OBJECTIVE: To investigate the anti-neuroinflammation effect of extract of Fructus Schisandrae chinensis (EFSC) on lipopolysaccharide (LPS)-induced BV-2 cells and the possible involved mechanisms. METHODS: Primary cortical neurons were isolated from embryonic (E17-18) cortices of Institute of Cancer Research (ICR) mouse fetuses. Primary microglia and astroglia were isolated from the frontal cortices of newborn ICR mouse. Different cells were cultured in specific culture medium. Cells were divided into 5 groups: control group, LPS group (treated with 1 μg/mL LPS only) and EFSC groups (treated with 1 μg/mL LPS and 100, 200 or 400 mg/mL EFSC, respectively). The effect of EFSC on cells viability was tested by methylthiazolyldiphenyltetrazolium bromide (MTT) colorimetric assay. EFSC-mediated inhibition of LPS-induced production of pro-inflammatory mediators, such as nitrite oxide (NO) and interleukin-6 (IL-6) were quantified and neuron-protection effect against microglia-mediated inflammation injury was tested by hoechst 33258 apoptosis assay and crystal violet staining assay. The expression of pro-inflammatory marker proteins was evaluated by Western blot analysis or immunofluorescence. RESULTS: EFSC (200 and 400 mg/mL) reduced NO, IL-6, inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression in LPS-induced BV-2 cells (P<0.01 or P<0.05). EFSC (200 and 400 mg/mL) reduced the expression of NO in LPS-induced primary microglia and astroglia (P<0.01). In addition, EFSC alleviated cell apoptosis and inflammation injury in neurons exposed to microglia-conditioned medium (P<0.01). The mechanistic studies indicated EFSC could suppress nuclear factor (NF)-?B phosphorylation and its nuclear translocation (P<0.01). The anti-inflammatory effect of EFSC occurred through suppressed activation of mitogen-activated protein kinase (MAPK) pathway (P<0.01 or P<0.05). CONCLUSION EFSC acted as an anti-inflammatory agent in LPS-induced glia cells. These effects might be realized through blocking of NF-κB activity and inhibition of MAPK signaling pathways.
Animals ; Astrocytes ; drug effects ; metabolism ; pathology ; Cell Line ; Cell Nucleus ; drug effects ; metabolism ; Chromatography, High Pressure Liquid ; Down-Regulation ; drug effects ; Inflammation ; pathology ; Inflammation Mediators ; metabolism ; Lipopolysaccharides ; MAP Kinase Signaling System ; drug effects ; Mice, Inbred ICR ; Microglia ; drug effects ; metabolism ; pathology ; NF-kappa B ; metabolism ; Nervous System ; pathology ; Neurons ; drug effects ; metabolism ; pathology ; Neuroprotective Agents ; pharmacology ; Plant Extracts ; pharmacology ; Schisandra ; chemistry ; Spectrometry, Mass, Electrospray Ionization

Fibroblast-like synoviocytes (FLS) play a pivotal role in Rheumatoid arthritis (RA) pathogenesis through aggressive migration and invasion. Madecassoside (Madec), a triterpenoid saponin present in Centella asiatica herbs, has a potent anti-inflammatory effect. In the present study, Madec exerted an obvious therapeutic effect in reversing the histological lesions in adjuvant-induced arthritis (AIA) rats. To recognize the anti-rheumatoid potentials of Madec, we further investigated whether Madec interfered with FLS invasion and metalloproteinase (MMP) expression. In cultures of primary FLS isolated from the AIA rats, Madec (10 and 30 μmol·L) was proven to considerably inhibit migration and invasion of FLS induced by interleukin 1β (IL-1β), but exhibiting no obvious effect on cell proliferation. Madec repressed IL-1β-triggered FLS invasion by prohibiting the expression of MMP-13. Additionally, Madec suppressed MMP-13 transcription via inhibiting the MMP-13 promoter-binding activity of NF-κB. Our results further showed that Madec down-regulated the translocation and phosphorylation of NF-κB as demonstrated by Western blotting and immunofluorescence assays. In conclusion, our results suggest that Madec exerts anti-RA activity via inhibiting the NF-κB/MMP-13 pathway.
Animals ; Antirheumatic Agents ; chemistry ; pharmacology ; therapeutic use ; Arthritis, Experimental ; chemically induced ; drug therapy ; pathology ; Cell Movement ; drug effects ; Cell Nucleus ; metabolism ; Cells, Cultured ; Gene Expression Regulation, Enzymologic ; drug effects ; Interleukin-1beta ; pharmacology ; Matrix Metalloproteinase 13 ; genetics ; NF-kappa B ; genetics ; metabolism ; Phosphorylation ; drug effects ; Protein Transport ; drug effects ; Rats ; Signal Transduction ; drug effects ; Synoviocytes ; drug effects ; metabolism ; Transcriptional Activation ; drug effects ; Triterpenes ; chemistry ; pharmacology ; therapeutic use

