Neuronal Differentiation and Outgrowth Effect of Thymol in Trachyspermum ammi Seed Extract via BDNF/TrkB Signaling Pathway in Prenatal Maternal Supplementation and Primary Hippocampal Culture.

Reviving the neuronal functions in neurodegenerative disorders requires the promotion of neurite outgrowth. Thymol, which is a principal component of Trachyspermum ammi seed extract (TASE), is reported to have neuroprotective effects. However, the effects of thymol and TASE on neuronal differentiation and outgrowth are yet to be studied. This study is the first report investigating the neuronal growth and maturation effects of TASE and thymol. Pregnant mice were orally supplemented with TASE (250 and 500 mg/kg), thymol (50 and 100 mg/kg), vehicle, and positive controls. The supplementation significantly upregulated the expression of brain-derived neurotrophic factor (BDNF) and early neuritogenesis markers in the pups' brains at post-natal day 1 (P1). Similarly, the BDNF level was significantly upregulated in the P12 pups' brains. Furthermore, TASE (75 and 100 µg/mL) and thymol (10 and 20 µM) enhanced the neuronal polarity, early neurite arborization, and maturation of hippocampal neurons in a dose-dependent manner in primary hippocampal cultures. The stimulatory activities of TASE and thymol on neurite extension involved TrkB signaling, as evidenced by attenuation via ANA-12 (5 µM), which is a specific TrkB inhibitor. Moreover, TASE and thymol rescued the nocodazole-induced blunted neurite extension in primary hippocampal cultures, suggesting their role as a potent microtubule stabilizing agent. These findings demonstrate the potent capacities of TASE and thymol in promoting neuronal development and reconstruction of neuronal circuitry, which are often compromised in neurodegenerative diseases and acute brain injuries.

Thymol in Trachyspermum ammi seed extract exhibits neuroprotection, learning, and memory enhancement in scopolamine-induced Alzheimer's disease mouse model.

Several reports have stated the neuroprotective and learning/memory effects of Tachyspermum ammi seed extract (TASE) and its principal component thymol; however, little is known about its underlying molecular mechanisms and neurogenesis potential. This study aimed to provide insights into TASE and a thymol-mediated multifactorial therapeutic approach in a scopolamine-induced Alzheimer's disease (AD) mouse model. TASE and thymol supplementation significantly reduced oxidative stress markers such as brain glutathione, hydrogen peroxide, and malondialdehyde in mouse whole brain homogenates. Tumor necrosis factor-alpha was significantly downregulated, whereas the elevation of brain-derived neurotrophic factor and phospho-glycogen synthase kinase-3 beta (serine 9) enhanced learning and memory in the TASE- and thymol-treated groups. A significant reduction in the accumulation of Aß 1-42 peptides was observed in the brains of TASE- and thymol-treated mice. Furthermore, TASE and thymol significantly promoted adult neurogenesis, with increased doublecortin positive neurons in the subgranular and polymorphic zones of the dentate gyrus in treated-mice. Collectively, TASE and thymol could  potentially act as natural therapeutic agents for the treatment of  neurodegenerative disorders, such as  AD.

Black Cumin (Nigella sativa L.): A Comprehensive Review on Phytochemistry, Health Benefits, Molecular Pharmacology, and Safety.

Mounting evidence support the potential benefits of functional foods or nutraceuticals for human health and diseases. Black cumin (Nigella sativa L.), a highly valued nutraceutical herb with a wide array of health benefits, has attracted growing interest from health-conscious individuals, the scientific community, and pharmaceutical industries. The pleiotropic pharmacological effects of black cumin, and its main bioactive component thymoquinone (TQ), have been manifested by their ability to attenuate oxidative stress and inflammation, and to promote immunity, cell survival, and energy metabolism, which underlie diverse health benefits, including protection against metabolic, cardiovascular, digestive, hepatic, renal, respiratory, reproductive, and neurological disorders, cancer, and so on. Furthermore, black cumin acts as an antidote, mitigating various toxicities and drug-induced side effects. Despite significant advances in pharmacological benefits, this miracle herb and its active components are still far from their clinical application. This review begins with highlighting the research trends in black cumin and revisiting phytochemical profiles. Subsequently, pharmacological attributes and health benefits of black cumin and TQ are critically reviewed. We overview molecular pharmacology to gain insight into the underlying mechanism of health benefits. Issues related to pharmacokinetic herb-drug interactions, drug delivery, and safety are also addressed. Identifying knowledge gaps, our current effort will direct future research to advance potential applications of black cumin and TQ in health and diseases.

