OBJECTIVETo examine the effects of brucine on the invasion, migration and bone resorption of receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis.

METHODSThe osteoclastogenesis model was builded by co-culturing human breast tumor MDA-MB-231 and mouse RAW264.7 macrophages cells. RANKL (50 ng/mL) and macrophage-colony stimulating factor (50 ng/mL) were added to this system, followed by treatment with brucine (0.02, 0.04 and 0.08 mmol/L), or 10 μmol/L zoledronic acid as positive control. The migration and bone resorption were measured by transwell assay and in vitro bone resorption assay. The protein expressions of Jagged1 and Notch1 were investigated by Western blot. The expressions of transforming growth factor-β1 (TGF-β1), nuclear factor-kappa B (NF-κB) and Hes1 were determined by enzyme-linked immunosorbent assay.

RESULTSCompared with the model group, brucine led to a dose-dependent decrease on migration of MDA-MB-231 cells, inhibited RANKL-induced osteoclastogenesis and bone resorption of RAW264.7 cells (P<0.01). Furthermore, brucine decreased the protein levels of Jagged1 and Notch1 in MDA-MB-231 cells and RAW264.7 cells co-cultured system as well as the expressions of TGF-β1, NF-κB and Hes1 (P<0.05 or P<0.01).

CONCLUSIONBrucine may inhibit osteoclastogenesis by suppressing Jagged1/Notch1 signaling pathways.

Animals ; Bone Neoplasms ; metabolism ; prevention & control ; secondary ; Breast Neoplasms ; drug therapy ; metabolism ; pathology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Female ; Humans ; Jagged-1 Protein ; metabolism ; Macrophages ; drug effects ; physiology ; Mice ; Osteoclasts ; drug effects ; physiology ; Receptor, Notch1 ; metabolism ; Signal Transduction ; drug effects ; Strychnine ; analogs & derivatives ; pharmacology ; therapeutic use

OBJECTIVETo investigate the effect of antibacterial peptide hCAP18/LL-37 on ovarian cancer microenvironment and the regulatory mechanism of its expression.

METHODSWe assessed the effect of macrophage-promoted ovarian cancer cells invasion using BioCoat Matrigel invasion chamber. The expressions of hCAP18/LL-37 and versican V1 were determined by real-time PCR and Western blot analysis. SKOV3 cells were transfected with shRNA plasmid to abrogate the expression of versican V1, and then the expression of hCAP18/LL-37 in macrophages and the invasiveness of SKOV3 cells were assayed.

RESULTSThe Matrigel invasion assay showed that after co-culture with macrophages for 4 days, the number of penetrated SKOV3 cells was 112.8±17.1/per high power field, significantly higher than that in the SKOV3 cells cultured alone (8.2±1.9/per high power field) (P<0.05). Addition of hCAP/LL-37 neutralizing antibody into the co-cultured macrophage-SKOV3 cells markedly inhibited the macrophage-promoted SKOV3 cells invasion. The penetrated SKOV3 cells was 22.2±5.6/per high power field, significantly lower than the 100.6±25.2/per high power field in the control macrophage- SKOV3 co-cultured cells (P<0.05). The expressions of hCAP18/LL-37 mRNA and protein in macrophages were remarkably enhanced upon co-culture with SKOV3 cells, but not changed in SKOV3 cells cultured alone. The expression and secretion of versican V1 in the ovarian cancer cells were also significantly increased after co-cultured with macrophages. Knockdown of versican V1 in SKOV3 cells by small interfering RNA significantly reduced the expression of hCAP18/LL-37 mRNA and protein in the macrophages, as well as decreased the invasiveness of SKOV3 cells (P<0.05).

CONCLUSIONSIn the cancer microenvironment, the macrophage-secreted hCAP18/LL-37 promote the invasiveness of ovarian cancer cells, and the hCAP18/LL-37 expression is regulated by versican V1 protein released by ovarian cancer cells.

