OBJECTIVE: To investigate the role of acetylated modification induced by coactivator p300 in lipopolysaccharide (LPS)- induced inflammatory mediator synthesis and its molecular mechanism. METHODS: Agilent SurePrint G3 Mouse Gene Expression V2 microarray chip and Western blotting were used to screen the molecules whose expression levels in mouse macrophages (RAW246.7) were correlated with the stimulation intensity of LPS. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (chip-qPCR) were used to verify the binding of the molecules to the promoters of IL-6 and TNF-α genes. The effects of transfection of RAW246.7 cells with overexpression or interfering plasmids on IL-6 and TNF-α synthesis were evaluated with ELISA, and the binding level of the target molecules and acetylation level of H3K27 in the promoter region of IL-6 and TNF-α genes were analyzed by chromatin immunoprecipitation sequencing technique (chip-seq). RESULTS: Gene microarray chip data and Western blotting both confirmed a strong correlation of p300 expression with the stimulation intensity of LPS. Immunocoprecipitation confirmed the binding between p300 and c-myb. The results of EMSA demonstrated that c-myb (P < 0.05), but not p300, could directly bind to the promoter region of IL-6 and TNF-α genes; p300 could bind to the promoters only in the presence of c-myb (P < 0.05). The expressions of p65, p300 and c-myb did not show interactions. Both p300 overexpression and LPS stimulation could increase the level of promoter-binding p300 and H3K27 acetylation level, thus promoting p65 binding and inflammatory gene transcription; such effects were obviously suppressed by interference of c-myb expression (P < 0.05). Interference of p65 resulted in inhibition of p65 binding to the promoters and gene transcription (P < 0.05) without affecting p300 binding or H3K27 acetylation level. CONCLUSION LPS can stimulate the synthesis of p300, whose binding to the promoter region of inflammatory genes via c-myb facilitates the cohesion of p65 by inducing H3K27 acetylation, thus promoting the expression of the inflammatory genes.
Acetylation ; Animals ; Inflammation Mediators ; Interleukin-6/metabolism* ; Lipopolysaccharides/pharmacology* ; Mice ; Tumor Necrosis Factor-alpha/metabolism*

It has become increasingly clear that cytokines play an important role in modulating neuroendocrine regulation, especially in the secretion of corticotropin (ACTH) in the pituitary. Oncostatin M (OSM), a cytokine of IL-6 family has been reported to increase ACTH secretion and pro-opiomelanocortin (POMC) transcription in murine corticotroph pituitary tumor cells (AtT20 cells). The present study was undertaken to determine the effects of OSM on hormonal release in primary culture of rat pituitary cells. Growth hormone or prolactin release was not affected by OSM. OSM (1 nM) stimulated ACTH release (35.1% increase versus control, p>0.001) in dispersed pituitary cells of rat to a lesser extent than in AtT20 cells. Corticotropin releasing hormone (CRH) (10 nM) also induced a 2.3-fold increase of ACTH secretion (p>0.001), but co-treatment of OSM and CRH did not exhibit any synergistic effect on ACTH secretion. We conclude OSM has a stimulatory effect on ACTH secretion in normal rat pituitary cell cultures, and OSM acts mainly on corticotroph, supporting the potential role of OSM to modulate immune-endocrine regulation in the pituitary.
Animal ; Cells, Cultured ; Corticotropin/secretion* ; Cytokines/pharmacology ; Cytokines/metabolism* ; Inflammation Mediators/pharmacology ; Inflammation Mediators/metabolism* ; Male ; Peptides/pharmacology ; Peptides/metabolism* ; Pituitary Gland/metabolism* ; Pituitary Gland/drug effects ; Pituitary Gland/cytology ; Prolactin/secretion* ; Rats ; Rats, Inbred WF ; Somatotropin/secretion*

It has become increasingly clear that cytokines play an important role in modulating neuroendocrine regulation, especially in the secretion of corticotropin (ACTH) in the pituitary. Oncostatin M (OSM), a cytokine of IL-6 family has been reported to increase ACTH secretion and pro-opiomelanocortin (POMC) transcription in murine corticotroph pituitary tumor cells (AtT20 cells). The present study was undertaken to determine the effects of OSM on hormonal release in primary culture of rat pituitary cells. Growth hormone or prolactin release was not affected by OSM. OSM (1 nM) stimulated ACTH release (35.1% increase versus control, p>0.001) in dispersed pituitary cells of rat to a lesser extent than in AtT20 cells. Corticotropin releasing hormone (CRH) (10 nM) also induced a 2.3-fold increase of ACTH secretion (p>0.001), but co-treatment of OSM and CRH did not exhibit any synergistic effect on ACTH secretion. We conclude OSM has a stimulatory effect on ACTH secretion in normal rat pituitary cell cultures, and OSM acts mainly on corticotroph, supporting the potential role of OSM to modulate immune-endocrine regulation in the pituitary.
Animal ; Cells, Cultured ; Corticotropin/secretion* ; Cytokines/pharmacology ; Cytokines/metabolism* ; Inflammation Mediators/pharmacology ; Inflammation Mediators/metabolism* ; Male ; Peptides/pharmacology ; Peptides/metabolism* ; Pituitary Gland/metabolism* ; Pituitary Gland/drug effects ; Pituitary Gland/cytology ; Prolactin/secretion* ; Rats ; Rats, Inbred WF ; Somatotropin/secretion*