Centella asiatica promotes early differentiation, axodendritic maturation and synaptic formation in primary hippocampal neurons.

BACKGROUND: Centella asiatica is a 'medhya-rasayana (nootrophic or memory booster)' herb that has been indicated in Ayurveda for improving memory function and treating dementia disorders. Although the neuroprotective effects of C. asiatica have been reported in earlier studies, the information on whether this nootropic herb could promote early differentiation and development of axon and dendrites in primary hippocampal neurons is currently limited. THE AIM OF THE STUDY: To investigate the effects of C. asiatica and asiatic acid, one of the principal active constituents of C. asiatica, on the various stages of neuronal polarity, including early neuronal differentiation, axonal outgrowth, dendritic arborization, axonal maturation, and synaptic formation. MATERIALS AND METHODS: Embryonic rat hippocampal neurons were incubated with C. asiatica leaf extract (CAE) or asiatic acid. After an indicated time, neurons were fixed and immunolabeled to visualize the neuronal morphology. Morphometric analyses for early neuronal differentiation, axonal and dendritic maturation and synaptogenesis were performed using Image J software. Neuronal viability was determined using trypan blue exclusion assay. RESULTS: CAE at varying concentrations ranging from 3.75 to 15 µg/mL enhanced neurite outgrowth with the highest optimal concentration of 7.5 µg/mL. The effects of CAE commenced immediately after cell seeding, as indicated by its accelerating effect on neuronal differentiation. Subsequently, CAE significantly elaborated dendritic and axonal morphology and facilitated synapse formation. Asiatic acid also facilitated neurite outgrowth, but to a lesser extent than CAE. CONCLUSION: These findings revealed that CAE exerted its modulatory effects in every stage of neuronal development, supporting its previously claimed neurotrophic function and suggest that this natural nootropic and its active component asiatic acid can be further investigated to explore a promising solution for degenerative brain disorders and injuries.

The potential LXRß agonist stigmasterol protects against hypoxia/reoxygenation injury by modulating mitophagy in primary hippocampal neurons.

BACKGROUND: Neuronal excitotoxicity induces a plethora of downstream signaling pathways, resulting in the calcium overload-induced excitotoxic cell death, a well-known phenomenon in cerebrovascular and neurodegenerative disorders. The naturally occurring phytosterol, stigmasterol (ST) is known for its potential role in cholesterol homeostasis and neuronal development. However, the ability of ST to protect against the induced excitotoxicity in hippocampal neurons has not been investigated yet. PURPOSE: The present study aimed to investigate whether ST could protect against hypoxia/reoxygenation (H/R)-induced excitotoxicity in hippocampal neurons. METHODS: After H/R, neurons were initially subjected to trypan blue exclusion assay for the assessment of cell viability. Live staining using fluorescence dyes namely JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide), DCFDA (2',7'-dichlorofluorescein diacetate) and FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) were used to measure MMP, ROS and synaptic vesicle pool size. Immunostaining was performed to analyze the expression levels of vesicular glutamate transporter 1 (VGLUT1), N-methyl-D-acetate receptor subunit 2B (GluN2B), LC3BII, p62, and PTEN induced protein kinase 1 (PINK1) in neuron after H/R. Western blotting was carried out to measure the protein expression of GluN2B. The molecular dynamics simulation was employed to elucidate the LXRß agonistic conformation of ST. RESULT: Pre-incubation of neuronal cultures with ST (20 µM) protected against excitotoxicity, and attenuated reactive oxygen species (ROS) generation, double-stranded DNA break, and mitochondrial membrane potential (MMP) loss. ST treatment also resulted in the downregulation of the expressions of VGLUT1 and GluN2B and the reduction of the size of recyclable synaptic vesicle (SV) pool. Like LXRß agonist GW3695, ST suppressed the expression of GluN2B. Furthermore, ST induced mitophagy through upregulating the expressions of LC3BII, p62, and PINK1. The molecular simulation study showed that ST interacted with the ligand binding domain of liver X receptor ß (LXRß), a known binding receptor of ST, through multiple hydrogen bonding. CONCLUSION: Collectively, these findings revealed that ST exhibited a promising neuroprotective effect by regulating both pre- and post-synaptic events following H/R, particularly, attenuation of GluN2B-mediated excitotoxicity and oxidative stress, and induction of mitophagy, and suggested that ST might be a therapeutic promise against ischemic stroke and its associated neurological disorders.

