Cholinergic modulation of the immune system - A novel therapeutic target for myocardial inflammation.

The immune system and the nervous system depend on each other for their fine tuning and working, thus cooperating to maintain physiological homeostasis and prevent infections. The cholinergic system regulates the mobilization, differentiation, secretion, and antigen presentation of adaptive and innate immune cells mainly through α7 nicotinic acetylcholine receptors (α7nAChRs). The neuro-immune interactions are established and maintained by the following mechanisms: colocalization of immune and neuronal cells at defined anatomical sites, expression of the non-neuronal cholinergic system by immune cells, and the acetylcholine receptor-mediated activation of intracellular signaling pathways. Based on these immunological mechanisms, the protective effects of cholinergic system in animal models of diseases were summarized in this paper, such as myocardial infarction/ischemia-reperfusion, viral myocarditis, and endotoxin-induced myocardial damage. In addition to maintaining hemodynamic stability and improving the energy metabolism of the heart, both non-neuronal acetylcholine and neuronal acetylcholine in the heart can alleviate myocardial inflammation and remodeling to exert a significant cardioprotective effect. The new findings on the role of cholinergic agonists and vagus nerve stimulation in immune regulation are updated, so as to develop improved approaches to treat inflammatory heart disease.

Nicotinic cholinergic system and COVID-19: In silico evaluation of nicotinic acetylcholine receptor agonists as potential therapeutic interventions.

SARS-CoV-2 infection was announced as a pandemic in March 2020. Since then, several scientists have focused on the low prevalence of smokers among hospitalized COVID-19 patients. These findings led to our hypothesis that the Nicotinic Cholinergic System (NCS) plays a crucial role in the manifestation of COVID-19 and its severe symptoms. Molecular modeling revealed that the SARS-CoV-2 Spike glycoprotein might bind to nicotinic acetylcholine receptors (nAChRs) through a cryptic epitope homologous to snake toxins, substrates well documented and known for their affinity to the nAChRs. This binding model could provide logical explanations for the acute inflammatory disorder in patients with COVID-19, which may be linked to severe dysregulation of NCS. In this study, we present a series of complexes with cholinergic agonists that can potentially prevent SARS-CoV-2 Spike glycoprotein from binding to nAChRs, avoiding dysregulation of the NCS and moderating the symptoms and clinical manifestations of COVID-19. If our hypothesis is verified by in vitro and in vivo studies, repurposing agents currently approved for smoking cessation and neurological conditions could provide the scientific community with a therapeutic option in severe COVID-19.

Influencia adrenérgica y colinérgica sobre la producción de especies reactivas de oxígeno en neutrófilos humanos* / Adrenergic and cholinergic influence on the production of reactive oxygen species in human neutrophils*

RESUMEN Objetivos. Determinar y comparar el efecto de fármacos agonistas adrenérgicos y colinérgicos sobre la producción de especies reactivas de oxígeno (ROS) en neutrófilos de individuos sanos. Materiales y métodos. Se tomaron muestras de sangre total de cinco participantes para purificar los neutrófilos mediante el método de gelatina. Se midió la producción de ROS por quimioluminiscencia (QLM) usando un contador de centelleo y forbol-12-miristato-13-acetato (PMA) como estímulo. También se realizaron pruebas sin PMA para medir la producción espontánea. Posteriormente, con el mismo método se midió la formación de ROS en presencia de nicotina (agonista colinérgico), salbutamol y clonidina (agonistas adrenérgicos), cada uno en concentraciones de 10-2 M, 10-3 M, 10-4 M y 10-5 M. Se calculó el área integrada bajo las curvas de QLM y se halló el porcentaje de inhibición o de estimulación según sea el caso. Se comparó el efecto provocado por las drogas con sus controles correspondientes y se realizó el análisis estadístico. Resultados. Se obtuvo una disminución de la producción de ROS como efecto de las sustancias estudiadas con una diferencia significativa entre los controles y el efecto producido a 10-2 M, 10-3 M y 10-4 M. Este efecto aumentó de intensidad conforme la concentración de las drogas se incrementó. Los mayores porcentajes de inhibición se mostraron a 10-2 M y 10-3 M. Salbutamol presentó los máximos valores con todas las concentraciones con diferencia significativa entre su inhibición y la generada por las demás drogas. Conclusiones. Los estímulos adrenérgico y colinérgico tienen un efecto inhibitorio de la producción de ROS en neutrófilos de individuos sanos.

