phospho-cAMP Response Element Binding Research Articles

Phoenixin-20 ameliorates Sevoflurane inhalation-induced post-operative cognitive dysfunction in rats via activation of the PKA/CREB signaling.

Post-operative cognitive dysfunction (POCD) is a common complication after surgery due to the usage of anesthetics, such as Sevoflurane, which severely impacts the life quality of patients. Currently, the pathogenesis of Sevoflurane-induced POCD has not been fully elucidated but is reportedly involved with oxidative stress (OS) injury and aggravated inflammation. Phoenixin-20 (PNX-20) is a PNX peptide consisting of 20 amino acids with promising inhibitory effects on OS and inflammation. Herein, we proposed to explore the potential protective function of PNX-20 on Sevoflurane inhalation-induced POCD in rats. Sprague-Dawley (SD) rats were treated with 100 ng/g PNX-20 for 7 days with or without pre-inhalation with 2.2% Sevoflurane. Markedly increased escape latency and decreased time in the target quadrant in the Morris water maze (MWM) test, and aggravated pathological changes and apoptosis in the hippocampus tissue were observed in Sevoflurane-treated rats, which were markedly attenuated by PNX-20. Furthermore, the aggravated inflammation and OS in the hippocampus observed in Sevoflurane-treated rats were notably abolished by PNX-20. Moreover, the brain-derived neurotrophic factor (BDNF), protein kinase A (PKA), and phospho-cAMP response element binding protein/cAMP response element binding protein (p-CREB/CREB) levels were markedly decreased in Sevoflurane-treated rats, which were memorably increased by PNX-20. Our results indicated that PNX-20 ameliorated Sevoflurane inhalation-induced POCD in rats via the activation of PKA/CREB signaling, which might supply a new treatment approach for POCD.

VEGF-A ameliorates ischemia hippocampal neural injury via regulating autophagy and Akt/CREB signaling in a rat model of chronic cerebral hypoperfusion

ObjectiveChronic cerebral hypoperfusion (CCH) can cause a series of pathophysiological processes, including neuronal autophagy and apoptosis. VEGF-A has been reported to affect angiogenesis and neurogenesis in many CNS diseases. However, its effects on neuronal autophagy and apoptosis, as well as the underlying mechanisms in CCH remain unclear. MethodsTo address these issues, the CCH model was established by permanent bilateral common carotid artery occlusion (2VO). Rats were sacrificed at different stages of CCH. Hippocampal morphological and ultrastructural changes were detected using HE staining and electron microscopy. The immunoreactivities of microtubule-associated protein 1 light chain 3 (LC3) and phospho-cAMP response element binding protein (p-CREB) were examined by immunofluorescence staining. The neuronal apoptosis was detected via TUNEL staining. The levels of LC3-II, Beclin-1, Akt, p-Akt, CREB, p-CREB, Caspase-3, and Bad were accessed by Western blotting. Furthermore, mouse hippocampal HT22 neurons received the oxygen and glucose deprivation (OGD) treatment, VEGF-A treatment, and GSK690693 (an Akt inhibitor) treatment, respectively. ResultsLC3-II protein started to increase at 3 days of CCH, peaked at 4 weeks of CCH, then decreased. CCH increased the levels of LC3-II, Caspase-3, and Bad, and decreased the levels of p-Akt, CREB, and p-CREB, which were reversed by VEGF-A treatment. VEGF-A also improved CCH-induced neuronal ultrastructural injuries and apoptosis in the hippocampus in vitro. In HT22, the anti-apoptosis and pro-phosphorylation of VEGF-A were reversed by GSK690693. ConclusionPresent results provide a novel neuroprotective effect of VEGF-A in CCH that is related to the inhibition of neuronal autophagy and activation of the Akt/CREB signaling, suggesting a potential therapeutic strategy for ischemic brain damage.

Acupuncture attenuates comorbid anxiety- and depressive-like behaviors of atopic dermatitis through modulating neuroadaptation in the brain reward circuit in mice

