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.

[Effect of transcutaneous auricular vagus nerve stimulation on the splenic α7nAchR/JAK2/STAT3 signaling pathway in LPS-induced depressive-like behavior rats].

OBJECTIVE: To observe the effect of transcutaneous auricular vagus nerve stimulation (taVNS) on the improvement of depressive-like behavior and the splenic α7 nicotinic acetylcholine receptor (α7nAchR) / Janus kinase 2 (JAK2 / signal transducer and activator of transcription 3 (STAT3) signaling pathway in lipopolysaccharide (LPS)-induced depressive-like behavior rats, so as to investigate the antidepressant mechanism of taVNS. METHODS: SD rats were randomly divided into SD control group, SD model group and SD taVNS group, and α7nAchR knockout rats were also randomly divided into α7 control group, α7 model group and α7 taVNS group, with 6 rats in each group. Rat model of depressive-like behavior was established by intraperitoneal injection of LPS (1 mg/kg). Rats in both SD taVNS and α7 taVNS groups received taVNS intervention once a day (2 Hz/15 Hz, 2 mA, 30 min) from 7 days before LPS injection to 2 days after LPS injection, respectively. The mean speed, activity time and side immobility time in the open field test were recorded after taVNS. The contents of interleukin 10 (IL-10) and chemokine (C-X-C motif) ligand 1 (CXCL1) in serum were detected by electrochemiluminescence multifactorial method. The splenic phosphorylated (p)-JAK2 and p-STAT3 protein expressions were detected by Western blot. RESULTS: Compared with their respective control groups, the mean speed and active time were reduced (P<0.01, P<0.05, P<0.001) and the side immobility time was increased (P<0.001) in the open field test, serum IL-10 and CXCL1 levels were up-regulated (P<0.01, P<0.05, P<0.001), and splenic p-JAK2 protein expressions were down-regulated (P<0.05, P<0.01) in SD and α7nAchR knockout rats, and splenic p-STAT3 protein expression were down-regulated (P<0.05) in SD rats after LPS injection. Following taVNS intervention and in comparison with the model group , the mean speed and active time were increased (P<0.01) and the side immobility time was decreased (P<0.001) in the open field test, serum IL-10 and CXCL1 levels down-regulated (P<0.05), while splenic p-JAK2 and p-STAT3 protein expressions were up-regulated (P<0.01, P<0.001) in the SD taVNS group rather than in the α7 taVNS group. Compared with SD taVNS group, the α7 taVNS group showed increased (P<0.001, P<0.05) side immobility time in the open field test and serum IL-10, decreased splenic p-JAK2 and p-STAT3 protein expressions (P<0.01, P<0.05). CONCLUSION: taVNS may exert anti-inflammatory effects through modulating the splenic α7nAchR/JAK2/STAT3 signaling pathway, thereby ameliorating LPS-induced depressive-like behavior in rats.

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.

Anti-neuroinflammation effects of transcutaneous auricular vagus nerve stimulation against depression-like behaviors via hypothalamic α7nAchR/JAK2/STAT3/NF-κB pathway in rats exposed to chronic unpredictable mild stress.

