Реферат
α7 nicotinic acetylcholine receptor(α7nAChR)is widely expressed in the central nervous system and immune system,and plays a neuro-immunoregulatory role.On the one hand,α7nAChR is involved in the transmission of neurotransmitters,the conduction of excitatory signals and the maintenance of synaptic plasticity,which is of great significance for maintaining the normal and stable neurocognitive function.On the other hand,as an important part of the cholinergic anti-inflammatory pathway,α7nAChR is involved in the regulation of physiological and pathological processes such as inflammatory response,oxidative stress,apoptosis and autophagy in the central system,and plays an immunomodulatory and neuroprotective role,thus being potential target for improving perioperative neurocognitive function.This article reviews the biological characteristics of α7nAChR and its effect on perioperative neurocognitive function,in order to provide ideas and methods for clinical improvement of perioperative neurocognitive function in surgical patients.
Реферат
Objective:To explore the effects of activating α7 nicotinic acetylcholine receptor(α7nAChR)on cognitive function and polarization of hippocampal microglia in traumatic brain injury (TBI) rats.Methods:Totally 36 male SD rats with 6-8 weeks old were randomly divided into Sham group ( n=12), TBI group ( n=12), TBI+ α7nAChR agonist group ( n=6) and TBI+ α7nAChR antagonist group( n=6). The TBI model was established by the " free fall impact" method. From the 4th to 6th day after modeling, mice in the TBI+ α7nAChR agonist group were intraperitoneally injected with α7nAChR agonist PNU-282987 (3 mg/kg). Rats in TBI+ α7nAChR antagonist group were intraperitoneally injected with α7nAChR antagonist methyllycaconitine citrate (5 mg/kg) first, then 45 minutes later they were injected with α7nAChR agonist PNU-282987 (3 mg/kg). Rats in the TBI group and Sham group were intraperitoneally injected with an equal volume of 0.9% sodium chloride solution. Morris water maze test was used to evaluate the learning and memory function of rats. Immunofluorescence staining was used to observe the ionized calcium binding adapter molecule 1 (Iba-1)(a marker for microglia) and arginase 1 (Arg-1)(a marker for M2 microglia). Western blot was used to detect the protein level of Arg-1 in hippocampal tissue. Statistical analysis was performed using GraphPad Prism 9 software. Independent sample t test was used for comparison between two groups, one-way ANOVA was used for comparison among multiple groups, and Tukey test was used for multiple comparison. Results:The results of the water maze test showed that after 7 days of modeling, there was a statistical difference in the escape latency among the 4 groups of rats ( F=6.134, P<0.05). There was no statistical difference in the escape latency between the TBI group and the TBI+ α7nAChR antagonist group( P>0.05), but the both were higher than that of the Sham group (both P<0.05). The escape latency of the TBI+ α7nAChR agonist group((31.87±9.01)s) was shorter than that of the TBI group((56.75±2.62)s) and the TBI+ α7nAChR antagonist group((60.00±0.00)s) (both P<0.05). The results of immunofluorescence staining showed that there were statistical differences in the fluorescence intensity and cell numbers of Arg-1 + /Iba-1 + among the four groups ( F=17.37, 9.33, both P<0.05). The immune fluorescence intensity (0.27±0.03) and cell numbers (21.67±4.41) of Arg-1 + /Iba-1 + in the TBI+ α7nAChR agonist group were higher than those in the TBI group((0.14±0.03), (11.33±2.60)) and TBI+ α7nAChR antagonist group((0.10±0.03), (7.67±1.20)) (all P<0.05). The results of Western blot showed that there was a statistical difference in the level of Arg-1 protein in hippocampus among the 4 groups ( F=8.323, P=0.001). There was no significant difference in the level of Arg-1 protein between the TBI group and the TBI+ α7nAChR antagonist group( P>0.05), and the level of Arg-1 protein in the TBI+ α7nAChR agonist group(1.06±0.22) was higher than that in the TBI group(0.60±0.13) and TBI+ α7nAChR antagonist group(0.35±0.10) (both P<0.05). Conclusion:Activating α7nAChR can promote the polarization of M2 type microglia in rat hippocampal tissue and improve the learning and memory function of TBI rats.
