Monocyte Chemotactic Protein-1 Protein Expression Research Articles

Changes in MCP-1, HGF, and IGF-1 expression in endometrial stromal cells, PBMCs, and PFMCs of endometriotic women following 1,25(OH)2D3 treatment.

1,25(OH)2D3 has anti-inflammatory and growth inhibitory effects. Our study explored the effect of 1,25(OH)2D3 treatment on the expression of monocyte chemotactic protein-1 (MCP-1), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1) by peripheral blood mononuclear cells (PBMCs), peritoneal fluid mononuclear cells (PFMCs), endometrial stromal cells (ESCs), and its effect on the proliferation of PBMCs and PFMCs of patients with endometriosis compared with controls. PBMCs, PFMCs, and ESCs were obtained from 10 endometriosis patients and 10 non-endometriotic individuals. After treating cells with 0.1μM of 1,25(OH)2D3 for 6, 24, and 48 h, the gene and protein expression of mentioned factors were evaluated by real-time PCR and ELISA methods, respectively. 1,25(OH)2D3 treatment significantly reduced the protein expression of MCP-1, HGF, and IGF-1 in PBMCs and PFMCs of endometriotic patients at 48 h (p< 0.05-<0.01). Also, this treatment significantly reduced MCP-1, HGF, and IGF-1 gene and/or protein expression in EESCs and EuESCs at 24 and 48 h (p< 0.05-<0.01). 1,25(OH)2D3 treatment also reduced the proliferation of PBMCs and PFMCs of endometriotic patients compared with controls (p < 0.01). 1,25(OH)2D3 can be considered as a potentially effective agent in the prevention and treatment of endometriosis along with other therapies.

Correlation between Increased Monocyte Chemotactic Protein 1 Level and Lung Function in Patients with Asthma

Background: The altered expression of monocyte chemotactic protein 1 (MCP1) in bronchoalveolar fluid was observed in patients and animal models of allergic asthma. However, the correlation between induced sputum MCP1 level and asthma clinical features remains poorly understood. Objective: This study aims to explore the relationship of MCP1 protein expression in induced sputum with Th2 inflammation and asthma clinical features. Methods: MCP1 protein expression in induced sputum and serum was detected by ELISA in patients with asthma, and its correlation with Th2 inflammation and lung function was analyzed. The effect of inhaled corticosteroids (ICSs) on MCP1 expression was also evaluated. Results: The MCP1 level in induced sputum (p = 0.0006) and serum (p = 0.0035) was markedly increased and negatively correlated with forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC)% (r = −0.3674, p = 0.0001) and PC-20 (r = −0.5746, p < 0.0001) in patients with asthma. The area under the curve (AUC) of MCP1 level receiver operating characteristic curve in induced sputum and serum was 0.7134 (p = 0.0007) and 0.7589 (p = 0.0042), respectively. The patients with asthma were grouped into four according to their induced sputum MCP1 level and Th2 signature categories (Th2^Hi MCP1^Hi, Th2^Hi MCP1^Low, Th2^Low MCP1^Hi, and Th2^Low MCP1^Low). The Th2^Low MCP1^Hi subgroup had the lowest FEV1/FVC% and histamine concentration required to cause a 20% decline in FEV1. After 4 weeks of ICS treatment, the MCP1 levels in induced sputum and serum were significantly reduced. Conclusions: The MCP1 level in induced sputum and serum increased in patients with asthma but decreased after ICS treatment. The Th2^Low MCP1^Hi subgroup had the worst lung function and highest airway hyperresponsiveness. The combination of MCP1 level in induced sputum and Th2 inflammation defines a new phenotype that can be used to predict lung function and treatment response in patients with asthma.

Resveratrol treatment reduces expression of MCP-1, IL-6, IL-8 and RANTES in endometriotic stromal cells.

Endometriosis is an inflammatory disease affecting reproductive‐aged women. Immunologic disturbance, as well as inflammation, have crucial roles in the pathogenesis of endometriosis. In this study, we evaluated the effects of resveratrol treatment on expression of monocyte chemotactic protein‐1 (MCP‐1), interleukin‐6 (IL‐6), IL‐8, and regulated upon activation, normal T cell expressed and secreted (RANTES) in endometrial stromal cells from patients with endometriosis compared with non‐endometriotic controls. Thirteen eutopic (EuESCs) and nine ectopic (EESCs) endometrial stromal cells from endometriotic patients as well as eleven endometrial stromal cells from non‐endometriotic controls (CESCs) were treated with resveratrol (100 μmol/L) or ethanol, and gene and/or protein expression of MCP‐1, IL‐6, IL‐8 and RANTES was examined at 6, 24 and 48 hours following treatment in the cells from all origins. Resveratrol treatment significantly reduced gene and protein expression of MCP‐1, IL‐6, and IL‐8 in EuESCs and EESCs compared with CESCs (P < .05‐.001, P < .05‐.001 and P < .05‐<.01, respectively), and this reduction was more noticeable in EESCs than EuESCs (P < .05‐<.001). Besides, resveratrol treatment significantly reduced RANTES protein expression in EESCs in all time intervals (P < .05). Resveratrol treatment significantly reduced the expression of MCP‐1, IL‐6, IL‐8 and RANTES in EESCs.