Asthma ; drug therapy ; metabolism ; Cytokines ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Humans ; Inflammation Mediators ; metabolism ; Phytotherapy ; Signal Transduction

OBJECTIVETo investigate the effect of oleic acid on the proliferation and secretion of pro-inflammatory mediators of human normal fibroblasts and scar fibroblasts.

METHODSHuman normal fibroblasts and scar fibroblasts were cultured in vitro and respectively divided into seven groups according to the random number table, with 8 samples in each group. Cells in blank control (BC) group were routinely cultured without addition of other agents. Cells in ethanol-control (EC) group were cultured with medium with the addition of 2% absolute ethanol. Cells in oleic acid groups were cultured with the addition of oleic acid in concentration of 0.25, 0.50, 1.00, 2.00, or 4.00 mmol/L in 2% absolute ethanol. The growth of cells in each group was observed with trypan blue staining on post culture day (PCD) 1-5. On PCD 2, structure of cells in BC, EC, and 1.00 mmol/L oleic acid groups was observed under inverted phase contrast microscope and transmission electron microscope; cell cycle of BC, EC, and 1.00 mmol/L oleic acid groups was measured by flow cytometer; cell proliferation activity in each group was measured by MTT assay; the level of NO in supernatant was assayed by Griess assay; the levels of TNF-α, IL-1β, IL-6, and IL-8 in supernatants in each group were determined by enzyme-linked immunosorbent assay. Data were processed with multifactor and repeated measurement design analysis of variance.

RESULTS(1) There was no significant difference in each index of normal fibroblasts and scar fibroblasts between BC group and EC group. (2) The numbers of normal fibroblasts and scar fibroblasts in 2.00 and 4.00 mmol/L oleic acid groups were significantly lower than those in corresponding BC and EC groups on PCD 2-5 (with F values respectively 13.773 and 11.344, P values all below 0.01). (3) On PCD 2, the numbers of normal fibroblasts and scar fibroblasts in 1.00 mmol/L oleic acid groups decreased, and the cells were aggregating, rounding, and easy to drop off. Cellular membrane disruption, vacuolar degeneration of mitochondrion, pyknosis, and lipid droplets were observed. (4) The percentages of G0/G1 and G2/M phases of normal fibroblasts in 1.00 mmol/L oleic acid group [(93.56 ± 9.98)%, (2.01 ± 0.75)%] were significantly higher than those in BC group [(84.23 ± 10.96)%, (0.37 ± 0.16)%, with F values respectively 3.026, 34.751, P < 0.05 or P < 0.01], while the percentage of normal fibroblasts in S phase [(4.42 ± 0.87)%] was markedly lower than that in BC group [(16.06 ± 1.74)%, F = 136.120, P < 0.01]. The percentages of scar fibroblasts of G0/G1 and G2/M phases in 1.00 mmol/L oleic acid group [(93.86 ± 13.90)%, (1.89 ± 0.66)%] were significantly higher than those in BC group [(83.88 ± 10.42)%, (0.41 ± 0.17)%, with F values respectively 3.529, 32.710, P < 0.05 or P < 0.01], and the percentage of scar fibroblasts in S phase [(3.87 ± 0.63)%] was markedly lower than that in BC group [(15.89 ± 2.02)%, F = 116.508, P < 0.01]. (5) The proliferation rates of normal fibroblasts and scar fibroblasts in 0.50-4.00 mmol/L oleic acid groups were significantly lower than those in corresponding BC and EC groups (with F values respectively 215.945 and 194.555, P < 0.05 or P < 0.01). (6) The content of NO in supernatant of normal fibroblasts in all oleic acid groups was obviously higher than that in BC and EC groups (F = 30.240, P < 0.05 or P < 0.01). The contents of NO in supernatants of scar fibroblasts in 1.00-4.00 mmol/L oleic acid groups were significantly higher than that in BC and EC groups (F = 12.495, P < 0.01). The contents of TNF-α and IL-6 in supernatants of normal fibroblasts and scar fibroblasts in 2.00 and 4.00 mmol/L oleic acid groups were obviously higher than those in corresponding BC and EC groups (with F(TNF-α) values respectively 6.911, 3.818, F(IL-6) values respectively 16.939, 11.600,P < 0.05 or P < 0.01). The contents of IL-1β in supernatants of normal fibroblasts and scar fibroblasts in groups of every concentration of oleic acid were significantly higher than those in corresponding BC and EC groups (with F values respectively 25.117, 9.137, P values all below 0.01). The contents of IL-8 in supernatants of normal fibroblasts in 1.00-4.00 mmol/L oleic acid groups were markedly higher than those in BC and EC groups (F = 2.717, P < 0.05 or P < 0.01). The contents of IL-8 in supernatants of scar fibroblasts in 2.00 and 4.00 mmol/L oleic acid groups were significantly higher than those in BC and EC groups (F = 3.338, P < 0.05). There was no statistically significant difference in above indexes between normal fibroblasts and scar fibroblasts in the same concentration of oleic acid group (with F values from 0.120 to 3.766, P values all above 0.05).

