BACKGROUND15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), one of the major metabolites from prostaglandin D2 in arachidonic acid metabolic pathway, has potential anti-inflammatory properties. The objective of this study was to explore the effects of 15d-PGJ2-loaded poly(D,L-lactide-co-glycolide) nanocapsules (15d-PGJ2-NC) on inflammatory responses and bone regeneration in local bone defect.

METHODSThe study was conducted on 96 Wistar rats from June 2014 to March 2016. Saline, unloaded nanoparticles, free 15d-PGJ2or 15d-PGJ2-NC, were delivered through a collagen vehicle inside surgically created transcortical defects in rat femurs. Interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) levels in the surrounding soft tissue were analyzed by Western blot and in the defect by quantitative real-time polymerase chain reaction over 14 days. Simultaneously, bone morphogenetic protein-6 (BMP-6) and platelet-derived growth factor-B (PDGF-B) messenger RNA (mRNA) in the defect were examined. New bone formation and EphrinB2 and osteoprotegerin (OPG) protein expression in the cortical defect were observed by Masson's Trichrome staining and immunohistochemistry over 28 days. Data were analyzed by one-way analysis of variance. Least-significant difference and Dunnett's T3 methods were used with a bilateral P< 0.05.

RESULTSApplication of l5d-PGJ2-NC (100 μg/ml) in the local bone defect significantly decreased IL-6, IL-1β, and TNF-α mRNA and protein, compared with saline-treated controls (P < 0.05). l5d-PGJ2-NC upregulated BMP-6 and PDGF-B mRNA (P < 0.05). New bone formation was observed in the cortical defect in l5d-PGJ2-NC-treated animals from 7th day onward (P < 0.001). Expression of EphrinB2 and OPG presented early on day 3 and persisted through day 28 in 15d-PGJ2-NC group (P < 0.05).

CONCLUSIONStable l5d-PGJ2-NC complexes were prepared that could attenuate IL-6, IL-1β, and TNF-α expression, while increasing new bone formation and growth factors related to bone regeneration.

Animals ; Bone Morphogenetic Protein 6 ; metabolism ; Bone Regeneration ; drug effects ; Inflammation ; drug therapy ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Male ; Platelet-Derived Growth Factor ; metabolism ; Prostaglandin D2 ; analogs & derivatives ; therapeutic use ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; metabolism

Objective To investigate the effect of 15-Deoxy-△(12,14)-prostaglandin J2 (15 d-PGJ2) on the expression of macrophage migration inhibitory factor (MIF) and its underlying mechanism in J774A.1. Methods The murine monocyte/macrophage cell line J774A.1 were divided into six groups:lipopolysaccharide (LPS) group,incubated with 1 μg/ml LPS for 1 h;normal control group,incubated with PBS for 1 h;negative control group,incubated with 5 μmol/L 15 d-PGJ2 for 1 h;15 d-PGJ2 group,incubated with 5 μmol/L 15 d-PGJ2 for 1 h followed by 1 μg/ml LPS for 1 h;GW9662 group,incubated with 5 μmol/L 15 d-PGJ2 for 1 h following GW9662 10 μmol/L for 1 h,and then incubated with 1 μg/ml LPS for 1 h;and Vehicle group,control of GW9662,GW9662 was replaced by its solvent DMSO. The expression of MIF was detected via immunofluorescence and agarose gel electrophoresis. RT-qPCR and Western blotting were used to test whether 15 d-PGJ2 could regulate mRNA and protein expression of MIF in J774A.1 upon LPS challenge. The effect of peroxisome proliferator-activated receptor-γ (PPAR-γ) antagonist GW9662 on the regulation of MIF by 15 d-PGJ2 was observed. The effects of 15 d-PGJ2 on the nuclear translocation of PPAR-γ upon LPS challenge were detected via high content screening analysis. Results MIF DNA and protein expressions were detected in J774A.1. MIF mRNA expression was up-regulated (1.75±0.09,P=0.037) when challenged with LPS and 15 d-PGJ2 inhibited its upregulation (0.84±0.08,P=0.026) in J774A.1. The protein level was consistent with the mRNA level. PPAR-γ antagonist GW9662 reversed the effect of 15 d-PGJ2 (mRNA,1.48±0.06,P=0.016;protein,1.28). Furthermore,nuclear translocation of PPAR-γ was regulated by 15 d-PGJ2 in J774A.1 upon LPS challenge(1.39±0.02 vs. 1.01±0.03,P=0.003). Conclusion 15 d-PGJ2 may down-regulate the MIF expression in J774A.1 in a PPAR-γ-dependent manner.
Anilides ; pharmacology ; Animals ; Cell Line ; Intramolecular Oxidoreductases ; metabolism ; Lipopolysaccharides ; Macrophage Migration-Inhibitory Factors ; metabolism ; Mice ; Monocytes ; drug effects ; PPAR gamma ; antagonists & inhibitors ; Prostaglandin D2 ; analogs & derivatives ; pharmacology