Atopic dermatitis (AD) is highly comorbid with negative emotions such as anxiety and depression. Although acupuncture has demonstrated efficacy in AD, its influence on comorbid anxiety and depression remains unclear. We sought to explore the impact and mechanisms of action of acupuncture on comorbid anxiety and depression of AD. AD-like skin lesions were induced by the topical application of MC903 to the mouse cheek. Acupuncture was performed at Gok-Ji (LI11) acupoints. AD-like phenotypes were quantified by lesion scores, scratching behavior, and histopathological changes. The effects of acupuncture on comorbid anxiety and depression-like behaviors were assessed using the elevated plus-maze (EPM), open-field tests (OFT), and tail-suspension test (TST). In addition, biochemical changes in the brain reward regions were investigated by immunoblotting for the expression of tyrosine hydroxylase (TH), dopamine D1 receptor (D1R), phospho-dopamine and cAMP-regulated phosphoprotein-32 kDa (pDARPP-32), phospho-cAMP response element binding protein (pCREB), ΔFosB, and brain-derived neurotrophic factor (BDNF) in the nucleus accumbens, dorsolateral striatum, and ventral tegmental area. Acupuncture effectively improved the chronic itching and robust AD-like skin lesions with epidermal thickening. Additionally, it considerably reduced comorbid anxiety- and depression-like symptoms, as indicated by more time spent in the open arms of the EPM and in the center of the open field and less time spent immobile in the TST. Higher pCREB, ΔFosB, BDNF, and pDARPP-32 levels, and reduced TH and D1R protein expression in the brain reward regions of AD mice were reversed by acupuncture treatment. The beneficial effects of acupuncture on clinical symptoms (scratching behavior) and comorbid psychological distress in AD strongly correlated with dorsal striatal ΔFosB levels. Collectively, these data indicate that acupuncture had a significant, positive impact on comorbid anxiety- and depression-like behaviors by modulating neuroadaptation in the brain reward circuit in mice with AD, providing a novel perspective for the non-pharmacological management of psychiatric comorbidities of AD.

CCR5 Activation Promotes NLRP1-Dependent Neuronal Pyroptosis via CCR5/PKA/CREB Pathway After Intracerebral Hemorrhage.

Neuronal pyroptosis is a type of regulated cell death triggered by proinflammatory signals. CCR5 (C-C chemokine receptor 5)-mediated inflammation is involved in the pathology of various neurological diseases. This study investigated the impact of CCR5 activation on neuronal pyroptosis and the underlying mechanism involving cAMP-dependent PKA (protein kinase A)/CREB (cAMP response element binding)/NLRP1 (nucleotide-binding domain leucine-rich repeat pyrin domain containing 1) pathway after experimental intracerebral hemorrhage (ICH). A total of 194 adult male CD1 mice were used. ICH was induced by autologous whole blood injection. Maraviroc (MVC)-a selective antagonist of CCR5-was administered intranasally 1 hour after ICH. To elucidate the underlying mechanism, a specific CREB inhibitor, 666-15, was administered intracerebroventricularly before MVC administration in ICH mice. In a set of naive mice, rCCL5 (recombinant chemokine ligand 5) and selective PKA activator, 8-Bromo-cAMP, were administered intracerebroventricularly. Short- and long-term neurobehavioral assessments, Western blot, Fluoro-Jade C, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and immunofluorescence staining were performed. The brain expression of CCL5 (chemokine ligand 5), CCR5, PKA-Cα (protein kinase A-Cα), p-CREB (phospho-cAMP response element binding), and NLRP1 was increased, peaking at 24 hours after ICH. CCR5 was expressed on neurons, microglia, and astrocytes. MVC improved the short- and long-term neurobehavioral deficits and decreased neuronal pyroptosis in ipsilateral brain tissues at 24 hours after ICH, which were accompanied by increased PKA-Cα and p-CREB expression, and decreased expression of NLRP1, ASC (apoptosis-associated speck-like protein containing a CARD), C-caspase-1, GSDMD (gasdermin D), and IL (interleukin)-1β/IL-18. Such effects of MVC were abolished by 666-15. At 24 hours after injection in naive mice, rCCL5 induced neurological deficits, decreased PKA-Cα and p-CREB expression in the brain, and upregulated NLRP1, ASC, C-caspase-1, N-GSDMD, and IL-1β/IL-18 expression. Those effects of rCCL5 were reversed by 8-Bromo-cAMP. CCR5 activation promoted neuronal pyroptosis and neurological deficits after ICH in mice, partially through the CCR5/PKA/CREB/NLRP1 signaling pathway. CCR5 inhibition with MVC may provide a promising therapeutic approach in managing patients with ICH.