BACKGROUND: Transcutaneous auricular vagus nerve stimulation (taVNS) is a vital neuromodulation for the treatment of depression, but its antidepressant molecular mechanism is unclear. The α7 nicotinic acetylcholine receptor (α7nAchR) is a key mediator of the vagus nerve that mediates its anti-inflammatory efficacy. Here, we investigated whether the antidepressant effect of taVNS in chronic unpredicted mild stress (CUMS)-exposed rats works through the α7nAchR/JAK2/STAT3/NF-κB pathway. METHODS: The depression model was established by CUMS for continuous 6 weeks in rats. From the 4th week of the experiment, CUMS-exposed rats were subjected to taVNS for 3 weeks. To clarify the role of α7nAchR in the antidepressant effect of taVNS, we used α7nAchR-/- gene knockout rats. The sucrose preference test (SPT), open field test (OFT), and forced swimming test (FST) were used to evaluate depression-like behaviors of rats. Immunofluorescent staining was used to observe the morphology of microglia in the hypothalamus. Western blot was used to examine the protein expression of α7nAchR, p-JAK2, p-STAT3, IL-1ß, NF-κB p65, and p-NF-κB p65 in the hypothalamus. RESULTS: Depression-like behaviors in CUMS-exposed rats were manifested by decreased SPT ratio, increased FST immobility time, decreased total distance, vertical movement score, and activity time of OFT. Hypothalamic neuroinflammation in CUMS-exposed rats was manifested by an amoebic-like activated state of microglia, downregulated expression of α7nAchR, p-JAK2, p-STAT3, and upregulated expression of NF-κB p65, p-NF-κB p65, and IL-1ß. TaVNS could significantly reverse the above-mentioned phenomena, but had a poor improvement effect for CUMS-exposed α7nAchR-/- rats. CONCLUSION: The hypothalamic α7nAchR/JAK2/STAT3/NF-κB signaling pathway may play an important role in the antidepressant-like behavior of taVNS.

[Acupuncture mitigates ocular surface inflammatory response via α7nAChR/NF-κB p65 signaling in dry eye guinea pigs].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the ocular surface inflammation and α7 nicotinic acetylcholine receptor (α7nAChR) / nuclear factor kappa-B (NF-κB) p65 signal pathway in guinea pigs with dry eye, so as to explore its underlying mechanism. METHODS: A total of 32 male British tricolor short haired guinea pigs were randomized into blank control, model, EA and sham acupuncture groups, with 8 guinea pigs in each group. The dry eye model was established by subcutaneous injection of scopolamine hydrobromide solution (0.6 mg/0.2 mL each time, 4 times a day for 10 days). Guinea pigs of the EA group was treated with EA at bilateral "Cuanzhu" (BL2) and "Taiyang" (HN5), and manual acupuncture at bilateral "Jingming" (BL1), "Sizhukong" (SJ23), "Tongziliao" (GB1) for 15 min, once daily for 14 days. For animals of the sham acupuncture group, a blunt needle was used to prick the skin surface of the acupoints, the acupoint selection and stimulation time were the same as those in the EA group. Before and after modeling and after the intervention, the breakup time (BUT) of lacrimal film, sodium fluorescein coloring (Fl) state of corneal epithelium and phenol red thread (PRT) moist length were recorded for assessing the severity of dry eye. The density of activated immune cells around the corneal epithelial stromal cells was determined by corneal confocal microscopy. The contents of interleukin-4 (IL-4), IL-6, IL-10, tumor necrosis factor α (TNF-α) in the cornea and lacri-mal gland tissues were determined by ELISA, and the expression levels of α7nAChR and NF-κB p65 in the cornea and lacrimal gland were detected by immunohistochemistry and Western blot, separately. RESULTS: Compared with the blank control group, the corneal Fl, density of activated immune cells of corneal epithelium, contents of IL-6, IL-10 and TNF-α in both corneal and lacrimal gland tissues, NF-κB p65 cell positive rate and protein expression of lacrimal gland and corneal tissues were significantly increased (P<0.01, P<0.05), while the BUT, PRT and lacrimal gland α7nAChR cell positive rate considerably decreased (P<0.01) in the model group. In comparison with the model group, the level of corneal Fl, density of the activated immune cells of corneal epithelium, contents of corneal and lacrimal IL-6 and TNF-α, and corneal and lacrimal NF-κB p65 cell positive rates and protein expressions were remarkably down-regulated in the EA group (P<0.01, P<0.05), rather than in the sham acupuncture group (P>0.05) except content of corneal IL-10, lacrimal NF-κB p65 cell positive rate and lacrimal α7nAChR protein expression, whereas the levels of BUT, PRT, corneal and lacrimal IL-10 and corneal and lacrimal α7nAChR cell positive rates and protein expressions significantly up-regulated in the EA group (P<0.01, P<0.05), rather than in the sham acupuncture group (P>0.05) except corneal TNF-α and corneal NF-κB p65 protein expression. CONCLUSION: EA can improve corneal and lacrimal function in dry eye guinea pigs, which may be associated with its actions in increasing the expression of α7nAChR, inhibiting the nuclear translocation of NF-κB, and reducing the activated immune cells and inflammatory reaction.

