PURPOSE: Experimental autoimmune uveoretinitis (EAU) is an animal model of posterior uveitis and heme oxygenase-1 (HO-1) is a well-known anti-oxidant factor. However, there is no report a protective role of HO-1 on EAU in vivo. To verify that HO-1 is induced in EAU by interphotoreceptor retinoid-binding protein (IRBP), that an HO-1 inducers ameliorates the associated inflammation, and that an HO-1 inhibitor exacerbates this inflammation. METHODS: Forty four Lewis rats were given either 40 mol/kg hemin or 40 mol/kg SnPP (tin protoporphyrin IX) by intraperitoneal injection and twenty two uveitis control rats were injected with 0.5 mL of saline once daily 5-20 days after IRBP immunization inducing EAU. Three normal control rats were used for Western blotting and ELISA assay of HO-1. The clinical uveitis signs of inflammation were scored in the three groups from 0 to 4 on alternate three days. To confirm the clinical results, histological and immunohistochemical stain of HO-1 were performed on the day of peak inflammation and Western blotting and ELISA assay of HO-1 were performed on 6th, 12th and 18th day after IRBP immunization. RESULTS: Hemin, an inducer of HO-1, ameliorated the clinical signs of EAU. In contrast, SnPP-treated rats show that the severity of the clinical sign were exacerbated at the peak period of the disease. These results are roughly compatible with histological, immunoblotting, and immunohistochemical evaluations and an ELISA assay of HO-1. CONCLUSIONS: We suggest that HO-1 plays an important protective role in EAU.
Animals ; Autoimmune Diseases/diagnosis/*drug therapy/metabolism ; Blotting, Western ; Disease Models, Animal ; Enzyme Inhibitors/*administration & dosage ; Enzyme-Linked Immunosorbent Assay ; Heme Oxygenase-1/*biosynthesis/drug effects ; Hemin/*administration & dosage ; Immunohistochemistry ; Injections, Intraperitoneal ; Male ; Metalloporphyrins/*administration & dosage ; Microscopy, Acoustic ; Protoporphyrins/*administration & dosage ; Rats ; Rats, Inbred Lew ; Retinitis/diagnosis/*drug therapy/metabolism ; Treatment Outcome ; Uveitis, Posterior/diagnosis/*drug therapy/metabolism

Spontaneous hypertensive rats (SHR) are an established model of genetic hypertension. Vascular smooth muscle cells (VSMC) from SHR proliferate faster than those of control rats (Wistar-Kyoto rats; WKY). We tested the hypothesis that induction of heme oxygenase (HO)-1 induced by aprotinin inhibits VSMC proliferation through cell cycle arrest in hypertensive rats. Aprotinin treatment inhibited VSMC proliferation in SHR more than in normotensive rats. These inhibitory effects were associated with cell cycle arrest in the G1 phase. Tin protoporphyrin IX (SnPPIX) reversed the anti-proliferative effect of aprotinin in VSMC from SHR. The level of cyclin D was higher in VSMC of SHR than those of WKY. Aprotinin treatment downregulated the cell cycle regulator, cyclin D, but upregulated the cyclin-dependent kinase inhibitor, p21, in VSMC of SHR. Aprotinin induced HO-1 in VSMC of SHR, but not in those of control rats. Furthermore, aprotinin-induced HO-1 inhibited VSMC proliferation of SHR. Consistently, VSMC proliferation in SHR was significantly inhibited by transfection with the HO-1 gene. These results indicate that induction of HO-1 by aprotinin inhibits VSMC proliferation through cell cycle arrest in hypertensive rats.
Animals ; Aprotinin ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Proliferation ; Cyclin D ; G1 Phase ; Heme ; Heme Oxygenase (Decyclizing) ; Heme Oxygenase-1 ; Hypertension ; Metalloporphyrins ; Muscle, Smooth, Vascular ; Phosphotransferases ; Protoporphyrins ; Rats ; Tin ; Transfection

