Search Results (92)

Teleost fish are the first evolutionary group to exhibit an innate and adaptive immune system. Within the mechanisms of adaptive immunity, fish possess, among others, T-helper cells (CD4-like) and their differentiation machinery, regulated by the master transcription factors T-bet, GATA3, Foxp3, and RORγ. Many studies support the existence of a non-neuronal cholinergic system involved in the immune response, named after the ability of leukocytes to synthesize de novo acetylcholine (ACh). Organophosphorus pesticides (OPs), such as diazoxon (DXN), are examples of compounds that act as cholinergic disruptors with immunotoxic effects. The present study aimed to evaluate the expression of transcription factors in leukocytes (spleen mononuclear cells, SMNCs) of Nile tilapia by modulating cholinergic pathways in immune cells using agonists, antagonists, and diazoxon (DXN), an anticholinesterase substance. The obtained data showed a significant increase in RORγ mRNA expression upon stimulation with the nicotinic agonist, whereas activation of the muscarinic receptor with its agonist increased T-bet mRNA expression. An alteration in RORγ expression levels induced by DXN exposure was also observed. The results suggest a probable directing of the immune response towards a pro-inflammatory profile orchestrated mainly by RORγ and T-bet transcription factors in response to cholinergic stimuli. Full article

This study investigates the neuroprotective and anxiolytic effects of Solanum macrocarpon L. leaf n-butanol extract (SMB) in a zebrafish model of scopolamine (SCOP; 100 μM)-induced cognitive and behavioral impairments. SCOP, a muscarinic receptor antagonist, is commonly used to mimic memory deficits and anxiety-like behaviors associated with neurodegenerative conditions. Zebrafish were chronically exposed to SMB at concentrations of 1, 3, and 6 mg/L. Behavioral assessments included anxiety-related paradigms, such as novel tank diving (NTT), novel approach (NA), and light–dark transition (LD) tests, as well as cognitive assays, including the Y-maze and novel object recognition (NOR) tests. SMB significantly mitigated SCOP-induced anxiety-like behaviors and cognitive deficits in a dose-dependent manner. Biochemical analyses demonstrated that SMB inhibited acetylcholinesterase (AChE) overactivity, indicating restoration of cholinergic function. Furthermore, SMB enhanced the activity of endogenous antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) and significantly reduced oxidative stress biomarkers, including malondialdehyde (MDA) and protein carbonyls. These findings suggest that SMB may exert neuroprotective effects through modulation of cholinergic signaling and oxidative stress. Overall, SMB represents a promising phytotherapeutic candidate for mitigating cognitive and anxiety-related symptoms linked to oxidative damage. Further investigations are warranted to characterize its active constituents and assess long-term efficacy and safety in models of neurodegeneration. Full article

The cholinergic system is one of the most ancient and widespread signaling systems in the body, implicated in a range of pathological conditions—from neurodegenerative disorders to cancer. Given its broad relevance, there is growing interest in characterizing this system across diverse cellular models to enable drug screening, mechanistic studies, and exploration of new therapeutic avenues. In this study, we investigated four cancer cell lines: one of neuroblastoma origin previously used in cholinergic signaling studies (SH-SY5Y), one non-small cell lung adenocarcinoma line (A549), and two small cell lung carcinoma lines (H69 and H82). We assessed the expression and localization of key components of the cholinergic system, along with the cellular capacity for acetylcholine (ACh) synthesis and release. Whole-cell flow cytometry following membrane permeabilization revealed that all cell lines expressed the ACh-synthesizing enzyme choline acetyltransferase (ChAT). HPLC-MS analysis confirmed that ChAT was functionally active, as all cell lines synthesized and released ACh into the conditioned media, suggesting the presence of autocrine and/or paracrine ACh signaling circuits, consistent with previous reports. The cell lines also demonstrated choline uptake, indicative of functional choline and/or organic cation transporters. Additionally, all lines expressed the ACh-degrading enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), as well as the alfa seven (α7) nicotinic and M1 muscarinic ACh receptor subtypes. Notably, flow cytometry of intact SH-SY5Y cells revealed two novel findings: (1) ChAT was localized to the extracellular membrane, a feature not observed in the lung cancer cell lines, and (2) BChE, rather than AChE, was the predominant membrane-bound ACh-degrading enzyme. These results were corroborated by both whole-cell and surface-confocal microscopy. In conclusion, our findings suggest that a functional cholinergic phenotype is a shared feature of several carcinoma cell lines, potentially serving as a survival checkpoint that could be therapeutically explored. The discovery of extracellular membrane-bound ChAT uniquely in neuroblastoma SH-SY5Y cells points to a novel form of in situ ACh signaling that warrants further investigation. Full article

