Activation Of α7nAChR Research Articles

Nicotinic Acetylcholine Receptor Subunit Alpha-7 Mediates PD-L1 and CTLA-4 Expression in HepG2 Cells

Background: The liver is the largest solid organ in the body and has several unique immunological roles. The alpha7 subtype of nicotinic acetylcholine receptor (α7nAChR) is expressed in the liver and has different immunoregulatory effects. Programmed death ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are two major checkpoints that act as a negative regulator of the immune system. In this study, we examined the effects of activation (with nicotine) and inhibition (with specific siRNA) of α7nAChR on the PD-L1 and CTLA-4 genes expression. Methods: Human hepatocellular carcinoma (HepG2) cell line was used to investigate the effects of treatment with low dose (1 μM) and high dose (10 μM) of nicotine on the PD-L1 and CTLA-4 expression. In addition to this, the effects of treatment with 100 nM concentration of specific siRNA targeting α7nAChR (α7-siRNA) were examined using quantitative real-time PCR. Results: Compared to untreated cells, the HepG2 cells treated with nicotine exhibited significant dose-dependent decreases in PD-L1 expression. Furthermore, nicotine has a biphasic effect on the CTLA-4 expression and decreased its expression in 10 μM concentration. The qRT-PCR revealed that α7-siRNA significantly reduced the mRNA levels of α7nAChR (P < 0.01). Also, compared to control cells, α7-siRNA treated cells exhibited significant increases in both PD-L1 and CTLA-4 expression. Conclusion: These experiments determined that nicotine treatment leads to decreases in PD-L1 and CTLA-4 expression. These effects are reversed by treatment with α7-siRNA, which indicates the involvement of α7nAChR related mechanisms in these processes.

Activation of α7 nicotinic acetylcholine receptors attenuates monocyte–endothelial adhesion through FUT7 inhibition

Cholinergic anti-inflammatory pathway (CAP) describes a neuronal-inflammatory reflex centered on systemic cytokine regulation by α7 nicotinic acetylcholine receptor (α7nAChR) activation of spleen-residue macrophage. However, the CAP mechanism attenuating distal tissue inflammation, inducing a low level of systemic inflammation, is lesser known. In this study, we hypothesized that CAP regulates monocyte accessibility by influencing their adhesion to endothelial cells. Using RNA-seq analysis, we identified that α1,3-Fucosyltransferase 7 (FucT-VII), the enzyme required for processing selectin ligands, was significantly downregulated by α7nAChR agonist among other cell–cell adhesion genes. The α7nAChR agonist inhibited monocytic cell line U-937 binding to P-selectin and adhesion to endothelial cells. Furthermore, α7nAChR agonist selectivity was confirmed by α7nAChR knockdown assays, showing that FUT7 inhibition and adhesion attenuation by the agonist was abolished by siRNA targeting α7nAChR encoding gene. Consistently, FUT7 knockdown inhibited the adhesive properties of U-937 and prevented them to adhere to endothelial cells. Overexpression of FUT7 also abrogated the adhesion attenuation induced by GTS-21 indicating that FUT7 inhibition was sufficient for inhibiting adhesion by α7nAChR activation. Our work demonstrated that α7nAChR activation regulates monocyte adhesion to endothelial cells through FUT7 inhibition, providing a novel insight into the CAP mechanism.

Activation of the alpha 7 nicotinic acetylcholine receptor mitigates osteoarthritis progression by inhibiting NF-κB/NLRP3 inflammasome activation and enhancing autophagy.