ABSTRACT Objectives. To determine and compare the effect of adrenergic and cholinergic agonist drugs on the production of reactive oxygen species (ROS) in neutrophils of healthy individuals. Materials and Methods. Whole blood samples were taken from five participants to purify neutrophils using the gelatin method. The production of chemiluminescent (QLM) ROS was measured using a scintillation counter and phorbol-12-myristat-13-acetate (PMA) as a stimulus. Non-PLA tests were also conducted to measure spontaneous production. Subsequently, with the same method, ROS formation was measured in the presence of nicotine (cholinergic agonist), salbutamol, and clonidine (adrenergic agonists), each in concentrations of 10-2 M, 10-3 M, 10-4 M, and 10-5 M. The area integrated under the QLM curves was calculated and the percentage of inhibition or stimulation was found as the case may be. The effect of the drugs was compared with their corresponding controls and statistical analysis was carried out. Results. A decrease in the production of ROS was obtained as an effect of the substances studied with a significant difference between the controls and the effect produced at 10-2 M, 10-3 M, and 10-4 M . This effect increased in intensity as drug concentration increased. The highest percentages of inhibition were shown at 10-2 M and 10-3 M. Salbutamol presented the maximum values with all the concentrations with a significant difference between its inhibition and that generated by the other drugs. Conclusions. Adrenergic and cholinergic stimuli have an inhibitory effect on the production of ROS in neutrophils of healthy individuals.

Role of α7nAChR in postoperative cognitive dysfunction in aged rats with tibial fracture / 中华麻醉学杂志

Objective To evaluate the role of α7 nicotinic acetylcholine receptor (α7nAChR) in postoperative cognitive dysfunction in aged rats with tibial fracture. Methods One hundred and fifty clean-grade healthy male Sprague-Dawley rats, aged 18-22 months, weighing 440-580 g, were divided into 5 groups ( n=30 each ) using a random number table method: control group ( group C ) , sham operation group ( group S) , tibial fracture group ( group T) , normal saline group ( group N) and α7nAChR agonist PUN282987 group (group P). Group C received no treatment. Ten percent chloral hydrate 0. 4 ml/100 g was injected intraperitoneally in group S. Group T underwent tibial fracture. PUN2829872. 4 mg/kg was in-traperitoneally injected at 5 min before tibial fracture in group P . The equal volume of normal saline was giv-en at 5 min before tibial fracture in group N. Morris water maze test was performed at day 7 after surgery. At days 1, 3 and 7 after surgery, the pathological changes of the hippocampal CA3 region were observed by haematoxylin and eosin staining, and the expression of α7nAChR, choline acetyltransferase ( ChAT ) , tumor necrosis factor-α( TNF-α) and interleukin-1β( IL-1β) in the hippocampal CA3 region was measured by Western blot. Results Compared with group C, the postoperative escape latency and swimming dis-tance were significantly prolonged, and the expression of α7nAChR, ChAT, TNF-α and IL-1β was up-regulated at each time point after operation in T, N and P groups ( P＜0. 05) , and no significant change was found in the parameters mentioned above in group S ( P＞0. 05) . Compared with group T, the postoper-ative escape latency and swimming distance were significantly shortened, and the expression of α7nAChR and ChAT was up-regulated and the expression of TNF-α and IL-1β was down-regulated at each time point after operation in group P ( P＜0. 05) , no significant change was found in the parameters mentioned above in group N ( P＞0. 05) , and the pathological changes of the hippocampal CA3 region were significantly at-tenuated in group P. Conclusion α7nAChR antagonism is involved in the development of postoperative cognitive dysfunction in aged rats with tibial fracture.

Effect of α7nAChR agonist on NLRP3 inflammasome during brain injury induced by cardiopulmo-nary bypass in rats / 中华麻醉学杂志

