cyclic-gmp and icariin

Great progress has been made in the basic researches on erectile dysfunction (ED) ever since the recognition of the close association of micromolecular nitric oxide (NO) with penile smooth muscle relaxation in 1990. NO-cGMP-PDE5 signaling pathway plays an important role in regulating the relaxation of corpus cavernosum smooth muscle, and its relevant studies have contributed greatly to the clinical treatment of ED. Chinese herbal drugs have long been used in the treatment of ED, and the action mechanisms of some of them clarified through the NO-cGMP-PDE5 signaling pathway. This article presents an overview on the recent advances in the studies of ED treatment with Chinese herbal drugs.

Topics: Animals; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Drugs, Chinese Herbal; Erectile Dysfunction; Flavonoids; Male; Nitric Oxide; Phytotherapy; Rabbits; Rats; Signal Transduction

Mechanical stimulation induces bone growth and remodeling by the secondary messenger, cyclic guanosine 3', 5'-monophosphate (cGMP), in osteoblasts. However, the role of cGMP in the regulation of estrogen biosynthesis, whose deficiency is a major cause of osteoporosis, remains unclear. Here, we found that the prenylated flavonoids, 3-

Topics: 3T3 Cells; Animals; Aromatase; Cell Differentiation; Cell Line; Cell Survival; Cyclic AMP; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Estrogens; Flavonoids; HEK293 Cells; Humans; Mice; Molecular Docking Simulation; Osteoblasts; Osteoporosis; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Stress, Mechanical

The natural product icariin inhibits human phosphodiesterase-5 (PDE5) and represents a unique pharmacophore for treating erectile dysfunction, pulmonary hypertension, and other diseases. In this study, we explore the available icariin-derived chemical scaffolds through medicinal chemistry to develop novel icariin PDE5 inhibitors with improved potency and specificity. We synthesized six novel semi-synthetic icariin analogs as well as three naturally occurring icariin analogs, and characterized the structure-activity relationship in the context of human PDE5 inhibition using in vitro enzyme inhibition and kinetics assays and molecular modeling. Mammalian-cell-based assays and in vitro enzyme inhibition assays against human PDE6C further helped to identify the most potent and selective icariin analogs. Our results reveal the synergistic contribution of functional groups at the C3 and C7 positions of the icariin backbone towards PDE5 inhibition. Whereas a hydrophobic and flexible alkanol group at the C7 position is sufficient to enhance icariin analog potency, combining this group with a hydrophilic sugar group at the C3 position leads to further enhancement of potency and promotes specificity towards PDE5 versus PDE6C. In particular, compounds 3 and 7 exhibit Ki values of 0.036 ± 0.005 μM and 0.036 ± 0.007 μM towards PDE5 respectively, which are approaching those of commercial PDE5 inhibitors, and can effectively reduce GMP levels in cultured human BJ-hTERT cells. This study identifies novel icariin analogs as potent and selective PDE5 inhibitors poised to become lead compounds for further pharmaceutical development.

Topics: Animals; Biocatalysis; Cell Line; Cell Line, Tumor; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Erectile Dysfunction; Flavonoids; Humans; Hypertension, Pulmonary; Male; Models, Chemical; Molecular Structure; Phosphodiesterase 5 Inhibitors; Structure-Activity Relationship