[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].

OBJECTIVE: 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. METHODS: 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-1ß (IL-1ß) 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. RESULTS: 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-1ß 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-1ß 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-1ß 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. CONCLUSION: 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.

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.

Functional characterization of multifunctional ligands targeting acetylcholinesterase and alpha 7 nicotinic acetylcholine receptor.

Alzheimer's disease (AD) is a neurodegenerative disorder associated with cholinergic dysfunction, provoking memory loss and cognitive dysfunction in elderly patients. The cholinergic hypothesis provided over the years with molecular targets for developing palliative treatments for AD, acting on the cholinergic system, namely, acetylcholinesterase and α7 nicotinic acetylcholine receptor (α7 nAChR). In our synthetic work, we used "click-chemistry" to synthesize two Multi Target Directed Ligands (MTDLs) MB105 and MB118 carrying tacrine and quinuclidine scaffolds which are known for their anticholinesterase and α7 nAChR agonist activities, respectively. Both, MB105 and MB118, inhibit human acetylcholinesterase and human butyrylcholinesterase in the nanomolar range. Electrophysiological recordings on Xenopus laevis oocytes expressing human α7 nAChR showed that MB105 and MB118 acted as partial agonists of the referred nicotinic receptor, albeit, with different potencies despite their similar structure. The different substitution at C-3 on the 2,3-disubstituted quinuclidine scaffold may account for the significantly lower potency of MB118 compared to MB105. Electrophysiological recordings also showed that the tacrine precursor MB320 behaved as a competitive antagonist of human α7 nAChR, in the micromolar range, while the quinuclidine synthetic precursor MB099 acted as a partial agonist. Taken all together, MB105 behaved as a partial agonist of α7 nAChR at concentrations where it completely inhibited human acetylcholinesterase activity paving the way for the design of novel MTDLs for palliative treatment of AD.

Myrtus communis subsp. communis improved cognitive functions in ovariectomized diabetic rats.

AIM: The purpose of this study was to investigate the possible effects of Myrtus communis subsp. communis (MC) on cognitive impairment in ovariectomized diabetic rats. MATERIAL AND METHOD: Female Sprague-Dawley rats were divided into 5 groups consisting of 15 rats each; Control (C), Diabetes (D), Ovariectomy and diabetes (OVX + D), Ovariectomy, diabetes and donepezil (OVX + D + Don), Ovariectomy, diabetes and Myrtus communis subsp. communis (OVX + D + MC). Blood glucose measurements were made at the beginning and end of the experiments. The animals underwent the novel object recognition test (NORT) and their performance was evaluated. In hippocampal tissues; amyloid beta (Aß) and neprilysin levels, acetylcholinesterase (AChE), and choline acetyltransferase (ChAT) activities, polysialylated neural cell adhesion molecule (PSA-NCAM), α7 subunit of neuronal nicotinic acetylcholine receptor (α7-nAChR) and brain derived neurotrophic factor (BDNF) gene expressions were examined. RESULTS: Animals with ovariectomy and diabetes showed increased levels of blood glucose, AChE activity and Aß levels, and decreased neprilysin levels, ChAT activity, α7-nAChR, PSA-NCAM and BDNF gene expressions in parallel with a decrease in NORT performance score. On the other hand, in the MC-treated OVX + D group, there was a significant decrease observed in blood glucose levels and AChE activities while there was improvement in NORT performances and an increase in hippocampal ChAT activity, neprilysin levels, α7-nAChR, PSA-NCAM and BDNF expressions. CONCLUSION: These results suggest that MC extract could improve cognitive and neuronal functions with its anticholinesterase and antihyperglycemic properties.

Synthesis, Pharmacological Characterization, and Structure-Activity Relationships of Noncanonical Selective Agonists for α7 nAChRs.