Objective To evaluate the effect of α7 nicotinic acetylcholine receptor (α7nAChR) agonist on inflammasome of NOD-like receptor pyrin domain containing 3 ( NLRP3) during brain injury in-duced by cardiopulmonary bypass ( CPB) in rats. Methods Twenty-four clean-grade adult male Sprague-Dawley rats, weighing 350-450 g, were randomly divided into sham operation group (group S), group CPB, and CPB plusα7nAChR agonist PHA568487 group (group CP) after 5-day Morris water maze train-ing, with 8 rats in each group. Group S was mechanically ventilated for 60 min without receiving CPB. Group CPB received CPB for 60 min. PHA 5684870. 8 mg/kg was intraperitoneally injected at 30 min be-fore CPB in group CP. Water maze test was performed on 3rd day after operation to record the escape laten-cy and times of crossing the original platform. The rats were sacrificed at 2 h after the behavioral test, and their hippocampi were harvested for determination of cell apoptosis ( by TUNEL) and contents of interleukin-1beta ( IL-1β) and IL-18 ( by enzyme-linked immunosorbent assay) , caspase-1 activity ( by using spectro-photometry) , expression of NLRP3, apoptosis-associated speck-like protein containing a CAR ( ASC) and pro-caspase-1 ( by Western blot or real-time polymerase chain reaction) , and expression of NLRP3, ASC and caspase-1 mRNA (using real-time polymerase chain reaction). Apoptotic index (AI) was calculated. Results Compared with group S, the escape latency was significantly prolonged, the times of crossing the original platform were decreased, the AI, contents of IL-1β and IL-18 and caspase-1 activity were in-creased, and the expression of NLRP3 and ASC protein and mRNA, pro-caspase-1 and caspase-1 mRNA was up-regulated in CPB and CP groups (P＜0. 05). Compared with group CPB, the escape latency was significantly shortened, the times of crossing the original platform were increased, the AI, contents of IL-1β and IL-18 and caspase-1 activity were decreased, and the expression of NLRP3 and ASC protein and mRNA, pro-caspase-1 and caspase-1 mRNA was down-regulated in group CP (P＜0. 05). Conclusion The mechanism by whichα7nAChR agonist alleviates CPB-induced brain injury may be related to inhibiting NLRP3 inflammasome and reducing inflammatory responses in brain tissues of rats.

Effect of α7nAChR agonists on acute lung injury caused by cardiopulmonary bypass in rats / 中华麻醉学杂志

Objective To evaluate the effect of α7 nicotinic acetylcholine receptor (α7nAChR)agonists on lung injury caused by cardiopulmonary bypass (CPB) in rats.Methods Eighteen healthy clean-grade adult male Sprague-Dawley rats,weighing 350-400 g,were divided into 3 groups (n =6 each)using a random number table method:sham operation group (group S),CPB group and α7nAChR agonist PHA568487 group (group PHA).The rats underwent no CPB and were mechanically ventilated for 60 min in group S.PHA568487 0.8 mg/kg (diluted to 2 ml in normal saline) was intraperitoneally injected at 30 min before CPB,and then CPB was performed for 60 min in group PHA.Normal saline 2 ml was intraperitoneally injected at 30 min before CPB,and then CPB was performed for 60 min in group CPB.Blood samples were collected from the internal jugular vein for determination of serum interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) concentrations by enzyme-linked immunosorbent assay.Lung tissues were obtained for microscopic examination of the pathologic changes and for determination of wet/dry weight ratio (W/D ratio) and matrix metalloproteinase-9 (MMP-9) expression (by Western blot).Results Compared with group S,the W/D ratio and serum concentrations of TNF-α and IL-6 were significantly increased,and the expression of MMP-9 was up-regulated in CPB and PHA groups (P＜0.05).Compared with group CPB,the W/D ratio and serum concentrations of TNF-α and IL-6 were significantly decreased,the expression of MMP-9 was down-regulated (P＜0.05),and the pathological changes of lung tissues were significantly attenuated in group PHA (P＜0.05).Conclusion α7nAChR agonists can reduce the acute lung injury caused by CPB in rats,and the mechanism may be related to down-regulating MMP-9 expression and inhibiting systemic inflammatory responses.

Acetylcholine chloride 1% usage for intraoperative cataract surgery miosis / Cloridrato de Acetilcolina 1% na obtenção da miose intraoperatória na cirurgia de catarata

Abstract Objective: To test the efficacy of Acetylcholine chloride use in obtaining intraoperative miosis on phacoemulsification cataract surgery. Methods: Patients with cataract diagnosis and elected for surgical phacoemulsification procedure were selected. All patients underwent conventional phacoemulsification procedure performed by a single surgeon and all patients had 0.2 ml of Acetylcholine chloride 1% irrigated in the anterior chamber at the end of the surgery. The pupillary diameter was measured immediately before the beginning of surgery, immediately before and two minutes after the use of acetylcholine chloride 1%. Results: A total of 30 eyes from 30 patients were included in the study. 18 were female, and mean age was of 69.5 years with a 7.2y standard deviation on the population study. The mean pupillary diameter immediately before the beginning of surgery was 7.5 mm with a standard deviation of 0.56 mm; the mean pupillary diameter immediately before the acetylcholine chloride 1% use (after the intraocular lens im-plantation) was 7.1 mm with a standard deviation of 0.57 mm. The mean pupillary diameter two minutes after the use of acetylcholine chloride 1% in the anterior chamber was 3.4 mm with standard deviation of 0.66 mm. The mean maximum action time of ACH chloride 1% was 64 seconds, with a standard deviation of 8 seconds. The mean intraocular pressure on the first postoperative day was 19.1 mmHg with a standard deviation of 2.45 mmHg. Conclusion: We conclude that acetylcholine chloride 1% is an important drug to obtaining intraoperative miosis in cataract surgery.

