Inhibition of phosphodiesterase 4 attenuates aquaporin 4 expression and astrocyte swelling following cerebral ischemia/reperfusion injury.

We have previously shown that phosphodiesterase 4 (PDE4) inhibition protects against neuronal injury in rats following middle cerebral artery occlusion/reperfusion (MCAO/R). However, the effects of PDE4 on brain edema and astrocyte swelling are unknown. In this study, we showed that inhibition of PDE4 by Roflumilast (Roflu) reduced brain edema and brain water content in rats subjected to MCAO/R. Roflu decreased the expression of aquaporin 4 (AQP4), while the levels of phosphorylated protein kinase B (Akt) and forkhead box O3a (FoxO3a) were increased. In addition, Roflu reduced cell volume and the expression of AQP4 in primary astrocytes undergoing oxygen and glucose deprivation/reoxygenation (OGD/R). Consistently, PDE4B knockdown showed similar effects as PDE4 inhibition; and PDE4B overexpression rescued the inhibitory role of PDE4B knockdown on AQP4 expression. We then found that the effects of Roflu on the expression of AQP4 and cell volume were blocked by the Akt inhibitor MK2206. Since neuroinflammation and astrocyte activation are the common events that are observed in stroke, we treated primary astrocytes with interleukin-1ß (IL-1ß). Astrocytes treated with IL-1ß showed decreased AQP4 and phosphorylated Akt and FoxO3a. Roflu significantly reduced AQP4 expression, which was accompanied by increased phosphorylation of Akt and FoxO3a. Furthermore, overexpression of FoxO3a partly reversed the effect of Roflu on AQP4 expression. Our findings suggest that PDE4 inhibition limits ischemia-induced brain edema and astrocyte swelling via the Akt/FoxO3a/AQP4 pathway. PDE4 is a promising target for the intervention of brain edema after cerebral ischemia.

Involvement of CKS1B in the anti-inflammatory effects of cannabidiol in experimental stroke models.

We have previously demonstrated that treatment with cannabidiol (CBD) ameliorates mitochondrial dysfunction and attenuates neuronal injury in rats following cerebral ischemia. However, the role of CBD in the progression of ischemic stroke-induced inflammation and the molecules involved remain unclear. Here, we found that CBD suppressed the production of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), reduced the activation of microglia, ameliorated mitochondrial deficits, and decreased the phosphorylation of nuclear factor κ-B (NF-κB) in BV-2 cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Cyclin-dependent kinase regulatory subunit 1B (CKS1B) expression was decreased in BV-2 cells following OGD/R and this reduction was blocked by treatment with CBD. Knockdown of CKS1B increased the activation of microglia and enhanced the production of IL-1ß and TNF-α in BV-2 cells treated with CBD. Moreover, CKS1B knockdown exacerbated mitochondrial deficits and increased NF-κB phosphorylation. CBD treatment also ameliorated brain injury, reduced neuroinflammation, and enhanced the protein levels of mitochondrial transcription factor A and CKS1B in rats following middle cerebral artery occlusion/reperfusion. These data identify CKS1B as a novel regulator of neuroinflammation; and reveals its involvement in the anti-inflammatory effects of CBD. Interventions targeting CKS1B expression are potentially promising for treating in ischemic stroke.

Advances in the development of phosphodiesterase 7 inhibitors.

Phosphodiesterase 7 (PDE7) specifically hydrolyzes cyclic adenosine monophosphate (cAMP), a second messenger that plays essential roles in cell signaling and physiological processes. Many PDE7 inhibitors used to investigate the role of PDE7 have displayed efficacy in the treatment of a wide range of diseases, such as asthma and central nervous system (CNS) disorders. Although PDE7 inhibitors are developed more slowly than PDE4 inhibitors, there is increasing recognition of PDE7 inhibitors as potential therapeutics for no nausea and vomiting secondary. Herein, we summarized the advances in PDE7 inhibitors over the past decade, focusing on their crystal structures, key pharmacophores, subfamily selectivity, and therapeutic potential. Hopefully, this summary will lead to a better understanding of PDE7 inhibitors and provide strategies for developing novel therapies targeting PDE7.

Mitofusin 2 confers the suppression of microglial activation by cannabidiol: Insights from in vitro and in vivo models.

