OBJECTIVE: To investigate the role of α7-nicotinic acetylcholine receptor (α7-nAChR) in the regulation of neuronal activity and expression of synapse-related proteins in the thalamic reticular nucleus (TRN) of insomnia rats treated by acupuncture. METHODS: A total of 36 male Sprague-Dawley (SD) rats of clean grade were randomly divided into a control group, a model group, an acupuncture group, and an acupuncture+antagonist group, with 9 rats in each group. The model group, the acupuncture group, and the acupuncture+antagonist group were treated with intraperitoneal injection of p-chlorophenylalanine (PCPA) to establish insomnia model. After successful modeling, the acupuncture group and the acupuncture+antagonist group received acupuncture at bilateral Neiguan (PC6) and Zusanli (ST36) once daily for 5 consecutive days. Thirty min before each acupuncture session, the acupuncture+antagonist group was intraperitoneally injected with methyllycaconitine citrate (MLA), an α7-nAChR antagonist, at a dosage of 5 mg/kg while the acupuncture group received the same volume of 0.9% sodium chloride solution. The rats' daytime spontaneous activity was observed. Neuronal discharge in the TRN was detected using neuroelectrophysiological methods. Immunofluorescence staining was used to detect parvalbumin-positive (PV⁺) neurons and co-expression of PV⁺ and postsynaptic density protein-95 (PSD-95) in the TRN. RESULTS: Compared with the control group, the model group showed increased daytime spontaneous activity (P<0.01); decreased average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); decreased neuronal discharge frequency (P<0.01), prolonged inter-discharge intervals (P<0.01) in the TRN; reduced number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.01). Compared with the model group, the acupuncture group showed decreased daytime spontaneous activity (P<0.01); increased average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); increased neuronal discharge frequency (P<0.01), shortened inter-discharge intervals (P<0.01) in the TRN; increased number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.05). Compared with the acupuncture group, the acupuncture+antagonist group exhibited increased daytime spontaneous activity (P<0.01); reduced average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); decreased neuronal discharge frequency (P<0.05), prolonged inter-discharge intervals (P<0.05) in the TRN; reduced number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.01). CONCLUSION α7-nAChR are involved in mediating the regulatory effect of acupuncture on circadian rhythm disturbances in PCPA-induced insomnia rats. Blocking α7-nAChR attenuates the activating effect of acupuncture on TRN neurons, and reduces the expression of PSD-95 protein on GABAergic neurons.
Animals ; Male ; Acupuncture Therapy ; alpha7 Nicotinic Acetylcholine Receptor/genetics* ; Rats, Sprague-Dawley ; Rats ; Sleep Initiation and Maintenance Disorders/physiopathology* ; Neurons/metabolism* ; Humans ; Thalamic Nuclei/physiopathology* ; Acupuncture Points ; Disks Large Homolog 4 Protein

The molecular mechanism of verbascoside(OC1) in promoting acetylcholine(ACh) release in the pathogenesis of Alzheimer's disease(AD) was studied. Adrenal pheochromocytoma cells(PC12) of rats induced by β-amyloid protein(1-42)(Aβ_(1-42)) were used as AD models in vitro and were divided into control group, model group(Aβ_(1-42) 10 μmol·L~(-1)), OC1 treatment group(2 and 10 μg·mL~(-1)). The effect of OC1 on phosphorylated proteins in AD models was analyzed by whole protein phosphorylation quantitative omics, and the selectivity of OC1 for calcium channel subtypes was virtually screened in combination with computer-aided drug design. The fluorescence probe Fluo-3/AM was used to detect Ca~(2+) concentration in cells. Western blot analysis was performed to detect the effects of OC1 on the expression of phosphorylated calmodulin-dependent protein kinase Ⅱ(p-CaMKⅡ, Thr286) and synaptic vesicle-related proteins, and UPLC/Q Exactive MS was used to detect the effects of OC1 on ACh release in AD models. The effects of OC1 on acetylcholine esterase(AChE) activity in AD models were detected. The results showed that the differentially modified proteins in the model group and the OC1 treatment group were related to calcium channel activation at three levels: GO classification, KEGG pathway, and protein domain. The results of molecular docking revealed the dominant role of L-type calcium channels. Fluo-3/AM fluorescence intensity decreased under the presence of Ca~(2+) chelating agent ethylene glycol tetraacetic acid(EGTA), L-type calcium channel blocker verapamil, and N-type calcium channel blocker conotoxin, and the effect of verapamil was stronger than that of conotoxin. This confirmed that OC1 promoted extracellular Ca~(2+) influx mainly through its interaction with L-type calcium channel protein. In addition, proteomic analysis and Western blot results showed that the expression of p-CaMKⅡ and downstream vesicle-related proteins was up-regulated after OC1 treatment, indicating that OC1 acted on vesicle-related proteins by activating CaMKⅡ and participated in synaptic remodeling and transmitter release, thus affecting learning and memory. OC1 also decreased the activity of AChE and prolonged the action time of ACh in synaptic gaps.
