BACKGROUND/AIMS: Asthma is characterized by airway hyperresponsiveness, inflammation, and remodeling. Peroxisome proliferator-activated receptors have been reported to regulate inflammatory responses in many cells. In this study, we examined the effects of intranasal rosiglitazone on airway remodeling in a chronic asthma model. METHODS: We developed a mouse model of airway remodeling, including smooth muscle thickening, in which ovalbumin (OVA)-sensitized mice were repeatedly exposed to intranasal OVA administration twice per week for 3 months. Mice were treated intranasally with rosiglitazone with or without an antagonist during OVA challenge. We determined airway inflammation and the degree of airway remodeling by smooth muscle actin area and collagen deposition. RESULTS: Mice chronically exposed to OVA developed sustained eosinophilic airway inflammation, compared with control mice. Additionally, the mice developed features of airway remodeling, including thickening of the peribronchial smooth muscle layer. Administration of rosiglitazone intranasally inhibited the eosinophilic inflammation significantly, and, importantly, airway smooth muscle remodeling in mice chronically exposed to OVA. Expression of Toll-like receptor (TLR)-4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) was increased in the OVA group and decreased in the rosiglitazone group. Co-treatment with GW9660 (a rosiglitazone antagonist) and rosiglitazone increased the expression of TLR-4 and NF-kappaB. CONCLUSIONS: These results suggest that intranasal administration of rosiglitazone can prevent not only air way inf lammation but also air way remodeling associated with chronic allergen challenge. This beneficial effect is mediated by inhibition of TLR-4 and NF-kappaB pathways.
Actins/metabolism ; Administration, Inhalation ; Airway Remodeling/*drug effects ; Animals ; Anti-Asthmatic Agents/*administration & dosage ; Asthma/chemically induced/*drug therapy/metabolism/physiopathology ; Chronic Disease ; Collagen/metabolism ; Disease Models, Animal ; Female ; Lung/*drug effects/metabolism/physiopathology ; Mice, Inbred BALB C ; NF-kappa B/metabolism ; Ovalbumin ; PPAR gamma/agonists/metabolism ; Pneumonia/chemically induced/physiopathology ; Pulmonary Eosinophilia/chemically induced/prevention & control ; Signal Transduction/drug effects ; Thiazolidinediones/*administration & dosage ; Toll-Like Receptor 4/metabolism

Renal tubulointerstitial fibrosis is the common ending of progressive renal disease. It is worth developing new ways to stop the progress of renal fibrosis. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have been studied to treat diabetic nephropathy, cisplatin-induced acute renal injury, ischemia reperfusion injury and adriamycin nephropathy. In this study, unilateral ureteral obstruction (UUO) was used to establish a different renal fibrosis model. PPAR? agonist pioglitazone was administrated by oral gavage and saline was used as control. At 7th and 14th day after the operation, mice were sacrificed for fibrosis test and T lymphocytes subsets test. Unexpectedly, through MASSON staining, immunohistochemistry for α-SMA, and Western blotting for a-SMA and PDGFR-β, we found that pioglitazone failed to attenuate renal fibrosis in UUO mice. However, flow cytometry showed that pioglitazone down-regulated Th1 cells, and up-regulated Th2 cells, Th17 cells and Treg cells. But the Th17/Treg ratio had no significant change by pioglitazone. Real-time PCR results showed that TGF-β and MCP-1 had no significant changes, at the same time, CD4(+) T cells associated cytokines were partially regulated by pioglitazone pretreatment. Taken together, pioglitazone failed to suppress renal fibrosis progression caused by UUO.
Animals ; Chemokine CCL2 ; metabolism ; Fibrosis ; Kidney ; pathology ; Kidney Diseases ; drug therapy ; etiology ; Male ; Mice ; Mice, Inbred C57BL ; PPAR gamma ; agonists ; T-Lymphocyte Subsets ; drug effects ; Thiazolidinediones ; administration & dosage ; pharmacology ; therapeutic use ; Transforming Growth Factor beta ; metabolism ; Urethral Obstruction ; complications

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

OBJECTIVETo investigate the effects of GW4064, a farnesoid X receptor (FXR) agonist, on adiponectin and its receptors during the differentiation of 3T3-L1 preadipocytes and on adiponectin receptors in HepG2 cells.

