α7nAChR Agonist Research Articles

Vagus Nerve Stimulation Relives Irritable Bowel Syndrome and the Associated Depression via α7nAChR-mediated Anti-inflammatory Pathway.

The present study is designed to investigate the role of vagus nerve in the treatments of irritable bowel syndrome (IBS) and the associated central nervous system disorders. An IBS animal model was established by giving acetic acid and chronic-acute stress (AA-CAS) treatment in adult male Wistar rats. Subdiaphragmatic vagotomy (SDV) and vagus nerve stimulation (VNS) were performed to intervene the excitability of vagus nerve. Permeability of blood brain barrier (BBB) was measured and agonist and antagonist of α7 nicotinic acetylcholine receptor (α7nAChR) were used to explore the relevant mechanisms. AA-CAS treatment resulted in abnormal fecal output, increased visceral sensitivity, depressive-like behaviors, and overexpression of inflammatory mediators, all of which were reversed by VNS treatment. The effects of VNS could also be observed when α7nAChR agonist was applied. Whereas α7nAChR antagonist (methyllycaconitine, MLA) reversed VNS's effects. Interestingly, VNS also reduced the increased permeability of blood brain barrier (BBB) following AA-CAS treatment in IBS rats. SDV treatment only show temporary efficacy on AA-CAS-induced symptoms and had no effect on the permeability of BBB. The intestinal abnormalities and depressive symptoms in IBS rats can be improved by VNS treatment. This positive effect of VNS was achieved through α7nAChR-mediated inflammatory pathway and may also be associated with the decreased of BBB permeability.

GTS-21 Enhances Regulatory T Cell Development from T Cell Receptor-Activated Human CD4+ T Cells Exhibiting Varied Levels of CHRNA7 and CHRFAM7A Expression.

Immune cells such as T cells and macrophages express α7 nicotinic acetylcholine receptors (α7 nAChRs), which contribute to the regulation of immune and inflammatory responses. Earlier findings suggest α7 nAChR activation promotes the development of regulatory T cells (Tregs) in mice. Using human CD4+ T cells, we investigated the mRNA expression of the α7 subunit and the human-specific dupα7 nAChR subunit, which functions as a dominant-negative regulator of ion channel function, under resting conditions and T cell receptor (TCR)-activation. We then explored the effects of the selective α7 nAChR agonist GTS-21 on proliferation of TCR-activated T cells and Treg development. Varied levels of mRNA for both the α7 and dupα7 nAChR subunits were detected in resting human CD4+ T cells. mRNA expression of the α7 nAChR subunit was profoundly suppressed on days 4 and 7 of TCR-activation as compared to day 1, whereas mRNA expression of the dupα7 nAChR subunit remained nearly constant. GTS-21 did not alter CD4+ T cell proliferation but significantly promoted Treg development. These results suggest the potential ex vivo utility of GTS-21 for preparing Tregs for adoptive immunotherapy, even with high expression of the dupα7 subunit.

Varenicline enhances recognition memory via α7 nicotinic acetylcholine receptors in the medial prefrontal cortex in male mice

Previous studies postulated that chronic administration of varenicline, a partial and full agonist at α4β2 and α7 nicotinic acetylcholine receptors (nAChRs), respectively, enhances recognition memory. However, whether its acute administration is effective, on which brain region(s) it acts, and in what signaling it is involved, remain unknown. To address these issues, we conducted a novel object recognition test using male C57BL/6J mice, focusing on the medial prefrontal cortex (mPFC), a brain region associated with nicotine-induced enhancement of recognition memory. Systemic administration of varenicline before the training dose-dependently enhanced recognition memory. Intra-mPFC varenicline infusion also enhanced recognition memory, and this enhancement was blocked by intra-mPFC co-infusion of a selective α7, but not α4β2, nAChR antagonist. Consistent with this, intra-mPFC infusion of a selective α7 nAChR agonist augmented object recognition memory. Furthermore, intra-mPFC co-infusion of U-73122, a phospholipase C (PLC) inhibitor, or 2-aminoethoxydiphenylborane (2-APB), an inositol trisphosphate (IP3) receptor inhibitor, suppressed the varenicline-induced memory enhancement, suggesting that α7 nAChRs may also act as Gq-coupled metabotropic receptors. Additionally, whole-cell recordings from mPFC layer V pyramidal neurons in vitro revealed that varenicline significantly increased the summation of evoked excitatory postsynaptic potentials, and this effect was suppressed by U-73122 or 2-APB. These findings suggest that varenicline might acutely enhance recognition memory via mPFC α7 nAChR stimulation, followed by mPFC neuronal excitation, which is mediated by the activation of PLC and IP3 receptor signaling. Our study provides evidence supporting the potential repositioning of varenicline as a treatment for cognitive impairment.

