Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.
Animals ; Antigens, Helminth ; isolation & purification ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Line ; Culture Media, Conditioned ; chemistry ; pharmacology ; Gene Expression Regulation ; Hedgehog Proteins ; agonists ; antagonists & inhibitors ; genetics ; immunology ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; metabolism ; parasitology ; prevention & control ; Macrophage Activation ; drug effects ; Macrophages ; cytology ; drug effects ; immunology ; Models, Biological ; Monocytes ; cytology ; drug effects ; metabolism ; Pentoxifylline ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; RNA, Messenger ; genetics ; immunology ; Schistosoma japonicum ; chemistry ; Signal Transduction ; Tetradecanoylphorbol Acetate ; pharmacology ; Zinc Finger Protein GLI1 ; genetics ; immunology ; Zygote ; chemistry

Osteoclast-like cells are known to inhibit arterial calcification. Receptor activator of NF-κB ligand (RANKL) is likely to act as an inducer of osteoclast-like cell differentiation. However, several studies have shown that RANKL promotes arterial calcification rather than inhibiting arterial calcification. The present study was conducted in order to investigate and elucidate this paradox. Firstly, RANKL was added into the media, and the monocyte precursor cells were cultured. Morphological observation and Tartrate resistant acid phosphatase (TRAP) staining were used to assess whether RANKL could induce the monocyte precursor cells to differentiate into osteoclast-like cells. During arterial calcification, in vivo and in vitro expression of RANKL and its inhibitor, osteoprotegerin (OPG), was detected by real-time PCR. The extent of osteoclast-like cell differentiation was also assessed. It was found RANKL could induce osteoclast-like cell differentiation. There was no in vivo or in vitro expression of osteoclast-like cells in the early stage of calcification. At that time, the ratio of RANKL to OPG was very low. In the late stage of calcification, a small amount of osteoclast-like cell expression coincided with a relatively high ratio of RANKL to OPG. According to the results, the ratio of RANKL to OPG was very low during most of the arterial calcification period. This made it possible for OPG to completely inhibit RANKL-induced osteoclast-like cell differentiation. This likely explains why RANKL had the ability to induce osteoclast-like cell differentiation but acted as a promoter of calcification instead.
Acid Phosphatase ; genetics ; metabolism ; Animals ; Aorta ; drug effects ; metabolism ; pathology ; Cell Differentiation ; Coculture Techniques ; Gene Expression Regulation ; Isoenzymes ; genetics ; metabolism ; Male ; Monocytes ; cytology ; drug effects ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; pathology ; Osteoclasts ; drug effects ; metabolism ; pathology ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tartrate-Resistant Acid Phosphatase ; Vascular Calcification ; genetics ; metabolism ; pathology

Eupatilin is the main active component of DA-9601, an extract from Artemisia. Recently, eupatilin was reported to have anti-inflammatory properties. We investigated the anti-arthritic effect of eupatilin in a murine arthritis model and human rheumatoid synoviocytes. DA-9601 was injected into collagen-induced arthritis (CIA) mice. Arthritis score was regularly evaluated. Mouse monocytes were differentiated into osteoclasts when eupatilin was added simultaneously. Osteoclasts were stained with tartrate-resistant acid phosphatase and then manually counted. Rheumatoid synoviocytes were stimulated with TNF-alpha and then treated with eupatilin, and the levels of IL-6 and IL-1beta mRNA expression in synoviocytes were measured by RT-PCR. Intraperitoneal injection of DA-9601 reduced arthritis scores in CIA mice. TNF-alpha treatment of synoviocytes increased the expression of IL-6 and IL-1beta mRNAs, which was inhibited by eupatilin. Eupatilin decreased the number of osteoclasts in a concentration dependent manner. These findings, showing that eupatilin and DA-9601 inhibited the expression of inflammatory cytokines and the differentiation of osteoclasts, suggest that eupatilin and DA-9601 is a candidate anti-inflammatory agent.
Animals ; Anti-Inflammatory Agents/pharmacology/*therapeutic use ; Arthritis, Experimental/chemically induced/*drug therapy ; Arthritis, Rheumatoid/drug therapy/pathology ; Cell Differentiation/*drug effects ; Cells, Cultured ; Collagen Type II ; Cytokines/biosynthesis ; Disease Models, Animal ; Drugs, Chinese Herbal/therapeutic use ; Female ; Flavonoids/pharmacology/*therapeutic use ; Humans ; Inflammation/drug therapy/immunology ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Lymph Nodes/cytology ; Mice ; Mice, Inbred DBA ; Monocytes/cytology ; Osteoclasts/*cytology ; Plant Extracts/pharmacology ; RNA, Messenger/biosynthesis ; Synovial Membrane/cytology ; T-Lymphocytes, Regulatory/cytology/immunology ; Tumor Necrosis Factor-alpha/pharmacology