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage degradation. Alpha 7 nicotinic acetylcholine receptor (α7nAChR) is associated with inflammatory and metabolic responses in OA. However, the mechanisms underlying the pathological process of OA remain unclear. The aim of the present study was to examine the role and mechanisms of α7nAChR-mediated autophagy and anti-inflammatory response in chondroprotection. Monosodium iodoacetate (MIA)-induced Wistar rat OA model was used to assess the in vivo effects of the ɑ7nAChR agonist (PNU-282987). The histopathological characteristics of OA were evaluated by immunohistochemistry (IHC), and the levels of autophagy markers were determined by western blotting and transmission electron microscopy. The anti-inflammatory effect of the ɑ7nAChR agonist was assessed by IHC, quantitative real-time polymerase chain reaction, and western blotting. Parallel experiments to determine the molecular mechanisms through which the ɑ7nAChR agonist prevents OA were performed using interleukin-1β (IL-1β)-treated chondrocytes. Our results showed that PNU-282987 reduced cartilage degeneration and matrix metalloproteinase (MMP)-1 and MMP-13 expressions. Activating α7nAChR with PNU-282987 significantly promoted MIA/IL-1β-induced chondrocyte autophagy, as demonstrated by the increase in LC3-II/LC3-I ratio, Beclin-1 levels, and autophagosome number. Furthermore, treating chondrocyte with ULK1 siRNA attenuated the PNU282987-induced enhancement of LC3-II/LC3-I ratio and Beclin-1 level. Additionally, PNU282987 suppressed NF-κB/NLRP3 inflammasome activation by inhibiting the ROS/TXNIP pathway and suppressed tumor necrosis factor-ɑ and IL-1β secretion in MIA/IL-1β-treated chondrocytes. Our results demonstrate that the activation of α7nAChR promotes chondrocyte autophagy and attenuates inflammation to mitigate OA progression, providing a novel target for the treatment of OA.

The Mechanisms Mediated by α7 Acetylcholine Nicotinic Receptors May Contribute to Peripheral Nerve Regeneration.

Due to the microenvironment created by Schwann cell (SC) activity, peripheral nerve fibers are able to regenerate. Inflammation is the first response to nerve damage and the removal of cellular and myelin debris is essential in preventing the persistence of the local inflammation that may negatively affect nerve regeneration. Acetylcholine (ACh) is one of the neurotransmitters involved in the modulation of inflammation through the activity of its receptors, belonging to both the muscarinic and nicotinic classes. In this report, we evaluated the expression of α7 nicotinic acetylcholine receptors (nAChRs) in rat sciatic nerve, particularly in SCs, after peripheral nerve injury. α7 nAChRs are absent in sciatic nerve immediately after dissection, but their expression is significantly enhanced in SCs after 24 h in cultured sciatic nerve segments or in the presence of the proinflammatory neuropeptide Bradykinin (BK). Moreover, we found that activation of α7 nAChRs with the selective partial agonist ICH3 causes a decreased expression of c-Jun and an upregulation of uPA, MMP2 and MMP9 activity. In addition, ICH3 treatment inhibits IL-6 transcript level expression as well as the cytokine release. These results suggest that ACh, probably released from regenerating axons or by SC themselves, may actively promote through α7 nAChRs activation an anti-inflammatory microenvironment that contributes to better improving the peripheral nerve regeneration.

Acetylcholine receptor agonists provide cardioprotection in doxorubicin-induced cardiotoxicity via modulating muscarinic M2 and α7 nicotinic receptor expression

The balance between cardiac sympathetic and parasympathetic activities has been intricately linked to mitochondrial function, cellular oxidative status, and immunomodulation in healthy and diseased myocardium. Cardiac autonomic neuropathy, along with the associated mitochondrial and cellular dysfunction, is an important pathophysiological feature of doxorubicin-induced cardiotoxicity (DIC). We tested the hypothesis that autonomic modulation by activation of acetylcholine receptors (AChR) effectively attenuates DIC. Rats were divided into control (0.9% sodium chloride solution) and doxorubicin groups (DOX, 3 mg/kg/d, 6 doses). Rats in the DOX group were equally subdivided into 4 interventional groups and treated for 30 days: vehicle, α7 nicotinic receptor agonist (PNU: PNU-282987, 3 mg/kg/d), muscarinic receptor agonist (BET: bethanechol, 12 mg/kg/d), and combined α7nAChR and mAChR agonists group (COM). Cardiac biochemical and functional analyses were done. The results show that AChR agonists protected the heart against DIC via improving mitochondrial and cardiac function, which was accompanied by reducing mitochondrial oxidative damage, apoptosis, and inflammation. Strikingly, PNU and BET exerted cardioprotection through different molecular pathways. PNU-mediated α7nAChR activation promoted mitochondrial fusion via upregulation of Mfn1-2 and attenuated DOX-induced autophagy. Contrarily, activation of mAChR by BET attenuated mitochondrial fission and mitophagy. The in vitro experiments confirmed the cytoprotective effects of AChR activation in DOX-treated H9c2 cells without compromising the anticancer effect of DOX in cancer cells. In conclusion, α7nAChR and mAChR agonists exerted cardioprotection against DIC via rebalancing autonomic function, improving mitochondrial function, reducing oxidative stress, and decreased cardiomyocyte apoptosis and inflammation, leading to improved cardiac function.