Currently, there is increasing attention on the regulatory effects of cannabidiol (CBD) on the inflammatory response and the immune system. However, the mechanisms have not yet been completely revealed. Mitofusin 2 (Mfn2) is a mitochondrial fusion protein involved in the inflammatory response. Here, we investigated whether Mfn2 confers the anti-inflammatory effects of CBD. We found that treatment with CBD decreased the levels of tumor necrosis factor α, interleukin 6, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and ionized calcium-binding adaptor molecule-1 (Iba1) in lipopolysaccharide (LPS)-challenged microglia. CBD also significantly suppressed the increase in reactive oxygen species (ROS) and the decline of mitochondrial membrane potential in BV-2 cells subjected to LPS. Interestingly, CBD treatment increased the expression of Mfn2, while knockdown of Mfn2 blocked the effect of CBD. By contrast, overexpression of Mfn2 reversed the increase in the levels of iNOS, COX-2, and Iba1 induced by Mfn2 small interfering RNA. In mice challenged with LPS, we found that CBD ameliorated the anxiety responses and cognitive deficits, increased the level of Mfn2, and decreased the expression of Iba1. Since neuro-inflammation and microglial activation are the common events that are observed in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, we treated EAE mice with CBD. Mice that received CBD showed amelioration of clinical signs, reduced inflammatory response, and increased myelin basic protein level. Most importantly, the adeno-associated virus delivery of short hairpin RNA against Mfn2 reversed the protective effects of CBD. Altogether, these results indicate that Mfn2 is an essential immunomodulator conferring the anti-inflammatory effects of CBD. Our results also shed new light on the mechanisms underlying the protective effects of CBD against inflammatory diseases including multiple sclerosis.

Roflupram attenuates α-synuclein-induced cytotoxicity and promotes the mitochondrial translocation of Parkin in SH-SY5Y cells overexpressing A53T mutant α-synuclein.

We have previously shown that inhibition of cAMP-specific 3',5'-cyclic phosphodiesterase 4 (PDE4) protects against cellular toxicity in neuronal cells. Since α-synuclein (α-syn) toxicity contributes to the neurodegeneration of Parkinson's disease (PD). The aim of this study was to explore the effects and mechanisms of PDE4 on α-syn-induced neuronal toxicity. Using mutant human A53T α-syn overexpressed SH-SY5Y cells, we found that PDE4B knockdown reduced cellular apoptosis. Roflupram (ROF, 20 µM), a selective PDE4 inhibitor, produced similar protective effects and restored the morphological alterations of mitochondria. Mechanistic studies identified that α-syn enhanced the phosphorylation of Parkin at Ser131, followed by the decreased mitochondrial translocation of Parkin. Whereas both PDE4B knockdown and PDE4 inhibition by ROF blocked the effects of α-syn on Parkin phosphorylation and mitochondrial translocation. Moreover, PDE4 inhibition reversed the increase in the phosphorylation of p38 mitogen-activated protein kinase (MAPK) induced by α-syn. ROF treatment also reduced the binding of p38 MAPK to Parkin. Consistently, overexpression of PDE4B blocked the roles of ROF on p38 MAPK phosphorylation, Parkin phosphorylation, and the subsequent mitochondrial translocation of parkin. Furthermore, PDE4B overexpression attenuated the protective role of ROF, as evidenced by reduced mitochondria membrane potential and increased cellular apoptosis. Interestingly, ROF failed to suppress α-syn-induced cytotoxicity in the presence of a protein kinase A (PKA) inhibitor H-89. Our findings indicate that PDE4 facilitates α-syn-induced cytotoxicity via the PKA/p38 MAPK/Parkin pathway in SH-SY5Y cells overexpressing A53T mutant α-synuclein. PDE4 inhibition by ROF is a promising strategy for the prevention and treatment of α-syn-induced neurodegeneration.

MiR-139-5p has an antidepressant-like effect by targeting phosphodiesterase 4D to activate the cAMP/PKA/CREB signaling pathway.