The Mechanism of miR-192 in Regulating High Glucose-Induced MCP-1 Expression in Rat Glomerular Mesangial Cells.

Although the pathogenetic mechanism of Diabetic Kidney Disease (DKD) has not been elucidated, an inflammatory mechanism may be a potential contributor. Monocyte chemotactic protein-1 (MCP-1) is suggested to be implicated in the development of DKD by playing a role in the infiltration of monocyte/macrophage. The aim of this study was to investigate the expression of MCP-1 under high glucose conditions, as well as the effects of microRNA-192 (miR-192) under these conditions, and to study the regulatory mechanism of MCP-1 in DKD. Rat glomerular mesangial cells were cultured in high glucose or isotonic mannitol. The messenger RNA(mRNA) expression of miR-192, miR-200b, miR-200c, E-box-binding homeobox 1 (Zeb1), and MCP-1 was then detected by real-time PCR, and the protein expression of Zeb1 and MCP- 1 was assessed by western blotting. The rat mesangial cells were transfected with an miR-192 inhibitor, NC inhibitor , and transfected with siRNA Zeb1, siNC. The cells were then cultured in high glucose to detect the mRNA expression of miR-192, miR-200b, miR-200c, Zeb1, and MCP-1 using realtime PCR, and Zeb1 and MCP-1 protein expression were determined by western blotting. MiR-192, miR-200b, miR-200c, and MCP-1 were overexpressed, whereas Zeb1 was downregulated when cultured in high glucose (P < 0.05). After transfection with an miR-192 inhibitor, the expression of miR-192, miR-200b, miR-200c, and MCP-1 was downregulated, whereas Zeb1 was increased, and these differences were statistically significant (P < 0.05). The observed changes in the expression in the NC inhibitor transfection group were similar to that of non-transfected cell lines. Silencing the expression of Zeb1 resulted in a significant increase in the expression of miR-192, miR- 200b, miR-200c, and MCP-1 (P < 0.05). The observed changes in the SiNC transfection group were similar to those of non-transfected cell lines. MiR-192 expression was upregulated to increase the expression of inflammatory factor MCP-1 by inhibiting the expression of Zeb1, which was mediated by breaking the regulatory loop of Zeb1 and miR-200b/c in rat mesangial cells cultured in high glucose.

Combination of curcumin and luteolin synergistically inhibits TNF-α-induced vascular inflammation in human vascular cells and mice

Emerging evidence shows that phytochemicals, the secondary plant metabolites present in a large variety of foods, have the potential ability in reducing the risk of cardiovascular diseases. However, the dosages of phytochemicals in the cellular and animal studies are too high to reach in humans by relevant foods or dietary supplement intake. The aims of this study were to investigate whether and how combined curcumin and luteolin synergistically inhibit tumor necrosis factor-alpha (TNF-α)-induced monocytes adhesion endothelium, a crucial step of the development of endothelial dysfunction, both in human vascular cells and mouse aortic endothelium. Our results show that combined curcumin (1 μM) and luteolin (0.5 μM) synergistically (combination index is 0.60) inhibited TNF-α-induced monocytes adhesion to human EA.hy926 endothelial cells while the individual chemicals did not have such effect at the selected concentrations. We also found that TNF-α-enhanced protein expressions of vascular cell adhesion molecule 1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1) and nuclear factor (NF)-κB translocation were synergistically reduced by the combined curcumin and luteolin in EA.hy 926 cells while the individual chemical did not have this inhibitory effect. Consistently, 2 weeks dietary intake of combined curcumin (500 mg/kg) and luteolin (500 mg/kg) in C57BL/6 mice synergistically prevented TNF-α-stimulated adhesion of mouse monocytes to aortic endothelium ex vivo as well as the TNF-α-increased aortic protein expression of MCP-1 and VCAM-1. Therefore, combined curcumin and luteolin at physiological concentrations synergistically inhibits TNF-α-induced monocytes adhesion to endothelial cells and expressions of MCP-1 and VCAM-1 via suppressing NF-κB translocation into the nucleus.