Effect of Intrahippocampal Administration of α7 Subtype Nicotinic Receptor Agonist PNU-282987 and Its Solvent Dimethyl Sulfoxide on the Efficiency of Hypoxic Preconditioning in Rats

We have previously suggested a key role of the hippocampus in the preconditioning action of moderate hypobaric hypoxia (HBH). The preconditioning efficiency of HBH is associated with acoustic startle prepulse inhibition (PPI). In rats with PPI > 40%, HBH activates the cholinergic projections of hippocampus, and PNU-282987, a selective agonist of α7 nicotinic receptors (α7nAChRs), reduces the HBH efficiency and potentiating effect on HBH of its solvent dimethyl sulfoxide (DMSO, anticholinesterase agent) when administered intraperitoneally. In order to validate the hippocampus as a key structure in the mechanism of hypoxic preconditioning and research a significance of α7nAChR activation in the hypoxic preconditioning, we performed an in vivo pharmacological study of intrahippocampal injections of PNU-282987 into the CA1 area on HBH efficiency in rats with PPI ≥ 40%. We found that PNU-282987 (30 μM) reduced HBH efficiency as with intraperitoneal administration, while DMSO (0.05%) still potentiated this effect. Thus, direct evidence of the key role of the hippocampus in the preconditioning effect of HBH and some details of this mechanism were obtained in rats with PPI ≥ 40%. The activation of α7nAChRs is not involved in the cholinergic signaling initiated by HBH or DMSO via any route of administration. Possible ways of the potentiating action of DMSO on HBH efficiency and its dependence on α7nAChRs are discussed.

Activation of α7nAChR Protects Against Gastric Inflammation and Dysmotility in Parkinson's Disease Rats.

The cholinergic anti-inflammatory pathway (CAIP) has been proposed to regulate gastrointestinal inflammation via acetylcholine released from the vagus nerve activating α7 nicotinic receptor (α7nAChR) on macrophages. Parkinson’s disease (PD) patients and PD rats with substantia nigra (SN) lesions exhibit gastroparesis and a decayed vagal pathway. To investigate whether activating α7nAChR could ameliorate inflammation and gastric dysmotility in PD rats, ELISA, western blot analysis, and real-time PCR were used to detect gastric inflammation. In vitro and in vivo gastric motility was investigated. Proinflammatory mediator levels and macrophage numbers were increased in the gastric muscularis of PD rats. α7nAChR was located on the gastric muscular macrophages of PD rats. The α7nAChR agonists PNU-282987 and GTS-21 decreased nuclear factor κB (NF-κB) activation and monocyte chemotactic protein-1 mRNA expression in the ex vivo gastric muscularis of PD rats, and these effects were abolished by an α7nAChR antagonist. After treatment with PNU-282987 in vivo, the PD rats showed decreased NF-κB activation, inflammatory mediator production, and contractile protein expression and improved gastric motility. The present study reveals that α7nAChR is involved in the development of gastroparesis in PD rats and provides novel insight for the treatment of gastric dysmotility in PD patients.

The Regulation Effect of α7nAChRs and M1AChRs on Inflammation and Immunity in Sepsis.

