Monocyte Chemotactic Protein-1 Protein Research Articles

Effect and mechanism of ubiquitin-like protein FAT10 on AngⅡ induced endothelial cell inflammation

Objective: To investigate the role and related mechanism of ubiquitin-like protein FAT10 in the angiotensin Ⅱ (AngⅡ)-induced endothelial cell inflammatory responses. Methods: The Western blot was used to detect the protein expression of FAT10 in 16-weeks old WKY rat carotid artery, thoracic aorta artery, renal artery and vascular smooth muscle cells (VSMC), human umbilical vein endothelial cells (HUVEC) and human breast cancer cells (MDA-MB-231). The optimal concentration and stimulation time of AngⅡ on inducing the highest FAT10 in HUVEC were determined. The following plasmids were constructed: control plasmid, overexpression FAT10 plasmid (Flag-FAT10), invalid interference plasmid, and interference FAT10 plasmid (sh-FAT10). These plasmids were then transfected into HUVEC cells and divided into following groups: control group, Flag-FAT10 group, invalid interference group, and sh-FAT10 group. After culturing with 100 nmol/L AngⅡ for 36 h, the control group and the Flag-FAT10 group were treated with reactive oxygen species scavenger N-acetyl-L-cysteine ​​(NAC), the protein expression levels of the inflammatory factor monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) were measured. Laser confocal microscopy was used to detect the generation levels of reactive oxygen species in the cells of vrious groups. Results: FAT10 was expressed in carotid artery, thoracic aorta, and renal artery of normal blood pressure rats and expressed in HUVEC, VSMC, MDA-MB-231. The expression level of FAT10 gradually increased in proportion to the increase of the time and concentration of AngⅡ stimulation in HUVEC, and the expression level of FAT10 was the highest when the HUVEC was treated with 100 nmol/L AngⅡ for 36 h (P<0.01). The protein expression level of MCP-1 (P<0.001) and TNF-α (P<0.01) was higher in AngⅡ treated HUVEC with FAT10 overexpression, while the expression level of MCP-1 and TNF-α protein was lower in AngⅡ treated HUVEC with FAT10 knockdown (all P<0.01). The level of intracellular reactive oxygen species (ROS) production was significantly increased with FAT10 overexpression (P<0.001), and the level of ROS was decreased when the expression of FAT10 was interfered (P<0.05). The increased level of MCP-1 and TNF-α proteins in FAT10 overexpressed HUVEC was reversed by NAC (all P<0.05). Conclusion: FAT10 promotes the release of inflammatory factors induced by AngⅡ in endothelial cells by increasing the level of intracellular ROS production.

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&lt; 0.05-&lt;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&lt; 0.05-&lt;0.01). 1,25(OH)2D3 treatment also reduced the proliferation of PBMCs and PFMCs of endometriotic patients compared with controls (p &lt; 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.

Monocyte Chemotactic Protein-1 (MCP1) Accumulation in Human Osteoclast Precursor Cultures.

In vitro osteoclast methods require constant treatment with macrophage colony stimulating factor (M-CSF) to support precursor survival and addition of the differentiation agent receptor activator of NF-κB ligand (RANKL). Constant exposure to granulocyte macrophage colony stimulating factor (GM-CSF) suppresses human osteoclast formation in vitro. Addition of the chemokine monocyte chemotactic protein-1 (MCP1) to such cultures dramatically increases osteoclast formation and overcomes GM-CSF mediated suppression. We investigated the effect of M-CSF, GM-CSF and the combination of M-CSF and GM-CSF treatment on the expression of chemokines in human CD14+ cells in culture. Of assayed chemokines, MCP1 was the most abundant in terms of mRNA transcript and protein in M-CSF treated cultures and was suppressed by GM-CSF. MCP1 protein accumulated up to 50 ng/mL in culture medium, greatly exceeding other assayed chemokines. C-C chemokine receptor-2 (CCR2) is the receptor for MCP1: the formation of osteoclast-like cells was inhibited by constant exposure to the CCR2 antagonist RS102895, in part by decreasing expression of RANK, the receptor for RANKL.

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.