Phosphodiesterase-5 (PDE5) inhibitors are predominantly used in the treatment of erectile dysfunction, and have been recently shown to have a potential therapeutic effect for the treatment of Alzheimer's disease (AD) through stimulation of nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signalling by elevating cGMP, which is a secondary messenger involved in processes of neuroplasticity. In the present study, the effects of a PDE5 inhibitor, icarrin (ICA), on learning and memory as well as the pathological features in APP/PS1 transgenic AD mice were investigated. Ten-month-old APP/PS1 transgenic mice overexpressing human amyloid precursor protein (APP695swe) and presenilin 1 (PS1-dE9) were given ICA (30 and 60 mg/kg) or sildenafil (SIL) (2 mg/kg), age-matched wild-type (WT) mice were given ICA (60 mg/kg), and APP/PS1 and WT control groups were given an isovolumic vehicle orally twice a day for four months. Results demonstrated that ICA treatments significantly improved learning and memory of APP/PS1 transgenic mice in Y-maze tasks. The amyloid precursor protein (APP), amyloid-beta (Aβ1-40/42) and PDE5 mRNA and/or protein levels were increased in the hippocampus and cortex of APP/PS1 mice, and ICA treatments decreased these physiopathological changes. Furthermore, ICA-treated mice showed an increased expression of three nitric oxide synthase (NOS) isoforms at both mRNA and protein levels, together with increased NO and cGMP levels in the hippocampus and cortex of mice. These findings demonstrate that ICA improves learning and memory functions in APP/PS1 transgenic mice possibly through the stimulation of NO/cGMP signalling and co-ordinated induction of NOS isoforms.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Cerebral Cortex; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Disease Models, Animal; Dose-Response Relationship, Drug; Flavonoids; Hippocampus; Humans; Male; Maze Learning; Mice, Transgenic; Nitric Oxide; Nitric Oxide Synthase; Nootropic Agents; Peptide Fragments; Phosphodiesterase 5 Inhibitors; Presenilin-1; Random Allocation

Icariin, a prenylated flavonol glycoside isolated from Epimedii herba, has been found to be a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of icariin is related to the nitric oxide (NO) signal pathway and PI3K/AKT pathway in its upstream. Rat bone marrow stromal cells (rBMSCs) were cultured in osteogenic medium and treated with icariin or together with L-NAME, ODQ, PDE5, and/or LY294002 (the inhibitor of NOS, sGC, cGMP, and PI3K respectively), and effects were examined on the expression of signal messengers (NOS, NO, sGC, cGMP, PKG and PI3K) and the levels of osteogenic markers (alkaline phosphatase or ALP, osteocalcin and calcified nodules). It was found that icariin dose-dependently increased ALP activity, and treatment at the optimal concentration (10(-5)M) increased NOS activity, iNOS and eNOS expression, NO production, sGC and cGMP contents and PKG expression besides the phosphorylation of AKT. The addition of L-NAME, ODQ and PDE5 significantly inhibited the icariin effects on above markers respectively. The addition of LY294002 decreased the p-AKT level, NOS activity, eNOS expression and NO production significantly, but had no significant effect on iNOS expression. The addition of any of the four inhibitors also abolished the osteogenic effect of icariin on rBMSCs as indicated by ALP activity, osteocalcin synthesis, calcium deposition and the number and areas of calcified nodules. These results suggest that the osteogenic effect of icariin involves the PI3K-AKT-eNOS-NO-cGMP-PKG signal pathway. Furthermore, dosage response studies showed that icariin at 10(-6)M (a physiologically achievable concentration in vivo) also activated this signal pathway.

Topics: Alkaline Phosphatase; Animals; Cell Differentiation; Chromones; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Male; Mesenchymal Stem Cells; Morpholines; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type III; Osteocalcin; Osteogenesis; Phenotype; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats, Wistar; Signal Transduction

To investigate effects of icariin on cardiac gene expression and the modulation of nitric oxide (NO) signal transduction during the differentiation of embryonic stem (ES) cells into cardiomyocytes in vitro.. The expression levels of cardiac developmental-dependent genes were measured using reverse transcription-polymerase chain reaction (RT-PCR). The chronotropic responses of cardiomyocytes to b-adrenoceptor stimulation were determined. The levels of cAMP and cGMP in ES cells were measured using radioimmunoassay. Endogenous NO levels were measured by using the Griess reaction. Aminoguanidine (AG) was used to confirm the influence of icariin on the endogenous NO signal pathway.. Icariin significantly elevated mRNA levels of cardiac transcription factors GATA4 and Nkx2.5, and cardiac-specific alpha-MHC, MLC-2v and beta-AR genes in a concentration- and time-dependent manner (P<0.05). Cardiomyocytes derived from embryoid body (EB) treated with icariin were more sensitive to isoprenaline (P<0.01). Treatment of ES cells with icariin resulted in a continued elevation in the cAMP/cGMP ratio before a shift to the cardiomyocyte phenotype (P<0.05). AG decreased the NO level, and delayed and decreased the incidence of contracting EB to only approximately 35% on d 5+11, an effect that could be rescued by icariin. When cells were cocultured with icariin and AG, the percentage of beating EB reached a peak level of 73% on d 5+11 (P<0.05).. The inducible effects of icariin were partly related to increase in the expression of cardiac developmental-dependent genes, and elevation of the cAMP/cGMP ratio in ES cells, as well as upregulation of endogenous NO generation during the early stages of cardiac development.

