OBJECTIVES: To investigate the protective effect of the probiotic bacterium Streptococcus salivarius K12 (K12) against Mycoplasma pneumoniae (Mp) infection in mice. METHODS: Forty male BALB/c mice were randomized into normal control group, K12 treatment group, Mp infection group, and K12 pretreatment prior to Mp infection group. The probiotic K12 was administered daily by gavage for 14 days before Mp infection induced by intranasal instillation of Mp. Three days after Mp infection, the mice were euthanized for analysis of bronchoalveolar lavage fluid (BALF) cell counts and serum levels of secretory immunoglobulin A (sIgA), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). RT-qPCR was performed to detect the P1 and community-acquired respiratory distress syndrome ( CARDS ) toxin of Mp in the lung tissues and the mRNA expressions of TNF-α, IL-6, chemokine 1 (CXCL1), matrix metalloproteinase 9 (MMP9), mucin 5ac (MUC5ac), collagen 3a1 (Col3a1), Toll-like receptor 2 (TLR2) and TLR4; the protein expressions of TLR2 and TLR4 in the lung tissue were detected using Western blotting. Pathological changes in the lung tissue and airway remodeling were examined with HE staining and AB/PAS staining. RESULTS: Compared with the Mp-infected mice with PBS treatment, the infected mice with K12 treatment showed significantly lowered mRNA levels of P1 and CARDS in the lung tissue and reduced white blood cell counts in the BALF (P<0.05). In spite of the absence of significant differences in serum levels of inflammatory factors between the two groups, the mRNA expressions of TNF‑α, IL-6, CXCL1, MMP9, MUC5ac and COL3A1 and the mRNA and protein levels of TLR2 and TLR4 in the lung tissues were significantly lower in K12-treated mice, in which AB/PAS staining showed obviously decreased mucus secretion. CONCLUSIONS K12 pretreatment can effectively reduce pulmonary inflammatory responses, improve airway remodeling and alleviate lung injury in Mp-infected mice.
Animals ; Mice ; Pneumonia, Mycoplasma/metabolism* ; Mice, Inbred BALB C ; Toll-Like Receptor 2/metabolism* ; Mycoplasma pneumoniae ; Male ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Lung/microbiology* ; Toll-Like Receptor 4/metabolism* ; Streptococcus salivarius ; Probiotics/administration & dosage* ; Bronchoalveolar Lavage Fluid ; Matrix Metalloproteinase 9/metabolism* ; Mucin 5AC/metabolism* ; Chemokine CXCL1/metabolism* ; Immunoglobulin A, Secretory/metabolism* ; Bacterial Toxins ; Bacterial Proteins

BACKGROUND: As a potent pro-inflammatory cytokine of the interleukin (IL)-1 family, IL-18 was elevated in early active and progressive plaque-type psoriatic lesions and that serum or plasma levels of IL-18 correlated with the Psoriasis Area and Severity Index (PASI). Although results from previous studies have established that IL-18 may aggravate psoriatic inflammation, the mechanisms of this process remain unknown. In this study, IL-18 knock out (KO) mice and wild-type (WT) mice were used to investigate the effects of IL-18 within a mouse model of psoriasis. METHODS: WT and IL-18 KO mice were divided into four groups, including imiquimod (IMQ)-treated IL-18 KO group (n = 11) and WT group (n = 13) as well as their respectively gene-matched control mice (receiving vaseline; n = 12). PASI scores were used to evaluate psoriatic lesions in IMQ-treated mice. Pathological features and dermal cellular infiltration were investigated by hematoxylin and eosin staining. The levels of psoriasis-related cytokines including IL-23, IL-17, IL-12, IL-1β, IFNγ, IL-15, IL-27, and IL-4 were tested by real-time polymerase chain reaction (PCR). The protein level of IL-1β, IL-27, CXCL1, and Ly6 g were investigated by immunohistochemistry (IHC). RESULTS: Acanthosis (98.46 ± 14.12 vs. 222.68 ± 71.10 μm, P < 0.01) and dermal cell infiltration (572.25 ± 47.45 vs. 762.47 ± 59.59 cells/field, P < 0.01) were significantly milder in IMQ-induced IL-18 KO mice compared with that in WT mice. IMQ-induced IL-18 KO mice manifested larger areas of Munro microabscesses (11,467.83 ± 5112.09 vs. 4093.19 ± 2591.88 μm, P < 0.01) and scales (100,935.24 ± 41,167.77 vs. 41,604.41 ± 14,184.10 μm, P < 0.01) as compared with WT mice. In skin lesions of IL-18 KO mice, the expressions of IL-1β, IL-4, and IL-27 were all significantly upregulated but IL-17 was decreased. Histologically, strong positive signals of Ly6g were observed within the epidermis of IL-18 KO mice but expressions of CXCL1 were decreased. CONCLUSIONS IL-18 may exacerbate prominent inflammation and influence pathological features in IMQ-induced mouse model of psoriasis. IL-18 may upregulate pro-inflammatory cytokines and reduce protective cytokines, thus aggravating psoriatic inflammation. In addition, IL-18 may be involved in the formation of Munro microabscesses and scales.
Animals ; Chemokine CXCL1 ; metabolism ; Cytokines ; metabolism ; Disease Models, Animal ; Imiquimod ; toxicity ; Interleukin-17 ; metabolism ; Interleukin-18 ; metabolism ; Mice ; Mice, Knockout ; Psoriasis ; chemically induced ; genetics ; metabolism ; Skin ; immunology ; metabolism

