Structure-activity relationship of pyrazol-4-yl-pyridine derivatives and identification of a radiofluorinated probe for imaging the muscarinic acetylcholine receptor M4

There is an accumulating body of evidence implicating the muscarinic acetylcholine receptor 4 (M4) in schizophrenia and dementia with Lewy bodies, however, a clinically validated M4 positron emission tomography (PET) radioligand is currently lacking. As such, the aim of this study was to develop a suitable M4 PET ligand that allows the non-invasive visualization of M4 in the brain. Structure-activity relationship studies of pyrazol-4-yl-pyridine derivates led to the discovery of target compound 12 - a subtype-selective positive allosteric modulator (PAM). The radiofluorinated analogue, [18F] 12, was synthesized in 28 ± 10% radiochemical yield, >37 GBq/μmol and an excellent radiochemical purity >99%. Initial in vitro autoradiograms on rodent brain sections were performed in the absence of carbachol and showed moderate specificity as well as a low selectivity of [18F] 12 for the M4-rich striatum. However, in the presence of carbachol, a significant increase in tracer binding was observed in the rat striatum, which was reduced by >60% under blocking conditions, thus indicating that orthosteric ligand interaction is required for efficient binding of [18F] 12 to the allosteric site. Remarkably, however, the presence of carbachol was not required for high specific binding in the non-human primate (NHP) and human striatum, and did not further improve the specificity and selectivity of [18F] 12 in higher species. These results pointed towards significant species-differences and paved the way for a preliminary PET study in NHP, where peak brain uptake of [18F] 12 was found in the putamen and temporal cortex. In conclusion, we report on the identification and preclinical development of the first radiofluorinated M4 PET radioligand with promising attributes. The availability of a clinically validated M4 PET radioligand harbors potential to facilitate drug development and provide a useful diagnostic tool for non-invasive imaging.

Associations of genetic variants in M3 receptor with blood pressure responses to salt and potassium intake / 西安交通大学学报(医学版)

【Objective】 M3 muscarinic acetylcholine receptor(M3 receptor), encoded by CHRM3 gene, is widely distributed in the cardiovascular system and plays an important role in cardiac regulation. The aim of this study was to assess the association of genetic variants in M3 receptor with blood pressure(BP) responses to controlled dietary sodium and potassium interventions. 【Methods】 A total of 333 subjects from 124 families were recruited from the rural areas of northern China. After a three-day baseline observation, they were sequentially on a seven-day low-salt diet, a seven-day high-salt diet, and a seven-day high-salt diet plus potassium supplementation. Thirteen CHRM3 single nucleotide polymorphisms(SNPs) were selected for analysis. 【Results】 SNP rs10802811 of the CHRM3 was significantly associated with diastolic BP(DBP) and mean arterial pressure(MAP) responses to both low-salt and high-salt diets while SNPs rs6429147, rs373288072, rs114677844 and rs663148 showed significant associations with systolic BP(SBP) and MAP responses to high-salt diet. In addition, SNP rs6692904 was significantly associated with SBP, DBP and MAP responses to high-salt diet with potassium supplementation. 【Conclusion】 Genetic variants in M3 receptor are significantly associated with BP responses to sodium and potassium intervention, suggesting that M3 receptor may be mechanistically involved in BP salt and potassium sensitivity.

Research progress on α7 nicotinic acetylcholine receptor in lung cancer / 中国胸心血管外科临床杂志

@#Lung cancer is one of the most prevalent and deadly malignances worldwide. Cigarette smoking has been identified to be the major risk factor of lung cancer, and nicotine is one of the most harmful components in tobacco smoke. Nicotinic acetylcholine receptors (nAChRs) are universally expressed in mammalian cells, including tumor cells, and perform various critical biological functions. α7nAChR, an important member of nAChRs family, possesses a high affinity for nicotine and plays a core role in the nicotine-mediated lung cancer cell proliferation, angiogenesis, invasion and metastasis. Nowadays, lots of α7nAChR antagonists have been found to inhibit lung cancer cell proliferation, invasion and angiogenesis in vitro and in vivo, and therefore prevented disease progression. These studies indicated that α7nAChR might be a potential target in treating lung cancer. In this review, we summarized the current researches on α7nAChR in the progress of lung cancer.

Expression and function of α7 nicotinic acetylcholine receptor in thymocytes of myasthenia gravis patients / 中国胸心血管外科临床杂志