Animals ; Rats ; Glucosides/administration & dosage* ; Acetylcholine/metabolism* ; Alzheimer Disease/genetics* ; PC12 Cells ; Phenols/chemistry* ; Neurotransmitter Agents/metabolism* ; Drugs, Chinese Herbal ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics* ; Humans ; Phosphorylation/drug effects* ; Calcium/metabolism* ; Polyphenols

Based on the α7 nicotinic acetylcholine receptor(α7nAChR), this study examined how tetrahydropalmatine(THP) affected BV2 microglia exposed to lipopolysaccharide(LPS), aiming to clarify the possible mechanism underlying the anti-depression effect of THP from the perspectives of preventing inflammation and regulating polarization. First, after molecular docking and determination of the content of Corydalis saxicola Bunting total alkaloids, THP was initially identified as a possible anti-depression component. The BV2 microglia model of inflammation was established with LPS. BV2 microglia were allocated into a normal group, a model group, low-and high-dose(20 and 40 μmol·L~(-1), respectively) THP groups, and a THP(20 μmol·L~(-1))+α7nAChR-specific antagonist MLA(1 μmol·L~(-1)) group. The CCK-8 assay was used to screen the safe concentration of THP. A light microscope was used to examine the morphology of the cells. Western blot and immunofluorescence were used to determine the expression of α7nAChR. qRT-PCR was performed to determine the mRNA levels of inducible nitric oxide synthase(iNOS), cluster of differentiation 86(CD86), suppressor of cytokine signaling 3(SOCS3), arginase-1(Arg-1), cluster of differentiation 206(CD206), tumor necrosis factor(TNF)-α, interleukin(IL)-6, and IL-1β. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of TNF-α, IL-6, and IL-1β in the cell supernatant. The experimental results showed that THP at concentrations of 40 μmol·L~(-1) and below had no effect on BV2 microglia. THP improved the morphology of BV2 microglia, significantly up-regulated the protein level of α7nAChR, significantly down-regulated the mRNA levels of iNOS, CD86, SOCS3, TNF-α, IL-6, and IL-1β, significantly up-regulated the mRNA levels of Arg-1 and CD206, and dramatically lowered the levels of TNF-α, IL-6, and IL-1β in the cell supernatant. However, the antagonist MLA abolished the above-mentioned ameliorative effects of THP on LPS-treated BV2 microglia. As demonstrated by the aforementioned findings, THP protected LPS-treated BV2 microglia by regulating the M1/M2 polarization and preventing inflammation, which might be connected to the regulation of α7nAChR on BV2 microglia.
