OBJECTIVETo investigate the change in the expression of tight junction protein ZO-1 in intestinal epithelial cells (Caco-2 cells) and the protective effect of eicosapentaenoic acid (EPA) after adherent-invasive Escherichia coli (E.coli) LF82 infection.

METHODSThe Caco-2 cell line was used to establish an in vitro model of tight junction of intestinal epithelial cells. Caco-2 cells were divided into EPA treatment groups (0, 25, 50, 100, and 200 μmol/L EPA) and EPA (0, 25, 50, 100, and 200 μmol/L EPA)+E.coli LF82 treatment (0, 6, and 12 hours) groups. A microscope was used to observe the morphological characteristics of the cells. MTT assay was used to determine the cell growth curve. The activity of alkaline phosphatase (ALP) at both sides of the cell membrane was compared to evaluate the Caco-2 cell model. MTT assay and flow cytometry were used to investigate the effects of different concentrations of EPA on the survival rate and apoptosis rate of Caco-2 cells. RT-qPCR was used to measure the mRNA expression of ZO-1 in Caco-2 cells after EPA and/or E.coli LF82 treatment. ELISA was used to measure the change in the level of tumor necrosis factor-α (TNF-α) in culture supernatant.

RESULTSAfter EPA treatment (25 and 50 μmol/L), the proliferation of Caco-2 cells was induced in a dose-dependent manner. The survival rates of the cells were significantly higher than those in the control group (P<0.05). The EPA treatment (100 and 200 μmol/L) groups had a significant inhibitory effect on the proliferation of Caco-2 cells in a dose-dependent manner. The survival rates of the cells were significantly lower than those in the control group (P<0.05). The EPA treatment (100 and 200 μmol/L) groups had a significant increase in cell apoptosis rate compared with the control group (P<0.05). The 6- and 12-hour E.coli LF82 treatment groups had decreasing mRNA expression of ZO-1 in Caco-2 cells over the time of treatment and had significantly lower mRNA expression of ZO-1 than the untreated group (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed an increase in the mRNA expression of ZO-1 with the increasing concentration of EPA, as well as significantly higher mRNA expression of ZO-1 than the Caco-2 cells treated with E.coli LF82 alone (P<0.05). The Caco-2 cells treated with E.coli LF82 alone for 6 or 12 hours had increasing secretion of TNF-&alpha; over the time of treatment and had significantly higher secretion than the untreated Caco-2 cells (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 μmol/L EPA for 6 or 12 hours showed a reduction in the secretion of TNF-&alpha; with the increasing concentration of EPA and had significantly lower secretion than the Caco-2 cells treated with E.coli LF82 alone (P<0.05).

CONCLUSIONSEPA can effectively prevent the destruction of tight junction of intestinal epithelial cells induced by E.coli LF82 infection and inhibit the secretion of inflammatory factors. Therefore, it has a certain protective effect on intestinal mucosal barrier.

Apoptosis ; drug effects ; Caco-2 Cells ; Eicosapentaenoic Acid ; pharmacology ; Escherichia coli ; pathogenicity ; Humans ; Intestinal Mucosa ; metabolism ; microbiology ; RNA, Messenger ; analysis ; Tight Junctions ; drug effects ; Tumor Necrosis Factor-alpha ; secretion ; Zonula Occludens-1 Protein ; genetics

OBJECTIVETo study whether the protective effects of glutamine against intestinal injury are associated with the regulation of intestinal secretion of NF-κB and TNF-&alpha; by glutamine.

METHODSTwenty-four 10-day-old Wistar rat pups were randomly intraperitoneally injected with normal sodium of 1 mL/kg (control group; n=8), LPS of 5 mg/kg (endotoxemia group; n=8) or LPS of 5 mg/kg plus glutamine of 10 mL/kg (glutamine group; n=8). The rats were sacrificed 3 hrs after injection. A segment of distal ileum was dissected. The pathologic changes of the small intestine were observed under an optical microscope (hematoxylin-eosin staining). The expression of intestinal NF-κB mRNA and protein were detected by RT-PCR and Western blot respectively. The TNF-&alpha; level in intestinal tissues was measured using ELISA.