In our previous study, we demonstrated that a standardized ethanol extract of Perilla frutescens var. acuta (PE) alleviates memory deficits in an Alzheimer’s disease mouse model by inhibiting amyloid β (Aβ) aggregation and promoting its disaggregation. However, the extent to which PE exerts additional cognitive benefits independent of Aβ pathology remained unclear. Here, we aimed to evaluate the effects of PE on synaptic plasticity and learning and memory functions. Male ICR mice were used, and cognitive impairment was induced by scopolamine administration. PE was orally administered at doses determined from previous studies, and cognitive performance was assessed using the passive avoidance, Y-maze, and Morris water maze tests. In parallel, hippocampal slices were employed to examine the effects of PE on synaptic plasticity. PE (100 and 300 μg/mL) significantly enhanced long-term potentiation (LTP) in a concentration-dependent manner without altering basal synaptic transmission. This facilitation of LTP was blocked by scopolamine (1 μM), a muscarinic acetylcholine receptor (mAChR) antagonist, and IEM-1460 (50 μM), a calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (CP-AMPAR) inhibitor, indicating the involvement of mAChR and CP-AMPAR pathways. In vivo, PE (100, 250, and 500 mg/kg) treatment improved memory performance across all behavioral tasks and upregulated hippocampal synaptic proteins including GluN2B, PSD-95, and CaMKII. Collectively, these results demonstrate that PE ameliorates scopolamine (1 mg/kg)-induced cognitive impairment by enhancing synaptic plasticity, likely through modulation of mAChR, CP-AMPAR, and NMDA receptor signaling. These findings highlight the therapeutic potential of PE for memory deficits associated with cholinergic dysfunction. Full article

Chlorella vulgaris is a nutrient-dense microalga with recognized antioxidant, anti-inflammatory, and metabolic regulatory properties, making it an attractive candidate for functional food applications. In such contexts, both chemical composition and particle size can influence dispersibility, bioactive release, and physiological effects. In this study, two commercial C. vulgaris powders from India (Sample 1) and the UK (Sample 2) were compared with respect to particle size, metabolite composition, and biological activity. Sample 1 exhibited finer particles, while Sample 2 was coarser. GC–MS profiling revealed distinct compositional differences: Sample 1 displayed a higher relative abundance of saturated fatty acids, β-sitosterol, β-amyrin, and glucitol, whereas Sample 2 contained higher levels of unsaturated fatty acids, betulin, salicylic acid, and specific carbohydrates. In vitro assays showed stronger inhibition of albumin denaturation by Sample 1 compared with Sample 2 and prednisolone. Ex vivo tests indicated that both samples induced tonic contraction of gastric smooth muscle through muscarinic acetylcholine receptors (mAChRs) and L-type calcium channels, as evidenced by the marked reduction in responses after atropine and verapamil treatment, with Sample 1 producing a more pronounced effect. Immunohistochemistry further demonstrated broader IL-1β upregulation with Sample 1 and localized nNOS modulation with Sample 2. Overall, the results demonstrate that the interplay between composition and particle size shapes the bioactivity of C. vulgaris, supporting its targeted use in digestive, neuroimmune, and cardiometabolic health. Full article

α7 nAChRs are known to modulate several physiological and pathological functions in glial cells, and their selective activation might have anti-inflammatory effects in the central and peripheral nervous system. OL progenitors (OPCs) respond to cholinergic stimuli via muscarinic receptors that are mainly involved in the modulation of their proliferation. Conversely, the role of nicotinic receptors, particularly α7 nAChRs, has been poorly investigated. In this study, we evaluated the expression of α7 nAChRs in a model of OPCs (Oli neu) and the potential effects mediated by their selective activation. Methods: Oli neu cells were used as a murine immortalized OPCs model. The effects of α7 nAChRs stimulation on cell proliferation and survival were assessed by the MTT assay. RT-PCR and Western blot analysis were used to analyze the expression of α7 nAChRs and proliferative and differentiative markers (PCNA, MBP). LPS exposure was used to induce the environment in which the antioxidant and anti-inflammatory properties of α7 nAChRs were analyzed, evaluating NFR2 and TNF-α expression, ROS levels through DCFDA staining while Oil Red O staining was used for the analysis of lipid droplet content as a marker of cellular inflammation response. Results: The α7 nAChR is expressed both in OPCs and OLs, and its stimulation by the selective agonist ICH3 increases cell proliferation without modifying the OLs’ differentiation capability. Moreover, ICH3 showed anti-inflammatory and antioxidant effects against LPS exposure. Conclusions: The results herein obtained confirm the role of α7 nAChR in the modulation of neuroinflammatory processes as well as their protective effects on OLs. Full article