A lack of selectivity of classical agonists for the nicotinic acetylcholine receptors (nAChR) has prompted us to identify and develop a distinct scaffold of α7 nAChR-selective ligands. Noncanonical 2,4,6-substituted pyrimidine analogues were framed around compound 40 for a structure-activity relationship study. The new lead compounds activate selectively the α7 nAChRs with EC50's between 30 and 140 nM in a PNU-120596-dependent, cell-based calcium influx assay. After characterizing the expanded lead landscape, we ranked the compounds for rapid activation using Xenopus oocytes expressing human α7 nAChR with a two-electrode voltage clamp. This approach enabled us to define the molecular determinants governing rapid activation, agonist potency, and desensitization of α7 nAChRs after exposure to pyrimidine analogues, thereby distinguishing this subclass of noncanonical agonists from previously defined types of agonists (agonists, partial agonists, silent agonists, and ago-PAMs). By NMR, we analyzed pKa values for ionization of lead candidates, demonstrating distinctive modes of interaction for this landscape of ligands.

Anti-inflammatory and autonomic effects of electroacupuncture in a rat model of diet-induced obesity.

OBJECTIVE: To study the effect of electroacupuncture (EA) on the cholinergic anti-inflammatory pathway (CAP) by measurement of vagal activity in rats with high-fat diet (HFD)-induced obesity. METHODS: Diet-induced obesity (DIO) was induced in 30 rats by feeding them a HFD for 12 weeks. A further 10 rats fed normal food comprised the lean diet (LD) control group. DIO rats were further subdivided into three groups that received a HFD only (HFD group, n=10), a HFD plus electroacupuncture (HFD+EA group, n=10) or a HFD plus minimal acupuncture (HFD+MA group, n=10). EA and MA treatments were continued for 8 weeks. Heart rate variability (HRV) was used to measure the function of the autonomic nervous system before and after treatment. ELISA was used to determine acetylcholine (ACh) and tumour necrosis factor (TNF)-α levels in the serum. Real-time PCR was used to assess the mRNA expression of α7-subtype nicotinic acetylcholine cholinergic receptors (α7nAChRs) and TNF-α in the mesenteric white adipose tissues (MWAT). RESULTS: EA but not MA significantly reduced rats' bodyweight. No difference was found in the low frequency (LF), high frequency (HF) and the balance between LF and HF (LF/HF) components of HRV before treatment. After the EA intervention, HF was elevated and LF/HF was reduced in the HFD+EA group comparedwith the HFD group. TNF-α in the serum and MWAT were increased in the HFD group, but were reduced in the HFD+EA group. Furthermore, EA promoted expression of α7nAChRs and ACh in the MWAT. There was no difference between the HFD and HFD+MA groups for any indices. CONCLUSIONS: EA enhanced vagal activity, promoted ACh release and activated α7nAChRs in the MWAT, leading to inhibition of proinflammatory cytokine production.

Sinomenine inhibits fibroblast-like synoviocyte proliferation by regulating α7nAChR expression via ERK/Egr-1 pathway.

Fibroblast like synoviocyte (FLS) is a crucial in the pathogenesis of rheumatoid arthritis (RA), and involved in inflammation and joint destruction. Sinomenine (SIN), an alkaloid derived from the plant Sinomenium acutum, has anti-inflammatory and analgesic effect and been used for RA treatment in China. Alpha 7 nicotinic acetylcholine receptors (α7nAChR), as the key receptor in cholinergic anti-inflammatory pathway (CAP) to inhibit inflammation, has been detected in RA patients synovium, but its role is still unclear. Here we investigated the association between the aggressive proliferation of FLS and α7nAChR expression and the effect of sinomenine. FLS was isolated from synovial tissues of adjuvant-induced-arthritis (AIA) rat. Tumor necrosis factor(TNF)-α was used to induce the aggressive proliferation of FLS. MTT assay was applied to evaluate the proliferation of FLS. The messenger RNA (mRNA) and protein levels of α7nAChR and early growth response gene-1 (Egr-1) were measured. The results showed that TNF-α induced FLS proliferation in vitro (P < .01) and increased the phosphorylation of ERK1/2 and the expression of Egr-1 and α7nAChR (P < .05 or P < .01). U0126, the inhibitor of ERK1/2 inhibited α7nAChR expression and FLS proliferation significantly (P < .05 or P < .01). Specific short interference RNA(siRNA) of α7nAChR decreased α7nAChR expression and inhibited FLS proliferation as well. SIN inhibited the proliferation of FLS and decreased the phosphorylation of ERK1/2, and the expression of Egr-1 and α7nAChR induced by TNF-α (P < .05). In conclusion, the expression of α7nAChR involved in the aggressive proliferation of FLS induced by TNF-α and was regulated by ERK/Egr-1 signal pathway. SIN inhibited FLS proliferation and α7nAChR expression through inhibiting ERK/Egr-1 signal pathway, this may contribute to the anti-inflammatory and anti-arthritic effect of SIN.

