Expression Of α7nAChR Research Articles

Anti-hyperalgesic and anti-inflammatory effects of 4R-tobacco cembranoid in a mouse model of inflammatory pain

4R is a tobacco cembranoid that binds to and modulates cholinergic receptors and exhibits neuroprotective and anti-inflammatory activity. Given the established function of the cholinergic system in pain and inflammation, we propose that 4R is also analgesic. Here, we tested the hypothesis that systemic 4R treatment decreases pain-related behaviors and peripheral inflammation via modulation of the alpha 7 nicotinic acetylcholine receptors (α7 nAChRs) in a mouse model of inflammatory pain. We elicited inflammation by injecting Complete Freund’s Adjuvant (CFA) into the hind paw of male and female mice. We then assessed inflammation-induced hypersensitivity to cold, heat, and tactile stimulation using the Acetone, Hargreaves, and von Frey tests, respectively, before and at different time points (2.5 h – 8d) after a single systemic 4R (or vehicle) administration. We evaluated the contribution of α7 nAChRs 4R-mediated analgesia by pre-treating mice with a selective antagonist of α7 nAChRs followed by 4R (or vehicle) administration prior to behavioral tests. We assessed CFA-induced paw edema and inflammation by measuring paw thickness and quantifying immune cell infiltration in the injected hind paw using hematoxylin and eosin staining. Lastly, we performed immunohistochemical and flow cytometric analyses of paw skin in α7 nAChR-cre::Ai9 mice to measure the expression of α7 nAChRs on immune subsets. Our experiments show that systemic administration of 4R decreases inflammation-induced peripheral hypersensitivity in male and female mice and inflammation-induced paw edema in male but not female mice. Notably, 4R-mediated analgesia and anti-inflammatory effects lasted up to 8d after a single systemic administration on day 1. Pretreatment with an α7 nAChR-selective antagonist prevented 4R-mediated analgesia and anti-inflammatory effects, demonstrating that 4R effects are via modulation of α7 nAChRs. We further show that a subset of immune cells in the hind paw expresses α7 nAChRs. However, the number of α7 nAChR-expressing immune cells is unaltered by CFA or 4R treatment, suggesting that 4R effects are independent of α7 nAChR-expressing immune cells. Together, our findings identify a novel function of the 4R tobacco cembranoid as an analgesic agent in both male and female mice that reduces peripheral inflammation in a sex-dependent manner, further supporting the pharmacological targeting of the cholinergic system for pain treatment.

Nicotine promotes development of bile duct ligation-induced liver fibrosis by increasing expression of nicotinic acetylcholine receptors in rats.

Liver fibrosis and cigarette smoking seem to be directly linked. Nicotine, as an agonist of nicotinic acetylcholine receptors (nAChRs), induces many downstream signaling pathways. The pathways through which nicotine affects the process of liver fibrosis have not been clarified. The present study aimed to investigate the nicotine-induced effects on fibrosis progression in cholestatic rats. First, the Wistar rats were subjected to sham or bile duct ligation (BDL) surgery. The rats were treated with low and high doses of nicotine (1 or 10 mg/kg) for three weeks. They were monitored for their body weights before and 21 days after BDL. Also, spleens were weighed to calculate the spleen/body weight ratio. Ductular proliferation and fibrosis were evaluated using hematoxylin and eosin (H&E) as well as Masson's trichrome staining. The mRNA expression of α4nAChR, α7nAChR, and fibrosis gene α-smooth muscle actin (α-SMA) was measured by real-time PCR. The findings showed that nicotine promotes the development of BDL-induced liver fibrosis. The ratio of spleen/body weight was significantly affected by nicotine exposure. H&E and Masson's trichrome staining showed that the level of liver fibrosis was higher in the cholestatic BDL groups, and this effect was significantly augmented in the nicotine-treated rats. Also, α4nAChR, α7nAChR, and α-SMA expression was observed in the BDL rats and increased following nicotine treatment. The activation of nAChR triggers biliary proliferation and liver fibrosis. Studying the intracellular mechanism of nicotine and alteration in the expression of nicotinic receptors following nicotine exposure can be useful both in diagnosing nicotine-related diseases and finding new treatment strategies.