The inflammatory storm in the early stage and immunosuppression in the late stage are responsible for the high mortality rates and multiple organ dysfunction in sepsis. In recent years, studies have found that the body's cholinergic system can spontaneously and dynamically regulate inflammation and immunity in sepsis according to the needs of the body. Firstly, the vagus nerve senses and regulates local or systemic inflammation by means of the Cholinergic Anti-inflammatory Pathway (CAP) and activation of α7-nicotinic acetylcholine receptors (α7nAChRs); thus, α7nAChRs play important roles for the central nervous system (CNS) to modulate peripheral inflammation; secondly, the activation of muscarinic acetylcholine receptors 1 (M1AChRs) in the forebrain can affect the neurons of the Medullary Visceral Zone (MVZ), the core of CAP, to regulate systemic inflammation and immunity. Based on the critical role of these two cholinergic receptor systems in sepsis, it is necessary to collect and analyze the related findings in recent years to provide ideas for further research studies and clinical applications. By consulting the related literature, we draw some conclusions: MVZ is the primary center for the nervous system to regulate inflammation and immunity. It coordinates not only the sympathetic system and vagus system but also the autonomic nervous system and neuroendocrine system to regulate inflammation and immunity; α7nAChRs are widely expressed in immune cells, neurons, and muscle cells; the activation of α7nAChRs can suppress local and systemic inflammation; the expression of α7nAChRs represents the acute or chronic inflammatory state to a certain extent; M1AChRs are mainly expressed in the advanced centers of the brain and regulate systemic inflammation; neuroinflammation of the MVZ, hypothalamus, and forebrain induced by sepsis not only leads to their dysfunctions but also underlies the regulatory dysfunction on systemic inflammation and immunity. Correcting the neuroinflammation of these regulatory centers and adjusting the function of α7nAChRs and M1AChRs may be two key strategies for the treatment of sepsis in the future.

Endogenous α7 nAChR Agonist SLURP1 Facilitates Escherichia coli K1 Crossing the Blood-Brain Barrier.

Alpha 7 nicotinic acetylcholine receptor (α7 nAChR) is critical for the pathogenesis of Escherichia coli (E. coli) K1 meningitis, a severe central nervous system infection of the neonates. However, little is known about how E. coli K1 manipulates α7 nAChR signaling. Here, through employing immortalized cell lines, animal models, and human transcriptional analysis, we showed that E. coli K1 infection triggers releasing of secreted Ly6/Plaur domain containing 1 (SLURP1), an endogenous α7 nAChR ligand. Exogenous supplement of SLURP1, combined with SLURP1 knockdown or overexpression cell lines, showed that SLURP1 is required for E. coli K1 invasion and neutrophils migrating across the blood-brain barrier (BBB). Furthermore, we found that SLURP1 is required for E. coli K1-induced α7 nAChR activation. Finally, the promoting effects of SLURP1 on the pathogenesis of E. coli K1 meningitis was significantly abolished in the α7 nAChR knockout mice. These results reveal that E. coli K1 exploits SLURP1 to activate α7 nAChR and facilitate its pathogenesis, and blocking SLURP1-α7 nAChR interaction might represent a novel therapeutic strategy for E. coli K1 meningitis.

Neuroinflammation Modulation via α7 Nicotinic Acetylcholine Receptor and Its Chaperone, RIC-3.

Nicotinic acetylcholine receptors (nAChRs) are widely expressed in or on various cell types and have diverse functions. In immune cells nAChRs regulate proliferation, differentiation and cytokine release. Specifically, activation of the α7 nAChR reduces inflammation as part of the cholinergic anti-inflammatory pathway. Here we review numerous effects of α7 nAChR activation on immune cell function and differentiation. Further, we also describe evidence implicating this receptor and its chaperone RIC-3 in diseases of the central nervous system and in neuroinflammation, focusing on multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Deregulated neuroinflammation due to dysfunction of α7 nAChR provides one explanation for involvement of this receptor and of RIC-3 in neurodegenerative diseases. In this review, we also provide evidence implicating α7 nAChRs and RIC-3 in neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) involving neuroinflammation. Besides, we will describe the therapeutic implications of activating the cholinergic anti-inflammatory pathway for diseases involving neuroinflammation.