BACKGROUND: Phosphodiesterase 4D (PDE4D) inhibitor is commonly used to treat depression, but side effects seriously decrease its efficacy. PDE4D was a downstream target mRNA of miR-139-5p. Therefore, we examined the effects of hippocampal miR-139-5p gain- and loss-of-function on depression-like behaviors, the expression level of PDE4D, and hippocampus neurogenesis. METHODS: Bioinformatic analyses were carried out to to screen differential genes. Quantitative real-time polymerase chain reaction (qRT-PCR) and luciferase reporter assay were used to confirm the relationship between miR-139-5p and PDE4D. MiR-139-5p mimics, miR-139-5p inhibitor, or miR-NC were used to explore the function of miR-139-5p in HT-22 cells. We further explored the role of miR-139-5p in vivo using AAV-injection. Elisa, western blotting, and fluorescence in situ hybridization (FISH) were used to detect the expression of miR-139-5p and PDE4D in CRC tissues. RESULTS: Here, we showed that PDE4D messenger RNA (mRNA) was a direct target of microRNA (miR)-139-5p, which was downregulated in a chronic ultra-mild stress (CUMS)-induced depression mouse model. Moreover, in experiments in vitro, miR-139-5p mimic repressed PDE4D expression in HT-22 cells, but promoted phosphorylated cyclic-AMP response element-binding protein (p-CREB) and brain-derived neurotrophic factor (BDNF) expression. Interestingly, adeno-associated virus (AAV)-miR-139-5p downregulated susceptibility to stress-induced depression-like behaviors in mice. AAV-miR-139-5p suppressed PDE4D in mouse hippocampal cells, increasing expression level of cyclic adenosine monophosphate (cAMP), p-CREB, and BDNF, and stimulating mouse hippocampal neurogenesis. CONCLUSIONS: Our findings suggested that miR-139-5p acted like an antidepressant by targeting PDE4D, thereby regulating the cAMP/protein kinase A (PKA)/CREB/BDNF pathway to improve depression.

Roflupram protects against rotenone-induced neurotoxicity and facilitates α-synuclein degradation in Parkinson's disease models.

We have previously shown that roflupram (ROF) protects against MPP+-induced neuronal damage in models of Parkinson's disease (PD). Since impaired degradation of α-synuclein (α-syn) is one of the key factors that lead to PD, here we investigated whether and how ROF affects the degradation of α-syn in rotenone (ROT)-induced PD models in vivo and in vitro. We showed that pretreatment with ROF (10 µM) significantly attenuated cell apoptosis and reduced the level of α-syn in ROT-treated SH-SY5Y cells. Furthermore, ROF significantly enhanced the lysosomal function, as evidenced by the increased levels of mature cathepsin D (CTSD) and lysosomal-associated membrane protein 1 (LAMP1) through increasing NAD+/NADH and the expression of sirtuin 1 (SIRT1). Pretreatment with an SIRT1 inhibitor selisistat (SELI, 10 µM) attenuated the neuroprotection of ROF, ROF-reduced expression of α-syn, and ROF-increased expression levels of LAMP1 and mature CTSD. Moreover, inhibition of CTSD by pepstatin A (20 µM) attenuated ROF-reduced expression of α-syn. In vivo study was conducted in mice exposed to ROT (10 mg·kg-1·d-1, i.g.) for 6 weeks; then, ROT-treated mice received ROF (0.5, 1, or 2 mg·kg-1·d-1; i.g.) for four weeks. ROF significantly ameliorated motor deficits, which was accompanied by increased expression levels of tyrosine hydroxylase, SIRT1, mature CTSD, and LAMP1, and a reduced level of α-syn in the substantia nigra pars compacta. Taken together, these results demonstrate that ROF exerts a neuroprotective action and reduces the α-syn level in PD models. The mechanisms underlying ROF neuroprotective effects appear to be associated with NAD+/SIRT1-dependent activation of lysosomal function.

Brahma-related gene-1 promotes tubular senescence and renal fibrosis through Wnt/ß-catenin/autophagy axis.

