Tabernanthalog and ibogainalog inhibit the α7 and α9α10 nicotinic acetylcholine receptors via different mechanisms and with higher potency than the GABAA receptor and CaV2.2 channel.

In this study, we have investigated the pharmacological activity and structural interaction of two novel psychoplastogens, tabernanthalog (TBG) and ibogainalog (IBG) at heterologously-expressed rat (r) and human (h) nicotinic acetylcholine receptors (nAChRs), the rα1ß2γ2L γ-aminobutyric acid type A receptor (GABAAR), and the human voltage-gated N-type calcium channel (CaV2.2 channel). Both compounds inhibited the nAChRs with the following receptor selectivity: α9α10 > α7 > α3ß2 â‰… α3ß4, indicating that ß2/ß4 subunits are relatively less important for their activity. The potencies of TBG and IBG were comparable at hα7 and hα9α10 subtypes, and comparable to their rat counterparts. TBG- and IBG-induced inhibition of rα7 was ACh concentration-independent and voltage-dependent, whereas rα9α10 inhibition was ACh concentration-dependent and voltage-independent, suggesting that they interact with the α7 ion channel pore and α9α10 orthosteric ligand binding site, respectively. These results were supported by molecular docking studies showing that at the α7 model TBG forms stable interactions with luminal rings at 9', 13', and 16', whereas IBG mostly interacts with the extracellular-transmembrane junction. In the α9α10 model, however, these compounds interacted with several residues from the principal (+) and complementary (-) sides in the transmitter binding site. Ibogaminalog (DM506) also interacted with a non-luminal site at α7, and one α9α10 orthosteric site. TBG and IBG inhibited the GABAAR and CaV2.2 channels with 10 to 30-fold lower potencies. In sum, we show that TBG and IBG inhibit the α7 and α9α10 nAChRs by noncompetitive and competitive mechanisms, respectively, and with higher potency than the GABAAR and CaV2.2 channel.

Electroacupuncture alleviates intestinal inflammation via a distinct neuro-immune signal pathway in the treatment of postoperative ileus.

BACKGROUND: The induction of intestinal inflammation as a result of abdominal surgery is an essential factor in postoperative ileus (POI) development. Electroacupuncture (EA) at ST36 has been demonstrated to relieve intestinal inflammation and restore gastrointestinal dysmotility in POI. This study aims to elucidate the neuroimmune pathway involved in the anti-inflammatory properties of EA in POI. METHODS: After intestinal manipulation (IM) was performed to induce POI, intestinal inflammation and motility were assessed 24 h post-IM, by evaluating gastrointestinal transit (GIT), cytokines expression, and leukocyte infiltration. Experimental surgery, pharmacological intervention, and genetic knockout mice were used to elucidate the neuroimmune mechanisms of EA. RESULTS: EA at ST36 significantly improved GIT and reduced the expression of pro-inflammatory cytokines and leukocyte infiltration in the intestinal muscularis following IM in mice. The anti-inflammatory effectiveness of EA treatment was abolished by sub-diaphragmatic vagotomy, whereas splenectomy did not hinder the anti-inflammatory benefits of EA treatment. The hexamethonium chloride (HEX) administration contributes to a notable reduction in the EA capacity to suppress inflammation and enhance motility dysfunction, and EA is ineffective in α7 nicotinic acetylcholine receptor (α7nAChR) knockout mice. CONCLUSIONS: EA at ST36 prevents intestinal inflammation and dysmotility through a neural circuit that requires vagal innervation but is independent of the spleen. Further findings revealed that the process involves enteric neurons mediating the vagal signal and requires the presence of α7nAChR. These findings suggest that utilizing EA at ST36 may represent a possible therapeutic approach for POI and other immune-related gastrointestinal diseases.

Hypothalamic inflammation and the development of an obese phenotype induced by high-fat diet consumption is exacerbated in alpha7 nicotinic cholinergic receptor knockout mice.

Hypothalamic inflammation and metabolic changes resulting from the consumption of high-fat diets have been linked to low grade inflammation and obesity. Inflammation impairs the hypothalamic expression of α7 nicotinic acetylcholine receptor (α7nAChR). The α7nAChR is described as the main component of the anti-inflammatory cholinergic pathway in different inflammation models. To assess whether the reduction in α7nAChR expression exacerbates hypothalamic inflammation induced by a high-fat diet (HFD), were used male and female global α7nAChR knockout mouse line in normal or high-fat diet for 4 weeks. Body weight gain, adiposity, glucose homeostasis, hypothalamic inflammation, food intake, and energy expenditure were evaluated. Insulin sensitivity was evaluated in neuronal cell culture. Consumption of an HFD for 4 weeks resulted in body weight gain and adiposity in male Chrna7-/- mice and the hypothalamus of male Chrna7-/- mice showed neuroinflammatory markers, with increased gene expression of pro-inflammatory cytokines and dysregulation in the nuclear factor kappa B pathway. Moreover, male Chrna7-/- mice consuming an HFD showed alterations in glucose homeostasis and serum of Chrna7-/- mice that consumed an HFD impaired insulin signalling in neuronal cell culture experiments. In general, female Chrna7-/- mice that consumed an HFD did not show the phenotypic and molecular changes found in male mice, indicating that there is sexual dimorphism in the analysed parameters. Thus, receptor deletion resulted in increased susceptibility to hypothalamic inflammation and metabolic damage associated with HFD consumption in male mice.