METHODSThe mRNA expressions of FXR, PPARγ2, adiponectin, AdipoR1, and AdipoR2 and the protein levels of adiponectin on days 0, 2, 4, 6, and 8 during the differentiation of 3T3-L1 preadipocytes treated with GW4064 were detected by fluorescent real-time PCR and ELISA, respectively. The mRNA expressions of AdipoR1 and AdipoR2 in HepG2 cells were also examined at 0, 12, 24, and 48 h after GW4064 treatment.

RESULTSThe mRNA expressions of FXR, PPAR&gamma;2, adiponectin, and AdipoR2 in 3T3-L1 preadipocytes and AdipoR2 in HepG2 cells treated with GW4064 was significantly increased compared with the control group (all P<0.05). The protein level of adiponectin was also significantly increased after GW4064 treatment. The expression of AdipoR1 in either 3T3-L1 preadipocytes or HepG2 cells showed no significant changes after GW4064 treatment.

CONCLUSIONGW4064 can up-regulate the expressions of FXR, PPAR&gamma;2, adiponectin, AdipoR2 in 3T3-L1 preadipocytes and AdipoR2 in HepG2 cells. As adiponectin and its receptors are two important factors in the treatment of non-alcoholic fatty liver disease, FXR agonist may potentially produce therapeutic effect on non-alcoholic fatty liver disease and can regulate adipocytes via up-regulating PPAR&gamma; during adipocyte differentiation.

3T3-L1 Cells ; Adiponectin ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Hep G2 Cells ; Humans ; Isoxazoles ; pharmacology ; Mice ; PPAR gamma ; metabolism ; Receptors, Adiponectin ; metabolism ; Receptors, Cytoplasmic and Nuclear ; agonists

Long-term treatment with an agonist of peroxisome proliferator-activated receptor (PPAR)-&gamma; is associated with bone fractures in the clinical practice. However, the mechanisms underlying the fractures are not fully understood. This study was aimed to examine the effect of rosiglitazone (an agonist of PPAR-&gamma;) of different doses on the proliferation, differentiation, and transforming growth factor beta 1 (TGF-β1)-induced expression of connective tissue growth factor (CTGF) in primary rat osteoblasts in vitro. Osteoblasts were isolated from newly born SD rats and treated with different doses of rosiglitazone (0-20 μmol/L). The proliferation and differentiation of osteoblasts were measured by MTT assay and NPP assay, respectively. The expression of CTGF was determined by RT-PCR and Western blotting. The results showed that most isolated osteoblasts displayed strong alkaline phosphatase (ALP) activity and treatment with different doses of rosiglitazone did not affect their proliferation, but significantly inhibited the differentiation of osteoblasts in a dose-dependent manner. Moreover, treatment with different doses of rosiglitazone significantly reduced the TGF-β1-induced CTGF mRNA transcription and protein expression in a dose-dependent manner in rat osteoblasts. It was concluded that the activation of PPAR-&gamma; may inhibit the differentiation of osteoblasts by reducing the TGF-β1-induced CTGF expression in vitro.
Animals ; Animals, Newborn ; Blotting, Western ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Connective Tissue Growth Factor ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Gene Expression ; drug effects ; Hypoglycemic Agents ; pharmacology ; Osteoblasts ; cytology ; drug effects ; metabolism ; PPAR gamma ; agonists ; metabolism ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Thiazolidinediones ; pharmacology ; Transforming Growth Factor beta1 ; pharmacology

A series of novel tetrahydrocarboline derivatives was designed and synthesized in order to discover more potent peroxisome proliferator-activated receptor (PPAR) alpha/gamma dual regulators. The structures of these compounds were confirmed by 1H NMR and HR-MS; their PPAR-regulating activities were evaluated in vitro. Compounds 6h, 6n, 6p and 6q exhibited more potent PPARalpha agonistic activities than the control drug WY14643, while compounds 60, 6g, 6i and 6q exhibited more potent PPARgamma agonistic activities than the control drug rosiglitazone. Compound 6q was discovered as a potent PPARalpha/gamma dual agonist and deserves further investigation.
Animals ; Carbolines ; chemical synthesis ; chemistry ; pharmacology ; Cells, Cultured ; Drug Design ; Hypoglycemic Agents ; chemical synthesis ; chemistry ; pharmacology ; Molecular Structure ; PPAR alpha ; agonists ; PPAR gamma ; agonists ; Peroxisome Proliferator-Activated Receptors ; agonists ; Pyrimidines ; metabolism ; Structure-Activity Relationship ; Thiazolidinediones ; metabolism ; Transfection

OBJECTIVETo investigate the effect of peroxisome proliferator-activated receptors &gamma; (PPAR&gamma;) on insulin receptor substrate-4 (IRS-4) gene expression in the brain.