Rapamycin liposome gutta inhibiting fungal keratitis of rats.

To study the therapeutic effect of rapamycin liposome eyedrops on fungal keratitis (FK) and its effect on the expression of monocyte chemotactic protein-1 (MCP-1). This study adopted the thin film dispersion method to prepare rapamycin liposomes eyedrops, as well as used the orthogonal design to analyze and study main influencing factors that affected the quality of liposomes. Totally 96 healthy Wistar rats were randomly divided into four groups: normal control group (A), FK blank control group (B), FK blank liposomes control group (C), and 30 FK rapamycin liposome treatment group (D). Groups B, C, and D were first prepared as FK animal models. The corneal response was recorded in details on day 1, 3, 5, 7, and 14 after modeling. Six rats were obtained and immunohistochemistry and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of MCP-1 protein and mRNA, respectively. The severity of corneal lesions in the rapamycin treatment group was reduced, and the clinical score of the slit lamp examination was lower than that of Groups B and C (P<0.01). The expression of MCP-1 in rapamycin treatment group was significantly inhibited, comparing to that of groups B and C (P<0.01). Liposome is a good drug carrier for rapamycin. Rapamycin has a good therapeutic effect on FK. It can reduce FK fungal burden and significantly inhibit the expression of MCP-1 protein and mRNA.

On the effects of NLRP3 inflammasome on mice pancreatic β-cell damage induced by advanced glycation end products

Objective To explore the role of the pyrin domain-containing 3 (NLRP3) inflammasome in advanced glycation end products (AGEs)-induced mice pancreatic β-cell damage. Methods AGEs were administered intraperitoneally for 6 weeks in NLRP3 knockout mice or C57BL/6J mice. Intraperitoneal glucose tolerance test and insulin releasing test were performed. Pancreatic sections were stained with haematoxylin and eosin, or with F4/80 and NLRP3 antibodies. Insulin and pancreatic tissue monocyte chemotactic protein 1 (MCP-1) as well as interleukin-1β (IL-1β) levels were measured with ELISA kits. Expression of MCP-1 protein was determined by western blot. MIN6 cells and mouse peritoneal macrophages cells were treated with AGEs and different interventions (antioxidant NAC, adenovirus NLRP3 shRNA or NLRP3 knockout). Reactive oxygen species production, NLRP3 mRNA expression, IL-1β secretion, caspase 1 activity, apoptosis and glucose stimulated insulin release were determined. Results Injection of AGEs induced an abnormal response to glucose, enhanced the insulitis score, and increased the levels of pancreatic tissue MCP-1 and IL-1β, as well as raised the expression of NLRP3 and F4/80 in pancreatic islet. Remarkably, co-localization of NLRP3 and macrophage marker F4/80 was observed in islet. The damages were improved in NLRP3 knockout mice. After incubation with AGEs, reactive oxygen species production and cell apoptosis was enhanced, NLRP3 inflammasome activated, with glucose-stimulated insulin release impaired in MIN6 cells. NAC treatment alliviated the above damages, but NLRP3 gene silencing had no effect on ROS level, apoptosis, and insulin secretion. Finally NAC treatment and NLRP3 gene knockout inhibited activation of NLRP3 inflammasome induced by AGEs in mouse peritoneal macrophages cells. Conclusion NLRP3 knockout ameliorates the islet β-cell damage induced by AGEs. These effects were associated with AGEs-induced islets macrophage infiltrating by up-regulation of MCP-1 expression, and AGEs-induced activation of NLRP3 inflammasome in macrophage through ROS pathway, which results in the release of active IL-1β and leads to the lesions of β-cell. (Chin J Endocrinol Metab, 2018, 34: 690-695) Key words: Advanced glycation end products; NLRP3 inflammasome; β-cell damage

Effects of Porphyromonas endodontalis lipopolysaccharides on the expression of monocyte chemotactic protein-1 in mouse osteoblasts