Topics: Animals; Cardiac Myosins; Cell Differentiation; Cell Line; Cyclic AMP; Cyclic GMP; Drugs, Chinese Herbal; Embryo, Mammalian; Epimedium; Flavonoids; GATA4 Transcription Factor; Gene Expression Regulation, Developmental; Homeobox Protein Nkx-2.5; Homeodomain Proteins; Mice; Myocytes, Cardiac; Myosin Light Chains; Nitric Oxide; Receptors, Adrenergic, beta; RNA, Messenger; Signal Transduction; Stem Cells; Transcription Factors

To further investigate the mechanisms of action of icariin (ICA), we assessed the effects of ICA on the in vitro formation of cGMP and cAMP in isolated rabbit corpus cavernosum. Isolated segments of rabbit corpus cavernosum were exposed to increasing concentrations of ICA and the dose-dependent accumulation of cGMP and cAMP was determined in the tissues samples by means of 125I radioimmunoassay. Responses of the isolated tissues preparations to ICA were compared with those obtained with the reference compounds sildenafil (Sild). Furthermore, the effects of ICA on the mRNA expression of specific cGMP-binding phosphodiesterase type V (PDE5) in rat penis were also observed. After incubation with ICA for 6 h or 14 h respectively, the levels of PDE5 mRNA were examined by reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that ICA increased cGMP concentrations directly (P < 0.05), but there was no significant effect on cAMP concentrations (P > 0.05). In the presence of sodium nitroprusside (SNP), a stimulatory agent of cGMP, both ICA and Sild increased cGMP concentrations with increasing dose (P < 0.01). Their EC50 was 4.62 (ICA) and 0.42 (Sild) micromol/L respectively. Under the same condition, ICA and Sild unaltered cAMP level significantly (P > 0.05). There were PDE5A1 and PDE5A2 mRNA expressions in rat corpus cavernosum with PDE5A2 being the dominant isoform. ICA could obviously inhibit these two isoforms mRNA expression in rat penis, and decrease PDE5A1 more pronouncedly (P < 0.01). The present study indicated that the aphrodisiac mechanisms of icariin involved the NO-cGMP signal transduction pathway, with increasing cGMP levels in the corpus cavernosum smooth muscle. The inhibitory effect of icariin on PDE5 mRNA expression, especially on PDE5A1, might account for its molecular mechanisms for its long-term activity.

Topics: Animals; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Flavonoids; In Vitro Techniques; Male; Penis; Rabbits; Radioimmunoassay; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