The Prenatal Hypoxic Pathology Associated with Maternal Stress Predisposes to Dysregulated Expression of the chrna7 Gene and the Subsequent Development of Nicotine Addiction in Adult Offspring

Introduction: Previous studies have shown that fetal hypoxia predisposes individuals to develop addictive disorders in adulthood. However, the specific impact of maternal stress, mediated through glucocorticoids and often coexisting with fetal hypoxia, is not yet fully comprehended. Methods: To delineate the potential effects of these pathological factors, we designed models of prenatal severe hypoxia (PSH) in conjunction with maternal stress and prenatal intrauterine ischemia (PII). We assessed the suitability of these models for our research objectives by measuring HIF1α levels and evaluating the glucocorticoid neuroendocrine system. To ascertain nicotine dependence, we employed the conditioned place aversion test and the startle response test. To identify the key factor implicated in nicotine addiction associated with PSH, we employed techniques such as Western blot, immunohistochemistry, and correlational analysis between chrna7 and nr3c1 genes across different brain structures. Results: In adult rats exposed to PSH and PII, we observed increased levels of HIF1α in the hippocampus (HPC). However, the PSH group alone exhibited reduced glucocorticoid receptor levels and disturbed circadian glucocorticoid rhythms. Additionally, they displayed signs of nicotine addiction in the conditioned place aversion and startle response tests. We also observed elevated levels of phosphorylated DARPP-32 protein in the nucleus accumbens (NAc) indicated compromised glutamatergic efferent signaling. Furthermore, there was reduced expression of α7 nAChR, which modulates glutamate release, in the medial prefrontal cortex (PFC) and HPC. Correlation analysis revealed strong associations between chrna7 and nr3c1 expression in both brain structures. Conclusion: Perturbations in the glucocorticoid neuroendocrine system and glucocorticoid-dependent gene expression of chrna7 associated with maternal stress response to hypoxia in prenatal period favor the development of nicotine addiction in adulthood.

Enhancing cognitive function in chronic TBI: The Role of α7 nicotinic acetylcholine receptor modulation

Traumatic brain injury (TBI) results in several pathological changes within the hippocampus that result in adverse effects on learning and memory. Therapeutic strategies to enhance learning and memory after TBI are still in the early stages of clinical development. One strategy is to target the α7 nicotinic acetylcholine receptor (nAChR), which is highly expressed in the hippocampus and contributes to the formation of long-term memory. In our previous study, we found that AVL-3288, a positive allosteric modulator of the α7 nAChR, improved cognitive recovery in rats after moderate fluid-percussion injury (FPI). However, whether AVL-3288 improved cognitive recovery specifically through the α7 nAChR was not definitively determined. In this study we utilized Chrna7 knockout mice and compared their recovery to wild-type mice treated with AVL-3288 after TBI. We hypothesized that AVL-3288 treatment would improve learning and memory in wild-type mice, but not Chrna7−/− mice after TBI. Adult male C57BL/6 wild-type and Chrna7−/− mice received sham surgery or moderate controlled cortical impact (CCI) and recovered for 3 months. Mice were then treated with vehicle or AVL-3288 at 30 min prior to contextual fear conditioning. At 3 months after CCI, expression of α7 nAChR, choline acetyltransferase (ChAT), high-affinity choline transporter (ChT), and vesicular acetylcholine transporter (VAChT) were found to be significantly decreased in the hippocampus. Treatment of wild-type mice at 3 months after CCI with AVL-3288 significantly improved cue and contextual fear conditioning, whereas no beneficial effects were observed in Chrna7−/− mice. Parietal cortex and hippocampal atrophy were not improved with AVL-3288 treatment in either wild-type or Chrna7−/− mice. Our results indicate that AVL-3288 improves cognition during the chronic recovery phase of TBI through modulation of the α7 nAChR.

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.