It has long been recognised that airway smooth muscle cells (ASMCs) possess an abundance of M2 muscarinic receptors (M2Rs). However, the contribution of postjunctional M2Rs to contractions of airway smooth muscle (ASM) induced by the release of acetylcholine (ACh) from parasympathetic nerves was thought to be minimal. Instead, it was believed that these responses were exclusively mediated by activation of M3Rs. However, evidence is emerging that postjunctional M2Rs may have a greater role than previously realised. In this review, we discuss ACh signalling in airways, highlighting the well-established autoinhibitory role of prejunctional M2Rs and the putative roles of postjunctional M2Rs to cholinergic contractions of ASM. The cellular mechanisms that underpin M2R-dependent contractions of ASM are reviewed, with a particular emphasis on the role of ion channels in these responses. The regulation of M2R signalling pathways by β-adrenoceptor activation is also considered, along with the potential involvement of postjunctional M2Rs in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). Full article

The cholinergic pathways in the central nervous system (CNS) play a pivotal role in different cognitive functions of the brain, such as memory and learning. This review takes a dive into the pharmacological side of this important part of CNS function, taking into consideration muscarinic receptors and cholinesterase enzymes. Targeting a specific subtype of five primary muscarinic receptor subtypes (M1-M5) through agonism or antagonism may benefit patients; thus, there is a great pharmaceutical research interest. Inhibition of AChE and BChE, orthosteric or allosteric, or partial agonism of M1 mAChR are correlated with Alzheimer’s disease (AD) symptoms improvement. Agonism or antagonism on different muscarinic receptor subunits may lessen schizophrenia symptoms (especially positive allosteric modulation of M4 mAChR). Selective antagonism of M4 mAChR is a promising treatment for Parkinson’s disease and dystonia, and the adverse effects are limited compared to inhibition of all five mAChR. Additionally, selective M5 antagonism plays a role in drug independence behavior. M3 mAChR overexpression is associated with malignancies, and M3R antagonists seem to have a therapeutic potential in cancer, while M1R and M2R inhibition leads to reduction of neoangiogenesis. Depending on the type of cancer, agonism of mAChR may promote cancer cell proliferation (as M3R agonism does) or protection against further tumor development (M1R agonism). Thus, there is an intense need to discover new potent compounds with specific action on muscarinic receptor subtypes. Chemical structures, chemical modification of function groups aiming at action enhancement, reduction of adverse effects, and optimization of Drug Metabolism and Pharmacokinetics (DMPK) will be further discussed, as well as protein–ligand docking. Full article

Background: Perfluorooctane sulfonic acid (PFOS), a widely used industrial chemical, was reported to induce memory and learning process dysfunction. Some studies tried to reveal the mechanisms that mediate these effects, but how they are produced is still unknown. Basal forebrain cholinergic neurons (BFCN) maintain cognitive function and their selective neurodegeneration induces cognitive decline, as observed in Alzheimer’s disease. PFOS was reported to disrupt cholinergic and glutamatergic transmissions and thyroid hormone action, which regulate cognitive processes and maintain BFCN viability. Objective/Methods: To evaluate PFOS neurodegenerative effects on BFCN and the mechanisms that mediate them, SN56 cells (a neuroblastoma cholinergic cell line from the basal forebrain) were treated with PFOS (0.1 µM to 40 µM) with or without thyroxine (T3; 15 nM), MK-801 (20 µM) or acetylcholine (ACh; 10 µM). Results: In the present study, we found that PFOS treatment (1 or 14 days) decreased thyroid receptor α (TRα) activity by decreasing its protein levels and increased T3 metabolism through increased deiodinase 3 (D3) levels. Further, we observed that PFOS treatment disrupted cholinergic transmission by decreasing ACh content through decreased choline acetyltransferase (ChAT) activity and protein levels and through decreasing muscarinic receptor 1 (M1R) binding and protein levels. PFOS also disrupted glutamatergic transmission by decreasing glutamate content through increased glutaminase activity and protein levels and through decreasing N-methyl-D-aspartate receptor subunit 1 (NMDAR1); effects mediated through M1R disruption. All these effects were mediated through decreased T3 activity and T3 supplementation partially restored to the normal state. Conclusions: These findings may assist in understanding how PFOS induces neurodegeneration, and the mechanisms involved, especially in BFCN, to explain the process that could lead to cognitive dysfunction and provide new therapeutic tools to treat and prevent its neurotoxic effects. Full article