Реферат
Cholinergic anti-inflammatory pathway in which acetylcholine released as the neurotransmitter, plays an important role in nerve-immune regulation. In this pathway, with the vagus nerve in the central nervous system as a starting point, the alpha 7 nicotinic acetylcholine receptor (α7 nAChR) on the surface of immune cell membrane is the key functional part. The interaction between electrical and chemical signals regulates the inflammation in the body via modulation of the JAK-STAT3, PI3K-Akt and other signaling pathways and the nuclear translocation of NF-κB, leading to inhibition of the release of pro-inflammatory factors and promotion of the release of anti-inflammatory factors. However, the detailed mechanism is far from clear. Studies have shown that the cholinergic anti-inflammatory pathway can be activated by drug targeting α7 nAChR and electrical stimulation of vagus nerve. Activation of α7 nAChR has the advantages of simple operation, less damage and significant effect. The commonly used drugs are selective agonists such as PNU282987 and GTS-21, and non-selective agonists such as nicotine. And this method has been found to play a role in the treatment of peripheral organ inflammatory diseases such as sepsis, ischemia-reperfusion injury, gastroenteritis, osteoarthritis and autoimmune diseases. As a key factor in the cholinergic anti-inflammatory pathways, α7 nAChR has become a potential therapeutic target for many inflammatory diseases. This paper reviewed the anti-inflammatory mechanism and activation mode of α7 nAChR involved in cholinergic anti-inflammatory pathway, as well as its application in inflammatory diseases in recent years, which may provide a reference for future research on its detailed mechanism of action and potential application as a new therapeutic target.
Реферат
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<sup>-1</sup> 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<sup>-1</sup>) 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 (IC<sub>50</sub>) being 6.03 g·L<sup>-1</sup>. 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<sup>-1</sup>) 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.
Реферат
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>)<sub>1-42</sub>. Method:Sixty male SD rats were divided into normal group, model group, huperzine A group (2.1×10<sup>-5</sup> g·kg<sup>-1</sup>), high-, medium- and low dose of Huanglian Jiedutang groups (6,3,1.5 g·kg<sup>-1</sup>). AD rat model was replicated by hippocampal injection of A<italic>β</italic><sub>1-42</sub>. 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><sub>1-42</sub>,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><sub>1-42</sub>.
Реферат
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.
Реферат
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.
Реферат
Objective:To investigate the effect of vagus nerve stimulation (VNS) on α7 nicotinic acetylcholine receptor (α7 nAChR) and inflammatory factor expressions and behavioristics in rats after traumatic brain injury (TBI).Methods:Seventy-two male Sprague-Dawley rats were randomly divided into sham-operated group, TBI group and VNS group ( n=24). The TBI models in the latter two groups were established by modified weight drop method; the dura mater in the sham-operated group was exposed without impingement. The VNS group received VNS treatment for 14 consecutive d (frequency: 30 Hz, pulse width: 100 μs, current: 0.8 mA, stimulation: 5 min, suspension: 5 min, repetition: 3 times). The neurological function was evaluated by Beam-Balance test and walking test 1, 3, 7, 14, 21, and 28 d after TBI; Morris Water Maze test was performed to observe the abilities of spatial learning and memory 24-28 d after TBI; the brain tissue sections were obtained for analyzing the volume of cerebral injury 28 d after TBI. Fourteen d after TBI, Nissel staining was used to observe the neuronal morphology and survival cell number of brain injured areas in each group, immunohistochemical staining was employed to detect the number of neuronalnuclear antigen (NeuN) positive neurons in the hippocampal dentate gyrus and CA3 area, and ELISA was used to detect the expressions of interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB. Western blotting was used to detect the α7 nAChR protein expression levels 3, 7, 14 d after TBI. Results:As compared with the TBI group, the VNS group showed statistically longer beam-balance latency and shorter beam-walking time 7, 14, 21, and 28 d after TBI, significantly shortened escape latency and prolonged exploration time in the target quadrant 27 and 28 d after TBI ( P<0.05). The volume of cerebral injury in the VNS group was significantly smaller than that in TBI group 28 d after TBI ( P<0.05). As compared with those in the TBI group, the number of Nissel positive neurons around the brain injury area was significantly larger, the number of NeuN positive cells in the hippocampal dentate gyrus and CA3 areas was significantly larger, and the expression levels of IL-1β, IL-6, TNF-α and NF-κB were significantly decreased in the VNS group 14 d after TBI ( P<0.05). The α7 nAChR protein expression in the injury area of VNS group was significantly higher than that of the TBI group 7 and 14 d after TBI ( P<0.05). Conclusion:VNS treatment can improve the neurobehavioral outcome of TBI rats, which may be linked with the increased α7 nAChR protein expression, and the decreased release of inflammation cytokines IL-1β, IL-6, TNF-α and NF-κB.