RESULTSThe structure of the small intestine in the control group remained normal. The inflammation cells infiltration and the edema of interstitial substance and epithelials were observed in the endotoxemia and glutamine groups. The pathologic changes in the glutamine group were significantly alleviated when compared with the endotoxemia group. The expression of NF-κB mRNA and protein in the glutamine group was significantly lower than that in the endotoxemia group (P<0.01). The secretion of TNF-&alpha; in the glutamine group was significantly lower than that in the endotoxemia group (P<0.01).

CONCLUSIONSThe protective effects of glutamine against intestinal injury may be associated with the down-regulation of intestinal NF-κB mRNA and protein expression and the reduction of the secretion of TNF-&alpha;.

Animals ; Endotoxemia ; metabolism ; Female ; Glutamine ; pharmacology ; Intestines ; drug effects ; metabolism ; Male ; NF-kappa B ; analysis ; genetics ; secretion ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo investigate the effect of ADMA on macrophage migration inhibitory factor (MIF) expression and tumor necrosis factor-&alpha; (TNF-&alpha;) and IL-8 secretion in THP-1 monocyte-derived macrophages. METHIDS: THP-1 monocytes were induced to differentiate into macrophages by a 24-h incubation with 160 nmol/L PMA. The THP-1 monocyte-derived macrophages were exposed to different concentrations of ADMA for 24 h, and the changes in MIF mRNA and protein expressions were analyzed with RT-PCR and Western blotting, respectively. Enzyme-linked immunosorbent assay was used to detect the levels of TNF-&alpha; and IL-8 in the supernatant of THP-1-derived macrophages following ADMA treatments.

RESULTSADMA obviously up-regulated MIF mRNA and protein expressions in THP-1-derived macrophages in a concentration- dependent manner. Exposure of the cells to 15 µmol/L ADMA for 24 h showed the most potent effect in up-regulating MIF mRNA and protein expressions. ADMA treatment also resulted in a dose-dependent increase of the levels of TNF-&alpha; and IL-8 in the culture supernatant of the macrophages, and the peak levels occurred following the treatment with 15 µmol/L ADMA.

CONCLUSIONADMA can up-regulate MIF expression and induce TNF-&alpha; and IL-8 secretion in THP-1 monocyte-derived macrophages.

Arginine ; analogs & derivatives ; pharmacology ; Cell Differentiation ; Cell Line ; Humans ; Interleukin-8 ; secretion ; Intramolecular Oxidoreductases ; genetics ; metabolism ; Macrophage Migration-Inhibitory Factors ; genetics ; metabolism ; Macrophages ; cytology ; metabolism ; Monocytes ; cytology ; Phenanthrenes ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo study the experimental efficacy of Qushi Huayu Decoction (祛湿化瘀方,QHD) on, protein and gene expression of cathepsin B (ctsb) in HepG2 cells induced by free fatty acids (FFAs).

METHODSThe model of HepG2 steatosis and tumor necrosis factor-&alpha; (TNF-&alpha;) secretion was induced by long-chain FFAs. HepG2 cells were divided into 4 groups: control group (group C), model group (group M), low-dose QHD group (group L) and high-dose QHD group (group H). Long-chain FFAs were added to groups M, L and H. The 10% blank-control serum was added to group C and M, while 5% and 10% QHD-containing sera were added to group L and H, respectively. The levels of serum TNF-&alpha; and cellular triglyceride (TG) were detected. Cellular p-IκB and ctsb expression were detected using Western blot and PCR. The expression and distribution of ctsb were observed by immunofluorescence.