Differential signalling induced by α7 nicotinic acetylcholine receptors in hippocampal dentate gyrus in vitro and in vivo

The activation of α7 nicotinic acetylcholine receptors (nAChRs) has been shown to improve hippocampus-dependent learning and memory. α7 nAChRs are densely expressed among several different cell types in the hippocampus, with high Ca2+ permeability, although it is unclear if α7 nAChRs mobilize differential signalling mechanisms among distinct neuronal populations. To address this question, we compared α7 nAChR agonist-induced responses (i.e. calcium and cAMP changes) between granule cells and GABAergic neurons in the hippocampal dentate gyrus both in vitro and in vivo. In cultured organotypic hippocampal slices, we observed robust intracellular calcium and cAMP increases in dentate granule cells upon activation of α7 nAChRs. In contrast, GABAergic interneurons displayed little change in either calcium or cAMP concentration after α7 nAChR activation, even though they displayed much larger α7 nAChR current responses than those of dentate granule cells. We found that this was due to smaller α7 nAChR-induced Ca2+ rises in GABAergic interneurons. Thus, the regulation of the Ca2+ transients in different cell types resulted in differential subsequent intracellular signalling cascades and likely the ultimate outcome of α7 nAChR activation. Furthermore, we monitored neuronal activities of dentate granule cells and GABAergic interneurons in vivo via optic fibre photometry. We observed enhancement of neuronal activities after nicotine administration in dentate granule cells, but not in GABAergic neurons, which was absent in α7 nAChR-deficient granule cells. In summary, we reveal a mechanism for α7 nAChR-mediated increase of neuronal activity via cell type-specific intracellular signalling pathways. KEY POINTS: α7 nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the central nervous system and regulate a variety of brain functions including learning and memory. Understanding the cellular signalling mechanisms of their activations among different neuronal populations is important for delineating their actions in cognitive function, and developing effective treatment strategies for cognitive deficits. We report that α7 nAChR activation leads to Ca2+ and cAMP increases in granule cells (but not in GABAergic interneurons) in hippocampal dentate gyrus in vitro, a key region for pattern separation during learning. We also found that nicotine enhanced granule cell (but not in GABAergic interneurons) activity in an α7 nAChR-dependent manner via in vivo fibre photometry recording. Based on our findings, we propose that differential responses to α7 nAChR activation between granule cells and GABAergic interneurons is responsible for the increase of excitation by α7 nAChR agonists in hippocampal circuits synergistically.

Geniposide protects pulmonary arterial smooth muscle cells from lipopolysaccharide‑induced injury via α7nAchR‑mediated TLR‑4/MyD88 signaling

Geniposide is a bioactive iridoid glucoside derived from Gardenia jasminoides that has proven anti-inflammatory effects against acute lung injury. The aim of this study was to determine whether geniposide could protect pulmonary arterial smooth muscle cells (PASMCs) from lipopolysaccharide (LPS)-induced injury and to explore the participation of α7 nicotinic acetylcholine receptor (α7nAChR), which was previously reported to suppress pro-inflammatory cytokine production in LPS-stimulated macrophages. In the present study, rat PASMCs were isolated and stimulated using LPS. The effect of geniposide on LPS-induced PASMC injury was then explored. Geniposide exerted anti-apoptotic and anti-inflammatory effects on LPS-treated PASMCs, as demonstrated by the downregulation of pro-apoptotic proteins and pro-inflammatory cytokines, respectively. Furthermore, the α7nAChR agonist PNU282987 accentuated the protective effect of geniposide against LPS-induced injury in PASMCs by inhibiting toll-like receptor-4/myeloid differentiation primary response 88 (TLR-4/MyD88) signaling and downregulating nuclear factor (NF)-κB expression. Conversely, methyllycaconitine, an inhibitor of α7nAChR, attenuated the effects of geniposide. These findings collectively suggested that in conjunction with geniposide, the activation of α7nAChR may contribute to further mitigating LPS-induced PASMC apoptosis and inflammation. In addition, the underlying mechanisms critically involve the NF-κB/MyD88 signaling axis. These results may provide novel insights into the treatment and management of lung diseases via geniposide administration.

The Allosteric Activation of α7 nAChR by α-Conotoxin MrIC Is Modified by Mutations at the Vestibular Site.

