OBJECTIVETo study the effect of oral administration of dimethyl dimethoxy biphenyl dicarboxylate (DDB) on adjusting angiogeneic/inflammatory mediators and ameliorating the pathology of bones in rats with collagen-induced arthritis (CIA).

METHODSWistar rat model of CIA was set up using bovine collagen type II. Fifty rats were divided into five groups randomly: normal, CIA model, DDB treatment, methotrexate (MTX) treatment, and combined DDB+MTX treatment. Ankle joints of rats were imaged with digital X-ray machine to show the destruction of joints. Fore and hind paw and knee joints were removed above the ankle joint then processed for haematoxylin and eosin staining. Plasma levels of vascular endothelial growth factor (VEGF), platelet derived growth factor, interleukin-8 (IL-8), IL-4, tumor necrosis factor α (TNF-α), and cyclooxygenase-2 (COX-2) were quantified by enzyme-linked immunosorbent assay. Nitric oxide levels were detected by Griess reagent.

RESULTSCompared with the CIA model group, a remarkable reduction in various angiogenic (VEGF and IL-8) and inflammatory mediators (TNF-α, IL-4 and COX-2) after treatment with DDB either alone or combined with MTX P<0.05 or P<0.01). Histopathological and X-ray findings were confirmatory to the observed DDB anti-arthritic effect. The DDB-treated group showed amelioration in signs of arthritis which appeared essentially similar to normal.

CONCLUSIONOur data shed light on the therapeutic efficacy of DDB in experimental rheumatoid arthritis (RA) compared with a choice drug (MTX) and it may be offered as a second-line drug in the treatment of RA.

Animals ; Arthritis, Experimental ; chemically induced ; diagnostic imaging ; drug therapy ; pathology ; Arthritis, Rheumatoid ; diagnostic imaging ; drug therapy ; pathology ; Collagen ; Cyclooxygenase 2 ; blood ; Dioxoles ; therapeutic use ; Enzyme-Linked Immunosorbent Assay ; Female ; Interleukin-4 ; blood ; Interleukin-8 ; blood ; Methotrexate ; therapeutic use ; Nitric Oxide ; biosynthesis ; Platelet-Derived Growth Factor ; analysis ; Radiography ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; blood ; Vascular Endothelial Growth Factor A ; blood

OBJECTIVETo investigate and compare the effects and mechanisms of three functional parts of Dahuang Zhechong Pill (DHZCP), including drugs with the function of removing blood stasis and promoting blood circulation (FP-I), drugs with the function of expelling heat and moistening dryness (FP-II), and drugs with the function of nourishing yin and replenishing blood (FP-III) of DHZCP, on platelet-derived growth factor (PDGF)-stimulated vascular smooth muscle cells (VSMCs) proliferation with the method of serum pharmacology.

METHODSVSMCs proliferation of rat was assayed by measuring the cell viability with the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) method. DNA synthesis in VSMCs was examined by detecting 5'-bromo-2'-deoxyuridine incorporation with the immunocytochemical method. Cycle of VSMCs was evaluated with flow cytometry. Expression of cyclin D1, p27, PKCα, and phosphorylated extracellular signal regulated kinase 1/2 (ERK1/2) was quantified by the Western blotting method.

RESULTSThe FP-I and FP-III containing serum was capable of inhibiting PDGF-stimulated proliferation and DNA synthesis of VSMCs, arrested VSMCs in G phase, downregulated cyclin D1, and upregulated p27 expression (P <0.01 or P <0.05). The FP-I and FP-III containing serum also inhibited the PDGF-induced phosphorylation of tyrosine of ERK1/2 and PKCα expression (P <0.01 or P <0.05).

CONCLUSIONSFP-I and FP-III of DHZCP are able to inhibit VSMCs proliferation via interrupting PKCα-ERK1/2 signaling, modulating the expression of cell cycle proteins to result in arresting the cells in G phase. The inhibitory effect is mainly related to the function of removing blood stasis and promoting blood circulation, slightly to the function of nourishing yin and replenishing blood, but not to the function of expelling heat and moistening dryness.

Animals ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; DNA ; biosynthesis ; Drugs, Chinese Herbal ; pharmacology ; Enzyme Activation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; MAP Kinase Signaling System ; drug effects ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; enzymology ; Platelet-Derived Growth Factor ; pharmacology ; Protein Kinase C-alpha ; metabolism ; Rats ; Rats, Sprague-Dawley ; Serum ; metabolism

OBJECTIVETo investigate effects of dahuang zhechong pill ( DHZCP) on the cell cycle and the related signal pathways in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF) with the method of serum pharmacology.