Monochasma savatieri Franch. protects against acute lung injury via α7nAChR-TLR4/NF-κB p65 signaling pathway based on integrated pharmacology analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Acute lung injury (ALI) is a life-threatening condition with high morbidity and mortality, underscoring the urgent need for novel treatments. Monochasma savatieri Franch. (LRC) is commonly used clinically to treat wind-heat cold, bronchitis, acute pneumonia and acute gastroenteritis. However, its role in the treatment of ALI and its mechanism of action are still unclear. AIM OF THE STUDY: This study aimed to demonstrate the pharmacological effects and underlying mechanisms of LRC extract, and provide important therapeutic strategies and theoretical basis for ALI. MATERIALS AND METHODS: In this study, a research paradigm of integrated pharmacology combining histopathological analysis, network pharmacology, metabolomics, and biochemical assays was used to elucidate the mechanisms underlaying the effects of LRC extract on LPS-induced ALI in BALB/c mice. RESULTS: The research findings demonstrated that LRC extract significantly alleviated pathological damage in lung tissues and inhibited apoptosis in alveolar epithelial cells, and the main active components were luteolin, isoacteoside, and aucubin. Lung tissue metabolomic and immunohistochemical methods confirmed that LRC extract could restore metabolic disorders in ALI mice by correcting energy metabolism imbalance, activating cholinergic anti-inflammatory pathway (CAP), and inhibiting TLR4/NF-κB signaling pathway. CONCLUSIONS: This study showed that LRC extract inhibited the occurrence and development of ALI inflammation by promoting the synthesis of antioxidant metabolites, balancing energy metabolism, activating CAP and suppressing the α7nAChR-TLR4/NF-κB p65 signaling pathway. In addition, our study provided an innovative research model for exploring the effective ingredients and mechanisms of traditional Chinese medicine. To the best of our knowledge, this is the first report describing the protective effects of LRC extract in LPS-induced ALI mice.

Hypothalamic α7 nicotinic acetylcholine receptor (α7nAChR) is downregulated by TNFα-induced Let-7 overexpression driven by fatty acids.

The α7nAChR is crucial to the anti-inflammatory reflex, and to the expression of neuropeptides that control food intake, but its expression can be decreased by environmental factors. We aimed to investigate whether microRNA modulation could be an underlying mechanism in the α7nAchR downregulation in mouse hypothalamus following a short-term exposure to an obesogenic diet. Bioinformatic analysis revealed Let-7 microRNAs as candidates to regulate Chrna7, which was confirmed by the luciferase assay. Mice exposed to an obesogenic diet for 3 days had increased Let-7a and decreased α7nAChR levels, accompanied by hypothalamic fatty acids and TNFα content. Hypothalamic neuronal cells exposed to fatty acids presented higher Let-7a and TNFα levels and lower Chrna7 expression, but when the cells were pre-treated with TLR4 inhibitor, Let-7a, TNFα, and Chrna7 were rescued to normal levels. Thus, the fatty acids overload trigger TNFα-induced Let-7 overexpression in hypothalamic neuronal cells, which negatively regulates α7nAChR, an event that can be related to hyperphagia and obesity predisposition in mice.

[Herbal cake-partitioned moxibustion regulates M1 and M2 macrophage polarization of colonic mucosa in rats with Crohn's disease].