α-conotoxins are 13–19 amino acid toxin peptides that bind various nicotinic acetylcholine receptor (nAChR) subtypes. α-conotoxin Mr1.7c (MrIC) is a 17 amino acid peptide that targets α7 nAChR. Although MrIC has no activating effect on α7 nAChR when applied by itself, it evokes a large response when co-applied with the type II positive allosteric modulator PNU-120596, which potentiates the α7 nAChR response by recovering it from a desensitized state. A lack of standalone activity, despite activation upon co-application with a positive allosteric modulator, was previously observed for molecules that bind to an extracellular domain allosteric activation (AA) site at the vestibule of the receptor. We hypothesized that MrIC may activate α7 nAChR allosterically through this site. We ran voltage-clamp electrophysiology experiments and in silico peptide docking calculations in order to gather evidence in support of α7 nAChR activation by MrIC through the AA site. The experiments with the wild-type α7 nAChR supported an allosteric mode of action, which was confirmed by the significantly increased MrIC + PNU-120596 responses of three α7 nAChR AA site mutants that were designed in silico to improve MrIC binding. Overall, our results shed light on the allosteric activation of α7 nAChR by MrIC and suggest the involvement of the AA site.

The anti-inflammatory role of α7 nicotinic acetylcholine receptor in microglial cells and its mechanisms

Objective To investigate the anti-inflammatory role of α7 nicotinic acetylcholine receptor (α7nAChR) under inflammatory stress and its mechanisms. Methods PNU282987 was used for the activation of α7nAChR and LPS was administrated as inflammatory stressor. Realtime PCR was used for the detection of IL-1β, IL-6, TNF-α, M1 macrophage marker CD68, CD86 and M2 macrophage marker CD206, Arg1. Cell immunofluorescence was used for the detection of M1/M2 ratio and Western blot was applied for the detection of autophagy-related proteins. Results Under the stimulation of LPS, the mRNA levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α, the proportion of M1 macrophage and autophagy process were increased in BV2 microglial cells. However, the administration of PNU282987 significantly decreased the mRNA levels of IL-1β, IL-6 and TNF-α and the proportion of M1 macrophage while increased the proportion of M2 macrophage and the level of autophagy process. Conclusion Activating α7nAChR plays an anti-inflammatory role in microglial cells under inflammatory stress due to the regulation of M1/M2 macrophage ratio and increase of autophagy level.

Nicotine promotes vascular calcification via intracellular Ca2+-mediated, Nox5-induced oxidative stress, and extracellular vesicle release in vascular smooth muscle cells

AimsSmokers are at increased risk of cardiovascular events. However, the exact mechanisms through which smoking influences cardiovascular disease resulting in accelerated atherosclerosis and vascular calcification are unknown. The aim of this study was to investigate effects of nicotine on initiation of vascular smooth muscle cell (VSMC) calcification and to elucidate underlying mechanisms.Methods and resultsWe assessed vascular calcification of 62 carotid lesions of both smoking and non-smoking patients using ex vivo micro-computed tomography (µCT) scanning. Calcification was present more often in carotid plaques of smokers (n = 22 of 30, 73.3%) compared to non-smokers (n = 11 of 32, 34.3%; P < 0.001), confirming higher atherosclerotic burden. The difference was particularly profound for microcalcifications, which was 17-fold higher in smokers compared to non-smokers. In vitro, nicotine-induced human primary VSMC calcification, and increased osteogenic gene expression (Runx2, Osx, BSP, and OPN) and extracellular vesicle (EV) secretion. The pro-calcifying effects of nicotine were mediated by Ca2+-dependent Nox5. SiRNA knock-down of Nox5 inhibited nicotine-induced EV release and calcification. Moreover, pre-treatment of hVSMCs with vitamin K2 ameliorated nicotine-induced intracellular oxidative stress, EV secretion, and calcification. Using nicotinic acetylcholine receptor (nAChR) blockers α-bungarotoxin and hexamethonium bromide, we found that the effects of nicotine on intracellular Ca2+ and oxidative stress were mediated by α7 and α3 nAChR. Finally, we showed that Nox5 expression was higher in carotid arteries of smokers and correlated with calcification levels in these vessels.ConclusionIn this study, we provide evidence that nicotine induces Nox5-mediated pro-calcific processes as novel mechanism of increased atherosclerotic calcification. We identified that activation of α7 and α3 nAChR by nicotine increases intracellular Ca2+ and initiates calcification of hVSMCs through increased Nox5 activity, leading to oxidative stress-mediated EV release. Identifying the role of Nox5-induced oxidative stress opens novel avenues for diagnosis and treatment of smoking-induced cardiovascular disease.

Activation of α7 nicotinic acetylcholine receptor ameliorates HIV-associated neurology and neuropathology.