Heat stress induced oxidative damage and perturbation in BDNF/ERK1/2/CREB axis in hippocampus impairs spatial memory

Heat exposure is an environmental stress that causes diverse heat related pathophysiological changes under extreme conditions. The brain including hippocampal region which is associated with learning and memory is significantly affected by heat stress resulting in memory impairment. However, the effect of heat on the spatial memory remains unclear. The present study aimed to explore the effect of heat stress on hippocampus and spatial memory in rats. Rat model of acute heat stress was used which was divided into two groups, viz. moderate heat stress (MHS) and severe heat stress (SHS). Redox parameters evaluation revealed that MHS and SHS exposure markedly increase the production of malondialdehyde (MDA), oxidised glutathione (GSSG), reactive oxidative species (ROS), protein oxidation level and decrease the reduced glutathione (GSH) levels in the hippocampal tissue. Furthermore, Cresyl Violet (CV) staining of hippocampal region showed higher pyknosis in rats exposed to SHS. Pronounced increase of caspase3 expression and Fluoro Jade-C (FJ-C) positive cells were observed in SHS resulting in neuronal injury and apoptosis in CA3 region of hippocampus culminating in spatial memory deficit. Our data also suggest that heat stress induces phospho Extracellular signal-regulated kinases (pERK)1/2 activation induced by Brain-derived neurotrophic factor (BDNF) leading to further activation of phospho cAMP-response element binding protein (pCREB) under MHS. However, during SHS, BDNF and pCREB expression were completely dysregulated and not sufficient to rescue cognitive decline in rats. In conclusion, SHS induces pathological alterations that include oxidative damage and apoptosis of hippocampal neurons, disturbing BDNF/ERK1/2/CREB axis that may affect spatial memory.

Cognitive enhancing effect of diapocynin in D-galactose-ovariectomy-induced Alzheimer's-like disease in rats: Role of ERK, GSK-3β, and JNK signaling

NADPH oxidase (NOX) has been identified as a crucial contender of oxidative damage in Alzheimer's disease (AD). However, the capability of diapocynin, a NOX inhibitor, to offer neuroprotection in AD models is still a matter of debate. Hence, the current work is dedicated to investigate the influence of diapocynin on cognitive impairment prompted by ovariectomy combined with D-galactose injection in rats (an AD animal model), and to elucidate the signaling mechanisms regulating diapocynin-induced effects. Female rats were exposed to ovariectomy or sham operation. Ovariectomized rats were injected intraperitoneally with D-galactose (150 mg/kg/day) for 70 days and, on day 43, they were orally treated with diapocynin (10 mg/kg/day) for 28 days. Diapocynin amended cognitive functions as confirmed using novel object recognition and Morris water maze tests along with histopathological improvement. It caused a prominent decrement in β-secretase, p-tau, and amyloid β, contrary to α-secretase elevation in hippocampus and hampered neuroinflammation and oxidative stress, manifested by declined levels of NOX1, tumor necrosis factor-α, and nuclear factor-kappa B p65. In addition, diapocynin augmented synaptophysin, brain-derived neurotrophic factor, and phospho-cAMP response element binding protein and enhanced protein expression of phosphorylated forms of phosphoinositide 3-kinase (PI3K), glycogen synthase kinase-3β (GSK-3β), protein kinase B (Akt), extracellular signal-regulated kinase (ERK) 1/2, ERK kinase kinase (Raf-1), and ERK kinase (MEK) 1/2, while inhibiting those of c-Jun and c-Jun N-terminal kinase (JNK). In conclusion, diapocynin attenuated memory impairment and AD-like anomalies via activating Raf-1/MEK/ERK and PI3K/Akt/GSK-3β, while inhibiting JNK/c-Jun signaling cascades.

Sex-Based Differences in the Cytokine Production and Intracellular Signaling Pathways in Patients With Rheumatoid Arthritis.

Objectives This study aims to investigate lymphoproliferation, cytokine production, and intracellular signaling molecules in peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals and rheumatoid arthritis (RA) patients to understand the extent of the involvement of these pathways in the pathogenesis of RA.Patients and methods The study included 65 participants (29 males, 36 females; mean age 51.8±10.3 years; range, 37 to 71 years) who were categorized into four groups as healthy males (n=22, mean age 49.8±10.6 years; range, 39 to 65 years), male RA patients (n=7, mean age 51.8±13.9 years; range, 37 to 68 years), healthy females (n=20, mean age 53.7±8.8 years; range, 42 to 67 years), and female RA patients (n=16, mean age 52.9±10.4 years; range, 40 to 71 years). PBMCs were collected from the participants and analyzed for Concanavalin A (Con A)-induced lymphoproliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cytokine production, and phospho-signal transducer and activator of transcription 3 (p-STAT-3), phospho-extracellular-signal-regulated kinase (p-ERK), phospho-cAMP response element binding (p-CREB), and phospho-protein kinase B expressions using enzyme-linked immunosorbent assay. Short form of the Arthritis Impact Measurement Scales 2 and multidimensional health assessment questionnaire were used to measure the level of disability and the quality of life.Results In RA patients, production of Con A-induced interleukin (IL)-2 and IL-17 was higher in both sexes while interferon-gamma levels decreased in RA females alone. Expression of p-STAT-3 in PBMCs increased in RA males while it was unaltered in RA females. p-ERK expression was not altered while p-CREB expression was enhanced in RA males and females. Protein-protein interaction analyses demonstrated that these and other key signaling molecules were dysregulated in RA patients.Conclusion Our results suggest that sex-based differences in RA pathogenesis result from differential alterations in signaling pathways to influence the inflammatory process.