Integrated System Pharmacology and In Silico Analysis Elucidating Neuropharmacological Actions of Withania somnifera in the Treatment of Alzheimer's Disease.

BACKGROUND: Withania somnifera (WS), also referred to as Medhya Rasayana (nootropic or rejuvenating), has traditionally been prescribed for various neurological ailments, including dementia. Despite substantial evidence, pharmacological roles of WS, neither as nootropic nor as an antidementia agent, are well-understood at the cellular and molecular levels. OBJECTIVES: We aimed at elucidating the pharmacological action mechanisms of WS root constituents against Alzheimer's Disease (AD) pathology. METHODS: Various bioinformatics tools and resources, including DAVID, Cytoscape, NetworkAnalyst and KEGG pathway database were employed to analyze the interaction of WS root bioactive molecules with the protein targets of AD-associated cellular processes. We also used a molecular simulation approach to validate the interaction of compounds with selected protein targets. RESULTS: Network analysis revealed that ß-sitosterol, withaferin A, stigmasterol, withanolide A, and withanolide D are the major constituents of WS root that primarily target the cellular pathways such as PI3K/Akt signaling, neurotrophin signaling and toll-like receptor signaling and proteins such as Tropomyosin receptor Kinase B (TrkB), Glycogen Synthase Kinase-3ß (GSK-3ß), Toll-Like Receptor 2/4 (TLR2/4), and ß-secretase (BACE-1). Also, the in silico analysis further validated the interaction patterns and binding affinity of the major WS compounds, particularly stigmasterol, withanolide A, withanolide D and ß-sitosterol with TrkB, GSK-3ß, TLR2/4, and BACE-1. CONCLUSION: The present findings demonstrate that stigmasterol, withanolide A, withanolide D and ß-sitosterol are the major metabolites that are responsible for the neuropharmacological action of WS root against AD-associated pathobiology, and TrkB, GSK-3ß, TLR2/4, and BACE-1 could be the potential druggable targets.

Neuroprotective Potentials of Marine Algae and Their Bioactive Metabolites: Pharmacological Insights and Therapeutic Advances.

Beyond their significant contribution to the dietary and industrial supplies, marine algae are considered to be a potential source of some unique metabolites with diverse health benefits. The pharmacological properties, such as antioxidant, anti-inflammatory, cholesterol homeostasis, protein clearance and anti-amyloidogenic potentials of algal metabolites endorse their protective efficacy against oxidative stress, neuroinflammation, mitochondrial dysfunction, and impaired proteostasis which are known to be implicated in the pathophysiology of neurodegenerative disorders and the associated complications after cerebral ischemia and brain injuries. As was evident in various preclinical studies, algal compounds conferred neuroprotection against a wide range of neurotoxic stressors, such as oxygen/glucose deprivation, hydrogen peroxide, glutamate, amyloid ß, or 1-methyl-4-phenylpyridinium (MPP+) and, therefore, hold therapeutic promise for brain disorders. While a significant number of algal compounds with promising neuroprotective capacity have been identified over the last decades, a few of them have had access to clinical trials. However, the recent approval of an algal oligosaccharide, sodium oligomannate, for the treatment of Alzheimer's disease enlightened the future of marine algae-based drug discovery. In this review, we briefly outline the pathophysiology of neurodegenerative diseases and brain injuries for identifying the targets of pharmacological intervention, and then review the literature on the neuroprotective potentials of algal compounds along with the underlying pharmacological mechanism, and present an appraisal on the recent therapeutic advances. We also propose a rational strategy to facilitate algal metabolites-based drug development.

Phytosterols of marine algae: Insights into the potential health benefits and molecular pharmacology.