α7 Nicotinic Acetylcholine Receptor Stimulation Attenuates Neuroinflammation through JAK2-STAT3 Activation in Murine Models of Intracerebral Hemorrhage.

Accounting for high mortality and morbidity rates, intracerebral hemorrhage (ICH) remains one of the most detrimental stroke subtypes lacking a specific therapy. Neuroinflammation contributes to ICH-induced brain injury and is associated with unfavorable outcomes. This study aimed to evaluate whether α7 nicotinic acetylcholine receptor (α7nAChR) stimulation ameliorates neuroinflammation after ICH. Male CD-1 mice and Sprague-Dawley were subjected to intracerebral injection of autologous blood or bacterial collagenase. ICH animals received either α7nAChR agonist PHA-543613 alone or combined with α7nAChR antagonist methyllycaconitine (MLA) or Janus kinase 2 (JAK2) antagonist AG490. Neurobehavioral deficits were evaluated at 24 hours, 72 hours, and 10 weeks after ICH induction. Perihematomal expressions of JAK2, signal transducer and activator of transcription 3 (STAT3), tumor necrosis factor-α (TNF-α), and myeloperoxidase (MPO) were quantified via Western blot. Histologic volumetric analysis of brain tissues was conducted after 10 weeks following ICH induction. PHA-543613 improved short-term neurobehavioral (sensorimotor) deficits and increased activated perihematomal JAK2 and STAT3 expressions while decreasing TNF-α and MPO expressions after ICH. MLA reversed these treatment effects. PHA-543613 also improved long-term neurobehavioral (sensorimotor, learning, and memory) deficits and ameliorated brain atrophy after ICH. These treatment effects were reduced by AG490. α7nAChR stimulation reduced neuroinflammation via activation of the JAK2-STAT3 pathway, thereby ameliorating the short- and long-term sequelae after ICH.

[Effects of Electroaupuncture Stimulation of "Xiajuxu" (ST 39), etc. on Duodenal Mucosal Injury, Serum Pro-inflammatory Factors Levels and Duodenal Nicotinic Acetylcholine Receptor alpha 7 Expression in Duodenal Ulcer Rats].