Escitalopram Ameliorates Cognitive Impairment in D-Galactose-Injected Ovariectomized Rats: Modulation of JNK, GSK-3\u03b2, and ERK Signalling Pathways

Though selective serotonin reuptake inhibitors (SSRIs) have been found to increase cognitive performance in some studies on patients and animal models of Alzheimer’s disease (AD), other studies have reported contradictory results, and the mechanism of action has not been fully described. This study aimed to examine the effect of escitalopram, an SSRI, in an experimental model of AD and to determine the involved intracellular signalling pathways. Ovariectomized rats were administered D-galactose (150 mg/kg/day, i.p) over ten weeks to induce AD. Treatment with escitalopram (10 mg/kg/day, p.o) for four weeks, starting from the 7th week of D-galactose injection, enhanced memory performance and attenuated associated histopathological changes. Escitalopram reduced hippocampal amyloid β 42, β-secretase, and p-tau, while increasing α-secretase levels. Furthermore, it decreased tumor necrosis factor-α, nuclear factor-kappa B p65, and NADPH oxidase, while enhancing brain-derived neurotrophic factor, phospho-cAMP response element binding protein, and synaptophysin levels. Moreover, escitalopram diminished the protein expression of the phosphorylated forms of c-Jun N-terminal kinase (JNK)/c-Jun, while increasing those of phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), glycogen synthase kinase-3β (GSK-3β), extracellular signal-regulated kinase (ERK) and its upstream kinases MEK and Raf-1. In conclusion, escitalopram ameliorated D-galactose/ovariectomy-induced AD-like features through modulation of PI3K/Akt/GSK-3β, Raf-1/MEK/ERK, and JNK/c-Jun pathways.

Combined bone marrow stromal cells and oxiracetam treatments ameliorates acute cerebral ischemia/reperfusion injury through TRPC6.

Ischemic stroke has become one of the leading causes of deaths and disabilities all over the world. In this study, we investigated the therapeutic effects of combined bone marrow stromal cells (BMSCs) and oxiracetam treatments on acute cerebral ischemia/reperfusion (I/R) injury. A rat model of middle cerebral artery occlusion (MCAO) followed by complete reperfusion, as well as a cortex neuron oxygen-glucose deprivation (OGD) model was established. When compared with BMSCs or oxiracetam monotherapy, combination therapy significantly improved functional restoration with decreased infarct volume in observed ischemic brain. We propose that it may occur through the transient receptor potential canonical (TRPC)6 neuron survival pathway. The increased expression of TRPC6 along with the reduction of neuronal cell death in the OGD cortex neurons and combination therapy group indicated that the TRPC6 neuron survival pathway plays an important role in the combined BMSCs and oxiracetam treatments. We further tested the activity of the calpain proteolytic system, and the results suggested that oxiracetam could protect the integrity of TRPC6 neuron survival pathway by inhibiting TRPC6 degradation. The protein levels of phospho-cAMP response element binding protein (p-CREB) were tested. It was found that BMSCs play a role in the activation of the TRPC6 pathway. Our study suggests that the TRPC6 neuron survival pathway plays a significant role in the protective effect of combined BMSCs and oxiracetam treatments on acute cerebral I/R injury. Combined therapy could inhibit the abnormal degradation of TRPC6 via decreasing the activity of calpain and increasing the activation of TRPC6 neuron survival pathway.

Multidrug resistance protein 4 (Mrp4) regulates nutrient and energy metabolism by altering the adipose tissue physiology