Resumo Objetivo: Demonstrar a eficácia do cloridrato de acetilcolina 1% na obtenção da miose intraoperatória na cirurgia de catarata pela técnica de facoemulsificação. Métodos: Pacientes com diagnóstico de catarata e indicação de cirurgia foram selecionados para participar do presente estudo. Todos os pacientes foram operados pela técnica de facoemulsificação convencional pelo mesmo cirurgião, todos foram submetidos à aplicação de 0,2 ml do cloridrato de acetilcolina 1% na câmara anterior ao final do procedimento cirúrgico. A medida do diâmetro pupilar foi realizada imediatamente antes do início da cirurgia, imediatamente antes do uso do cloridrato de acetilcolina 1% e após 2 minutos. Resultados: Foram estudados 30 olhos de 30 pacientes, destes, 18 eram do sexo feminino, a média de idade do estudo foi de 69,5 anos com desvio padrão de 7,2 anos. A média do diâmetro pupilar imediatamente antes do início da cirurgia foi 7,55 mm com desvio padrão de 0,56mm, a média do diâmetro pupilar imediatamente antes do uso do cloridrato de acetilcolina 1% (após implante da lente intraocular no saco capsular) foi 7,1mm com desvio padrão de 0,57mm. A média do diâmetro pupilar após 2 minutos da aplicação da acetilcolina na câmara anterior foi de 3,4 mm com desvio padrão de 0,66mm. O tempo médio de ação máxima do medicamento foi de 64 segundos, com desvio padrão de 8 segundos. A média da pressão intraocular no primeiro dia do pós-operatório foi de 19,1 mmHg com desvio padrão de 2,45mmHg. Conclusão: O estudo acima mostrou que a acetilcolina apresenta boa eficácia na obtenção de miose intraoperatória na cirurgia de facoemulsificação, permitindo uma maior facilidade na confecções das suturas corneanas ou corneo-escleral, reduzindo a incidência de sinéquias anteriores periféricas. Concluimos que o cloridrato de acetilcolina 1% é um importante medicamento na obtenção da miose intraoperatória na cirurgia de catarata.

Effect of PUN282987 on global cerebral ischemia-reperfusion injury in aged rats / 中华麻醉学杂志

Objective To evaluate the effect of PUN282987 on global cerebral ischemia-reperfusion (I/R) injury in aged rats.Methods One hundred and twenty pathogen-free healthy male SpragueDawley rats,aged 18-22 months,weighing 450-600 g,were divided into 3 groups (n=40 each) using a random number table:sham operation group (group S),global cerebral I/R group (group I/R) and α7 nicotinic acetylcholine receptor (α7nAChR) agonist PNU282987 group (group PUN).The animals were anesthetized with intraperitoneal 10％ chloral hydrate 0.4 ml/100g,and global cerebral I/R was produced by 4-vessel occlusion technique in I/R and PUN groups.PUN282987 2.4 mg/kg was injected intraperitoneally before ischemia in group PUN.At 1,5,12 and 24 h of reperfusion,10 rats were randomly selected in each group and then sacrificed,and the brains were removed for detection of the neuronal apoptosis and for determination of the expression of α7nAChR,choline acetyltransferase (ChAT),tumor necrosis factor-α (TNF-α) and intedeukin-1β (IL-1β) in the hippocampal CA1 region.Apoptosis rate was calculated.Results Compared with group S,the apoptosis rate was significantly increased,and the expression of α7nAChR,ChAT,TNF-α and IL-1β was up-regulated at each time point in I/R and PUN groups (P＜0.05).Compared with group I/R,the apoptosis rate was significantly decreased,the expression of α7nAChR and ChAT was up-regulated,and the expression of TNF-α and IL-1β was down-regulated at each time point in group PUN (P＜0.05).Conclusion PUN282987 can reduce global cerebral I/R injury in aged rats.

Morphological changes in the salivary acini after in vivo cholinergic stimulation.