Antimicrobial Cationic Peptides ; metabolism ; pharmacology ; Cell Line, Tumor ; Coculture Techniques ; Collagen ; Drug Combinations ; Female ; Humans ; Laminin ; Macrophages ; metabolism ; Neoplasm Invasiveness ; Neoplasm Proteins ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; physiopathology ; Plasmids ; Proteoglycans ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; Transfection ; Tumor Microenvironment ; drug effects ; Versicans ; metabolism

Rheumatoid arthritis (RA) is the most common inflammatory arthritis and a major cause of disability. Presently, the clinical therapeutic medicines for inflammatory and arthritic diseases are unsatisfactory due to severe adverse effects or ineffectiveness. The Guge Fengtong formula (GGFT), containing the standardized extracts of Dioscoreae Nipponicae Rhizoma, Spatholobi Caulis, and Zingiberis Rhizoma, has long been used for RA treatment by Chinese doctorsin China. However, the detailed anti-inflammatory and anti-arthritic activity of GGFT has not been reported so far. In the present work, we aimed to evaluate the anti-inflammatory and anti-arthritic effects of GGFT using three in vivo animal models, and tried to uncover its preliminarythe underlying mechanism of action mechanism in RAW 264.7 macrophages. The obtained results indicated that GGFT significantly attenuated ear edema, decreased carrageenan-induced paw edema, reduced the arthritis score, and reversed the weight loss of the complete Freund's adjuvant (CFA)CFA-injected rats. Additionally, marked decrease of in synovial inflammatory infiltration and synovial lining hyperplasia in the joints and decline of inflammatory factors (TNF-α and IL-1β) in the serum were observed in the GGFT-treated rats. In lipopolysaccharide-activated RAW264.7 macrophages, GGFT reduced the production of NO, PGE2, and IL-6, and inhibited the expression of iNOS, COX-2, and NF-κB expression. Our results demonstrated that GGFT possessed considerable anti-inflammatory activity and have had potential therapeutic effects on adjuvant induced arthritis in rats, which provided providing experimental evidences for its traditional application in the treatment of RA and other inflammatory diseases.
Animals ; Anti-Inflammatory Agents ; pharmacology ; therapeutic use ; Antirheumatic Agents ; pharmacology ; therapeutic use ; Arthritis ; Arthritis, Rheumatoid ; drug therapy ; metabolism ; pathology ; Carrageenan ; Cytokines ; blood ; Dioscorea ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Fabaceae ; Freund's Adjuvant ; Inflammation ; chemically induced ; drug therapy ; metabolism ; Inflammation Mediators ; metabolism ; Macrophages ; drug effects ; Male ; Mice ; Mice, Inbred ICR ; Phytotherapy ; RAW 264.7 Cells ; Rats, Sprague-Dawley ; Zingiberaceae

OBJECTIVETo investigate effect of Chaiqin Chengqi Decoction (, CQCQD) on changes of neuronal acetylcholine receptor alpha 7 (nAChRα7) of peritoneal macrophages in acute pancreatitis (AP).

METHODSEighteen Kunming mice were equally randomized into the control group, AP group and CQCQD treatment group. AP was induced by two intraperitoneal injections of 4 g/kg L-arginine at 1 h apart, while control mice received saline injections. At 72 h after the first injection of L-arginine, mice in the treatment group were intragastrically administered 0.1 mL/10 g CQCQD every 2 h for 3 times, whilst mice in the other two groups received the same amount of saline feeding. Mice were sacrificed by cervical dislocation 2 h after the last feeding of either CQCQD or saline. Peritoneal macrophages were collected for determination of nAChRα7 mRNA and protein expression. Serum was collected for detection of interleukin-6 (IL-6), IL-10 and acetylcholine (ACh) levels, and pancreas was for histopathology analysis.

RESULTSThe CQCQD treatment significantly ameliorated the severity of AP as evidenced by reducing the pancreatic histopathology score (4.5±0.5 vs. 6.2±1.7, P<0.05) and the serum IL-6 levels (1228.3±419.2 pg/mL vs. 1589.6±337.3 pg/mL, P<0.05). The mRNA and protein expression of nAChRα7 of the peritoneal macrophages in the AP group were similar to the control group (P>0.05), but were significantly up-regulated after the CQCQD treatment (P<0.05). The serum ACh levels in the AP group were significantly lower than those in the control group (3.1±0.6 μg/mL vs 4.8±0.7 μg/mL P<0.05), but were significantly increased after the CQCQD treatment (5.6±1.5 μg/mL vs 3.1±0.6 μg/mL, P<0.05).

CONCLUSIONCQCQD is protective against L-arginine-induced AP through mechanisms involving nAChRα7 of peritoneal macrophages.

Acetylcholine ; pharmacology ; Acute Disease ; Animals ; Blotting, Western ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Interleukin-10 ; blood ; Interleukin-6 ; blood ; Macrophages, Peritoneal ; drug effects ; metabolism ; pathology ; Mice ; Neurons ; drug effects ; metabolism ; Pancreas ; drug effects ; pathology ; Pancreatitis ; blood ; drug therapy ; pathology ; RNA, Messenger ; genetics ; metabolism ; alpha7 Nicotinic Acetylcholine Receptor ; genetics ; metabolism

OBJECTIVETo investigate the role of oxidative stress in the endoplasmic reticulum stress-induced apoptosis of alveolar macrophages triggered by quartz dust.