@#Objective    To investigate the expression of α7 nicotinic acetylcholine receptor (α7 nAChR) in thymocytes of patients with myasthenia gravis (MG) and its effect on cytokine secretion and T cell proliferation. Methods    Patients with MG who underwent expanded thoracoscopic thymectomy in the Comprehensive Diagnosis and Treatment Center of the Henan Provincial People’s Hospital from June 2021 to June 2022 were selected and allocated to a MG group. Patients who underwent partial thymectomy to expose the surgical field during the cardiac disease surgery from June 2021 to September 2022 in the Department of Adult Cardiac Surgery of Fuwai Huazhong Cardiovascular Hospital were selected as the control group. Thymic single cell suspensions were prepared from MG and control groups, and the expression of α7 nAChR in thymocytes of the two groups was detected by real-time polymerase chain reaction and Western blotting. Then CD3/CD28 monoclonal antibody coupled with magnetic beads was used to induce T cell activation, and the levels of cytokines interferon-gamma (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), IL-6, IL-10, IL-17, and IL-21 in thymocytes of the two groups were detected by enzyme-linked immunosorbent assay (ELISA). The activated T cells of the MG group were divided into a blank control group, an α7 nAChR antagonist group, and an α7 nAChR agonist group according to different treatment methods. After 72 hours of culture, IFN-γ, TNF-α, IL-4, IL-6, IL-10, IL-17, and IL-21 expression levels in the culture supernatant were measured by ELISA. Afterwards, CD4-PE and CD8-APC antibodies were added, and the proliferation of T cell subsets was detected by flow cytometry. Results    A total of 10 MG patients were collected, including 3 males and 7 females with an average age of 19.25±6.28 years; and 15 control patients were collected, including 6 males and 9 females with an average age of 26.18±6.77 years. Compared with the control group, the mRNA and protein levels of α7 nAChR in the thymocytes of MG group were decreased, and the expression levels of IFN-γ, TNF-α, IL-4, IL-6 and IL-21 in the supernatant were increased (P<0.05), but there was no statistical difference in the expression of IL-10 and IL-17 (P>0.05). The cell-culture experiment showed that compared with the blank control group, the levels of IFN-γ, TNF-α, IL-6 and IL-21 secreted by T cells in the α7 nAChR antagonist group were increased (P<0.05), while they were decreased in the α7 nAChR agonist group (P<0.05). There was no statistical difference in the secretion levels of IL-4, IL-10 or IL-17 among the three groups (P>0.05). CD4+ T and CD8+ T cells in the α7 nAChR agonist group were significantly less than those in the blank control group and α7 nAChR antagonist group (P<0.001), while they were significantly more in the α7 nAChR antagonist group than those in the blank control group (P<0.001). Conclusion    The expression of α7 nAChR in thymocytes of MG patients is decreased, and α7 nAChR may be involved in the inflammatory response in thymocytes and thus in thymic function.

Role of α7nAChR in penehyclidine hydrochloride-induced reduction of endotoxin-induced acute lung injury in mice / 中华麻醉学杂志

Objective:To evaluate the role of α7 nicotinic acetylcholine receptor (α7nAChR) in penehyclidine hydrochloride-induced reduction of endotoxin-induced acute lung injury (ALI) in mice.Methods:Forty SPF healthy male C57BL/6 mice, aged 6-8 weeks, weighing 18-25 g, were divided into 4 groups ( n=10 each) using a random number table method: control group (group C), ALI group, penehyclidine hydrochloride group (PHC group), and α7nAChR inhibitor MLA group (MLA group). ALI was induced by intraperitoneal injection of lipopolysaccharide 15 mg/kg in anesthetized animals, while normal saline was given instead in group C. In PHC group, penehyclidine hydrochloride 2 mg/kg was intraperitoneally injected at 30 min before developing the model. MLA 10 mg/kg was intraperitoneally injected at 10 min before administration of penehyclidine hydrochloride in MLA group. Mice were sacrificed at 6 h after lipopolysaccharide administration, and lung tissues were collected for microscopic examination of the pathological changes (by HE staining) and for determination of the wet/dry weight ratio (W/D ratio), content of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-10 (by enzyme-linked immunosorbent assay) and expression of α7nAChR (by Western blot). Results:Compared with C group, the W/D ratio and contents of TNF-α and IL-1β were significantly increased, the content of IL-10 was decreased, and the expression of α7nAChR was up-regulated in ALI, PHC and MLA groups ( P<0.05). Compared with ALI group, the W/D ratio and contents of TNF-α and IL-1β were significantly decreased, the content of IL-10 was increased, and the expression of α7nAChR was up-regulated in PHC group ( P<0.05). Compared with PHC group, the W/D ratio and contents of TNF-α and IL-1β were significantly increased, the content of IL-10 was decreased, and the expression of α7nAChR was down-regulated in MLA group ( P<0.05). Compared with ALI group, the pathological changes of lung tissues were significantly mitigated in PHC group, while this effect of PHC was partially reversed by α7nAChR inhibitor MLA. Conclusions:α7nAChR is involved in penehyclidine hydrochloride-induced reduction of endotoxin-induced ALI in mice.

Diagnostic accuracy and clinical utility of bed side tests versus laboratory tests in the diagnosis of ocular myasthenia

Purpose: To assess the diagnostic accuracy of forced eyelid closure test (FECT), ice pack test (IPT), repetitive nerve stimulation test (RNS), and acetylcholine receptor (AchR) antibody test in patients with suspected ocular myasthenia. To assess the clinical utility of AchR antibody test in predicting disease progression. Methods: Retrospective cohort study of patients diagnosed with ocular myasthenia at a South?Indian neuro?ophthalmology tertiary eye clinic. Baseline characteristics; ocular myasthenia symptoms; results of FECT, IPT, RNS, and AchR antibody test; and progression time to generalized myasthenia (GM) over 36 months from the time of diagnosis were recorded and analyzed using receiver operator curve analysis, multiple logistic regression, and Kaplan朚eier survival analysis. Results: FECT had a sensitivity of 96.7% (95% CI: 88.5�.6) and a specificity of 75% (95% CI: 34.9�.8). Combination of FECT and IPT, using the positivity of at least one test, increased the sensitivity to 98.3% (95% CI: 91�0), reducing the specificity to 50% (95% CI: 15.7�.3), whereas using the positivity of both tests, we obtained a sensitivity of 71.7% (95% CI: 58.6�.5) and a specificity of 100% (95% CI: 63.1�0). In the subset of patients with double negative RNS and AchR antibodies, the positive predictive value of combined FECT and IPT (double positive) was 100%. Patients who developed GM were more likely to have a positive AchR antibody test result (P = 0.001). Conclusion: Combined FECT and IPT (double positive) has high diagnostic accuracy even among patients with normal RNS and negative AchR antibodies. Despite low sensitivity, AchR?antibody test has a significant predictive value in disease progression.

