Nicotinic Acetylcholine Receptor Α7 Subunit Research Articles

Activation of nicotinic acetylcholine receptor α7 subunit limits Zika viral infection via promoting autophagy and ferroptosis

Vagus nerve regulates viral infection and inflammation via the alpha 7 nicotinic acetylcholine receptor (α7 nAChR); however, the role of α7 nAChR in ZIKA virus (ZIKV) infection, which can cause severe neurological diseases such as microcephaly and Guillain-Barré syndrome, remains unknown. Here, we first examined the role of α7 nAChR in ZIKV infection in vitro. A broad effect of α7 nAChR activation was identified in limiting ZIKV infection in multiple cell lines. Combined with transcriptomics analysis, we further demonstrated that α7 nAChR activation promoted autophagy and ferroptosis pathways to limit cellular ZIKV viral loads. Additionally, activation of α7 nAChR prevented ZIKV-induced p62 nucleus accumulation, which mediated an enhanced autophagy pathway. By regulating proteasome complex and an E3 ligase NEDD4, activation of α7 nAChR resulted in increased amount of cellular p62, which further enhanced ferroptosis pathway to reduce ZIKV infection. Moreover, utilizing in vivo neonatal mouse models, we showed that α7 nAChR is essential in controlling the disease severity of ZIKV infection. Taken together, our findings identify an α7 nAChR-mediated effect that critically contributes to limiting ZIKV infection, and α7 nAChR activation offers a novel strategy for combating ZIKV infection and its complications.

Transcutaneous auricular vagus nerve stimulation promotes gastric motility by up-rgulating α7nAChR and suppressing NF-κB p65 expression in duodenum in rats with functional dyspepsia

To study the effect of transcutaneous auricular vagus nerve stimulation (taVNS) on gastric sensitivity and motility in rats with functional dyspepsia (FD), so as to explore its underlying mechanism in improving FD. A total of 48 young SD rats were randomly divided into control (n=10), model (n=9), taVNS (n=9), subdiaphragmatic vagus nerve stimulation (SDVNS, n=9) and sham SDVNS (n=7) groups. The FD model was established by gavage of 0.1% iodoa-cetamide+2% glucose, once daily for 6 days. Rats in the taVNS group received taVNS (0.5 mA) of optopoint "Heart" and "Stomach" for 30 min, once daily for 14 days, while rats in the SDVNS group received subdiaphragmatic vagus nerve stimulation through the implanted electrode, and those of the sham SDVNS group received only application of the same electrodes without electrical stimulation. Electromyogram (EMG) of the cervical trapezius muscle (reflecting gastric sensitivity) was recorded before and after intragastric expansion via an air ballon and the gastric emptying rate was calculated for assessing the gastric motility. The contents of acetylcholine (ACh), nicotinic acetylcholine receptor α7 subunit (α7nAChR), interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the duodenum tissue were detected by enzyme-linked immunosorbent assay (ELISA). The expression of nuclear factor kappa B (NF-κB) p65 in the duodenum tissue was determined by Western blot. In comparison with the control group, the EMG change rate at intragastric pressure levels of 40, 60 and 80 mm Hg, expression of NF-κB p65 protein, and contents of IL-6, IL-1β and TNF-α were significantly increased (P<0.05,P<0.01, P<0.001), while the gastric emptying rate, ACh and α7nAChR contents considerably decreased (P<0.05, P<0.001) in the model group. After interventions, the EMG change rate, contents of IL-6, IL-1β and TNF-α, and expression of NF-κB p65 were notably decreased (P<0.05, P<0.01, P<0.001), and the gastric emptying rate, ACh and α7nAChR contents obviously increased (P<0.05, P<0.001) in both taVNS and SDVNS groups relevant to the model group. In comparison with the sham SDVNS group, the EMG change rate, contents of IL-6, IL-1β and TNF-α, and expression of NF-κB p65 were notably decreased (P<0.01, P<0.05,P<0.001), and the gastric emptying rate, ACh and α7nAChR contents obviously increased (P<0.01, P<0.001) in the both SDVNS and taVNS groups. taVNS can reduce gastric sensitivity and promote gastric emptying in FD model rats, which may be closely related to its functions in up-regulating ACh and α7nAChR contents and inhibiting the activation of NF-κB p65 signaling in the duodenum.