BACKGROUND: Marine algae are rich in some unique biologically active secondary metabolites having diverse pharmacological benefits. Of these, sterols comprise a group of functional lipid compounds that have attracted much attention to natural product scientists. PURPOSE: This review was aimed to update information on the health effects of algae-derived phytosterols and their molecular interactions in various aspects of human health and diseases and to address some future perspectives that may open up a new dimension of pharmacological potentials of algal sterols. METHODS: A literature-based search was carried out to retrieve published research information on the potential health effects of algal phytosterols with their pharmacological mechanisms from accessible online databases, such as Pubmed, Google Scholar, Web of Science, and Scopus, using the key search terms of 'marine algae sterol' and 'health potentials such as antioxidant or anti-inflammatory or anti-Alzheimer's or anti-obesity or cholesterol homeostasis or hepatoprotective, antiproliferative, etc.' RESULTS: Phytosterols of marine algae, particularly fucosterol, have been investigated for a plethora of health benefits, including anti-diabetes, anti-obesity, anti-Alzheimer's, antiaging, anticancer, and hepatoprotection, among many others, which are attributed to their antioxidant, anti-inflammatory, immunomodulatory and cholesterol-lowering properties, indicating their potentiality as therapeutic leads. These sterols interact with enzymes and various other proteins that are actively participating in different cellular pathways, including antioxidant defense system, apoptosis and cell survival, metabolism, and homeostasis. CONCLUSION: In this review, we briefly overview the chemistry, pharmacokinetics, and distribution of algal sterols, and provide critical insights into their potential health effects and the underlying pharmacological mechanisms, beyond the well-known cholesterol-lowering paradigm.

Calotropis gigantea Promotes Neuritogenesis and Synaptogenesis through Activation of NGF-TrkA-Erk1/2 Signaling in Rat Hippocampal Neurons.

Calotropis gigantea (L.) R. Br (Apocynaceae) (commonly known as milkweed or crown flower) is a large shrub native to temperate regions of Asia, including China, Bangladesh and India and has a long history of use in traditional medicines. In this study, we investigated the neuromodulatory effects of the ethanol extracts of C. gigantea leaves (CGE) during synaptogenesis in the late stage of neuronal development and during early stage neuritogenesis in cultured rat hippocampal neurons. Maximum neuritogenic activity was achieved at a CGE concentration of 7.5 µ g/ml. At this concentration, CGE facilitated the early development of cytoarchitecture, as evidenced by increases in morphometric parameters, such as, the numbers, lengths, and number of branches of initial neurites, axon and dendrites. During the synaptogenic stage (DIV 14), immunocytochemistry (ICC) showed that CGE upregulated synaptic vesicle 2 (SV2, a marker of axon terminals) and postsynaptic density-95 (PSD-95, a postsynaptic marker) and their colocalization. CGE upregulated nerve growth factor (NGF) and activated extracellular signal-regulated kinase 1/2 (Erk1/2), which is blocked by a TrkA-specific inhibitor suggesting the neuritogenic and synaptogenic potential of CGE was due to the activation of NGF-TrkA-Erk1/2 signaling. Moreover, UPLC of CGE did not detect stigmasterol, an active component of C. gigantea. However, the chloroform-methanol and ethyl acetate subfractions of CGE exhibited initial neuritogenic activity, suggesting that multiple active components were responsible for the neurotrophic-mimetic properties of CGE. Our data prove the neuromodulatory ability of CGE and provide a means of identifying new active phytochemicals with potential nootropic, preventative or therapeutic effects on the human brain.

Neurotrophic Activity of the Carrageenophyte Kappaphycus alvarezii Cultivated at Different Depths and for Different Growth Periods in Various Areas of Indonesia.

The carrageenophyte Kappaphycus alvarezii (Rhodophyta) has neurotrophic activity in primary hippocampal neurons. This seaweed is abundant and easily cultivated in tropical coastal areas. To determine the best growth conditions for neurotrophic activity, thalli were grown at different depths and for different periods in various areas of Indonesia. Neurotrophic activity was measured based on the number of primary neurites, the total length of the primary neurites, and the length of the longest neurite. K. alvarezii had higher neurotrophic activity than carrageenophytes K. striatum and Eucheuma denticulatum cultured under the same conditions. K. alvarezii grown at the surface for 45 days had higher (1.4- to 1.8-fold) neurotrophic activity than thalli grown at depth (2 m) or harvested sooner (15 days) (P < 0.05). Relatively high activities were detected in thalli cultured at Ternate and Garut, Indonesia. Therefore, from a commercial perspective, the culture conditions at the surface for 45 days were optimal for the production of both neurotrophic compounds and carrageenan. K. alvarezii produced neurotrophic compounds under various environmental conditions, although some conditions were optimal.