This study investigated the effect of advanced glycation end products (AGEs) on differentiation of naïve CD4(+) T cells and the role of the receptor of AGEs (RAGE) and peroxisome proliferator-activated receptors (PPARs) activity in the process in order to gain insight into the mechanism of immunological disorders in diabetes. AGEs were prepared by the reaction of bovine serum albumin (BSA) with glucose. Human naïve CD4(+) T cells, enriched from blood of healthy adult volunteers with negative selection assay, were cultured in vitro and treated with various agents including AGEs, BSA, high glucose, PGJ2 and PD68235 for indicated time. In short hairpin (sh) RNA knock-down experiment, naïve CD4(+) T cells were transduced with media containing shRNA-lentivirus generated from lentiviral packaging cell line, Lent-X(TM) 293 T cells. Surface and intracellular cytokine stainings were used for examination of CD4(+) T cell phenotypes, and real-time PCR and Western blotting for detection of transcription factor mRNA and protein expression, respectively. The suppressive function of regulatory T (Treg) cells was determined by a [(3)H]-thymidine incorporation assay. The results showed that AGEs induced higher pro-inflammatory Th1/Th17 cells differentiated from naïve CD4(+) T cells than the controls, whereas did not affect anti-inflammatory Treg cells. However, AGEs eliminated suppressive function of Treg cells. In addition, AGEs increased RAGE mRNA expression in naïve CD4(+) T cells, and RAGE knock-down by shRNA eliminated the effect of AGEs on the differentiation of CD4(+) T cells and the reduction of suppressive function of Treg cells. Furthermore, AGEs inhibited the mRNA expression of PPARγ, not PPARα PPARγ agonist, PGJ2, inhibited the effect of AGEs on naïve CD4(+) T cell differentiation and reversed the AGE-reduced suppressive function of Treg cells; on the other hand, PPARγ antagonist, PD68235, attenuated the blocking effect of RAGE shRNA on the role of AGEs. It was concluded that AGEs may promote CD4(+) T cells development toward pro-inflammatory state, which is associated with increased RAGE mRNA expression and reduced PPARγ activity.
Adult ; Animals ; Blotting, Western ; CD4-Positive T-Lymphocytes ; drug effects ; metabolism ; Cattle ; Cell Differentiation ; drug effects ; Cells, Cultured ; Glucose ; pharmacology ; Glycation End Products, Advanced ; pharmacology ; HEK293 Cells ; Humans ; Interferon-gamma ; metabolism ; Interleukin-17 ; metabolism ; PPAR gamma ; agonists ; genetics ; metabolism ; Prostaglandin D2 ; analogs & derivatives ; pharmacology ; RNA Interference ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Serum Albumin, Bovine ; pharmacology ; T-Lymphocytes, Regulatory ; drug effects ; metabolism ; Th1 Cells ; drug effects ; metabolism ; Th17 Cells ; drug effects ; metabolism

BACKGROUNDA proinflammatory milieu emerging in the lung due to neutrophil accumulation and activation is a key in the pathogenesis of acute lung injury (ALI). 15-deoxy-Δ(12, 14)-prostaglandin J2 (15d-PGJ2), one of the terminal products of the cyclooxygenase-2 pathway, is known to be the endogenous ligand of peroxisome proliferator-activated receptor γ (PPAR-γ) with multiple physiological properties. Growing evidence indicates that 15d-PGJ2 has anti-inflammatory, antiproliferative, cytoprotective and pro-resolving effects. We investigated whether 15d-PGJ2 has a protective effect against endotoxin-induced acute lung injury in rats.