Effects of ERα gene overexpression on bone mineral density and calcium and phosphorus metabolism inovariectomized osteoporosis mice

Objective: To investigate the effects of estrogen receptor α (ERα) gene overexpression on bone metabolism and calcium and phosphorus metabolism in ovariectomized osteoporosis mice, and to provide experimental basis for targeted gene therapy of osteoporosis. Methods: Thirty SPF female mice were randomly divided into sham operation group, model group and ERα overexpression group with 10 mice in each group. After the model was established, the ERα overexpression group was transfected with recombinant adenovirus vector carrying mouse ERα gene by intraspinal injection. The model group was transfected with empty virus, and the sham operation group was not treated. The expression of ERα gene in bone tissue of mice was detected by quantitative Real-time PCR (qRT-PCR). Bone mineral density (BMD) of mouse femur was measured after modeling. Trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular segregation (Tb.Sp), bone volume fraction (BV/TV) and biomechanical strength of femur were measured by micro-CT scanning. Serum levels of calcium (Ca), phosphorus (P), osteocalcin (BGP) and alkaline phosphatase (ALP) were measured by automatic biochemical analyzer. The expressions of tissue inhibitor of metalloproteinases 1 (TIMP-1) and monocyte chemotactic protein 1 (MCP-1) in bone homogenate were detected by Immunohistochemistry. Results: Compared with sham operation group, the expression level of ERα gene in bone tissue of model group was decreased significantly, the levels of BMD, BV/TV, Tb. Th, maximum load, rigidity coefficient, Ca and P were decreased, while the levels of Tb. Sp, BGP and ALP were increased significantly (P＜0.05). Compared with the sham operation group, the expression level of TIMP-1 protein in the bone tissue of the model group was significantly decreased, while that of MCP-1 protein was increased, while that of the ERα overexpression group was increased while that of MCP-1 was decreased (P＜0.05).The levels of ERα gene expression, BMD, BV/TV, TB. Th, maximum load, rigidity coefficient, Ca and P in the ERα overexpression group were significantly higher than those in the model group, while Tb. Sp, BGP and ALP were significantly lower (P＜0.05). Compared with the sham operation group, mean optical density of TIMP-1 in the bone tissue of the model group was significantly decreased, while that of MCP-1 was significantly increased, and that of the ERα overexpression group was significantly increased while that of MCP-1 was significantly decreased (P＜0.05). Conclusion: ERα gene overexpression can improve osteoporosis by regulating bone mineral density, bone parameters, bone metabolism, calcium and phosphorus metabolic indicators and the expression levels of TIMP-1 and MCP-1 in tissues.

Effect of complement C5a on the expression of MCP-1 and NGAL in immune kidney injury of trichloroethylene sensitized mice

Objective: To explore the possible role of C5a in the pathogenesis of renal injury in TCE- sensitized mice, to analyze the impact of expression of neutrophil gelatinase-associated lipocalin (NGAL) and monocyte chemotactic protein-1 (MCP-1) in the presence or absence of C5a receptor antagonist (C5aRA) pretreatment. Methods: A total of 50 female specific pathogens free(SPF) BALB/c mice were randomly divided into blank control group (n=5) , solvent control group (n=5) , TCE group (n=20) , and TCE+C5aRA group (n= 20) . After one week for adaptive feeding, a mouse model of TCE-induced skin sensitization was established by treating with 50% TCE and 30% TCE in turn. The mice in solvent control group accept same reagents without TCE and the mice in blank control group underwent nothing. In TCE +C5aRA group, except for the TCE solution treatment, mice were intraperitoneally injected with 0.5 mg/kg C5aRA solution at the time of challenge. And the skin erythema and edema reaction were scored 24 h after the last challenge. The mice were divided into sensitization positive group and sensitization negative group according to the scoring result. The mice were aseptically sacrificed 72 h after the last challenge to obtain the kidneys. The structural damage of kidney was observed after histopathological staining. The levels of NGAL and MCP-1 mRNA and proteins were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) , respectively. Results: The sensitization rate of mice in TCE group and TCE+C5aRA group was 45.0% (9/20) and 40.0% (8/20) , respectively. No skin lesions was found in the mice of blank control group and solvent control group. The results of histopathological staining showed that the TCE sensitization positive mice showed renal tubular dilatation, vacuolar degeneration of renal tubular epithelial cells, and infiltration of interstitial cells. The pathological damage of the kidney in TCE sensitization positive group was mild, and no inflammatory cell infiltration was seen. The data of qRT-PCR showed that the expression levels of NGAL and MCP-1 mRNA in the TCE sensitization positive group were significantly increased than in solvent control group and TCE sensitization negative group (P<0.05) , while the levels of NGAL and MCP-1 mRNA in TCE+C5aRA sensitization positive group were decreased than TCE sensitization positive group (P <0.05) . The results of IHC showed that the expression levels of NGAL and MCP-1 in TCE protein sensitization positive group were significantly higher than those in solvent control group and TCE sensitization negative group (P<0.05) . After C5aRA pretreatment, the expression levels of NGAL and MCP-1 protein were decreased than the mice in TCE sensitization positive group (P<0.05) . Conclusion: The regulation of C5a on the expression of MCP-1 and NGAL may participate in TCE- induced mice kidney damage, and pharmacological inhibition of C5a seems to be an effective way to protect the kidney injury in TCE-sensitized mice.