Acetylcholine receptor agonists effectively attenuated multiple program cell death pathways and improved left ventricular function in trastuzumab-induced cardiotoxicity in rats

AimsCardiotoxicity is a seriously debilitating complication of trastuzumab (TRZ) therapy in patients with cancer as a consequence of overexpression of the human epidermal growth factor receptor 2. Although most TRZ-induced cardiotoxicity (TIC) cases are reversible, some patients experience chronic cardiac dysfunction, and these irreversible concepts may be associated with cardiomyocyte death. Acetylcholine receptor (AChR) activation has been shown to exert cardioprotection in several heart diseases, but the effects of AChR agonists against TIC have not been investigated. Main methodForty adult male Wistar rats were randomized into 5 groups: (i) CON (0.9 % normal saline), (ii) TRZ (4 mg/kg/day), (iii) TRZ + α7nAChR agonist (PNU-282987: 3 mg/kg/day), (iv) TRZ + mAChR agonists (bethanechol: 12 mg/kg/day), and (v) TRZ + combined treatment (Combined PNU-282987 and bethanechol). Key findingsThe progression of TIC was driven by mitochondrial dysfunction, autophagic deficiency, and excessive myocyte death including by pyroptosis, ferroptosis, and apoptosis, which were significantly alleviated by α7nAChR and mAChR agonists. Interestingly, necroptosis was not associated with development of TIC. More importantly, the in vitro study validated the cytoprotective effects of AChR activation in TRZ-treated H9c2 cells, while not interfering with the anticancer properties of TRZ. All of these findings indicated that TRZ induced mitochondrial dysfunction, autophagic deficiency, and excessive myocyte death including pyroptosis, ferroptosis, and apoptosis, leading to impaired cardiac function. These pathological alterations were attenuated by α7nAChR and mAChR agonists. Significanceα7nAChR and mAChR agonists might be used as a future therapeutic target in the mitigation of TIC.

GTS-21 alleviates murine collagen-induced arthritis through inhibition of peripheral monocyte trafficking into the synovium

Emerging preclinical and clinical evidence reveals a critical role for the cholinergic anti-inflammatory pathway (CAP) in mediating rheumatoid arthritis (RA). Activation of CAP via vagus nerve stimulation or alpha 7 nicotinic acetylcholine receptor (α7nAChR) agonists has previously been shown to significantly reduce inflammation and improve outcomes in animal models of experimental arthritis. In this study, we sought to determine the protective mechanism of CAP on inflammatory arthritis, specifically RA, by using a selective α7nAChR agonist, GTS-21, to examine the role of CAP in the recruitment of monocytes/macrophages into the synovium in a collagen-induced arthritis (CIA) mouse model.We found that GTS-21 ameliorated systemic and local synovial inflammation, thereby reducing synovial macrophage infiltration in CIA mice. Using in vivo imaging, we further demonstrated that GTS-21 suppressed the trafficking of monocytes into inflamed joints, while our in vitro Transwell assay data confirmed that GTS-21 reduced the migratory ability of monocytes. In addition, we found that GTS-21 reduced the number of peripheral inflammatory monocytes and down-regulated expression of the chemokines CCR2 and CCR5 on monocytes and CCL2 in the paw tissue. GTS-21 also mediated the expression levels of the adhesion molecules LFA-1 and VLA-4 on monocytes and VCAM-1 in the paw tissue, thereby blocking monocyte adhesion to the extracellular matrix.Together, our data demonstrate that GTS-21 alleviates arthritis by inhibiting peripheral monocyte trafficking into the synovium. Our findings describe a novel mechanism through which the cholinergic signaling pathway can reduce synovial inflammation in RA patients.

Cholinergic anti-inflammatory pathway mediates diesel exhaust PM2.5-induced pulmonary and systemic inflammation