METHODSTwenty-four male Wistar rats were randomly assigned into four groups (n = 6 per group): sham+vehicle group, sham+15d-PGJ2 group, LPS+vehicle group, and LPS+15d-PGJ2 group. The rats were given either lipopolysaccharide (LPS, 6 mg/kg intravenously) or saline, and pretreated with 15d-PGJ2 (0.3 mg/kg intravenously) or its vehicle (dimethyl sulphoxide) 30 minutes before LPS. Histological alterations, wet/dry weight (W/D) ratio and myeloperoxidase (MPO) activity as well as tumor necrosis factor (TNF)-α and cytokine-induced neutrophil chemoattractant-1 (CINC-1) levels were determined in lung tissues four hours after LPS injection. Immunohistochemical analysis for intercellular adhesion molecule-1 (ICAM-1) expression and Western blotting analysis for nuclear factor (NF)-κB p65 translocation and IκBα protein levels were also studied.

RESULTS15d-PGJ2 pretreatment significantly attenuated LPS-induced lung injury, and reduced the increased W/D ratio, MPO activity, TNF-α, CINC-1 levels, and ICAM-1 expression in the lung. 15d-PGJ2 also suppressed the nuclear NF-κB p65 translocation and increased cytosolic IκBα levels.

CONCLUSIONS15d-PGJ2 protects against endotoxin-induced acute lung injury, most likely through the reduction of proinflammatory protein levels during endotoxemia subsequent to the inhibition of NF-κB activation.

Acute Lung Injury ; chemically induced ; drug therapy ; immunology ; Animals ; Chemokine CXCL1 ; metabolism ; I-kappa B Proteins ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Lipopolysaccharides ; toxicity ; Male ; NF-KappaB Inhibitor alpha ; NF-kappa B ; metabolism ; Prostaglandin D2 ; analogs & derivatives ; therapeutic use ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; metabolism

15-Deoxy-Δ(12), 14-prostaglandin J2 (15d-PGJ2), a well known peroxisome proliferator activated receptor (PPAR) γ ligand, has been shown to inhibit cellular proliferation and induce apoptosis and differentiation. However, whether 15d-PGJ2 influences the cytokines in the culture supernatant of bone marrow mesenchymal stem cells (BM-MSC) is unknown. This study was purposed to investigate the influence of 15d-PGJ2 on cytokines in the culture supernatant of BM-MSC. The fibroblast-like cells attached to the culture dish from bone marrow of healthy donors were isolated. The immunophenotype and differentiation potential of the obtained cells were detected by flow cytometry and oil red O and von kassa staining respectively to confirm that these cells were BM-MSC. Thereafter, the BM-MSC were cultured with complete medium supplemented with 10, 20, 40 and 60 µmol/L 15d-PGJ2 for 24 hours respectively. The real-time PCR was used to assay the PPARγ mRNA level, the confocal immuno fluorescence technique was used to detect the expression level of PPARγ. The results showed that the BM-MSC underwent apoptosis and got detached from the culture dish when the concentration of 15d-PGJ2 was no less than 20 µmol/L. The PPARγ mRNA level of BM-MSCs cultured with medium containing 10 µmol/L 15d-PGJ2 was higher than that cultured without 15d-PGJ2, and the difference was statistically significant (P < 0.05). The enhancement of PPARγ expression was observed after stimulated by 15d-PGJ2. The protein chip detecting the culture supernatants of BM-MSC cultured with 10 µmol/L 15d-PGJ2 or without 15d-PGJ2 for 24 hours demonstrated that expression levels of some of the cytokines varied. It is concluded that the down-regulation of TIMP-2 exists after treatment of 15d-PGJ2, which is statistical significant.
Adult ; Bone Marrow Cells ; drug effects ; metabolism ; Cells, Cultured ; Culture Media ; chemistry ; Cytokines ; metabolism ; Humans ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; Prostaglandin D2 ; analogs & derivatives ; pharmacology ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Young Adult

OBJECTIVETo observe the effect of ligand of peroxisome proliferators-activated receptor gamma (PPAR gamma) 15d-PGJ2 on the proliferation and activation of hepatic stellate cells (HSC) and to study the role played by PPAR gamma during the process of HSC activation.