PURPOSE: Nitric oxide (NO) is a short-lived bioactive molecule that is known to play an important role in the pathogenesis of periodontal disease. In the current study, we investigated the effect of the flavonoid quercetin on the production of NO in murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen related to inflammatory periodontal disease, and tried to elucidate the underlying mechanisms of action. METHODS: LPS was isolated from P. intermedia ATCC 25611 cells by the standard hot phenol-water method. The concentration of NO in cell culture supernatants was determined by measuring the accumulation of nitrite. Inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) protein expression, phosphorylation of c-Jun N-terminal kinase (JNK) and p38, inhibitory kappaB (IkappaB)-alpha degradation, and signal transducer and activator of transcription 1 (STAT1) phosphorylation were analyzed via immunoblotting. RESULTS: Quercetin significantly attenuated iNOS-derived NO production in RAW246.7 cells activated by P. intermedia LPS. In addition, quercetin induced HO-1 protein expression in cells activated with P. intermedia LPS. Tin protoporphyrin IX (SnPP), a competitive inhibitor of HO-1, abolished the inhibitory effect of quercetin on LPS-induced NO production. Quercetin did not affect the phosphorylation of JNK and p38 induced by P. intermedia LPS. The degradation of IkappaB-alpha induced by P. intermedia LPS was inhibited when the cells were treated with quercetin. Quercetin also inhibited LPS-induced STAT1 signaling. CONCLUSIONS: Quercetin significantly inhibits iNOS-derived NO production in murine macrophages activated by P. intermedia LPS via anti-inflammatory HO-1 induction and inhibition of the nuclear factor-kappaB and STAT1 signaling pathways. Our study suggests that quercetin may contribute to the modulation of host-destructive responses mediated by NO and appears to have potential as a novel therapeutic agent for treating inflammatory periodontal disease.
Cell Culture Techniques ; Heme Oxygenase-1 ; I-kappa B Proteins ; JNK Mitogen-Activated Protein Kinases ; Lipopolysaccharides ; Macrophages ; Metalloporphyrins ; Nitric Oxide ; Nitric Oxide Synthase ; Periodontal Diseases ; Phosphorylation ; Prevotella ; Prevotella intermedia ; Protoporphyrins ; Quercetin ; STAT1 Transcription Factor ; Tin

OBJECTIVETo prepare a zinc porphyrinated polyimide nanofibrous membrane for rapid detection of trace amount of ammonia.

METHODSZinc porphyrin chromophore was copolymerized into polyimide backbones and the according nanofibrous membrane was prepared by electrospinning technique. Ammonia detection was achieved by recording the color and spectral changes of the membrane before and after exposing to the target gas. The sensitivity, selectivity and detection limit of prepared membrane were further studied.

RESULTSThe obtained nanofibrous membrane preserved typical photophysical properties of zinc porphyrin chromophores. When exposed to ammonia, a dual chromo and spectrum responses of the nanofibrous membrane were observed. The binding affinity constant and the detection limit of zinc porphyrinated polyimide nanofibrous membrane calculated from surface plasmon resonance (SPR) analysis and UV-vis were 3.33 X10³ L/mol and 3.13 mg/m³, respectively.

CONCLUSIONThe membrane prepared in this study exhibits good sensitivity, selectivity and reproducibility towards ammonia detection.

Ammonia ; analysis ; Imides ; Membranes, Artificial ; Metalloporphyrins ; Nanostructures ; Sensitivity and Specificity ; Surface Plasmon Resonance ; methods

OBJECTIVETo observe the ability of triple helix-forming oligonucleotides (TFOs) modified with manganese porphyrin to combine with and cleave HBV DNA fractions.

METHODSTFO were modified with manganese porphyrin and acridines, and then reacted with the (32)P labeled HBV DNA fragments at 37 degrees C in vitro (pH 7.4). Electrophoretic mobility shift assays and DNase I footprinting tests were used to show the affinity and specificity of TFO to bind to target sequences. The ability of TFO to cleave HBV DNA fragments was tested by cleavage experiments.

RESULTSTFO modified with manganese porphyrin and acridine could bind to the target sequence in a sequence-dependent manner, with a Kd value of 3.5 x 10(-7) mol/L and a relative affinity of 0.008. In the presence of potassium monopersulfate (KHSO(5)), TFO modified with manganese porphyrin and acridine could cleave the target sequence where the triplex DNA was formed.

CONCLUSIONIn the presence of KHSO(5), TFO modified with manganese porphyrin and acridine could bind and cleave the target HBV-DNA in a sequence-dependent manner.

DNA ; drug effects ; pharmacology ; DNA, Viral ; chemistry ; drug effects ; Hepatitis B virus ; genetics ; Manganese ; pharmacology ; Metalloporphyrins ; pharmacology ; Potassium Compounds ; pharmacology ; Sulfates ; pharmacology

OBJECTIVETo observe the ability of triple helix-forming oligonucleotides (TFO) modified with manganese porphyrin to combine with and cleave HBV DNA fractions.