Photoperiods are crucial environmental factors in the growth and health of modern intensive broiler chicken production. To date, the effects of different photoperiods on glucose metabolism, acetylcholine (ACh), and its relative acetylcholine receptor modulation in broilers remain elusive. Herein, we aimed to identify the effects of different photoperiods on regulating glucose metabolism, ACh, nicotinic acetylcholine receptor alpha 4 (α4 nAChR) mRNA, and M3 muscarinic acetylcholine receptor (M3 mAChR) modulation in broilers. A total of 216 healthy 5-day-old Arbor Acres (AA) male broilers was randomly assigned to 12L:12D, 18L:6D, and 24L:0D photoperiods for 4 weeks. The results show that, compared with the 12L:12D photoperiod, the 18L:6D and 24L:0D photoperiods significantly increase the average daily gain (ADG) and average daily feed intake (ADFI) of broilers (p < 0.05). However, the feed efficiency (FE) of broilers significantly decreased in the 18L:6D and 24L:0D photoperiods (p < 0.05). Moreover, compared with the 12L:12D photoperiod, the ACh concentrations and α4 nAChR mRNA expression levels in the hypothalamus and medulla oblongata of broilers significantly increased (p < 0.05); M3 mAChR mRNA expression levels in cecum significantly reduced in the 18L:6D photoperiod and the 24L:0D photoperiod (p < 0.05). Compared with the 12L:12D photoperiod, the serum glucose (GLU), serum insulin (INS), serum triglyceride (TG) levels, and homeostasis model assessment of insulin resistance (HOMA-IR) of broilers significantly enhanced in the 18L:6D and 24L:0D photoperiods (p < 0.05). Our results indicate that extending the photoperiod can promote the growth rate, ACh expression, and α4 nAChR mRNA expression of broilers while reducing the feed efficiency, inhibiting M3 mAChR mRNA expression, and inducing glucose metabolism disorders in broilers. Full article

Despite the great effort that has gone into developing new molecules as multitarget compounds to treat Alzheimer’s disease (AD), none of these have been approved to treat this disease. Therefore, it will be interesting to determine whether benzazoles such as benzimidazole, benzoxazole, and benzothiazole, employed as pharmacophores, could act as multitarget drugs. AD is a multifactorial disease in which several pharmacological targets have been identified—some are involved with amyloid beta (Aβ) production, such as beta secretase (BACE1) and beta amyloid aggregation, while others are involved with the cholinergic system as acetylcholinesterase (AChE) and butirylcholinesterase (BChE) and nicotinic and muscarinic receptors, as well as the hyperphosphorylation of microtubule-associated protein (tau). In this review, we describe the in silico and in vitro evaluation of benzazoles on three important targets in AD: AChE, BACE1, and Aβ. Benzothiazoles and benzimidazoles could be the best benzazoles to act as multitarget drugs for AD because they have been widely evaluated as AChE inhibitors, forming π–π interactions with W286, W86, Y72, and F338, as well as in the AChE gorge and catalytic site. In addition, the sulfur atom from benzothiazol interacts with S286 and the aromatic ring from W84, with these compounds having an IC₅₀ value in the μM range. Also, benzimidazoles and benzothiazoles can inhibit Aβ aggregation. However, even though benzazoles have not been widely evaluated on BACE1, benzimidazoles evaluated in vitro showed an IC₅₀ value in the nM range. Therefore, important chemical modifications could be considered to improve multitarget benzazoles’ activity, such as substitutions in the aromatic ring with electron withdrawal at position five, or a linker 3 or 4 carbons in length, which would allow for better interaction with targets. Full article

Neuroinflammation is a critical factor that contributes to neurological impairment and is closely associated with the onset and progression of neurodegenerative diseases. In the central nervous system (CNS), microglia play a pivotal role in the regulation of inflammation through various signaling pathways. Therefore, mitigating microglial inflammation is considered a promising strategy for restraining neuroinflammation. Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the CNS and exhibit clear neuroprotective effects in various disease models. However, whether the activation of mAChRs can harness benefits in neuroinflammation remains largely unexplored. In this study, the anti-inflammatory effects of mAChRs were found in a neuroinflammation mouse model. The expression of various cytokines and chemokines was regulated in the brains and spinal cords after the administration of mAChR agonists. Microglia were the primary target cells through which mAChRs exerted their anti-inflammatory effects. The results showed that the activation of mAChRs decreased the pro-inflammatory phenotypes of microglia, including the expression of inflammatory cytokines, morphological characteristics, and distribution density. Such anti-inflammatory modulation further exerted neuroprotection, which was found to be even more significant by the direct activation of neuronal mAChRs. This study elucidates the dual mechanisms through which mAChRs exert neuroprotective effects in central inflammatory responses, providing evidence for their application in inflammation-related neurological disorders. Full article