Vagus nerve stimulation promotes resolution of inflammation by a mechanism that involves Alox15 and requires the α7nAChR subunit

Nonresolving inflammation underlies a range of chronic inflammatory diseases, and therapeutic acceleration of resolution of inflammation may improve outcomes. Neural reflexes regulate the intensity of inflammation (for example, through signals in the vagus nerve), but whether activation of the vagus nerve promotes the resolution of inflammation in vivo has been unknown. To investigate this, mice were subjected to electrical vagus nerve stimulation (VNS) or sham surgery at the cervical level followed by zymosan-induced peritonitis. The duration of inflammation resolution was significantly reduced and efferocytosis was significantly increased in mice treated with VNS as compared with sham. Lipid mediator (LM) metabololipidomics revealed that mice treated with VNS had higher levels of specialized proresolving mediators (SPMs), particularly from the omega-3 docosahexaenoic (DHA) and docosapentaenoic (n-3 DPA) metabolomes, in peritoneal exudates. VNS also shifted the ratio between proinflammatory and proresolving LMs toward a proresolving profile, but this effect by VNS was inverted in mice deficient in 12/15-lipoxgenase (Alox15), a key enzyme in this SPM biosynthesis. The significant VNS-mediated reduction of neutrophil numbers in peritoneal exudates was absent in mice deficient in the cholinergic α7-nicotinic acetylcholine receptor subunit (α7nAChR), an essential component of the inflammatory reflex. Thus, VNS increased local levels of SPM and accelerated resolution of inflammation in zymosan-induced peritonitis by a mechanism that involves Alox15 and requires the α7nAChR.

Electroacupuncture Ameliorates Intestinal Barrier Destruction in Mice With Bile Duct Ligation–Induced Liver Injury by Activating the Cholinergic Anti-Inflammatory Pathway

ObjectivesElectroacupuncture (EA) at Zusanli (ST36) can attenuate inflammation in different rodent models. However, the therapeutic mechanisms underlying its action in inhibiting intestinal barrier destruction and liver injury in cholestasis mice have not been clarified. This study aimed at investigating whether EA at ST36 could activate the cholinergic anti-inflammatory pathway to inhibit intestinal barrier destruction and liver injury in cholestasis mice. Materials and MethodsMale Hmox1floxp/floxp C57BL/6 mice were randomized and subjected to a sham or bile duct ligation (BDL) surgery. The BDL mice were randomized and treated with, or without (BDL group), sham EA at ST36 (BDL+sham-ST36) or EA at ST36 (BDL+ST36), or received α-bungarotoxin (α-BGT), a specific inhibitor of nicotinic acetylcholine receptor α7 subunit (α7nAChR), before stimulation (BDL+ST36+α-BGT). These mice, together with a group of intestine-specific heme oxygenase-1 (HO-1) knockout (KO) Villin-Cre-HO-1−/− mice, were monitored for their body weights before and 14 days after BDL. The levels of plasma cytokines and liver injury–related alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by enzyme-linked immunoassay, and pathological changes in the intestinal mucosa and liver fibrosis as well as intestinal barrier permeability in individual mice were examined by histology and immunohistochemistry. The levels of α7nAChR, HO-1, ZO-1, Occludin, Claudin-1, and NF-κBp65 expression and NF-κBp65 phosphorylation in intestinal tissues were quantified. ResultsCompared with the sham group, BDL significantly increased the levels of plasma interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor α, ALT, and AST and caused intestinal mucosal damages, high permeability, and liver fibrosis in mice, which were remarkably mitigated, except for further increased levels of plasma IL-10 in the BDL+ST36 group of mice. Similarly, EA at ST36 significantly up-regulated α7nAChR and HO-1 expression; mitigated the BDL-decreased ZO-1, Occludin, and Claudin-1 expression; and attenuated the BDL-increased NF-κBp65 phosphorylation in intestinal tissues of mice. The therapeutic effects of EA at ST36 were significantly abrogated by pretreatment with α-BGT or HO-1 KO. ConclusionEA at ST36 inhibits the BDL-induced intestinal mucosal damage and liver fibrosis by activating the HO-1 cholinergic anti-inflammatory pathway in intestinal tissues of mice.

Neuroprotective effect of alpha-pinene is mediated by suppression of the TNF-α/NF-κB pathway in Alzheimer's disease rat model.

