Luteolin prevents f MLP-induced neutrophils adhesion via suppression of LFA-1 and phosphodiesterase 4 activity

Luteolin is an active ingredient found early from Fofium perillae and Flos Ionicerae, and has a specific inhibition on phosphodiesterase 4 （PDE4） activity in vitro. Researches show luteolin has pharmacological effects of anti-inflammation, anti-anaphylaxis, antitumor, antioxidant, protection of nervous system and so on, and has mainly been used for the treatment of respiratory inflammatory diseases, cancer and cardiovascular disease in clinic. PDE4, specific to hydrolyze cyclic AMP （cAMP）, is considered to be a new anti-inflammatory target due to the decisive role on cAMP signal in inflammatory cells such as neutrophils. In order to explore the anti-inflammatory mechanism, we further studied the effects of luteolin on the activity and expression of PDE4, the expression of lymphocyte function-associated antigen-1 （LFA-1） and macrophage-1 （MAC-l） in neutrophils, and the adhesion of neutrophils and endothelial cells. The results showed that luteolin had a dose-dependent inhibition on both bare PDE4 activity and PDE4 in cultured neutrophils, and had an obviously promotive effect on gene expressions of PDE4A, 4B and 4D in later period. Luteolin had a significant inhibitory effect on neutrophils adhesion and LFA-1 expression in early stage, and had no obvious effect on MAC-1 expression. Therefore, luteolin can inhibit LFA-1 expression of neutrophils, then inhibit the adhesion of neutrophils and endothelial cells, and the mechanism is at least related with the inhibition of PDE4 activity.

Influence of gallic acid on porcine neutrophils phosphodiesterase 4,IL-6,TNF-α and rat arthritis model

Our previous studies showed that the anti-inflammatory effects of Paeonia lactiflora roots extract may be mediated, at least in part, through its gallic acid content, and this effect may be regulated in part by an inhibition on c AMP-phosphodiesterase（PDE）. To explore the anti-inflammatory effect and mechanism, the influence of gallic acid on neutrophils PDE4 activity and expression, TNF-α and IL-6 content and rat arthritis model were further studied. PDE4 activity and gene express were calculated respectively by substrate c AMP change examined with HPLC and real-time RT-PCR. The concentration of IL-6 and TNF-α in supernatant were assayed by ELISA method. Model of rat arthritis was caused by complete Freund＇s adjuvant. Results showed that gallic acid had a dose-dependent restraint on PDE4 activity of neutrophils in vitro, promoted significantly PDE4 A expression（P〈0.01）, and had no influence on the expressions of PDE4 B and 4D. However, PDE4 C expression was not detected. Gallic acid could promote IL-6 release（P〈0.05）, and inhibit TNF-α release of neutrophils（P〈0.05）. The experiment in vivo showed that gallic acid had obvious restraint on local inflammation of animal model（P〈0.05）. Therefore, the anti-inflammatory effect of gallic acid may be mediated in part through an inhibition on PDE4 activity and further an increase of IL-6 and a decrease of TNF-α of neutrophils, and this effect seemed to have no relationship with PDE4 expression.

Association of ALOX5AP and PDE4D with the risk of lacunar infarct in people from Jiangsu Province,China

The genes for 5-1ipoxygenase activating protein （ALOX5AP） and phosphodiesterase 4D （PDE4D） have been demonstrated as susceptibility genes for lacunar in the Icelandic and Pakistani populations, but little is known about the role of these genes in Chinese populations. The present study utilized polymerase chain reaction and ligase detection reaction to detect single nucleotide polymorphisms （SNPs） in 280 consecutive stroke patients and 258 unrelated population-based controls from Nanjing, Jiangsu Province, China. The allele frequency, genotypes, and haplotypes of the two SNPs （rs456009 and rs966221） in PDE4D were similar between the two groups. However, A allele frequency of rs4073259 （A/G） and rs4769055 （A/C） in the ALOX5AP gene exhibited differences in two groups, and especially the haplotype of the SNP was significantly different between the two groups. Results suggested that the ALOX5AP gene might be involved in lacunar infarct, while PDE4D gene was not a risk factor for lacunar infarct in individuals from Jiangsu Province, China.