METHODSDNA synthesis in VSMCs was examined by detecting 5'-bromo-2'-deoxyuridine incorporation with the immunocytochemical method. The cycle of VSMCs was evaluated with flow cytometry. Expressions of cyclin D1, p27, protein kinase Cα (PKCα), and phosphorylated extracellular signal regulated kinase 1/2 (ERK1/2) were quantified by Western blot method.

RESULTSDHZCP containing serum significantly inhibited DNA synthesis of PDGF-stimulated VSMCs, arrested the cells in G G(1) phase, modulated the protein expressions of cyclin D D(1) and p27, and suppressed the activation of PKCα and ERK1/2.

CONCLUSIONDHZCP containing serum inhibits VSMCs proliferation via modulating the expressions of cell cycle proteins to arrest the cell in G G(1) phase, which is attributed to, at least in part, suppressing PKCα-ERK1/2 signaling in VSMCs.

Animals ; Aorta, Thoracic ; cytology ; Blood Proteins ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; DNA ; biosynthesis ; Drugs, Chinese Herbal ; pharmacology ; G1 Phase ; drug effects ; physiology ; MAP Kinase Signaling System ; physiology ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; enzymology ; Platelet-Derived Growth Factor ; pharmacology ; Protein Kinase C-alpha ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effects of adenovirus-mediated human tissue kallikrein (Ad-hKLK1) gene transfer on platelet-derived growth factor-BB (PDGF-BB)-induced migration of vascular smooth muscle cells from spontaneously hypertensive rats (VSMC(SHR)).

METHODSA bicistronic recombinant adenovirus vector (Ad-hKLK1) carrying the target hKLK1 gene and the reporter gene EGFP was constructed. VSMCs isolated from the thoracic aorta of male SHR were passaged, and the quiescent VSMC(SHR) in passages 3-6 seeded in 6-well plates were treated with Ad-hKLK1 and control virus. Human PDGF-BB or icatibant Hoe140, a BK B2 antagonistat, was used as the chemoattractant and placed in the bottom chamber of the Boyden chamber. The mRNA expressions of bradykinin B1 receptor and B2 receptor were detected by RT-PCR in VSMC(SHR).

RESULTShKLK1 gene transfer significantly inhibited PDGF-BB-induced migration of VSMC(SHR), with the peak inhibition rate of 34.6% (P<0.001). PDGF-BB significantly increased the mRNA expression of B2 receptor but not B1 receptor in VSMC(SHR).

CONCLUSIONShKLK1 gene transfer can inhibit the migration of VSMC(SHR) induced by PDGF-BB, and the inhibitory effects may be not mediated by bradykinin B2 receptor.

Adenoviridae ; genetics ; metabolism ; Animals ; Aorta, Thoracic ; cytology ; Cell Movement ; drug effects ; genetics ; Cells, Cultured ; Gene Transfer Techniques ; Humans ; Hypertension ; pathology ; Male ; Muscle, Smooth, Vascular ; cytology ; Platelet-Derived Growth Factor ; pharmacology ; Proto-Oncogene Proteins c-sis ; Rats ; Rats, Inbred SHR ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Tissue Kallikreins ; biosynthesis ; genetics

OBJECTIVEConstruction of a small interfering RNA (siRNA) eukaryotic expression vector specific to mouse myocardin gene and study on the role of myocardin-siRNA on differentiation of mouse bone mesenchymal stem cells (MSCs) into smooth muscle-like cells induced by PDGF-BB in vitro.

METHODSMouse MSCs were isolated from bone marrow and cultured with 50 mg/L PDGF-BB and fetal bovine serum (20%). Specific myocardin-siRNA sequence was cloned into a plasmid pGenesil-1.0 vector, which contained U6 promoter. The recombinant plasmid and control plasmid were transfected into MSCs which had been cultured with PDGF-BB for 6 days beforehand. The expression of myocardin mRNA was detected by RT-PCR 48 hours after the transfection. Immunohistochemistry was used to detect the SM-MHC and to identify the smooth muscle-like cells.

RESULTSThe recombinant plasmids carrying myocardin-siRNA sequences were constructed successfully and the myocardin mRNA was reduced 42.86% by pGen-myo-shRNA in comparing with that of the controls (P<0.01); and the expression of SM-MHC protein was down-regulated (P<0.01).

CONCLUSIONSubset of mouse MSCs have the potential to differentiate into smooth muscle-like cells, a possible cell source responsible for atherosclerotic plaque formation, and myocardin expression may play an important role during this process.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Down-Regulation ; Gene Silencing ; Genetic Vectors ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Myosin Heavy Chains ; metabolism ; Nuclear Proteins ; biosynthesis ; genetics ; physiology ; Plaque, Atherosclerotic ; pathology ; Plasmids ; Platelet-Derived Growth Factor ; pharmacology ; Proto-Oncogene Proteins c-sis ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Trans-Activators ; biosynthesis ; genetics ; physiology ; Transfection

OBJECTIVETo investigate the role of extracellular signal-regulated kinase 1/2 on the inhibition of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) on the proliferation and collagen synthesis of cultured rat pulmonary fibroblasts induced by platelet-derived growth factor (PDGF).