OBJECTIVE: To observe the relatively specific effect of electroacupuncture (EA) of "Xiajuxu" (ST 39, the lower hesea paint of the small intestine), etc. on the level of serum TNF-alpha, lnterleukin-1 P (IL-1 P) and high mobility group protein B 1 (HMGB 1) contents, and duodenum a7 nicotinic acetyicholine receptor (nAchR) expression in duodenal ulcer rats, so as to explore its mechanisms underlying improving duodenal ulcer. METHODS: Sixty SD rats were randomly divided into 6 groups: normal control, model, Xiajuxu (ST 39), Zusanli (ST 36), Shangjuxu (ST 37) and Yanglingquan (GB 34). The duodenal ulcer model was established by subcutaneous injection of 10% Cysteamine Hydrochloride (300 mg/kg), following by giving the rats with access to water containing Cysteamine. EA (10 Hz/50 Hz, 1- 3 mA) was applied to bilateral ST 39, ST 36, ST 37 and GB 34 for 30 min, once daily for 10 days. The ulcer scores (0-5 points) of the duodenal mucosa were assessed according to modified Moraes' methods. Serum TNF-alpha, IL-1 beta and HMGB 1 levels were assayed by ELISA and the expression of neuronal a7 nAchR in the duodenal tissue was detected by Western blot. RESULTS: After modeling, the ulcer score, serum TNF-alpha, IL-i p and HMGB 1 contents were significantly increased (P<0.01) and the expression level of a7 nAchR in the duodenal tissue was significantly down- regulated in comparison with the normal control group (P<0.01). Following EA intervention, the serum TNF-alpha and HMGB 1 con- tents in the Xiajuxu(ST 39), Zusanli (ST 36), Shangjuxu (ST 37) and Yanglingquan (GB 34) groups, and the ulcer scores and IL-1 beta content of the Xiajuxu(ST 39), Zusanli (ST 36) and Shangjuxu (ST 37) groups were considerably reduced, and the expression of alpha7 nAchR in both Xiajuxu (ST 39) and Zusanli (ST 36) groups was evidently increased (P<0.05, P<0.0.1). No significant changes were found in the ulcer score, serum IL-1 beta content, and a7 nAchR expression in the Yanglingquan (GB 34) group and a 7 nAchR expression in the Shangjuxu (ST 37) group in comparison with the model group (P>0.05). CONCLUSION: EA stimulation of ST 36, ST 37 and ST 39 can reduce ulcer injury in duodenal ulcer model rats, which may be associated with their effects in down-regulating serum TNF-alpha, IL-1 beta and HMGB 1 contents and up-regulating alpha7 nAchR expression of the duodenal tissue, possibly by suppressing immune and inflammatory reactions and regulating nicotinic activity.

Perinatal Phosphatidylcholine Supplementation and Early Childhood Behavior Problems: Evidence for CHRNA7 Moderation.

OBJECTIVE: α7-Nicotinic receptors are involved in the final maturation of GABA inhibitory synapses before birth. Choline at levels found in the amniotic fluid is an agonist at α7-nicotinic receptors. The authors conducted a double-blind placebo-controlled trial to assess whether high-dose oral phosphatidylcholine supplementation during pregnancy to increase maternal amniotic fluid choline levels would enhance fetal development of cerebral inhibition and, as a result, decrease childhood behavior problems associated with later mental illness. METHOD: The authors previously reported that newborns in the phosphatidylcholine treatment group have increased suppression of the cerebral evoked response to repeated auditory stimuli. In this follow-up, they report parental assessments of the children's behavior at 40 months of age, using the Child Behavior Checklist. RESULTS: At 40 months, parent ratings of children in the phosphatidylcholine group (N=23) indicated fewer attention problems and less social withdrawal compared with the placebo group (N=26). The improvement is comparable in magnitude to similar deficits at this age associated with later schizophrenia. The children's behavior is moderated by CHRNA7 variants associated with later mental illness and is related to their enhanced cerebral inhibition as newborns. CONCLUSIONS: CHRNA7, the α7-nicotinic acetylcholine receptor gene, has been associated with schizophrenia, autism, and attention deficit hyperactivity disorder. Maternal phosphatidylcholine treatment may, by increasing activation of the α7-nicotinic acetylcholine receptor, alter the development of behavior problems in early childhood that can presage later mental illness.

Selectivity optimization of substituted 1,2,3-triazoles as α7 nicotinic acetylcholine receptor agonists.

Three series of substituted anti-1,2,3-triazoles (IND, PPRD, and QND), synthesized by cycloaddition from azide and alkyne building blocks, were designed to enhance selectivity and potency profiles of a lead α7 nicotinic acetylcholine receptor (α7-nAChR) agonist, TTIn-1. Designed compounds were synthesized and screened for affinity by a radioligand binding assay. Their functional characterization as agonists and antagonists was performed by fluorescence resonance energy transfer assay using cell lines expressing transfected cDNAs, α7-nAChRs, α4ß2-nAChRs, and 5HT3A receptors, and a fluorescence cell reporter. In the IND series, a tropane ring of TTIn-1, substituted at N1, was replaced by mono- and bicyclic amines to vary length and conformational flexibility of a carbon linker between nitrogen atom and N1 of the triazole. Compounds with a two-carbon atom linker optimized binding with Kd's at the submicromolar level. Further modification at the hydrophobic indole of TTIn-1 was made in PPRD and QND series by fixing the amine center with the highest affinity building blocks in the IND series. Compounds from IND and PPRD series are selective as agonists for the α7-nAChRs over α4ß2-nAChRs and 5HT3A receptors. Lead compounds in the three series have EC50's between 28 and 260 nM. Based on the EC50, affinity, and selectivity determined from the binding and cellular responses, two of the leads have been advanced to behavioral studies described in the companion article (DOI: 10.1021/acschemneuro.5b00059).

A combination of neostigmine and anisodamine protects against ischemic stroke by activating α7nAChR.

BACKGROUND: Increasing endogenous acetylcholine by neostigmine decreased the ischemic cerebral injury. The off-target action on muscarinic receptor produced a variety of adverse effects and limited the clinical application on stroke. AIM: We combined neostigmine with anisodamine and investigated the neuroprotection and mechanism. METHODS: Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion. Neuroprotective action of neostigmine in combination with anisodamine at varying ratios was examined to determine the optimal combination as well as ideal therapeutic window. Potential involvement of α7 nicotinic acetylcholine receptor was examined by measuring the infarct size, the expression of proinflammatory cytokines, and the biomarkers of apoptosis in α7 nicotinic acetylcholine receptor knockout mice. A set of in vitro experiments was conducted in RAW264.7 cells to probe into potential molecular mechanisms. RESULTS: The neostigmine/anisodamine combination conferred neuroprotection. The protection was most potent at a ratio of 1:500. At such a ratio, the combination increased the binding of acetylcholine to α7 nicotinic acetylcholine receptor and reduced proinflammatory cytokines. The neuroprotection was evident only in wild-type and not in α7 nicotinic acetylcholine receptor knockout mice. The combination significantly decreased the expression of Bad and Bax, and increased Bcl-2 and Bcl-xl in α7 nicotinic acetylcholine receptor wild-type mice but not in knockout mice. The combination did not affect caspase-8, cleaved caspase-8, or caspase-12. CONCLUSIONS: Current study identified the optimal combination of neostigmine and anisodamine against ischemic stroke, and indicated that the acetylcholine-α7 nicotinic acetylcholine receptor is involved in the protective effects.

Maximizing the effect of an α7 nicotinic receptor PAM in a mouse model of schizophrenia-like sensory inhibition deficits.

Positive allosteric modulators (PAMs) for the α7 nicotinic receptor hold promise for the treatment of sensory inhibition deficits observed in schizophrenia patients. Studies of these compounds in the DBA/2 mouse, which models the schizophrenia-related deficit in sensory inhibition, have shown PAMs to be effective in improving the deficit. However, the first published clinical trial of a PAM for both sensory inhibition deficits and related cognitive difficulties failed, casting a shadow on this therapeutic approach. The present study used both DBA/2 mice, and C3H Chrna7 heterozygote mice to assess the ability of the α7 PAM, PNU-120596, to improve sensory inhibition. Both of these strains of mice have reduced hippocampal α7 nicotinic receptor numbers and deficient sensory inhibition similar to schizophrenia patients. Low doses of PNU-120596 (1 or 3.33mg/kg) were effective in the DBA/2 mouse but not the C3H Chrna7 heterozygote mouse. Moderate doses of the selective α7 nicotinic receptor agonist, choline chloride (10 or 33mg/kg), were also ineffective in improving sensory inhibition in the C3H Chrna7 heterozygote mouse. However, combining the lowest doses of both PNU-120596 and choline chloride in this mouse model did improve sensory inhibition. We propose here that the difference in efficacy of PNU-120596 between the 2 mouse strains is driven by differences in hippocampal α7 nicotinic receptor numbers, such that C3H Chrna7 heterozygote mice require additional direct stimulation of the α7 receptors. These data may have implications for further clinical testing of putative α7 nicotinic receptor PAMs.