Previous research has indicated that the cholinergic anti-inflammatory pathway (CAP) can regulate the duration and intensity of inflammatory responses. A wide range of research has demonstrated that PM2.5 exposure may induce various negative health effects via pulmonary and systemic inflammations. To study the potential role of the CAP in mediating PM2.5-induced effects, mice were treated with vagus nerve electrical stimulation (VNS) to activate the CAP before diesel exhaust PM2.5 (DEP) instillation. Analysis of pulmonary and systemic inflammations in mice demonstrated that VNS significantly reduced the inflammatory responses triggered by DEP. Meanwhile, inhibition of the CAP by vagotomy aggravated DEP-induced pulmonary inflammation. The flow cytometry results showed that DEP influenced the CAP by altering the Th cell balance and macrophage polarization in spleen, and in vitro cell co-culture experiments indicated that this DEP-induced change on macrophage polarization may act via the splenic CD4+ T cells. To further confirm the effect of alpha7 nicotinic acetylcholine receptor (α7nAChR) in this pathway, mice were then treated with α7nAChR inhibitor (α-BGT) or agonist (PNU282987). Our results demonstrated that specific activation of α7nAChR with PNU282987 effectively alleviated DEP-induced pulmonary inflammation, while specific inhibition of α7nAChR with α-BGT exacerbated the inflammatory markers. The present study suggests that PM2.5 have an impact on the CAP, and CAP may play a critical function in mediating PM2.5 exposure-induced inflammatory response. Availability of data and materialsThe datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Dose-dependent effects of GAT107, a novel allosteric agonist-positive allosteric modulator (ago-PAM) for the α7 nicotinic cholinergic receptor: a BOLD phMRI and connectivity study on awake rats.

Alpha 7 nicotinic acetylcholine receptor (α7nAChR) agonists have been developed to treat schizophrenia but failed in clinical trials due to rapid desensitization. GAT107, a type 2 allosteric agonist-positive allosteric modulator (ago-PAM) to the α7 nAChR was designed to activate the α7 nAChR while reducing desensitization. We hypothesized GAT107 would alter the activity of thalamocortical neural circuitry associated with cognition, emotion, and sensory perception. The present study used pharmacological magnetic resonance imaging (phMRI) to evaluate the dose-dependent effect of GAT107 on brain activity in awake male rats. Rats were given a vehicle or one of three different doses of GAT107 (1, 3, and 10 mg/kg) during a 35 min scanning session. Changes in BOLD signal and resting state functional connectivity were evaluated and analyzed using a rat 3D MRI atlas with 173 brain areas. GAT107 presented with an inverted-U dose response curve with the 3 mg/kg dose having the greatest effect on the positive BOLD volume of activation. The primary somatosensory cortex, prefrontal cortex, thalamus, and basal ganglia, particularly areas with efferent connections from the midbrain dopaminergic system were activated as compared to vehicle. The hippocampus, hypothalamus, amygdala, brainstem, and cerebellum showed little activation. Forty-five min post treatment with GAT107, data for resting state functional connectivity were acquired and showed a global decrease in connectivity as compared to vehicle. GAT107 activated specific brain regions involved in cognitive control, motivation, and sensory perception using a BOLD provocation imaging protocol. However, when analyzed for resting state functional connectivity there was an inexplicable, general decrease in connectivity across all brain areas.

Analogs of 6-Bromohypaphorine with Increased Agonist Potency for α7 Nicotinic Receptor as Anti-Inflammatory Analgesic Agents.

Hypaphorines, tryptophan derivatives, have anti-inflammatory activity, but their mechanism of action was largely unknown. Marine alkaloid L-6-bromohypaphorine with EC50 of 80 μM acts as an agonist of α7 nicotinic acetylcholine receptor (nAChR) involved in anti-inflammatory regulation. We designed the 6-substituted hypaphorine analogs with increased potency using virtual screening of their binding to the α7 nAChR molecular model. Fourteen designed analogs were synthesized and tested in vitro by calcium fluorescence assay on the α7 nAChR expressed in neuro 2a cells, methoxy ester of D-6-iodohypaphorine (6ID) showing the highest potency (EC50 610 nM), being almost inactive toward α9α10 nAChR. The macrophages cytometry revealed an anti-inflammatory activity, decreasing the expression of TLR4 and increasing CD86, similarly to the action of PNU282987, a selective α7 nAChR agonist. 6ID administration in doses 0.1 and 0.5 mg/kg decreased carrageenan-induced allodynia and hyperalgesia in rodents, in accord with its anti-inflammatory action. Methoxy ester of D-6-nitrohypaphorine demonstrated anti-oedemic and analgesic effects in arthritis rat model at i.p. doses 0.05-0.26 mg/kg. Tested compounds showed excellent tolerability with no acute in vivo toxicity in dosages up to 100 mg/kg i.p. Thus, combining molecular modelling and natural product-inspired drug design improved the desired activity of the chosen nAChR ligand.