Crocetin, a naturally occurring carotenoid, possesses antioxidant and antiatherosclerotic properties, of which the underlying mechanism remains unclear. In the present study, we examined the effects of crocetin (0.1, 1, 10 μmol·L(-1)) on angiotensin II (Ang II, 0.1 μmol·L(-1)) induced expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) and monocyte-endothelial cell adhesion. The effects of crocetin on the activation of nuclear factor kappa B (NF-κB) and intracellular reactive oxygen species (ROS) were also observed. The results demonstrated that crocetin notably suppressed Ang II induced NF-κB activation (P<0.01) and VCAM-1 expression (P<0.05, P<0.01) in HUVECs, accompanied by a markedly reduced monocyte-endothelial cell adhesion (P<0.05, P<0.01). In addition, preincubation with crocetin resulted in a significant enhancement of cellular antioxidant capacity (P<0.05, P<0.01), while Ang II induced intracellular ROS decreased markedly (P<0.05, P<0.01). These results indicated that crocetin was capable of suppressing Ang II induced VCAM-1 expression and monocyte-endothelial cell adhesion by suppression of NF-κB activation, which might be derived from the enhancement of antioxidant capacity and subsequent reduction of intracellular ROS.
Angiotensin II ; metabolism ; Antioxidants ; pharmacology ; Carotenoids ; pharmacology ; Cell Adhesion ; drug effects ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Monocytes ; cytology ; NF-kappa B ; metabolism ; Reactive Oxygen Species ; metabolism ; Vascular Cell Adhesion Molecule-1 ; metabolism

OBJECTIVETo investigate whether I-tetrahydropalmatine (I-THP), an alkaloid mainly present in Corydalis family, could ameliorate early vascular inflammatory responses in atherosclerotic processes.

METHODSFluorescently labeled monocytes were co-incubated with human umbilical vein endothelial cells (HUVECs), which were pretreated with I-THP and then simulated with tumor necrosis factor (TNF)-α in absence of I-THP to determine if I-THP could reduce thecytokine-induced adhesion of monocytes to HUVECs. Then I-THP were further studied the underlying mechanisms through observing the transcriptional and translational level of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and the nuclear translocation of nuclear factor (NF)-κ B in HUVECs.

RESULTSL-THP could block TNF-α-induced adhesion of monocytes to HUVECs and could significantly inhibited the expression of ICAM-1 and VCAM-1 on cell surface by 31% and 36% at 30 μ mol/L. L-THP pretreatment could also markedly reduce transcriptional and translational level of VCAM-1 as well as mildly reduce the total protein and mRNA expression levels of ICAM-1. Furthermore, I-THP attenuated TNF-α-stimulated NF-κ B nuclear translocation.

CONCLUSIONThese results provide evidences supporting that I-THP could be a promising compound in the prevention and treatment of the early vascular inflammatory reaction in atherosclerosis by inhibiting monocyte adhesion to vascular endothelial cell through downregulating ICAM-1 and VCAM-1 in vascular endothelial cell based on suppressing NF-κ B.