To investigate the effects of lipopolysaccharide (LPS) extracted from Porphyromonas endodontalis (P.endodontalis) on expression of monocyte chemotactic protein-1 (MCP-1) mRNA and protein in MC3T3-E1 cells and the role of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kB(NF-kB）in the process. MC3T3-E1 cells were treated with different concentrations of P.endodontalis LPS(0-50mg/L) and 20 mg/L P.endodontalis LPS for different hours (0-48 h). The expression of MCP-1 mRNA was detected by real-time reverse transcription PCR(RT-PCR) and protein was detected by enzyme-1inked immunosorbent assay (ELISA). MC3T3-E1 cells were pretreated with SB203580 (inhibitor of p38MAPK) and BAY11-7082 (inhibitor of NF-kB) for 1h, and then were treated with 20 mg/L P.endodontalis LPS for 24 h, the expression of MCP-1 mRNA was also detected by RT-PCR. Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 13.0 software package. The level of MCP-1 mRNA and protein increased significantly after treatment with different concentrations of P.endodontalis LPS (0-50 mg/L), which indicated that P.endodontalis LPS induced osteoblasts to express MCP-1 in a dose dependent manners. During the observation time (0-48 h), the impact of 20 mg/L P.endodontalis LPS on induction of MCP-1 in MC3T3-E1 cells exhibited a time-dependent manner. The expression of MCP-1 mRNA decreased significantly after pretreated with 10 mol/L SB203580 and BAY11-7082 for 1 h,and the inhibitory effect of SB203580 was stronger than BAY11-7082. P.endodontalis LPS may induce the expression of MCP-1 mRNA and protein in MC3T3-E1 cells through the signaling pathway of p38MAPK and NF-kB.

Chlorella Protein Hydrolysate Attenuates Glucose Metabolic Disorder and Fatty Liver in High-fat Diet-induced Obese Mice.

Chlorella (Parachlorella beijerinckii) powder is reported to show a preventive effect against metabolic syndromes such as arteriosclerosis, hyperlipidemia, and hypertension. Approximately 60% of the chlorella content is protein. In order to understand the role of chlorella protein, we prepared a chlorella protein hydrolysate (CPH) by protease treatment. Male C57BL/6 mice were divided into three groups: a normal diet group, high-fat diet (HFD) group, and high-fat diet supplemented with CPH (HFD+CPH) group. The CPH administration improved glucose intolerance, insulin sensitivity, and adipose tissue hypertrophy in the high-fat diet-fed mice. In addition, the HFD+CPH group had significantly decreased liver total cholesterol and triglyceride levels compared with those in the HFD group. Furthermore, the HFD+CPH group had a decreased level of monocyte chemotactic protein-1 (MCP-1) in serum and a lower MCP-1 mRNA expression level in adipose tissue compared with the HFD group. The present study suggests that chlorella protein hydrolysate can prevent a high-fat diet-induced glucose disorder and fatty liver by inhibiting adipocyte hypertrophy and reducing the MCP-1 protein and gene expression.

MCP/CCR2 signaling is essential for recruitment of mesenchymal progenitor cells during the early phase of fracture healing.

ObjectiveThe purpose of this study was to investigate chemokine profiles and their functional roles in the early phase of fracture healing in mouse models.MethodsThe expression profiles of chemokines were examined during fracture healing in wild-type (WT) mice using a polymerase chain reaction array and histological staining. The functional effect of monocyte chemotactic protein-1 (MCP-1) on primary mouse bone marrow stromal cells (mBMSCs) was evaluated using an in vitro migration assay. MCP-1−/− and C-C chemokine receptor 2 (CCR2)−/− mice were fractured and evaluated by histological staining and micro-computed tomography (micro-CT). RS102895, an antagonist of CCR2, was continuously administered in WT mice before or after rib fracture and evaluated by histological staining and micro-CT. Bone graft exchange models were created in WT and MCP-1−/− mice and were evaluated by histological staining and micro-CT.Results MCP-1 and MCP-3 expression in the early phase of fracture healing were up-regulated, and high levels of MCP-1 and MCP-3 protein expression observed in the periosteum and endosteum in the same period. MCP-1, but not MCP-3, increased migration of mBMSCs in a dose-dependent manner. Fracture healing in MCP-1−/− and CCR2−/− mice was delayed compared with WT mice on day 21. Administration of RS102895 in the early, but not in the late phase, caused delayed fracture healing. Transplantation of WT-derived graft into host MCP-1−/− mice significantly increased new bone formation in the bone graft exchange models. Furthermore, marked induction of MCP-1 expression in the periosteum and endosteum was observed around the WT-derived graft in the host MCP-1−/− mouse. Conversely, transplantation of MCP-1−/− mouse-derived grafts into host WT mice markedly decreased new bone formation.ConclusionsMCP-1/CCR2 signaling in the periosteum and endosteum is essential for the recruitment of mesenchymal progenitor cells in the early phase of fracture healing.