Effects of kynurenic acid and choline on lipopolysaccharide-induced cyclooxygenase pathway

Abstract Objectives Inflammation can be endogenously modulated by the cholinergic anti-inflammatory pathway via calcium (Ca2+)-permeable alpha-7 nicotinic acetylcholine receptor (α7nAChR) ion channel expressed in immune cells. α7nAChR agonist choline and tryptophan metabolite kynurenic acid (KYNA) produces immunomodulatory effects. This study aimed to determine the effects of the choline and KYNA on the lipopolysaccharide (LPS)-induced cyclooxygenase (COX)-2 pathway. Methods In vitro inflammation model was produced via LPS administration in macrophage cells. To determine the effective concentrations, choline and KYNA were applied with increasing concentrations and LPS-induced inflammatory parameters investigated. The involvement of nAChR mediated effects was investigated with the use of non-selective nAChR and selective α7nAChR antagonists. The effects of choline and KYNA on COX-2 enzyme, PGE2, TNFα, NF-κB and intracellular Ca2+ levels were analyzed. Results LPS-induced COX-2 expression, PGE2 TNFα and NF-κB levels were decreased with choline treatment while intracellular calcium levels via α7nAChRs increased. KYNA also showed an anti-inflammatory effect on the same parameters. Additionally, KYNA administration increased the effectiveness of choline on these inflammatory mediators. Conclusions Our data suggest a possible interaction between the kynurenine pathway and the cholinergic system on the modulation of LPS-induced inflammatory response in macrophages.

Acetylcholine potentiates glutamate transmission from the habenula to the interpeduncular nucleus in losers of social conflict

Switching behaviors from aggression to submission in losers at the end of conspecific social fighting is essential to avoid serious injury or death. We have previously shown that the experience of defeat induces a loser-specific potentiation in the habenula (Hb)-interpeduncular nucleus (IPN) and show here that this is induced by acetylcholine. Calcium imaging and electrophysiological recording using acute brain slices from winners and losers of fighting behavior in zebrafish revealed that the ventral IPN (vIPN) dominates over the dorsal IPN in the neural response to Hb stimulation in losers. We also show that GluA1 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits on the postsynaptic membrane increased in the vIPN of losers. Furthermore, these loser-specific neural properties disappeared in the presence of an α7 nicotinic acetylcholine receptor (nAChR) antagonist and, conversely, were induced in brain slices of winners treated with α7 nAChR agonists. These data suggest that acetylcholine released from Hb terminals in the vIPN induces activation of α7 nAChR followed by an increase in postsynaptic membrane GluA1. This results in an increase in active synapses on postsynaptic neurons, resulting in the potentiation of neurotransmissions to the vIPN. This acetylcholine-induced neuromodulation could be the neural foundation for behavioral switching in losers. Our results could increase our understanding of the mechanisms of various mood disorders such as social anxiety disorder and social withdrawal.

MP03-10 BRAIN α7 NICOTINIC ACETYLCHOLINE RECEPTORS SUPPRESS THE RAT MICTURITION REFLEX VIA BRAIN HYDROGEN SULFIDE

You have accessJournal of UrologyCME1 Apr 2023MP03-10 BRAIN α7 NICOTINIC ACETYLCHOLINE RECEPTORS SUPPRESS THE RAT MICTURITION REFLEX VIA BRAIN HYDROGEN SULFIDE Nobutaka Shimizu, Takahiro Shimizu, Shogo Shimizu, Youichirou Higashi, Suo Zou, Hideo Fukuhara, Takashi Karashima, Keiji Inoue, and Motoaki Saito Nobutaka ShimizuNobutaka Shimizu More articles by this author , Takahiro ShimizuTakahiro Shimizu More articles by this author , Shogo ShimizuShogo Shimizu More articles by this author , Youichirou HigashiYouichirou Higashi More articles by this author , Suo ZouSuo Zou More articles by this author , Hideo FukuharaHideo Fukuhara More articles by this author , Takashi KarashimaTakashi Karashima More articles by this author , Keiji InoueKeiji Inoue More articles by this author , and Motoaki SaitoMotoaki Saito More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003214.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We previously reported that brain α7 nicotinic acetylcholine receptors (α7 nAChRs) and brain hydrogen sulfide (H2S) suppressed the rat micturition reflex via brain GABA receptors. However, it is unclear how the brain α7 nAChRs and brain H2S are related to each other in regulation of the micturition reflex. In this study, we examined whether brain H2S is involved in the suppression of micturition induced by stimulation of brain α7 nAChR. METHODS: Urethane anesthetized (0.8 g/kg, ip) male Wistar rats (350-420 g) were used. A catheter was inserted into the bladder to perform continuous cystometry (12 ml/h saline infusion). We examined effects of intracerebroventricularly (icv) pretreated GYY4137 (GYY, H2S donor, 1 or 3 nmol/rat) or AOAA (non-selective inhibitor of H2S synthesis, 3 or 10 μg/rat) on PHA568487 (PHA, α7 nAChR agonist, 0.3 or 1 nmol/rat, icv)-induced intercontraction intervals (ICI) prolongation. Continuous cystometry and evaluation of ICI and maximal voiding pressure (MVP) was started 120 min after the surgery and the first icv administration was performed 60 min after starting cystometry. RESULTS: PHA at a lower dose (0.3 nmol/rat) showed no significant effect on ICI, while under pretreatment with GYY, PHA (0.3 nmol/rat) significantly prolonged ICI even at a lower dose (Figure 1). PHA at a higher dose (1 nmol/rat) induced ICI prolongation and the PHA-induced prolongation was significantly suppressed by AOAA (Figure 2). The AOAA-induced suppression of the PHA-induced ICI prolongation was cancelled by supplementation of brain H2S via GYY (Figure 3). PHA at each dose showed no effect on MVP (data not shown). CONCLUSIONS: Brain α7 nAChRs suppressed the rat micturition reflex via brain H2S, therefore, brain α7 nAChRs and H2S might be new therapeutic targets for neurogenic bladder overactivity. Source of Funding: Smoking Research Foundation in Japan, KAKENHI (#20K07827), The Kochi Medical School Hospital President’s Discretionary Grant © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e25 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nobutaka Shimizu More articles by this author Takahiro Shimizu More articles by this author Shogo Shimizu More articles by this author Youichirou Higashi More articles by this author Suo Zou More articles by this author Hideo Fukuhara More articles by this author Takashi Karashima More articles by this author Keiji Inoue More articles by this author Motoaki Saito More articles by this author Expand All Advertisement PDF downloadLoading ...