OBJECTIVE: To observe the effect of herbal cake-partitioned moxibustion (Moxi) on the expressions of inflammatory factors and M1/M2 polarization in colonic mucosal macrophages in Crohn's disease (CD) rats, so as to explore its underlying mechanisms in the treatment of CD. METHODS: Forty male SD rats were randomly divided into normal, model, Moxi and medication groups (n=10). The CD model was established by enema of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) solution (5%TNBS∶50% alcohol=2∶1, 3 mL/kg), once every 7 days, 4 times altogether. For rats of the Moxi group, cake-partitioned moxibustion was given to "Tianshu" (ST25) and "Qihai" (CV6), two moxa-cones for each acupoint every time, once daily for 10 days. For rats of the medication group, intragastric perfusion of mesalazine solution was given twice daily for 10 days. After the treatment, the colonic mucosa tissue was sampled, and the macrophages were isolated, purified and cultured. The pathological changes of colon tissues were observed by H.E. staining. The ultrastructure of colon tissue was observed by transmission electron microscopy. The expression levels of α7nAChR, NF-κB p65 and TNF-α in colon mucosal macrophages were detected by Western blot. The number of M1 and M2 macrophages in colon mucosa was detected by flow cytometry and immunofluorescence assay. RESULTS: Compared with the normal group, the colon tissue of rats presented huge ulceration and inflammatory manifestations, the junction of colon epithelial cells was loose, the structure of organelles was damaged; the expression level of α7nAChR in macrophages of colon mucosa was significantly decreased (P<0.01), while the expression levels of NF-κB p65 and TNF-α, and the number of M1 and M2 macrophages were increased (P<0.01, P<0.05) in the model group. In comparison with the model group, the morphology and structure of colon mucosa tissues of rats in Moxi and medication groups were improved; the expression level of α7nAChR, the number of M2 macrophage in colon mucosa were significantly increased (P<0.01, P<0.05), while the expression levels of NF-κB p65 and TNF-α, and the number of M1 macrophage were significantly decreased (P<0.01, P<0.05) in both the Moxi and medication groups. CONCLUSION: Herbal cake-partitioned moxibustion may inhibit NF-κB activation by up-regulating the expression level of α7nAChR to promote the polarization of macrophages from M1 to M2 type, and reduce the proportion of M1 macrophages, inhibit the expression of TNF-α in colonic mucosa of CD rats, so as to relieve the intestinal inflammation.

Swimming exercise and clove oil can improve memory by molecular responses modification and reduce dark cells in rat model of Alzheimer's disease.

Alzheimer's disease (AD) is marked by reduced acetylcholine receptor (AChR) density and an increase in nucleotide oligomerization domain (NOD)-like receptors NLR family, pyrin domain containing 1 (NLRP1). We examined the effect of swimming and consumption of clove supplements on memory, dark cells, and α7nAChR and NLRP1 mRNA and protein expression in the hippocampus of the rat model of AD. Forty-eight rats were divided into six groups: sham (sh), healthy-control (HC), Alzheimer (-control (AC), -training (AT), -training-supplement (ATS), and -supplement (AS)). Alzheimer was induced by injection of amyloid ß1-42 (Aß1-42). Swimming exercise protocol (30 min) and gavaging clove supplement (0.1 mg/kg) were administered daily for three weeks. The results indicated that in response to AD, α7 nicotinic acetylcholine receptor (α7nAChR) mRNA and protein rate (p = 0.001) and memory (p = 0.003) were significantly decreased. In contrast, NLRP1 mRNA and protein rate (p = 0.001) and dark cells (p = 0.001) were significantly increased. This is while exercise and clove supplementation improved Alzheimer-induced changes in α7nAChR, NLRP1, memory, and dark cells (p < 0/05). The present study indicated that exercising and consuming clove supplementation could improve memory by increasing α7nAChR and decreasing NLRP1 and dark cells.

Electroacupuncture at Neiguan (PC6) attenuates cardiac dysfunction caused by cecal ligation and puncture via the vagus nerve.

PURPOSE: Previous studies proved the benefits of electroacupuncture (EA) on heart in ischemia reperfusion injury and chronic heart failure. However, the role of EA on sepsis-induced cardiac dysfunction has rarely been elucidated before. In this study, we aimed to investigate the effects of EA on cardiac dysfunction in a rat model of sepsis and to speculate the underlying mechanisms. METHODS: Sepsis was induced by cecum ligation and puncture in anesthetized rats. EA at the acupoint "Neiguan (PC6)" was applied 0.5 h after the induction of sepsis for 20 min. Heart rate variability was obtained immediately after EA to evaluate autonomic balance. Echocardiography was performed at 6 h and 24 h after sepsis induction in vivo. Measurements of hemodynamics, blood gases, cytokines and biochemistry were collected at 24 h. Cardiac tissue underwent immunofluorescence staining to determine the expression of α7 nicotinic acetylcholine receptor (α7nAChR) on macrophages. RESULTS: EA increased vagus nerve activity, prevented the development of hyperlactatemia, attenuated the decline of left ventricle ejection fraction, suppressed systemic and cardiac inflammation and alleviated the histopathological manifestations of heart in sepsis rats. Furthermore, the cardiac tissue from EA treated rats showed increased expressions of α7nAChR on macrophages. The cardio-protective and anti-inflammatory effects of EA were partly or completely prevented in rats with vagotomy. CONCLUSION: EA at PC6 attenuates left ventricle dysfunction and decreases inflammation in sepsis-induced cardiac dysfunction. The cardio-protective effects of EA are mediated through vagus nerve mediated cholinergic pathway.