A novel mechanism underlying the protective effect of PDE4 inhibitor against cognitive impairment:inhibiting neuroinflammation through inducing autophagy in microglial cells

OBJECTIVE Inhibition of phosphodiesterase 4(PDE4) improves the learning and memory abilities in Alzheimer disease animal models. The cognition-enhancing effects of PDE4 inhibition involve reduced inflammatory responses in the brain. However,the underlying mechanisms are illunderstood. cA MP induces autophagy,and deficiency of autophagy leads to elevated inflammatory factors.In the present study,we aimed to investigate the contribution of autophagy to the anti-inflammatory effect of PDE4 inhibitor ROF. METHODS Acidic vesicles were traced by Lysotracker(LYT) red and acridine orange(AO) staining. Autophagosomes in BV-2 cells was observed by immunofluorescence staining of microtubule-associated protein 1 light chain 3(LC3). Aβ_(25-35) or lipopolysaccharide(LPS) with ATP were used to activate microglial cells and inflammasome. Cytokine levels were measured by ELISA method. The levels of pro-inflammatory factors and essential proteins involved in the formation of autophagosome were detected by Western blotting. RESULTS ROF increased the level of LC3-Ⅱ,while the level of p62 was decreased. Enhanced fluorescent signals were observed in BV-2 cells treated with ROF by AO and LYT red staining. In addition,immunofluorescence indicated a significant increase in punctate LC3. Both LPS plus ATP and Aβ_(25-35) enhanced the conversion of pro-caspase-1 to cleaved-caspase-1 and increased the production of mature IL-1β. Interestingly,these effects were blocked by the treatment of ROF. Moreover,ROF decreased the apoptosis of neuronal N2 a cells in conditioned media from BV-2 microglia. These effects were reversed by inhibition of microglial autophagy.Treatment with ROF also showedenhanced autophagy in mcie treated with LPS. CONCLUSION PDE4 inhibitor ROF inhibits inflammasome activities and reduces the release of IL-1β by inducing autophagy.

Role of PDE4D splice variants in the mediation of antidepressant and cognition-enhancing actions and its mechanisms