METHODSSeventy-two healthy adult Wistar rats were randomly divided into control group, quartz dust group, quartz dust plus N-acetyl cysteine (NAC) group, and NAC group, with 18 rats in each group. One milliliter of sterile saline (for the control and NAC groups) or 1 ml of saline with 5%ultrafine quartz dust (for dust group and dust plus NAC group) was given to each rat by non-exposed endotracheal infusion. From the second day after dust infusion, rats in dust plus NAC group and NAC group received intragastric administration of NAC (100 mg/kg). In each week, the treatment with NAC lasted for 5 consecutive days, followed by 2 days' interval. For each group, 6 rats were randomly selected on the 14th, 28th, or 56th day after dust exposure; they were sacrificed by bloodletting from the femoral artery, and the lungs were collected. Bronchoalveolar lavage fluid was collected to separate macrophages. The protein expression of caspase-12 in alveolar macrophages, the apoptosis rate and reactive oxygen species (ROS) content of alveolar macrophages, and the protein carbonyl content of alveolar macrophages were determined by Western blot, flow cytometry, and colorimetry, respectively.

RESULTSIncreased protein expression of caspase-12, apoptosis rate, and content of ROS and protein carbonyl were discovered on the 14th day in the dust group, in comparison with the control group (P < 0.05), and the increase lasted till the 28th and 56th days. (P < 0.05). Compared with the dust group, the dust plus NAC group showed significant decreases in the content of ROS on the 14th, 28th, and 56th days (P < 0.05), significant decreases in the content of protein carbonyl on the 28th and 56th days (P < 0.05), and significant decreases in the protein expression of caspase-12 and apoptosis rate (P < 0.05).

CONCLUSIONOxidative stress is potentially involved in the endoplasmic reticulum stress-induced apoptosis of alveolar macrophages triggered by quartz dust. Oxidative damage of protein in the endoplasmic reticulum may play an important role in the process.

Animals ; Caspase 12 ; metabolism ; Dust ; Endoplasmic Reticulum Stress ; drug effects ; Macrophages, Alveolar ; drug effects ; pathology ; Male ; Oxidative Stress ; drug effects ; Protein Carbonylation ; Quartz ; toxicity ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism

OBJECTIVETo explore the mechanism of tanshinol on alleviate the inflammatory injury of lung tissue in rat hepatopulmonary syndrome (HPS).

METHODSSD rats were randomly divided into normal control group (n = 8), hepatopulmonary syndrome (HPS) group (n = 11) and tanshinol intervention group (n = 9). HE staining was used to observe the histopathology changes of pulmonary and hepatic tissues, and to count the number of macrophages in lung tissues. The activity of alanine transferase (ALT) and concentrations of endotoxin, tumor necrosis factor-a (TNF-alpha) and homocystein (Hcy) in plasma were detected. The concentrations of TNF-alpha, nitric oxide (NO) and malondialdehyde (MDA) and the activity of inducible nitric oxide synthase (iNOS) in the lung tissues were measured, respectively.

RESULTSThickened alveolar septum and increased macrophages were observed in lungs in HPS rat. After administered with tanshinol, the pulmonary pathological changes were alleviated and the number of macrophages in lung tissue was decreased compared with HPS group. The activity of ALT and the concentrations of endotoxin, TNF-alpha and Hcy in plasma ,and TNF-alpha, iNOS, NO and MDA in lung tissue in HPS group were higher than those of normal control group; meanwhile, those tanshinol group were less those that of HPS group.

CONCLUSIONTanshinol may play an important role in delaying the development of HPS through protecting liver or directly antagonizing the effect of intestinal endotoxemia so as to alleviate the inflammatory reaction in lung tissue.

Alanine Transaminase ; metabolism ; Animals ; Caffeic Acids ; pharmacology ; Disease Models, Animal ; Endotoxins ; blood ; Hepatopulmonary Syndrome ; drug therapy ; pathology ; Homocysteine ; blood ; Liver ; drug effects ; pathology ; Lung ; drug effects ; pathology ; Macrophages ; drug effects ; pathology ; Male ; Malondialdehyde ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVEThe aim of this study was to investigate the role of macrophages in promotion of ovarian tumor cell proliferation mediated by over-expression of antimicrobial peptide LL-37.