Multidrug resistance protein 4 (Mrp4; encoded by Abcc4) belongs to the ATP‐binding cassette efflux transporter superfamily and transports several xenobiotics and endogenous molecules, such as prostaglandins and cyclic nucleotides. Mrp4 is highly expressed in the kidney and bladder, with minimal expression detected in the liver. We recently determined that the lack of Mrp4 increases adipose tissue and body weights in mice. Interestingly, the expression of Mrp4 and its role in adipose tissue physiology, nutrient metabolism and the pathogenesis of metabolic diseases are unknown. The current study was aimed at characterizing these specific roles of Mrp4 employing wildtype (WT) and knockout (Mrp4−/−) mice. Our studies determined that Mrp4 is expressed in high levels in adipose tissue and that the lack Mrp4 resulted in adipocyte hypertrophy. Along with increases in body and adipose tissue weights, lack of Mrp4 in mice also resulted in increased blood glucose and leptin levels, and impaired glucose tolerance. Additionally, Mrp4−/− mice have increased levels of adipose tissue phospho‐cAMP response element binding protein (p‐Creb), which is known to regulate adipocyte glucose and lipid metabolism. In contrast to altered glucose metabolism, Mrp4−/− mice did not show any significant changes in plasma triglycerides, free fatty acids or total cholesterol levels compared to WT mice. Indirect calorimetry measurements under basal conditions showed that Mrp4−/− mice have decreased volumetric oxygen consumption (VO2), respiratory exchange ratio (RER), energy expenditure (EE), ambulatory activity (AA) and wheel counts compared to WT mice. Decreased indirect calorimetry parameters and impaired glucose metabolism in Mrp4−/− mice can be attributed to increased adipose tissue weights, which complements the observed obese and diabetic phenotype in these mice. Together, our data indicate that Mrp4 regulates nutrient and energy metabolism by altering the adipose tissue physiology. In conclusion, the absence of Mrp4 function in mice results in the development of obesity and diabetes.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The memory-enhancing effects of 7,8,4’-trihydroxyisoflavone, a major metabolite of daidzein, are associated with activation of the cholinergic system and BDNF signaling pathway in mice

Daidzein is one of the dietary isoflavones present in soybean-based products. After ingestion, daidzein is bioconverted into its major metabolite, 7,8,4’-trihydroxyisoflavone (THIF). Given the pharmacological importance of daidzein, 7,8,4’-THIF has also attracted the interest of researchers. However, there are no reports on the effects of 7,8,4’-THIF on cognition and memory with regard to the cholinergic system. Therefore, this study sought to evaluate the memory-enhancing effects of 7,8,4’-THIF in mice. Treatment with 7,8,4’-THIF ameliorated the cognitive impairments induced by scopolamine, a muscarinic acetylcholine receptor antagonist, in the Y-maze and passive avoidance tests. Interestingly, 7,8,4’-THIF treatment also improved cognitive function in normal mice. This treatment was also able to reverse acetylcholinesterase (AChE) and thiobarbituric acid reactive substance (TBARS) activities in the hippocampus. Finally, 7,8,4’-THIF significantly increased the expression levels of the following molecules in the hippocampus: brain-derived neurotrophic factor (BDNF); phospho extracellular signal-regulated kinase (ERK); phospho cAMP response element binding (CREB); and choline acetyltransferase (ChAT). Our data suggest that 7,8,4’-THIF, a metabolized product of daidzein, improves cognitive function by activating the cholinergic system and the BDNF/ERK/CREB signaling pathway in mice.

Anti-melanogenic activity of phytosphingosine via the modulation of the microphthalmia-associated transcription factor signaling pathway

BackgroundMicrophthalmia-associated transcription factor (MITF) suppresses the expression of enzymes controlling the production of melanin. Phytosphingosine is a well-known cosmetic agent, but its anti-melanogenic activity and mechanism of action remain unclear. ObjectiveThis study was designed to investigate the effects of phytosphingosine on melanin synthesis and elucidate the plausible mechanism of actions in vitro and ex vivo systems. MethodsMelanin content, cell viability, tyrosinase activity, p-CREB DNA binding activity, and the protein gene expression levels of the enzymes and proteins involved in melanogenesis were measured with the treatment of phytosphingosine. ResultsPhytosphingosine inhibits melanin synthesis in cultured melan-a cells and a reconstructed human skin model. One possible mechanism of the anti-melanogenic activity of phytosphingosine appears to be associated with the modulation of MITF, which suppresses the expression of tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2. Further analysis revealed that phytosphingosine suppressed paired box 3 and SRY-related HMG-box 10, critical transcription factors of MITF. Phytosphingosine also effectively downregulated the protein levels of β-catenin and the phospho-cAMP response element binding protein, an upstream regulatory factor of MITF. These results are closely related to the suppression of MITF gene expression. In addition, treatment with phytosphingosine for over 12h, which is a relatively long period of time, did not directly suppress these MITF transcriptional factors. Instead, phytosphingosine induced ERK activation, which led to MITF phosphorylation, followed by its degradation. Therefore, the downregulation of MITF protein levels by phytosphingosine with a long time exposure is in part associated with MITF protein degradation through the MAPK kinase activation pathway. ConclusionThe modulation of MITF by phytosphingosine is closely related with the signaling pathways, such as the suppression of the MITF gene expression and the degradation of the MITF protein, depending on the duration of treatment time. These results suggest that phytosphingosine might serve as an effective melanogenesis inhibitor in melanocytes via the regulation of the MITF signaling pathways.