Stigmasterol upregulates immediate early genes and promotes neuronal cytoarchitecture in primary hippocampal neurons as revealed by transcriptome analysis.

BACKGROUND: The hippocampus is a vulnerable brain region that is implicated in learning and memory impairment by two pathophysiological features, that is, neurite regression and synaptic dysfunction, and stigmasterol (ST), a cholesterol-equivalent phytosterol, is known to facilitate neuromodulatory effects. PURPOSE: To investigate the neuromodulatory effects of ST on the development of central nervous system neurons and the molecular bases of these effects in primary hippocampal neurons. METHODS: Rat embryonic (E18-19) brain neurons were cultured in the absence or presence of ST (75 µM). Neuritogenic activities of ST were evident by increases in various morphometric parameters. To identify underlying affected genes, total RNA was isolated on day in vitro 12 (DIV 12) and mRNA high throughput sequencing (mRNA-Seq) was performed. Affected key genes for neuronal development were identified using bioinformatics tools and their upregulations were confirmed by immunocytochemistry. RESULTS: Among the differentially expressed 17,337 RefSeq genes, 445 genes (up/down 293/157) passed the p-value < 0.05 criterion, 52 genes (up/down; 37/13) had a p-value < 0.05 and a false discovery rate (FDR) q-value of < 0.2, and 24 genes (up/down; 20/4) passed the more stringent criterion of both p < 0.05 and q < 0.05. After applying a stringent FDR q-value cutoff of < 0.2, it was found ST induced many immediate early genes (IEGs), and that a major proportion of upregulated genes were related to central nervous system (CNS) development (neurite outgrowth or synaptic transmission). In a Venn diagram for CNS development Gene Ontologies (GOs) (i.e., axon development, dendrite development, modulation of synaptic transmission), Reln emerged as a central player in these processes, and highly interconnected 'hub' genes, including Dcx, Egr1, Ntrk2, and Slc24a2, were revealed by gene co-expression networks. Finally, transcriptomic data was confirmed by immunocytochemistry of primary hippocampal neurons. CONCLUSION: The study indicates that ST upregulates genes for neuritogenesis and synaptogenesis, and suggests ST be viewed as a potential resource for improving brain functions.

Alleviation of Hippocampal Endoplasmic Reticulum Stress by Allomyrina dichotoma Larvae Extract.

In the brain, endoplasmic reticulum (ER) stress results in synaptic dysfunction and eventually leads to neurodegeneration. Allomyrina dichotoma larvae are a Chinese ethnomedicine and are widely used in East Asia. In the present study, we investigated the ability of ethanol extract of A. dichotoma larvae (ADE) to improve synaptic structure and function by activating unfolded protein response (UPR) under ER stress in animal and neuron culture models. ER stress was induced in obese mice fed a high fat diet (HFD) or by treating dissociated cultures of rat embryonic (E19) hippocampal neurons with tunicamycin (TM). Western blot and real-time or conventional RT-PCR were performed to analyze the expressions of ER stress marker proteins. In dissociated hippocampal cultures, immunocytochemistry was performed for synaptic proteins, and cultures were stained with styryl dye FM1-43 to assess presynaptic activities. In HFD-fed obese mice, ADE efficiently reduced the expressions of ER stress markers, such as, xbp-1, chop, atf4, erdi4, and eIf2a, and those of the ER chaperone/foldases Bip/grp78, Ero-1l, and PDI. Unconventionally spliced xbp-1s mRNA was not detected. In primary rat hippocampal cultures under ER stress, ADE significantly lowered the nuclear expression of CHOP, inhibited the downregulations of postsynaptic proteins, such as, GluN2A, GluN2B, and PSD-95, and maintained the pool size of recycling presynaptic vesicles. The study shows that ADE potently suppressed the induction of ER stress and maintained the structure and function of hippocampal neurons, and suggests that ADE is a potentially valuable food supplement and preventive therapeutic for ER stress-related nervous disorders.