METHODSTo co-culture ovarian tumor cells SKOV3, 3AO and HO-8910 with macrophages. The Transwell(®) inserts system was used in the co-culture model. The effect of macrophages promoted ovarian tumor cell proliferation was assessed by BrdU-ELISA and cell number counting. Expressions of mRNA and protein of LL-37 in the macrophages and SKOV3 cells were determined by RT-PCR and Western blot analysis. To observe that LL-37 is responsible for macrophage-promoted ovarian tumor cells growth, LL-37 neutralizing antibody was added to abrogate the LL-37 activation.

RESULTSThe cell number assay showed that after 4 days coincubation with macrophages in the proportion of 1:0.5, the number of SKOV3 cells increased from (6.0 ± 0.5)×10(4) to (11.8 ± 1.3)×10(4), showing a significant difference (P < 0.05). It also showed that the growth of the SKOV3 cells was dependent on the macrophage number (P < 0.05). The number variability of 3AO and HO-8910 cells was as the same as SKOV3 cells upon co-culture with macrophages. As determined by BrdU-ELISA, the resulted proliferation of ovarian tumor cells was similar to the result of cell number counting. RT-PCR and Western blot results showed that the expression of LL-37 mRNA and protein in the macrophages was remarkably enhanced in a time dependent manner upon coincubation with SKOV3 cells, but did not work in SKOV3 cells. BrdU-ELISA assay exhibited that treatment of cells with LL-37 significantly stimulated HO-8910 and 3AO cell proliferation. Addition of LL-37 neutralizing antibody markedly inhibited macrophage-promoted ovarian tumor cell (SKOV3, 3AO and HO-8910 cells) proliferation. The OD values of these three cells were decreased from 2.95 ± 0.11 to 1.45 ± 0.04, from 3.39 ± 0.36 to 1.32 ± 0.09 and from 3.93 ± 0.17 to 1.68 ± 0.23, respectively (P < 0.05).

CONCLUSIONSOver-expression and release of LL-37 from macrophages is responsible for proliferation of ovarian tumor cells in co-culture condition. The data presented indicate that LL-37 may be critical for macrophage-induced tumor progression.

Antibodies, Neutralizing ; pharmacology ; Cathelicidins ; genetics ; metabolism ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Coculture Techniques ; Female ; Humans ; Macrophages ; cytology ; physiology ; Ovarian Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism

OBJECTIVETo investigate the anti-inflammatory and immunoregulatory effects of Yupingfeng (, YPF) Powder and its components in rats.

METHODSA rat chronic bronchitis (CB) model was developed using lipopolysaccharide (LPS) combined with bacillus Calmette Guerin (BCG). YPF, simple recipe Astragalus membranaceus (Fisch.) Bge (AM) and Astragalus membranaceus (Fisch.) Bge plus rhizome of Atractylodes macrocephala Koidz (AM+RA) decoction were administered (intragastric administration, once a day for 21 days) to rats, to prevent and treat CB. Immunoregulatory and anti-inflammatory effects of YPF, AM and AM+RA were tested by serum pharmacology in vitro on splenic lymphocytes of normal rats and alveolar macrophages of CB rats.

RESULTSInflammation in the pulmonary tissue and the bronchus of CB rats was significantly reduced in the YPF-treatment groups, AM and AM+RA groups demonstrating the efficacy of YPF. Serum samples collected at different times from rats after administration of YPF, AM and AM+RA demonstrated increased proliferation of splenic lymphocytes with area under the effect curve (AUE) of 552.6%, 336.3% and 452.0%, respectively. Treatment of alveolar macrophages with serum samples in YPF, AM or AM+RA group inhibited interleukin-8 (IL-8) in the cell culture media, and the effect was much better in the YPF group compared with AM or AM+RA group, with a higher maximal effect (Emax, P<0.05) and larger AUE (P <0.01 and P<0.05). Moreover, serum from rats treated with AM or AM+RA had similar efficacy, while the efficiency was lower than that treated with YPF.

CONCLUSIONYPF demonstrated anti-inflammatory and immunoregulatory effects in a rat model of CB, and timedependent relationships were demonstrated in vitro.