To investigate the effect of icariin on the cyclic guanosine monophosphate (cGMP)-hydrolytic activity of phosphodiesterase-5 (PDE5) isoforms and the cGMP levels in cavernous smooth muscle cells treated with sodium nitroprusside (SNP).. PDE5 isoforms (PDE5A1, A2, and A3) were isolated from sf9 insect cells infected with baculoviruses carrying PDE5 isoform cDNA. Icariin was isolated from Epimedii herba. Varying amounts (10(-6) to 10(-11) M) of icariin or zaprinast were added to reaction mixtures containing PDE5 isoforms and cGMP. The inhibitory effects of icariin and zaprinast were analyzed by GraphPad Software and are expressed as concentration that inhibits 50% (IC50) values. Cavernous smooth muscle cells were isolated from 3-month-old rats, treated with icariin (100 and 200 microM) or zaprinast (200 microM) for 15 minutes, and then with 10 microM SNP for 30, 60, 120, 240, and 360 minutes. The cells were then analyzed for the cGMP concentration using an enzyme immunoassay system.. Icariin inhibited PDE5A1, A2, and A3 with an IC50 value of 1.0, 0.75, and 1.1 microM, respectively. The corresponding IC50 values for zaprinast were 0.33, 0.23, and 0.32 microM. Icariin consistently outperformed the control (SNP-only treatment) in maintaining greater cGMP levels, particularly at the greater concentration of 200 microM. In contrast, zaprinast at 200 microM did better than the control only at 60 and 360 minutes.. Icariin was inhibitory to all three PDE5 isoforms with similar IC50 values, which were approximately three times greater than those for zaprinast. Icariin was able to enhance cGMP levels in SNP-treated cavernous smooth muscle cells.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Drugs, Chinese Herbal; Flavonoids; Immunoenzyme Techniques; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Penis; Rats

To clarify the mechanism of the therapeutic action of icariin on erectile dysfunction (ED).. PDE5 was isolated from the human platelet and PDE4 from the rat liver tissue using the FPLC system (Pharmacia, Milton Keynes, UK) and the Mono Q column. The inhibitory effects of icariin on PDE5 and PDE4 activities were investigated by the two-step radioisotope procedure with [(3)H]-cGMP/[(3)H]-cAMP. Papaverine served as the control drug.. Icariin and papaverine showed dose-dependent inhibitory effects on PDE5 and PDE4 activities. The IC(50) of Icariin and papaverine on PDE5 were 0.432 micromol/L and 0.680 micromol/L, respectively and those on PDE4, 73.50 micromol/L and 3.07 micromol/L, respectively. The potencies of selectivity of icariin and papaverine on PDE5 (PDE4/PDE5 of IC(50)) were 167.67 times and 4.54 times, respectively.. Icariin is a cGMP-specific PDE5 inhibitor that may be developed into an oral effective agent for the treatment of ED.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Cyclic AMP; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 4; Cyclic Nucleotide Phosphodiesterases, Type 5; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Flavonoids; Humans; In Vitro Techniques; Male; Papaverine; Penile Erection; Phosphodiesterase Inhibitors; Rats; Tritium

Icariin (ICA) is a kind of new biological respone modifier(BRM) and differentiational agent. In order to further elucidate the reversion of malignant phenotypes of tumor cells and the mechanism of its action, highly metastatic human lung cancer cells(PG) were treated with ICA in vitro. In this study, MTT assay, radioimmune assay, flow cytometry(FCM) and invasion assay were used. The results showed that ICA could influence the distribution of PG cells cycle and reduce S phase. Moreover, ICA increased the level of cAMP in PG cells, reduced the level of cGMP and increased the cAMP/cGMP ratio. On the other hand, ICA decreased PG cells adhesive ratio to laminin substrate and decreased the ability of invasion or migration. These data demonstrate that ICA maybe a kind of effective anticancer drug.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Adhesion; Cell Division; Cyclic AMP; Cyclic GMP; Flavonoids; Flow Cytometry; Humans; Lung Neoplasms; Mice; Tumor Cells, Cultured

In order to search for new tumor differentiation inducer, the effects and mechanism of Icariin were studied.. Icariin is an monomer purified from Epimedium Koreanum Nakai. The differentiation of induction effects of Icariin were observed on the human promyelocytic leukemic cell (HL-60) by NBT reduction test, 125I-cAMP and 125I-cGMP double antibody radioimmune methods, scanning electronic microscopy technique.. After treating HL-60 cells with Icariin at concentration 0.1 g/L, reduction of NBT and cAMP/cGMP ratio were increased. There were many rugosities and ball-like processes on the cell surface.. Icariin had the effects of induction of differentiation on HL-60 cells, the mechanism might be related to elevating the cAMP/cGMP ratio.

Topics: Cell Transformation, Neoplastic; Cyclic AMP; Cyclic GMP; Drugs, Chinese Herbal; Flavonoids; HL-60 Cells; Humans