Berberine Alkaloids/chemistry* ; alpha7 Nicotinic Acetylcholine Receptor/chemistry* ; Microglia/metabolism* ; Mice ; Animals ; Cell Line ; Corydalis/chemistry* ; Humans ; Molecular Docking Simulation ; Inflammation/drug therapy* ; Nitric Oxide Synthase Type II/immunology* ; Tumor Necrosis Factor-alpha/immunology*

Sepsis is a common clinical syndrome in intensive care unit (ICU) with high morbidity and high mortality, making it a global health issue. The estimated global incidence of sepsis is 437/100 000, with an in-hospital mortality of 17%, which is higher in developing countries and underdeveloped regions. Despite some progress in sepsis treatment in recent years, the complexity of its pathophysiology limits therapeutic effectiveness. The cholinergic anti-inflammatory pathway (CAP), a neuro-immune regulatory pathway, plays a crucial role in sepsis through key components such as the vagus nerve, central M-type muscarinic receptor, the spleen and splenic sympathetic nerves, acetylcholine, and the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR). This article explores the potential mechanisms and roles of CAP in sepsis, focusing on CAP-related cell signaling pathways, including nuclear factor-κB (NF-κB) signaling pathway, Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling pathway, phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, and cyclooxygenase (COX) and prostaglandin E2 (PGE2) signaling pathways. Potential applications of CAP in sepsis treatment include stimulating the vagus nerve (e.g., through pharmacological, electrical, or acupuncture stimulation), using α7nAChR agonists (e.g., nicotine, GTS-21, and PNU-282987), adrenergic receptor agonists (e.g., dexmedetomidine and salbutamol), or other drugs and bioactive substances (e.g., buprenorphine and traditional Chinese medicine components). These approaches aim to activate CAP, suppress inflammatory responses, and improve sepsis prognosis, providing a theoretical basis for treatment and promoting the development of related drugs.
Sepsis/metabolism* ; Humans ; Signal Transduction ; alpha7 Nicotinic Acetylcholine Receptor ; NF-kappa B/metabolism* ; Anti-Inflammatory Agents ; Acetylcholine

Traumatic brain injury (TBI), as a significant central nervous system damage disease with high frequency in the world, leads to a huge number of patients with impaired health and lower quality of life every year. Lung injury is a common and dangerous consequence, which dramatically raises the mortality of patients. Discovering the pathophysiology of lung injury after TBI and discovering viable therapeutic targets has become an important need for clinical diagnosis and therapy. Neurotransmitters, as the fundamental chemical agents of the nervous system for signal transmission, not only govern neuronal activity and apoptosis in TBI but also significantly influence the pathophysiological mechanisms of lung injury subsequent to TBI. The imbalance is intricately linked to the onset and progression of lung damage. This paper systematically reviews the clinical characteristics and predominant pathogenesis of lung injury following TBI, emphasizing the role of key neurotransmitters, including glutamate (Glu), γ-aminobutyric acid (GABA), norepinephrine (NE), dopamine (DA), and acetylcholine (ACh), in lung injury post-TBI. It examines their influence on inflammatory response, vascular permeability, and pulmonary circulation function. Additionally, the paper evaluates the research advancements and potential applications of targeted therapeutic strategies for various neurotransmitter systems, such as receptor antagonists, transporter inhibitors, and neurotransmitter analogues. This research aims to offer a theoretical framework for clarifying the neural regulatory mechanisms of lung injury following TBI and to establish a basis for the development of novel therapeutic strategies and enhancement of the prognosis of the patients.