Animals ; Anti-Inflammatory Agents ; pharmacology ; therapeutic use ; Body Weight ; drug effects ; Bronchitis, Chronic ; drug therapy ; pathology ; Cell Proliferation ; drug effects ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Immunologic Factors ; pharmacology ; therapeutic use ; Interleukin-8 ; metabolism ; Lung ; drug effects ; pathology ; ultrastructure ; Lymphocytes ; drug effects ; Macrophages, Alveolar ; drug effects ; metabolism ; Powders ; Rats ; Rats, Sprague-Dawley ; Spleen ; pathology ; Time Factors

Recent studies have documented that Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT) pathway can modulate the apoptotic program in a myocardial ischemia/reperfusion (I/R) model. To date, however, limited studies have examined the role of JAK3 on myocardial I/R injury. Here, we investigated the potential effects of pharmacological JAK3 inhibition with JANEX-1 in a myocardial I/R model. Mice were subjected to 45 min of ischemia followed by varying periods of reperfusion. JANEX-1 was injected 1 h before ischemia by intraperitoneal injection. Treatment with JANEX-1 significantly decreased plasma creatine kinase and lactate dehydrogenase activities, reduced infarct size, reversed I/R-induced functional deterioration of the myocardium and reduced myocardial apoptosis. Histological analysis revealed an increase in neutrophil and macrophage infiltration within the infarcted area, which was markedly reduced by JANEX-1 treatment. In parallel, in in vitro studies where neutrophils and macrophages were treated with JANEX-1 or isolated from JAK3 knockout mice, there was an impairment in the migration potential toward interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), respectively. Of note, however, JANEX-1 did not affect the expression of IL-8 and MCP-1 in the myocardium. The pharmacological inhibition of JAK3 might represent an effective approach to reduce inflammation-mediated apoptotic damage initiated by myocardial I/R injury.
Animals ; Apoptosis/drug effects ; Cell Movement/drug effects ; Chemokines/pharmacology ; Heart Function Tests/drug effects ; Inflammation/pathology ; Janus Kinase 3/*antagonists & inhibitors/metabolism ; Macrophages/drug effects/metabolism/pathology ; Male ; Mice ; Mice, Inbred C57BL ; Myocardial Reperfusion Injury/drug therapy/*enzymology/physiopathology/*prevention & control ; Myocardium/enzymology/pathology ; Myocytes, Cardiac/drug effects/metabolism/pathology ; Neutrophils/drug effects/metabolism/pathology ; Quinazolines/pharmacology/therapeutic use

KR-31543, (2S, 3R, 4S)-6-amino-4-[N-(4-chlorophenyl)-N-(2-methyl-2H-tetrazol-5-ylmethyl) amino]-3,4-dihydro-2-dimethyoxymethyl-3-hydroxy-2-methyl-2H-1-benz opyran is a new neuroprotective agent for ischemia-reperfusion damage. It has also been reported that KR-31543 has protective effects on lipid peroxidation and H2O2-induced reactive oxygen species production. In this study, we investigated the anti-inflammatory and anti-atherogenic properties of KR-31543. We observed that KR-31543 treatment reduced the production of MCP-1, IL-8, and VCAM-1 in HUVECs, and of MCP-1 and IL-6 in THP-1 human monocytes. We also examined the effect of KR-31543 on monocytes migration in vitro. KR-31543 treatment effectively reduced the migration of THP-1 human monocytes to the HUVEC monolayer in a dose-dependent manner. We next examined the effects of this compound on atherogenesis in LDL receptor deficient (Ldlr-/-) mice. After 10 weeks of western diet, the formation of atherosclerotic lesion in aorta was reduced in the KR-31543-treated group compared to the control group. The accumulation of macrophages in lesion was also reduced in KR-31543 treated group. However, the plasma levels of total cholesterol, HDL, LDL, and triglyceride were not affected by KR-31543 treatment. Taken together, these results show that KR-31543 has anti-inflammatory properties on human monocytes and endothelial cells, and inhibits fatty streak lesion formation in mouse model of atherosclerosis, suggesting the potential of KR-31543 for the treatment for atherosclerosis.
Animals ; Aorta/pathology ; Atherosclerosis/blood/*drug therapy/pathology ; Benzopyrans/*pharmacology/therapeutic use ; Cholesterol, HDL/blood ; Cholesterol, LDL/blood ; Diet ; Disease Models, Animal ; Human Umbilical Vein Endothelial Cells/drug effects/metabolism ; Inflammation Mediators/*metabolism ; Interleukin-6/metabolism ; Interleukin-8/metabolism ; Macrophages/metabolism ; Mice ; Mice, Transgenic ; Monocytes/drug effects/*metabolism ; Neuroprotective Agents/*pharmacology/therapeutic use ; Receptors, CCR2/metabolism ; Receptors, LDL/genetics ; Tetrazoles/*pharmacology/therapeutic use ; Transendothelial and Transepithelial Migration/drug effects ; Triglycerides/blood ; Vascular Cell Adhesion Molecule-1/metabolism