Acupuncture combined with western medication for ocular myasthenia gravis: a randomized controlled trial / 中国针灸

OBJECTIVE@#To compare the clinical efficacy between acupuncture combined with western medication and simple western medication for ocular myasthenia gravis (OMG), and to explore its possible mechanism.@*METHODS@#A total of 60 patients of ocular myasthenia gravis were randomized into an acupuncture combined with western medication group (30 cases, 1 case dropped off) and a western medication group (30 cases, 2 cases dropped off). Oral pyridostigmine bromide tablet and prednisone acetate tablet were given in the western medication group. On the basis of the treatment in the western medication group, Tongdu Tiaoqi acupuncture (acupuncture for unblocking the governor vessel and regulating qi ) was applied at Baihui (GV 20), Fengfu (GV 16), Hegu (LI 4), Zusanli (ST 36), etc. in the acupuncture combined with western medication group, once a day, 6 days a week. The treatment was given 8 weeks in both groups. Before and after treatment, the OMG clinical absolute score was observed, electrophysiological indexes of orbicularis oculi (value of mean jitter, percentage of jitter >55 μs and percentage of blocks) were measured by single-fiber electromyography (SFEMG), serum levels of acetylcholine receptor antibody (AChR-Ab), interferon-gamma (IFN-γ) and interleukin-4 (IL-4) were detected by ELISA method.@*RESULTS@#After treatment, the OMG clinical absolute scores, values of mean jitter, percentages of jitter >55 μs, percentages of blocks and serum levels of AChR-Ab, IFN-γ and IL-4 were decreased compared before treatment in both groups (P<0.05), and those in the acupuncture combined with western medication group were lower than the western medication group (P<0.05).@*CONCLUSION@#Acupuncture combined with western medication can effectively improve ptosis, palpebra superior fatigability, eye movement disorder and neuromuscular junction dysfunction in patients with ocular myasthenia gravis, the therapeutic effect is superior to simple western medication. Its mechanism may be related to down-regulating serum levels of AChR-Ab, IFN-γ and IL-4 and promoting the recovery of orbicularis oculi function.

Effect of Huanglian Jiedutang on Learning and Memory Ability and Cholinergic System in AD Rats Induced by Aβ1-42 / 中国实验方剂学杂志

Objective:To investigate the effects of Huanglian Jiedutang on learning and memory ability and the cholinergic system in Alzheimer's disease（AD） rats induced by amyloid <italic>β</italic>-protein（A<italic>β</italic>）_1-42. Method:Sixty male SD rats were divided into normal group， model group， huperzine A group （2.1×10^-5 g·kg^-1）， high-， medium- and low dose of Huanglian Jiedutang groups （6，3，1.5 g·kg^-1）. AD rat model was replicated by hippocampal injection of A<italic>β</italic>_1-42. After 4 weeks of treatment， Morris water maze test was performed. Hematoxylineosin （HE） staining was used to observe the pathological changes of rat hippocampus. Sampling blood from abdominal aorta was taken. Acetylcholine （ACh）， acetylcholinesterase （AchE） and choline acetyltransferase （ChAT） in serum and hippocampus were detected by enzyme-linked immunosorbent assay （ELISA）. The expression of hippocampal <italic>α</italic>7 nicotinic acetylcholine receptor （<italic>α</italic>7nAChR） protein was detected by Western blot. The expression of hippocampal <italic>α</italic>7nAChR mRNA was detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Result:Compared with the normal group， there were obvious pathological changes in the model group，such as neuron necrosis in the cerebral cortex，pyramidal cell or granular cell necrosis in the hippocampus，disorder of arrangement and inflammatory cell infiltration，prolonged escape latency，decreased escape platform times，decreased residence time in the effective area and swimming path in the effective area （<italic>P<</italic>0.05，<italic>P<</italic>0.01）. The contents of <italic>α</italic>7nAChR mRNA，ACh，AchE，ChAT，<italic>α</italic>7nAChR in the hippocampus decreased （<italic>P<</italic>0.01）. Compared with the model group，the escape latency of the middle dose group was shorter （<italic>P<</italic>0.05）， the escape platform times，the swimming path in the effective area and the residence time in the effective area increased （<italic>P<</italic>0.05，<italic>P<</italic>0.01）， the contents of serum ACh，ChAT， hippocampal AchE，ChAT and <italic>α</italic>7nAChR increased （<italic>P<</italic>0.05，）. The expression of hippocampal <italic>α</italic>7nAChR protein significantly increased （<italic>P<</italic>0.01）， the residence time of effective area in high dose group was prolonged （<italic>P<</italic>0.01）， the times of escape platform increased，and the contents of serum ACh，ChAT and hippocampal ACh，AchE，<italic>α</italic>7nAChR protein and <italic>α</italic>7nAChR mRNA increased （<italic>P<</italic>0.05）. Conclusion:Huanglian Jiedutang can significantly improve the learning and memory ability of AD rats induced by A<italic>β</italic>_1-42，and its mechanism may be related to the improvement of cholinergic system damage and enhancement of cholinergic system function induced by A<italic>β</italic>_1-42.