Humans ; Brain Injuries, Traumatic/metabolism* ; Neurotransmitter Agents/metabolism* ; Lung Injury/metabolism* ; gamma-Aminobutyric Acid/metabolism* ; Glutamic Acid/metabolism* ; Norepinephrine/metabolism* ; Dopamine/metabolism* ; Acetylcholine/metabolism*

OBJECTIVE: The occurrence and development of atrial fibrillation (AF) are influenced by the autonomic nervous system and inflammation. Acupuncture is an effective treatment for AF. This study explored the protective effects of acupuncture in a rat model of paroxysmal AF and investigated its mechanisms. METHODS: Male Sprague-Dawley rats (n = 130) were randomly divided into blank control (Con), sham operation (Sham), AF, and acupuncture treatment (Acu) groups. A paroxysmal AF model was established by rapid atrial pacing through the jugular vein. Rats in the Acu group were immobilized to receive acupuncture treatment at Neiguan acupoint (PC6) for 20 min daily for seven days. The other groups were immobilized for the same duration over the treatment period but did not receive acupuncture. The AF induction rate, AF duration, cardiac electrophysiological parameters, and heart rate variability were evaluated by monitoring surface electrocardiogram and vagus nerve discharge signals. After the intervention, the rats were euthanized, and atrial morphology was assessed using haematoxylin and eosin staining. The expression of macrophage F4/80 antigen (F4/80) and cluster of differentiation (CD) 86 in atrial myocardial tissue was detected using immunohistochemistry, immunofluorescence and flow cytometry. The expression levels or contents of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), α7 nicotinic acetylcholine receptor (α7nAChR), phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in atrial myocardial tissue were detected using Western blotting, reverse transcription-quantitative polymerase chain reaction, or enzyme-linked immunosorbent assay. The role of α7nAChR in acupuncture treatment was verified by intraperitoneal injection of the α7nAChR antagonist methyllycaconitine (MLA). RESULTS: Compared with the AF group, acupuncture significantly reduced AF duration and induction rate, improved cardiac electrophysiology by enhancing vagus nerve activity and regulating autonomic balance. It also decreased the pro-inflammatory M1 macrophage proportion, alleviating myocardial injury and infiltration. MLA weakened acupuncture's electrophysiological improvement and anti-inflammatory effect. Results suggest that acupuncture triggers the α7nAChR-JAK2/STAT3 pathway and exerts cardioprotection via neuroimmune regulation. CONCLUSION Acupuncture significantly reduced the AF induction rate, shortened AF duration, improved cardiac electrophysiological parameters, enhanced vagus nerve activity, and decreased the expression of pro-inflammatory M1 macrophages and inflammatory factors in rats with paroxysmal AF. Its positive effects are related to the activation of the α7nAChR-mediated JAK2/STAT3 signalling pathway, indicating that the interaction between cardiac vagus nerve and macrophages may be a potential target for acupuncture in the prevention and treatment of AF. Please cite this article as: Li ZH, Yang WM, Huang Q, Shi GX, Liu CZ, Zhang YQ. Acupuncture activates vagus nerve-macrophage axis and improves cardiac electrophysiology and inflammatory response in rats with atrial fibrillation via α7nAChR-JAK2/STAT3 pathway. J Integr Med. 2025; 23(4): 398-414.
Animals ; Male ; Rats, Sprague-Dawley ; STAT3 Transcription Factor/metabolism* ; alpha7 Nicotinic Acetylcholine Receptor/metabolism* ; Janus Kinase 2/metabolism* ; Atrial Fibrillation/metabolism* ; Vagus Nerve/physiopathology* ; Rats ; Acupuncture Therapy ; Signal Transduction ; Macrophages/metabolism* ; Inflammation/therapy*