Synthesis and Antagonist Activity of Methyllycaconitine Analogues on Human α7 Nicotinic Acetylcholine Receptors.

Methyllycaconitine (MLA), 1, is a naturally occurring norditerpenoid alkaloid that is a highly potent (IC50 = 2 nM) selective antagonist of α7 nicotinic acetylcholine receptors (nAChRs). Several structural factors affect its activity such as the neopentyl ester side-chain and the piperidine ring N-side-chain. The synthesis of simplified AE-bicyclic analogues 14-21 possessing different ester and nitrogen side-chains was achieved in three steps. The antagonist effects of synthetic analogues were examined on human α7 nAChRs and compared to that of MLA 1. The most efficacious analogue (16) reduced α7 nAChR agonist responses [1 nM acetylcholine (ACh)] to 53.2 ± 1.9% compared to 3.4 ± 0.2% for MLA 1. This demonstrates that simpler analogues of MLA 1 possess antagonist effects on human α7 nAChRs but also indicates that further optimization may be possible to achieve antagonist activity comparable to that of MLA 1.

α7 nicotinic acetylcholine receptor agonist improved brain injury and impaired glucose metabolism in a rat model of ischemic stroke.

Vagus nerve stimulation through the action of acetylcholine can modulate inflammatory responses and metabolism. α7 Nicotinic Acetylcholine Receptor (α7nAChR) is a key component in the biological functions of acetylcholine. To further explore the health benefits of vagus nerve stimulation, this study aimed to investigate whether α7nAChR agonists offer beneficial effects against poststroke inflammatory and metabolic changes and to identify the underlying mechanisms in a rat model of stroke established by permanent cerebral ischemia. We found evidence showing that pretreatment with α7nAChR agonist, GTS-21, improved poststroke brain infarction size, impaired motor coordination, brain apoptotic caspase 3 activation, dysregulated glucose metabolism, and glutathione reduction. In ischemic cortical tissues and gastrocnemius muscles with GTS-21 pretreatment, macrophages/microglia M1 polarization-associated Tumor Necrosis Factor-α (TNF-α) mRNA, Cluster of Differentiation 68 (CD68) protein, and Inducible Nitric Oxide Synthase (iNOS) protein expression were reduced, while expression of anti-inflammatory cytokine IL-4 mRNA, and levels of M2 polarization-associated CD163 mRNA and protein were increased. In the gastrocnemius muscles, stroke rats showed a reduction in both glutathione content and Akt Serine 473 phosphorylation, as well as an elevation in Insulin Receptor Substrate-1 Serine 307 phosphorylation and Dynamin-Related Protein 1 Serine 616 phosphorylation. GTS-21 reversed poststroke changes in the gastrocnemius muscles. Overall, our findings, provide further evidence supporting the neuroprotective benefits of α7nAChR agonists, and indicate that they may potentially exert anti-inflammatory and metabolic effects peripherally in the skeletal muscle in an acute ischemic stroke animal model.