Alzheimer’s disease (AD) is one of the most common causes of dementia, accounting for more than 60% of all cases. It is a neurodegenerative disease in which symptoms such as a decline in memory, thinking, learning, and organizing skills develop gradually over many years and eventually become more severe. To date, there is no effective treatment for the cause of Alzheimer’s disease, and the existing pharmacological options primarily help manage symptoms. Treatment is mainly based on acetylcholinesterase (AChE) inhibitors such as donepezil, rivastigmine, and galantamine, which exhibit numerous adverse cardiovascular and gastrointestinal effects due to excessive stimulation of peripheral cholinergic activity involving muscarinic receptors. Therefore, in addition to the obvious drugs that act on the cause of the disease, new drugs based on AChE inhibition that show the fewest side effects are needed. One potential drug could be a new compound under study, tetrahydroacridine derivative (CHDA), which showed significant potential to inhibit the AChE enzyme in previous in vitro studies. The present study shows that while having very potent AChE inhibitory properties, CHDA is a compound with low toxicity to nerve cell culture and living organisms. In addition, it exhibits dissociative activity against amyloid β fibrils, which is extremely important for applications in Alzheimer’s disease therapy. Full article

The synthesized compound 1-(2-chlorophenyl) 6-7-dimethoxy-3-methyl-3,4-dihydroisoquinoline (DIQ) was investigated as a biological agent. Its potential to affect muscle contractility was predicted through in silico PASS analysis. Based on the in silico analysis, its capabilities were experimentally investigated. The study aimed to investigate the effects of DIQ on the ex vivo spontaneous contractile activity (CA) of smooth muscle (SM) tissue. DIQ was observed to reduce the strength of Ca²⁺-dependent contractions in SM preparations (SMP), possibly by increasing cytosolic Ca²⁺ levels through the activation of a voltage-gated L-type Ca²⁺ channel. DIQ potently affected calcium currents by modulating the function of muscarinic acetylcholine receptors (mAChRs) and 5-hydroxytryptamine (5-HT) receptors at a concentration of 50 μM. Immunohistochemical tests showed a 47% reduction in 5-HT_2A and 5-HT_2B receptor activity in SM cells and neurons in the myenteric plexus (MP), further confirming the effects of DIQ. Furthermore, a significant inhibition of neuronal activity was observed when the compound was co-administered with 5-HT to SM tissues. The conducted experiments confirm the ability of the isoquinoline analog to act as a physiologically active molecule to control muscle contractility and related physiological processes. Full article

Age-related conditions, such as sarcopenia, cause physical disabilities for an increasing section of society. At the neuromuscular junction, the postsynaptic-derived neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT-4) have neuroprotective functions and contribute to the correct regulation of the exocytotic machinery. Similarly, presynaptic muscarinic signalling plays a fundamental modulatory function in this synapse. However, whether or not these signalling pathways are compromised in ageing neuromuscular system has not yet been analysed. The present study analyses, through Western blotting, the differences in expression and activation of the main key proteins of the BDNF/NT-4 and muscarinic pathways related to neurotransmission in young versus ageing Extensor digitorum longus (EDL) rat muscles. The main results show an imbalance in several sections of these pathways: (i) a change in the stoichiometry of BDNF/NT-4, (ii) an imbalance of Tropomyosin-related kinase B receptor (TrkB)-FL/TrkB-T1 and neurotrophic receptor p 75 (p75^NTR), (iii) no changes in the cytosol/membrane distribution of phosphorylated downstream protein kinase C (PKC)βI and PKCε, (iv) a reduction in the M2-subtype muscarinic receptor and P/Q-subtype voltage-gated calcium channel, (v) an imbalance of phosphorylated mammalian uncoordinated-18-1 (Munc18-1) (S313) and synaptosomal-associated protein 25 (SNAP-25) (S187), and (vi) normal levels of molecules related to the management of acetylcholine (Ach). Based on this descriptive analysis, we hypothesise that these pathways can be adjusted to ensure neurotransmission rather than undergoing negative alterations caused by ageing. However, further studies are needed to assess this hypothetical suggestion. Our results contribute to the understanding of some previously described neuromuscular functional age-related impairments. Strategies to promote these signalling pathways could improve the neuromuscular physiology and quality of life of older people. Full article