CONCLUSIONSAlthough oleic acid in high concentration inhibits the proliferation of scar fibroblasts, it also inhibits the proliferation of normal fibroblasts. Oleic acid in high concentration can cause excessive and continued inflammatory reaction by promoting the secretion of pro-inflammatory mediators of normal fibroblasts and scar fibroblasts, and they are detrimental to wound healing.

Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix ; metabolism ; Fibroblasts ; cytology ; drug effects ; secretion ; Humans ; Inflammation Mediators ; metabolism ; Oleic Acid ; pharmacology

The Uncariae Ramulus Cum Uncis is a commonly used traditional Chinese medicine. In recent years, many studies have revealed its prominent neuroprotection function. The active ingredients in Uncariae Ramulus Cum Uncis could protect the nervous system in a multi-path and multi-target manner. Uncariae Ramulus Cum Uncis shows the neuroprotective effect by resisting oxidation, scavenging free radicals, modulating neurotransmitters and their related receptors, regulating the inflammatory factors and their related pathways, attenuating neuron apoptosis, reducing intracellular Ca2+ overloads and mitigating neurodegeneration. In this paper, the authors summarized the advance in studies on neuroprotective mechanisms of Uncariae Ramulus Cum Uncis.
Animals ; Calcium ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Free Radical Scavengers ; pharmacology ; Humans ; Inflammation Mediators ; metabolism ; Neuroprotective Agents ; pharmacology ; Neurotransmitter Agents ; metabolism ; Uncaria ; chemistry

Diosgenin, a well-known steroid sapogenin derived from plants, has been used as a starting material for production of steroidal hormones. The present review will summarize published literature concerning pharmacological potential of diosgenin, and the underlying mechanisms of actions. Diosgenin has shown a vast range of pharmacological activities in preclinical studies. It exhibits anticancer, cardiovascular protective, anti-diabetes, neuroprotective, immunomodulatory, estrogenic, and skin protective effects, mainly by inducing apoptosis, suppressing malignant transformation, decreasing oxidative stress, preventing inflammatory events, promoting cellular differentiation/proliferation, and regulating T-cell immune response, etc. It interferes with cell death pathways and their regulators to induce apoptosis. Diosgenin antagonizes tumor metastasis by modulating epithelial-mesenchymal transition and actin cytoskeleton to change cellular motility, suppressing degradation of matrix barrier, and inhibiting angiogenesis. Additionally, diosgenin improves antioxidant status and inhibits lipid peroxidation. Its anti-inflammatory activity is through inhibiting production of pro-inflammatory cytokines, enzymes and adhesion molecules. Furthermore, diosgenin drives cellular growth/differentiation through the estrogen receptor (ER) cascade and transcriptional factor PPARγ. In summary, these mechanistic studies provide a basis for further development of this compound for pharmacotherapy of various diseases.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Antioxidants ; pharmacology ; Cell Proliferation ; drug effects ; Diosgenin ; pharmacology ; Humans ; Inflammation Mediators ; metabolism ; Oxidative Stress ; drug effects ; Phytoestrogens ; pharmacology ; Plant Extracts ; pharmacology