S-adenosylmethionine improves cognitive impairment in D-galactose-induced brain aging by inhibiting oxidative stress and neuroinflammation.

Oxidative stress and neuroinflammation play crucial roles in aging. S-adenosylmethionine (SAM), a popular supplement, is a potential antioxidant and candidate therapy for depression. This study aimed to evaluate the neuroprotective effects of SAM on D-galactose-induced brain aging and explore its underlying mechanisms. Brain aging model was established with D-galactose (180 mg/kg/day) for 8 weeks. During the last 4 weeks, SAM (16 mg/kg) was co-administrated with D-galactose. Behavior tests were used to assess cognitive function and depression-like behaviors of rats. Results showed that cognitive impairment and depression-like behaviors were reversed by SAM. SAM reduced neuronal cell loss, increased brain-derived neurotrophic factor level in the hippocampus, inhibited amyloid-ß level and microglia activation, as well as pro-inflammatory factors levels in the hippocampus and serum. Further, SAM enhanced antioxidant capacity and attenuated cholinergic damage by reducing malondialdehyde levels, increasing acetylcholine levels, expression levels of α7 nicotinic acetylcholine receptor (α7nAChR), nuclear factor erythrocyte 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in the hippocampus. Above all, SAM has a potential neuroprotective effect on ameliorating cognitive impairment in brain aging, which is related to inhibition of oxidative stress and neuroinflammation, as well as α7nAChR signals. DATA AVAILABILITY: Data will be made available on request.

Activation of the α7 Nicotinic Acetylcholine Receptor Prevents against Microglial-Induced Inflammation and Insulin Resistance in Hypothalamic Neuronal Cells.

Neuronal hypothalamic insulin resistance is implicated in energy balance dysregulation and contributes to the pathogenesis of several neurodegenerative diseases. Its development has been intimately associated with a neuroinflammatory process mainly orchestrated by activated microglial cells. In this regard, our study aimed to investigate a target that is highly expressed in the hypothalamus and involved in the regulation of the inflammatory process, but still poorly investigated within the context of neuronal insulin resistance: the α7 nicotinic acetylcholine receptor (α7nAchR). Herein, we show that mHypoA-2/29 neurons exposed to pro-inflammatory microglial conditioned medium (MCM) showed higher expression of the pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α, in addition to developing insulin resistance. Activation of α7nAchR with the selective agonist PNU-282987 prevented microglial-induced inflammation by inhibiting NF-κB nuclear translocation and increasing IL-10 and tristetraprolin (TTP) gene expression. The anti-inflammatory role of α7nAchR was also accompanied by an improvement in insulin sensitivity and lower activation of neurodegeneration-related markers, such as GSK3 and tau. In conclusion, we show that activation of α7nAchR anti-inflammatory signaling in hypothalamic neurons exerts neuroprotective effects and prevents the development of insulin resistance induced by pro-inflammatory mediators secreted by microglial cells.

Sinomenine inhibits macrophage M1 polarization by downregulating α7nAChR via a feedback pathway of α7nAChR/ERK/Egr-1.

BACKGROUND: Sinomenine (SIN) is an anti-inflammatory drug that has been used for decades in China to treat arthritis. In a previous study, SIN acted on α7 nicotinic acetylcholine receptor (α7nAChR) to inhibit inflammatory responses in macrophages, which indicates a new anti-inflammatory mechanism of SIN. However, the level of α7nAChR was increased in the inflammatory responses and was downregulated by SIN in vitro, so the underlying mechanisms of SIN acting on α7nAChR remain unclear. PURPOSE: To analyze the role of α7nAChR in inflammation and the effect and mechanism of SIN regulation of α7nAChR. METHODS: The effects of SIN on α7nAChR in endotoxemic mice and LPS-stimulated macrophages were observed. Nicotine (Nic) was used as a positive control, and berberine (Ber) was used as a negative control targeting α7nAChR. The antagonists of α7nAChR, α-bungarotoxin (BTX) and mecamylamine (Me), were used to block α7nAChR. In RAW264.7 macrophage cells in vitro, α7nAChR short hairpin RNA (shRNA) was used to knock down α7nAChR. Macrophage polarization was analyzed by the detection of TNF-α, IL-6, iNOS, IL-10, Arg-1, and Fizz1. U0126 was used to block ERK phosphorylation. The cytokines α7nAChR, ERK1/2, p-ERK1/2 and Egr-1 were detected. RESULTS: SIN decreased the levels of TNF-α, IL-6 and the expression of α7nAChR increased by LPS in endotoxemic mice. The above effects of SIN were attenuated by BTX. In the α7nAChR shRNA transfected RAW264.7 cells, compared with the control, α7nAChR was knocked down, and M1 phenotype markers (including TNF-α, IL-6, and iNOS) were significantly downregulated, whereas M2 phenotype markers (including IL-10, Arg-1, and Fizz1) were significantly upregulated when stimulated by LPS. SIN inhibited the expression of p-ERK1/2 and the transcription factor Egr-1 induced by LPS in RAW264.7 cells, and the above effects of SIN were attenuated by BTX. The expression of α7nAChR was suppressed by U0126, which lessened the expression of p-ERK1/2 and Egr-1. CONCLUSIONS: SIN acts on α7nAChR to inhibit inflammatory responses and downregulates high expression of α7nAChR in vivo and in vitro. The increase of α7nAChR expression is correlated with inflammatory responses and participates in macrophage M1 polarization. SIN downregulates α7nAChR via a feedback pathway of α7nAChR/ERK/Egr-1, which contributes to inhibiting macrophage M1 polarization and inflammatory responses.