HIV-associated neurocognitive disorders (HAND) in the era of combination antiretroviral therapy are primarily manifested as impaired behaviours, glial activation/neuroinflammation and compromised neuronal integrity, for which there are no effective treatments currently available.In the current study, we used doxycycline-inducible astrocyte-specific HIV Tat transgenic mice (iTat), a surrogate HAND model, and determined effects of PNU-125096, a positive allosteric modulator of α7 nicotinic acetylcholine receptor (α7 nAChR) on Tat-induced behavioural impairments and neuropathologies.We showed that PNU-125096 treatment significantly improved locomotor, learning and memory deficits of iTat mice while inhibited glial activation and increased PSD-95 expression in the cortex and hippocampus of iTat mice. Using α7 nAChR knockout mice, we showed that α7 nAChR knockout eliminated the protective effects of PNU-125096 on iTat mice. In addition, we showed that inhibition of p38 phosphorylation by SB239063, a p38 MAPK-specific inhibitor exacerbated Tat neurotoxicity in iTat mice. Last, we used primary mouse cortical individual cultures and neuron-astrocytes co-cultures and in vivo staining of iTat mouse brain tissues and showed that glial activation was directly involved in the interplay among Tat neurotoxicity, α7 nAChR activation and the p38 MAPK signalling pathway.Taken together, these findings demonstrated for the first time that α7 nAChR activation led to protection against HAND and suggested that α7 nAChR modulator PNU-125096 holds significant promise for development of therapeutics for HAND.

Cytisine Exerts an Anti-Epileptic Effect via α7nAChRs in a Rat Model of Temporal Lobe Epilepsy.

Background and Purpose: Temporal lobe epilepsy (TLE) is a common chronic neurological disease that is often invulnerable to anti-epileptic drugs. Increasing data have demonstrated that acetylcholine (ACh) and cholinergic neurotransmission are involved in the pathophysiology of epilepsy. Cytisine, a full agonist of α7 nicotinic acetylcholine receptors (α7nAChRs) and a partial agonist of α4β2nAChRs, has been widely applied for smoking cessation and has shown neuroprotection in neurological diseases. However, whether cytisine plays a role in treating TLE has not yet been determined. Experimental Approach: In this study, cytisine was injected intraperitoneally into pilocarpine-induced epileptic rats for three weeks. Alpha-bungarotoxin (α-bgt), a specific α7nAChR antagonist, was used to evaluate the mechanism of action of cytisine. Rats were assayed for the occurrence of seizures and cognitive function by video surveillance and Morris water maze. Hippocampal injuries and synaptic structure were assessed by Nissl staining and Golgi staining. Furthermore, levels of glutamate, γ-aminobutyric acid (GABA), ACh, and α7nAChRs were measured. Results: Cytisine significantly reduced seizures and hippocampal damage while improving cognition and inhibiting synaptic remodeling in TLE rats. Additionally, cytisine decreased glutamate levels without altering GABA levels, and increased ACh levels and α7nAChR expression in the hippocampi of TLE rats. α-bgt antagonized the above-mentioned effects of cytisine treatment. Conclusion and Implications: Taken together, these findings indicate that cytisine exerted an anti-epileptic and neuroprotective effect in TLE rats via activation of α7nAChRs, which was associated with a decrease in glutamate levels, inhibition of synaptic remodeling, and improvement of cholinergic transmission in the hippocampus. Hence, our findings not only suggest that cytisine represents a promising anti-epileptic drug, but provides evidence of α7nAChRs as a novel therapeutic target for TLE.

α7 Nicotinic acetylcholine receptor mediates right ventricular fibrosis and diastolic dysfunction in pulmonary hypertension.