OBJECTIVETo investigate the effect of total flavone of haw leaves (TFHL) on the expression of nuclear factor erythroid-2 related factor (Nrf2) and other related factors in nonalcoholic steatohepatitis (NASH) rats induced by high-fat diet and then to further discuss the mechanism of TFHL's prevention against NASH.

METHODSHigh-fat diet was fed to 40 rats to establish the NASH model. Then model rats were intragastrically administrated with 40, 80, 160 mg/(kg•day) TFHL, respectively. The pathological changes of liver tissues in NASH rats were detected by oil red O and hematoxylin-eosin (HE) stainings. The expression of Nrf2 in rat liver was examined through immunohistochemistry. The level of 8-iso-prostaglandin F2α in serum was detected through enzyme linked immunosorbent assay (ELISA). The mRNA and protein levels of Nrf2 and other related factors in liver tissue were measured by real-time reverse transcriptionpolymerase chain reaction and western blot.

RESULTSLipid deposition, hepatic steatosis, focal necrosis in lobular inflammation and ballooning degeneration were emerged in livers of NASH rats. The 8-iso-prostaglandin F2α in the serum of NASH rats increased significantly compared with the control group (P<0.05). The mRNA of Nrf2, hemeoxyenase1 (HO-1) and the mRNA and protein levels of quinine oxidoreductase (NQO1) in NASH rats liver tissue showed a striking increase, while the mRNA levels of Keap1, r-glutamylcysteine synthethase (rGCS) and glutathione S-transferase (GST) were significantly decreased compared with the control group (P<0.05). After TFHL treatment, 8-iso-prostaglandin F2α level in serum significantly decreased, and Nrf2 mRNA and protein levels in hepatocytes nucleus enhanced compared with the model group (P<0.05 or 0.01). Meanwhile the Keap1 mRNA, the mRNA and protein levels of HO-1, NQO1 antibody, rGCS antibody, GST increased after TFHL treatment (P<0.05 or 0.01).

CONCLUSIONSNrf2 and other related factors were involved in development of NASH, and they also served as an important part in its occurrence. By regulating expression of Nrf2 and other related factors, TFHL may play a role in antioxidative stress and prevention of NASH.

Animals ; Cell Nucleus ; drug effects ; metabolism ; Crataegus ; chemistry ; Dinoprost ; metabolism ; Flavones ; pharmacology ; therapeutic use ; Lipids ; chemistry ; Liver ; drug effects ; metabolism ; pathology ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Non-alcoholic Fatty Liver Disease ; drug therapy ; genetics ; pathology ; Phytotherapy ; Plant Leaves ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley

Annexin A2, a multifunctional tumor associated protein, promotes nuclear factor-kappa B (NF-κB) activation by interacting with NF-κB p50 subunit and facilitating its nuclear translocation. Here we demonstrated that two ginsenosides Rg5 (G-Rg5) and Rk1 (G-Rk1), with similar structure, directly bound to Annexin A2 by molecular docking and cellular thermal shift assay. Both Rg5 and Rk1 inhibited the interaction between Annexin A2 and NF-κB p50 subunit, their translocation to nuclear and NF-κB activation. Inhibition of NF-κB by these two ginsenosides decreased the expression of inhibitor of apoptosis proteins (IAPs), leading to caspase activation and apoptosis. Over expression of K302A Annexin A2, a mutant version of Annexin A2, which fails to interact with G-Rg5 and G-Rk1, effectively reduced the NF-κB inhibitory effect and apoptosis induced by G-Rg5 and G-Rk1. In addition, the knockdown of Annexin A2 largely enhanced NF-κB activation and apoptosis induced by the two molecules, indicating that the effects of G-Rg5 and G-Rk1 on NF-κB were mainly mediated by Annexin A2. Taken together, this study for the first time demonstrated that G-Rg5 and G-Rk1 inhibit tumor cell growth by targeting Annexin A2 and NF-κB pathway, and G-Rg5 and G-Rk1 might be promising natural compounds for targeted cancer therapy.
Active Transport, Cell Nucleus ; drug effects ; Annexin A2 ; chemistry ; deficiency ; genetics ; metabolism ; Antineoplastic Agents ; chemistry ; metabolism ; pharmacology ; Apoptosis ; drug effects ; Biological Products ; chemistry ; metabolism ; pharmacology ; Cell Nucleus ; drug effects ; metabolism ; Down-Regulation ; drug effects ; Drug Discovery ; Gene Knockdown Techniques ; Ginsenosides ; chemistry ; Hep G2 Cells ; Humans ; Molecular Docking Simulation ; Molecular Targeted Therapy ; NF-kappa B p50 Subunit ; metabolism ; Protein Conformation