Berberine Alkaloids ; pharmacology ; Cell Adhesion ; drug effects ; Cell Nucleus ; drug effects ; metabolism ; Down-Regulation ; drug effects ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Monocytes ; cytology ; drug effects ; metabolism ; NF-kappa B ; metabolism ; Protein Transport ; drug effects ; RNA, Messenger ; genetics ; metabolism ; Signal Transduction ; drug effects ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology ; Vascular Cell Adhesion Molecule-1 ; genetics ; metabolism

OBJECTIVETo study the effects of drug plasma of musk and olibanum (DP-M&O) on the release of inflammatory cytokines from monocytes and the expressions of the proteins associated with inflammation of prostatic or endothelial cells induced by prostate antigen (PAg) stimulation.

METHODSWe prepared DP-M&O using SD rats and monocytes and PAgs using BALB/c mice. We pre-treated the monocytes with DP-M&O at the gradient concentrations of 0, 2.5, 5, 10, and 20% for 1 hour, activated them with PAgs, and then cultured them for 96 hours, followed by detection of the release of inflammatory cytokines. We co-cultured the prostate RWPE-1 cells with the endothelial EA. hy926 cells, pre-treated them with the same gradient concentrations of DP-M&O as above for 1 hour, activated with PAgs, and cultured for 96 hours. Then we determined the expression levels of the proteins associated with inflammation of RWPE-1 and EA. hy926 cells by Western blot.

RESULTSDP-M&O decreased the levels of TNF-alpha, IL-1beta, IL-6, and IL-8 and increased that of IL-10 in a concentration-dependent manner. Significant differences were found between the 20% P-M&O and PAg groups in the release of the inflammatory cytokines TNF-alpha (70.8 +/- 22.3 vs. 277.1 +/- 65.5, P < 0.01) , IL-113 (277.5 +/- 22.6 vs. 630.4 +/- 89.7, P <0.01), IL-6 (232.7 +/- 62.7 vs. 994.2 vs. 182.3, P < 0.01), IL-8 (227.3 +/- 79.2 vs. 769.3 +/- 284.1, P < 0.01), and IL-10 (640.2 +/- 201.2 vs. 271.1 +/- 55.8, P < 0.01). Compared with the PAg group, the 10 and 20% P-M&O groups showed remarkable decreases in the protein expression of MCP-1/CCL2 in the RWPE-1 cells (1.12 +/- 0.34 vs. 0.56 +/- 0.11 and 0.34 +/- 0.08) and that of VCAM-1 in the EA. hy926 cells (0.94 +/- 0.22 vs. 0.52 +/- 0.17 and 0.38 +/- 0.12) (P < 0.05 or 0.01).

CONCLUSIONThe compatibility of musk and olibanum can decrease the expression of MCP-1/CCL2 in prostate cells and VCAM-1 in vascular endothelial cells, blocking the adhesion of leucocytes and suppressing inflammatory response.

Animals ; Blotting, Western ; Cytokines ; metabolism ; Endothelial Cells ; drug effects ; metabolism ; Fatty Acids, Monounsaturated ; pharmacology ; Frankincense ; pharmacology ; Inflammation ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Interleukin-8 ; Male ; Mice ; Mice, Inbred BALB C ; Monocytes ; drug effects ; metabolism ; Prostate ; cytology ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; metabolism ; Vascular Cell Adhesion Molecule-1 ; metabolism

OBJECTIVETo observe the changes in the adhesive function of vascular endothelial cells (VEC) and rat monocytes induced by lipopolysaccharide (LPS) and co-cultured with smooth muscle cells (SMC) and the intervention effect of paeonol (Pae).