Changes of neuronal acetylcholine receptor alpha 7 of peritoneal macrophage in experimental acute pancreatitis treated by Chaiqin Chengqi Decoction ().

OBJECTIVE: To investigate effect of Chaiqin Chengqi Decoction (, CQCQD) on changes of neuronal acetylcholine receptor alpha 7 (nAChRα7) of peritoneal macrophages in acute pancreatitis (AP). METHODS: Eighteen Kunming mice were equally randomized into the control group, AP group and CQCQD treatment group. AP was induced by two intraperitoneal injections of 4 g/kg L-arginine at 1 h apart, while control mice received saline injections. At 72 h after the first injection of L-arginine, mice in the treatment group were intragastrically administered 0.1 mL/10 g CQCQD every 2 h for 3 times, whilst mice in the other two groups received the same amount of saline feeding. Mice were sacrificed by cervical dislocation 2 h after the last feeding of either CQCQD or saline. Peritoneal macrophages were collected for determination of nAChRα7 mRNA and protein expression. Serum was collected for detection of interleukin-6 (IL-6), IL-10 and acetylcholine (ACh) levels, and pancreas was for histopathology analysis. RESULTS: The CQCQD treatment significantly ameliorated the severity of AP as evidenced by reducing the pancreatic histopathology score (4.5±0.5 vs. 6.2±1.7, P<0.05) and the serum IL-6 levels (1228.3±419.2 pg/mL vs. 1589.6±337.3 pg/mL, P<0.05). The mRNA and protein expression of nAChRα7 of the peritoneal macrophages in the AP group were similar to the control group (P>0.05), but were significantly up-regulated after the CQCQD treatment (P<0.05). The serum ACh levels in the AP group were significantly lower than those in the control group (3.1±0.6 µg/mL vs 4.8±0.7 µg/mL P<0.05), but were significantly increased after the CQCQD treatment (5.6±1.5 µg/mL vs 3.1±0.6 µg/mL, P<0.05). CONCLUSION: CQCQD is protective against L-arginine-induced AP through mechanisms involving nAChRα7 of peritoneal macrophages.

Membrane permeability-guided identification of neuroprotective components from Polygonum cuspidatun.

CONTEXT: Polygonum cuspidatum Sieb et Zucc. (Polygonaceae) possesses various pharmacological activities and has been widely using as one of the most popular and valuable Chinese herbal medicines in clinics. Its usage has increasingly attracted much of our attention and urges investigation on its bioactive components. OBJECTIVE: To establish a rapid and valid approach for screening potential neuroprotective components from P. cuspidatum. MATERIALS AND METHODS: Potential neuroprotective components from P. cuspidatum were screened utilizing liposome equilibrium dialysis followed by high-performance liquid chromatography (HPLC) analysis. Their neuroprotective effects on modulation of protein expression of α7 nAChR, α3 nAChR and synaptophysin (SPY) on SH-SY5Y human neuroblastoma cell line (SH-SY5Y) were evaluated by means of Western blotting. RESULTS: Two potential compounds, polydatin (C1) and emodin-8-O-ß-D-glucoside (C2), were detected and identified in our study. The biological tests showed that both compounds C1 and C2, respectively, at concentrations of 0.1 and 0.25 mg/mL significantly increased protein expression of α7 and α3 nicotinic acetylcholine receptors (nAChRs) in SH-SY5Y cells. Moreover, C1 and C2 at 0.1 mg/mL significantly reversed the Aß1₋42-induced decrease of α7 and α3 nAChRs protein expression in SH-SY5Y cells. In addition, C2 at 0.1 mg/mL significantly increased protein expression of SPY in SH-SY5Y cells and Aß11₋42-induced SH-SY5Y cells whereas C1 did not provide any positive effects. DISCUSSION AND CONCLUSION: In conclusion, our approach utilizing liposome equilibrium dialysis combined with HPLC analysis and cell-based assays is a prompt and useful method for screening neuroprotective agents.