In patients with advanced cancer, cancer-induced bone pain (CIBP) is a severe and common problem that is difficult to manage and explain. As c-Jun N-terminal kinase (JNK) and chemokine (C-X-C motif) ligand 1 (CXCL1) have been shown to participate in several chronic pain processes, we investigated the role of JNK and CXCL1 in CIBP and the relationship between them. A rat bone cancer pain model was established by intramedullary injection of Walker 256 rat gland mammary carcinoma cells into the left tibia of Sprague-Dawley rats. As a result, intramedullary injection of Walker 256 carcinoma cells induced significant bone destruction and persistent pain. Both phosphorylated JNK1 (pJNK1) and pJNK2 showed time-dependent increases in the ipsilateral spinal cord from day 7 to day 18 after tumor injection. Inhibition of JNK activation by intrathecal administration of SP600125, a selective pJNK inhibitor, attenuated mechanical allodynia and heat hyperalgesia caused by tumor inoculation. Tumor cell inoculation also induced robust CXCL1 upregulation in the ipsilateral spinal cord on day 18 after tumor injection. Inhibition of CXCL1 by intrathecal administration of CXCL1 neutralizing antibody showed a stable analgesic effect. Intrathecal administration of SP600125 reduced CXCL1 increase in the spinal cord, whereas inhibition of CXCL1 in the spinal cord showed no influence on JNK activation. Taken together, these results suggested that JNK activation in spinal cord contributed to the maintenance of CIBP, which may act through modulation of CXCL1. Inhibition of the pJNK/CXCL1 pathway may provide a new choice for treatment of CIBP.
Animals ; Antibodies, Neutralizing ; immunology ; therapeutic use ; Bone Neoplasms ; complications ; metabolism ; Cancer Pain ; drug therapy ; etiology ; metabolism ; Cell Line, Tumor ; Chemokine CXCL1 ; immunology ; metabolism ; Female ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; metabolism

Recent studies indicated that interleukin (IL)-17, growth-related oncogene (GRO)-α and IL-8 play an important role in the pathogenesis of nasal polyps. However, the effects of the increased amount of IL-17 and the production of GRO-α and IL-8 in human nasal polyp fibroblasts are not completely understood. This study aimed to determine the effects of the increased IL-17 on the changes of GRO-α and IL-8 expression in human nasal polyp fibroblasts and further investigate the mechanism of neutrophil infiltration in nasal polyps. Nasal polyp fibroblasts were isolated from six cases of human nasal polyps, and the cells were stimulated with five different concentrations of IL-17. Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of GRO-α and IL-8. The mRNA of GRO-α and IL-8 was expressed in unstimulated controls and remarkably increased by stimulation with IL-17. Moreover, the levels of GRO-α and IL-8 produced by fibroblasts were increased gradually with the increases in IL-17 concentrations. The present study showed that nasal fibroblasts can produce GRO-α and IL-8, and their production is remarkably enhanced by IL-17 stimulation, thereby clarifying the mechanism of the IL-17 mediated neutrophil infiltration in nasal polyps. These findings might provide a rationale for using IL-17 inhibitors as a treatment for nasal inflammatory diseases such as nasal polyps.
Adult ; Cells, Cultured ; Chemokine CXCL1 ; biosynthesis ; Female ; Fibroblasts ; metabolism ; pathology ; Humans ; Interleukin-17 ; pharmacology ; Interleukin-8 ; biosynthesis ; Male ; Middle Aged ; Nasal Polyps ; metabolism ; pathology ; Neutrophil Infiltration ; drug effects ; RNA, Messenger ; biosynthesis

BACKGROUNDA proinflammatory milieu emerging in the lung due to neutrophil accumulation and activation is a key in the pathogenesis of acute lung injury (ALI). 15-deoxy-Δ(12, 14)-prostaglandin J2 (15d-PGJ2), one of the terminal products of the cyclooxygenase-2 pathway, is known to be the endogenous ligand of peroxisome proliferator-activated receptor γ (PPAR-γ) with multiple physiological properties. Growing evidence indicates that 15d-PGJ2 has anti-inflammatory, antiproliferative, cytoprotective and pro-resolving effects. We investigated whether 15d-PGJ2 has a protective effect against endotoxin-induced acute lung injury in rats.