The Red Alga Gracilariopsis chorda and Its Active Constituent Arachidonic Acid Promote Spine Dynamics via Dendritic Filopodia and Potentiate Functional Synaptic Plasticity in Hippocampal Neurons.

Exogenous neurotrophins can induce neuronal differentiation, outgrowth, survival, and synaptic function in the central nervous system. In primary cultures of rat hippocampal neurons, an ethanol extract of the red alga Gracilariopsis chorda (GCE) and its active compound arachidonic acid (AA) significantly increased the densities of dendritic filopodia and spines, promoted the expression of presynaptic vesicle protein 2 (SV2) and postsynaptic density protein 95 (PSD-95), induced robust synaptogenesis, and increased the expression of cell division control protein 42 (CDC42) and actin-related protein 2 (ARP2), which are important for actin organization in dendritic protrusions, and facilitated presynaptic plasticity by increasing the size of the synaptic vesicle pool at presynaptic nerve terminals. In addition, oral administration of GCE and AA for 10 days, at concentrations of 1 mg/g and 2.2 µg/g body weight, respectively, significantly protected against scopolamine-induced memory impairment in mice by increasing the latency time in the passive avoidance test. These results provide strong scientific evidence that these natural products can be used as neurotrophic substances and/or dietary supplements for the prevention and treatment of memory-related neurological disorders via the reconstruction of axo-dendrites and its synapses.

Radix Puerariae modulates glutamatergic synaptic architecture and potentiates functional synaptic plasticity in primary hippocampal neurons.

ETHNOPHARMACOLOGICAL RELEVANCE: Neurologic disorders are frequently characterized by synaptic pathology, including abnormal density and morphology of dendritic spines, synapse loss, and aberrant synaptic signaling and plasticity. Therefore, to promote and/or protect synapses by the use of natural molecules capable of modulating neurodevelopmental events, such as, spinogenesis and synaptic plasticity, could offer a preventive and curative strategy for nervous disorders associated with synaptic pathology. Radix Puerariae, the root of Pueraria monatana var. lobata (Willd.) Sanjappa&Pradeep, is a Chinese ethnomedicine, traditionally used for the treatment of memory-related nervous disorders including Alzheimer's disease. In the previous study, we showed that the ethanolic extracts of Radix Puerariae (RPE) and its prime constituent, puerarin induced neuritogenesis and synapse formation in cultured hippocampal neurons, and thus could improve memory functions. AIMS OF THE STUDY: In the present study, we specifically investigated the abilities of RPE and puerarin to improve memory-related brain disorders through modulating synaptic maturation and functional potentiation. MATERIALS AND METHODS: Rat embryonic (E19) brain neurons were cultured in the absence or presence of RPE or puerarin. At predetermined times, cells were live-stained with DiO or fixed and immunostained to visualize neuronal morphologies, or lysed for protein harvesting. Morphometric analyses of dendritic spines and synaptogenesis were performed using Image J software. Functional pre- and postsynaptic plasticity was measured by FM1-43 staining and whole-cell patch clamping, respectively. RPE or puerarin-mediated changes in actin-related protein 2 were assessed by Western blotting. Neuronal survivals were measured using propidium iodide exclusion assay. RESULTS: RPE and puerarin both: (1) promoted a significant increase in the numbers, and maturation, of dendritic spines; (2) modulated the formation of glutamatergic synapses; (3) potentiated synaptic transmission by increasing the sizes of reserve vesicle pools at presynaptic terminals; (4) enhanced NMDA receptor-mediated postsynaptic currents, and (5) increased cell viability against naturally occurring cell death. Moreover, upregulation of actin-related protein 2 (ARP2) in RPE and puerarin treated brain neurons suggest that RPE and puerarin induced synaptic plasticity might be associated, at least in part, with ARP2-mediated actin-dependent regulation of spinogenesis. CONCLUSIONS: Our findings indicate that RPE and puerarin might play a substantial role in the morphological and functional maturation of brain neurons and suggest that RPE and puerarin are potentially valuable preventative therapeutics for memory-related nervous disorders.