AIMS: Overactive bladder syndrome treated by muscarinic receptor antagonists may be complicated by reduced salivation. Cholinergic agonists may reverse this effect. The aim of the present study was to determine the antagonizing effect of a cholinergic agonist (carbachol) on a muscarinic receptor antagonist (oxybutynin) in the submandibular acini in a rat model. METHODS: Forty male Sprague Dawley rats were divided into three groups: Group I (control), Group II (vehicle), and Group III (treatment). Group III was subdivided so Group IIIa was treated with a muscarinic receptor antagonist (oxybutynin) for 1 week, Group IIIb was treated with oxybutynin for 3 weeks, and Group IIIc was treated with oxybutynin for 1 week and oxybutynin and a cholinergic agonist (carbachol) for 2 weeks. Histological and ultrastructural studies were performed on submandibular glands. RESULTS: Group IIIa showed moderate atrophic changes in the serous acini and ducts. Group IIIb showed serous acini with distorted wall, widening of the inter-lobar space, and deposition of mononuclear cells in the connective tissue. Group IIIc had serous acini similar to Group I, with mildly dilated inter-lobar ducts, but some serous acini revealed double nuclei and the inter-lobar duct showed luminal vacuolations. Ultrastructural studies confirmed histological results. CONCLUSIONS: Muscarinic receptor antagonist administration led to changes in the submandibular gland of rats, while concomitant administration of cholinergic agonists seemed to counteract these atrophic changes. Additional studies should assess carbachol as a cholinergic agonist in treating dry mouth in patients with overactive bladder syndrome who are taking the muscarinic receptor. Neurourol. Urodynam. 35:574-581, 2016. © 2015 Wiley Periodicals, Inc.

Nicotinic and muscarinic acetylcholine receptors shape ganglion cell response properties.

The purpose of this study was to evaluate the expression patterns of nicotinic and muscarinic ACh receptors (nAChRs and mAChRs, respectively) in relation to one another and to understand their effects on rabbit retinal ganglion cell response properties. Double-label immunohistochemistry revealed labeled inner-retinal cell bodies and complex patterns of nAChR and mAChR expression in the inner plexiform layer. Specifically, the expression patterns of m1, m4, and m5 muscarinic receptors overlapped with those of non-α7 and α7 nicotinic receptors in presumptive amacrine and ganglion cells. There was no apparent overlap in the expression patterns of m2 muscarinic receptors with α7 nicotinic receptors or of m3 with non-α7 nicotinic receptors. Patch-clamp recordings demonstrated cell type-specific effects of nicotinic and muscarinic receptor blockade. Muscarinic receptor blockade enhanced the center responses of brisk-sustained/G4 On and G4 Off ganglion cells, whereas nicotinic receptor blockade suppressed the center responses of G4 On-cells near the visual streak but enhanced the center responses of nonstreak G4 On-cells. Blockade of muscarinic or nicotinic receptors suppressed the center responses of brisk-sustained Off-cells and the center light responses of subsets of brisk-transient/G11 On- and Off-cells. Only nicotinic blockade affected the center responses of G10 On-cells and G5 Off-cells. These data indicate that physiologically and morphologically identified ganglion cell types have specific patterns of AChR expression. The cholinergic receptor signatures of these cells may have implications for understanding visual defects in disease states that result from decreased ACh availability.

Mechanisms underlying the reversal of acantholysis in pemphigus by a cholinergic receptor agonist / 中华皮肤科杂志

Objective To evaluate the reversal effect of a cholinergic receptor agonist on acantholysis in pemphigus,and to investigate its mechanism.Methods Human HaCaT keratinocytes were co-cultured with pemphigus vulgaris immunoglobulin G (PV-IgG) to establish a cell model of pemphigus,then classified into two groups to be incubated with the cholinergic receptor agonist carbachol for 12 hours (test group) or remain untreated (control group).Cell dissociation assay was performed to quantitatively estimate the reversal effect of carbachol on acantholysis,and immunofluorescence assay to qualitatively assess the changes of desmosomal proteins.Radio-immunoprecipitation assay (RIPA) lysis buffer and Triton X-100 were used to lyse HaCaT cells to obtain total proteins and cytoplasmic proteins,and Western blot was conducted to determine the expression levels of adhesion-related proteins desmoglein 3 (Dsg3) and plakoglobin (PG) on the surface of HaCaT cells,as well as the phosphorylation levels of p38 mitogen activated protein kinase (p38 MAPK) and epidermal growth factor receptor (EGFR) at different time points.Quantitative polymerase chain reaction (qPCR) was performed to detect the mRNA expressions of the above surface proteins,and coimmunoprecipitation assay to qualitatively evaluate the interaction between Dsg3 and PG.Results The number of cell debris was significantly lower in the test group than in the control group (18.67 ± 2.52 vs.46.67 ± 2.03,t =11.22,P＜0.01).Immunofluorescence assay showed that carbachol could reverse the internalization of desmosomal molecules induced by PV-IgG.In the pemphigus cell model,the levels of total Dsg3 and PG as well as non-desmosomal Dsg3 were decreased,while the level of non-desmosomal PG increased,and the interaction between Dsg3 and PG was attenuated.When the pemphigus cell model was co-cultured with carbachol,these above changes were reversed.Carbachol also increased the mRNA levels (expressed as 2-△△Ct) of Dsg3 and PG from 1.428 ± 0.215 and 1.563 ± 0.247 in the control group to 4.974 ± 0.948 (t =3.65,P =0.01) and 13.420 ± 1.715 (t =6.85,P ＜ 0.01) in the test group respectively.In phosphorylation assay,carbachol inhibited the phosphorylation of EGFR,but had no significant effect on that of p38 MAPK.Conclusions The cholinergic receptor agonist carbachol can reverse acantholysis in pemphigus,likely by inhibiting the internalization of Dsg3 and PG,enhancing their expressions and interaction,and suppressing the phosphorylation of the key signaling molecule for acantholysis,EGFR.