Monoterpene alpha-pinene possesses antioxidant, cardioprotective, and neuroprotective properties. We evaluated the effect of alpha-pinene on oxidative/nitrosative stress, neuroinflammation, and molecular and behavioral changes induced by beta-amyloid (Aβ)1-42 in rats and investigated the possible mechanisms of these outcomes. Male Wistar rats received alpha-pinene (50 mg/kg intraperitoneally) for 14 consecutive days after intrahippocampal injection of Aβ1-42 . Alpha-pinene decreased malondialdehyde and nitric oxide levels, increased glutathione content, and enhanced catalase activity in Aβ-injected rats. Also, messenger RNA expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, nuclear factor κB, and N-methyl- d-aspartate receptor subunits 2A and 2B reduced in the hippocampus of these animals. Besides this, alpha-pinene repressed the Aβ1-42 -induced reduction of nicotinic acetylcholine receptor α7 subunit and brain-derived neurotrophic factor expression. Treatment with alpha-pinene caused Aβ-receiving rats to spend more time in the target quadrant in the Morris water maze test and led to an increase in percentages of open arm entrance and time spent in the open arm in the elevated plus-maze test. We concluded that alpha-pinene strengthens the antioxidant system and prevents neuroinflammation in the hippocampus of rats receiving Aβ. It improves spatial learning and memory and reduces anxiety-like behavior in these animals. Consequently, alpha-pinene alleviates Aβ-induced oxidative/nitrosative stress, neuroinflammation, and behavioral deficits. It is probably a suitable candidate for the treatment of neurodegenerative diseases.

Interaction of α9α10 Nicotinic Receptors With Peptides and Proteins From Animal Venoms.

Unlike most neuronal nicotinic acetylcholine receptor (nAChR) subunits, α7, α9, and α10 subunits are able to form functional homo- or heteromeric receptors without any β subunits. While the α7 subtype is widely distributed in the mammalian brain and several peripheral tissues, α9 and α9α10 nAChRs are mainly found in the cochlea and immune cells. α-Conotoxins that specifically block the α9α10 receptor showed anti-nociceptive and anti-hyperalgesic effects in animal models. Hence, this subtype is considered a drug target for analgesics. In contrast to the α9α10-selective α-conotoxins, the three-finger toxin α-bungarotoxin inhibits muscle-type and α7 nAChRs in addition to α9α10 nAChRs. However, the selectivity of α-neurotoxins at the α9α10 subtype was less intensively investigated. Here, we compared the potencies of α-conotoxins and α-neurotoxins at the human α9α10 nAChR by two-electrode voltage clamp analysis upon expression in Xenopus oocytes. In addition, we analyzed effects of several α9α10-selective α-conotoxins on mouse granulocytes from bone marrow to identify possible physiological functions of the α9α10 nAChR subtype in these cells. The α-conotoxin-induced IL-10 release was measured upon LPS-stimulation. We found that α-conotoxins RgIA, PeIA, and Vc1.1 enhance the IL-10 expression in granulocytes which might explain the known anti-inflammatory and associated analgesic activities of α9α10-selective α-conotoxins. Furthermore, we show that two long-chain α-neurotoxins from the cobra Naja melanoleuca venom that were earlier shown to bind to muscle-type and α7 nAChRs, also inhibit the α9α10 subtype at nanomolar concentrations with one of them showing a significantly slower dissociation from this receptor than α-bungarotoxin.

Nicotinic Acetylcholine Receptor α7 Subunit Is an Essential Regulator of Seizure Susceptibility.

A large body of data has confirmed that α7 nicotinic acetylcholine receptors (nAChRs) play a pivotal role in cognition, memory, and other neuropsychiatric diseases, but their effect on seizure susceptibility in C57BL/6 wild-type mice is not fully understood. Here, we showed that decreased activity of α7 nAChRs could increase the excitability of CA1 pyramidal neurons and shorten the onset time of epilepsy in pilocarpine-induced mouse models. However, compared with the control group, there was no apparent effect of increasing the activity of α7 nAChRs. Moreover, the expression of α7 nAChRs is downregulated in human epileptogenic tissues. Taken together, our findings indicate that α7 nAChR is an essential regulator of seizure susceptibility.

토끼 방향특이성 망막 신경절세포의 니코티닉 아세틸콜린 수용체 α7과 β4 소단위의 위치 측정

토끼 방향특이성 망막 신경절세포의 니코티닉 아세틸콜린 수용체 α7과 β4 소단위의 위치 측정

Expression levels of the α7 nicotinic acetylcholine receptor in the brains of patients with Alzheimer's disease and their effect on synaptic proteins in SH-SY5Y cells.

Alzheimer's disease (AD) is a chronic neurodegenerative, and abnormal aggregation of the neurotoxic β amyloid (Aβ) peptide is an early event in AD. The present study aimed to determine the correlation between the nicotinic acetylcholine receptor α7 subunit (α7 nAChR) and Aβ in the brains of patients with AD, and to investigate whether the increased expression levels of the α7 nAChR could alter the neurotoxicity of Aβ. The expression levels of α7 nAChR and Aβ in the brains of patients with AD and healthy brains were analyzed using immunofluorescence. Moreover, SH-SY5Y cells were used to stably overexpress or silence α7 nAChR expression levels, prior to the treatment with or without 1 µmol/l Aβ1-42 oligomer (AβO). The mRNA and protein expression levels of α7 nAChR, synaptophysin (SYP), postsynaptic density of 95 kDa (PSD-95) and synaptosomal-associated protein of 25 kDa (SNAP-25) were subsequently analyzed using reverse transcription-quantitative PCR and western blotting. In addition, the concentration of acetylcholine (ACh) and the activity of acetylcholinesterase (AChE) were analyzed using spectrophotometry, while the cell apoptotic rate was determined using flow cytometry. The expression of Aβ in the brains of patients with AD was found to be significantly increased, whereas the expression of α7 nAChR was significantly decreased compared with the healthy control group. In vitro, the expression levels of α7 nAChR were significantly increased or decreased following the overexpression or silencing of the gene, respectively. Consistent with these observations, the mRNA and protein expression levels of SYP, PSD-95 and SNAP-25 were also significantly increased following the overexpression of α7 nAChR and decreased following the genetic silencing of the receptor. In untransfected or negative control cells, the expression levels of these factors and the apoptotic rate were significantly reduced following the exposure to AβO, which was found to be attenuated by α7 nAChR overexpression, but potentiated by α7 nAChR RNA silencing. However, no significant differences were observed in either the ACh concentration or AChE activity following transfection. Collectively, these findings suggested that α7 nAChR may protect the brains of patients with AD against Aβ, as α7 nAChR overexpression increased the expression levels of SYP, SNAP-25 and PSD-95, and attenuated the inhibitory effect of Aβ on the expression of these synaptic proteins and cell apoptosis. Overall, this indicated that α7 nAChR may serve an important neuroprotective role in AD.

RETRACTED: Nicotinic acetylcholine receptor α7 subunit maybe a risk factor of seizure susceptibility

RETRACTED: Nicotinic acetylcholine receptor α7 subunit maybe a risk factor of seizure susceptibility

Short-Term High-Fat Diet Consumption Reduces Hypothalamic Expression of the Nicotinic Acetylcholine Receptor α7 Subunit (α7nAChR) and Affects the Anti-inflammatory Response in a Mouse Model of Sepsis.

Sepsis is one of the leading causes of death in hospitalized patients and the chronic and low-grade inflammation observed in obesity seems to worsen susceptibility and morbidity of infections. However, little is known with respect to a short-term high-fat diet (HFD) and its role in the development of sepsis. Here, we show for the first time, that short-term HFD consumption impairs early nicotinic acetylcholine receptor α7 subunit (α7nAChR)- mediated signaling, one of the major components of the cholinergic anti-inflammatory pathway, with a focus on hypothalamic inflammation and innate immune response. Mice were randomized to a HFD or standard chow (SC) for 3 days, and sepsis was subsequently induced by a lethal intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) or by cecal ligation and puncture (CLP) surgery. In a separate experiment, both groups received LPS (i.p.) or LPS (i.p.) in conjunction with the selective α7nAChR agonist, PNU-282987 (i.p. or intracerebroventricular; i.c.v.), and were sacrificed 2 h after the challenge. Short-term HFD consumption significantly reduced the α7nAChR mRNA and protein levels in the hypothalamus and liver (p < 0.05). Immunofluorescence microscopy demonstrated lower cholinergic receptor nicotinic α7 subunit (α7nAChR)+ cells in the arcuate nucleus (ARC) (α7nAChR+ cells in SC = 216 and HFD = 84) and increased F4/80+ cells in the ARC (2.6-fold) and median eminence (ME) (1.6-fold), which can contribute to neuronal damage. Glial fibrillary acidic protein (GFAP)+ cells and neuronal nuclear antigen (NeuN)+ cells were also increased following consumption of HFD. The HFD-fed mice died quickly after a lethal dose of LPS or following CLP surgery (2-fold compared with SC). The LPS challenge raised most cytokine levels in both groups; however, higher levels of TNF-α (Spleen and liver), IL-1β and IL-6 (in all tissues evaluated) were observed in HFD-fed mice. Moreover, PNU-282987 administration (i.p. or i.c.v.) reduced the levels of inflammatory markers in the hypothalamus following LPS injection. Nevertheless, when the i.c.v. injection of PNU-282987 was performed the anti-inflammatory effect was much smaller in HFD-fed mice than SC-fed mice. Here, we provide evidence that a short-term HFD impairs early α7nAChR expression in central and peripheral tissues, contributing to a higher probability of death in sepsis.

Sensitization-dependent nicotine place preference in the adult zebrafish

Sensitization of motor activity is a behavioural test to evaluate the effects of psychostimulants. Conditioned place preference (CPP) is an associative learning procedure to examine the rewarding properties of drugs. We aimed to assess whether motor sensitization to drugs of abuse can make zebrafish more vulnerable to establishing drug-induced CPP. We first evaluated sensitization of locomotor activity of zebrafish to repeated administrations of nicotine and cocaine during 5 days and after 5 days of withdrawal. After withdrawal, when zebrafish were re-exposed to the same dose of nicotine or cocaine locomotor activity was increased by 103% and 166%, respectively.Different groups of zebrafish were sensitized to nicotine or cocaine and trained on a nicotine-CPP task the day after withdrawal. The nicotine dose selected for sensitization was not effective for developing CPP in naïve zebrafish whereas it elicited CPP in zebrafish that were previously sensitized to nicotine or cocaine. Levels of nicotinic acetylcholine receptor β2, α6 and α7 subunit, Pitx3, and tyrosine hydroxylase 1 (TH1) mRNAs were increased in the brain of nicotine- and cocaine-sensitized zebrafish. Nicotine-CPP performed with drug-sensitized zebrafish provoked further enhancements in the expression of α6 and α7 subunit, Pitx3, and TH1 mRNAs suggesting that the expression of these molecules in the reward pathway is involved in both processes. Our findings indicate that repeated exposures to low doses of drugs of abuse can increase subject's sensitivity to the rewarding properties of the same or different drugs. This further suggests that casual drug intake increases the probability of becoming addict.

Electro-acupuncture regulates the cholinergic anti-inflammatory pathway in a rat model of chronic obstructive pulmonary disease

ObjectiveAcupuncture has a definite therapeutic effect on chronic obstructive pulmonary disease (COPD), and the cholinergic anti-inflammatory pathway (CAP) has been shown to be involved in regulation of inflammation. In this study, we investigated whether electro-acupuncture (EA) affects the CAP in COPD. MethodsSprague–Dawley rats were induced into COPD through exposure to cigarette smoke combined with lipopolysaccharide. EA treatment was applied at Zusanli (ST36) and Feishu (BL13) points for 30 min/d for 7 d. Seventy-two rats were randomly divided into six study groups, including normal, normal + EA, normal + α-bungarotoxin (α-BGT) (the antagonist of the nicotinic acetylcholine receptor α7 subunit (α7nAChR)) + EA, COPD, COPD + EA, and COPD + α-BGT + EA. Lung function, pathology and vagus nerve discharge were tested. The levels of acetylcholine (ACh), acetylcholinesterase (AChE), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid (BALF) and lung tissue were measured by enzyme-linked immunosorbent assay. The mRNA and protein expression and immunoreactivity of α7nAChR and its postreceptor inflammation signal pathway, including janus kinase 2 (JAK2), signal transducers and activators of transcription 3 (STAT3), nuclear factor-κB (NF-κB), were observed by quantitative reverse transcription-polymerase chain reaction, Western blot and immunohistochemistry. ResultsCompared with normal rats, there were a significant decline in lung function and discharge of the vagus nerve (P < 0.01), a marked sign of lung inflammation and an increase of ACh, AChE, IL-6 and TNF-α level in BALF or lung tissue (P < 0.05, P < 0.01) and higher expression of α7nAChR, JAK2, STAT3 and NF-κB (P < 0.05, P < 0.01) in the COPD rats. In rats receiving EA, the lung function and vagal discharge were enhanced (P < 0.01), lung inflammation was improved and the levels of ACh, AChE, IL-6 and TNF-α were decreased (P < 0.01). Further, the expression of α7nAChR, JAK2, STAT3 and NF-κB was downregulated (P < 0.05, P < 0.01). However, the above effects of EA were blocked in rats injected with α-BGT (P < 0.01). ConclusionEA treatment can reduce the lung inflammatory response and improve lung function in COPD, which may be related to its involvement in the regulation of CAP.

Curcumin Acts as a Positive Allosteric Modulator of α7-Nicotinic Acetylcholine Receptors and Reverses Nociception in Mouse Models of Inflammatory Pain.

Effects of curcumin, a major ingredient of turmeric, were tested on the function of the α7-subunit of the human nicotinic acetylcholine (α7-nACh) receptor expressed in Xenopus oocytes and on nociception in mouse models of tonic and visceral pain. Curcumin caused a significant potentiation of currents induced by acetylcholine (ACh; 100 μM) with an EC50 value of 0.2 µM. The effect of curcumin was not dependent on the activation of G-proteins and protein kinases and did not involve Ca2+-dependent Cl- channels expressed endogenously in oocytes. Importantly, the extent of curcumin potentiation was enhanced significantly by decreasing ACh concentrations. Curcumin did not alter specific binding of [125I]α-bungarotoxin. In addition, curcumin attenuated nociceptive behavior in both tonic and visceral pain models without affecting motor and locomotor activity and without producing tolerance. Pharmacological and genetic approaches revealed that the antinociceptive effect of curcumin was mediated by α7-nACh receptors. Curcumin potentiated the antinociceptive effects of the α7-nACh receptor agonist N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-4-chlorobenzamide (PNU282987). Collectively, our results indicate that curcumin is a positive allosteric modulator of α7-nACh receptor and reverses nociception in mouse models of tonic and visceral pain.

The association between the nicotinic acetylcholine receptor α4 subunit gene (CHRNA4) rs1044396 and Internet gaming disorder in Korean male adults.

The primary aim of this study was to investigate the genetic predisposition of Internet gaming disorder (IGD), and the secondary aim was to compare the results to those of alcohol dependence (AD). Two independent case-control studies were conducted. A total of 30 male participants with IGD, diagnosed according to the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria, and 30 sex-matched controls participated in study 1. We designed targeted exome sequencing (TES) to test for 72 candidate genes that have been implicated in the pathogenesis of addiction. The genes included seven neurotransmitter (dopamine, serotonin, glutamate, r-aminobutyric acid (GABA), norepinephrine, acetylcholine, and opioid) system genes. A total of 31 male in-patients with AD and 29 normal male controls (NC) were enrolled in study 2. The same 72 genes included in study 1 and ten additional genes related to alcohol-metabolic enzyme were selected as the target genes, and we identified the genetic variants using the same method (TES). The IGD group had a lower frequency of the T allele of rs1044396 in the nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4), and this variant represents a protective allele against IGD. However, we did not find a significant difference in the polymorphisms of the 72 genes that encode neurotransmitter systems between the AD and NC groups. This study demonstrated that rs1044396 of CHRNA4 was significantly associated with IGD.

Nicotinic acetylcholine receptor α7 subunit improves energy homeostasis and inhibits inflammation in nonalcoholic fatty liver disease

ObjectiveNonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide; yet, the pathogenesis of the disorder is not completely understood. The nicotinic acetylcholine receptor α7 subunit (α7nAChR) plays an indispensable role in the vagus nerve-regulated cholinergic anti-inflammatory pathway. MethodsIn the present study, we investigated the key role of α7nAChR in NAFLD development. Male wild-type (WT) and α7nAChR knockout (α7nAChR−/−) mice were fed a normal chow or a high-fat diet (HFD) for 16weeks to induce NAFLD. ResultsWe found that both the mRNA and protein levels of α7nAChR in the liver tissue of NAFLD mice were significantly higher than those in mice fed normal chow. There were no differences in food intake, body weight, hepatic cholesterol and triglyceride contents, and insulin sensitivity between WT and α7nAChR−/− mice under normal condition. When the WT and α7nAChR−/− mice were challenged with HFD, the body weight of α7nAChR−/− mice became higher than that of WT mice. The oxygen consumption and energy expenditure in HFD-fed α7nAChR−/− mice were significantly lower than that in HFD-fed WT mice. The HFD-fed α7nAChR−/− mice also showed more aggravated hepatic lipid accumulation, steatosis and oxidative stress than HFD-fed WT mice. Macrophage infiltration; mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β; and liver fibrosis were significantly accelerated in HFD-fed α7nAChR−/− mice compared to that in HFD-fed WT mice. In addition, the bolus insulin injection-activated insulin signaling pathway, which was reflected by the phosphorylation of insulin receptor at Tyr1162/Tyr1163 site (p-IRTyr1162/Tyr1163), insulin receptor substrate-1 at Tyr612 site (p-IRS-1Tyr612) and Akt at Ser473 (p-AktSer473), was significantly compromised in liver tissues of HFD-fed α7nAChR−/− mice relative to HFD-fed WT mice. Finally, pharmacologically activation of α7nAChR in HFD-fed mice, with a selective agonist PNU-282987, remarkably ameliorated the hepatic steatosis, inflammatory cell infiltration and fibrosis. ConclusionIn conclusion, our results demonstrate that activation of α7nAChR improves energy homeostasis and inhibits inflammation in nonalcoholic fatty liver disease.

Involvement of arterial baroreflex and nicotinic acetylcholine receptor α7 subunit pathway in the protection of metformin against stroke in stroke-prone spontaneously hypertensive rats

Stroke is a leading cause of mortality and disability worldwide. There is growing evidence that metformin (Met) has potent neuroprotective effects; however, its mechanisms remain unclear. We examined the role of the arterial baroreflex and cholinergic-α7 nicotinic acetylcholine receptor (α7nAChR) anti-inflammory pathway in the beneficial effects of Met against stroke. Stroke-prone spontaneously hypertensive rats (SHRSP) were used to observe stroke development indicated by lifespan of SHRSP and the ischemic injury induced by permanent middle cerebral artery occlusion (MCAO). Sinoaortic denervation was used to inactivate the arterial baroreflex. MCAO were also performed in α7nAChR knockout (KO) mice. Briefly, Met increased the life span of SHRSP and reduced the infarct area induced by MCAO. Met also improved the function of arterial baroreflex. The beneficial effects of Met on stroke were markedly attenuated by blunting the arterial baroreflex. Met up-regulated the expression of vesicular acetylcholine transporter (VAChT) and α7nAChR, down-regulated the level of pro-inflammtory cytokines in serum and peri-infarct of ischemic brain. Arterial baroreflex dysfunction decreased the expression of VAchT and α7nAChR, showed upward tendency in the level of pro-inflammtory cytokines. Most importantly, arterial baroreflex dysfunction nearly abolished such effect of Met on cholinergic signaling. In addition, the α7nAChR KO mice also had significantly worse ischemic damage induced by MCAO, and neuroprotection of Met disappeared in α7nAChR KO mice. In conclusion, Met improved the arterial baroreflex function, and then enhancing cholinergic anti-inflammatory pathway in an α7nAChR-dependent manner, thereby effectively prevent ischemic induced brain injury and delayed stroke onset in SHRSP.

Effect of nicotinic acetylcholine receptor α7 subunit gene on liver inflammatory reaction in mice with nonalcoholic steatohepatitis and related mechanisms

Objective: To investigate the effect of nicotinic acetylcholine receptor α7 (α7nAChR) subunit gene on liver inflammation in mice with nonalcoholic steatohepatitis (NASH) and related mechanisms. Methods: C57BL/6J mice and α7nAChR gene knockout mice were fed for 24 weeks to establish the NASH model, and the mice were sacrificed to isolate and culture the primary liver macrophages. After the treatment with nicotine and endotoxin, ELISA was used to measure the levels of the inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in supernatant; indirect immunofluorescence assay and Western blot were used to observe the effect on the NF-κB signaling pathway, and quantitative PCR was used to measure the mRNA expression of Toll-like receptor-4 (TLR-4) in macrophages. An analysis of variance was used for comparison of means between multiple groups. Results: The results of ELISA showed that compared with the endotoxin+nicotine group of C57 NASH mice, the endotoxin+nicotine group of gene knockout NASH mice had significantly higher levels of IL-6 and TNF-α in supernatant (IL-6: 1 599±65 pg/ml vs 1 465±45 pg/ml, P < 0.05; TNF-α: 1 567±66 pg/ml vs 1 433±50 pg/ml, P < 0.05). The results of Western blot showed that compared with the endotoxin+nicotine group of C57 NASH mice, the endotoxin+nicotine group of gene knockout NASH mice had significantly higher relative protein expression of phosphorylated NF-κB and TLR-4 (NF-κB: 69 425±600 vs 51 133±200, P < 0.05; TLR-4: 93 387±684 vs 64 198±630, P < 0.05). The results of indirect immunofluorescence assay showed that the endotoxin+nicotine group of gene knockout NASH mice had a significantly higher fluorescence intensity of NF-κB than the endotoxin+nicotine group of C57 NASH mice. The results of PCR showed that the endotoxin+nicotine group of gene knockout NASH mice had significantly higher relative mRNA expression of TLR-4 than the endotoxin+nicotine group of C57 NASH mice (4.13±0.13 vs 2.93±0.14, P < 0.05). Conclusion: The α7nAChR gene knockout can aggravate the degree of inflammatory reaction in NASH, and its mechanism may be related to the fact that the NF-κB signaling pathway cannot be inhibited, which aggravates inflammatory reaction.