RESULTSAfter incubating with FFA for 24 h, TG deposition in HepG2, TNF-&alpha; content in cell supernatant, the protein expression of cellular ctsb and P-IκB, as well as mRNA expression of ctsb increased markedly in group M compared with group C (P<0.05, P<0.01). Compared with group M, TG deposition, the expression of cellular ctsb, P-IκB and ctsb mRNA in groups L and H, as well as TNF-&alpha; content in group H, decreased significantly (P<0.05). Cell immunochemical fluorescence studies showed that ctsb was released from lysosomes and distributed in the cytoplasm extensively and diffusedly after being stimulated with FFA. In this study, these above-mentioned changes were inhibited markedly in groups L and H.

CONCLUSIONQHD might have a direct inhibitory effect on the ctsb target in the FFA-ctsb-TNF&alpha; pathway of hepatic lipotoxicity.

Cathepsin B ; genetics ; metabolism ; Cell Death ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Fatty Acids ; pharmacology ; Gene Expression Regulation, Enzymologic ; drug effects ; Hep G2 Cells ; Humans ; I-kappa B Proteins ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; NF-KappaB Inhibitor alpha ; RNA, Messenger ; genetics ; metabolism ; Subcellular Fractions ; drug effects ; enzymology ; Triglycerides ; metabolism ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo evaluate the effect of Jianpi Huoxue decoction (JHD)-containing serum on tumor necrosis factor-alpha (TNF-alpha) secretion and endotoxin receptor gene expression in RAW264.7 cells induced by lipopolysaccharide (LPS).

METHODSThe cytotoxicity of blank-control serum and JHD-containing serum at different concentrations were evaluated through the lactate dehydrogenase (LDH) assay in RAW264.7 cells. RAW264.7 cells were divided into six groups: 5% blank-control serum group (C1, n=3), 5% blank-control serum plus LPS group (L1, n=4), 5% JHD-containing serum plus LPS group (J1, n=4), 10% blank-control serum group (C2, n=3), 10% blank-control serum plus LPS group (L2, n=4), and 10% JHD-containing serum plus LPS group (J2, n=4). After cultured with the corresponding serum for 1 h, cells in L1, L2, J1 and J2 were treated with LPS (0.1 microg/mL) for 12 h without rinse. The supernate, cells, protein and RNA were collected for assay. TNF-alpha in the culture supernate was assayed by the enzyme linked immunosorbent assay (ELISA). Protein expression of TNF-alpha in RAW cells was detected by Western-blot. TNF-alpha, Toll-like receptor 2 (TLR2), TLR4 and CD14 mRNA expression in RAW cells were detected by real-time RT-PCR.

RESULTSThe LDH assay supported that cultured for 24 h or less with the JHD-containing serum at the concentration of 10% or lower, RAW264.7 cells showed no cytotoxicity. After stimulation with LPS for 2 h, TNF-alpha in the culture supernate of the 5% blank-control serum plus LPS group (L1, P=0.03), 10% blank-control serum plus LPS group (L2, P=0.002) and in the cell layer (P=0.01) of these groups increased remarkably. After stimulation with LPS for 1 h, the mRNA expression of TNF-alpha (P=0.004), TLR (P=0.03), CD14 (P=0.004) was up-regulated obviously. In the 10% JHD-containing serum plus LPS group (J2), the protein expression of TNF-alpha in both supernate (P=0.04) and cell layer (P=0.04), gene expression of TNF-alpha (P=0.03), TLR4 (P=0.001), CD14 (P=0.001) were all inhibited. On the other hand, the TLR2 mRNA expression was not up-regulated after LPS stimulation in the 10% blank-control serum plus LPS group (L2).

CONCLUSIONJHD-containing serum inhibited the LPS-induced cytokines expression in RAW264.7 which was probably associated with its inhibitory effect on the mRNA expression of LPS receptors TLR and CD14.