Alpha-7 Nicotinic Acetylcholine Receptor Activation Inhibits Trauma Induced Pronociceptive Autoimmune Responses

Both autonomic nervous system dysfunction and immune system activation are characteristic of chronic pain after limb injuries. Cholinergic agonists reduce immune system activation in many settings. We hypothesized, therefore, that alpha-7 nicotinic acetylcholine receptor (α7nAChR) agonist administration would reduce nociceptive and immune changes after tibia fracture and cast immobilization in mice. Fracture mice were treated with either vehicle, a low (.2 mg/kg) dose, or a high (1 mg/kg) dose of the selective α7nAChR agonist PNU-282987 for 4 weeks. We assessed hindpaw allodynia and weight bearing as behavioral outcomes. The assessment of adaptive immune responses included regional lymph node hypertrophy, germinal center formation, α7nAChR expression, and IgM deposition. Assessment of innate immune system activation focused on IL-1β and IL-6 generation in fractured hindlimb skin. We observed that mechanical allodynia and unweighting were alleviated by PNU-282987 treatment. Drug treatment also reduced popliteal lymph node hypertrophy and germinal center formation. Immunohistochemical studies localized α7nAChR to germinal center B lymphocytes, and this expression increased after fracture. Analysis of fracture limb hindpaw skin demonstrated increased inflammatory mediator (IL-1β and IL-6) levels and IgM deposition, which were abrogated by PNU-282987. Serum analyses demonstrated fracture-induced IgM reactivity against keratin 16, histone 3.2, GFAP, and NMDAR-2B. Administration of PNU-282987 reduced the enhancement of IgM reactivity. Collectively, these data suggest that the α7nAChR is involved in regulating posttraumatic innate and adaptive immune responses and the associated nociceptive sensitization. PerspectiveThese studies evaluate the effects of a selective α7nAChR agonist in a tibial fracture/cast immobilization model of limb pain. Administration of the drug reduced nociceptive and functional changes 4 weeks after injury. These novel findings suggest that well-tolerated α7nAChR agonists may be viable analgesics for chronic pain after limb injuries.

Monosodium Glutamate Effect on The Expression of ɑ7nACHR and ɑ4nACHR Subunits in The Testicular Tissue.

Monosodium glutamate (MSG) is a popular food flavor enhancer, and a glutamate subset that induces different toxicities such as hepatotoxicity, neurotoxicity, reproductive toxicity, and nephrotoxicity. This study was conducted to assess the effects of MSG on the α7 and α4 nicotinic acetylcholine receptor (nACHR) protein subunits expression of adult rat testis and the safety role of vitamin C. For this experimental research, 24 rats were haphazardly grouped into four equal groups (n=6) and orally gavaged for 30 days as follows: control group (distilled water gavage), MSG group (3 g/kg/b.w/ day), vitamin C group (150 mg/kg/b.w/day), and MSG+vitamin C group (3 g/kg/b.w/day+150 mg/kg/b.w/day, respectively) that rats of all groups on the 30th day were anesthetized, and the left testes were used for of α4 and α7 nACHR protein subunit evaluation by immunohistochemistry (IHC). Statistical computations were performed using Graph Pad Prism software. The present study revealed a significant reduction in the expression and optical density (OD) of α7 nACHR and α4 nACHR in the seminiferous tubules and intertubular connective tissue in the MSG group compared to the control group. In the MSG+vitamin C group, the expression and OD of α7 nACHR and α4 nACHR increased in the seminiferous tubules and intertubular connective tissue but this improvement was not significant compared to the MSG group. MSG decreased the expression level of nACHR protein subunits, α7 and α4, in the seminiferous tubules and interstitial testicular tissue. Vitamin C in the MSG+vitamin C group could not significantly improve the expression of α7 and α4 nACHR subunits in testicular tissue. Probably, MSG toxicity can be compensated with higher doses of vitamin C.

Ultrasound stimulation of the vagal nerve improves acute septic encephalopathy in mice.

Septic encephalopathy (SE) is characterized by symptoms such as coma, delirium, and cognitive dysfunction, and effective therapeutic interventions for SE remain elusive. In this study, we aimed to investigate the potential alleviating effects of vagal nerve stimulation (VNS) on SE-associated signs. To evaluate our hypothesis, we utilized a mouse model of SE induced by intraperitoneal injection of lipopolysaccharide (0.3 mg per mouse) and administered noninvasive, high-frequency ultrasound VNS. To assess the efficacy of ultrasound VNS, we measured inflammation-related molecules, including the α7 nicotinic acetylcholine receptor (α7nAChR) expression in peritoneal macrophages and plasma interleukin 1β (IL-1β) levels. Consistent with our hypothesis, SE mice exhibited reduced α7nAChR expression in macrophages and elevated IL-1β levels in the blood. Remarkably, VNS in SE mice restored α7nAChR expression and IL-1β levels to those observed in control mice. Furthermore, we evaluated the effects of VNS on survival rate, body temperature, and locomotor activity. SE mice subjected to VNS demonstrated a modest, yet significant, improvement in survival rate, recovery from hypothermia, and increased locomotor activity. To investigate the impact on the brain, we examined the hippocampus of SE mice. In control mice, VNS increased the expression of c-fos, a marker of neuronal electrical excitability, in the hippocampus. In SE mice, VNS led to the restoration of aberrant firing patterns in hippocampal neurons. Additionally, proteomic analysis of hippocampal tissue in SE mice revealed abnormal increases in two proteins, tissue factor (TF) and acyl-CoA dehydrogenase family member 9 (ACAD9), which returned to control levels following VNS. Collectively, our findings support the value of exploring the beneficial effects of ultrasound VNS on SE.

Salvianolic acids from Salvia miltiorrhiza Bunge and their anti-inflammatory effects through the activation of α7nAchR signaling

Ethnopharmacological relevanceCardiovascular disease (CVD) is a serious disease with a high incidence rate and mortality. Inflammation is closely related to the occurrence of CVDs. As an essential medicine of promoting blood circulation and removing blood stasis in China, Salvia miltiorrhiza Bunge (Danshen) is widely used to treat CVDs due to its anti-inflammatory and cardiovascular protective effects. Salvianolic acids are the most abundant component in the water extract of S. miltiorrhiza, which has a significant effect on the treatment of CVDs. However, due to the complex composition of salvianolic acids, the active molecules and their underlying mechanisms have not been fully explored. Aim of this studyThe present study aims to isolate and identify salvianolic acids from Danshen with anti-inflammatory activity and explore the potential mechanisms of isolates. MethodsThe structures of isolated salvianolic acids were elucidated by UV, IR, NMR, MS and electronic circular dichroism (ECD) calculations. Then anti-inflammatory activities of isolates were screened out by the zebrafish inflammation models. The most active compound was further used to explore the anti-inflammatory mechanisms on LPS-stimulated RAW 264.7 cells. The key inflammatory cytokines IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of STAT3, p-STAT3 (Tyr705), NF-κB p65, IκBα, p-IκBα (Ser32) and α7nAchR were determined by Western blotting. The nuclear translocation of p-STAT3 (Tyr705) and NF-κB p65 was evaluated by immunofluorescence assays. Finally, the in vivo anti-inflammatory mechanisms were investigated by observation of neutrophil migration, H&E staining, survival analysis and quantitative PCR (Q-PCR) in LPS-microinjected zebrafish. ResultsTwo new and four known compounds were isolated from Danshen. Among them, isosalvianolic acid A-1 (C1) and ethyl lithospermate (C5) inhibited neutrophil migrations in three zebrafish inflammation models and C1 with the best activities decreased the secretion of IL-6 and TNF-α and inhibited the expression level of p-IκBα (Ser32) in LPS stimulated RAW 264.7 cells. In addition, C1 also reduced the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Moreover, C1 significantly upregulated the protein expression of α7nAchR, and the knockdown of α7nAchR counteracted the effects of C1 on the production of IL-6 and TNF-α and the expression levels of p-STAT3 (Tyr705), NF-κB p65 and p-IκBα (Ser32). In vivo experiments, C1 decreased the migration and infiltration of inflammatory cells, increased the survival ratio and inhibited the mRNA level of IL-6, TNF-α, STAT3, NF-κB and IκBα in LPS-microinjected zebrafish. ConclusionTwo new and four known compounds were isolated from Danshen. Among them, C1 exerted anti-inflammatory activities by activating α7nAchR signaling and subsequently inhibiting STAT3 and NF-κB pathways. This study provided evidence for the clinical application of Danshen and contributed to the development of C1 as a novel in the treatment of cardiovascular disease.

α7 Nicotinic Acetylcholine Receptors May Improve Schwann Cell Regenerating Potential via Metabotropic Signaling Pathways.

Schwann cells (SCs) are glial cells involved in peripheral axon myelination. SCs also play a strategic role after peripheral nerve injury, regulating local inflammation and axon regeneration. Our previous studies demonstrated the presence of cholinergic receptors in SCs. In particular, the α7 nicotinic acetylcholine receptors (nAChRs) are expressed in SCs after peripheral axotomy, suggesting their involvement in the regulation of SC-regenerating properties. To clarify the role that α7 nAChRs may play after peripheral axon damage, in this study we investigated the signal transduction pathways triggered by receptor activation and the effects produced by their activation. Both ionotropic and metabotropic cholinergic signaling were analyzed by calcium imaging and Western blot analysis, respectively, following α7 nAChR activation. In addition, the expression of c-Jun and α7 nAChRs was evaluated by immunocytochemistry and Western blot analysis. Finally, the cell migration was studied by a wound healing assay. Activation of α7 nAChRs, activated by the selective partial agonist ICH3, did not induce calcium mobilization but positively modulated the PI3K/AKT/mTORC1 axis. Activation of the mTORC1 complex was also supported by the up-regulated expression of its specific p-p70 S6KThr389 target. Moreover, up-regulation of p-AMPKThr172, a negative regulator of myelination, was also observed concomitantly to an increased nuclear accumulation of the transcription factor c-Jun. Cell migration and morphology analyses proved that α7 nAChR activation also promotes SC migration. Our data demonstrate that α7 nAChRs, expressed by SCs only after peripheral axon damage and/or in an inflammatory microenvironment, contribute to improve the SCs regenerating properties. Indeed, α7 nAChR stimulation leads to an upregulation of c-Jun expression and promotes Schwann cell migration by non-canonical pathways involving the mTORC1 activity.

Involvement of baroreflex deficiency in the age-related loss of estrogen efficacy against cerebral ischemia.

For post-menopausal women, stroke is complicated by the variable effects of estrogen therapy and the age-related therapeutic consequences involved. Estrogen therapy has been shown to have an age-dimorphic effect, which is neuroprotective in young females, but non-neuroprotective, even neurotoxic in acyclic females. We hypothesized that arterial baroreflex (ABR) and its downstream acetylcholine-α7 nicotinic acetylcholine receptor (α7nAChR) anti-inflammatory pathways are involved in estrogen efficacy toward cerebral ischemic damage. Our data showed that estrogen supplements contributed to ABR improvement and neuroprotection in adult, not aged, ovariectomized (OVX) rats. In adult rats, OVX-induced estrogen deficiency aggravated middle cerebral artery occlusion (MCAO), which induced brain infarction and reduced ABR function, with decreased α7nAChR expression of the brain and exaggerated inflammation following MCAO; these effects were significantly prevented by supplementation with estrogen. ABR impairment by sinoaortic denervation partly attenuated the estrogen effect on baroreflex sensitivity (BRS) and ischemic damage in adult rats, as well as α7nAChR expression and inflammatory response. These data suggested that ABR and acetylcholine-α7nAChR anti-inflammatory pathways are involved in the neuroprotection of estrogen in adult OVX rats. In contrast, aged rats exhibited more severe ischemic damage and inflammatory response than adult rats, as well as poorer baroreflex function and lower α7nAChR expression. Estrogen supplements did not improve BRS or confer neuroprotection in aged rats without affecting brain α7nAChR and post-ischemic inflammation. Most importantly, ketanserin restored ABR function and significantly postponed the onset of stroke in aged female strokeprone spontaneously hypertensive rats, whereas estrogen treatment failed to delay the development of stroke. Our findings reveal that estrogen is protective against ischemic stroke (IS) in adult female rats and that ABR played a role in this beneficial action. Dysfunction of ABR and unresponsiveness to estrogen in aged female rats may contribute to a reduced estrogen efficacy against cerebral ischemia.

Role of cholinergic anti-inflammatory pathway in the regulation of vascular endothelial permeability and inflammation in type 2 diabetes

Abstract Despite tremendous advancement in glycemic control, anti-diabetic medications have failed to revert vascular impairment once triggered by the metabolic disorder. The cholinergic anti-inflammatory pathway (CAP) is a neurophysiological mechanism that regulates the immune system. Studies show that the CAP inhibits inflammation by suppressing cytokine synthesis via release of acetylcholine in organs of the reticuloendothelial system, including the heart, lungs and gastrointestinal tract. Upon release, acetylcholine interacts with α7 nicotinic acetylcholine receptors (α7nAChR), down-regulate pro-inflammatory cytokine synthesis and prevent tissue damage and inflammation. Although a CAP modulated the response to severe inflammation during sepsis, the role of this pathway in the regulation of type-2 diabetes (T2D)-induced increase in endothelial permeability is completely unknown. Our data showed that human coronary artery endothelial cells (HCAECs) from T2D displayed increased endothelial permeability and enhanced pro-inflammatory signaling in response to pro-coagulant thrombin and endotoxin lipopolysaccharide (LPS), respectively. We also found increased expression and activity of acetylcholinesterase enzyme, and no change in expression of α7nAChR in T2D endothelial cells (ECs) compared to ECs from normal subjects. Our research findings showed that the T2D increased the expression and activity of AChE enzyme in HCAECs, and thereby decreasing the availability of ACh at α7nAChR. In summary, our work offered novel insights and therapeutic targets that we think will lead to the development of more effective treatments with improved outcomes. Grant from CTRE Chicago State University, Chicago, IL

Nicotine-mediated signaling in glomerular podocytes

Introduction: Although nicotine’s harmful effects on renal function are established, the precise cellular mechanisms of smoking-related damage are understudied. Smoking-Related Glomerulopathy (SRG) is a renal disease phenotype associated with smoking. This condition histologically mimics diabetic nephropathy, however, SRG patients present with proteinuria and renal insufficiency without diabetes. Here we investigated the acute and chronic nicotine-related oxidative and nitrosative stress in glomerular podocytes. We hypothesized that nicotine may promote nitrosative stress in the kidney cells through the rapid production of peroxynitrite (ONOO - ) and a decline in nitric oxide (NO) bioavailability. Methods: To test the hypothesis, we used a conditionally immortalized human podocyte line and confocal imaging to detect the production of ONOO - (Hydroxyl Radical and Peroxynitrite Sensor; HPF), intracellular Ca 2+ (Fluo-8), and NO (DAF-FM). The presence of nicotinic acetylcholine receptors (nAChR) receptor subunits in podocytes was confirmed with immunocytochemistry and live imaging using commercially available pharmacology. NOS activity was analyzed in response to Ang II with the specific blockers for NOS1 (NΩ-Propyl-L-arginine hydrochloride) and NOS2 (L-NIL) subunits. One-way ANOVA (OriginPro) was used for statistical analysis. Results: Immunostaining indicated that human and rat kidneys express nicotinic acetylcholine receptors (nAChR). Notably, we detected specific expression of α7 nAChR in glomerular podocytes. In podocytes, acute nAChR activation promoted the mobilization of intracellular Ca 2+ controlled by intracellular store activation, and fast ONOO - transients. Multiple consecutive applications of nicotine resulted in repeated intracellular Ca 2+ and ONOO - transients. Nicotine-mediated ONOO - response was efficiently blocked in the presence of superoxide dismutase (SOD). The application of specific α7 or α4β2, α2β4, α4β4 and α3β4 nAChR agonists elicited Ca 2+ transients but did not reproduce the ONOO - response to nicotine, suggesting that nitrosative processes may occur independently from Ca 2+ influx or nAChR function. Chronic exposure to nicotine (12-hrs) resulted in a significant decline in podocytes’ NO bioavailability (p<0.05). While under normal conditions, NO is produced primarily by NOS1 (70%), and the rest is attributed to NOS2, chronic nicotine exposure led to an elevation of NOS2 (75%) and a decline in NOS1 activity (p<0.001). We observed similar NOS remodeling under hyperglycemic conditions, suggesting similar nitrosative processes in response to nicotine and high glucose. Conclusions: Nicotine promotes superoxide-stimulated nitrosative stress and peroxynitrite formation in podocytes leading to decline in NO bioavailability and pathological activation of NOS2. R01 NIDDK DK126720 (OP), DK129227 (OP), HL148114 (DVI), NIH/NCATS/SCTR UL1TR001450/SCTR 2214 (OP). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alpha 7-nicotinic cholinergic regulation of pericyte-containing retinal capillaries.

Local blood flow regulation relies mostly on the coordination between neurons and pericyte-containing capillaries. Pericyte relaxation and contraction are influenced by various vasoactive substances and regulated by neurotransmitters. α7 nicotinic acetylcholine receptors (α7-nAChRs), involved in the regulation of vascular function and inhibitory GABA systems, have neuroprotective effects against central nervous system diseases. Although α7-nAChRs are found throughout the retina, their contribution to the retinal capillary tone remains unknown. Here we investigate the neurovascular coupling mechanism underlying α7-nAChR-mediated retinal capillary tone regulation. Changes in capillary diameter and transverse diameter of pericytes during drug perfusion were observed using differential interference contrast microscopy (DIC) to elucidate signaling pathways underlying α7-nAChR-mediated regulation of capillary blood flow at the whole retinal level. Patch clamp technique was used to investigate α7-nAChRs-mediated regulation of the γ-aminobutyric acid (GABA) synaptic circuit. Immunofluorescence was used to explore the expression of α7-nAChRs and GABA receptors. Activating α7-nAChRs on the endothelial cell membrane caused perinuclear accumulation of eNOS. This resulted in dilated retinal capillaries and pericytes via the NOS/NO-cGMP signaling pathway. Neuronal α7-nAChRs activation relaxed retinal capillaries and pericytes via a neurovascular coupling mechanism. α7-nAChR increased the vesicular release of GABA, possibly promoting the release of NO by binding to GABAA receptors in retinal ganglionic cells (RGCs), and relaxation of blood vessels via eNOS-NO by binding to GABAB receptors on retinal capillary endothelial cells. α7-nAChR activation causes vasorelaxation of retinal capillaries.

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.

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. 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-1β, NF-κB p65, and p-NF-κB p65 in the hypothalamus. 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-1β. TaVNS could significantly reverse the above-mentioned phenomena, but had a poor improvement effect for CUMS-exposed α7nAchR-/- rats. The hypothalamic α7nAchR/JAK2/STAT3/NF-κB signaling pathway may play an important role in the antidepressant-like behavior of taVNS.

Low-dose ketamine inhibits neuronal apoptosis and neuroinflammation in PC12 cells via α7nAChR mediated TLR4/MAPK/NF-κB signaling pathway.

Ketamine is commonly used for sedation, analgesia and anesthetics. Much evidence has shown that it has an immune-regulatory effect. The cholinergic anti-inflammatory pathway mediated by α7nAChR is a prominent target of anti-inflammatory therapy. However, whether ketamine suppresses inflammatory levels in nerve cells by activating α7nAChR remains unknown. Lipopolysaccharide (LPS) was used to establish the neuroinflammation model in PC12 cells in vitro, and α7nAChR siRNA was transfected into PC12 cells 30min before LPS to inhibit gene expression of α7nAChR. PC12 cells were stimulated with LPS for 24h, and the indicators were detected at 2h after GTS-21 and ketamine were added. The results showed that LPS increased the proportion of PC12 cells apoptosis, activated TLR4/MAPK/NF-κB signaling pathway, and increased the expression of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Ketamine reduced the ratio of early apoptosis and late apoptosis of PC12, inhibited the entry of P65 into the nucleus, decreased the activation of TLR4/MAPK/NF-κB and improved neuroinflammation. However, the ameliorating effects of ketamine on neuronal apoptosis and neuroinflammation were inhibited in the α7nAChRi group. This indicated that α7nAChR played a key role in the anti-inflammatory process of ketamine. Low-dose ketamine inhibited TLR4/MAPK/NF-κB by activating the α7nAChR-mediated cholinergic anti-inflammatory pathway, thereby producing the protective effect on neuronal apoptosis and neuroinflammation.

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

PurposePrevious 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. MethodsSepsis 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. ResultsEA 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. ConclusionEA 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.

Inflammatory and oxidative endophenotypes in borderline personality disorder: A biomarker cluster analysis

Objectives This study is designed to search for aggrupation of inflammatory/oxidative biomarker alterations in borderline personality disorder (BPD) and their association with phenotypic features. Methodology Inflammatory/nitrosative proteins were measures in plasma and peripheral blood mononuclear cells obtained from BPD patients. Patients were assessed on different clinical dimensions of BPD. Oxidative damage was tested by measuring TBARS, nitrites, catalase, GPx and SOD. Protein expression of IκBα, NFκB, iNOS, COX-2, PPARγ, Keap1, NQO1, Nrf2 and α7nAChR was also determined. Western blot and ELISA were used for measurements and a cluster analysis of inflammatory/oxidative biomarkers alterations was performed to investigate subgroups of patients with similar alterations and its relationship with clinical features of BPD. Results 69 patients were included in the study. Two inflammatory/nitrosative clusters of patients were found: Cluster 1 patients showed significantly higher levels of GPx, IκBα, keap1, NQO1, PPARγ, α7nAChR and Nrf2 than cluster 2 patients. These patients had significantly longer duration of illness, milder anxiety symptoms and lower prescription of antipsychotic drugs than cluster 2. Conclusions Two clusters of BPD patients according to the inflammatory/nitrosative profiles were identified. Cluster 1 had increased antioxidant and anti-inflammatory biomarkers and was characterised by greater chronicity of illness but less acute symptomatic severity.