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.

Abstract 661: Adipose specific monocyte chemotactic protein-1 knockdown reduces pulmonary metastasis of Lewis lung carcinoma in mice

Abstract Adipose accumulation is a strong indicator of detrimental health outcomes because adipose tissue produces pro-inflammatory adipokines that participate in many pathological processes including cancer. Monocyte chemotactic protein-1 (MCP-1) is a potent adipokine and body adiposity is positively correlated with adipose MCP-1 expression. This study tested the hypothesis that adipose-produced MCP-1 contributes to metastasis. In a metastasis model of Lewis lung carcinoma (LLC), male adipose MCP-1 knockdown (MCP-1-/-) and wild-type (WT) mice were fed the standard AIN93G diet or a high-fat diet (HFD) containing 16% or 45% of energy from soybean oil. The duration of experimental feeding was 11 weeks. The expression of MCP-1 in epididymal adipose tissue was analyzed by quantitative real-time PCR and ELISA. Pulmonary metastases from a primary tumor, established by subcutaneous injection of LLC cells, were the primary endpoints. There was no significant difference in fat body mass between MCP-1-/- and WT mice fed the same diet. There were no differences in energy intake among the four groups. The HFD increased and adipose MCP-1 knockdown decreased MCP-1 mRNA and protein in adipose tissue compared to their respective controls. The HFD increased the number of metastases formed in the lungs in WT mice. The number of metastasis was lower in MCP-1-/- mice fed the HFD than in WT mice fed the HFD but was higher compared to WT mice fed the AIN93G diet. The volume of metastases was smaller in MCP-1-/- mice than in WT mice, regardless of diet. Adipose MCP-1-/- mice, compared to WT mice, exhibited lower concentrations of insulin, pro-inflammatory adipokines (leptin, plasminogen activator inhibitor-1, and resistin), and angiogenic markers (vascular endothelial growth factor, hepatic growth factor, and angiopoietin-2) in plasma. These results indicate that adipose MCP-1 knockdown may lead to the down-regulation of inflammatory and angiogenic pathways during malignant spread. We conclude that adipose MCP-1 deficiency attenuates HFD-enhanced pulmonary metastasis of LLC and supports our hypothesis that adipose-produced MCP-1 contributes to malignant spread. Citation Format: Sneha Sundaram, Lin Yan. Adipose specific monocyte chemotactic protein-1 knockdown reduces pulmonary metastasis of Lewis lung carcinoma in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 661.

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.

Bone marrow stromal cells transplantation promotes the resolution and recanalization of deep vein thrombosis in rabbits through regulating macrophage infiltration and angiogenesis.

This study aims to validate whether bone marrow stromal cells (BMSCs) transplantation could promote the resolution and recanalization of deep vein thrombosis (DVT) and to explore the underlying mechanism. The right hind femoral vein was embolized to establish the DVT rabbit model. BMSCs from New Zealand white rabbits were isolated and identified, and then injected into DVT rabbits. After that, the extent of angiogenesis was determined by the amount of capillaries that were positive for antibody against vWF. Macrophage infiltration was measured by immunohistochemistry with F4/80 antibody. M1 or M2 macrophages were identified as F4/80 + CD11c + or F4/80 + CD206 + cells by using flow cytometry analysis, respectively. BMSCs were successfully isolated and identified. BMSCs transplantation promotes macrophage infiltration and angiogenesis in DVT rabbits. BMSCs transplantation causes M1/M2 polarization, altered cytokine production and increased monocyte chemotactic protein 1 (MCP-1) protein expression in DVT rabbits. However, injection of MCP-1 protein not only reversed the effects of BMSCs transplantation on macrophage infiltration and angiogenesis, but also reversed the effects of BMSCs transplantation on M1/M2 polarization and cytokine production in DVT rabbits. BMSCs transplantation promotes the resolution and recanalization of DVT in rabbits through regulating macrophage infiltration and angiogenesis, the underlying mechanism is associated with MCP-1 expression.

Long noncoding RNA MALAT1 acts as a competing endogenous RNA to regulate Amadori-glycated albumin-induced MCP-1 expression in retinal microglia by a microRNA-124-dependent mechanism.