Toxicity and Efficacy of Water-eluted Fraction from Siegesbeckiae Herba Against Human Embryonic Lung Fibroblasts / 中国实验方剂学杂志

Objective:To detect the toxicity of water-eluted fraction from Siegesbeckiae Herba （SWEF） at different concentrations against MRC-5 human embryonic lung fibroblasts and its impacts on the expression of <italic>α</italic>7 nicotinic acetylcholine receptor （<italic>α</italic>7nAChR） and inflammatory factors， so as to figure out the active components responsible for toxicity and efficacy. Method:The toxicities of SWEF at 1， 6， 10， 20， and 50 g·L^-1 against MRC-5 cells were determined by cell counting kit-8 （CCK-8） assay combined with flow cytometry and Trypan blue staining. The changes in <italic>α</italic>7nAChR expression and inflammatory factor levels before and after <italic>α</italic>7nAChR gene silencing were detected to reveal the pharmacodynamic effect of SWEF on MRC-5 cells. Result:SWEF （≥6 g·L^-1） obviously inhibited the viability of MRC-5 cells （<italic>P</italic><0.01） and promoted their apoptosis and necrosis （<italic>P</italic><0.01）， with the half-maximal inhibitory concentration （IC₅₀） being 6.03 g·L^-1. The determination of <italic>α</italic>7nAChR expression and inflammatory factor levels in MRC-5 cells showed that SWEF contained <italic>α</italic>7nAChR agonist-like substance， which enhanced <italic>α</italic>7nAChR mRNA and protein expression （<italic>P</italic><0.05， <italic>P</italic><0.01） and decreased the inflammatory factor levels （<italic>P</italic><0.05， <italic>P</italic><0.01）. SWEF down-regulated the inflammatory factors possibly by re-regulating <italic>α</italic>7nAChR mRNA expression， exhibiting a negative correlation between them （<italic>P</italic><0.01）. Conclusion:SWEF （≥6 g·L^-1） is highly toxic to MRC-5 cells. Pharmacodynamic studies have confirmed that <italic>α</italic>7nAChR agonist-like substance contained in SWEF was responsible for the elevated <italic>α</italic>7nAChR expression and reduced inflammatory cytokines. It is inferred that excessive <italic>α</italic>7nAChR agonist-like substance may trigger the toxicity of<italic> </italic>Siegesbeckiae Herba.

Left-sided vagus nerve stimulation improves cardiopulmonary resuscitation outcomes in rats as effectively as right-sided vagus nerve stimulation / 世界急诊医学杂志（英文）

@#BACKGROUND: Our group previously reported that right-sided vagus nerve stimulation (RVNS) significantly improved outcomes after cardiopulmonary resuscitation (CPR) in a rat model of cardiac arrest (CA). However, whether left-sided vagus nerve stimulation (LVNS) could achieve the same effect as RVNS in CPR outcomes remains unknown. METHODS: A rat model of CA was established using modified percutaneous epicardial electrical stimulation to induce ventricular fibrillation (VF). Rats were treated with LVNS or RVNS for 30 minutes before the induction of VF. All animals were observed closely within 72 hours after return of spontaneous circulation (ROSC), and their health and behavior were evaluated every 24 hours. RESULTS: Compared with those in the RVNS group, the hemodynamic measurements in the LVNS group decreased more notably. Vagus nerve stimulation (VNS) decreased the serum levels of tumor necrosis factor-alpha (TNF-α) and the arrhythmia score, and attenuated inflammatory infiltration in myocardial tissue after ROSC, regardless of the side of stimulation, compared with findings in the CPR group. Both LVNS and RVNS ameliorated myocardial function and increased the expression of α-7 nicotinic acetylcholine receptor in the myocardium after ROSC. Moreover, a clear improvement in 72-hour survival was shown with VNS pre-treatment, with no significant difference in efficacy when comparing the laterality of stimulation. CONCLUSIONS: LVNS may have similar effects as RVNS on improving outcomes after CPR.

Design, synthesis, and activity study of α-conotoxin A10LPnIA fluorescent probe / 药学学报

italic>α7 nicotinic acetylcholine receptor (nAChR) is widely distributed in the central and peripheral nervous systems, and is closely related to a variety of neurological diseases and inflammation response. α-Conotoxin [A10L]PnIA, as an antagonist targeting α7 nAChR, plays an important role in studying the physiological and pathological processes involved in α7 nAChR. [A10L]PnIA was labeled with fluorescein 5-carboxytetramethylrhodamine, and the active peptide ([A10L]PnIA-F) was obtained by a two-step oxidative folding procedure in vitro. The Xenopus oocyte expression system and the two-electrode voltage clamp technique were used to identify the potency of [A10L]PnIA-F fluorescent peptide, and its cytotoxicity was detected by mouse macrophages and CCK8 method. The molecular weight of [A10L]PnIA-F fluorescent peptide was identified by mass spectrometry as 2 077.28 Da, which was consistent with the theoretical value. Electrophysiological determination of its half-maximal inhibitory concentration (IC50) for α7 nAChR is 17.32 nmol·L-1, which is consistent with [A10L]PnIA (IC50, 13.84 nmol·L-1). The cytotoxicity test results showed that within the concentration range of 5 nmol·L-1 to 10 μmol·L-1, there was no significant inhibition on the growth of mouse macrophages. The results showed that the α-conotoxin fluorescent probe [A10L]PnIA could provide pharmacological tools for the research of α7 nAChR-related neurophysiological and pathological mechanisms.

