids and phagocytic macrophages.
ATP-Binding Cassette Transporters/metabolism ; Age Factors ; Animals ; Blood Chemical Analysis ; Cholesterol/metabolism ; Diabetes Mellitus, Type 2/*drug therapy/genetics ; Dyslipidemias/drug therapy/genetics ; Female ; Hyperglycemia/drug therapy/genetics ; Hypoglycemic Agents/*therapeutic use ; Injections, Intraperitoneal ; *Lipid Metabolism/drug effects ; Listeria monocytogenes/*drug effects/immunology ; Listeriosis/*drug therapy/immunology/microbiology ; Macrophages/drug effects/*metabolism ; Mice ; Obesity/drug therapy/genetics ; Peptides/*therapeutic use ; Phagocytosis/drug effects ; Venoms/*therapeutic use

OBJECTIVETo investigate the contamination state of Legionella in cooling water samples from different places in Wuxi city and reveal the molecular biological characteristics of Legionella strains.

METHODS112 parallel water samples (500 ml each) were collected from 56 sites in Wuxi city during year 2009 - 2010. The samples were used for Legionella test and quantitative culture. The isolated Legionella strains were used for serotyping, pulsed-field gel electrophoresis (PFGE), sequence-based typing (SBT), and intracellular growth were tested.

RESULTSThe positive proportion of Legionella was 39. 3% (22/56) among all sampling sites. A total of 29 Legionella strains were isolated, and the serotypes include LP1, LP3, LP5 and LP6. LP1 serotype was the major one with a proportion of 65.5% (19/29). 29 Legionella strains got 17 PFGE types. There were 10 SBT types among 10 Legionella strains with different PFGE types. Comparing to LP1 strain (ATCC 33152), WX2011062 (LP6) and WX2011067 (LP5) had strong intracellular growth ability in mouse peritoneal macrophages J774 cell line (the amount of intracellular bacteria on day 0 after infection were (5.5 +/- 1.32) x 10(5), (3.9 +/- 0.60) x 10(5), (7.8 +/- 0.76) x 10(5) CFU/ml, respectively; the amount of intracellular bacteria on day 3 after infection were (58.3 +/- 1.61) x 10(5), (2700.0 +/- 655.74) x 10(5), (3066.7 +/- 208.17) x 10(5) CFU/ml, respectively).

CONCLUSIONThe Legionella contamination existed in cooling water samples from different places in Wuxi city. Legionella strains isolated showed high genetic variation. Some Legionella strains had vigorous intracellular growth ability.

Air Conditioning ; Animals ; Cells, Cultured ; Environmental Microbiology ; Legionella ; genetics ; growth & development ; isolation & purification ; Legionella pneumophila ; growth & development ; isolation & purification ; Macrophages ; microbiology ; Mice ; Serotyping ; Water Microbiology

OBJECTIVETo determine the distribution of integrins and calcium channels on major human and mouse host cells of Leptospira species.

METHODSThe expression of β1, β2 and β3 integrins was detected with immunofluorescence assay on the surface of human monocyte line THP-1, mouse mononuclear-macrophage-like cell line J774A.1, human vascular endothelial cell line HUVEC, mouse vascular endothelial cell EOMA, human hepatocyte line L-02, mouse hepatocyte line Hepa1-6, human renal tubular epithelial cell line HEK-293, mouse glomerular membrane epithelial cell line SV40-MES13, mouse collagen blast line NIH/3T3, human and mouse platelets. The distribution of voltage gate control calcium channels Cav3.1, Cav3.2, Cav3.3 and Cav2.3, and receptor gate calcium channels P(2)X(1), P(2)2X(2), P(2)X(3), P(2)X(4), P(2)X(5), P(2)X(6) and P(2)X(7) were determined with Western blot assay.

RESULTSβ1 integrin proteins were positively expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, L-02, Hepa1-6 and HEK-239 cells as well as human and mouse platelets. β2 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, and NIH/3T3 cells. β3 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, Hepa1-6, HEK-239 and NIH/3T3 cells as well as human and mouse platelets. P(2)X(1) receptor gate calcium channel was expressed on the membrane surface of human and mouse platelets, while P(2)X(5) receptor gate calcium channel was expressed on the membrane surface of J774A.1, THP-1, L-02, Hepa1-6, HEK-239 and HUVEC cells. However, the other calcium channels were not detected on the tested cell lines or platelets.

CONCLUSIONThere is a large distribution diversity of integrins and calcium channel proteins on the major human and mouse host cells of Leptospira species, which may be associated with the differences of leptospira-induced injury in different host cells.

Animals ; Blood Platelets ; metabolism ; microbiology ; Calcium Channels ; metabolism ; Cell Line ; HEK293 Cells ; Host-Pathogen Interactions ; Human Umbilical Vein Endothelial Cells ; Humans ; Integrins ; metabolism ; Leptospira ; pathogenicity ; Macrophages ; metabolism ; microbiology ; Mice ; Monocytes ; metabolism ; microbiology ; NIH 3T3 Cells