Although accelerated cellular senescence is closely related to the progression of chronic kidney disease (CKD) and renal fibrosis, the underlying mechanisms remain largely unknown. Here, we reported that tubular aberrant expression of Brahma-related gene 1 (BRG1), an enzymatic subunit of the SWItch/Sucrose Non-Fermentable complex, is critically involved in tubular senescence and renal fibrosis. BRG1 was significantly up-regulated in the kidneys, predominantly in tubular epithelial cells, of both CKD patients and unilateral ureteral obstruction (UUO) mice. In vivo, shRNA-mediated knockdown of BRG1 significantly ameliorated renal fibrosis, improved tubular senescence, and inhibited UUO-induced activation of Wnt/ß-catenin pathway. In mouse renal tubular epithelial cells (mTECs) and primary renal tubular cells, inhibition of BRG1 diminished transforming growth factor-ß1 (TGF-ß1)-induced cellular senescence and fibrotic responses. Correspondingly, ectopic expression of BRG1 in mTECs or normal kidneys increased p16INK4a, p19ARF, and p21 expression and senescence-associated ß-galactosidase (SA-ß-gal) activity, indicating accelerated tubular senescence. Additionally, BRG1-mediated pro-fibrotic responses were largely abolished by small interfering RNA (siRNA)-mediated p16INK4a silencing in vitro or continuous senolytic treatment with ABT-263 in vivo. Moreover, BRG1 activated the Wnt/ß-catenin pathway, which further inhibited autophagy. Pharmacologic inhibition of the Wnt/ß-catenin pathway (ICG-001) or rapamycin (RAPA)-mediated activation of autophagy effectively blocked BRG1-induced tubular senescence and fibrotic responses, while bafilomycin A1 (Baf A1)-mediated inhibition of autophagy abolished the effects of ICG-001. Further, BRG1 altered the secretome of senescent tubular cells, which promoted proliferation and activation of fibroblasts. Taken together, our results indicate that BRG1 induces tubular senescence by inhibiting autophagy via the Wnt/ß-catenin pathway, which ultimately contributes to the development of renal fibrosis.

Discovery of novel trimethoxyphenylbenzo[d]oxazoles as dual tubulin/PDE4 inhibitors capable of inducing apoptosis at G2/M phase arrest in glioma and lung cancer cells.

To discover PDE4/tubulin dual inhibitors with novel skeleton structures, 7-trimethoxyphenylbenzo[d]oxazoles 4a-u and 4-trimethoxyphenylbenzo[d]oxazoles 5a-h were designed and synthesized by migrating the trimethoxyphenyl group of TH03 to the benzo[d]oxazole moiety. Among these compounds, approximately half of them displayed good antiproliferative activities against glioma (U251) and lung cancer (A549 and H460) cell lines. The structure-activity relationships of trimethoxyphenylbenzo[d]oxazoles led to the identification of 4r bearing indol-5-yl side-chain as a novel dual PDE4/tubulin inhibitor, which exhibited satisfactory antiproliferative activities against glioma (IC50 = 300 ± 50 nM) and lung cancer (average IC50 = 39.5 nM) cells. Further investigations revealed that 4r induced apoptosis at G2/M phase arrest and disrupted the microtubule network. The preliminary mechanism of action showed that 4r down-regulated the expression of cyclin B1 and its upstream regulator gene cdc25C in A549.

Inhibition of phosphodiesterase-4 suppresses HMGB1/RAGE signaling pathway and NLRP3 inflammasome activation in mice exposed to chronic unpredictable mild stress.

Inhibition of phosphodiesterase-4 (PDE4) produces robust anti-inflammatory and antidepressant-like effects in multiple animal models. However, the detailed mechanisms have not been well studied. Receptor for advanced glycation endproducts (RAGE) and inflammasome activation are implicated in the etiology of depression. Here, we aimed to investigate the involvement of RAGE and nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome in the antidepressant-like effects of PDE4 inhibition in mice. We found that inhibition of PDE4 by roflupram (ROF, 0.5, and 1.0 mg/kg, i.g.) exerted antidepressant-like effects in mice subjected to chronic unpredictable mild stress (CUMS). Simultaneously, ROF inhibited CUMS-induced microglial activation and restored the morphology of microglial cells in the hippocampus, as evidenced by reduced total process length, area, volume, number of branching points, number of terminal points and total sholl intersections of microglia. ROF also decreased the expression of ionized calcium-binding adapter molecule-1 and the level of interleukin-1ß. Western blot analysis showed that PDE4 inhibition suppressed the high-mobility group box 1 protein (HMGB1)/RAGE signaling pathway, as the levels of HMGB1, RAGE, toll-like receptor 4, phosphorylated p38 mitogen-activated protein kinase, and nuclear factor κ-B were decreased in both hippocampus and cortex in mice after treatment with ROF. Moreover, ROF also attenuated the protein levels of NLRP3, the apoptosis-associated speck-like protein containing (ASC), and cysteine-requiring aspartate protease-1 (Caspase-1), which are key proteins in the NLRP3-mediated inflammasome signaling pathway. In summary, these results demonstrate that the down-regulation of HMGB1/RAGE signaling pathway and inflammasome suppression possibly contribute to the antidepressant-like effects of PDE4 inhibitors. And, ROF has potential as a candidate drug in the treatment of depression.