METHODPrimary rat vascular endothelial cells (VECs) and rat vascular smooth muscle cells (VSMCs) were cultured by predigesting and adhering tissue blocks. The VEC-VSMC co-culture model was established by Transwell chamber. LPS was used to induce VEC injury. MTT assay and LDH assay were used to determine the VEC activity. ELISA assay was used to detect IL-1beta and TNF-alpha secreted by the VEC. The immunocytochemistry assay was carried out to detect the expression of ICAM-1. The Rose Bengal Staining was used to test adhesive function between VECs and monocytes.

RESULTThe concentration of LPS-induced VEC injury was 100 microg x L(-1), and the time was 7 h. after the intervention on the above cell model for 24 h, Paeonol (15, 30, 60 micromol x L(-1)) could effectively inhibit LPS-induced VEC injury and VEC injury, significantly enhance the survival rate of LPS-injured VECs, decrease IL-1beta and TNF-alpha secreted by the injured VEC, and reduce the expression of ICAM-1, so as to inhibit the adhesion of LPS-induced VECs and monocytes.

CONCLUSIONPaeonol could inhibit IL-1beta and TNF-alpha expression to protect VECs from being injured by LPS, and reduce ICAM-1 expression to inhibit the adhesion between VECs and monocytes.

Acetophenones ; pharmacology ; Animals ; Cell Adhesion ; drug effects ; Coculture Techniques ; Endothelial Cells ; cytology ; drug effects ; metabolism ; secretion ; Gene Expression Regulation ; drug effects ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-1beta ; metabolism ; Lipopolysaccharides ; pharmacology ; Male ; Monocytes ; cytology ; drug effects ; Myocytes, Smooth Muscle ; cytology ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo investigate the effects of non-neuronal muscarinic receptors (NNMR) stimulation on atherosclerosis and endothelial cells activation.

METHODSAtherosclerosis model was established in ApoE-/- mice by a high fat diet for 7 weeks. During the experimental periods, animals were received a low (7 mg/kg/d) or a high (21 mg/kg/d) dose of arecoline by gavage. At the termination of the treatments, serum total cholesterol and NO levels were measured, and the aorta morphology was analyzed by hematoxylin and eosin staining. The gene expression of monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules in the thoracic aortas was determined by RT-PCR, and the MCP-1 protein expression and NF-κB activity were detected by Western blot analysis. NO production, MCP-1 secretion in cultured rat aortic endothelial cells (RAECs), and monocyte-endothelium adhesion assay were also performed after arecoline treatments.

RESULTSArecoline efficiently decreased atherosclerotic plaque areas, increased serum nitric oxide (NO) content, suppressed the mRNA and protein expression of MCP-1, and modulated the IκB-α degradation and P65 phosphorylation in the aortae of ApoE-/- mice. Furthermore, arecoline promoted NO production and suppressed MCP-1 secretion in cultured RAECs after ox-LDL exposure, and either atropine or NG-nitro-L-arginine methylester could abrogate these effects. Arecoline also significantly inhibited the adherence of U937 monocytes to the ox-LDL injured human umbilical vein endothelial cells, which could be abolished by atropine.

CONCLUSIONOur results indicate that arecoline attenuates the progression of atherosclerosis and inhibits endothelial cells activation and adherence by stimulating endothelial NNMR. These effects, at least in part, are due to its modulation on NF-κB activity.

Animals ; Aorta ; cytology ; Apolipoproteins E ; Arecoline ; pharmacology ; Atherosclerosis ; physiopathology ; prevention & control ; Cell Adhesion Molecules ; metabolism ; Chemokine CCL2 ; metabolism ; Cholesterol ; blood ; Disease Progression ; Endothelial Cells ; cytology ; drug effects ; Endothelium, Vascular ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; I-kappa B Proteins ; metabolism ; Lipoproteins, LDL ; Mice ; Mice, Knockout ; Monocytes ; cytology ; NF-KappaB Inhibitor alpha ; Nitric Oxide ; blood ; Nitroarginine ; pharmacology ; Rats ; Receptors, Muscarinic ; physiology ; Transcription Factor RelA ; metabolism