METHODSTwenty-four male Wistar rats were randomly assigned into four groups (n = 6 per group): sham+vehicle group, sham+15d-PGJ2 group, LPS+vehicle group, and LPS+15d-PGJ2 group. The rats were given either lipopolysaccharide (LPS, 6 mg/kg intravenously) or saline, and pretreated with 15d-PGJ2 (0.3 mg/kg intravenously) or its vehicle (dimethyl sulphoxide) 30 minutes before LPS. Histological alterations, wet/dry weight (W/D) ratio and myeloperoxidase (MPO) activity as well as tumor necrosis factor (TNF)-α and cytokine-induced neutrophil chemoattractant-1 (CINC-1) levels were determined in lung tissues four hours after LPS injection. Immunohistochemical analysis for intercellular adhesion molecule-1 (ICAM-1) expression and Western blotting analysis for nuclear factor (NF)-κB p65 translocation and IκBα protein levels were also studied.

RESULTS15d-PGJ2 pretreatment significantly attenuated LPS-induced lung injury, and reduced the increased W/D ratio, MPO activity, TNF-α, CINC-1 levels, and ICAM-1 expression in the lung. 15d-PGJ2 also suppressed the nuclear NF-κB p65 translocation and increased cytosolic IκBα levels.

CONCLUSIONS15d-PGJ2 protects against endotoxin-induced acute lung injury, most likely through the reduction of proinflammatory protein levels during endotoxemia subsequent to the inhibition of NF-κB activation.

Acute Lung Injury ; chemically induced ; drug therapy ; immunology ; Animals ; Chemokine CXCL1 ; metabolism ; I-kappa B Proteins ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Lipopolysaccharides ; toxicity ; Male ; NF-KappaB Inhibitor alpha ; NF-kappa B ; metabolism ; Prostaglandin D2 ; analogs & derivatives ; therapeutic use ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the effect of CXCL1 gene expression on biological function of ovarian cancer cells and its mechanism of action.

METHODSRT-PCR was used to amplify the full-length sequence of CXCL1, which was used to construct the recombinant lentiviral plasmids, and then the plasmids were packaged with human renal epithelial cell line 293T cells, and the ovarian cancer 2780 cells were transfected with CXCL1 gene by virus supernatant particles. The expression of CXCL1 mRNA and protein in 2780 cells with lentivirus-mediated CXCL1expression were determined by RT-PCR and ELISA, respectively. The growth curve, cell cycle and colony formation in vitro were measured by MTT assay, flow cytometry and colony formation counting, respectively. The cell invasion and migration in vitro was detected by Transwell chambers and migration attack methods, respectively.

RESULTSThe sequencing results showed that the recombinant lentiviral plasmid of CXCL1-pWPI had 100% homology with the CXCL1 gene, identified by BLAST analysis. RT-PCR and ELISA results showed positive expression of CXCL1 mRNA and protein in the CXCL1-PWPI group. The doubling time of the CXCL1-PWPI group was significantly shorter than that in the A2780 and PWPI groups (P < 0.05). The rate of colony-formation in the CXCL1-PWPI group was also significantly higher than that of the A2780 and PWPI groups (P < 0.05). The proportion of cells in G1 phase (44.0%) in the CXCL1-PWPI group was also significantly lower compared with that in the PWPI and A2780 groups (61.4% and 62.7%), with a significant difference (P < 0.001). There was no significant difference between the cell migration capacity (P > 0.05) of the CXCL1-PWPI, PWPI and A2780 groups, but the invasion capacity of the CXCL1-PWPI group was significantly higher than that of the PWPI and A2780 groups (P < 0.05).

CONCLUSIONThe over-expression of CXCL1 gene may promote the proliferation and invasion ability of A2780 cells in vitro.

Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Chemokine CXCL1 ; genetics ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lentivirus ; genetics ; Neoplasm Invasiveness ; Neoplasms, Glandular and Epithelial ; genetics ; metabolism ; pathology ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Plasmids ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo explore the role of early growth response factor-1 (Egr-1) signal pathway in acute intrahepatic cholestatic liver injury in rats.