OBJECTIVE Phosphodiesterase 4(PDE4),specific for cyclicAMP(cAMP)-hydrolyzing,has four isoforms(PDE4A-D) with at least 25 splice variants. PDE4 inhibitors produce definite antidepressant-like and cognitive-enhancing effects. However,none of PDE4 inhibitors has yet been approved for clinical utility so far due to the concomitant side effects. The present research is to explore the splice variants of PDE4 D responsible for antidepressant-like and cognitive-enhancing effects of PDE4 inhibitors but not side effects. METHODS Long-form PDE4 Ds were silenced by the bilateral microinfusion of lentiviral vector containing mi RNAs(4Dmi R) into the prefrontal cortex(PFC),PDE4D4 or D5 was overexpressed by the bilateral microinfusion of lentiviral vector containing full c DNA into hippocampus. Antidepressant-like behaviors were measured by tail-suspension test(TST),forced swimming test(FST)and chronic unpredictable stress model. Cognitive behaviors were measured by the novel object recognition test(NOR) and Morris water maze test(MWM) in both normal mice and the mice with chronic unpredictable stress-induced memory deficits. The emetic potential was evaluated by the assessment of the anaesthetic reversal effect,a surrogate of the emesis test in non-vomiting species. The expressions of PDE4 isoforms/splice variants and cAMP level were examined by Western-blot and ELISA analysis. The dendritic complexity and spine density were assessed by Golgi staining. RESULTS(1)High and specific expression of EGFP(green,indicator of 4Dmi R expression) in PFC was observed under fluorescence microscopy.(2) 4Dmi R significantly down-regulated PDE4D4/5 splice variants,but not PDE4 A,PDE4 B or PDE4D1/2/3.(3) 4Dmi R treatments significantly increased cAMP signaling and dendritic complexity in PFC.(4) Rolipram and/or 4Dmi R treatments significantly decreased immobility in TST and FST.(5) Rolipram and/or 4Dmi R treatments reversed the depressive-like behaviors in chronically stressed mice,including the reduced sucrose preference,prolonged latency to novelty-suppressed feeding and increased immobility in FST.(6) Rolipram and/or 4Dmi R treatments significantly increased the recognition index in NOR task and both the entries and durations in MWM task.(7) Rolipram and/or 4Dmi R treatments reversed the memory deficits in chronically stressed mice,including the reduced the recognition index in NOR task and the decreased durations in MWM task.(8) Rolipram and/or 4DmiR treatments reversed the decreased cA MP signaling,dendritic complexity and spine density.(9) Rolipram or plus 4Dmi R treatment significantly decreased the duration of anaesthesia in the alpha2 adrenergic receptor-mediated anesthesia,but not 4Dmi R treatment alone.(10)Hippocampal overexpression of PDE4D5,but not PDE4D4,produced depressive-like and cognitive defect behaviors,which were reversed by rolipram.The measurements including cAMP signaling,dendritic complexity and in vivo hippocampal LTP,showed the same changes. CONCLUSION Long-form PDE4 Ds,especially the PDE4D5,are the major isoforms responsible for antidepressant-like and cognitive-enhancing effects with little side effects. The critical roles of long-form PDE4 Ds are mediated by their regulation of cAMP signaling pathway and neuroplasticity.

4-型磷酸二酯酶与局部cAMP信号调节（英文）

Of the eleven families of cyclic nucleotide phosphodiesterases(PDEs) present in the human body,PDE4s represent the most widely expressed family of PDEs. A large body of work has been published on the expression and function of these PDEs,which preferentially hydrolyze cAMP in all cells studied,including neurons and supporting cells of the CNS. Four distinct genes termed PDE4 A,PDE4B,PDE4C and PDE4D encode PDE4 proteins. However,the number of PDE4s identified in different tissues and cells is estimated to be more than 30. Differences in regulation and localization explain this extreme heterogeneity. PDE4 hydrolytic activity is regulated by phosphorylation,and protein kinase A(PKA) was the first kinase identified. This PKA-dependent regulation establishes a feedback loop where cAMP regulates its own degradation to control the intensity and localization of the hormone and neurotransmitter signal. In addition,numerous additional kinases phosphorylate PDE4s to modulate the PKA-dependent activation and fine tune cAMP levels by growth factors and other extracellular cues. Thus,PDE4 can be considered a coincidence detector that integrates multiple signaling pathways. Finally,different PDE4s are involved in numerous macromolecular complexes targeting the cAMP hydrolytic activity to different subcellular domains. Thus,PDE4s function in different subcellular compartments,and inhibition of different isoforms affects cAMP levels in different subdomains with consequently different functions. The dyad space and the control of excitation/contraction will be used as examples of these localized regulations.

Endothelial phosphodiesterase 4B inactivation ameliorates endothelial-to-mesenchymal transition and pulmonary hypertension