Реферат
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.
Реферат
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.
Реферат
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.
Тема - темы
Animals , Mice , Disease Models, Animal , Glycoproteins , Mice, Knockout , Mice, Transgenic , Tumor Necrosis Factor-alpha , alpha7 Nicotinic Acetylcholine Receptor , MetabolismРеферат
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.
Реферат
Objective To observe dexmedetomidine coordinate with mlelatonin attenuate the scopolamine-induced delirium in rats and its mechanism.Methods Thirty male adult SD rats aged 6-8 weeks were randomly divided into six groups:normal saline control group (group C),scopolamine-induced delirious model group (group S),dexmedetomidine group (group D),mlelaton group (group M),α-bungarotoxin antagonism group (group BM),joint protection group (group DM).A model of delirium was reproduced by intraperitoneal inj ection of scopolamine 1.8 mg/kg.The rats in group C was given equal sterile normal saline instead,the rats in group D was intraperitoneal injected of dexmedetomidine 40 μg/kg 15 minutes before scopolamine injection,the rats in group M was intrap-eritoneal injected of mlelaton 5 mg/kg in the contralateral abdominal at the same time with scopolamine injection,the rats in group BM was intraperitoneal injected ofα-BGT 1 μg/kg 15 minutes before scopolamine injection and mlelaton 5 mg/kg in the contralateral abdominal at the same time with scopolamine injection,the rats in group DM was intraperitoneal injected of dexmedetomidine 40 μg/kg 15 minutes before scopolamine injection and mlelaton 5 mg/kg in the contralateral abdominal at the same time with scopolamine injection.The rats were assigned for open field test 15 minutes before and 10 minutes after model reproduction for 15 minutes.The level ofα7nAchR in serum was deter-mined by enzyme linked immunosorbent assay.Results When compared with group C,rats in group S ran significant longer total distance and space distance,had faster total speed and space speed (P<0.05).When compared with group S,rats in group D,group M,group DM ran significant shorter total distance and space distance,had significant slower total speed and space speed (P<0.01 );when compared with group D,rats in group DM ran significant shorter total distance and space dis- tance (P<0.05 ),had slower total speed and space speed,however without significant statistical difference.When compared with that in group C,the level of α7nAChR in serum were significantly decreased in group S (P<0.05).When compared with group S,the level of α7nAChR were signifi-cantly increased in group D (P<0.01).There were no significant difference between group M and group S (P=0.96).When compared with group D,the level of α7nAChR had an elevated trend in group DM.Conclusion Dexmedetomidine can improve the symptoms of delirium,possibly by in-creasing the activity of alpha 7nAChR.Melatonin may improve the effect of dextromidine on delirium.