Electroacupuncture Alleviates LPS-Induced ARDS Through α7 Nicotinic Acetylcholine Receptor-Mediated Inhibition of Ferroptosis.

Acute respiratory distress syndrome (ARDS) is an uncontrollable, progressive pulmonary inflammatory disease, and as a common clinical critical disease, there is no effective treatment available. Electroacupuncture (EA) therapy is a type of traditional Chinese medicine physiotherapy that can alleviate the inflammatory response. However, the potential mechanism of EA in the treatment of ARDS is not yet clear. Ferroptosis is a new type of programmed cell death characterized by intracellular iron accumulation and lipid peroxidation. Recently, emerging evidence has shown that ferroptosis is closely related to the occurrence and development of ARDS caused by various pathological factors. Here, we further investigated whether EA-mediated inhibition of ferroptosis in lung tissue could attenuate lipopolysaccharide (LPS)-induced ARDS and explored its underlying mechanisms. In this study, mice were administered LPS intraperitoneally to establish a model of LPS-induced ARDS. We found that EA stimulation could not only reduce the exudation of inflammatory cells and proteins in the alveolar lumen but also significantly alleviate the pathological changes of lung tissue, inhibit the production of proinflammatory cytokines and improve the survival rate of mice. Concurrently, we also found that ferroptosis events occurred in the lung tissue of LPS-induced ARDS mice, manifested by elevated iron levels, ROS production and lipid peroxidation. Intriguingly, our results showed that EA stimulation at the Zusanli (ST36) acupoint activated α7 nicotinic acetylcholine receptor (α7nAchR) in lung tissue mainly through the sciatic nerve and cervical vagus nerve, thus exerting anti-ferroptosis and pulmonary protective effects. Additionally, these effects were eliminated by methyllycaconitine (MLA), a selective antagonist of α7nAchR. In vitro experiments, activation of α7nAchR protected alveolar epithelial cells from LPS-induced ferroptosis. Furthermore, our experiments showed that the pulmonary protective effects of EA stimulation were effectively reversed by erastin, a ferroptosis activator. Collectively, we demonstrated that EA stimulation could alleviate LPS-induced ARDS by activating α7nAchR to inhibit LPS-induced ferroptosis in alveolar epithelial cells. Targeting and regulating ferroptosis in alveolar epithelial cells may be a potential intervention approach for the treatment of LPS-induced ALI/ARDS in the future.

Genistein-3'-sodium sulfonate ameliorates cerebral ischemia injuries by blocking neuroinflammation through the α7nAChR-JAK2/STAT3 signaling pathway in rats.

BACKGROUND: Neuroinflammation plays a pivotal role in the acute progression of cerebral ischemia/reperfusion injury (I/RI). We previously reported that genistein-3'-sodium sulfonate (GSS), a derivative from the extract of the phytoestrogen genistein (Gen), protects cortical neurons against focal cerebral ischemia. However, the molecular mechanism underlying the neuroprotective effects exerted by GSS remains unclear. PURPOSE: The present study focused on the anti-inflammatory effects of GSS following I/RI in rats. STUDY DESIGN: Randomized controlled trial. METHODS: The tMCAO rat model and LPS-stimulated BV2 in vitro model were used. Longa's scare was used to observe neurological function. TTC staining and Nissl staining were used to evaluate brain injury. ELISA, qRT-PCR, Western blotting and immunofluorescent staining methods were used to detect cytokine concentration, mRNA level, protein expression and location. RESULTS: GSS treatment improves neurological function, reduces the volume of cerebral infarction, attenuates proinflammatory cytokines and inactivates the phosphorylation of JAK2 and STAT3 in I/RI rats. Furthermore, GSS increased the expression of α7nAChR. More importantly, the neuroprotective, anti-inflammatory and inhibiting JAK2/STAT3 signaling pathway effects of GSS were counteracted in the presence of alpha-bungarotoxin (α-BTX), an α7nAChR inhibitor, suggesting that α7nAChR is a potential target associated with the anti-inflammatory effects of GSS in the I/RI rats. GSS also inhibited BV2 cells from releasing IL-1ß via the α7nAChR pathway after LPS stimulation. CONCLUSION: GSS protects against cerebral I/RI through the expression of α7nAChR and inhibition of the JAK2/STAT3 pathway. Our findings provide evidence for the role of the cholinergic anti-inflammatory pathway in neuroinflammation and uncover a potential novel mechanism for GSS treatment in ischemic stroke. The downstream signals of GSS, α7nAChR- JAK2/STAT3 could also be potential targets for the treatment of I/RI.