Copper induces – and copper chelation by tetrathiomolybdate inhibits – endothelial activation in vitro

Endothelial activation with increased expression of cellular adhesion molecules and chemokines critically contributes to vascular inflammation and atherogenesis. Redox-active transition metal ions play an important role in vascular oxidative stress and inflammation. Therefore, the goal of the present study was to investigate the role of copper in endothelial activation and the potential anti-inflammatory effects of copper chelation by tetrathiomolybdate (TTM) in human aortic endothelial cells (HAECs). Incubating HAECs with cupric sulfate dose- and time-dependently increased mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and monocyte chemotactic protein-1 (MCP-1). Copper also activated the redox-sensitive transcription factors, nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1), which was inhibited by pretreatment of the cells with TTM. Furthermore, TTM dose-dependently inhibited tumor necrosis factor α (TNFα)-induced activation of NF-κB and AP-1, as well as mRNA and protein expression of VCAM-1, ICAM-1, and MCP-1, which was abolished by preincubating the cells with 5 µM TTM and 15 µM cupric sulfate. The inhibitory effect of TTM on TNFα-induced NF-κB activation was associated with decreased phosphorylation and degradation of IκBα. These data suggest that intracellular copper causes activation of redox-sensitive transcription factors and upregulation of inflammatory mediators in endothelial cells. Copper chelation by TTM may attenuate TNFα-induced endothelial activation and, hence, inhibit vascular inflammation and atherosclerosis.

Effects of cyclic stretch on expression of cytokines and intercellular adhesion molecule-1 in human pulmonary artery endothelial cell

To observe the effects of cyclic stretch on expression of cytokines and adhesion molecules in human pulmonary artery endothelial cells (HPAECs), herein to provide a theoretical basis to ventilator-induced lung injury (VILI). HPAECs were subjected to cyclic stretch by the Flexcell FX-5000T system at 0.5 Hz of 10% or 20% elongation for 3, 6, 12, 24 hours respectively. The mRNA and protein expression of interleukin (IL-6, IL-8), monocyte chemotactic protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) was determined by fluorescent quantitation reverse transcription-polymerase chain reaction (qRT-PCR), enzyme linked immunosorbent assay (ELISA) or Western blotting. Increasing the stretch force, the mRNA and protein expression of IL-8, MCP-1, ICAM-1 were up regulated with increasing stretch time. Compared with the control (set 1), after 20% cyclic stretch for 24 hours, IL-8 mRNA expression was up regulated to 1.58±0.10, MCP-1 mRNA expression was up regulated to 2.85±0.52, and ICAM-1 mRNA expression was up regulated to 1.90±0.14 (all P<0.05). Compared with control group, after 20% cyclic stretch for 24 hours, the protein expression of IL-8 and MCP-1 in HPAEC was significantly increased (IL-8: 3401.08±439.60 ng/L vs. 1422.60±66.98 ng/L, MCP-1: 1117.64±237.54 ng/L vs. 307.88±80.84 ng/L, both P<0.05), ICAM-1 protein expression was up regulated to 2.15±0.40 (P<0.05), while the expression of IL-6 mRNA and protein had no statistic difference compared with control group. Cyclic stretch enhanced the expression of IL-8, MCP-1 and ICAM-1 in an intensity-dependent fashion, so it may be involved in the pathogenesis of lung injury induced by mechanical ventilation.

The anti-inflammatory effect of kaempferol on early atherosclerosis in high cholesterol fed rabbits.

BackgroundAtherosclerosis has been widely accepted as an inflammatory disease of vascular, adhesion molecules play an important role in the early progression of it. The aim of the present study was to evaluate the effect of kaempferol on the inflammatory molecules such as E-selectin (E-sel), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesionmolecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1) in high cholesterol induced atherosclerosis rabbit models.MethodsThirty male New Zealand white (NZW) rabbits were randomly divided into five groups, control group, model group, fenofibrate (12mg/kg) group and kaempferol groups (150 mg/kg and 30 mg/kg). The rabbits were fed with a normal diet or a high cholesterol diet for 10 weeks. Levels of blood lipids, serum tumour-necrosis factor-alpha (TNF-α) and serum interleukin-1beta (IL-1β) were detected at the end of the sixth and tenth week. Malonaldehyde (MDA) level and superoxide dismutase (SOD) activity in serum were also determined. Lesion areas of the aorta were measured with morphometry analysis after ten weeks. Gene expression of E-sel, ICAM-1, VCAM-1 and MCP-1 in aortas was determined by RT-PCR (reverse transcription-polymerase chain reaction). Immunohistochemical staining was employed to measure protein expression of E-sel, ICAM-1, VCAM-1 and MCP-1.ResultsModel rabbits fed with ten weeks of high-cholesterol diet developed significant progression of atherosclerosis. Compared with the control, levels of blood lipids, TNF-α, IL-1β and MDA increased markedly in serum of model rabbits, while SOD levels decreased. Gene and protein expressions of E-sel, ICAM-1, VCAM-1 and MCP-1 in atherosclerotic aortas increased remarkably in model group. However, comparing to the model rabbits, levels of TNF-α, IL-1β and MDA decreased significantly and serum SOD activity increased, gene and protein expressions of E-sel, ICAM-1, VCAM-1 and MCP-1 in aortas decreased significantly with the treatment of kaempferol.ConclusionKaempferol shows anti-atherosclerotic effect by modulating the gene and protein expression of inflammatory molecules.