Roflumilast prevents ischemic stroke-induced neuronal damage by restricting GSK3ß-mediated oxidative stress and IRE1α/TRAF2/JNK pathway.

Inhibition of phosphodiesterase 4 (PDE4) protects against neuronal apoptosis induced by cerebral ischemia. However, the exact mechanisms responsible for the protection of PDE4 inhibition have not been completely clarified. Roflumilast (Roflu) is an FDA-approved PDE4 inhibitor for the treatment of chronic obstructive pulmonary disease. The potential protective role of Roflu against ischemic stroke-associated neuronal injury remains unexplored. In this study, we investigated the effect and mechanism of Roflu against ischemic stroke using in vitro oxygen-glucose deprivation reperfusion (OGD/R) and in vivo rat middle cerebral artery occlusion (MCAO) models. We demonstrated that Roflu significantly reduced the apoptosis of HT-22 cells exposed to OGD/R, enhanced the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2), and reduced oxidative stress. Treatment with Roflu increased the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3ß (GSK3ß) but decreased the level of phosphorylated inositol requiring enzyme 1α (IRE1α). Interestingly, constitutively active GSK3ß (S9A) mutation abolished the effects of Roflu on oxidative stress and IRE1α phosphorylation. Moreover, Roflu decreased the binding of IRE1α to tumor necrosis factor receptor-associated factor 2 (TRAF2) and attenuated the phosphorylation of c-Jun N-terminal kinase (JNK). We also found that PDE4B knockdown reduced the phosphorylation of both IRE1α and JNK, while overexpression of PDE4B antagonized the role of PDE4B knockdown on the activation of IRE1α and JNK. Besides, the inhibition of PDE4 by Roflu produced similar effects in primary cultured neurons. Finally, Roflu ameliorated MCAO-induced cerebral injury by decreasing infarct volume, restoring neurological score, and reducing the phosphorylation of IRE1α and JNK. Collectively, these data suggest that Roflu protects neurons from cerebral ischemia reperfusion-mediated injury via the activation of GSK3ß/Nrf-2 signaling and suppression of the IRE1α/TRAF2/JNK pathway. Roflu has the potential as a protective drug for the treatment of cerebral ischemia.

Roflupram, a novel phosphodiesterase 4 inhibitor, inhibits lipopolysaccharide-induced neuroinflammatory responses through activation of the AMPK/Sirt1 pathway.

Roflupram (ROF) is a novel phosphodiesterase 4 inhibitor. We previously found that ROF suppressed the production of pro-inflammatory factors in microglial cells; however, the underlying mechanisms are largely unknown. The present study aimed to elucidate the underlying molecular mechanisms of the anti-neuroinflammatory effects of ROF in lipopolysaccharide (LPS)-activated microglial cells and LPS-challenged mice. Treatment with ROF suppressed LPS-induced expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in BV-2 microglia cell line. Immunofluorescence and Western blotting analysis showed that ROF significantly inhibited the activation of microglia, as evidenced by decreased expression of ionized calcium binding adaptor molecule-1 (Iba1). Similar results were obtained in primary cultured microglial cells. ROF induced the phosphorylation of AMP-activated protein kinase (AMPK) and the expression of Sirtuin 1 (Sirt1). Interestingly, the AMPK inhibitor, compound C, blocked the role of ROF in both the phosphorylation of AMPK and the expression of Sirt1 in BV-2 cells stimulated with LPS. More importantly, the Sirt1 inhibitor, EX527, abolished the inhibitory role of ROF on the production of pro-inflammatory factors, and reactivated BV-2 cells. In mice challenged with LPS, ROF improved cognition and decreased the levels of IL-6 and TNF-α in both the cortex and hippocampus. In contrast, EX527 weakened the effects of ROF on cognitive enhancement and reduction of pro-inflammatory factors in the cortex and hippocampus. Furthermore, EX527 blocked the inhibitory role of ROF in the activation of microglial cells in both the hippocampus and cortex. Taken together, our results indicated that ROF attenuated LPS-induced neuroinflammatory responses in microglia, and the AMPK/Sirt1 pathway is essential for the anti-inflammatory effects of ROF.