Genistein-3'-sodium sulfonate Attenuates Neuroinflammation in Stroke Rats by Down-Regulating Microglial M1 Polarization through α7nAChR-NF-κB Signaling Pathway.

Microglial M1 depolarization mediated prolonged inflammation contributing to brain injury in ischemic stroke. Our previous study revealed that Genistein-3'-sodium sulfonate (GSS) exerted neuroprotective effects in ischemic stroke. This study aimed to explore whether GSS protected against brain injury in ischemic stroke by regulating microglial M1 depolarization and its underlying mechanisms. We established transient middle cerebral artery occlusion and reperfusion (tMCAO) model in rats and used lipopolysaccharide (LPS)-stimulated BV2 microglial cells as in vitro model. Our results showed that GSS treatment significantly reduced the brain infarcted volume and improved the neurological function in tMCAO rats. Meanwhile, GSS treatment also dramatically reduced microglia M1 depolarization and IL-1ß level, reversed α7nAChR expression, and inhibited the activation of NF-κB signaling in the ischemic penumbra brain regions. These effects of GSS were further verified in LPS-induced M1 depolarization of BV2 cells. Furthermore, pretreatment of α7nAChR inhibitor (α-BTX) significantly restrained the neuroprotective effect of GSS treatment in tMCAO rats. α-BTX also blunted the regulating effects of GSS on neuroinflammation, M1 depolarization and NF-κB signaling activation. This study demonstrates that GSS protects against brain injury in ischemic stroke by reducing microglia M1 depolarization to suppress neuroinflammation in peri-infarcted brain regions through upregulating α7nAChR and thereby inhibition of NF-κB signaling. Our findings uncover a potential molecular mechanism for GSS treatment in ischemic stroke.

Electroacupuncture ameliorates intestinal inflammation by activating α7nAChR-mediated JAK2/STAT3 signaling pathway in postoperative ileus.

Inflammatory cytokines produced by muscularis macrophages largely contribute to the pathological signs of postoperative ileus (POI). Electroacupuncture (EA) can suppress inflammation, mainly or partly via activation of vagal efferent. The goal of this study was to investigate the mechanisms by which EA stimulation at an hindlimb region ameliorates inflammation in POI. Methods: Intestinal motility and inflammation were examined after 24 h after intestinal manipulation (IM)-induced POI in mice. Local immune response in the intestinal muscularis, expression of macrophages, α7 nicotinic acetylcholine receptor (α7nAChR), Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) were determined by flow cytometry, Western Blot, qPCR and immunofluorescence. The effects of α7nAChR antagonists (methyllycaconitine and α-bungarotoxin) and JAK2/STAT3 inhibitors (AG490 and WP1066) were also administered in a subset of mice prior to EA. In the parasympathetic pathways, intestinal motility and inflammation were determined after cervical vagotomy and sub-diaphragmatic vagotomy. The expression of gamma absorptiometry aminobutyric acid (GABAA) receptor in dorsal motor nucleus of vagal (DMV) cholinergic neurons was assessed by immunofluorescence and the response to DMV microinjection of bicuculine (antagonist of GABAA receptor) or muscimol (agonist of GABAA receptor) were assessed. Results: EA suppressed intestinal inflammation and promoted gastrointestinal motility. Mechanistically, EA activated the α7nAChR-mediated JAK2/STAT3 signaling pathway in macrophages which reduced the production of inflammatory cytokines. Furthermore, we also demonstrated that hindlimb region stimulation drove vagal efferent output by inhibiting the expression of GABAA receptor in DMV to ameliorate inflammation. Conclusions: The present study revealed that EA of hindlimb regions inhibited the expression of GABAA receptor in DMV neurons, whose excited vagal nerve, in turn suppressed IM-induced inflammation via activation of α7nAChR-mediated JAK2/STAT3 signaling pathway.

Excessive ROS production and enhanced autophagy contribute to myocardial injury induced by branched-chain amino acids: Roles for the AMPK-ULK1 signaling pathway and α7nAChR.