To determine whether the long noncoding RNA MALAT1 may be involved in the inflammatory effect of Amadori-glycated albumin (AGA) in retinal microglia via a microRNA-124 (miR-124)-dependent mechanism. Diabetes mellitus was induced by streptozotocin (STZ) injection. The expression of monocyte chemotactic protein-1 (MCP-1) in the retinas of rats was determined using quantitative reverse transcription-PCR (qRT-PCR) analyses and enzyme-linked immunosorbent assay (ELISA). Both qRT-PCR and ELISA were used to detect the levels of MCP-1 mRNA and soluble MCP-1 protein in the primary rat retinal microglia treated with AGA. The regulation of a putative target of miR-124 was validated by luciferase reporter assays. MALAT1 knockdown ameliorated diabetic retinopathy (DR) and inhibited MCP-1 release in the retinas of STZ-induced diabetic rats. The cultured retinal microglial cells treated with AGA-released MCP-1 in a dose- and time-dependent manner. In addition, AGA consistently induced MALAT1 expression in the retinal microglial cells. Next, we demonstrated that the expression of MCP-1 is controlled by miR-124, which binds to the 3'-UTR of MCP-1 in microglial cells. Luciferase reporter assays and RNA-binding protein immunoprecipitation assays showed that MALAT1 targets miR-124. Finally, we demonstrated that MALAT1 acts as a competing endogenous RNA by directly binding to miR-124 to regulate AGA-induced MCP-1 expression in microglial cells. MALAT1-miR-124-MCP-1 signaling pathway may be involved in AGA-induced MCP-1 expression in microglial cells, which may provide a new approach for the treatment of DR.

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.

Effect of berberine on lipopolysaccharide-induced monocyte chemotactic protein-1 and interleukin-8 expression in a human retinal pigment epithelial cell line

PurposeIn this study, we elucidated the effects of berberine, a major alkaloid component contained in medicinal herbs, such as Phellodendri Cortex and Coptidis Rhizoma, on expression of monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) in a human retinal pigment epithelial cell line (ARPE-19) caused by lipopolysaccharide (LPS) stimulation.MethodsARPE-19 cells were cultured to confluence. Berberine and LPS were added to the medium. MCP-1 and IL-8 mRNA were measured by real-time polymerase chain reaction. MCP-1 and IL-8 protein concentrations in the media were measured using enzyme-linked immunosorbent assay.ResultsAfter stimulation with LPS, MCP-1 and IL-8 mRNA in ARPE-19 cells reached maximum levels at 3 h, and MCP-1 and IL-8 protein in the culture media reached maximum levels at 24 h. Berberine dose-dependently inhibited MCP-1 and IL-8 mRNA expression of the cells and protein levels in the media stimulated with LPS.ConclusionsThese findings indicate that berberine inhibited the expression of MCP-1 and IL-8 induced by LPS.

Infrapatellar fat pad features in osteoarthritis: a histopathological and molecular study.

The infrapatellar fat pad (IFP) is considered a local producer of adipocytokines, suggesting a potential role in OA. The objective of this study was to evaluate the histopathological and molecular characteristics of OA IFPs compared with controls. The histopathological characteristics of IFPs were evaluated in patients undergoing total knee replacements and in control patients (without OA), considering the following parameters: presence of inflammatory cells, vascularization, adipose lobules dimension and thickness of the interlobular septa. Immunohistochemistry was performed to evaluate VEGF, monocyte chemotactic protein 1 (MCP-1) and IL-6 proteins. Quantitative real time PCR was performed to evaluate the expression levels of adipocytokines in the OA IFPs. OA IFPs showed an increase in inflammatory infiltration, vascularization and thickness of the interlobular septa compared with controls. VEGF, MCP-1 and IL-6 proteins were higher in OA IFPs compared with in controls. Inflammatory infiltration, hyperplasia, vascularization and fibrosis were increased in OA IFP synovial membranes compared with in those of controls. VEGF protein levels were associated with an increased number of vessels in the OA IFPs, while MCP-1 and IL-6 protein levels were associated with higher grades of inflammatory infiltration. Leptin levels were positively correlated with adiponectin and MCP-1expression, while adiponectin positively correlated with peroxisome proliferative activated receptor gamma, MCP-1 and IFP vascularity. MCP-1 showed a positive correlation with peroxisome proliferative activated receptor gamma. IFP lobules dimensions were positively correlated with IL-6 expression and negatively with thickness of interlobular septa. VEGF mRNA levels were positively correlated with increased synovial vascularity. OA IFPs and synovial membranes are more inflamed, vascularized and fibrous compared with those of control patients (without OA).