METHODSThe ends of TFO were modified with manganese porphyrin and acridine; At 37 degrees C and pH 7.4 condition in vitro, TFO modified with manganese porphyrin and acridine were bound with 32P labeled HBV DNA fragments, the affinity and specificity binding to target sequence were tested by electrophoretic mobility shift and DNase 1 footprinting assays, the ability to cleave HBV DNA fractions was observed with cleavage experiments.

RESULTSTFO modified with manganese porphyrin and acridine could bind to target sequence in a sequence-dependent manner with Kd values of 3.5 x 10(-7) mol/L and a relative affinity of 0.008. In the presence of KHSO5, TFO modified with manganese porphyrin and acridine could cleave target sequence in the region forming triple DNA.

CONCLUSIONIn the presence of KHSO5, TFO modified with manganese porphyrin and acridine could cleave target HBV-DNA in sequence-dependent manner.

Binding, Competitive ; DNA Fingerprinting ; Deoxyribonuclease I ; metabolism ; Electrophoretic Mobility Shift Assay ; Hepatitis B virus ; genetics ; Manganese ; chemistry ; Metalloporphyrins ; chemistry ; Nucleic Acid Conformation ; Oligodeoxyribonucleotides ; chemistry ; genetics ; metabolism

OBJECTIVE: To identify and evaluate the lateral border zone by comparing the size and distribution of the abnormal signal area demonstrated by MR imaging with the infarct area revealed by pathological examination in a reperfused myocardial infarction cat model. MATERIALS AND METHODS: In eight cats, the left anterior descending coronary artery was occluded for 90 minutes, and this was followed by 90 minutes of reper-fusion. ECG-triggered breath-hold turbo spin-echo T2-weighted MR images were initially obtained along the short axis of the heart before the administration of contrast media. After the injection of Gadomer-17 and Gadophrin-2, contrast-enhanced T1-weighted MR images were obtained for three hours. The size of the abnormal signal area seen on each image was compared with that of the infarct area after TTC staining. To assess ultrastructural changes in the myocardium at the infarct area, lateral border zone and normal myocardium, electron microscopic examination was performed. RESULTS: The high signal area seen on T2-weighted images and the enhanced area seen on Gadomer-17-enhanced T1WI were larger than the enhanced area on Gadophrin-2-enhanced T1WI and the infarct area revealed by TTC staining; the difference was expressed as a percentage of the size of the total left ventricle mass (T2= 39.2 %; Gadomer-17 =37.25 % vs Gadophrin-2 = 29.6 %; TTC staining = 28.2 %; p < 0.05). The ultrastructural changes seen at the lateral border zone were compatible with reversible myocardial damage. CONCLUSION: In a reperfused myocardial infarction cat model, the presence and size of the lateral border zone can be determined by means of Gadomer-17- and Gadophrin-2-enhanced MR imaging.
Animal ; Cats ; Contrast Media ; Gadolinium ; Magnetic Resonance Imaging/*methods ; Mesoporphyrins/diagnostic use ; Metalloporphyrins/diagnostic use ; Myocardial Infarction/*pathology ; Myocardial Reperfusion ; Myocardium/pathology ; Support, Non-U.S. Gov't

OBJECTIVETo investigate the effect of the photodynamic therapy (PDT) with the new water-soluble metalloporphyrin compound on human prostate cancer PC-3 cells in vitro and the anticancer mechanism of PDT.

METHODSThe new water-soluble manganese, 5,10,15, 20-tetra (N-methyl4-pyridyl) porphinato (2-) tetraiodide salt, was synthesized. The PC-3 cells were treated with the compound of serial concentrations(0, 0.1, 1, 1.0 micromol/L) followed by irradiation of different dosages of visible light. The techniques of MTT and Annexin-V/propidium iodide double-labeled flow cytometry (FCM) were applied to measuring the inhibitory effect of the compound on the growth activity and apoptosis of the cells.

RESULTSWhen the metalloporphyrin compound concentration was within 10 micromol/L and the irradiation time was within 30 min, the water-soluble metalloporphyrin compound had a significant inhibitory effect on the proliferation of PC-3 cells and induced PC-3 cell apoptosis, and the effects depended greatly on metalloporphyrin concentration and illumination dosages. Higher concentrations and dosages induced the death of the majority of PC-3 cells.

CONCLUSIONThe PDT of the water-soluble metalloporphyrin compound followed by light irradiation has a distinctive killing effect on PC-3 cells in vitro, and the rates of proliferation inhibition and cell apoptosis are correlated with metalloporphyrin concentration and the dosages of light irradiation. The results suggest that the mechanism of metalloporphyrin PDT may be involved with the induction of apoptosis in human prostate cancer cells.