Lycii Cortex, a popular herb medicine in traditional Chinese medicine, is used to treat different inflammation-related diseases. The aim of our work is to find the key constituents inhibiting NF-kappaB, a key regulator of inflammation. In the investigations of cell-based in vitro assays of extracts, we found that both ethyl acetate extract and methanol extract of Lycii Cortex inhibited the TNF-alpha-induced activation of NF-kappaB. Through bioassay-guided fractionation, we identified 4 phenolic amides including trans-N-(p-coumaroyl) tyramine (1), trans-N-feruloyltyramine (2), trans-N-caffeoyltyramine (3), and dihydro-N-caffeoyltyramine (4). Four phenolic amides showed differently inhibitory activities on TNF-alpha-induced NF-kappaB activation. Trans-N-caffeoyltyramine (3) was identified as the key component with an IC50 of 18.41 micromol x L(-1). It was suggested that the hydroxyl group at C-3 in trans-N-caffeoyltyramine might be a key binding site and its C-7,8-double bond might play an important role on NF-kappaB inhibitory activities as the link of the conjugation of pi electrons leading to a partial planar conformation. It might be inferred that the biological activity of compound 3 is attributed to the structure of Michael reaction acceptor containing alpha, beta-unsaturated ketones and benzene along with hydroxyl group in o-diphenol.
Biological Assay ; Cell Line ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Humans ; Inflammation Mediators ; antagonists & inhibitors ; immunology ; Lycium ; chemistry ; Molecular Structure ; NF-kappa B ; antagonists & inhibitors ; immunology

OBJECTIVETo probe the recovery mechanism of Zhenggu powder (ZGP) on acute soft tissue injury in cell levels.

METHODSForty rabbits which established animal model of acute soft tissue injury with hammer hitting,were divided randomly into normal group (A), model group (B), vaseline group (C)and ZGP group (D). Injured part was applied external drug daily after model was made. All animals were killed after using drug for 4 days. The local tissue of injured part was taken pathologic study, and was measured the content of IL-1beta, IL-6, TNFalpha by ELISA method and TXB2, 6-keto-PGF1alpha by RIA method.

RESULTSMuscular tissue of group A was normal. But that of group B and C was aberrant,such as swelling and broken of muscular fiber or infiltration of inflammatory cell. Such histological change of group D was lightly and hyperplasia of blood vessel was found. The contents of IL-1beta, TNFalpha, TXB2 and TXB2/6-keto-PGF1alpha in group D were lower than that of group B and group C. On the contrary, the contents of 6-keto-PGF1alpha in the group D were higher than that of group B and group C. The difference of content of IL-6 between groups was not obvious.

CONCLUSIONZGP could promote not only the dilution and the transportation of inflammatory cell factors,but also the repair and the regeneration of the injured tissue structures. The therapeutic effect of ZGP was not relative to IL-6.

Animals ; Cytokines ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Female ; Humans ; Inflammation Mediators ; immunology ; Male ; Rabbits ; Random Allocation ; Soft Tissue Injuries ; drug therapy ; immunology ; pathology

OBJECTIVETo study the chemical composition, anticancer, anti-neuroinflflammatory, and antioxidant activities of the essential oil of Patrinia scabiosaefolia (EO-PS).

METHODSPatrinia scabiosaefolia was analyzed by gas chromatography-mass spectrometry. Eight human carcinoma cell lines, including SGC-7901, AGS, HepG2, HT-29, HCT-8, 5-FU/HCT-8, HeLa, and MDA-MB-231, were assessed by methylthiazolyldiphenyltetrazolium bromide (MTT) assay. Anti-neuroinflflammatory activity was assessed by production of interleukin (IL)-1β and IL-6 induced by lipopolysaccharide in BV-2 cells (microglia from mice). The antioxidant activity was evaluated with a 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay.

RESULTSForty-four components, representing 83.919% of the total oil, were identifified in the EO-PS. The major constituents were caryophyllene oxide (12.802%), caryophyllene (6.909%), α-caryophyllene (2.927%), β-damascenone (3.435%), calarene (5.621%), and phenol (3.044%). The MTT assay showed that the EO-PS exhibited significant dose-dependent growth inhibition in the 50-200 μg/mL dilution range. The EO-PS exhibited a dose-dependent scavenging activity against the DPPH radical, with an half of maximal inhibitory concentration 1.455 mg/mL.

CONCLUSIONSThe EO-PS possesses a wide range of antitumor, anti-neuroinflflammatory and antioxidant activities, suggesting that it may be a good candidate for further investigations of new bioactive substances.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Antineoplastic Agents ; pharmacology ; Antioxidants ; pharmacology ; Cell Death ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cytokines ; metabolism ; Free Radical Scavengers ; pharmacology ; Humans ; Inflammation Mediators ; metabolism ; Mice ; Oils, Volatile ; chemistry ; pharmacology ; Patrinia ; chemistry