Expression and significance of monocyte chemotactic protein 1 in early radiation-induced lung injury tissue of mice

Objective To study the expression and significance of monocyte chemotactic protein 1 (MCP-1) in the process of early radiation-induced lung injury in mice. Methods 40 mice were randomly divided into control group (10 cases) and post-irradiation group (30 cases). A large dose (15 Gy) irradiation on chest was performed in post-irradiation group. Every 10 mice in this group were killed on the 1st day (RT1d), the 10th day (RT10d), the 30th day (RT30d), respectively, and the specimens were collected. The expression of MCP-1 mRNA was detected by quantitative real-time PCR(RT-PCR), and MCP-1 and IL-6 proteins in BALF were detected by ELISA. The pathological changes of lung tissue were observed with HE staining. Results Under the microscope, lung tissue began to show inflammation on the 10th day and was more seriousness on the 30th day in post-irradiation group. Immunohistochemistry staining showed that the expression of MCP-1 in lung tissue irradiated was increased. RT-PCR results showed that the relative expressions of MCP-1 mRNA in control group, RT1d group, RT10d group and RT30d group were 7.63±0.77, 6.81±0.75, 5.75±0.73 and 4.61±0.75, respectively. The differences between RT1d group and control group, RT10d and RT1d group, and RT30d and RT10d were significantly different (all P < 0.05). ELISA results showed that the levels of MCP-1 and IL-6 proteins in post-irradiation group were significantly higher than those in the control group (P < 0.05). Conclusion The expression of MCP-1 in mice after irradiation is abnormal, and MCP-1 may participate in early radiation-induced lung injury. Key words: Radiation; Lung injury; Monocyte chemotactic protein 1; Interleukin-6

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.

Catechin-7-O-β-D-glucopyranoside isolated from the seed of Phaseolus calcaratus Roxburgh ameliorates experimental colitis in rats

The seeds of Phaseolus calcaratus Roxburgh (PHCR) are common legumes that comprise part of the daily diet in Chinese and Korean culture. Recent findings highlight anti-inflammatory and anti-septic potentials of catechin-7-O-β-D-glucopyranoside (CGP) isolated from PHCR seeds. We investigated the intestinal anti-inflammatory activity and associated mechanisms of CGP using a rat model of trinitrobenzenesulfonic acid (TNBS)-induced colitis. Oral treatment with CGP (10mg/kg body weight) suppressed body weight loss and intestinal inflammatory damages in TNBS-induced colitic rats. This treatment reduced myeloperoxidase activity and malondialdehyde level, but increased glutathione level in the TNBS colitic rats. CGP treatment also inhibited the TNBS-mediated increases in nitric oxide synthase, cyclooxygenase-2, interleukin-1β, tumor necrosis factor-α, intercellular adhesion molecule-1, and monocyte chemotactic protein-1 proteins or mRNA levels. This inhibition was accompanied by the increased mRNA levels of mucins MUC2 and MUC3. The CGP treatment prevented phosphorylation of p38 mitogen-activated protein kinase, IκB-α, and DNA-nuclear factor-κB binding, all of which were increased in the inflamed colons of TNBS-treated rats. Furthermore, oral administration with a crude PHCR butanol extract (100mg/kg body weight) which contains 1.5% of CGP showed intestinal anti-inflammatory potentials similar to that of CGP. Collectively, our current findings suggest that CGP or CGP-containing PHCR seeds may have favorable effects on intestinal inflammatory diseases.

Melanocyte pigmentation inversely correlates with MCP-1 production and angiogenesis-inducing potential.

The incidence of certain angiogenesis-dependent diseases is higher in Caucasians than in African Americans. Angiogenesis is amplified in wound healing and cornea models in albino C57 mice compared with black C57 mice. Moreover, mouse and human melanocytes with low pigmentation stimulate endothelial cell (EC) proliferation and migration in vitro more than melanocytes with high pigmentation. This effect is due, in part, to the secretion of an angiogenic protein called fibromodulin (FMOD) from lowly pigmented melanocytes. Herein, we expand upon the mechanism contributing to increased angiogenesis in lighter skin and report that monocyte chemotactic protein-1 (MCP-1) is secreted by nonpigmented mouse melanocytes by 5- to 10-fold more than pigmented melanocytes. MCP-1 protein stimulates EC proliferation and migration in vitro and angiogenesis in vivo. Mechanistic studies determine that FMOD is upstream of MCP-1 and promotes its secretion from both melanocytes and activated ECs via stimulation of NF-κB activity. Mice injected with FMOD-neutralizing antibodies show 2.3-fold decreased levels of circulating MCP-1. Human studies confirmed that, on average, Caucasians have 2-fold higher serum levels of MCP-1 than African Americans. Taken together, this study implicates the FMOD/MCP-1 pathway in the regulation of angiogenesis by local melanocytes and suggests that melanogenic activity may protect against aberrant angiogenic diseases.

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.