Epithelial-mesenchymal transition (EMT) has been implicated in tumor invasion and metastasis and provides novel strategies for cancer therapy. Hypaconitine (HpA), a diester-diterpenoid alkaloid isolated from the root of the Aconitum species, exhibits anti-inflammatory, analgesic, and especially, cardiotoxic activities. Here, we reported the anti-metastatic potentials of HpA in transforming growth factor-β1 (TGF-β1)-induced EMT in lung cancer A549 cells. The cytotoxic effect of HpA was determined by MTT assay. A549 cells were treated with TGF-β1 with or without HpA co-treatment, and the morphological alterations were observed with a microscopy. The expression of E-cadherin, N-cadherin, and NF-κB was determined by both Western blotting and immunofluorescence analyses. The adhesion, migration, and invasion were detected with Matrigel, wound-healing, and transwell assays, respectively. The expression of Snail was determined by Western blotting. The expression of NF-κB p65, IκBα, and p-IκBα in nuclear and cytosolic extracts was assessed by Western blotting. The results showed that low concentration of HpA (<16 μmol·L) had no obvious cytotoxicity to A549 cells. Morphologically, TGF-β1 treatment induced spindle-shaped alteration in the cells. The upregulation of N-cadherin, NF-κB, and Snail and the downregulation of E-cadherin were detected after TGF-β1 treatment. The adhesion, migration and invasion abilities were also increased by TGF-β1. Besides, TGF-β1 induced expression of Snail in a time-dependent manner. Furthermore, TGF-β1 induced nuclear translocation of NF-κB p65. All these alterations were dramatically inhibited by HpA co-treatment. In addition, the NF-κB inhibitor PDTC showed similar inhibitory effect. In conclusion, these results showed that HpA inhibited TGF-β1-induced EMT in A549 cells, which was possibly mediated by the inactivation of the NF-κB signaling pathway, providing an evidence for anti-cancer effect of HpA.
A549 Cells ; Aconitine ; analogs & derivatives ; pharmacology ; Active Transport, Cell Nucleus ; drug effects ; Antineoplastic Agents, Phytogenic ; pharmacology ; Cadherins ; analysis ; Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Dose-Response Relationship, Drug ; Epithelial-Mesenchymal Transition ; drug effects ; Humans ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Neoplasm Invasiveness ; Transforming Growth Factor beta1 ; antagonists & inhibitors ; physiology

Angiotensin II (Ang II) induces the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. In kidneys, Ang II plays an important role in the development of proteinuria by the modification of podocyte molecules. We have previously found that Ang II suppressed podocyte AMP-activated protein kinase (AMPK) via Ang II type 1 receptor and MAPK signaling pathway. In the present study, we investigated the roles of AMPK on the changes of p130Cas of podocyte by Ang II. We cultured mouse podocytes and treated them with various concentrations of Ang II and AMPK-modulating agents and analyzed the changes of p130Cas by confocal imaging and western blotting. In immunofluorescence study, Ang II decreased the intensity of p130Cas and changed its localization from peripheral cytoplasm into peri-nuclear areas in a concentrated pattern in podocytes. Ang II also reduced the amount of p130Cas in time and dose-sensitive manners. AMPK activators, metformin and AICAR, restored the suppressed and mal-localized p130Cas significantly, whereas, compound C, an AMPK inhibitor, further aggravated the changes of p130Cas. Losartan, an Ang II type 1 receptor antagonist, recovered the abnormal changes of p130Cas suppressed by Ang II. These results suggest that Ang II induces the relocalization and suppression of podocyte p130Cas by the suppression of AMPK via Ang II type 1 receptor, which would contribute to Ang II-induced podocyte injury.
AMP-Activated Protein Kinases/antagonists & inhibitors/chemistry/*metabolism ; Aminoimidazole Carboxamide/analogs & derivatives/pharmacology ; Angiotensin II/*pharmacology ; Angiotensin II Type 1 Receptor Blockers/pharmacology ; Animals ; Blotting, Western ; Cell Line ; Cell Nucleus/metabolism ; Crk-Associated Substrate Protein/*metabolism ; Cytoplasm/metabolism ; Focal Adhesion Kinase 1/metabolism ; Losartan/pharmacology ; Metformin/pharmacology ; Mice ; Microscopy, Confocal ; Podocytes/cytology/drug effects/metabolism ; Protein Kinase Inhibitors/*pharmacology ; Ribonucleotides/pharmacology ; Signal Transduction/*drug effects