Effects of β-sheet breaker peptide H102 on synaptic plasticity associated proteins in double transgenic AD mice

To investigate the effects ofβ-sheet breaker peptide H102 on expression of synaptic plasticity associated proteins and learning and memory functions in double transgenic Alzheimer's disease(AD) mice,and to discuss its mechanisms. Thirty APP-swe/PS1dE9 double transgenic male mice of 8 weeks were randomly divided into model group and H102 treatment group (15 mice per group). In addition,a group of C57BL/6J mice with the same age and background was set as normal. H102 (5.8 mg/kg) 5 μl was infused by in-tranasal administration to mice in H102 treatment group,and equal volume of blank solution of H102 (chitosan,BSA) was given to mice in con-trol group and model group. The ability of spatial reference memory was tested by Morris Water Maze after 16 weeks treatment,then immunohis-tochemistry tests and Western blot technique were used to detect the content ofβ-amyloid peptide(Aβ1-42) protein and phospho protein kinase C α、β2、γ(p-PKCα, p-PKCβ2, p-PKCγ), phospho-N-methyl-D-aspartate receptor1(p-NMDAR1), phospho-Calcium/Calmodulin dependent pro-tein kinaseⅡα(p-CaMKⅡα) and phospho-cAMP response element binding protein(p-CREB) of synaptic plasticity associated proteins in mice brain. The ability of learning and memory was significantly improved in H102 treatment group than that in model group by the test of Morris Water Maze. The contents of Aβ1-42 proteins and p-PKCα, p-PKCβ2, p-PKCγ, p-NMDAR1, p-CaMKⅡαand p-CREB of synaptic plas-ticity associated proteins in mice brain were improved significantly in H102 treatment group than those in model group by the test of immunohis-tochemistry tests and Western blot technique. β-sheet breaker peptide H102 can significantly improve synaptic plasticity and the ability of learning and memory in double transgenic AD mice.

N-(4-bromophenethyl) Caffeamide Inhibits Melanogenesis by Regulating AKT/Glycogen Synthase Kinase 3 Beta/Microphthalmia-associated Transcription Factor and Tyrosinase-related Protein 1/Tyrosinase.

Skin color is primarily produced by melanin, which is a crucial pigment that protects the skin from UV-induced damage and prevents carcinogenesis. However, accumulated melanin in the skin may cause hyperpigmentation and related disorders. Melanin synthesis comprises consecutive oxidative reactions, and tyrosinase is the enzyme that catalyzes the rate-limiting process of melanogenesis. In this study, tyrosinase-related protein 1 (TRP-1) and TRP-2 contributed to melanin formation. N-(4-bromophenethyl) caffeamide ((E)-N-(4-bromophenethyl)-3-(3,4-dihydroxyphenyl)acrylamide; K36H), a caffeic acid phenyl amide derivative, inhibited α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis and tyrosinase activity in B16F0 cells. In addition, K36H reduced the protein expression of the phospho-cAMP response element binding protein (p-CREB), microphthalmia-associated transcription factor (MITF), tyrosinase, and TRP-1. Moreover, K36H promoted AKT and glycogen synthase kinase 3 beta (GSK3β) phosphorylation, thereby inhibiting MITF transcription activity. Thus, K36H attenuated α-MSH-induced cAMP pathways, contributing to hypopigmentation. The results of a safety assay revealed that K36H did not exhibit cytotoxicity or irritate the skin or eyes. According to these results, K36H may have the potential to be used as a whitening agent in the cosmetic and pharmaceutical industries.

Co-Treatment with Anthocyanins and Vitamin C Ameliorates Ethanol- Induced Neurodegeneration via Modulation of GABAB Receptor Signaling in the Adult Rat Brain.