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Neiguan" (PC 6) on myocardial fibrosis in spontaneously hypertensive rats (SHR), and explore preliminarily the mediating role of cholinergic anti-inflammatory pathway (CAP) and its downstream nuclear factor κB (NF-κB) signaling pathway. METHODS: Six 12-week-old WKY male rats were employed as the normal group. Eighteen 12-week-old SHR were randomly divided into 3 groups, i.e. a model group, an EA group and a blocking group (EA after blocking α7 nicotinic acetylcholine receptor [α7nAchR]), with 6 rats in each one. In the EA group, EA was delivered at "Neiguan"(PC 6) and the site 0.5 cm from its left side, with disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in current intensity. One intervention took 30 min and was given once every 2 days, lasting 8 weeks. In the blocking group, prior to each EA, the α7nAchR specific blocker, α-bungartoxin was injected intravenously in the tails of the rats. After EA intervention, the systolic blood pressure (SBP), the diastolic blood pressure (DBP) and the mean arterial pressure (MAP) were measured with non-invasive blood pressure monitor. Using echocardiogram, the left ventricular (LV) anterior wall end-diastolic thickness (LVAWd) , LV posterior wall end-diastolic thickness (LVPWd) and the LV end-diastolic internal diameter (LVIDd) were measured. The level of hydroxyproline (Hyp) in the myocardial tissue was determined by using alkaline hydrolysis, and that of acetylcholine (Ach) was detected by ELISA. With the real-time PCR adopted, the mRNA expression of NF-κB p65, tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 were determined. RESULTS: Compared with the normal group, SBP, DBP, MAP, LVAWd and LVPWd were increased (P<0.01), and LVIDd was decreased (P<0.01) in the rats of the model group. SBP, DBP, MAP and LVAWd were dropped (P<0.01, P<0.05), and LVIDd rose (P<0.01) in the EA group when compared with those in the model group. The differences in the above indexes were not statistically significant between the blocking group and the model group (P>0.05). Compared with the normal group, Hyp level and the mRNA expression of NF-κB p65, TNF-α, IL-1β and IL-6 in the myocardial tissue increased (P<0.01, P<0.05) and Ach level decreased (P<0.01) in the model group. Hyp level, the mRNA expression of NF-κB p65, TNF-α, IL-1β and IL-6 in the myocardial tissue were reduced (P<0.05, P<0.01) and Ach level rose (P<0.01) in the EA group when compared with those in the model group. These indexes were not different statistically between the blocking group and the model group (P>0.05). CONCLUSION CAP may be involved in ameliorating the pathological damage of myocardial fibrosis during EA at "Neiguan"(PC 6). The underlying effect mechanism is associated with up-regulating the neurotransmitter, Ach and down-regulating mRNA expression of NF-κB p65 and pro-inflammatory factors such as TNF-α, IL-1β and IL-6 in myocardial tissue.
Rats ; Male ; Animals ; Rats, Inbred SHR ; NF-kappa B/metabolism* ; Rats, Inbred WKY ; Electroacupuncture ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Neuroimmunomodulation ; alpha7 Nicotinic Acetylcholine Receptor ; Acetylcholine ; Fibrosis ; RNA, Messenger

In this study, we used a rat model of pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT) to investigate the role and mechanism of angiotensin (Ang)-(1-7) in regulating pulmonary artery diastolic function. Three weeks after subcutaneous injection of MCT or normal saline, the right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) of rats were detected using a right heart catheter. Vascular endothelium-dependent relaxation was evaluated by acetylcholine (ACh)-induced vasodilation. The relaxation function of vascular smooth muscle was evaluated by sodium nitroprusside (SNP)-induced vasodilation. Human pulmonary artery endothelial cells (HPAECs) were incubated with Ang-(1-7) to measure nitric oxide (NO) release levels. The results showed that compared with control rats, RVSP and RVHI were significantly increased in the MCT-PAH rats, and both ACh or SNP-induced vasodilation were worsened. Incubation of pulmonary artery of MCT-PAH rats with Ang-(1-7) (1 × 10^-9-1 × 10^-4 mol/L) caused significant vaso-relaxation. Pre-incubation of Ang-(1-7) in the pulmonary artery of MCT-PAH rats significantly improved ACh-induced endothelium-dependent relaxation, but had no significant effect on SNP-induced endothelium-independent relaxation. In addition, Ang-(1-7) treatment significantly increased NO levels in HPAECs. The Mas receptor antagonist A-779 inhibited the effects of Ang-(1-7) on endothelium-dependent relaxation and NO release from endothelial cells. The above results demonstrate that Ang-(1-7) promotes the release of NO from endothelial cells by activating Mas receptor, thereby improving the endothelium-dependent relaxation function of PAH pulmonary arteries.