Synthesis, Biological Evaluation, and Molecular Docking of Arylpyridines as Antiproliferative Agent Targeting Tubulin.

Mimicking different pharmacophoric units into one scaffold is a promising structural modification tool to design new drugs with enhanced biological properties. To continue our research on the tubulin inhibitors, the synthesis and biological evaluation of arylpyridine derivatives (9-29) are described herein. Among these compounds, 6-arylpyridines (13-23) bearing benzo[d]imidazole side chains at the 2-position of pyridine ring displayed selective antiproliferative activities against HT-29 cells. More interestingly, 2-trimethoxyphenylpyridines 25, 27, and 29 bearing benzo[d]imidazole and benzo[d]oxazole side chains displayed more broad-spectrum antitumor activities against all tested cancer cell lines. 29 bearing a 6-methoxybenzo[d]oxazole group exhibited comparable activities against A549 and U251 cells to combretastatin A-4 (CA-4) and lower cytotoxicities than CA-4 and 5-Fu. Further investigations revealed 29 displays strong tubulin polymerization inhibitory activity (IC50 = 2.1 µM) and effectively binds at the colchicine binding site and arrests the cell cycle of A549 in the G2/M phase by disrupting the microtubules network.

Surface engineering of magnetic iron oxide nanoparticles by polymer grafting: synthesis progress and biomedical applications.

Magnetic iron oxide nanoparticles (IONPs) have wide applications in magnetic resonance imaging (MRI), biomedicine, drug delivery, hyperthermia therapy, catalysis, magnetic separation, and others. However, these applications are usually limited by irreversible agglomeration of IONPs in aqueous media because of their dipole-dipole interactions, and their poor stability. A protecting polymeric shell provides IONPs with not only enhanced long-term stability, but also the functionality of polymer shells. Therefore, polymer-grafted IONPs have recently attracted much attention of scientists. In this tutorial review, we will present the current strategies for grafting polymers onto the surface of IONPs, basically including "grafting from" and "grafting to" methods. Available functional groups and chemical reactions, which could be employed to bind polymers onto the IONP surface, are comprehensively summarized. Moreover, the applications of polymer-grafted IONPs will be briefly discussed. Finally, future challenges and perspectives in the synthesis and application of polymer-grafted IONPs will also be discussed.

Roflupram exerts neuroprotection via activation of CREB/PGC-1α signalling in experimental models of Parkinson's disease.

BACKGROUND AND PURPOSE: Roflupram improves cognition and limits neuroinflammation in the brain. However, the beneficial effects of roflupram on Parkinson's disease (PD) remain unknown. Therefore, we aimed to elucidate the pharmacological effects and mechanisms of action of ROF in experimental models of PD. EXPERIMENTAL APPROACH: We used an in vitro PD model of SH-SY5Y cells exposed to 1-methyl-4-phenylpyridinium iodide (MPP+ ). Cell viability and apoptosis were analysed via the MTT assay and flow cytometry. Mitochondrial morphology, mitochondrial respiratory capacity, and ROS were measured by a mitochondrial tracker, Seahorse Analyzer, and a MitoSOX-Red dye. For in vivo PD model, behavioural tests, Nissl staining, and immunohistochemistry were used to evaluate protection by roflupram. The levels of TH, cAMP response element-binding protein (CREB), and PPARγ coactivator-1α (PGC-1α) were analysed by western blotting. KEY RESULTS: Roflupram decreased MPP+ -induced apoptosis in SH-SY5Y cells and human dopaminergic neurons. Roflupram also increased mitochondrial respiratory capacity, decreased ROS production, and restored mitochondrial morphology. Roflupram reversed the MPP+ -induced reductions of phosphorylated CREB, PGC-1α and TH. These protective effects were blocked by the PKA inhibitor H-89 or by PGC-1α siRNA. In mice treated with MPTP, roflupram significantly improved motor functions. Roflupram prevented both dopaminergic neuronal loss and the reduction of phosphorylated CREB and PGC-1α in the substantia nigra and striatum. CONCLUSION AND IMPLICATIONS: Roflupram protected dopaminergic neurons from apoptosis via the CREB/PGC-1α pathway in PD models. Hence, roflupram has potential as a protective drug in the treatment of PD.