Effect of herba centellae on the expression of HGF and MCP-1

The aim of this study was to explore the effects of herba centellae on protein and mRNA expression of hepatocyte growth factor (HGF), and monocyte chemotactic protein-1 (MCP-1) in renal tubulointerstitial fibrosis (TIF). A unilateral ureteral obstruction (UUO) model was established in 50 male Sprague Dawley (SD) rats. Blood samples were collected and the blood urea nitrogen (BUN) levels, serum creatinine (Scr), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured. Immunohistochemistry (IHC) detected the localization and expression levels of HGF and MCP-1. In addition, quantitative polymerase chain reaction (qPCR) detected the mRNA expression of HGF and MCP-1. Thirty rats were used to prepare the rat serum containing drug by cell culture, and qPCR and immunocytochemistry (ICC) were performed to examine the mRNA and protein expression of HGF and MCP-1. MCP-1 and its mRNA expression was significantly higher in rat renal interstitium of the UUO group and cells of the transforming growth factor-β1 (TGF-β1) stimulation group compared with that of the control group (P<0.01). MCP-1 and its mRNA expression in the drug intervention group were significantly reduced compared with that of the UUO model group (P<0.01). However, MCP-1 and its mRNA expression in the high-dose herba centellae group was significantly lower compared with that of the low-dose herba centellae group (P<0.01). Furthermore, HGF and its mRNA expression significantly increased in the drug intervention group (P<0.01), and expression in the high-dose group was significantly higher compared with that of the low-dose group (P<0.01), but similar to that of the fosinopril group (P>0.05). The levels of BUN decreased in the drug intervention group; however, no significant differences were determined in Scr, ALT and AST levels. Herba centellae may have inhibited MCP-1 and its mRNA expression through the upregulation of HGF and its mRNA expression, in order to achieve resistance to TIF without showing evident hepatorenal toxicity.

Abstract 447: Foam Cell Formation and Triglyceride Production After Incubation of Human Monocytes and Endothelial Cells with MAA-Modified Proteins

Introduction: Oxidized proteins and the formation of foam cells are central aspects in the development and progression of atherosclerosis. Malondialdehyde acetaldehyde (MAA) modified low density lipoprotein (LDL) is generated when lipoproteins are exposed to the oxidative product malondialdehyde. These MAA-adducted proteins have been shown to bind scavenger receptors, up-regulate adhesion molecules, induce cytokine expression, and initiate antibody and T-cell responses in both human and animal models of atherosclerotic disease. However, the induction of foam cells using these antigens has been untested. Objective: The purpose of this study was to evaluate the ability of MAA-adducted proteins on LDL to induce foam cell formation and upregulate innate inflammatory mediators of atherosclerosis in human peripheral monocytes, mouse macrophage, and human aortic endothelial cell lines. Methods: Mouse aortic endothelial (2167), mouse macrophage (J774) and human peripheral monocytes were incubated with LDL, oxidized-LDL, MAA-LDL, or MAA-human albumin (25 and 50 ng/ml) for 24 or 48 hours and evaluated. Cells were accessed for uptake using Oil Red O, triglyceride production, and cholesterol esters using filipin. Real-time semi-quantitative RT-PCR and ELISA was performed to evaluate IL-6 and monocyte chemotactic protein-1 (MCP-1) mRNA expression and protein secretion. MAA binding was also assessed using an anti-MAA monoclonal antibody. Results: LDL uptake is increased in aortic endothelial cells, J774 and human monocytes as evidenced by filipin and Oil Red O staining for esterified cholesterol LDL as compared to sham controls. Triglyceride data show a 5.1 fold increase in monocytes and a 6 fold increase in aortic endothelial cells increase following MAA-LDL incubation over control p&lt;0.001. Additionally, IHC for MAA-adducted proteins after incubation with MAA-modified demonstrate surface binding and intracellular uptake of MAA-adducted proteins. Incubation of MAA-albumin and MAA-LDL with aortic endothelial cells results in significant IL-6 and MCP-1 mRNA synthesis and protein expression 5 fold for IL-6 p&lt;0.001 and 3 fold for MCP-1 p&lt;0.01 Conclusions: These data show that MAA-modified proteins bind to and are processed by endothelial cells, macrophage, and human monocytes. MAA-modified proteins result in the production of innate immune mediators of tissue inflammation and inflammation recruitment. The direct effect of MAA in forming “foam cells” as evidenced by increased triglyceride and cholesterol is supportive for a role of MAA-modified proteins in the progression of atherosclerosis. The nature and impact of MAA-modification is hypothesized to be central to cellular apoptosis, autophagy and/or necrosis. Implications: MAA-modified proteins may be directly pathogenic in the development of plaque inflammation and progression of atherosclerosis.