Effects of nicotine or electric vagal stimulation on injury to the transplanted lungs in rats / 中华麻醉学杂志

Objective To evaluate the effects of nicotine or electric vagal stimulation on injury to transplanted lungs in rats.Methods Twenty-four male Wistar rats with transplanted lung,weighing 250-300 g,were randomly divided into 3 groups (n =8 each) using a random number table:control group (group C),nicotine group (group NI) and electric vagal stimulation group (group VS).Nicotine 2 mg/kgwas injected intraperitoneally at 15 min after lung transplantation in group NI.Right cervical vagus nerve trank was stimulated for 20 min with continuous electric rectangular pulses (5 V,2 ms,1 Hz) at 10 min interval twice in total starting from 15 min after lung transplantation in group VS.Arterial blood samples were then collected for blood gas analysis at 15,30,45,75,105 and 135 min after lung transplantation.Arterial blood samples were then collected at 135 min after lung transplantation for determination of the plasma concentration of interleukin-6 (IL-6),IL-8 and tumor necrosis factor-α (TNF-α) by ELISA.The rats were sacrificed at 135 min after lung transplantation and the tissue of transplanted lung was removed for determination of lung injury score (LIS) according to the pathological changes obtained with light microscope,and of wet/dry lung weight ratio (W/D ratio).Results Compared with group C,PaO2 and pH value were significantly increased,and LIS,W/D ratio,and the concentrations of plasma IL-6,IL-8 and TNF-α were decreased in NI and VS groups.Conclusion Nicotine or electric vagal stimulation can attenuate injury to transplanted lungs in rats.

Changes in cholinergic anti-inflammatory pathway in hippocampi in aged rats with global cerebral ischemia/reperfusion injury / 中华麻醉学杂志

Objective To evaluate the changes in cholinergic anti-inflammatory pathway in hippocampi global in aged rats with cerebral ischemia/reperfusion (I/R ) injury .Methods One hundred and twenty male Sprague-Dawley rats , aged 18-22 months ,weighing 450-600 g ,were randomly divided into 2 groups ( n= 60 each):sham operation group (group S) and global cerebral I/R group (group I/R) .The animals were anesthetized with intraperitoneal 10% chloral hydrate 0.4 ml/100 g .Global cerebral I/R was induced by 4-vessel occlusion method described by Pulsinelli .Fifteen rats were sacrificed at 1 ,3 ,5 and 7 days of reperfusion ,and brains were removed for determination of neuronal apoptosis and expression of α7 nicotinic acetylcholine receptor (α7nAChR ) , choline acetyltransferase (ChAT ) ,tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in the hippocampal CA1 region .The apoptosis rate was calculated .Results Compared with group S ,the apoptosis rate was increased and the expression of α7nAChR ,ChAT ,TNF-αand IL-1βwas up-regulated in group I/R ( P<0.05 or 0.01 ) . The expression of α7nAChR and ChAT was up-regulated gradually during reperfusion and peaked at 5 day of reperfusion ( P< 0.05 ) .Conclusion Global cerebral I/R injury can activate cholinergic anti-inflammatory pathway in aged rat hippocampi ,and the activation of this pathway is the endogenous mechanism of inhibition of excessive inflammatory responses in brain tissues .