METHODSA single dose (50 mg/kg) of alpha-naphthylisothiocyanate (ANIT) was administered by gavage to each experimental rat to induce intrahepatic cholestasis. Liver tissue and serum specimens were collected from rats at 24, 48 and 72 h after the intoxication. The values of Egr-1, cytokine induced neutrophil chemoattractant 1(CINC-1), macrophage inflammatory protein-2 (MIP-2) mRNA, the protein expression of inducible nitricoxide synthase (iNOS) and the values of tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6) were assayed by real-time PCR, immunohistochemistry, and ELISA, respectively. The levels of MDA, SOD, NO and MPO were assayed by thiobarbituric acid method, xanthine oxidase method, nitric acid deoxidizing assay, and colorimetric method, respectively.

RESULTSIn the model group at 24, 48, 72 h after intoxication, the values of CINC-1 and MIP-2 mRNA were higher than those of the controls (P < 0.05). In the model group at 24, 48 h after intoxication, the value of Egr-1 mRNA was higher than that of the controls (P < 0.05), but there was no significant difference at 72 h (P < 0.05). Of the model group, the absorbance value of iNOS was lower than that of the controls at 24, 48 and 72 h (P < 0.05). Of the model group at 24, 48, 72 h after intoxication, the values of TNF alpha, IL-6, MPO and MDA were higher than those of the controls (P < 0.05), but the values of NO and SOD were lower than those of the controls (P < 0.05).

CONCLUSIONSEgr-1 signal pathway is involved in acute intrahepatic cholestatic liver injury induced by ANIT. After Egr-1 was activated, CINC-1 and MIP-2 are activated consequently and attract neutrophils into the liver. TNF alpha and IL-6 are activated at the same time, so neutrophils are activated and the resulting lipid peroxidation and MDA increased, injuring the liver. iNOS and NO may play a protective role in acute intrahepatic cholestatic liver injury induced by ANIT.

1-Naphthylisothiocyanate ; Acute Disease ; Animals ; Chemokine CXCL1 ; metabolism ; Chemokine CXCL2 ; metabolism ; Cholestasis, Intrahepatic ; chemically induced ; metabolism ; pathology ; Early Growth Response Protein 1 ; metabolism ; Interleukin-6 ; metabolism ; Liver ; chemistry ; pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the time course of nuclear factor-kappaB (NF-kappaB) activation in the process of stress ulcer formation.

METHODSModel of stress ulcer was reproduced by subjecting male Sprague-Dawley rats to water-immersion restraint (WIR) stress. At indicated time after the beginning of WIR stress, animals were sacrificed and cytoplasmic and nuclear protein and total RNA were prepared from gastric corpus mucosal tissues. DNA-binding activity of NF-KB was assessed as an index of NF-kappaB activation with electrophoretic mobility shift assay. Degradation of IkappaBalpha and IkappaBbeta, the inhibitory proteins of NF-kappaB, was analyzed by Western blot analysis. Expression of NF-kappaB dependent genes including tumor necrosis factor-alpha (TNF-alpha) , interleukin-1beta (IL-1beta), cytokine-inducible neutrophil chemoattractant-1 ( CINC-1), intercellular adhesion molecule-1 (ICAM-1), and inducible nitric oxide synthase (iNOS) was detected with Northern blot analysis.

RESULTSWIR stress induced a rapid biphasic activation of gastric mucosal NF-kappaB within 15 min of the beginning of stress, peaking at 45 min and 360 min. Compared with baseline, NF-kappaB activation by stress was increased (10.6 +/- 1.3) and (8.9 +/- 1.2) fold at 45 min and 360 min, respectively (P < 0.01). Antibody supershift assays revealed that p50/p65 heterodimer was the major active component of mucosal NF-kappaB. Western blot analysis showed that degradation of IkappaBalpha and IkappaBbeta occurred at first and second wave of NF-kappaB activation. Corresponding with the rapid and persistent activation of NF-kappaB, the levels of TNF-alpha, IL-1beta, CINC-1 and ICAM-1 mRNA in gastric mucosa were markedly increased 15 to 30 min after stress, respectively. Up-regulation of iNOS mRNAs was observed 30 to 90 min after stress, and the expression of all of these genes was increased consistently until the end of stress.

CONCLUSIONNF-kappaB activation is an early event and may play an important role in proinflammatory gene over-expression in rat gastric mucosa during WIR stress.

Animals ; Chemokine CXCL1 ; metabolism ; Disease Models, Animal ; Gastric Mucosa ; metabolism ; Gene Expression ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-1beta ; metabolism ; Male ; NF-kappa B ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Stress Disorders, Traumatic ; Tumor Necrosis Factor-alpha ; metabolism ; Ulcer ; etiology ; metabolism