Disruption of Smad4 impairs TGF-β/Smad3 and Smad7 transcriptional regulation during renal inflammation and fibrosis in vivo and in vitro

The mechanism by which TGF-β regulates renal inflammation and fibrosis is largely unclear; however, it is well accepted that its biological effects are mediated through Smad2 and Smad3 phosphorylation. Following activation, these Smads form heteromeric complex with Smad4 and translocate into the nucleus to bind and regulate the expression of target genes. Here we studied the roles of Smad4 to regulate TGF-β signaling in a mouse model of unilateral ureteral obstruction using conditional Smad4 knockout mice and in isolated Smad4 mutant macrophages and fibroblasts. Disruption of Smad4 significantly enhanced renal inflammation as evidenced by a greater CD45(+) leukocyte and F4/80(+) macrophage infiltration and upregulation of IL-1β, TNF-α, MCP-1, and ICAM-1 in the obstructed kidney and in IL-1β-stimulated macrophages. In contrast, deletion of Smad4 inhibited renal fibrosis and TGF-β1-induced collagen I expression by fibroblasts. Further studies showed that the loss of Smad4 repressed Smad7 transcription, leading to a loss of functional protein. This, in turn, inhibited IκBα expression but enhanced NF-κB activation, thereby promoting renal inflammation. Interestingly, deletion of Smad4 influenced Smad3-mediated promoter activities and the binding of Smad3 to the COL1A2 promoter, but not Smad3 phosphorylation and nuclear translocation, thereby inhibiting the fibrotic response. Thus, Smad4 may be a key regulator for the diverse roles of TGF-β1 in inflammation and fibrogenesis by interacting with Smad7 and Smad3 to influence their transcriptional activities in renal inflammation and fibrosis.

Regulation of Monocyte Chemotactic Protein-1 Expression in Human Endometrial Endothelial Cells by Sex Steroids: A Potential Mechanism for Leukocyte Recruitment in Endometriosis

The main aim of this study is to describe the in vivo temporal and spatial expression of monocyte chemotactic protein 1 (MCP-1) in human endometrial endothelial cells (HEECs) and to compare the in vitro regulation of MCP-1 expression by sex steroids in HEECs from women with or without endometriosis. Eutopic endometrial tissues and endometriosis implants were grouped according to the menstrual cycle phase and were examined by immunohistochemistry for MCP-1 expression. No significant difference was observed for MCP-1 immunoreactivity in the endothelial cells of eutopic endometrium of women with endometriosis when compared to endometrium of women without endometriosis and to endometriosis implants. For in vitro studies, the purity of cultured HEECs (90%-95%) was confirmed by immunocytochemistry using endothelium-specific markers CD31 and CD146. The effects of estradiol (5 x 10(- 8) mol/L), progesterone (10(-7) mol/L), or both on MCP-1 messenger RNA (mRNA) and protein levels were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and enzyme-linked immunosorbent serologic assay (ELISA), respectively. Sex steroids did not have significant effect on MCP-1 mRNA and protein expression in HEECs from women without endometriosis. However, we observed that the sex steroid treatment stimulated MCP-1 mRNA and protein expression in HEECs from women with endometriosis (P < .05). We postulate that the stimulation of chemokine expression by sex steroids in the endometrial endothelial cells in women with endometriosis may play a central role in recruiting mononuclear cells, therefore contributing to the inflammatory aspect of endometriosis.