Human-specific duplicate CHRFAM7A gene is associated with more severe osteoarthritis and amplifies pain behaviours

ObjectivesCHRFAM7A is a uniquely human fusion gene that functions as a dominant negative regulator of alpha 7 acetylcholine nicotinic receptor (α7nAChR) in vitro. This study determined the impact of CHRFAM7A...

GTS-21, a selective alpha7 nicotinic acetylcholine receptor agonist, ameliorates diabetic nephropathy in Leprdb/db mice

Diabetic nephropathy (DN) is a serious complicating factor in human type 2 diabetes mellitus (T2DM), and it commonly results in end-stage renal disease (ESRD) that requires kidney dialysis. Here, we report that the α7 nicotinic acetylcholine receptor (α7nAChR) agonist GTS-21 exerts a novel anti-inflammatory action to ameliorate DN, as studied using an inbred strain of Leprdb/db mice in which hyperglycemia and obesity co-exist owing to defective leptin receptor (Lepr) signaling. For this analysis, GTS-21 was administered to 10–12 week-old male and female mice as a 4 mg/kg intraperitoneal injection, twice-a-day, for 8 weeks. Kidney function and injury owing to DN were monitored by determination of plasma levels of BUN, creatinine, KIM-1 and NGAL. Histologic analysis of glomerular hypertrophy and mesangial matrix expansion were also used to assess DN in these mice. Concurrently, renal inflammation was assessed by measuring IL-6 and HMGB1, while also quantifying renal cell apoptosis, and apoptotic signaling pathways. We found that Leprdb/db mice exhibited increased markers of BUN, creatinine, NGAL, KIM-1, IL-6, cytochrome C, and HMGB-1. These abnormalities were also accompanied by histologic kidney injury (mesangial matrix expansion and apoptosis). Remarkably, all such pathologies were significantly reduced by GTS-21. Collectively, our results provide new evidence that the α7nAChR agonist GTS-21 has the ability to attenuate diabetes-induced kidney injury. Additional studies are warranted to further investigate the involvement of the vagal cholinergic anti-inflammatory reflex pathway (CAP) in ameliorating diabetic nephropathy.

Outcomes of nicotinic modulation on markers of intestinal IgA antibody response.

Acetylcholine (ACh), as a ligand of nicotinic acetylcholine receptors (nAChRs), plays a key role in the cholinergic anti-inflammatory pathway; however, its role in the immunoglobulin A (IgA) response remains unknown. Therefore, the present study aimed to investigate the role of ACh in the intestinal biomarkers involved in IgA synthesis and the polymeric immunoglobulin receptor (pIgR) involved in IgA transcytosis. Groups of mice were administered GTS-21 (an α7nAChR agonist) or mecamylamine (a non-selective nAChR antagonist) intraperitoneally for 7 days. Intestinal fluids were used for antibody concentration assessment by ELISA, cell suspensions from Peyer's patches and the lamina propria were obtained for flow cytometric analysis of plasma cells, and CD4+ T-cells expressing intracellular transforming growth factor (TGF)-β and IgA-producing interleukin (IL)-4, -5, -6 and -10, and isolated epithelial cells to determine the levels of pIgR mRNA using reverse transcription-quantitative PCR. Regarding to the untreated control group, the concentration of IgA was reduced in the mecamylamine group and unaltered in the GTS-21 group while IgM levels exhibited no differences; the percentage of IgA+ plasma cells from Peyer's patches and the lamina propria, and the percentage of TGF-β+/CD4+ T-cells from Peyer's patches were greater in the GTS-21-group. In both treatment groups, the percentages of IgM+ plasma cells and IL-6+/IL-10+ CD4+ T cells were greater in both compartments; pIgR mRNA expression levels decreased in epithelial cells. The percentage of IL-4 CD4+ T-cells were greater in Peyer's patches and lower in the lamina propria in the mecamylamine group, and the percentage of IL-5 CD4+ T-cells in the lamina propria were decreased in both treatment groups. These findings require further examination to address the impact of cholinergic modulation on IgA-transcytosis via pIgR. The present study may be an experimental reference for clinical trials that address the role of nicotinic system in intestinal dysfunctions as postoperative ileus.