Functional characterization of acetylcholine receptors and calcium signaling in rat testicular capsule contraction.

The motor activity of mammalian testicular capsule (TC) contributes to male fertility and infertility, but the acetylcholine receptors related to the contractions induced by cholinergic drugs are poorly known. Indeed to characterize the acetylcholine receptors and cellular signaling by Ca(2+) involved in TC motor activity of rats, the potency of agonists (pD2) and antagonists (pA2) of acetylcholine receptors, and effects of Ca(2+) cellular transport blockers on the cholinergic contractions were evaluated. pD2 values of acetylcholine (5.98) were ten-fold higher than that of carbachol (4.99). Efficacy (Emax) of acetylcholine and carbachol to induce contractions corresponded to 95% and 97% of Emax for KCl, but Emax for nicotine was very low (8% of Emax for KCl). Further, physostigmine did not affect the acetylcholine potency. Contractions induced by acetylcholine or carbachol were antagonized by muscarinic but not nicotinic antagonist. The order of pA2 values obtained for muscarinic antagonists, namely atropine>4-DAMP>AF-DX116>pirenzepine, corresponded to a typical profile of M3 receptors. Contractions induced by acetylcholine or carbachol were inhibited by blockers of Ca(2+) influx through voltage-dependent calcium channels (nifedipine and Ni(2+)), Ca(2+) reuptake by sarco-endoplasmic reticulum (cyclopiazonic acid) and mitochondria (FCCP). The protein kinase C (PKC) inhibitor chelerythrine only affected the acetylcholine-induced contraction. These results suggest that TC motor activity of rats are mediated mainly by M3 receptors followed by the increase of cytosolic Ca(2+) concentration regulated by voltage-dependent calcium channels, sarco-endoplasmic reticulum and mitochondria. Furthermore, the differential effects of chelerythrine in the acetylcholine or carbachol-induced contractions are discussed.

Regulatory role of acetylcholine receptor in muramyl dipeptide-induced activation of NLR2/RIP2 pathway in macrophages of mice / 中华麻醉学杂志

Objective To evaluate the regulatory role of acetylcholine receptor in muramyl dipeptide (MDP)-induced activation of Nod-like receptor 2/receptor-interacting protein 2 (2NLR2/RIP2) pathway in macrophages of mice.Methods RAW264.7 cells at the logarithmic growth phase were seeded in 12-well plates (density 1 × 106 cells/ml,2 ml/well),a total of 108 wells.The cells were randomly divided into 3 groups (n =36 each) using a random number table:control group (group C),MDP group (group M),and GTS-21 (a7nAChR specific agonist) group (group G).The cells were routinely cultured in group C.MDP with the final concentration of 10 μg/ml was added to the culture medium in group M.MDP with the final concentration of 10μg/ml and GTS21 with the final concentration of 50 μg/ml were added to the culture medium in group G.The cells were incubated for 24 h.At 1,6 and 24 h of incubation with MDP,12 wells were chosen and the cell suspension was obtained for measurement of NLR2 mRNA expression (by real-time fluorescent quantitative PCR),RIP2 expression (by Western blot),and concentrations of tumor necrosis factor-alpha (TNF-α) and high mobility group box-1 (HMGB1) in the culture media (by ELISA).Results Compared with group C,the levels of NLR2 mRNA,RIP2,TNFα and HMGB1 were significantly increased at each time point in group M (P ＜ 0.05).Compared with group M,the levels of NLR2 mRNA,RIP2,TNF-α and HMGB1 were significantly decreased at each time point in group G (P ＜ 0.05).Conclusion Acetylcholine receptor can suppress MDP-induced transduction of NLR2/RIP2 pathway in macrophages of mice.

Effect of muscarinic acetylcholine receptor agonist on glycinergic spontaneous inhibitory postsynaptic currents and micro-inhibitory postsynaptic currents in spinal lamina Ⅱ neurons of rats / 中华麻醉学杂志