METHODSPrimarily cultured cortical neurons from E17-18 Sprague Dawley rats, after 1 week of plating, were exposed to 10 µmol/L PPAR&gamma; agonist rosiglitazone for 0, 1, 4 or 24 h. Adult C57BL/6J mice or conditional brain PPAR&gamma; knock-out mice (B-PPAR&gamma;-KO, BKO) received an intraperitoneal injection of rosiglitazone in 10% DMSO at 12 mg/kg or injection of the same volume of saline containing 10% DMSO. The effect of rosiglitazone on the survival of the neurons was examined by MTT assay. The expression of IRS-4 mRNA was analyzed by real-time quantitative PCR.

RESULTSThe survival of the cortical neurons showed no significant difference between the agonist groups and the control group. The expression of IRS-4 mRNA was significantly up-regulated in the cortical tissues and neurons of the agonist groups compared with the control groups (P<0.05), but in BKO mice without treatment, IRS-4 mRNA expression was significantly down-regulated (P<0.05).

CONCLUSIONPPAR&gamma; can enhance the expression of IRS-4 mRNA in the brain.

Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Cerebral Cortex ; cytology ; metabolism ; Female ; Gene Transfer Techniques ; Insulin Receptor Substrate Proteins ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Neurons ; cytology ; metabolism ; PPAR gamma ; agonists ; genetics ; metabolism ; Pregnancy ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Thiazolidinediones ; pharmacology ; Up-Regulation

OBJECTIVETo study the effects of peroxisome proliferator-activated receptor gamma agonists on angiotensin II-induced cellular response in cultured fibroblasts derived from patients with hypertrophic scars, so as to investigate its effects on preventing the formation of hypertrophic scars.

METHODSFibroblasts were freshly isolated from hypertrophic scars and cultured with angiotensin II, rosiglitazone and GW9662 at a certain concentration. Fibroblasts proliferation were assessed via Cell Counting Kit-8; the mRNA and protein expressions of Collagen I and Fibronectin (FN) were determined by quantitative real-time RT-PCR and Western blotting.

RESULTSThe absorbance of CCK-8 and relative expression of Collagen I, FN mRNA and protein were 1.082 5 +/- 0.007, 6.45 +/- 0.97, 4.92 +/- 0.86, 2.92 +/- 0.41, 2.78 +/- 1.04 in Ang II group; 0.722 4 +/- 0.012, 1.82 +/- 0.34, 1.78 +/- 0.27, 1.57 +/- 0.46, 1.68 +/- 0.39 in Ros + Ang II group; 0.554 7 +/- 0.012, 0.97 +/- 0.12, 1.07 +/- 1.08, 1.05 +/- 0.43, 1.14 +/- 0.36 in Ros group; 1.056 0 +/- 0.005, 5.83 +/- 0.24, 4.47 +/- 0.32, 2.69 +/- 0.35, 2.62 +/- 0.27 in GW9662 + ros + Ang II group. The results showed a significant difference between the Ang II group and the control group (P < 0.05). The effect of Ang II could be markedly inhibited by Ros (P < 0.05). In addition, Ros did not influence cell proliferation and production of extracellular matrix (P > 0.05). There was a significant difference between the GW9662 + Ros + Ang II group and the Ros + Ang II (P < 0.05).

CONCLUSIONSPPAR-gamma agonists inhibit Ang II-induced proliferation and extracellular matrix synthesis effectively in the hypertrophic scar fibroblasts. Thus PPAR-gamma agonists may have potential therapeutic effect for hypertrophic scar.

Angiotensin II ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; Collagen Type I ; biosynthesis ; Extracellular Matrix ; drug effects ; Fibroblasts ; drug effects ; metabolism ; pathology ; Fibronectins ; biosynthesis ; Humans ; PPAR gamma ; agonists