Animals ; Cell Line ; Culture Media ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression ; drug effects ; Lipopolysaccharide Receptors ; genetics ; Lipopolysaccharides ; pharmacology ; Macrophages ; cytology ; drug effects ; metabolism ; Male ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Immunologic ; genetics ; Serum ; Signal Transduction ; drug effects ; Toll-Like Receptor 2 ; genetics ; Toll-Like Receptor 4 ; genetics ; Tumor Necrosis Factor-alpha ; metabolism ; secretion

OBJECTIVETo study the effect of a new obesity-related gene NYGGF4 on the insulin sensitivity and secretory function of adipocytes.

METHODS3T3-L1 preadipocytes transfected with either an empty expression vector (pcDNA3.1; control group) or an NYGGF4 expression vector (NYGGF4-pcDNA3.1) were cultured in vitro and differentiated into the matured adipocytes with the standard insulin plus dexamethasone plus 3-isobutyl-methylxanthine (MDI) induction cocktail. 2-deoxy-D-[3H] glucose uptake was determined by liquid scintillation counting. Western blot was performed to detect the protein content and translocation of glucose transporter 4 (GLUT4). The supernatant concentrations of TNF-alpha, IL-6, adiponectin and resistin were measured using ELISA.

RESULTSNYGGF4 over-expression in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. NYGGF4 over-expression impaired insulin-stimulated GLUT4 translocation without affecting the total protein content of GLUT4. The concentrations of TNF-alpha, IL-6, adiponectin and resistin in the culture medium of 3T3-L1 transfected with NYGGF4 were not significantly different from those in the control group.

CONCLUSIONSNYGGF4 over-expression impairs the insulin sensitivity of 3T3-L1 adipocytes through decreasing GLUT4 translocation and had no effects on the secretory function of adipocytes.

3T3-L1 Cells ; Adipocytes ; drug effects ; secretion ; Adiponectin ; secretion ; Animals ; Carrier Proteins ; genetics ; physiology ; Glucose ; metabolism ; Glucose Transporter Type 4 ; analysis ; metabolism ; Insulin ; pharmacology ; Interleukin-6 ; secretion ; Mice ; Resistin ; analysis ; Transfection ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo investigate whether low molecular weight heparin (LMWH) may suppress the expression and secretion of vascular endothelial growth factor (VEGF) from tumor cells in vitro and inhibit the VEGF-induced proliferation of human tumor vascular endothelial cells.

METHODSHuman lung cancer cell line A549, human liver cancer cell line HepG2, human colon carcinoma cell lines HCT116 and HCT8 were used in this study. The expression levels of VEGF and TNF-alpha (tumor necrosis factor-alpha) in the tumor cells with or without pretreatment of LMWH/heparin were measured by standard sandwich ELISA technique. The VEGF mRNA level of HepG2 cells cultured with or without LMWH/heparin was determined by RT-PCR and real time PCR. Human umbilical vein endothelial cells (HUVEC) were cultured in tissue culture medium (TCM) with or without LMWH/heparin for 3 days. Then non-radioactive cell proliferation assay (MTS) kit and cell cycle assay by flow cytometry were performed to measure the proliferation of HUVEC.

RESULTSThe VEGF levels in the control, LMWH, and heparin groups of the pulmonary adenocarcinoma cell line A549 were (1045.89 +/- 165.30) pg/ml, (782.45 +/- 67.17) pg/ml and (916.54 +/- 71.25) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups of the colon adenocarcinoma cell line HCT116 were (955.76 +/- 51.14) pg/ml, (822.89 +/- 142.39) pg/ml and (951.77 +/- 188.22) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups in the colon adenocarcinoma cell line HCT8 were (1290.62 +/- 41.23) pg/ml, (1063.34 +/- 63.82) pg/ml and (1257.14 +/- 11.40) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups in the liver cancer cell line HepG2 were (1083.00 +/- 134.35) pg/ml, (758.00 +/- 84.85) pg/ml and (874.00 +/- 22.62) pg/ml, respectively. The VEGF expression levels in the above mentioned cell lines cultured in TCM were significantly reduced in the LMWH-treated groups compared with that of the control group (P < 0.05). But the level of TNF-alpha in TCM-cultured cells was unaffected by LMWH. The VEGF mRNA was reduced in the LMWH-treated HepG2 cell line. Moreover, TCM exhibited stimulating effect on proliferation of HUVEC and the effect was significantly impaired by LMWH treatment. Flow cytometric analysis revealed that LMWH treatment arrested HUVECs at the G1 phase of cell cycle.