Pulmonary hypertension (PH) is a fatal disorder characterized by pulmonary vascular remodeling and obstruction. The phosphodiesterase 4 (PDE4) family hydrolyzes cyclic AMP (cAMP) and is comprised of four subtypes (PDE4A–D). Previous studies have shown the beneficial effects of pan-PDE4 inhibitors in rodent PH;however, this class of drugs is associated with side effects owing to the broad inhibition of all four PDE4 isozymes. Here, we demonstrate that PDE4B is the predominant PDE isozyme in lungs and that it was upregulated in rodent and human PH lung tissues. We also confirmed that PDE4B is mainly expressed in the lung endothelial cells (ECs). Evaluation of PH in Pde4b wild type and knockout mice confirmed that Pde4b is important for the vascular remodeling associated with PH. In vivo EC lineage tracing demonstrated that Pde4b induces PH development by driving endothelial-to-mesenchymal transition (EndMT), and mechanistic studies showed that Pde4b regulates EndMT by antagonizing the cAMP-dependent PKA–CREB–BMPRII axis. Finally, treating PH rats with a PDE4B-specific inhibitor validated that PDE4B inhibition has a significant pharmacological effect in the alleviation of PH. Collectively, our findings indicate a critical role for PDE4B in EndMT and PH, prompting further studies of PDE4B-specific inhibitors as a therapeutic strategy for PH.

环核苷酸磷酸二酯酶:神经精神疾病的药物作用靶标(英文)

OBJECTIVE To investigate the relationship between the neurochemical and behavioral effects of inhibitors of cyclic nucleotide phosphodiesterases 2(PDE2) and PDE4 in the hope of identifying promising therapeutic targets and indications. METHODS Depression-and anxiety-like behaviors were evaluated by forced swimming,tail suspending and elevated plus maze tests. The memory enhancing effects were evaluated by Morris water maze test. RESULTS Inhibition of PDE4 results in antidepressant-like and memory-enhancing effects,in part due to increased cyclicAMP signaling.While targeting PDE4 subtypes(PDE4A-D) pharmacologically has proven challenging due to a highly conserved inhibitor-binding site in the catalytic region of the enzyme,some promise is offered by recently identified negative allosteric modulators of PDE4,which can act in a subtype-specific manner.While PDE2 has not been studied as extensively,it has been shown to be coded for by a single mammalian gene and to be highly expressed in the brain. Its inhibition results in anxiolytic-and antidepressant-like effects as wells as reducing oxidative stress and affording some degree of neuroprotection;these actions appear to be due to increased cyclicGMP signaling,even though PDE2 catalyzes the hydrolysis of both cyclicAMP and cyclicGMP. CONCLUSION Inhibitors targeting specific family of PDE may exhibit differential pharmacological effects and aid a more efficient pharmacotherapy towards neuropsychological conditions,while drug discovery efforts focusing on PDE2 are not as advanced as those for PDE4,some selective inhibitors are being developed and evaluated.

Sex-dependent association of phosphodiesterase 4D gene polymorphisms with ischemic stroke in Henan Han population

Background Recent evidence has implicated the gene for phosphodiesterase 4D （PDE4D） as susceptibility gene for ischemic stroke （IS） in Icelandic population. However, there are few reports on the associations between PDE4D gene polymorphisms and IS in Chinese individuals. The present study aimed to investigate the possible association of genetic polymorphisms in PDE4D gene with IS in Henan Han population. Methods A total of 400 patients with IS and 400 matched controls were examined using a case-control design. Two single nucleotide polymorphism （SNPs） （rs918592 and rs2910829） in PDE4D gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） method. Odds ratios （OR） and 95% confidence intervals （95% CI） were calculated to test the association between the genetic factors and IS. Genetic parameter and association studies were carried out with SPSS 16.0. Results Among the two SNPs tested, the rs918592 was significantly associated with IS （OR： 1.351, 95% CI： 1.110-1.645）, especially in male patients （OR： 1.427, 95% CI： 1.105-1.844）. Haplotype analysis showed that A-T was associated with an increased risk of the IS （OR： 2.114, 95% CI： 2.005-2.230） while G-T was associated with decreased risk of IS （OR： 0.419, 95% CI： 0.302--0.583）. Protecting effect of haplotype G-T was also significant in males （OR： 0.264, 95% Cl： 0.162-0.431）. Conclusions The present study demonstrated a strong association of rs918592 with IS. Haplotype A-T increased the risk of IS while haplotype G-T had a protective effect in Henan Han population. The association was sex-dependent with male patients showing stronger effect.