BACKGROUNDS AND AIMS: Leucine, isoleucine, and valine are diet derived and essential amino acids that are termed branched-chain amino acids (BCAA). BCAA are widely used as dietary supplements to boost muscle growth and enhance exercise performance. However, the effects of BCAA on myocardial function are largely unknown. This study was designed to investigate whether BCAA affect heart function and, if so, to further explore the underlying molecular basis for the observed effects. METHODS AND RESULTS: C57BL/6J mice were randomly divided into two groups, the control group received solvent (water) and the BCAA group received 2% BCAA dissolved in water, for a successive period of 12 weeks. Compared with control, BCAA treatment significantly increased water consumption without changing body weight or diet consumption; heart tissue BCAA levels were increased, markers representative of myocardial injury in heart tissue including c-reactive protein and cardiac muscle troponin were increased ; and creatine kinase, creatine kinase-MB, and lactate dehydrogenase were increased in serum; severe myocardial fibrosis was observed by Masson staining, which was accompanied by increased reactive oxygen species (ROS) production and decreased superoxide dismutase activity in heart tissue; both p-AMPK and p-ULK1 were significantly increased as was autophagy, judged by the presence of LC3 by western blotting and immunofluorescence, increased numbers of autophagosomes were found by transmission electron microscopy in the BCAA group. In vitro, 20 mmol/L BCAA significantly decreased cell viability and increased the production of ROS, as well as the expression of p-AMPK/AMPK and p-ULK1/ULK1 in cultured H9C2 cells. Treatment with the ROS scavenger N-acetyl-L-cysteine (NAC) improved cell viability and reversed ROS changes. Decreased H9C2 cell viability induced with 20 mmol/L BCAA was reversed by either blocking AMPK or inhibition of ULK1. Furthermore, blocking AMPK significantly decreased p-ULK1/ULK1, while inhibition of ULK1 reversed the enhanced expression of LC3-II/LC3-I induced by BCAA. Excessive ROS production and decreased cell viability induced by BCAA were further confirmed in primary cultured murine cardiomyocytes. Pharmacological activation of α7nAChR with PNU-282987 attenuated BCAA-induced injury in primary murine cardiomyocytes. However, this compound failed to suppress BCAA activation of AMPK and autophagy (LC3-II/I ratio). CONCLUSION: These results provide the first evidence that treatment of mice with BCAA induced myocardial injury by triggering excessive ROS production and by enhancing AMPK-ULK1 pathway-dependent autophagy. These findings suggested that inhibition of either ROS production or autophagy may alleviate myocardial injury induced by BCAA.

Acupuncture attenuates cognitive deficits through α7nAChR mediated anti-inflammatory pathway in chronic cerebral hypoperfusion rats.

AIMS: Chronic cerebral hypoperfusion (CCH) elicits inflammatory response, which contributes to the pathology of cognitive impairment. Several studies demonstrate that the alpha-7 nicotinic acetylcholine receptor (α7nAChR) can be a key component to modulate the inflammatory responses. We have reported previously that acupuncture attenuated cognitive deficits induced by CCH. In present study, whether effect of acupuncture was related to α7nAChR mediated anti-inflammatory pathway in CCH rats was further explored. MAIN METHODS: Acupuncture was performed in CCH rats induced by bilateral common carotid arteries occlusion. Neuronal injury, the activation of microglia, the release of inflammatory cytokines, the expression of α7nAChR, and the activation of JAK2/STAT3 signaling pathways were detected. Cognitive function and central inflammation were evaluated after the intraperitoneal injection of an α7nAChR agonist PNU282987, or intracerebroventricular injection of an α7nAChR antagonist α-bungarotoxin (α-BGT). KEY FINDINGS: We found that there were neuronal damage and inflammation, accompanied with the decreased expressions of α7nAChR in the hippocampus under CCH condition. Acupuncture inhibited neuronal damage, activation of microglia, and inflammatory cytokines. The expressions of α7nAChR, together with its downstream JAK2/STAT3 pathways were up regulated by acupuncture. PNU282987 mimicked the anti-inflammatory and neuroprotective effects as well as the cognitive improvements of acupuncture. Meanwhile, the benefit effects of acupuncture above were blocked by α-BGT. SIGNIFICANCE: It was demonstrated that acupuncture promoted cognitive function and afforded neuroprotective effects against inflammation via activation of α7nAChR and its downstream JAK2-STAT3 pathway in CCH rats. It provides a new insight for acupuncture as an anti-inflammatory intervention for cognitive impairment.

Chinese Herbal Formula Ermiao Powder () Regulates Cholinergic Anti-inflammatory Pathway in Rats with Rheumatoid Arthritis.