The anti-inflammatory role of α7 nicotinic acetylcholine receptor in microglial cells and its mechanisms / 药学实践杂志

Objective To investigate the anti-inflammatory role of α7 nicotinic acetylcholine receptor (α7nAChR) under inflammatory stress and its mechanisms. Methods PNU282987 was used for the activation of α7nAChR and LPS was administrated as inflammatory stressor. Realtime PCR was used for the detection of IL-1β, IL-6, TNF-α, M1 macrophage marker CD68, CD86 and M2 macrophage marker CD206, Arg1. Cell immunofluorescence was used for the detection of M1/M2 ratio and Western blot was applied for the detection of autophagy-related proteins. Results Under the stimulation of LPS, the mRNA levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α, the proportion of M1 macrophage and autophagy process were increased in BV2 microglial cells. However, the administration of PNU282987 significantly decreased the mRNA levels of IL-1β, IL-6 and TNF-α and the proportion of M1 macrophage while increased the proportion of M2 macrophage and the level of autophagy process. Conclusion Activating α7nAChR plays an anti-inflammatory role in microglial cells under inflammatory stress due to the regulation of M1/M2 macrophage ratio and increase of autophagy level.

Effects of electrical stimulation of vagus nerve on α7 nicotinic acetylcholine receptor and inflammatory factors in ischemic stroke model mice / 中华行为医学与脑科学杂志

Objective:To explore the effect of α7 nicotinic acetylcholine receptor (α7nAchR) and changes of inflammatory factors during vagus nerve electrical stimulation in ischemic stroke model mice.Methods:Ninety SPF grade mice were randomly divided into sham operation+ vagus nerve electrical stimulation group (sham+ VNS group), model group (permanent middle cerebral artery occlusion group, pMCAO group), model+ vagus nerve electrical stimulation (pMCAO+ VNS group) and model group+ α7nAchR agonist group (pMCAO+ P group), model + vagus nerve electrical stimulation+ α7nAchR antagonist (pMCAO+ VNS+ A group), model+ vagus nerve electrical stimulation+ α7nAchR antagonist placebo group (pMCAO+ VNS+ AC group), with 15 mice in each group. The changes of vital signs of mice in each group were monitored during modeling.At 7, 14 and 21 days after successful modeling, neurological severity score (NSS) and adhesive removal test were used to evaluate the neurological deficit of mice.Immunofluorescence and Western blot were used to detect the expression of α7nAchR and its neural cell localization. ELISA was used to detect the level of TNF-α and IL-6. Prism 8.0 software was used for statistical analysis, and one-way ANOVA was used to analyze the physiological parameters during modeling.Repeated measurement ANOVA was used to compare the neurobehavioral score results, and t-test was used to compare the protein level and fluorescence intensity. Results:(1)The results of repeated measurement ANOVA showed that the interaction between group and time in NSS score was not significant ( F=0.91, P>0.05), and the group main effect ( F=46.68, P<0.05) and time main effect ( F=25.56, P<0.05) were significant. The results of Tukey's test showed that the NSS scores of pMCAO+ VNS group and pMCAO+ P group were significantly lower than those of pMCAO group (both P<0.05). There was no significant difference between pMCAO+ P group and pMCAO+ VNS group ( P>0.05). The NSS scores in pMCAO+ VNS+ A group were higher than that in pMCAO+ VNS group ( P<0.05), and significantly lower than that in pMCAO+ VNS+ AC group ( P<0.05). In the adhesive removal test, the interaction between group and time in the adhesive tape contact time and tear off time of mice was not significant ( F=0.67, 0.71, all P>0.05), and the group main effect ( F=30.12, 42.46, all P<0.05) and time main effect ( F=52.18, 47.34, all P<0.05) of mice in each group were significant.Tukey's test showed that the adhesive removal test on the 21st day, the adhesive tape contact time and tear off time of pMCAO+ VNS group were significantly shorter than those of pMCAO group (both P<0.05), and there was no significant difference between pMCAO+ P group and pMCAO+ VNS group (both P>0.05). (2)Western blot showed that compared with pMCAO group (0.36±0.01), the expression of α7nAchR in pMCAO+ VNS group (0.83±0.03) and pMCAO+ P group (0.67±0.02) increased ( t=13.53, 16.08, both P<0.01). The expression of α7nAchR in PCAO+ VNS+ A group (0.37±0.01) was significantly lower than that in PCAO+ VNS group ( t=12.88, P<0.01). Immunofluorescence results also showed that compared with pMCAO group (3.75±0.19), the expression of α7nAchR in pMCAO+ VNS group (8.96±0.48) and pMCAO+ P group increased (8.17±0.64) ( t=10.04, 6.67, both P<0.05). Immunofluorescence results also showed that compared with the pMCAO group (3.75±0.19), the expression of α7nAchR protein in the brain tissue of mice in the pMCAO+ VNS group and pMCAO+ P group increased ((8.96±0.48), (8.17±0.64), t=10.04, 6.67, all P<0.05). (3)The results of ELISA showed that compared with pMCAO group, the levels of TNF-a and IL-6 in serum and tissue supernatant of pMCAO+ VNS group and pMCAO+ P group were significantly lower than those of pMCAO group ( t=23.28, 15.30, 12.26, 11.08; all P<0.05). TNF in serum and tissue supernatant of mice in pMCAO+ VNS+ A group The levels of IL-6 in serum and tissue supernatant were significantly higher than those in pMCAO+ VNS group ( t=12.70, 11.01, 11.69, 17.37; all P<0.05) and pMCAO+ VNS+ AC group ( t=12.29, 11.07, 14.61, 29.27; all P<0.05). Conclusion:VNS may reduce inflammation by increasing the expression of α7nAchR protein in brain tissue, thereby playing a certain neuroprotective effect on ischemic stroke.