CONCLUSIONLMWH can suppress the expression and secretion of VEGF by tumor cell lines and therefore have a potential inhibiting effect on angiogenesis induced by VEGF.

Adenocarcinoma ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media, Conditioned ; Endothelial Cells ; cytology ; HCT116 Cells ; Hep G2 Cells ; Heparin ; pharmacology ; Heparin, Low-Molecular-Weight ; pharmacology ; Humans ; Lung Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; secretion

Cell Line ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; genetics ; metabolism ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Mucin 5AC ; genetics ; secretion ; RNA, Messenger ; genetics ; secretion ; Respiratory System ; cytology ; Tumor Necrosis Factor-alpha ; pharmacology ; Up-Regulation ; drug effects

OBJECTIVETo investigate the influences of Shensu Yin to RAW 264.7 on the expression of TLR3, TLR4 and the factors of the downstream in RAW 264. 7 cells.

METHODRAW 264.7 cell line was stimulated with Lipopolysaccharide and POLY I: C, respectively, and treated with the drug serum of Shensuyin simultaneously. 24 hours later, collected the supernatant and measured the inflammatory factors TNF-alpha and IFN-beta, extracted mRNA and measured the expression of TLR3, TLR4 and other correlated indexes of the downstream, analyzed and evaluated Shensu Yin's substance basis of pharmacodynamic actions.

RESULTShensu Yin drug serum depressed the expression of TLR4, MyD88, TRAF-6, TRAM and TRIF mRNA, as a result, it decreased the amount of TNF-alpha and IFN-beta.

CONCLUSIONDepressing the expression of TLR3, MyD88, TRAM and TRIF mRNA may be the elementary basis of Shensu Yin to play heat-clearing and detoxicating effect.

Adaptor Proteins, Vesicular Transport ; genetics ; Animals ; Cell Line ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Interferon-beta ; secretion ; Lipopolysaccharides ; pharmacology ; Macrophages ; cytology ; drug effects ; metabolism ; Male ; Mice ; Myeloid Differentiation Factor 88 ; genetics ; Plants, Medicinal ; chemistry ; Poly I-C ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Interleukin ; genetics ; Signal Transduction ; drug effects ; Toll-Like Receptor 3 ; genetics ; Toll-Like Receptor 4 ; genetics ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo explore the effects of traditional Tibetan medicine, Fructus Lonicerae microphyllae (FLM) on phagecytosis and cytokines production of murine macrophages.

METHODThe phagecytosis of murine macrophages was analyzed by neutral red phagecytosis assay. The activities of IL-1 and TNF-alpha were measured by biological methods. The mRNA of TNF-alpha and INF-gamma expressed by macrophages was detected by RT-PCR.

RESULTThe phagecytosis of murine macrophages was significantly enhanced by FLM at a concentration from 1 microg x mL(-1) to 100 microg x mL(-1) and the secretions of IL-1, and TNF-alpha from macrophages were markedly induced by FLM. Meanwhile, FLM also increased the expression of TNF-alpha mRNA and INF-gamma mRHA from macrophages in vitro.

CONCLUSIONFLM could promote phagecytosis and cytokines production of murine macrophages.

Animals ; Cell Line ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Female ; Fibroblasts ; cytology ; Fruit ; chemistry ; Interferon-gamma ; biosynthesis ; genetics ; Interleukin-1 ; secretion ; Lonicera ; chemistry ; Macrophages, Peritoneal ; metabolism ; physiology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Phagocytosis ; drug effects ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics