Expression Of α7nAChR Research Articles

THE ROLE OF ASTROCYTIC α7 NICOTINIC ACETYLCHOLINE RECEPTORS IN NEUROINFLAMMATION-MEDIATED COGNITIVE IMPAIRMENT

An important component of Alzheimer's disease (AD) is neuroinflammation. In the periphery, α7 nAChR activation inhibits macrophage production of pro-inflammatory cytokines. In the CNS, α7 nAChRs are enriched in brain regions affected early in AD. However, α7 nAChR expression is not restricted to neurons; these receptors are highly enriched within the CNS immune system. We have previously reported important roles for astrocytic α7 nicotinic acetylcholine receptor (nAChR)-Aβ interaction with subsequent effects on cognition. To refine these studies, we sought to test the hypothesis that astrocytic α7 nAChRs serve an analogous anti-inflammatory role to that in the periphery. To test our hypothesis, we generated mice in which the fourth exon of the α7 nAChR gene (Chrna7) is flanked by loxP sites (α7nAChRflox). We then crossed α7nAChRflox mice with mice expressing Cre recombinase driven by the glial acidic fibrillary protein (GFAP) and studied neuroinflammatory responses and cognition following intracerebroventricular (ICV) injection of LPS (10 ng); a dose that induces transient neuroinflammatory responses without measurable peripheral inflammation. We studied hemizygous astrocytic α7nAChR (GFAP-A7+/-) knockdown mice compared to α7nAChRflox+/- littermates. Whereas sham and LPS ICV-injected α7nAChRflox+/- littermates performed similarly in hippocampus-dependent associative learning and memory, GFAP-A7+/- mice were impaired. Bio-Plex analysis of serum, hippocampus and cholinergic basal forebrain revealed that α7nAChRflox+/- littermates mounted both a pro- and anti-inflammatory response to ICV LPS; GFAP-A7+/- mice did not. However, GFAP-A7+/- mice exhibited pronounced microglia activation and tau hyperphosphorylation in hippocampal structures. Morphometric analysis showed that GFAP-A7+/- mice presented with blunted astrocyte response to LPS challenge. Currently, we are using cultured astrocytes from hemizygous GFAP-Cre α7nAChR knockdown mice and α7nAChRflox littermates to decipher mechanistic details. Blunted astrocyte response, absence of an anti-inflammatory response, microglia activation and tau pathology resulting from ICV LPS injection suggests that knockdown of α7 nAChRs in astrocytes triggers a feed-forward pro-inflammatory response to elicit AD-like cognitive deficits. Thus, similar to their role in the periphery, astrocytic α7 nAChRs serve an anti-inflammatory role in the CNS. This has significant implications for AD since Aβ binds α7 nAChRs to silence these receptors.

\u03b17 nicotinic acetylcholine receptor upregulation by anti-apoptotic Bcl-2 proteins

Nicotinic acetylcholine receptors (nAChRs) mediate and modulate synaptic transmission throughout the brain, and contribute to learning, memory, and behavior. Dysregulation of α7-type nAChRs in neuropsychiatric as well as immunological and oncological diseases makes them attractive targets for pharmaceutical development. Recently, we identified NACHO as an essential chaperone for α7 nAChRs. Leveraging the robust recombinant expression of α7 nAChRs with NACHO, we utilized genome-wide cDNA library screening and discovered that several anti-apoptotic Bcl-2 family proteins further upregulate receptor assembly and cell surface expression. These effects are mediated by an intracellular motif on α7 that resembles the BH3 binding domain of pro-apoptotic Bcl-2 proteins, and can be blocked by BH3 mimetic Bcl-2 inhibitors. Overexpression of Bcl-2 member Mcl-1 in neurons enhanced surface expression of endogenous α7 nAChRs, while a combination of chemotherapeutic Bcl2-inhibitors suppressed neuronal α7 receptor assembly. These results demonstrate that Bcl-2 proteins link α7 nAChR assembly to cell survival pathways.

Role of α7nAChR in postoperative cognitive dysfunction in aged rats with tibial fracture

Objective To evaluate the role of α7 nicotinic acetylcholine receptor (α7nAChR) in postoperative cognitive dysfunction in aged rats with tibial fracture. Methods One hundred and fifty clean-grade healthy male Sprague-Dawley rats, aged 18-22 months, weighing 440-580 g, were divided into 5 groups (n=30 each) using a random number table method: control group (group C), sham operation group (group S), tibial fracture group (group T), normal saline group (group N) and α7nAChR agonist PUN282987 group (group P). Group C received no treatment.Ten percent chloral hydrate 0.4 ml/100 g was injected intraperitoneally in group S. Group T underwent tibial fracture.PUN282987 2.4 mg/kg was intraperitoneally injected at 5 min before tibial fracture in group P. The equal volume of normal saline was given at 5 min before tibial fracture in group N. Morris water maze test was performed at day 7 after surgery.At days 1, 3 and 7 after surgery, the pathological changes of the hippocampal CA3 region were observed by haematoxylin and eosin staining, and the expression of α7nAChR, choline acetyltransferase (ChAT), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the hippocampal CA3 region was measured by Western blot. Results Compared with group C, the postoperative escape latency and swimming distance were significantly prolonged, and the expression of α7nAChR, ChAT, TNF-α and IL-1β was up-regulated at each time point after operation in T, N and P groups (P 0.05). Compared with group T, the postoperative escape latency and swimming distance were significantly shortened, and the expression of α7nAChR and ChAT was up-regulated and the expression of TNF-α and IL-1β was down-regulated at each time point after operation in group P (P 0.05), and the pathological changes of the hippocampal CA3 region were significantly attenuated in group P. Conclusion α7nAChR antagonism is involved in the development of postoperative cognitive dysfunction in aged rats with tibial fracture. Key words: Cholinergic agonists; Aged; Cognition disorders; Postoperative complications

Etiopathology and psychosomatic background of awake bruxism – A review

Convulsive status epilepticus (CSE) is a neurological disease with contraction and extension of limbs, leading to damage of hippocampus and cognition. This study aimed to explore the effects of dexmedetomidine (DEX) on the cognitive function and neuroinflammation in CSE rats. All rats were divided into control group, CSE group and DEX group. Morris water maze test was used to measure cognitive function. Acute hippocampal slices were made to detect long-term potentiation (LTP). Immunohistochemistry was used to determine the expression of α7-nicotinic acetylcholine receptor (α7-nAChR) and interleukin-1β (IL-1β). Enzyme-linked immunosorbent assay (ELISA) was used to measure serum levels of IL-1β, tumor necrosis factor-α (TNF-α), S-100β and brain-derived neurotrophic factor (BDNF). Our results showed that DEX improved the memory damage caused by CSE. DEX reduced seizure severity and increased the amplitudes and sustainable time of LTP, and also inhibited the hippocampal expression of α7-nAChR and IL-1β in CSE rats. DEX treatment decreased serum IL-1β, TNF-α and S-100β levels and increased BDNF levels. The effects of DEX on seizure severity and LTP could be simulated by nicotine or attenuated by concurrent α-bungarotoxin (α-BGT) treatment. In conclusions, DEX significantly improved spatial cognitive dysfunction, reduced seizure severity and increased LTP in CSE rats. Improvements by DEX were closely related to enhancement of cholinergic anti-inflammatory pathway.

Alpha7 nAChR Expression Is Correlated with Arthritis Development and Inhibited by Sinomenine in Adjuvant-Induced Arthritic Rats.

Sinomenine (SIN) is the active ingredient of the Chinese herb Sinomenium acutum that has been used to treat rheumatoid arthritis (RA) for about 30 years in China. Marked expression of the alpha7 nicotinic acetylcholine receptor (α7nAChR) in the joint synovium of RA patients suggested a relationship between α7nAChR and RA. This study investigated the relationship between α7nAChR and RA development and the effects of SIN on α7nAChR expression in vivo and in vitro. Sprague-Dawley rats were injected with complete Freund's adjuvant to induce arthritis and then treated with SIN or methotrexate (MTX) from day 0 to day 30. Four clinical parameters—paw volume, arthritic index (AI), serum TNF-α concentration, and erythrocyte sedimentation rate (ESR)—were measured. Splenic lymphocytes were isolated for Bacille Calmette Guerin (BCG) stimulation. α7nAChR expression in tissues and cells was examined by RT-PCR, western blot, immunofluorescence, flow cytometry, and immunohistochemistry. Cell proliferation was evaluated by the CCK-8 assay. The relationship between α7nAChR expression and the four clinical parameters was analyzed by single-factor correlation analysis. Our results showed that the paw volume, AI, TNF-α concentration, and ESR in adjuvant-induced arthritic (AIA) rats were reduced by SIN or MTX treatment. SIN decreased α7nAChR expression in tissues and cells compared to the model group, while MTX had no significant effect on α7nAChR expression. Moreover, there was a positive relationship between α7nAChR expression and paw swelling, AI, and TNF-α concentration. Splenic lymphocyte activation was accompanied by increased α7nAChR expression, while SIN treatment inhibited cell activation and downregulated α7nAChR expression. α7nAChR expression showed a positive correlation with the progression of RA in AIA rats that may involve lymphocyte activation. Different from MTX, the inhibition of SIN on α7nAChR expression might contribute to its antiarthritic effect, suggesting that SIN could be an important supplement to the treatment strategy for RA.

Mining and validating the expression pattern and prognostic value of acetylcholine receptors in non-small cell lung cancer.

Acetylcholine receptors (AChRs), including nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs), are highly expressed in bronchial epithelial cells.We used The Cancer Genome Atlas (TCGA) data set to evaluate the expression pattern and prognostic value of the AChR gene family in non-small cell lung cancer (NSCLC). The mined data was validated by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC).The survival analysis of TCGA data set showed that only CHRNA7 in the AChR gene family affected prognosis in both lung adenocarcinoma and lung squamous cell carcinoma. Furthermore, qRT-PCR proved that CHRNA7 was significantly upregulated in tumor tissues compared with matched normal tissues at mRNA level (P = .001). The expression level of α7 nAChR (encoded by CHRNA7) in 141 patients was measured by IHC and a high expression of α7 nAChR was associated with unfavorable prognosis (P = .008). Multivariate analysis showed that α7 nAChR was an independent prognostic factor (HR = 2.041; 95% CI 1.188-3.506; P = .007).α7 nAChR was upregulated in NSCLC and was associated with unfavorable prognosis. This gene may be a potential target for lung cancer treatment.

α7-Nicotinic Acetylcholine Receptor Promotes Cholangiocarcinoma Progression and Epithelial-Mesenchymal Transition Process.

Cholangiocarcinoma is one of the most deadly malignant tumors characterized by a tendency of local invasiveness and metastasis at the early phase, high recurrence rate, and difficulty in treatment. Alpha7-nicotinic acetylcholine receptor (α7-nAChR) is highly expressed in a variety of tumors, including cholangiocarcinoma, and may promote tumor progression, but the mechanisms are largely unknown. Our study is the first to expound upon the role that α7-nAChR plays in cholangiocarcinoma. We assessed 50 human cholangiocarcinoma tissue samples and 20 normal biliary samples using immunohistochemical staining to find the correlation between α7-nAChR expression and clinicopathological characteristics. We used human cholangiocarcinoma cell lines QBC939 and RBE and α7-nAChR gene knockdown RBE cell lines generated by shRNA lentivirus transfection to investigate the biological functions of α7-nAChR in proliferation, apoptosis, migration, and invasiveness in vitro. Further, western blotting was used to detect apoptosis and epithelial-mesenchymal transition (EMT)-related signaling proteins. Cholangiocarcinoma xenografts in nude mice were used for tumorigenicity assays in vivo. The expression of α7-nAChR was high in cholangiocarcinoma tissues and was closely related to a shorter survival time in patients. α7-nAChR knockdown decreased cell proliferation ability, increased early apoptosis, and weakened cell migration and invasion. Apoptosis-related proteins and components of the EMT process were altered after α7-nAChR knockdown. Moreover, nude mice xenograft experiments confirmed that α7-nAChR could promote cholangiocarcinoma in vitro. Overexpression of α7-nAChR induces cholangiocarcinoma progression by blocking apoptosis and promoting the EMT process. As an effective molecular biomarker and prognostic factor, α7-nAChR is a promising therapeutic target in cholangiocarcinoma.

GTS-21 has cell-specific anti-inflammatory effects independent of α7 nicotinic acetylcholine receptors.

α7 Nicotinic acetylcholine receptors (nAChRs) reportedly reduce inflammation by blocking effects of the important pro-inflammatory transcription factor, nuclear factor kappa-light chain-enhancer of B cells (NFκB). The α7 nAChR partial agonist GTS-21 reduces secretion of pro-inflammatory cytokines including interleukin-6 (IL6) and tumor-necrosis factor (TNF) in models of endotoxemia and sepsis, and its anti-inflammatory effects are widely ascribed to α7 nAChR activation. However, mechanistic details of α7 nAChR involvement in GTS-21 effects on inflammatory pathways remain unclear. Here, we investigate how GTS-21 acts in two cell systems including the non-immune rat pituitary cell line GH4C1 expressing an NFκB-driven reporter gene and cytokine secretion by ex vivo cultures of primary mouse macrophages activated by lipopolysaccharide (LPS). GTS-21 does not change TNF-stimulated NFκB signaling in GH4C1 cells expressing rat α7 nAChRs, suggesting that GTS-21 requires additional unidentified factors besides α7 nAChR expression to allow anti-inflammatory effects in these cells. In contrast, GTS-21 dose-dependently suppresses LPS-induced IL6 and TNF secretion in primary mouse macrophages endogenously expressing α7 nAChRs. GTS-21 also blocks TNF-induced phosphorylation of NFκB inhibitor alpha (IκBα), an important intermediary in NFκB signaling. However, α7 antagonists methyllycaconitine and α-bungarotoxin only partially reverse GTS-21 blockade of IL6 and TNF secretion. Further, GTS-21 significantly inhibited LPS-induced IL6 and TNF secretion in macrophages isolated from knockout mice lacking α7 nAChRs. These data indicate that even though a discrete component of the anti-inflammatory effects of GTS-21 requires expression of α7 nAChRs in macrophages, GTS-21 also has anti-inflammatory effects independent of these receptors depending on the cellular context.

α7‐nAChR‐Mediated Therapeutic Angiogenesis Accounts for the Advantageous Effect of Low Nicotine Doses Against Myocardial Infarction in Rats

Angiogenesis accelerates tissue regeneration in a variety of ischemic conditions including myocardial infarction (MI) and constitutes a valuable approach for post‐MI repair. Here we investigated whether angiogenesis promotion induced by α7‐nicotinic acetylcholine receptors (α7‐nAChRs) mitigates histopathological, electrocardiographic, and molecular consequences of MI in rats. These profiles were evaluated in rats with isoprenaline (85 mg/kg/day i.p. for 2 days)‐induced MI and co‐treated subcutaneously with nicotine (20 μg/kg/day s.c.), PHA‐543613 (PHA, selective α7‐nAChR agonist, 20 μg/kg/day), methyllycaconitine (MLA, selective α7‐nAChR antagonist, 40 μg/kg/day) plus nicotine, or the vehicle for 16 consecutive days. Isoprenaline‐insulted rats showed (i) ECG signs of MI such as significant ST‐segment elevations and prolonged QT‐intervals, (ii) deteriorated left ventricular histopathological scoring and elevated inflammatory cell infiltration, (iii) reduced immunohistochemical expression of cardiac CD34, a biomarker of capillary density, (iv) decreased cardiac expression of iNOS and α7‐nAChRs, and (v) adaptive increases in cardiac HO‐1 expression and plasma angiogenic markers such as vascular endothelial growth factor (VEGF) and nitric oxide (NO). These effects of isoprenaline, except cardiac iNOS and α7‐nAChRs downregulation, were ameliorated in rats co‐treated with nicotine or the selective α7‐nAChR agonist PHA for 16 consecutive days. We also show that concurrent α7‐nAChR blockade by MLA reversed the ECG, histopathological, and capillary density effects of nicotine, thereby reinforcing the advantageous cardioprotective and anti‐ischemic roles of α7‐nAChRs in this setting. In conclusion, the activation of α7‐nAChRs with PHA or doses of nicotine in the microgram scale promotes neovascularization and offers a promising therapeutic strategy for MI.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bipolar Cell Type-Specific Expression and Conductance of Alpha-7 Nicotinic Acetylcholine Receptors in the Mouse Retina.

PurposeMotion detection is performed by a unique neural network in the mouse retina. Starburst amacrine cells (SACs), which release acetylcholine and gamma-aminobutyric acid (GABA) into the network, are key neurons in the motion detection pathway. Although GABA contributions to the network have been extensively studied, the role of acetylcholine is minimally understood. Acetylcholine receptors are present in a subset of bipolar, amacrine, and ganglion cells. We focused on α7-nicotinic acetylcholine receptor (α7-nAChR) expression in bipolar cells, and investigated which types of bipolar cells possess α7-nAChRs.MethodsRetinal slice sections were prepared from C57BL/6J and Gus8.4-GFP mice. Specific expression of α7-nAChRs in bipolar cells was examined using α-bungarotoxin (αBgTx)-conjugated Alexa dyes co-labeled with specific bipolar cell markers. Whole-cell recordings were conducted from bipolar cells in retinal slice sections. A selective α7-nAChR agonist, PNU282987, was applied by a puff and responses were recorded.ResultsαBgTx fluorescence was observed primarily in bipolar cell somas. We found that α7-nAChRs were expressed by the majority of type 1, 2, 4, and 7 bipolar cells. Whole-cell recordings revealed that type 2 and 7 bipolar cells depolarized by PNU application. In contrast, α7-nAChRs were not detected in most of type 3, 5, 6, and rod bipolar cells.ConclusionsWe found that α7-nAChRs are present in bipolar cells in a type-specific manner. Because these bipolar cells provide synaptic inputs to SACs and direction selective ganglion cells, α7-nAChRs may play a role in direction selectivity by modulating these bipolar cells' outputs.

Activation of α7-nAChRs protects SH-SY5Y cells from 1-methyl-4-phenylpyridinium-induced apoptotic cell death via ERK/p53 signaling pathway.

Epidemiologic studies have shown a reduced risk of developing Parkinson's disease (PD) among cigarette smokers. Nicotine, as a key component in tobacco products, is thought as a possible candidate for action of smoking in neuroprotection. α7 nicotinic acetylcholine receptors (α7-nAChRs) is one of the most abundant nAChRs in the mammalian brain. Although nicotine is thought to exert this protective action by acting on nicotinic receptors, including the α7-nAChRs; the mechanisms underlying how α7-nAChRs protect against dopaminergic neuron loss are highly complex. Using nicotine and a selective α7-nAChR agonist PNU-282987, we first confirmed that their addition to SH-SY5Y cells challenged with 1-methyl-4-phenylpyridinium (MPP+ ) could afford neuroprotection and result in a reduction in apoptotic cell death. Then, we found that the pretreatment with nicotine and PNU-282987 showed the neuroprotective antiapoptotic effects via activating the α7-nAChRs/MAPK/p53 axis. Furthermore, we used RNA interference to silence the expression of α7-nAChRs in SH-SY5Y cells and found that suppressing α7-nAChR expression diminished the antiapoptotic effects of nicotine and PNU-282987, not the toxic effects of MPP+ . Moreover, α7-nAChR knockdown could only decrease the inhibitory effects of nicotine and PNU-282987 on the phosphorylated extracellular signal-regulated kinase (ERK), not c-Jun amino-terminal kinase and p38. Therefore, our findings indicate the important roles of ERK/MAPK signaling in the neuroprotective effects of α7-nAChRs and suggest that α7-nAChR agonists may be validated as novel treatments for PD.

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.

Acute Activation of α7-Nicotinic Receptors by Nicotine Improves Rodent Skin Flap Survival Through Nitrergic System.

Recent reports have identified angiogenic, anti-inflammatory, and antioxidant properties of acute treatment with nicotine via activation of nicotinic acetylcholine receptors (nAChRs). In addition, the nitric oxide (NO) pathway is involved in ischemic reperfusion injuries. We investigated the effects of acute pretreatment with nicotine in a rat model of random-pattern skin flap and the potential role of the NO pathway. The Sprague-Dawley rats received increasing doses of (-)-nicotine (0.5, 1, 1.5, 2, and 3 mg/kg) before the procedure. Dorsal skin flaps with caudal pedicles were elevated at the midline, and flap survival was evaluated 7 days after surgery. In addition, animals received an α7-nAChR antagonist, methyllycaconitine, with nicotine. Quantitative reverse transcription polymerase chain reaction was also applied to measure the dermal expression of α7-nAChR. Next, a nonselective NO synthase inhibitor, N-nitro-L-arginine methyl ester hydrochloride; a selective inducible NO synthase inhibitor, aminoguanidine; and an NO precursor, L-arginine, were administered with nicotine. Nicotine at doses of 1, 1.5, and 2 mg/kg significantly increased flap survival, whereas the protective effects of nicotine disappeared at higher doses. Methyllycaconitine completely reversed the protective effects of nicotine and the elevated cutaneous expression of α7-nAChR in nicotine-pretreated rats. In addition, systemic administration of N-nitro-L-arginine methyl ester hydrochloride or aminoguanidine with an effective dose of nicotine caused a significant decrease in flap survival. Conversely, coinjection of a subeffective dose of L-arginine with the subeffective dose of nicotine significantly boosted its protective effects. Acute pretreatment with nicotine by stimulating the expression and activation of cutaneous α7-nAChR improves skin flap survival, which is partially mediated through modulation of the NO pathway.

Eleutheroside E attenuates isoflurane-induced cognitive dysfunction by regulating the α7-nAChR-NMDAR pathway.

There is growing evidence that cognitive dysfunction induced by anesthetics is adversely affecting a large number of elderly surgical patients. Eleutheroside E (EE), a principal component of Eleutherococcus senticosus, exerts obvious protective effects on cognition. The aim of this study was to investigate the neuroprotective effect of EE on isoflurane (ISO)-induced cognitive dysfunction and explore the possible mechanisms. Learning and memory are assessed in novel object recognition and Morris water maze. We found that with ISO exposure, aged rats had a lower preference for the new object and spent less time in the target quarter. However, the amnesia can be alleviated by EE (50 mg/kg, intraperitoneally). Further research focused on the possible protective molecules associated with learning and memory, such as acetylcholine (ACh) and choline acetyltransferase (ChAT), nicotinic acetylcholine receptors (α7-nAChR), and NR2B, is required. The ACh in the hippocampus and serum was decreased after ISO exposure; meanwhile, the expression of ChAT, α7-nAChRs, and NR2B was downregulated. This abnormal state can be reversed by the administration of EE. Here, our results suggested that EE may be a potential therapeutic agent against ISO-induced cognitive dysfunction. The possible mechanism can be attributed to its neuroprotection through enhancing ChAT, which promotes the synthesis of ACh, further influencing the expression of the α7-nAChR-NR2B complex.

Decreased α7nAChR mRNA levels in peripheral blood monocytes are associated with enhanced inflammatory cytokine production in patients with lupus nephritis

The cholinergic anti-inflammatory pathway modulates cytokine release by activating alpha-7 nicotinic acetylcholine receptors (α7nAChR) in monocytes/macrophages. We aimed to determine the role of α7nAChR in lupus nephritis (LN). We enrolled 36 inactive and 35 active LN patients, 34 primary glomerulonephritis patients, and 35 healthy controls. Peripheral blood monocytes were isolated, and mRNA expression of α7nAChR, interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α) in monocytes was measured. α7nAChR and IL-10 mRNA levels were significantly decreased, but IL-6 was increased, in LN patients compared with healthy controls or glomerulonephritis patients (all P < 0.01). Interestingly, α7nAChR mRNA levels were negatively correlated to SLEDAI (r = −0.68, P < 0.01), anti-dsDNA (r = −0.38, P < 0.05), and proteinuria (r = −0.49, P < 0.01) levels, and positively correlated to serum complement C3 levels (r = 0.38, P < 0.05) in patients with active LN. Furthermore, α7nAChR mRNA levels were negatively correlated to TNF-α (r = −0.50, P < 0.01), IL-1β (r = −0.42, P < 0.05), IL-6 (r = −0.69, P < 0.01) mRNA levels, and positively correlated to IL-10 (r = 0.45, P < 0.01). TNF-α, IL-1β, and IL-6 protein levels in the supernatant of cultured monocytes from active LN patients were significantly higher, while IL-10 was lower, than that of healthy controls. PNU-282987, an α7nAChR agonist, significantly decreased TNF-α, IL-1β, and IL-6 but increased IL-10 in the monocyte culture supernatant of active LN patients, which were abolished by an α7nAChR antagonist methyllycaconitine. The effects of PNU-282987 were confirmed in lipopolysaccharides-stimulated monocytes. Taken together, these findings suggest that decrease in α7nAChR mRNA levels may play a role in LN and that activation of α7nAChR may inhibit inflammation in LN.

Relationship between α7nAChR signaling pathway and regulatory T cells during vagus nerve stimulation-induced reduction of endotoxin-caused acute lung injury in mice

Objective To evaluate the relationship between α7 nicotinic acetylcholine receptor (α7nAChR) signaling pathway and regulatory T cells (Tregs) during vagus nerve stimulation-induced reduction of endotoxin-caused acute lung injury (ALI) in mice. Methods Clean-grade healthy male C57BL/6 mice, aged 6-8 weeks, weighing 22-25 g, were divided into 5 groups (n=10 each) using a random number table method: control group (group C), group ALI, vagus nerve stimulation group (group VNS), α-BGT group (group α-BGT) and vagus nerve stimulation plus α-BGT group (group VNS + α-BGT). After successful establishment of the model, the vagus nerve was stimulated for 30 s with a stimulus intensity of 0.5 mA, frequency of 20 Hz, an interval of 5 min, 60 min in total.Sterile normal saline 100 μl was injected into the trachea, and the vagus nerve was only exposed but not stimulated in group C. In group ALI, the ALI model was established, and the vagus nerve was isolated but not stimulated.In group α-BGT, α-BGT 1 μg/kg was intraperitoneally injected, the ALI model was established 1 h later, and the vagus nerve was isolated but not stimulated.In group VNS+ α-BGT, α-BGT 1 μg/kg was intraperitoneally injected, the ALI model was established 1 h later, and the vagus nerve was stimulated at 1 h after the end of establishment.Animals were sacrificed at 72 h after establishing the model, and lungs were removed for determination of lung water content, percentage of Tregs (using flow cytometry), myeloperoxidase (MPO) activity (by colorimetric assay), expression of α7nAChR (by Western blot) and contents of interleukin-10 (IL-10), transforming growth factor-β (TGF-β) and IL-1β (by enzyme-linked immunosorbent assay). Results Compared with group C, the lung water content, MPO activity and IL-1β content were significantly increased in the other four groups, the expression of α7nAChR was significantly down-regulated in ALI, α-BGT and VNS+ α-BGT groups, the percentage of Tregs was significantly increased in group VNS, the IL-10 content was significantly decreased in ALI and α-BGT groups and increased in VNS and VNS+ α-BGT groups, and TGF-β contents were significantly decreased in ALI, α-BGT and VNS+ α-BGT groups and increased in group VNS (P<0.05). Compared with group ALI, the lung water content, MPO activity and IL-1β content were significantly decreased, the expression of α7nAChR was up-regulated, and the percentage of Tregs and contents of IL-10 and TGF-β were increased in group VNS, and the TGF-β content in group α-BGT and contents of IL-10 and TGF-β in group VNS+ α-BGT were significantly increased (P<0.05). Compared with group VNS, the lung water content, MPO activity and IL-1β content were significantly increased, the expression of α7nAChR was down-regulated, and the percentage of Tregs and contents of IL-10 and TGF-β were decreased in α-BGT and VNS+ α-BGT groups (P<0.05). The contents of IL-10 and TGF-β were significantly higher in group VNS+ α-BGT than in group α-BGT (P<0.05). Conclusion Vagus nerve stimulation can activate α7nAChR signaling pathway and raise the percentage of Tregs, thus reducing ALI in mice. Key words: Vagus nerve; Respiratory distress syndrome, adult; T-Lymphocytes, regulatory; Receptors, nicotinic

Abstract 254: Ultrasound Improves the Outcomes After Cardiopulmonary Resuscitation by Stimulating the Cholinergic Anti-Inflammatory Pathway

Introduction: Recently it was shown that the ultrasound (US) treatment prevented renal ischemia- reperfusion injury (IRI) in mice. In the present study, we investigated the effects of the US treatment against IRI after CPR . Hypothesis: US would improve the outcomes after resuscitation in a rat model of CPR, and the protective effect of US would be dependent on the cholinergic anti-inflammatory pathway (CAP) via α7nAChR. Methods: The animals were randomized into 5 groups (n=8 each): (1) CPR group: the rats underwent 6 mins of untreated ventricular fibrillation (VF) followed by CPR and defibrillation; (2) US group: the treatment was identical to the CPR group except that rats were exposed to US treatment 24h before CPR; (3) MLA group: the treatment was identical to the US group except thatα7nAChR antagonist MLA (4mg/kg) was administered 30 mins prior to US treatment and VF respectively; (4) GTS group: the treatment was identical to the CPR group except that α7nAChR agonist GTS-21(4mg/kg) was injected 30 mins before VF. (5) SHAM group: the rats were exposed to surgical preparation without CPR and US application. One day before CPR, the US treatment was delivered to the left kidney by US pulses (contrast general mode with 9 MHz) with a bursting mechanical index of 0.72. US pulses were 1 second in duration and were applied once every 6 seconds for 2 mins. After the treatment of the left kidney, the right kidney was exposed to US treatment for another 2 mins. Results: Significantly longer duration of survival was observed in US-treated animals. And US treatment also suppressed the TNF-α and IL-6 following resuscitation, and improved neurological function. The expression of α7nAChR were promoted in hippocampal neurons in US group. Moreover, the protective effect of US treatment could be abolished by MLA and imitated by GTS-21. Conclusions: US treatment before IRI improves the outcomes after CPR, and CAP via α7nAChR plays an important role during CPR.

Variation of nicotinic subtype α7 and muscarinic subtype M3 acetylcholine receptor expression in three main types of leukemia.

Cholinergic receptors, such as α7-nicotinic acetylcholine receptor (α7-nAChR) and M3-muscarinic acetylcholine receptor (M3-mAChR), have been demonstrated to serve a significant role in the proliferation, differentiation and apoptosis of leukemic cells. However, the expression of these receptors in samples from patients with leukemia remains unclear. The present study aimed to determine the expression of M3-mAChR and α7-nAChR in the bone marrow or peripheral blood of 51 patients with leukemia, including acute myeloid leukemia (AML; n=33), acute lymphoblastic leukemia (ALL; n=13), and chronic myeloid leukemia (CML; n=5). Peripheral blood mononuclear cells (PBMCs) were also isolated from healthy subjects (n=5) for comparison. Western blot analysis was performed to determine the protein expression profiles, and a pattern of decreased α7-nAChR levels in patients with leukemia was observed. Among the leukemia types, the lowest expression of α7-nAChR and M3-mAChR were identified in patients with T-cell ALL/lymphoma (T-ALL). CML exhibited the highest level of M3-mAChR, which was significantly different from APL and AML-M4, yet not from healthy subjects (P<0.05). Therefore, different expression profiles of α7-nACR and M3-mAChR were detected amongst the leukemia types. Collectively, the present study supports the potential role of cholinergic signaling in mediating leukemogenesis. However, further studies in larger cohorts are required to validate these findings.

3-Dehydroandrographolide protects against lipopolysaccharide-induced inflammation through the cholinergic anti-inflammatory pathway

Acute lung injury (ALI) is a deadly disease without effective chemotherapy, so far. Traditional Chinese medicine andrographis herba is frequently used in the treatment of respiratory diseases. In searching for natural anti-ALI components from andrographis herba, the activities of 3-dehydroandrographolide (3-DA), a new natural andrographolide product from andrographis herba were evaluated. In this study, murine macrophage RAW 264.7 cells and BALB/c mice were treated with LPS (lipopolysaccharide, 100 ng/ml in vitro; 3 mg/kg, intratracheal) to establish inflammation models. 3-DA attenuated the release of pro-inflammatory cytokines IL-6 and TNF-α, inhibited the degradation and phosphorylation of IκBα, and suppressed the nuclear translocation of NF-κB p65 as well as the phosphorylation of Akt at Ser473 in LPS-stimulated RAW 264.7 macrophage cells. Furthermore, 3-DA increased α7nAchR expression level and bound with α7nAchR. More importantly, the anti-inflammatory effects of 3-DA were counteracted in the presence of α7nAchR siRNA or methyllycaconitine (MLA, a α7nAchR specific inhibitor), suggesting that α7nAchR is a potential target in the anti-inflammatory effects of 3-DA. Besides, 3-DA significantly inhibited inflammation in LPS-induced ALI mice, which was associated with the decrease of lung water content and inflammatory cytokines, the inhibition of neutrophil and macrophage infiltration, and activation of the NF-κB/Akt signaling pathway. Moreover, these protective effects were attenuated by the treatment of MLA. Taken together, 3-DA alleviates LPS-induced inflammation via the cholinergic anti-inflammatory pathway in vitro and in vivo. These findings provide a rationale for the role of the cholinergic anti-inflammatory pathway in inflammation and the promising clinical application of 3-DA to treat ALI.

Auricular Vagus Nerve Stimulation Exerts Antiinflammatory Effects and Immune Regulatory Function in a 6-OHDA Model of Parkinson's Disease.

According to epidemiologic studies, smoking appears to downregulate the prevalence of Parkinson's disease (PD), possibly due to antiinflammatory mechanisms via activation of α7 nicotinic acetylcholine receptors (α7 nAChRs). This receptor also appears to play a role in T-cell differentiation. Recently, it has become apparent that the innate immune system participates in PD pathogenesis. The aim of this study was to evaluate the effects of auricular vagus nerve stimulation (aVNS) on substantia nigra (SN) dopaminergic neurodegeneration and the associated neuroinflammation and immune responses in a rat PD model. Adult male Wistar rats were unilaterally administered 6-hydroxydopamine (6-OHDA) to the medial forebrain bundle, followed by aVNS treatment after surgery. Following motor behavioral tests, the expression of tyrosine hydroxylase (TH) in the SN and the levels of inflammatory cytokines in the ventral midbrain were evaluated. In addition, changes in the trends of subsets of CD4+ T lymphocytes in the SN were measured by immunofluorescence staining. Western blotting was used to evaluate the α7 nAChR protein level. Compared with 6-OHDA treats rats, aVNS treatment significantly improved motor deficits, increased TH and α7 nAChR expression, and reduced the levels of inflammatory cytokines (tumor necrosis factor-a (TNF-α) and interleukin-1β (IL-1β)) (p < 0.05). Additionally, aVNS increased the numbers of regulatory T (Treg) cells while decreasing T helper (Th)17 cells. aVNS exerted neuroprotective effects against dopaminergic damage, possibly by suppressing the evolution of inflammation and modulating innate immune responses. Thus, aVNS may be a potential promising therapy in the future.