Microglia Regulating Neuronal Apoptosis Via Alpha7 Nicotinic Acetylcholine Receptor in HIV-Associated Neurocognitive Disorders

Background: Microglia, the innate immune cells of the central nervous system (CNS), originate from erythromyeloid progenitor cells in the embryonic yolk sac. As the innate immune cells of the CNS, microglia play an important role in innate immunity, neurogenesis, and brain tissue repair by activating the alpha7 nicotinic acetylcholine receptor (α7nAChR). They are the key target cells for HIV-1 infection in the CNS and have been shown to produce a chronic inflammatory response in the brain which underlies HIV-associated neurocognitive disorders (HAND). Apart from the microglial neuroinflammation, recent studies have reported that activated microglia could induce neuronal apoptosis through the release of neurotoxic factors (e.g., ROS and NLRP3 inflammasome) in HAND. In addition, α7nAChR was significantly up-regulated in the striatum which is the most serious part of neuron apoptosis in gp120 transgenic mice brains. However, the underlying mechanism of neuronal cell apoptosis by gp120-activated microglia remains unclear. Here, this study aimed to explore the mechanism by which HIV-1 gp120-activated microglia induce neuronal apoptosis. Materials and methods: The expression of α7nAChR was examined by immunohistochemistry in the brain of HIV-1 LAV gp120 transgenic (Tg) mice (C57BL/6J background, 12-month-old). Methyllycaconitine (MLA, specific α7nAChR agonists) and Nicotine (NT, specific α7nAChR antagonists) were used to evaluate the effect of α7nAChR on apoptosis induced by gp120. Immortalized microglial cells (BV2) were incubated for 24 hours with gp120, NT, and gp120+NT, respectively, with or without preincubation with MLA for 15 minutes to obtain their supernatants. Next, the neuronal cells (SH-SY5Y) were treated with the same as BV2 or with collected BV2 culture supernatants. The apoptosis effect of SH-SY5Y and the level of ROS in the BV2 culture supernatants were assessed via quantitative analysis of flow cytometry. Comparisons between supernatant groups and drug groups were conducted to evaluate the effects of the SH-SY5Y apoptosis induced by microglia. Western blotting of microglia cell lysates was employed for the analysis of cellular signaling pathways. Student's t-tests and ANOVA were applied to determine statistical significance. Results: The expression of α7nAChR in HIV-1 gp120 transgenic mice was significantly upregulated (P < 0.001). The effect of apoptosis was more significant in microglia culture supernatants groups compared to that in drug-treated groups (P < 0.001). Neuronal activity was not changed in the NT group or MLA group than in the control, but HIV-1 gp120 could increase the number of apoptotic neurons (P < 0.01). Significant neuronal apoptosis was observed when treated with BV2 supernatants from the group treated by combining the gp120 with NT (P < 0.001), and this effect was in turn inhibited by MLA (P < 0.001). These results suggested that gp120-activated microglia might induce neuronal apoptosis invitro owing to gp120 upregulating α7nAChR. In addition, the ROS level in microglia was significantly upregulated following gp120 exposure (P < 0.001), and this effect could be enhanced by NT (P < 0.001). Notably, there was a positive correlation between the levels of ROS in microglia culture supernatants and the levels of neuronal apoptosis treated in the same supernatants. This positive relevance indicated that microglia might induce neuronal apoptosis by producing excessive ROS. The expressions of p53, OMI, and caspase-3 significantly upregulated following gp120 exposure with the down-regulate expression of XIAP (P < 0.001). And this effect could be enhanced by NT (P < 0.001). The results of western blotting indicated that gp120 and NT can up-regulate the ROS to activate p53 by α7nAChR, which could lead to an increased OMI and decreased XIAP levels. (P < 0.001). Conclusions: Microglia may play a critical role in regulating neuronal apoptosis in HAND. HIV-1 gp120 and NT could increase the generation of ROS through α7nAChR in microglia, which leads to neuronal apoptosis. Moreover, our results showed that the combined action of NT and gp120 may upregulate p53 and Omi / HtrA2 protein expression, and the downregulation of XIAP protein may significantly activate the level of caspase-3. (Acknowledgements:Corresponding author: Hong Cao, gzhcao@smu.edu.cn; National Natural Science Foundation of China, No. 82172259 to H.C.) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Alpha7 nicotinic acetylcholine receptor agonist PHA-543613 improves memory deficits in presenilin 1 and presenilin 2 conditional double knockout mice

Cholinergic system dysfunction has been considered as a critical feature of neurodegenerative progression in Alzheimer's disease (AD). The α7 nicotinic acetylcholine receptors (α7-nAChRs) are widely expressed in the hippocampus cortex and play an important role in memory formation, considered as potential therapeutic agents targets. However, underlying mechanisms have not been fully elucidated. Here, we combine behavioral, molecular biological methods with in vitro slice and in vivo multichannel electrophysiological recording techniques to investigate the molecular, cellular synaptic and neuronal mechanisms of activating α7-nAChR by PHA-543613 (a selective α7-nAChR agonist), which influences the impaired cognitive function using presenilin 1 (PS1) and presenilin 2 (PS2) conditional double knockout (cDKO) mice. Our results demonstrated that PHA-543613 treatment significantly improved the impaired hippocampus-related memory via recovering the reduced the hippocampal synaptic protein levels of α7-nAChR, NMADAR and AMPAR, thereby restoring the impaired post-tetanic potentiation (PTP), long-term potentiation (LTP), activation of molecular signaling pathway for neuronal protection, theta power and strength of theta-gamma phase-amplitude coupling (PAC) at hippocampus in 6-month-old cDKO mice. For the first time, we systematically reveal the mechanisms by which PHA-543613 improves memory deficits at different levels. Therefore, our findings may be significant for the development of therapeutic strategies for AD.