Vascular smooth muscle cells (VSMCs) undergo phenotypic changes in response to vascular injury such as angioplasty. Protein kinase G (PKG) has an important role in the process of VSMC phenotype switching. In this study, we examined whether rosiglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma agonist, could modulate VSMC phenotype through the PKG pathway to reduce neointimal hyperplasia after angioplasty. In vitro experiments showed that rosiglitazone inhibited the phenotype change of VSMCs from a contractile to a synthetic form. The platelet-derived growth factor (PDGF)-induced reduction of PKG level was reversed by rosiglitazone treatment, resulting in increased PKG activity. This increased activity of PKG resulted in phosphorylation of vasodilator-stimulated phosphoprotein at serine 239, leading to inhibited proliferation of VSMCs. Interestingly, rosiglitazone did not change the level of nitric oxide (NO) or cyclic guanosine monophosphate (cGMP), which are upstream of PKG, suggesting that rosiglitazone influences PKG itself. Chromatin immunoprecipitation assays for the PKG promoter showed that the activation of PKG by rosiglitazone was mediated by the increased binding of Sp1 on the promoter region of PKG. In vivo experiments showed that rosiglitazone significantly inhibited neointimal formation after balloon injury. Immunohistochemistry staining for calponin and thrombospondin showed that this effect of rosiglitazone was mediated by modulating VSMC phenotype. Our findings demonstrate that rosiglitazone is a potent modulator of VSMC phenotype, which is regulated by PKG. This activation of PKG by rosiglitazone results in reduced neointimal hyperplasia after angioplasty. These results provide important mechanistic insight into the cardiovascular-protective effect of PPARgamma.
Animals ; Aorta/injuries/metabolism/*pathology ; Calcium-Binding Proteins/genetics/metabolism ; Cell Proliferation ; Cyclic GMP/metabolism ; Cyclic GMP-Dependent Protein Kinases/genetics/*metabolism ; Hyperplasia/metabolism ; Microfilament Proteins/genetics/metabolism ; Muscle, Smooth, Vascular/metabolism/pathology ; Myocytes, Smooth Muscle/drug effects/*metabolism ; Nitric Oxide/metabolism ; PPAR gamma/agonists/*metabolism ; Promoter Regions, Genetic ; Rats ; Rats, Sprague-Dawley ; Sp1 Transcription Factor/metabolism ; Thiazolidinediones/pharmacology ; Thrombospondins/genetics/metabolism ; Tunica Intima/metabolism/*pathology ; Vascular System Injuries/*metabolism/pathology

OBJECTIVE: To investigate the effect of administration of rosiglitazone, an artificial ligand of PPARγ, on the expression and secretion of apolipoprotein (apoM) in fat-fed, streptozotocin-treated rats, an animal model for type 2-like diabetes. METHODS: Healthy male SD rats were divided into 4 groups: a control group (n=7), a high-fat chow group (HF group, n=8), a diabetes mellitus group (DM group, n=7), and a diabetes mellitus group with rosiglitazone intervention group (RSG group, n=7). Fasting blood glucose (FBG), fasting insulin (FINS), triglyceride (TG) and total cholesterol (TC) were measured at the beginning of the study. The diabetic rats model was established by feeding high fat chow and intraperitoneal injection of streprozotocin. Then the randomly selected treatment group was given rosiglitazone by daily gavage for 8 weeks. All the rats were killed at the fifteenth week, at which time blood and tissues (liver, kidney, adipose) were collected and prepared. The levels of FBG, FINS, TG and TC were assayed. The level of apoM in serum was measured by enzyme-linked immunosorbent assay (ELISA). Reverse transcription polymerase chain reaction (RT-PCR) was used to determine apoM mRNA expression in liver, kidney, and adipose tissues. RESULTS: Compared with either control group or HF group, serum apoM concentration in the DM group was reduced significantly (P<0.05); compared with the DM group, however, serum apoM concentrations in RSG group were increased (P<0.05). The expression of apoM mRNA in liver was highest, in kidney medium, and in adipose tissue extremely low (P<0.05). ApoM mRNA expression in liver and kidney was decreased in both DM and HF groups compared to control group (P<0.05). But, as with serum apoM concentration, apoM mRNA in the liver, kidney and adipose tissues of the RSG group were all increased markedly (P<0.05). The level of serum apoM in SD rats correlated negatively with TG (r=-0.466, P=0.011), TC (r=-0.568, P= 0.001), FBS (r =-0.371, P<0.001), and FINS(r=-0.768, P= 0.048 ). CONCLUSION These results suggest that apoM may participate in the glucose and lipid metabolism by the regulation of PPARγ.
Animals ; Apolipoproteins ; blood ; genetics ; metabolism ; Apolipoproteins M ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Dietary Fats ; administration & dosage ; Lipocalins ; blood ; genetics ; metabolism ; Male ; PPAR gamma ; agonists ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Rosiglitazone ; Thiazolidinediones ; therapeutic use