METHODSPulmonary fibroblasts were prepared from lungs of neonatal Wistar rats as described previously. Cells were divided into 4 groups: (1) control group (0.4% FBS group); (2) PDGF (10 ng/ml) stimulated group; (3) PD98059+PDGF group (25 micromol/L PD98059+10 ng/ml PDGF); (4) AcSDKP+PDGF group (10(-8) mol/L AcSDKP+10 ng/ml PDGF). All experiments were performed in the fourth passages. Metabolic activity of fibroblasts was observed by MTT, and expressions of type I and type III collagen were measured by immunocytochemistry and western blot. Expressions of phospho-ERK1/2 and ERK1/2 were detected by western blot.

RESULTSCompared with control group, exposure of pulmonary fibroblasts to 10 ng/ml PDGF increased cell metabolic activity, expression of type I and type III collagen and phosphorylation of ERK1/2. 25 micromol/L PD98059 and AcSDKP both could inhibit the metabolic activity of pulmonary fibroblasts, type I and type III collagen synthesis and phosphorylation of ERK1/ 2 induced by PDGF, with significant differences (P < 0.05). AcSDKP+PDGF group compared with PDGF stimulated group, metabolic activity of pulmonary fibroblasts decreased to 77.4%. Immunocytochemistry result showed that in AcSDKP+PDGF group, expressions of type I and type III collagen decreased to 69.3% and 67.2% compared with those of PDGF stimulated group. Western blot result showed that in AcSDKP+PDGF group, expressions of type I and type III collagen decreased to 92.4% and 78.0%, phospho-ERK1/2 decreased to 83.5% compared with those of PDGF stimulated group, with significant differences (P < 0.05).

CONCLUSIONERK1/2 plays an important role in the inhibition of AcSDKP on the proliferation and collagen synthesis of cultured rat pulmonary fibroblasts induced by PDGF.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen ; biosynthesis ; Fibroblasts ; drug effects ; metabolism ; physiology ; MAP Kinase Signaling System ; physiology ; Oligopeptides ; pharmacology ; Platelet-Derived Growth Factor ; pharmacology ; Rats ; Rats, Wistar

OBJECTIVETo investigate the signal transduction mechanism underlying the effects of angiotensin II (AngII) on extracellular signal-regulated kinase 1/2 (ERK1/2), early growth response-1 (EGR-1) and platelet-derived growth factor-B (PDGF-B) in hepatic stellate cells (HSCs).

METHODSHSC-T6 cells treated with AngII for 10 or 30 min were examined for phospho-P42/44 protein expression using Western blotting. In another experiment, the cells were preincubated for 1 h in the presence of U0126 (an inhibitor of the MAPK/ERK kinase), irbesartan (an AT-1 receptor blocker), or antioxidant-N-acetylcysteine (NAC) prior to AngII exposure, and the protein expression of phospho-P42/44 and PDGF-B were measured with Western blotting. The DNA binding activity of EGR-1 was analyzed using electrophoretic gel mobility shift assay (EMSA), and the expression of PDGF-B was detected immunohistochemically.

RESULTSAngII induced phospho-P42/44 expression in HSC-T6, which was abrogated by U0126 or irbesartan. NAC did not inhibit phospho-P42/44 expression. EMSA showed that AngII exposure of the HSC cells markedly increased EGR-1 DNA binding activity, reaching the maximum after 60 min of exposure followed by progressive declination; irbesartan and U0126 significantly suppressed AngII-induced EGR-1 activity enhancement. ACEI at 1 micromol/L and 10 nmol/L inhibited EGR-1 activity, but ACEI at the concentration of 0.1 nmol/L resulted in enhanced EGR-1 activity. NAC showed no obvious effect in suppressing EGR-1 activity. AngII increased PDGF-B protein level in the HSCs, the effect of which was inhibited by irbesartan. U0126, NAC and ACEI did not attenuate PDGF-BB protein level in the HSCs.

CONCLUSIONStimulation of the HSCs with AngII results in EGR-1 activation via the ERK1/2 pathway, leading to up-regulation of PDGF-B expression.