Preliminary study on the efficacy of ultrasound therapy in the rat model of sepsis / 中华危重病急救医学

Objective:To investigate the possible mechanism of ultrasound therapy in the rat model of sepsis.Methods:Seventy-eight male Sprague-Dawley (SD) rats were randomly divided into Sham group ( n = 12), septic model group ( n = 22), ultrasound treatment group ( n = 22), methyllycaconitine citrate (MLA) combined with ultrasound treatment group ( n = 22). In the Sham group, only the abdomen was opened, the cecum was found to be free, without cecal ligation and puncture (CLP). In the septic model group, CLP was used to replicate the septic rat model. After operation, each group of rats were subcutaneously injected with preheated 37 ℃ normal saline. The rats in the ultrasound treatment group were treated with ultrasound [Philips IU22 L9-3 ultrasound instrument and 9 MHz probe were used to break the sequence in the spleen area once every 6 seconds, with 1 second for each time, the mechanical index (MI) was 0.72, and the treatment time was 10 minutes]. In the MLA combined with ultrasound treatment group, α7 nicotinic acetylcholine receptor (α7nAChR) specific blocker MLA 4 mg/kg was injected intraperitoneally 30 minutes before operation, and ultrasound treatment was performed 2 hours after operation. The levels of tumor necrosis factor-α (TNF-α) and interleukin (IL-1β, IL-6) in serum of each group were measured by enzyme-linked immunosorbent assay (ELISA) at 24 hours after operation. The 10-day survival rate of each group was recorded, and the symptoms of each group were evaluated by clinical disease score (CDS). The histopathological changes of lung and colon were observed under light microscope. Results:Compared with the Sham group, the 10-day survival rate of rats in the septic model group was decreased significantly [40% (4/10) vs. 100% (6/6)], the CDS was (10.73±2.19 vs. 6.17±0.58) and the levels of TNF-α, IL-6, and IL-1β were increased significantly at 24 hours after operation [TNF-α (ng/L): 42.00±8.92 vs. 13.16±3.19, IL-6 (ng/L): 129.37±25.04 vs. 63.99±12.92, IL-1β(ng/L): 254.98±67.27 vs. 76.83±25.39, all P < 0.01]. Compared with the septic model group, the survival rate in the ultrasound treatment group was improved [70% (7/10) vs. 40% (4/10)], but there was no significant difference ( P > 0.05). The CDS (7.64±2.68 vs. 10.73±2.19) and the expressions of TNF-α, IL-6, and IL-1β were significantly reduced at 24 hours after operation [TNF-α(ng/L): 16.93±6.02 vs. 42.00±8.92, IL-6 (ng/L): 73.65±24.38 vs. 129.37±25.04, IL-1β(ng/L): 111.86±14.08 vs. 254.98±67.27, all P < 0.01]. Compared with the ultrasound treatment group, the survival rate in the MLA combined with ultrasound treatment group was reduced [60% (6/10) vs. 70% (7/10)], but the difference was not statistically significant ( P > 0.05). CDS was significantly increased (9.55±2.72 vs. 7.64±2.68), and the levels of TNF-α, IL-6 and IL-1β were significantly increased at 24 hours after operation [TNF-α(ng/L): 34.61±7.89 vs. 16.93±6.02, IL-6 (ng/L): 112.92±10.42 vs. 73.65±24.38, IL-1β(ng/L): 212.57±32.16 vs. 111.86±14.08, all P < 0.01]. Microscopically, in the septic model group, the alveolar septum was thickened, a large number of inflammatory cells infiltrated, normal pulmonary reticular structure disappeared, and pulmonary interstitium showed obvious hemorrhage and edema, meanwhile, the structure of colonic villi was obviously abnormal, with cells were edema and inflammatory cell infiltration, and the arrangement was disordered, so that the subepithelial space and the top of it fell off. After ultrasound treatment, the thickness of the alveolar interval in rats was similar to that in Sham group, without obvious inflammatory cell infiltration, and the pulmonary reticular structure was relatively intact. At the same time, the morphology of colonic villi was basically normal and orderly, the edema of cell was not obvious, and subcutaneous space and tip fall off were not obvious. After being antagonized by MLA, the rat lung tissue showed thickened alveolar septum, inflammatory cell infiltration, incomplete pulmonary network structure, hemorrhage and edema in the interstitium. The villi structure of the colon was faintly visible, with obvious cell edema and inflammatory cell infiltration, and the arrangement was abnormal. Conclusion:Ultrasound treatment improves the prognosis of septic rats, MLA can reverse the anti-inflammatory effect of ultrasound therapy by antagonizing α7nAChR, suggesting that the protective mechanism of ultrasound in sepsis may be related to activating the cholinergic anti-inflammatory pathway mediated by α7nAChR.