METHODSBy using RT-PCR and cell culture, we investigated the effects of 5 micro mol/L and 10 micro mol/L 15d-PGJ2 on culture-activated HSC and on PDGF-induced HSC proliferation, production of extracellular matrix and expression of chemokines.

RESULTSThe expression of alpha-SMA was significantly suppressed by 5mumol/L 15d-PGJ2, and the expression of PPAR gamma was significantly higher in the 15d-PGJ2 treated group than in the untreated group (0.64+/-0.03 vs 0.09+/-0.01, t=36.0517, P<0.01); PDGF-induced HSC proliferation was dose-dependently suppressed by 15d-PGJ2; the expressions of PPAR gamma in 5 micro mol/L and also in 10 micro mol/L 15d-PGJ2 plus PDGF pre-treated group increased much more than those in the PDGF-treated group (0.03+/-0.02 vs 0.60+/-0.03, t=42.6616, P<0.01 and 0.03+/-0.02 vs 0.69+/-0.04, t=33.83, P<0.01); the expressions of alpha-SMA, alpha 1 (I)-collagen and MCP-1 were suppressed.

CONCLUSIONActivation of PPAR gamma can modulate pro-fibrotic and pro-inflammatory roles of HSC and the increased expression of PPAR gamma may become a new target for antifibrosis.

Animals ; Cell Differentiation ; Cell Proliferation ; drug effects ; Cells, Cultured ; Hepatic Stellate Cells ; cytology ; metabolism ; Male ; PPAR gamma ; metabolism ; Prostaglandin D2 ; analogs & derivatives ; pharmacology ; Rats ; Rats, Wistar

Peroxisome proliferator-activated receptors-gamma (PPAR-gamma) is critical for phenotype determination at early differentiation stages of mesenchymal cells, whereas its physiological role is unclear. Therefore, we investigated the role of 15-deoxy-delta 12,14-prostaglandinJ2 (15d-PGJ2), the natural receptor ligand for PPAR-gamma, on dedifferentiation and inflammatory responses, such as COX-2 expression and PGE2 production, in articular chondrocytes. Our data indicate that the 15d-PGJ2 caused a loss of differentiated chondrocyte phenotype as demonstrated by inhibition of type II collagen and proteoglycan synthesis. 15d-PGJ2 also induced COX-2 expression and PGE2 production. The 15d-PGJ2-induced dedifferentiation effect seems to be dependent on PPAR-gamma activation, as the PPRE luciferase activity increased and PPAR-gamma antagonist, BADGE, abolished type II collagen expression. However, BADGE did not block 15d-PGJ2-induced COX-2 expression. Collectively, our findings suggest that PPAR-gamma-dependent and -independent mechanisms of 15d-PGJ2-induced dedifferentiation and inflammatory responses in articular chondrocytes, respectively. Additionally, these data suggest that targeted modulation of the PPAR-gamma pathway may offer a novel approach for therapeutic inhibition of joint tissue degradation.
Animals ; Arteries/*metabolism ; Cell Differentiation ; Chondrocytes/*metabolism ; Cyclooxygenase 2/*metabolism ; Dinoprostone/metabolism ; Dose-Response Relationship, Drug ; *Gene Expression Regulation, Enzymologic ; Genes, Reporter ; Immunoblotting ; PPAR gamma/*metabolism ; Prostaglandin D2/*analogs & derivatives/metabolism ; Rabbits ; Time Factors ; Transfection