A new polyhydroxylated oleanane triterpenoid from the roots of Rhodomyrtus tomentosa.

A new oleanane triterpenoid, 2α,3ß,6ß,23,29-pentahydroxyolean-12-en-28- oic acid (1), was isolated from the roots of Rhodomyrtus tomentosa, together with four known oleanane triterpenoids (2-5) and two known ursane triterpenoids (6-7). The structure of compound 1 was determined by extensive NMR and HR-ESI-MS data analysis. Compounds 4-5 showed cytotoxicity against PC12 cell lines at a concentration of 50 µM, and compound 1 exhibited moderate neuroprotective activity against corticosterone induced PC12 cell death at the same concentration.

Inhibition of PDE4 protects neurons against oxygen-glucose deprivation-induced endoplasmic reticulum stress through activation of the Nrf-2/HO-1 pathway.

Inhibition of phosphodiesterase 4 (PDE4) produces neuroprotective effects against cerebral ischemia. However, the involved mechanism remains unclear. Augmentation of endoplasmic reticulum (ER) stress promotes neuronal apoptosis, and excessive oxidative stress is an inducer of ER stress. The present study aimed to determine whether suppression of ER stress is involved in the protective effects of PDE4 inhibition against cerebral ischemia. We found that exposing HT-22 cells to oxygen-glucose deprivation (OGD) significantly activated ER stress, as evidenced by increased expression of the 78-kDa glucose-regulated protein (GRP78), phosphorylated eukaryotic translation-initiation factor 2α (eIF2α), and C/EBP-homologous protein (CHOP). Overexpression of PDE4B increased ER stress, while knocking down PDE4B or treatment with the PDE4 inhibitor, FCPR03, prevented OGD-induced ER stress in HT-22 cells. Furthermore, FCPR03 promoted the translocation of nuclear factor erythroid 2-related factor 2 (Nrf-2) from the cytoplasm to the nucleus. Importantly, the Nrf-2 inhibitor, ML385, blocked the inhibitory role of FCPR03 on OGD-induced ER stress. ML385 also abolished the protective role of FCPR03 in HT-22 cells subjected to OGD. Knocking down heme oxygenase-1 (HO-1), which is a target of Nrf-2, also blocked the protective role of FCPR03, enhanced the level of reactive oxygen species (ROS), and increased ER stress and cell death. We then found that FCPR03 or the antioxidant, N-Acetyl-l-cysteine, reduced oxidative stress in cells exposed to OGD. This effect was accompanied by increased cell viability and decreased ER stress. In primary cultured neurons, we found that FCPR03 reduced OGD-induced production of ROS and phosphorylation of eIF2α. The neuroprotective effect of FCPR03 against OGD in neurons was blocked by ML385. These results demonstrate that inhibition of PDE4 activates Nrf-2/HO-1, attenuates the production of ROS, and thereby attenuates ER stress in neurons exposed to OGD. Additionally, we conclude that FCPR03 may represent a promising therapeutic agent for the treatment of ER stress-related disorders.

Inhibition of PDE4 Attenuates TNF-α-Triggered Cell Death Through Suppressing NF-κB and JNK Activation in HT-22 Neuronal Cells.