Реферат
This paper briefly introduces the structure and function of α7nAChR, and mainly reviews the relevant researches about the relationship between a7nAChR and cognition of schizophrenia in the last ten years from molecule genetics and new targets of medication two aspects. It suggests that the abnormal α7AChR is involved in cognition impairment of schizophrenia, and the a7nAChR agonist is probably able to improve the impaired cognition in patients with schizophrenia. Furthermore, KAT-2 as a key enzyme acting on its endogenous antagonist KYNA is also promising to become a new drug target
Реферат
Objective To investigate the effects of α7 nicotinic acetylcholine receptor (α7nAChR) on the inflammatory response induced by lipopolysaccharide (LPS) in RAW264.7 macrophages and its molecular mechanisms. Methods RAW264.7 macrophages were culturedin vitro. Inflammatory cell model was constructed by LPS stimulation. Cells were challenged by LPS (1, 10, 100 and 500μg/L) for 5 hours or 100μg/L LPS for 0, 2, 4, 8, 12, 24, 48 and 72 hours, and the release of tumor necrosis factor-α (TNF-α) was detected by the enzyme linked immunosorbent assay (ELISA). The location of α7nAChR was examined in RAW264.7 macrophages by immunofluorescence. Then the cell proliferation and toxicity kit (CCK-8) was used to detect 1, 10, 100, 1000μmol/L GTS-21, a α7nAchR agonist, on the cell viability after LPS stimulation. ELISA was used to detect 1, 10, 100, 1000μmol/L GTS-21 on the levels of TNF-α, interleukin 1β (IL-1β) after LPS stimulation. Cells were challenged with 100μg/L LPS and 100μmol/L GTS-21, then, the level of high mobility group box 1 (HMGB1) was detected by Western Blot and the intracellular location of HMGB1 and nuclear factor-κB p65 (NF-κB p65) was tested by immunofluorescence.Results LPS increased the level of TNF-α to a peak at the concentration of 100μg/L and at 24 hours after stimulation. Theα7nAChR expressed on the macrophages. The cell viability was decreased in a dose-dependent manner [(96.2±1.0)%, (92.0±1.1)% vs. (86.5±2.2)%, bothP < 0.05]. Compared with the control group, the levels of TNF-α and IL-1βin the supernatant of LPS group were significantly increased [TNF-α (ng/L): 453.0±60.6 vs. 100.8±3.2, IL-1β(μg/L): 8.21±0.31 vs. 0.87±0.16, bothP < 0.05]. TNF-α and IL-1β were significantly decreased by 10μmol/L and 100μmol/L GTS-21 in a dose-dependent manner [TNF-α (ng/L): 227.5±17.5, 81.0±8.8 vs. 453.0±60.6;IL-1β (μg/L): 4.86±0.72, 2.32±0.45 vs. 8.21±0.31, allP < 0.05]. GTS-21 significantly reduced the expression of HMGB1 which was induced by LPS management (gray value: 0.788±0.130 vs. 2.061±0.330,P < 0.05) and reversed LPS-induced HMGB1 cytoplasmic transfer. GTS-21 also reversed LPS-induced nuclear translocation of NF-κB p65. Conclusion GTS-21 reduces the inflammatory response via inhibiting the activation of NF-κB.
Реферат
AIM: To explore the influence of clonidine on inflammatory response in lung injury mice and its possible mechanism.METHODS: Clonidine solution was intravenously injected into the mice with lung injury induced by LPS.The left upper lobe of the lung was collected to detect lung wet/dry weight ratio (W/D) and total lung water content (TLW).The concentrations of IL-6, IL-1β and TNF-α were measured by ELISA.The expression of α7 nicotinic acetylcholine receptor (α7nAChR) and high-mobility group box protein 1 (HMGB1) at mRNA and protein levels was determined by RT-PCR and Western blot.After importing α7nAChR siRNA lentiviral vector or injecting exogenous HMGB1 protein, the inflammatory cytokines were detected.RESULTS: Clonidine attenuated lung injury and inhibited inflammatory reaction.Clonidine promoted the activation of cholinergic anti-inflammatory pathway by promoting α7nAChR expression.Clonidine inhibited HMGB1 expression, which promoted the secretion of IL-6, IL-1β and TNF-α.HMGB1 was negatively regulated by α7nAChR.CONCLUSION: Clonidine functions as an anti-inflammatory reagent to the lung injury mice.The mechanism may be related to activating the cholinergic anti-inflammatory pathway and inhibiting the expression of HMGB1.