Rats ; Humans ; Animals ; Vasodilation ; Pulmonary Arterial Hypertension ; Monocrotaline/toxicity* ; Rats, Sprague-Dawley ; Hypertension, Pulmonary/chemically induced* ; Endothelial Cells ; Pulmonary Artery ; Endothelium ; Acetylcholine/pharmacology* ; Nitroprusside/pharmacology*

Acetylcholine (ACh) is an important neuromodulator in various cognitive functions. However, it is unclear how ACh influences neural circuit dynamics by altering cellular properties. Here, we investigated how ACh influences reverberatory activity in cultured neuronal networks. We found that ACh suppressed the occurrence of evoked reverberation at low to moderate doses, but to a much lesser extent at high doses. Moreover, high doses of ACh caused a longer duration of evoked reverberation, and a higher occurrence of spontaneous activity. With whole-cell recording from single neurons, we found that ACh inhibited excitatory postsynaptic currents (EPSCs) while elevating neuronal firing in a dose-dependent manner. Furthermore, all ACh-induced cellular and network changes were blocked by muscarinic, but not nicotinic receptor antagonists. With computational modeling, we found that simulated changes in EPSCs and the excitability of single cells mimicking the effects of ACh indeed modulated the evoked network reverberation similar to experimental observations. Thus, ACh modulates network dynamics in a biphasic fashion, probably by inhibiting excitatory synaptic transmission and facilitating neuronal excitability through muscarinic signaling pathways.
Cholinergic Agents/pharmacology* ; Acetylcholine/metabolism* ; Neurons/metabolism* ; Synaptic Transmission/physiology*

OBJECTIVES: To evaluate the effect of echinacoside (ECH) on cognitive dysfunction in post cerebral stroke model rats. METHODS: The post stroke cognitive impairment rat model was created by occlusion of the transient middle cerebral artery (MCAO). The rats were randomly divided into 3 groups by a random number table: the sham group (sham operation), the MCAO group (received operation for focal cerebral ischemia), and the ECH group (received operation for focal cerebral ischemia and ECH 50 mg/kg per day), with 6 rats in each group. The infarct volume and spatial learning were evaluated by triphenyl tetrazolium chloride staining and Morris water maze. The expression of α7nAChR in the hippocampus was detected by immunohistochemistry. The contents of acetylcholine (ACh), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), activities of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and catalase (CAT) were evaluated by enzyme linked immunosorbent assay. The neural apoptosis and autophagy were determined by TUNEL staining and LC3 staining, respectively. RESULTS: ECH significantly lessened the brain infarct volume and ameliorated neurological deficit in infarct volume and water content (both P<0.01). Compared with MCAO rats, administration of ECH revealed shorter escape latency and long retention time at 7, 14 and 28 days (all P<0.01), increased the α7nAChR protein expression, ACh content, and ChAT activity, and decreased AChE activity in MCAO rats (all P<0.01). ECH significantly decreased MDA content and increased the GSH content, SOD, and CAT activities compared with MCAO rats (all P<0.05). ECH suppressed neuronal apoptosis by reducing TUNEL-positive cells and also enhanced autophagy in MCAO rats (all P<0.01). CONCLUSION ECH treatment helped improve cognitive impairment by attenuating neurological damage and enhancing autophagy in MCAO rats.
Acetylcholinesterase ; Animals ; Autophagy ; Brain Ischemia/metabolism* ; Cerebral Infarction ; Cognitive Dysfunction/drug therapy* ; Glutathione/metabolism* ; Glycosides ; Infarction, Middle Cerebral Artery/drug therapy* ; Neuroprotective Agents/therapeutic use* ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury/drug therapy* ; Stroke/drug therapy* ; Superoxide Dismutase/metabolism* ; alpha7 Nicotinic Acetylcholine Receptor