Right ventricular (RV) fibrosis is a key feature of maladaptive RV hypertrophy and dysfunction and is associated with poor outcomes in pulmonary hypertension (PH). However, mechanisms and therapeutic strategies to mitigate RV fibrosis remain unrealized. Previously, we identified that cardiac fibroblast α7 nicotinic acetylcholine receptor (α7 nAChR) drives smoking-induced RV fibrosis. Here, we sought to define the role of α7 nAChR in RV dysfunction and fibrosis in the settings of RV pressure overload as seen in PH. We show that RV tissue from PH patients has increased collagen content and ACh expression. Using an experimental rat model of PH, we demonstrate that RV fibrosis and dysfunction are associated with increases in ACh and α7 nAChR expression in the RV but not in the left ventricle (LV). In vitro studies show that α7 nAChR activation leads to an increase in adult ventricular fibroblast proliferation and collagen content mediated by a Ca2+/epidermal growth factor receptor (EGFR) signaling mechanism. Pharmacological antagonism of nAChR decreases RV collagen content and improves RV function in the PH model. Furthermore, mice lacking α7 nAChR exhibit improved RV diastolic function and have lower RV collagen content in response to persistently increased RV afterload, compared with WT controls. These finding indicate that enhanced α7 nAChR signaling is an important mechanism underlying RV fibrosis and dysfunction, and targeted inhibition of α7 nAChR is a potentially novel therapeutic strategy in the setting of increased RV afterload.

Cynandione A and PHA-543613 inhibit inflammation and stimulate macrophageal IL-10 expression following α7 nAChR activation

Cynandione A, an acetophenone isolated from Cynanchum Wilfordii Radix, attenuates inflammation. The present study aimed to study the mechanisms underlying cynandione A-induced antiinflammation. Treatment with cynandione A and the specific α7 nicotinic acetylcholine receptor (α7 nAChR) agonist PHA-543613 remarkably reduced overexpression of proinflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β in lipopolysaccharide (LPS)-treated RAW264.7 cells and primary peritoneal macrophages, and endotoxemic mice. Both cynandione A and PHA-543613 also stimulated IL-10 expression in naïve and LPS-treated macrophages and endotoxemic mice. Cynandione A- and PHA-543613-inhibited proinflammatory cytokine expression was completely blocked by the α7 nAChR antagonist methyllycaconitine and the IL-10 antibody. The stimulatory effect of cynandione A and PHA-543613 on IL-10 expression were suppressed by methyllycaconitine and knockdown of α7 nAChRs using siRNA/α7 nAChR. Cynandione A significantly stimulated STAT3 phosphorylation, which was attenuated by methyllycaconitine and the IL-10 neutralizing antibody. The STAT3 activation inhibitor NSC74859 also blocked cynandione A-inhibited proinflammatory cytokine expression. Taken together, our results, for the first time, demonstrate that cynandione A and PHA-543613 inhibit inflammation through macrophageal α7 nAChR activation and subsequent IL-10 expression.

Autophagy is Involved in Neuroprotective Effect of Alpha7 Nicotinic Acetylcholine Receptor on Ischemic Stroke.

The α7 nicotinic acetylcholine receptor (α7nAChR) belongs to the superfamily of cys loop cationic ligand-gated channels, which consists of homogeneous α7 subunits. Although our lab found that activation of α7nAChR could alleviate ischemic stroke, the mechanism is still unknown. Herein, we explored whether autophagy is involved in the neuroprotective effect mediated by α7nAChR in ischemic stroke. Transient middle cerebral artery occlusion (tMCAO) and oxygen and glucose deprivation (OGD/R) exposure were applied to in vivo and in vitro models of ischemic stroke, respectively. Neurological deficit score and infarct volume were used to evaluate outcomes of tMCAO in the in vivo study. Autophagy-related proteins were detected by Western blot, and autophagy flux was detected by using tandem fluorescent mRFP-GFP-LC3 lentivirus. At 24 h after tMCAO, α7nAChR knockout mice showed worse neurological function and larger infarct volume than wild-type mice. PNU282987, an α7nAChR agonist, protected against OGD/R-induced neuronal injury, enhanced autophagy, and promoted autophagy flux. However, the beneficial effects of PNU282987 were eliminated by 3-methyladenine (3-MA), an autophagy inhibitor. Moreover, we found that PNU282987 treatment could activate the AMPK-mTOR-p70S6K signaling pathway in the in vitro study, while the effect was attenuated by compound C, an AMPK inhibitor. Our results demonstrated that the beneficial effect on neuronal survival via activation of α7nAChR was associated with enhanced autophagy, and the AMPK-mTOR-p70S6K signaling pathway was involved in α7nAChR activation–mediated neuroprotection.