Angiotensin II ; pharmacology ; Blotting, Western ; Cells, Cultured ; Early Growth Response Protein 1 ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Immunohistochemistry ; Platelet-Derived Growth Factor ; biosynthesis ; Proto-Oncogene Proteins c-sis ; Signal Transduction ; drug effects

OBJECTIVETo investigate the effects of Haikun Shenxi on the expression of platelet-derived growth factor-BB (PDGF-BB) and mRNA in renal tissue of rats with adriamycin nephropathy.

METHODRat model was established by unilateral nephrectomy and injecting adriamycin intraperitoneally. The adriamycin-induced nephrotic rats were randomly divided into 6 groups: normal group, sham operation group, model group, lotensin treatment group, Haikun Shenxi low and high dose treatment groups (0.77, 0.08 mg x kg(-1). Ten weeks later, the 24 hour urine protein and blood biochemistry examinations and renal pathologic changes were observed, and the expression of PDGF-BB and mRNA was measured using immunohistochemical method.

RESULTCompared with model group, proteinuria and the levels of serum creatinine (Scr) , urea nitrogen (BUN) were decreased obviously in both Haikun Shenxi low and high dose groups. The expression of PDGF-BB and mRNA was mostly presented in cytoplasm of renal tubular epithelial cells and mesangial area, and it could be reduced significantly after treatment (P < 0. 05).

CONCLUSIONThe level of PDGF-BB and mRNA is high in renal tissue of adriamycin-induced nephrotic rats. This progress could be effectively inhibited by Haikun Shenxi and the mechanism may be that it can control the excessive expression of PDGF-BB and mRNA.

Animals ; Blood Urea Nitrogen ; Creatinine ; blood ; Doxorubicin ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Gene Expression Regulation ; drug effects ; Glomerular Mesangium ; drug effects ; metabolism ; pathology ; Glomerulosclerosis, Focal Segmental ; chemically induced ; genetics ; metabolism ; Immunohistochemistry ; In Situ Hybridization ; Kidney ; drug effects ; metabolism ; pathology ; Male ; Medicine, Chinese Traditional ; Phaeophyta ; chemistry ; Platelet-Derived Growth Factor ; biosynthesis ; genetics ; Polysaccharides ; chemistry ; isolation & purification ; pharmacology ; Proto-Oncogene Proteins c-sis ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Wistar

AIMTo investigate whether AcSDKP can inhibit proliferation and collagen synthesis in cultured rat cardiac fibroblasts mediated by PDGF.

METHODSNeonatal rat cardiac fibroblasts were isolated. The cell proliferation was observed by 3H-proline incorporation assay.

RESULTSOn the culture of 0.4% FBS, PDGF stimulated cardiac fibroblasts proliferation and collagen synthesis with a dose-dependent manner at the concentrations from 1 ng/ml to 20 ng/ml, in which 10 ng/ml PDGF reached its peak. AcSDKP at the concentration from 10(-10) mol/L to 10(-8) mol/L could inhibit cardiac fibroblasts proliferation and collagen synthesis mediated by PDGF. 10(-9) mol/L AcSDKP attained its peak on inhibiting cardiac fibroblasts proliferation and collagen synthesis.

CONCLUSIONAcSDKP can inhibit proliferation and collagen synthesis in cultured rat cardiac fibroblasts mediated by PDGF.

Animals ; Cell Proliferation ; Cells, Cultured ; Collagen ; biosynthesis ; Fibroblasts ; cytology ; drug effects ; metabolism ; Myoblasts, Cardiac ; cytology ; drug effects ; metabolism ; Oligopeptides ; pharmacology ; Platelet-Derived Growth Factor ; pharmacology ; Rats ; Rats, Wistar

OBJECTIVETo investigate the expression and clinical significance of platelet-derived growth factor receptor alpha (PDGFR-alpha) in gastrointestinal stromal tumor (GIST).

METHODSParaffin embedded tissue specimens from 119 GISTs were analyzed for PDGFR-alpha expression by immunohistochemical method, and specimens from non-GISTs were used as controls. Positive signals were shown in cytoplasma and cell membrane.

RESULTSOf 119 GISTs,78 (65.5%) were positive for PDGFR-alpha . The expression rate of PDGFR-alpha protein in very low risk, low risk, intermediate risk and high risk cases was 52.9 % (9/17), 71.0% ( 22/31), 67.9% (19/28) and 65.1% (28/43), respectively (P>0.05). PDGFR-alpha protein expression rate was significantly different between GISTs and non-GISTs (P<0.05).

CONCLUSIONPDGFR-alpha combined with CD117 can be used in diagnosis and differential diagnosis of GIST, but it may not be used as a prognostic index.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; biosynthesis ; Female ; Gastrointestinal Stromal Tumors ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Proto-Oncogene Proteins c-kit ; biosynthesis ; Receptor, Platelet-Derived Growth Factor alpha ; biosynthesis