α4β2-Nicotine acetylcholine receptor: advances in relevant diseases and drugs / 药学学报

The α4β2-nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that is distributed throughout the nervous system. It is involved in the regulation of various neurotransmitters including acetylcholine, dopamine, γ-aminobutyric acid, and norepinephrine. α4β2-nAChR plays an important role in learning, memory, cognition, attention, inflammation, and pain. A large number of studies have shown that α4β2-nAChR is an important therapeutic target for neurological diseases such as Alzheimer's disease, Parkinson's disease, epilepsy, depression, nicotine dependence, pain, etc. It is an important target in the early diagnosis and curative effect detection of neurodegenerative diseases including Alzheimer's disease. This review summarizes the role, mechanisms and related drug research advances on α4β2-nAChR ligand drugs in neurological diseases, as well as providing a theoretical basis for identifying and developing more suitable α4β2-nAChR-related compounds.

Establishment of a gp120 transgenic mouse model with 7 nAChR knockout / 南方医科大学学报

OBJECTIVE@#To construct a HIV-1 gp120 transgenic mouse model (gp120) with 7 nicotinic acetylcholine receptor (7nAChR) gene knockout.@*METHODS@#The 7nAChR gene knockout mice (7R) were crossed with HIV-1gp120 transgenic mice (gp120) to generate F1 generation mice. We selected the F1 mice with the genotype of 7R/gp120 to mate to obtain the F2 mice. The genotypes of the F3 mice were identified by PCR, and the protein expressions in the double transgenic animal model was analyzed by immunohistochemistry. BV2 cells were treated with gp120 protein and 7nAChR inhibitor, and the expressions of IL-1β and TNF- were detected using ELISA.@*RESULTS@#The results of PCR showed the bands of the expected size in F3 mice. Two F3 mice with successful double gene editing (7R/gp120) were obtained, and immunohistochemistry showed that the brain tissue of the mice did not express 7 nAChR but with high gp120 protein expression. In the cell experiment, treatment with gp120 promoted the secretion of IL-1β and TNF- in BV2 cells, while inhibition of 7nAChR significantly decreased the expression of IL-1β and TNF- ( < 0.001).@*CONCLUSIONS@#By mating gp120 Tg mice with 7R mice, we obtained gp120 transgenic mice with 7nAChR gene deletion, which serve as a new animal model for exploring the role of 7nAChR in gp120-induced neurotoxicity.

Enhancement of T Follicular Helper Cell-Mediated Humoral Immunity Reponses During Development of Experimental Autoimmune Myasthenia Gravis / 神经科学通报·英文版

Myasthenia gravis (MG) is a prototypical antibody-mediated neurological autoimmune disease with the involvement of humoral immune responses in its pathogenesis. T follicular helper (Tfh) cells have been implicated in many autoimmune diseases. However, whether and how Tfh cells are involved in MG remain unclear. Here, we established and studied a widely-used and approved animal model of human MG, the rat model with acetylcholine receptor alpha (AChRα) subunit (R-AChR)-induced experimental autoimmune myasthenia gravis (EAMG). This model presented mild body-weight loss 10 days after the first immunization (representing the early stage of disease) and more obvious clinical manifestations and body-weight loss 7 days after the second immunization (representing the late stage of disease). AChR-specific pre-Tfh cells and mature Tfh cells were detected in these two stages, respectively. In co-cultures of Tfh cells and B cells, the number of IgG2b-secreting B cells and the level of anti-AChR antibodies in the supernatant were higher in the cultures containing EAMG-derived Tfh cells. In immunohistochemistry and immunofluorescence assays, a substantial number of CD4/Bcl-6 T cells and a greater number of larger germinal centers were observed in lymph node tissues resected from EAMG rats. Based on these results, we hypothesize that an AChR-specific Tfh cell-mediated humoral immune response contributes to the development of EAMG.

Fecal microbiota transplantation regulates the cholinergic anti-inflammatory pathway in cerebral cortex of septic rats through intestinal microbiota / 中华危重病急救医学