Resveratrol reduces glutamate‐mediated monocyte chemotactic protein‐1 expression via inhibition of extracellular signal‐regulated kinase 1/2 pathway in rat hippocampal slice cultures

Published evidence has linked glutamate with the pathogenesis of Alzheimer's disease (AD) and the up-regulation of a variety of chemokines, including monocyte chemotactic protein-1 (MCP-1)/chemokine ligand 2, with AD-associated pathological changes. In this study, we assessed the potential molecular basis for the role of glutamate in hippocampal inflammation by determining its effects on MCP-1 induction. We also attempted to identify the mechanism by which resveratrol (trans-3,5,4'-trihydroxystilbene), a polyphenolic phytostilbene, modulates the expression of MCP-1 in the glutamate-stimulated hippocampus. An ex vivo study using rat hippocampal slices demonstrated a time- and dose-dependent increase in MCP-1 release from glutamate-exposed hippocampus. This increase was accompanied by enhanced MCP-1 gene expression via the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway and interleukin-1beta (IL-1beta) expression. The inhibition of the MEK/ERK pathway with SL327, which is capable of crossing the blood-brain barrier, nearly abolished the observed glutamate-induced effects. Furthermore, anti-IL-1beta antibodies suppressed the glutamate-induced expression of MCP-1 mRNA and protein, whereas an isotype-matched antibody exerted only minimal effects. It is worthy of note that resveratrol, to a similar degree as SL327, down-regulated glutamate-induced IL-1beta expression and reduced the expression of MCP-1 mRNA and protein release via the inactivation of ERK1/2. These results indicate that the activation of the MEK/ERK pathway and the consequent IL-1beta expression are essential for glutamate-stimulated MCP-1 production in the hippocampus. Additionally, our data reveal an anti-inflammatory mechanism of resveratrol involving the inactivation of the ERK1/2 pathway in the hippocampus, which is linked principally to AD-associated cognitive dysfunction.

CCR3, CCR2A and macrophage inflammatory protein (MIP)‐1α, monocyte chemotactic protein‐1 (MCP‐1) in the mouse hippocampus during and after pilocarpine‐induced status epilepticus (PISE)

To investigate protein and gene expressions of chemokine subtypes CCR3, CCR2A and their respective ligands macrophage inflammatory protein 1-alpha (MIP-1alpha), monocyte chemotactic protein-1 (MCP-1) in the normal mouse central nervous system (CNS) and in the hippocampus at different time points during and after pilocarpine-induced status epilepticus (PISE). CCR3 and MIP-1alpha protein expressions were mapped in the mouse CNS. The protein and gene expressions of CCR3 and CCR2A and their respective ligands MIP-1alpha, MCP-1 in the hippocampus were studies by immunocytochemical and quantitative real-time RT-PCR during and after PISE. CCR3 and MIP-1alpha gene expression and immunopositive neurones were broadly distributed in the CNS. CCR3 and CCA2A gene and their protein expression were downregulated in the hippocampus at 1 h during PISE. The protein expression of MIP-1alpha, MCP-1 decreased but gene expression increased at 2 h during PISE. In the hilus of the dentate gyrus, significant reduction of the numbers of CCR3, CCR2A, MCP-1 immunopositive neurones occurred from 1 h during to 2 months after PISE, but the number of MIP-1alpha neurones reduced from 2 h during to 2 months after PISE. Induced expression of CCR3 at 1 week, CCR2A, MCP-1 or MIP-1alpha at 1 week and 2 months after PISE was found in reactive astrocytes. MCP-1 was also demonstrated in the blood vessels of the hippocampus at 2 months after PISE. CCR3 and MIP-1alpha may play important functional roles in the mouse brain. The downregulation of CCR3, CCR2A, MIP-1alpha and MCP-1 in the hippocampal neurones at the acute stage during and after PISE may weaken the neuroprotective mechanisms. However, induced expression of MCP-1 in hippocampal blood vessel may be related to changes in permeability of the blood-brain barrier during epileptogenesis.

Resveratrol inhibits foam cell formation via NADPH oxidase 1-mediated reactive oxygen species and monocyte chemotactic protein-1

Resveratrol is a polyphenolic compound in red wine that has anti-oxidant and cardioprotective effects in animal models. Reactive oxygen species (ROS) and monocyte chemotactic protein-1 (MCP-1) play key roles in foam cell formation and atherosclerosis. We studied LPS-mediated foam cell formation and the effect of resveratrol. Resveratrol pretreatment strongly suppressed LPS-induced foam cell formation. To determine if resveratrol affected the expression of genes that control ROS generation in macrophages, NADPH oxidase 1 (Nox1) was measured. Resveratrol treatment of macrophages inhibited LPS-induced Nox1 expression as well as ROS generation, and also suppressed LPS-induced MCP-1 mRNA and protein expression. We investigated the upstream targets of Nox1 and MCP-1 expression and found that Akt-forkhead transcription factors of the O class (FoxO3a) is an important signaling pathway that regulates both genes. These inhibitory effects of resveratrol on Nox1 expression and MCP-1 production may target to the Akt and FoxO3a signaling pathways.