Objective To investigate the effect of the mascarinic acetylcholine receptor (mAChR) agonist Oxo-tremorine-M (Oxo-M) on glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) and micro-inhibitory postaynaptic currents (mIPSCs) in lamina Ⅱ neurons in the spinal cord of rats. Methods Glycinergic IPSCs (sIPSCs and mIPSCs) in lamina Ⅱ neurons of spinal slices were recorded using the whole-cell voltage-clamp method. The non-selective mAchR ngonist Oxo-M was applied through bath perfusian. The effects of Oxo-M 1, 3, 5 and 10 μmol/L on sIPSCs and mIPSCs were examined. Results Oxo-M at the concentrations of 3-10 μmol/L significantly increased the frequency of sIPSCs without changing the amplitude in 16 lamina Ⅱ neurons tested. Interestingly, when the concentration of Oxo-M was increased to 10 μmol/L, the potentiating effect of Oxo-M on the frequency of slPSCs was decreased as compared with 3 μmol/L Oxo-M in the above 16 neurons. The slPSCs were completely abolished by 2 μmol/L strychnine. Atropine, the specific mAChR antagonist, completely blocked the effect of Oxo-M on the frequency of sIPSCs. In 9 additional lamina Ⅱ neurons, 1-10 μmol/L oxo-M failed to alter significantly the frequency and amplitude of glycinergic mIPSCs. Conclusion Activation of mAChRs in the somatodendritic site of glycinergic interneurous increases the synaptic glycine input to spinal dorsal horn neurons, but not in a dose-dependent manner.

Recent advances in pharmacotherapy of glaucoma.

Glaucoma is a slow progressive degeneration of the retinal ganglion cells (RGCs) and the optic nerve axons, leading to irreversible blindness if left undiagnosed and untreated. Although increased intraocular pressure is a major risk factor of glaucoma, other factors include increased glutamate levels, alterations in nitric oxide (NO) metabolism, vascular alterations and oxidative damage caused by reactive oxygen species. Glaucoma is the second leading cause of blindness globally, accounting for 12.3% of the total blindness. Glaucoma has been broadly classified as primary or secondary open-angle or angle-closure glaucoma. The primary goal in management of glaucoma is to prevent the risk factor, especially elevated intraocular pressure (IOP), using medications, laser therapy or conventional surgery. The first-line treatment of glaucoma usually begins with the use of a topical selective or nonselective blocker or a prostaglandin analog. Second-line drugs of choice include alpha-agonists and topical carbonic anhydrase inhibitors. Cholinergic agonists are considered third-line treatment options. When a single therapy is not sufficient to lower the IOP, a combination therapy is indicated. To enhance the patient compliance, drug delivery systems like electronic devices, ocular inserts, tansdermal and mechanical drug delivery systems have been developed. Use of viscoelastic agents in ophthalmic formulations, emulsions and soluble ophthalmic drug inserts (SODI) enhance patience compliance and ocular drug delivery in patients in long-term glaucoma therapy. For patients who do not respond to antiglaucoma medications, laser trabeculoplasty and incisional surgery are recommended. Several nutrients and botanicals hold promise for the treatment of glaucoma, but most studies are preliminary, and larger, controlled studies are required. Future directions for the development of a novel therapy glaucoma may target glutamate inhibition, NMDA receptor blockade, exogenously applied neurotrophins, open channel blockers, antioxidants, protease inhibitors and gene therapy.

Cholinergic receptor nicotinic acetyleholine receptor α7 agonist ameliorates trinitrobenzene sulfonic acid induced colitis in mice / 中华消化杂志

Objective To study the therapeutic effect of cholinergie receptor,an nicotinic acetylcholine receptor(nAChR)α7 agonist,on trinitrobeazene sulfonic acid(TNBS)-induced colitis in mice.Methods BALB/C mice were randomly divided into control group,TNBS group,anabaseine(AN)as the agonist of nAChRα7(AN group),and chlorisondamine diiodide(CHD)as the antagonist of nAChRα7(CHD group).TNBS-induced colitis was produced at day 1,either 10 μg anabaseine or 1.5 μg chlorisondamine diiodide was administrated after the induction of colitis,and repeated on interval day till the mice were sacrificed at day 8.The myeloperoxidase(MPO)activity and level of tumor necrosis factors(TNF)-α in colonic tissue were examined by histological method and enzyme-linked immunosorbent assay (ELISA),respectively.Lamina propria mononuclear cells(LPMCs)were isolated,and NF-κB activation was further detected by Western blot.Results Compared with TNBS group,the tissue damage,MPO activity and concentration of TNF-α in mice treated with anabaseine were decreased[MPO activity:(7.6±2.1)U/mg vs(12.2±2.6)U/mg,TNF-α level:(396±98)pg/g vs(627±112)Pg/g],and NF-κB activation in LPMCs was inhibited.Whereas the MPO activity[(14.1±1.8) U/mg)]and concentration of TNF-α[(692±79)pg/g)]in mice treated with chlorisondamine diiodide were increased and NF-κB activation in LPMCs were amplified. Conclusion nAChRa7 agonist can inhibit colonic inflammatory response by down-regulating the consentration of TNF-α and inhibiting NF-κB activation.