Abstract 11231: Acetylcholine Receptor Agonists Exert Cardioprotection Against Trastuzumab-Induced Cardiotoxicity by Attenuating NLRP3/GSDMD-Mediated Pyroptosis

Introduction: The most significant adverse effect of trastuzumab (TRZ) is cardiotoxicity. Multiple factors are involved in the pathogenesis of TRZ-induced cardiotoxicity (TIC), including mitochondrial dysfunction, inflammation, and pyroptosis. Although vagal modulation using acetylcholine receptors (AChR) agonists have been shown to exert cardioprotection in various heart diseases, the effects of AChR agonists against TIC have not been investigated. Hypothesis: We hypothesized that AChR agonists exert cardioprotective effects against TIC by attenuating cardiac mitochondrial dysfunction, inflammation, and cardiomyocyte pyroptosis, leading to improved left ventricular (LV) function. Methods: Wistar rats were divided into 5 groups, including i) control (0.9%NSS), ii) TRZ+VEH (TRZ, 4 mg/kg+VEH), iii) TRZ+α7 nicotinic AChR agonist (α7nAChR: PNU-282987, 3 mg/kg/day), iv) TRZ+muscarinic AChR agonist (mAChR: bethanechol, 12 mg/kg/day), and v) combined PNU+BET treatment groups (COM). At the end of the experiments, cardiac biochemical and functional analyses were determined. Results: TRZ-treated rats showed a decreased %LV ejection fraction (%LVEF) (Fig. 1A). Moreover, mitochondrial reactive oxygen species (ROS) levels and mitochondrial depolarization were increased in TIC rats (Fig. 1B-C). TRZ-treated rats also exhibited the upregulation of interleukin-6 mRNA, NLR family pyrin domain containing, and cleaved-gasdermin D/gasdermin expression (Fig. 1D-F). All AChR agonists similarly protected the hearts by attenuating cardiac mitochondrial dysfunctions, inflammation, and pyroptosis, leading to improved LV function in TIC rats (Fig. 1A-F). Conclusions: Either α7nAChR or mAChR agonist equally exerted cardioprotective effects against TIC by reducing cardiac mitochondrial dysfunction, inflammation, and pyroptosis, culminating in improved LV function. These findings provide potential novel treatment strategies against TIC.

The α7 nicotinic acetylcholine receptor agonist PNU-282987 ameliorates sepsis-induced acute kidney injury through CD4+CD25+ regulatory T cells in rats.

The ameliorative effects of α7 nicotinic acetylcholine receptor (α7nAChR) agonists have been demonstrated in acute kidney injury (AKI) caused by multiple stimulations. However, the ameliorative effect of α7nAChR on sepsis-induced acute kidney injury (SAKI) in the cecal ligation and puncture (CLP) model is unclear. Previous studies have demonstrated that α7nAChR is highly expressed on the surface of CD4+CD25+ regulatory T cells (Tregs). However, the role of Tregs in SAKI is unclear. We hypothesized that Tregs might play a role in the ameliorative effect of α7nAChR on SAKI. Hence, in this study, we determined the effects of PNU-282987 (a selective α7nAchR agonist) on SAKI and evaluated whether PNU-282987 would attenuate SAKI via regulating Tregs. Our study showed that immediate administration of PNU-282987 after CLP surgery in rats improved renal function, reduced levels of systemic inflammatory factors (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), etc.), inflammatory cell infiltration and tubular apoptosis in renal tissues, and increased forkhead/winged helix transcription factor p3 (Foxp3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression indicating activated Tregs. Moreover, in in vitro experiments, isolated Tregs co-cultured with PNU-282987 also displayed enhanced expression of CTLA-4 and Foxp3. Furthermore, Tregs were co-cultured with PNU-282987 for 24 hours and then reinfused into rats through the tail vein immediately after CLP surgery, and a significant renal protective effect was observed 24 hours postoperatively. These results demonstrate that PNU-282987 exerts its renal protective effects on SAKI through activation of Tregs.