Blocking mPTP on Neural Stem Cells and Activating the Nicotinic Acetylcholine Receptor α7 Subunit on Microglia Attenuate Aβ-Induced Neurotoxicity on Neural Stem Cells.

β-Amyloid (Aβ) can stimulate microglia to release a variety of proinflammatory cytokines and induce neurotoxicity. Nicotine has been reported to inhibit TNF-α, IL-1, and ROS production in microglia. Mitochondrial permeability transition pore (mPTP) plays an important role in neurotoxicity as well. Here, we investigated whether activating the microglial α7-nAChR has a neuroprotective role on neural stem cells (NSCs) and the function of mPTP in NSCs in this process. The expression of α7-nAChR in rat NSCs was detected by immunocytochemistry and RT-PCR. The viability of microglia and NSCs was examined by MTT assay. The mitochondrial membrane potential (ΔΨm) and morphological characteristics of NSCs was measured by JC-1 staining and transmission electron microscopy respectively. The distribution of cytochrome c in the subcellular regions of NSCs was visualized by confocal laser scanning microscopy, and the expression levels of cyclophilin D and cleaved caspase-3 were assayed by western blot. The apoptotic rate of NSCs was measured by flow cytometry. The expression of α7-nAChR was detected in microglial cells, but no expression was found in NSCs. The viability of rat microglial cells and NSCs was not affected by reagents or coculture itself. Aβ1-42-mediated microglial activation impaired the morphology and the ΔΨm of mitochondria of NSCs as well as increased cell apoptosis. However, the damage was attenuated when the α7-nAChRs on microglial cells were activated or the mPTPs on NSCs were blocked. Blockade of mPTPs on NSCs and activation of α7-nAChRs on microglia exhibit neuroprotective roles in Aβ-induced neurotoxicity of NSCs.

Nicotine-induced plasticity in the retinocollicular pathway: Evidence for involvement of amyloid precursor protein

During early postnatal development retinocollicular projections undergo activity-dependent synaptic refinement that results in the formation of precise topographical maps in the visual layers of the superior colliculus (SC). Amyloid Precursor Protein (APP) is a widely expressed transmembrane glycoprotein involved in the regulation of several aspects of neural development, such as neurite outgrowth, synapse formation and plasticity. Stimulation of cholinergic system has been found to alter the expression and processing of APP in different cell lines. Herein, we investigated the effect of nicotine on the development of retinocollicular pathway and on APP metabolism in the SC of pigmented rats. Animals were submitted to intracranial Elvax implants loaded with nicotine or phosphate-buffered saline (vehicle) at postnatal day (PND) 7. The ipsilateral retinocollicular pathway of control and experimental groups was anterogradely labeled either 1 or 3weeks after surgery (PND 14 or PND 28). Local nicotine exposure produces a transitory sprouting of uncrossed retinal axons outside their main terminal zones. Nicotine also increases APP content and its soluble neurotrophic fragment sAPPα. Furthermore, nicotine treatment upregulates nicotinic acetylcholine receptor α7 and β2 subunits. Taken together, these data indicate that nicotine disrupts the ordering and topographic mapping of axons in the retinocollicular pathway and facilitates APP processing through the nonamyloidogenic pathway, suggesting that sAPPα may act as a trophic agent that mediates nicotine-induced morphological plasticity.