Chronic ethanol exposure is known to cause neuronal damage in both humans and experimental animal models. Ethanol treatment induces neurotoxicity via the generation of reactive oxygen species (ROS), while anthocyanins (extracted from black soybean) and ascorbic acid (vitamin C) are free radical scavengers that can be used as neuroprotective agents against ROS. In this study the underlying neuroprotective potential of black soybean anthocyanins and vitamin C was determined. For this purpose, adult rats were exposed to 10% (v/v) ethanol for 8 weeks, followed by co-treatment with anthocyanins (24 mg/kg) and vitamin C (100 mg/kg) during the last 4 weeks. Our results showed that ethanol administration increased the expression of γ -aminobutyric acid B1 receptor (GABAB1R) and induced neuronal apoptosis via alterations to the Bax/Bcl-2 ratio, release of cytochrome C and activation of caspase-3 and caspase-9. Anthocyanins alone and supplementation with vitamin C showed an additive effect in reversing the trend of apoptotic signals induced by ethanol in the cortex and hippocampus. Consequently, anthocyanins also decreased the expression of poly (ADP ribose) polymerase-1 induced by ethanol and prevented DNA damage. Furthermore, anthocyanins and vitamin C reversed the ethanol-induced expression of GABAB1R and its downstream signaling molecule phospho-cAMP response element binding protein. Moreover, histopathology and immunohistochemistry results showed that anthocyanins and vitamin C significantly reduced ethanol-induced neuronal cell death. Our study revealed a neuroprotective role of anthocyanins and vitamin C via modulation of GABAB1R expression in the adult brain. Hence, we suggest that anthocyanins or co-treatment with anthocyanins and vitamin C may be a new and potentially effective neuroprotective agent for alcohol abuse.

Sex differences in the adult HPA axis and affective behaviors are altered by perinatal exposure to a low dose of bisphenol A

Bisphenol A (BPA), an estrogen-mimicking endocrine disrupter, when administered perinatally can affect affective behaviors in adult rodents, however the underlying mechanisms remain largely unclear. Postnatal day (PND) 80 vehicle-injected control female rats showed more obvious depression- and anxiety-like behaviors than males, indicative of sexually dimorphic affective behaviors. When female breeders were subcutaneously injected with BPA (2µg/kg) from gestation day 10 to lactation day 7, sex difference of affective behaviors was impaired in their offspring (PND80 BPA-rats), as results that female BPA-rats showed a visible “antianxiety-like” behavior, and male BPA-rats increased depression-like behavior compared to vehicle-injected controls. Notably, basal levels of serum corticosterone and adrenocorticotropin (ACTH), and corticotropin-releasing hormone mRNA were increased in male BPA-rats, but not in female BPA-rats, in comparison with vehicle-injected controls. Following mild-stressor the elevation of corticosterone or ACTH levels was higher in male BPA-rats, whereas it was lower in female BPA-rats than vehicle-injected controls. In comparison with vehicle-injected controls, the level of glucocorticoid receptor (GR) mRNA in hippocampus or hypothalamic paraventricular nucleus was increased in female BPA-rats, while decreased in male BPA-rats. In addition, the levels of hippocampal mineralocorticoid receptor (MR) mRNA, neuronal nitric oxide synthase (nNOS) and phospho-cAMP response element binding protein (p-CREB) were increased in female BPA-rats, but were decreased in male BPA-rats. Furthermore, the testosterone level was reduced in male BPA-rats. The results indicate that the perinatal exposure to BPA through altering the GR and MR expression disrupts the GR-mediated feedback of hypothalamic–pituitary–adrenal (HPA) axis and MR-induced nNOS–CREB signaling, which alters sex difference in affective behaviors.

Nardosinone Improves the Proliferation, Migration and Selective Differentiation of Mouse Embryonic Neural Stem Cells

In this study, we investigated the impact of Nardosinone, a bioactive component in Nardostachys root, on the proliferation and differentiation of neural stem cells. The neural stem cells were isolated from cerebrums of embryonic day 14 CD1 mice. The proliferation of cells was monitored using the cell counting kit-8 assay, bromodeoxyuridine incorporation and cell cycle analysis. Cell migration and differentiation were investigated with the neurosphere assay and cell specific markers, respectively. The results showed that Nardosinone promotes cells proliferation and increases cells migration distance in a dose-dependent manner. Nardosinone also induces the selective differentiation of neural stem cells to neurons and oligodendrocytes, as indicated by the expression of microtubule-associated protein-2 and myelin basic protein, respectively. Nardosinone also increases the expression of phospho-extracellular signal-regulated kinase and phospho-cAMP response element binding protein during proliferation and differentiation. In conclusion, this study reveals the regulatory effects of Nardosinone on neural stem cells, which may have significant implications for the treatment of brain injury and neurodegenerative diseases.