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a ligand-activated transcription factor and plays an important role in growth, differentiation, and inflammation in different tissues. In this study, we investigated the effects of 15d-PGJ2, a high-affinity ligand of PPAR-gamma, on dedifferentiation and on inflammatory responses such as COX-2 expression and PGE2 production in rabbit articular chondrocytes with a focus on ERK-1/-2, p38 kinase, and PPAR-gamma activation. We report here that 15d-PGJ2 induced dedifferentiation and/or COX-2 expression and subsequent PGE2 production. 15d-PGJ2 treatment stimulated activation of ERK-1/-2, p38 kinase, and PPAR-gamma. Inhibition of ERK-1/-2 with PD98059 recovered 15d-PGJ2-induced dedifferentiation and enhanced PPAR-gamma activation, whereas inhibition of p38 kinase with SB203580 potentiated dedifferentiation and partially blocked PPAR-gamma activation. Inhibition of ERK-1/-2 and p38 kinase abolished 15d-PGJ2-induced COX-2 expression and subsequent PGE2 production. Our findings collectively suggest that ERK-1/-2 and p38 kinase oppositely regulate 15d-PGJ2-induced dedifferentiation through a PPAR-gamma-dependent mechanism, whereas COX-2 expression and PGE2 production is regulated by ERK-1/-2 through a PPAR-gamma-independent mechanism but not p38 kinase in articular chondrocytes. Additionally, these data suggest that targeted modulation of the PPAR-gamma and mitogen-activated protein kinase pathway may offer a novel approach for therapeutic inhibition of joint tissue degradation.
Animals ; Cartilage, Articular/*cytology ; Cell Differentiation/drug effects ; Chondrocytes/cytology/*drug effects/metabolism ; Cyclooxygenase 2/*analysis ; Dinoprostone/biosynthesis ; Mitogen-Activated Protein Kinase 1/*physiology ; Mitogen-Activated Protein Kinase 3/*physiology ; PPAR gamma/*physiology ; Prostaglandin D2/*analogs & derivatives/pharmacology ; Rabbits ; p38 Mitogen-Activated Protein Kinases/*physiology

AIMTo investigate the effect of PPARgamma activators on inhibition of cardiac non myocytes (CNM) proliferation and the PPARgamma-dependent pathway possibly involved.

METHODSAngiotensin II was used to induce proliferation of primarily cultured CNM from neonatal rats, and pioglitazone or 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) was applied to these CNM in various dosages in vitro. MTT assay and 3H-TdR uptake were determined to estimate proliferation of CNM, and transient transfection of reporter gene containing PPRE from ACO promoter (PPRE-pGL3) with or without PPARgamma expression plasmid (PPARgamma-pSG5) to CNM was performed to determine the control of target-gene transcription.

RESULTSAngiotensin II caused a significant increase in MTT value and 3H-TdR uptake in CNM, which could be significantly reversed by pioglitazone and 15d-PGJ2 in a dose-dependent manner. Transient cotransfection of PPRE-pGL3 with PPARgamma-pSG5 to CNM resulted in significant increase in luciferase activity compared with that without PPARgamma-pSG5 cotransfection. Pioglitazone and 15d-PGJ2 induced increase in luciferase activity also in a dose-dependent manner.

CONCLUSIONPioglitazone and 15d-PGJ2, as the activators of PPARgamma, inhibit proliferation of CNM from neonatal rats, the effect may be related to the activation of PPARgamma.

Angiotensin II ; pharmacology ; Animals ; Cell Proliferation ; Cells, Cultured ; Heart ; Male ; Myocardium ; cytology ; PPAR gamma ; metabolism ; Prostaglandin D2 ; analogs & derivatives ; pharmacology ; Rats ; Rats, Wistar ; Thiazolidinediones ; pharmacology

Apoptosis ; Cell Line, Tumor ; Colorectal Neoplasms ; drug therapy ; pathology ; HT29 Cells ; Humans ; PPAR gamma ; genetics ; physiology ; PTEN Phosphohydrolase ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; Phosphoric Monoester Hydrolases ; analysis ; Prostaglandin D2 ; analogs & derivatives ; pharmacology ; Protein-Serine-Threonine Kinases ; analysis ; Proto-Oncogene Proteins ; analysis ; Proto-Oncogene Proteins c-akt ; Thiazolidinediones ; pharmacology ; Tumor Suppressor Proteins ; analysis ; Up-Regulation