Glucocorticoid receptor (GR) is identified as a member of nuclear receptor family. To exert its biological action, the ligand bound GR is translocated from the cytoplasm into the nucleus by regulating transcriptional signals of related genes. In clinical practice, the effects of glucocorticoid are often mediated by GR signaling pathways. An increasing number of studies have indicated that GR signaling pathways play an essential role in the proliferation, invasion and prognosis of bladder cancer. Meanwhile, the new-generation selective GR activator improves its anti-tumor effects, and at the same time reduces the adverse reactions of hormones, which probably raises the prospect for the treatment of bladder cancer.
Animals ; Antineoplastic Agents ; pharmacology ; Cell Nucleus ; genetics ; Humans ; Prognosis ; Protein Transport ; genetics ; Receptors, Glucocorticoid ; agonists ; physiology ; Signal Transduction ; genetics ; Transcriptional Activation ; drug effects ; physiology ; Urinary Bladder Neoplasms ; genetics ; physiopathology

OBJECTIVEThis study observed attenuating effect of hydroxysafflor yellow A (HSYA), an effective ingredient of aqueous extract of Carthamus tinctorius L, on lipopolysaccharide (LPS)-induced endothelium inflammatory injury.

METHODSEahy926 human endothelium cell (EC) line was used; thiazolyl blue tetrazolium bromide (MTT) test was assayed to observe the viability of EC; Luciferase reporter gene assay was applied to measure nuclear factor-κB (NF-κB) p65 subunit nuclear binding activity in EC; Western blot technology was used to monitor mitogen activated protein kinase (MAPKs) and NF-κB activation. Reverse transcription polymerase chain reaction (RT-PCR) method was applied to observe intercellular cell adhesion molecule-1 (ICAM-1) and E-selectin mRNA level; EC surface ICAM-1 expression was measured with flow cytometry and leukocyte adhesion to EC was assayed with Rose Bengal spectrophotometry technology.

RESULTSHSYA protected EC viability against LPS-induced injury (P <0.05). LPS-induced NF-κB p65 subunit DNA binding (P <0.01) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) phosphorylation was inhibited by HSYA. HSYA attenuated LPS triggered ICAM-1 and E-selectin mRNA levels elevation and phosphorylation of p38 MAPK or c-Jun N-terminal kinase MAPK. HSYA also inhibited LPS-induced cell surface ICAM-1 protein expression P <0.01) and leukocyte adhesion to EC (P <0.05).

CONCLUSIONHSYA is effective to protect LPS-induced high expression of endothelium adhesive molecule and inflammatory signal transduction.

Cell Adhesion ; drug effects ; Cell Nucleus ; drug effects ; metabolism ; Cell Survival ; drug effects ; Chalcone ; analogs & derivatives ; chemistry ; pharmacology ; therapeutic use ; E-Selectin ; genetics ; metabolism ; Endothelium, Vascular ; drug effects ; pathology ; Gene Expression Regulation ; drug effects ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; pathology ; Humans ; I-kappa B Proteins ; metabolism ; Inflammation ; drug therapy ; pathology ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Leukocytes ; cytology ; drug effects ; Lipopolysaccharides ; MAP Kinase Signaling System ; drug effects ; NF-KappaB Inhibitor alpha ; Phosphorylation ; drug effects ; Protective Agents ; pharmacology ; Protein Binding ; drug effects ; Quinones ; chemistry ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism

Carbon monoxide (CO), as a vital small molecule in signaling pathways, is found to be involved in ischemia-reperfusion injury (IRI) in renal transplantation. CO-releasing molecule-2 (CORM-2), a CO-releasing molecule, is a type of metal carbonyl complexes which can quickly release CO in vivo. In this study, an in vitro oxidative stress injury model was established to examine the effect of CORM-2 pretreatment on the nuclear-cytoplasmic translocation of high mobility group box 1 protein (HMGB1) in mouse primary renal proximal tubular epithelial cells (RPTECs). Immunofluorescence staining showed that HMGB1 in the medium- and CORM-2-treated groups was predominantly localized in the nucleus of the cells, whereas higher amounts of HMGB1 translocated to the cytoplasm in the HO- and inactive CORM-2 (iCORM-2)-treated groups. Western blotting of HMGB1 showed that the total amounts of cytoplasmic HMGB1 in the HO-treated (0.59±0.27) and iCORM-2-treated (0.57±0.22) groups were markedly higher than those in the medium-treated (0.19±0.05) and CORM-2-treated (0.21±0.10) groups (P<0.05). Co-immunoprecipitation showed that the levels of acetylated HMGB1 in the HO-treated (642.98±57.25) and iCORM-2-treated (342.11±131.25) groups were markedly increased as compared with the medium-treated (78.72±74.17) and CORM-2-treated (71.42±53.35) groups (P<0.05), and no significant difference was observed between the medium-treated and CORM-2-treated groups (P>0.05). In conclusion, our study demonstrated that in the in vitro oxidative stress injury model of primary RPTECs, CORM-2 can significantly inhibit the nuclear-cytoplasmic translocation of HMGB1, which is probably associated with the prevention of HMGB1 acetylation.
Active Transport, Cell Nucleus ; drug effects ; Animals ; Carbon Monoxide ; pharmacology ; Cell Nucleus ; metabolism ; Cells, Cultured ; Epithelial Cells ; drug effects ; metabolism ; HMGB1 Protein ; metabolism ; Kidney Tubules ; cytology ; Mice ; Organometallic Compounds ; pharmacology ; Oxidative Stress

OBJECTIVETo investigate the effect and the potential mechanism of Senegenin (Sen) against injury induced by hypoxia/reoxygenation (H/R) in highly differentiated PC12 cells.

METHODSThe cultured PC12 cells were treated with H/R in the presence or absence of Sen (60 μmol/L). Four groups were included in the experiment: control group, H/R group, H/R+Sen group and Sen group. Cell viability of each group and the level of lactate dehydrogenase (LDH) in culture medium were detected for the pharmacological effect of Sen. Hoechst 33258 staining and annexin V/propidium iodide double staining were used to analyze the apoptosis rate. Moreover, mitochondrial membrane potential (△Ψm), reactive oxygen species (ROS) and intracellular free calcium ([Ca(2+)]i) were measured by fluorescent staining and flow cytometry. Cleaved caspase-3 and activity of NADPH oxidase (NOX) were determined by colorimetric protease assay and enzyme linked immunosorbent assay, respectively.

RESULTSSen significantly elevated cell viability (P<0.05), decreased the leakage of LDH (P<0.05) and apoptosis rate (P<0.05) in H/R-injured PC12 cells. Sen maintained the value of △Ψm (P<0.05) and suppressed the activity of caspase-3 (P<0.05). Moreover, Sen reduced ROS accumulation P<0.05) and [Ca(2+)]i increment (P<0.05) by inhibiting the activity of NOX (P<0.05).

CONCLUSIONSen may exert cytoprotection against H/R injury by decreasing the levels of intracellular ROS and [Ca(2+)]i, thereby suppressing the mitochondrial pathway of cellular apoptosis.

Animals ; Apoptosis ; drug effects ; Calcium ; metabolism ; Caspase 3 ; metabolism ; Cell Hypoxia ; drug effects ; Cell Nucleus ; drug effects ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Flow Cytometry ; Fluorescence ; Intracellular Space ; metabolism ; Membrane Potential, Mitochondrial ; drug effects ; NADPH Oxidases ; metabolism ; Neuroprotective Agents ; pharmacology ; Oxygen ; pharmacology ; PC12 Cells ; Rats ; Reactive Oxygen Species ; metabolism ; Staining and Labeling