Apoptosis ; drug effects ; radiation effects ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Dose-Response Relationship, Radiation ; Humans ; Male ; Metalloporphyrins ; pharmacology ; Photochemotherapy ; Prostatic Neoplasms ; pathology

Aprotinin is used clinically in cardiopulmonary bypass surgery to reduce transfusion requirements and the inflammatory response. The mechanism of action for the anti-inflammatory effects of aprotinin is still unclear. We examined our hypothesis whether inhibitory effects of aprotinin on cytokine-induced inducible nitric oxide synthase (iNOS) expression (IL-1beta plus TNF-alpha), reactive oxygen species (ROS) generation, and vascular smooth muscle cell (VSMC) proliferation were due to HO-1 induction in rat VSMCs. Aprotinin induced HO-1 protein expression in a dose-dependent manner, which was potentiated during inflammatory condition. Aprotinin reduced cytokine mixture (CM)-induced iNOS expression in a dose dependent manner. Furthermore, aprotinin reduced CM-induced ROS generation, cell proliferation, and phosphorylation of JNK but not of P38 and ERK1/2 kinases. Aprotinin effects were reversed by pre-treatment with the HO-1 inhibitor, tin protoporphyrin IX (SnPPIX). HO-1 is therefore closely involved in inflammatory-stimulated VSMC proliferation through the regulation of ROS generation and JNK phosphorylation. Our results suggest a new molecular basis for aprotinin anti-inflammatory properties.
Animals ; Aprotinin ; Cardiopulmonary Bypass ; Cell Proliferation ; Inflammation ; Metalloporphyrins ; Muscle, Smooth, Vascular ; Nitric Oxide Synthase Type II ; Phosphorylation ; Phosphotransferases ; Protoporphyrins ; Rats ; Reactive Oxygen Species ; Tin

In vascular smooth muscle cells (VSMCs), induction of the heme oxygenase-1 (HO-1) confers vascular protection against cellular proliferation mainly via its up-regulation of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) that is involved in negative regulation of cellular proliferation. In the present study, we investigated whether the phytochemical curcumin and its metabolite tetrahydrocurcumin could induce HO-1 expression and growth inhibition in rat VSMCs and, if so, whether their antiproliferative effect could be mediated via HO-1 expression. At non-toxic concentrations, curcumin possessing two Michael-reaction acceptors induced HO-1 expression by activating antioxidant response element (ARE) through translocation of the nuclear transcription factor E2-related factor-2 (Nrf2) into the nucleus and also inhibited VSMC growth triggered by 5% FBS in a dose-dependent manner. In contrast, tetrahydrocurcumin lacking Michael-reaction acceptor showed no effect on HO-1 expression, ARE activation and VSMC growth inhibition. The antiproliferative effect of curcumin in VSMCs was accompanied by the increased expression of p21(WAF1/CIP1). Inhibition of VSMC growth and expression of p21(WAF1/CIP1) by curcumin were partially, but not completely, abolished when the cells were co- incubated with the HO inhibitor tin protoporphyrin. In human aortic smooth muscle cells (HASMCs), curcumin also inhibited growth triggered by TNF-alpha and increased p21(WAF1/CIP1) expression via HO-1-dependent manner. Our findings suggest that curcumin has an ability to induce HO-1 expression, presumably through Nrf2-dependent ARE activation, in rat VSMCs and HASMCs, and provide evidence that the antiproliferative effect of curcumin is considerably linked to its ability to induce HO-1 expression.
Active Transport, Cell Nucleus ; Animals ; Aorta/cytology ; Cell Nucleus/metabolism ; Cell Proliferation/*drug effects ; Cells, Cultured ; Curcumin/analogs & derivatives/*pharmacology ; Cyclin-Dependent Kinase Inhibitor p21/biosynthesis/metabolism ; Gene Expression Regulation ; Heme Oxygenase (Decyclizing)/biosynthesis/genetics/*physiology ; Heme Oxygenase-1/biosynthesis/genetics/*physiology ; Humans ; Metalloporphyrins/pharmacology ; Muscle, Smooth, Vascular/drug effects/*physiology ; Myocytes, Smooth Muscle/drug effects/*physiology ; NF-E2-Related Factor 2/metabolism ; Protoporphyrins/pharmacology ; Rats ; Regulatory Sequences, Nucleic Acid ; Response Elements ; Tumor Necrosis Factor-alpha/pharmacology