Norartocarpetin from a folk medicine Artocarpus communis plays a melanogenesis inhibitor without cytotoxicity in B16F10 cell and skin irritation in mice

BackgroundMany natural products used in preventive medicine have also been developed as cosmeceutical ingredients in skin care products, such as Scutellaria baicalensis and Gardenia jasminoides. Norartocarpetin is one of the antioxidant and antityrosinase activity compound in Artocarpus communis; however, the cytotoxicity, skin irritation and antimelanogenesis mechanisms of norartocarpetin have not been investigated yet.MethodsIn the present study, cell viability in vitro and skin irritation in vivo are used to determine the safety of norartocarpetin. The melanogenesis inhibition of norartocarpetin was determined by cellular melanin content and tyrosinase in B16F10 melanoma cell. Moreover, we examined the related-melanogenesis protein by western blot analysis for elucidating the antimelanogenesis mechanism of norartocarpin.ResultsThe result of the present study demonstrated that norartocarpetin not only present non-cytotoxic in B16F10 and human fibroblast cells but also non-skin irritation in mice. Moreover, our result also first found that norartocarpetin downregulated phospho-cAMP response element-binding (phospho-CREB) and microphthalmia-associated transcription factor (MITF) expression, which in turn decreased both synthesis of tyrosinases (TRP-1 and TRP-2) and cellular melanin content. This process is dependent on norartocarpetin phosphorylation by mitogen-activated protein kinases such as phospho-JNK and phospho-p38, and it results in decreased melanogenesis.ConclusionThe present study suggests that norartocarpetin could be used as a whitening agent in medicine and/or cosmetic industry and need further clinical study.

Enrichment of Retinal Ganglion Cells in Rat Retinal Lysate by Excimer Laser Ablation of the Outer Retina

Retinal ganglion cells (RGC) are a relatively small cell population in the retina. This leads to an unfavorable signal-to-noise ratio when analyzing RGC proteins in whole retina lysate. We present a novel technique to obtain RGC-enriched rat retinal lysate by removing the outer retinal layers with an excimer laser before lysation. Outer retinal layers were ablated with an excimer laser on flat mounted retinas from adult albino rats. 4'6-Diamidino-2-phenylindole dihydrochloride hydrate (DAPI) nuclear staining was used to assess the ablation efficacy (n = 6). Western blot for layer specific markers (rhodopsin, parvalbumin, β-III-tubulin) was performed to quantify changes in protein composition (n = 7). Excimer-ablated (EX) and full-thickness (FT) retinas 48 hours after optic nerve crush (ONC) were compared regarding the effect on phospho-cAMP response element binding protein (pCREB) and Thy1 levels (n = 5). Area quantification of dapi-stained retinas showed that 73% 4.9% of the ablation area was free of photoreceptor and bipolar cell nuclei. In Western blot, laser ablation led to a significant reduction of the photoreceptor marker rhodopsin and increase of the ganglion cell layer (GCL) marker -iiitubulin (relative quantity: rhodopsin 0.47 ± 0.05, P < 0.0001; β-III-tubulin 2.35 ± 0.37, P = 0.02). Changes of pCREB and Thy1 after ONC were significantly different between FT and EX retinas (relative quantity pCREB: FT 1.4 ± 0.16, EX 0.78 ± 0.07, P = 0.008; Thy1: FT 0.95 ± 0.02, EX 0.63 ± 0.07, P = 0.006). We demonstrated that excimer laser ablation of outer retinal layers is feasible, producing RGC-enriched retinal lysate. Laser ablation may allow a more specific detection of RGC responses to experimental stimuli.

Acamprosate Suppresses Ethanol-Induced Place Preference in Mice With Ethanol Physical Dependence

The present study investigated the effect of acamprosate on ethanol (EtOH)-induced place preference in mice with EtOH physical dependence. The expression of EtOH (2 g/kg, intraperitoneally)-induced place preference in mice without EtOH treatment before the experiment was dose-dependently suppressed by acamprosate. The levels of protein kinase A (PKA) and phospho-cAMP response element binding protein (p-CREB) in the limbic forebrain after EtOH-conditioning in naïve mice was unchanged. Furthermore, mice on the 4th day of withdrawal from continuous EtOH vapor inhalation for 9 days showed transient and significant enhancement of EtOH (1 g/kg, intraperitoneally)-induced place preference, which was significantly suppressed by acamprosate (300 mg/kg, oral administration; p.o., once a day) administered daily for 3 days after withdrawal from EtOH inhalation and during EtOH-conditioning. PKA and p-CREB proteins in the limbic forebrain of EtOH-conditioned mice on 4th day of withdrawal from continuous EtOH inhalation for 9 days significantly increased, which were completely abolished by acamprosate. These findings suggest that the signal transduction pathway via the PKA–p-CREB pathway in the limbic forebrain may be functionally related to the development of sensitization of EtOH-induced place preference and provide a possible molecular basis for the pharmacological effect of acamprosate to prevent or reduce the relapse of alcohol dependence.