OBJECTIVE: To investigate the effect of Chinese herbal formula Ermiao Powder (, EMP) on the expression of cholinergic anti-inflammatory pathway in rats with rheumatoid arthritis (RA). METHODS: Seventy-two rats were randomly divided into 6 groups according to body weight, including normal control group, collageninduced arthritis (CIA) group, three doses EMP groups, and methotrexate (MTX) group (n=12 per group). All of the rats except for those in the normal control group were given multipoint subcutaneous injection of bovine type II collagen to establish a CIA model. Three EMP groups received a high- (4.5 g/kg), medium- (3.0 g/kg), and low- (1.5 g/kg) doses of EMP by intragavage, respectively. MTX group was injected intraperitoneally MTX at 0.9 mg/kg once a week as the positive control. The administration was 3 consecutive weeks. Joint swelling, arthritis index, and body weight changes in different experimental groups of rats were tested. The joint damage was evaluated by masson staining. Quantitative real-time polymerase chain reaction, Western blot, and immunohistochemistry (IHC) were performed to evaluate the expression of CHRNA7, encoding α7 nicotinic acetylcholine receptor in the cholinergic anti-inflammatory pathway, in different tissues and their localization in the spleen and joints. RESULTS: CHRNA7 expression levels were significantly higher in the joints and spleens of CIA group than those in normal control group (both P<0.05). Moreover, the CHRNA7 mRNA and protein levels in the spleen and joints of MTX and three doses of EMP groups were significantly lower than CIA group (all P<0.05). Compared with the MTX group, treatment with low-dose EMP resulted in significant reduction of CHRNA7 mRNA and protein expression levels (P<0.05 or P<0.01). IHC showed positive signals of CHRNA7 in the white pulp and red pulp of the spleens of rats; CHRNA7 was expressed on fibroblast-like synoviocytes, macrophages, and endothelial cells in the joints of rats, and the expression in the joints of low-dose EMP group was significantly lower than that in the CIA group (P<0.01). CONCLUSIONS: Cholinergic anti-inflammatory pathway was involved in the generation of the inflammatory reaction in CIA rats, and EMP exerted therapeutic effect on RA through cholinergic anti-inflammatory pathway.

CHRFAM7A: A human specific fusion gene, accounts for the translational gap for cholinergic strategies in Alzheimer's disease.

BACKGROUND: Cholinergic neuronal loss is one of the hallmarks of AD related neurodegeneration; however, preclinical promise of α7 nAChR drugs failed to translate into humans. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of α7 nAChR and was unaccounted for in preclinical models. METHODS: Molecular methods: Function of CHRFAM7A alleles was studied in vitro in two disease relevant phenotypic readouts: electrophysiology and Aß uptake. Genome edited human induced pluripotent stem cells (iPSC) were used as a model system with the human context. Double blind pharmacogenetic study: We performed double-blind pharmacogenetic analysis on the effect of AChEI therapy based on CHRFAM7A carrier status in two paradigms: response to drug initiation and DMT effect. Mini Mental Status Examination (MMSE) was used as outcome measure. Change in MMSE score from baseline was compared by 2-tailed T-test. Longitudinal analysis of clinical outcome (MMSE) was performed using a fitted general linear model, based on an assumed autoregressive covariance structure. Model independent variables included age, sex, and medication regimen at the time of the first utilized outcome measure (AChEI alone or AChEI plus memantine), APOE4 carrier status (0, 1 or 2 alleles as categorical variables) and CHRFAM7A genotype. FINDINGS: The direct and inverted alleles have distinct phenotypes. Functional CHRFAM7A allele classifies the population as 25% non-carriers and 75% carriers. Induced pluripotent stem cell (iPSC) models α7 nAChR mediated Aß neurotoxicity. Pharmacological readout translates into both first exposure (p = 0.037) and disease modifying effect (p = 0.0048) in two double blind pharmacogenetic studies. INTERPRETATION: CHRFAM7A accounts for the translational gap in cholinergic strategies in AD. Clinical trials not accounting for this uniquely human genetic factor may have rejected drug candidates that would benefit 25% of AD. Reanalyses of the completed trials using this pharmacogenetic paradigm may identify effective therapy.

Mechanism of calcium potentiation of the α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor (nAChR) is among the most abundant types of nAChR in the brain, yet the ability of nerve-released ACh to activate α7 remains enigmatic. In particular, a major population of α7 resides in extra-synaptic regions where the ACh concentration is reduced, owing to dilution and enzymatic hydrolysis, yet ACh shows low potency in activating α7. Using high-resolution single-channel recording techniques, we show that extracellular calcium is a powerful potentiator of α7 activated by low concentrations of ACh. Potentiation manifests as robust increases in the frequency of channel opening and the average duration of the openings. Molecular dynamics simulations reveal that calcium binds to the periphery of the five ligand binding sites and is framed by a pair of anionic residues from the principal and complementary faces of each site. Mutation of residues identified by simulation prevents calcium from potentiating ACh-elicited channel opening. An anionic residue is conserved at each of the identified positions in all vertebrate species of α7. Thus, calcium associates with a novel structural motif on α7 and is an obligate cofactor in regions of limited ACh concentration.