SARS-CoV-2 Spike Protein Downregulates Cell Surface α7nAChR through a Helical Motif in the Spike Neck.

A deficiency of the functional α7 nicotinic acetylcholine receptor (α7nAChR) impairs neuronal and immune systems. The SARS-CoV-2 spike protein (S12) facilitates virus cell entry during COVID-19 infection and can also independently disrupt cellular functions. Here, we found that S12 expression significantly downregulated surface expression of α7nAChR in mammalian cells. A helical segment of S12 (L1145-L1152) in the spike neck was identified to be responsible for the downregulation of α7nAChR, as the mutant S12AAA (L1145A-F1148A-L1152A) had minimal effects on surface α7nAChR expression. This S12 segment is homologous to the α7nAChR intracellular helical motif known for binding chaperone proteins RIC3 and Bcl-2 to promote α7nAChR surface expression. Competition from S12 for binding these proteins likely underlies suppression of surface α7nAChR. Considering the critical roles of α7nAChR in cellular functions, these findings provide a new perspective for improving mRNA vaccines and developing treatment options for certain symptoms related to long COVID.

Exploration of the mechanism by which Huangqi Guizhi Wuwu decoction inhibits Lps-induced inflammation by regulating macrophage polarization based on network pharmacology

BackgroundHuangqi Guizhi Wuwu decoction (HQGZWWD) is a traditional Chinese herbal medicine formulation with significant anti-inflammatory activity. However, its underlying mechanism remains unknown. Through network pharmacology and experimental validation, this study aimed to examine the potential mechanism of HQGZWWD in regulating macrophage polarization and inflammation.MethodsThe active components were obtained from the Traditional Chinese Medicine Systems Pharmacology database and Analysis Platform (TCMSP), whereas the corresponding targets were obtained from the TCMSP and Swiss Target Prediction database. The GeneCards database identified targets associated with macrophage polarization and inflammation. Multiple networks were developed to identify the key compounds, principal biological processes, and pathways of HQGZWWD that regulate macrophage polarization and inflammation. Autodock Vina is utilized to assess the binding ability between targets and active compounds. Finally, confirm the experiment’s central hypothesis. Human histiocytic lymphoma (U-937) cells were transformed into M1 macrophages following stimulation with Lipopolysaccharide (LPS) to evaluate the effect of HQGZWWD drug-containing mouse serum (HQGZWWD serum) on regulating macrophage polarization and inflammation.ResultsA total of 54 active components and 859 HQGZWWD targets were obtained. There were 9972 targets associated with macrophage polarization and 11,109 targets associated with inflammation. After screening, 34 overlapping targets were identified, of which 5 were identified as central targets confirmed by experiments, including the α7 nicotinic acetylcholine receptor (α7 nAchR), interleukin 6 (IL-6), Interleukin-1 beta (IL-1β), interleukin 10 (IL-10) and growth factor beta (TGF-β1). Pathway enrichment analysis revealed that 34 overlapping targets were enriched in multiple pathways associated with macrophage polarization and inflammation, including the TGF beta signaling pathway, NF-kappa B signaling pathway, JAK-STAT signaling pathway, and TNF signaling pathway. Molecular docking confirmed that the majority of HQGZWWD’s compounds can bind to the target. In vitro experiments, HQGZWWD serum was shown to up-regulate the expression of α7 nAchR, reduce the number of M1 macrophages, stimulate the production of M2 macrophages, inhibit the expression of pro-inflammatory cytokines IL-6 and IL1-β, and increase the expression of anti-inflammatory cytokines IL-10 and TGF-β1.ConclusionHQGZWWD can regulate the number of M1/M2 macrophages and the level of inflammatory cytokines, and the underlying mechanism may be related to the up-regulation of α7 nAchR expression.