Tumor necrosis factor-α (TNF-α) is a critical pro-inflammatory cytokine regulating neuroinflammation. At high concentrations, it is toxic to neurons, and such damage is positively correlated with acute and chronic neurological diseases. Our previous studies showed that inhibition of phosphodiesterase 4 (PDE4) attenuated the production of TNF-α induced by lipopolysaccharides in microglial cells. However, whether PDE4 inhibition can block the neurotoxic effects of TNF-α in neuronal cells is unknown. In this study, we investigated the protective effects of FCPR16, a novel PDE4 inhibitor, against TNF-α-induced cellular apoptosis in HT-22 hippocampal neuronal cells. We demonstrated that FCPR16 dose-dependently increased the viability of HT-22 cells exposed to TNF-α insult. Propidium iodide/calcein staining and flow cytometry analysis showed that FCPR16 decreased cell apoptosis triggered by TNF-α. Western blot analysis showed that FCPR16 decreased the level of cleaved caspase 3 and caspase 8, but had no effect on caspase 9. Mechanistically, FCPR16 blocked the TNF-α-induced phosphorylation of c-Jun N-terminal kinase (JNK) in HT-22 cells, and inhibition of JNK showed a similar protective effect as FCPR16. Furthermore, FCPR16 decreased the translocation of nuclear factor-κB (NF-κB) p65 from the cytosol into the nucleus. In addition, FCPR16 decreased the expression of inducible nitric oxide synthase and the production of reactive oxygen species in HT-22 cells exposed to TNF-α. Moreover, knockdown of PDE4B by specific small interfering RNA reduced the apoptosis of HT-22 cells treated with TNF-α. Taken together, our findings suggest that FCPR16 promotes the survival of neuronal cells exposed to TNF-α by suppressing the activation of JNK and NF-κB.

Copper Regulates the Susceptibility of Zebrafish Larvae to Inflammatory Stimuli by Controlling Neutrophil/Macrophage Survival.

Copper has been revealed to negatively affect the hematopoietic system, which has an important function in immune pathogen defense, but little is known about the potential mechanism. In this study, copper-stressed larvae exhibited significantly increased mortality as well as reduced percentages of GFP-labeled macrophages and neutrophils after Aeromonas hydrophila (A. hydrophila) infection. However, those copper-stressed GFP-labeled macrophages and neutrophils showed more rapid responses to A. hydrophila infection. The transcriptional profiles in copper-stressed macrophages or neutrophils were unveiled by RNA-Sequencing, and KEGG pathway analysis revealed enrichment of differentially expressed genes (DEGs) in lysosome, apoptosis, oxidative phosphorylation, phagosome, etc. The copper-stressed macrophages or neutrophils were revealed to have an increase in reactive oxygen species (ROS) and mitochondria ROS (mROS)-mediated apoptosis, and a reduction in phagocytosis. Furthermore, the A. hydrophila-infected copper-stressed macrophages or neutrophils were found to be unable to maintain a consistently increased expression in immune responsive genes. This study demonstrated for the first time that copper might induce the susceptibility of fish larvae to inflammatory stimuli via triggering macrophage or neutrophil apoptosis, leading to reduced phagocytic activities and non-sustainable immune responses in immune macrophages or neutrophils.

Discovery of arylbenzylamines as PDE4 inhibitors with potential neuroprotective effect.

Growing evidence confirms the potential of PDE4 inhibitors for the treatment of Parkinson's disease. Our reported PDE4 inhibitors FCPR16 and FCPR03 have displayed neuroprotective effects in SH-SY5Y cells, but have very low oral bioavailability. To access analogues with improved bioavailability, a new series of arylbenzylamine derivatives were designed and synthesized. Preliminary screening results of the series showed that arylbenzylamine derivatives bearing a pyridin-3-amine side chain displayed good inhibitory activities against human PDE4B1 and PDE4D7 isoforms. Moreover, kinetic studies revealed that the most potent compounds 11r and 11s with mid-nanomolar IC50 values partially bind to PDE4B1 (Imax = 93% and 90% respectively). Molecular docking results revealed the possible interactions of compounds 11r and 11s with upstream conserved region 2 (UCR2) of PDE4B1, which illuminate possible reasons for their partial inhibition against PDE4. Using a cell-based model of PD, compounds 11r and 11s were found to alleviate cellular apoptosis in SH-SY5Y cells induced by MPP+ (1-methyl-4-phenylpyridinium), with this neuroprotective effect being greater than PDE4 inhibitor rolipram. Furthermore, compound 11r displayed nearly sevenfold oral bioavailability (8.20%) than FCPR03 (1.23%).