Objective@#To investigate the effects of fecal microbiota transplantation on septic gut flora and the cortex cholinergic anti-inflammatory pathway in rats.@*Methods@#Sixty clean grade male Sprague-Dawley (SD) rats were divided into normal saline (NS) control group, sepsis model group and fecal microbiota transplantation group by random number table, with 20 rats in each group. The rat model of sepsis was reproduced by injection of 10 mg/kg lipopolysaccharide (LPS) via tail vein, the rats in the NS control group was given the same amount of NS. The rats in the fecal microbiota transplantation group received nasogastric infusion of feces from healthy donor on the 1st day, 2 mL each time, for 3 times a day, the other two groups were given equal dose of NS by gavage. Fecal samples were collected on the 7th day after modeling, the levels of intestinal microbiota composition was determined using the 16SrDNA gene sequencing technology. The brain function was evaluated by electroencephalogram (EEG), and the proportion of each waveform in EEG was calculated. After sacrifice of rats, the brain tissues were harvested, the levels of protein expression of α7 nicotinic acetylcholine receptor (α7nAChR) were determined by Western Blot, and positive cells of Iba-1 in brain tissue were detected by immunohistochemistry method. The levels of interleukins (IL-6 and IL-1β) and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA).@*Results@#Seven days after the reproduction of the model, all rats in the NS control group survived, while 10 rats and 8 rats died in the sepsis model group and fecal microbiota transplantation group, respectively, with mortality rates of 50% and 40% respectively. Finally, there were 20 rats in the NS control group, 10 in the sepsis model group and 12 in the fecal microbiota transplantation group. Compared with the NS control group, the diversity and composition of intestinal flora were changed, the incidence of abnormal EEG increased significantly, the expression of α7nAchR in the cortex decreased significantly, and the levels of Iba-1, TNF-α, IL-6 and IL-1β were significantly increased in the model group, suggested that the intestinal flora was dysbiosis, and severe inflammatory reaction occurred in the cerebral cortex, and brain function was impaired. Compared with the model group, the diversity of intestinal flora in the fecal microbiota transplantation group was significantly increased (species index: 510.24±58.76 vs. 282.50±47.42, Chao1 index: 852.75±25.24 vs. 705.50±46.50, both P < 0.05), the dysbiosis of intestinal flora at phylum, family, genus level induced by LPS were also significantly reversed, and with the improvement of intestinal flora, the incidence of abnormal EEG waveforms was lower in the fecal microbiota transplantation group compared with that in the model group [25.0% (3/12) vs. 80.0% (8/10), P < 0.05], and the expression of α7nAChR protein in the cerebral cortex was significantly increased (α7nAChR/β-actin: 1.56±0.05 vs. 0.82±0.07, P < 0.05), immunohistochemistry analysis showed that Iba-1 positive expression of microglia decreased significantly, and cerebral cortex TNF-α, IL-6, IL-1β levels were significantly decreased [TNF-α (ng/L): 6.28±0.61 vs. 12.02±0.54, IL-6 (ng/L): 28.26±3.15 vs. 60.58±4.62, IL-1β (ng/L): 33.63±3.48 vs. 72.56±2.25, all P < 0.05].@*Conclusion@#The results reveal that fecal microbiota transplantation has remarkably modulated the dysbiosis of intestinal microbiota and activated cholinergic anti-inflammatory pathway, and ameliorate the brain dysfunction in septic rats.

Fecal microbiota transplantation regulates the cholinergic anti-inflammatory pathway in cerebral cortex of septic rats through intestinal microbiota / 中华危重病急救医学

Objective To investigate the effects of fecal microbiota transplantation on septic gut flora and the cortex cholinergic anti-inflammatory pathway in rats. Methods Sixty clean grade male Sprague-Dawley (SD) rats were divided into normal saline (NS) control group, sepsis model group and fecal microbiota transplantation group by random number table, with 20 rats in each group. The rat model of sepsis was reproduced by injection of 10 mg/kg lipopolysaccharide (LPS) via tail vein, the rats in the NS control group was given the same amount of NS. The rats in the fecal microbiota transplantation group received nasogastric infusion of feces from healthy donor on the 1st day, 2 mL each time, for 3 times a day, the other two groups were given equal dose of NS by gavage. Fecal samples were collected on the 7th day after modeling, the levels of intestinal microbiota composition was determined using the 16SrDNA gene sequencing technology. The brain function was evaluated by electroencephalogram (EEG), and the proportion of each waveform in EEG was calculated. After sacrifice of rats, the brain tissues were harvested, the levels of protein expression of α7 nicotinic acetylcholine receptor (α7nAChR) were determined by Western Blot, and positive cells of Iba-1 in brain tissue were detected by immunohistochemistry method. The levels of interleukins (IL-6 and IL-1β) and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). Results Seven days after the reproduction of the model, all rats in the NS control group survived, while 10 rats and 8 rats died in the sepsis model group and fecal microbiota transplantation group, respectively, with mortality rates of 50% and 40% respectively. Finally, there were 20 rats in the NS control group, 10 in the sepsis model group and 12 in the fecal microbiota transplantation group. Compared with the NS control group, the diversity and composition of intestinal flora were changed, the incidence of abnormal EEG increased significantly, the expression of α7nAchR in the cortex decreased significantly, and the levels of Iba-1, TNF-α, IL-6 and IL-1β were significantly increased in the model group, suggested that the intestinal flora was dysbiosis, and severe inflammatory reaction occurred in the cerebral cortex, and brain function was impaired. Compared with the model group, the diversity of intestinal flora in the fecal microbiota transplantation group was significantly increased (species index: 510.24±58.76 vs. 282.50±47.42, Chao1 index: 852.75±25.24 vs. 705.50±46.50, both P < 0.05), the dysbiosis of intestinal flora at phylum, family, genus level induced by LPS were also significantly reversed, and with the improvement of intestinal flora, the incidence of abnormal EEG waveforms was lower in the fecal microbiota transplantation group compared with that in the model group [25.0% (3/12) vs. 80.0% (8/10), P < 0.05], and the expression of α7nAChR protein in the cerebral cortex was significantly increased (α7nAChR/β-actin: 1.56±0.05 vs. 0.82±0.07, P < 0.05), immunohistochemistry analysis showed that Iba-1 positive expression of microglia decreased significantly, and cerebral cortex TNF-α, IL-6, IL-1β levels were significantly decreased [TNF-α (ng/L): 6.28±0.61 vs. 12.02±0.54, IL-6 (ng/L): 28.26±3.15 vs. 60.58±4.62, IL-1β (ng/L): 33.63±3.48 vs. 72.56±2.25, all P < 0.05]. Conclusion The results reveal that fecal microbiota transplantation has remarkably modulated the dysbiosis of intestinal microbiota and activated cholinergic anti-inflammatory pathway, and ameliorate the brain dysfunction in septic rats.