To observe a PPAR-alpha agonist effect of N-oleoylethanolamine (OEA) on CB2 (cannabinoid receptor 2), an anti-inflammatory receptor in vascular endothelial cell, healthy HUVECs and TNF-alpha induced HUVECs were used to establish a human vascular endothelial cell inflammatory model. Different doses of OEA (10, 50 and 100 micromol x L(-1)) had been given to HUVECs, cultured at 37 degrees C for 7 h and then collected the total protein and total mRNA. CB2 protein expression was detected by Western blotting and CB2 mRNA expression was assayed by real-time PCR. As the results shown, OEA (10 and 50 micromol x L(-1)) could induce the CB2 protein and mRNA expression, but not 100 micromol x L(-1). To detect if anti-inflammation effect of OEA is partly through CB2, CB2 inhibitor AM630 was used to inhibit HUVEC CB2 expression, then the VCAM-1 expression induced by TNF-alpha was detected, or THP-1 adhere to TNF-alpha induced HUVECs was examined. OEA (50 micromol x L(-1)) could inhibit TNF-alpha induced VCAM-1 expression and THP-1 adhere to HUVECs, these effects could be partly inhibited by a CB2 inhibitor AM630. The anti-inflammation effect of OEA is induced by PPAR-alpha and CB2, suggesting that CB2 signaling could be a target for anti-atherosclerosis, OEA have wide effect in anti-inflammation, it may have better therapeutic potential in anti-inflammation in HUVECs, thus achieving anti-atherosclerosis effect.
Anti-Inflammatory Agents ; pharmacology ; Atherosclerosis ; pathology ; Cell Adhesion ; drug effects ; Cells, Cultured ; Endocannabinoids ; pharmacology ; Endothelial Cells ; cytology ; metabolism ; Ethanolamines ; pharmacology ; Humans ; Indoles ; pharmacology ; Monocytes ; drug effects ; Oleic Acids ; pharmacology ; PPAR alpha ; antagonists & inhibitors ; RNA, Messenger ; metabolism ; Receptor, Cannabinoid, CB2 ; antagonists & inhibitors ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology ; Vascular Cell Adhesion Molecule-1 ; metabolism

The study was aimed to investigate the mechanism of mannan-binding lectin (MBL) on bacterial lipopolysaccharide (LPS)-induced human peripheral blood monocyte-derived dendritic cell (DC) maturation. The monocytes were prepared from the peripheral blood of healthy adult volunteers. The immature dendritic cells (imDC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4. FACS was used to investigate the interaction of MBL with imDC and the impact of MBL on LPS binding to imDC. ELISA and Western blot was used to analyze the interaction of MBL with soluble TLR4 ectodomain protein (sTLR4); Western blot was used to detect LPS-induced NF-κB translocation in imDC. The results showed that MBL could directly bind to imDC in the presence of calcium. sTLR4 protein or LPS could competitively inhibit the binding of MBL to imDC. ELISA and Western blot showed that MBL could evidently bind to sTLR4 protein in a concentration-dependent manner. FACS showed that MBL could competitively inhibit the binding of LPS to imDC by binding to imDC directly. Western blot showed that MBL decreased LPS-induced NF-κB translocation in imDC. It is concluded that MBL may competitively inhibit the binding of LPS to imDC by binding to TLR4 expressed on imDC, resulted in inhibition of LPS-induced DC maturation, suggesting that MBL can regulate DC maturation through ligand-binding. This study provides the good foundation to clarify the mechanism of MBL inhibiting the LPS-induced DC maturation.
Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Humans ; Ligands ; Lipopolysaccharides ; adverse effects ; Mannose-Binding Lectin ; pharmacology ; Monocytes ; cytology ; metabolism ; Toll-Like Receptor 4 ; metabolism