Реферат
OBJECTIVE Individuals vary in sensitivity to the behavioral effects of nicotine, resulting in differences in their vulnerability to addiction. The role of rearing environment in determining individual sensitivity to nicotine is unclear. The neuropharmacological mechanisms mediating the effect of rearing environment on the actions of nicotine are also understood. Thus, the contribution of rearing environment in determining the sensitivity to the locomotor effects of nicotine and regulating α4β2*- and α7-nicotinic acetylcholine (nACh) receptor expressionwas determined in rats reared in isolated (IC) or enriched (EC) conditions. METHODS To measure locomotor activity, adolescent rats (postnatal day 21- 51) were injected with saline (1 mL·kg-1) or nicotine (0.3 mg·kg-1) subcutaneously, then placed in chamber?swhere ambulatory activity was monitored for 30-min by computer for 14 daily sessions. α4β2*- andα7- nACh receptor expression in the mesolimbic dopamine pathway was determined by quantitative autoradiography of [125I]-epibatidine and [125I]-bungarotoxinbinding, respectively, in 16 μmol·L- 1 coronal sections. Values for receptor expression in fmol are x ± s of 8 brains and compared by two- tailed, unpaired t-test with P<0.05 considered significant. RESULTS EC-rats are similarly sensitive as IC-rats to the locomotor effects of nicotine. [125I]-epibatidine binding in the ventral tegmental area of EC-rats was reduced (2.8 ± 0.3 fmoL) compared to IC-rats (4.0 ± 0.4 fmoL); there was no difference in the nucleus accumbens. There was no difference between EC- and IC-rats in α7-nACh receptor expression in the mesolimbic dopamine pathway. CONCLUSION Rearing environment differentially regulates nACh receptor subtypes in EC and IC rats. These data suggest regulation of nACh receptors by environmental factors may be a mechanism for the protective effect of enrichment against altered sensi?tivity to nicotine in genetically vulnerable individuals. The characterization of these mechanisms will aid in development of novel pharmacological tools mimicking the protection afforded by environmental enrichment in nicotine-sensitive individuals.
Реферат
Objective To observe the role of α7 nicotinic acetylcholine receptor (α7nAChR) in the protection against delirium by the use of dexmedetomidine (DEX) in endotoxin derived delirium and its mechanism. Methods 100 male adult C57BL/6 mice were randomly divided into normal saline control group (NS group), DEX control group, lipopolysaccharide (LPS) induced endotoxemia model group (LPS group), DEX protection group (DEX+LPS group), and α-bungarotoxin antagonism group (α-BGT+DEX+LPS group), with 20 mice in each group. A model of endotoxemia was reproduced by intraperitoneal injection of LPS 20 mg/kg, and the mice in NS group and DEX control group were given equivalent sterile normal saline. The mice in DEX control group, DEX+LPS group, and α-BGT+DEX+LPS group were intraperitoneally injected with DEX 40 μg/kg 15 minutes before LPS injection. The mice in α-BGT+DEX+LPS group were intraperitoneally injected with α7nAChR inhibitor α-BGT 1 μg/kg 15 minutes before DEX injection. The mice in NS group were given equivalent sterile normal saline. Ten mice in each group were assigned for open field test before and 24 hours after model reproduction, and the mice were then sacrificed to obtain the specimens. The levels of tumor necrosis factor-α (TNF-α) and neuron-specific enolase (NSE) in serum were determined by enzyme-linked immune sorbent assay (ELISA). Western Blot method was used to determine the expression of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in hippocampus. Another 10 mice were subjected to new object recognition test to observe the total exploration time during training period and preference index at 3 hours and 24 hours after LPS challenge. Results There were no significant differences in all parameters between NS group and DEX control group. ① It was shown by the open field test results that there were no significant differences in all parameters of open field test before model reproduction among all the groups. Twenty-four hours after model reproduction, when compared with NS group, the mice in LPS group showed that they had the ability of cognition of new environment, but learning and memory abilities were lowered, and tension was increased. DEX could significantly attenuate the degree of delirium, however, the protection of DEX from the delirious syndrome was antagonized partly by α-BGT. ② The new object recognition test results showed that compared with NS group, the ability of exploring new object was decreased in LPS group. DEX could significantly improve the exploration ability. However, DEX failed to control the delirious syndrome in α-BGT+DEX+LPS group. ③ The results of ELISA showed that the levels of TNF-α and NSE in serum were significantly increased in LPS groups as compared with that in NS group, and the levels of TNF-α and NSE were significantly decreased in DEX+LPS group. However, α-BGT could antagonise the protective effect of DEX [TNF-α (ng/L) in NS, LPS, DEX+LPS and α-BGT+DEX+LPS groups was 23.72±3.13, 808.78±87.86, 192.96±31.47, 829.99±80.98, respectively, and NSE (μg/L) was 8.70±0.74, 25.90±3.03, 18.10±2.14, and 23.12±2.21, respectively, all P < 0.01]. ④ The results of Western Blot showed that compared with NS group, the protein expression of ChAT in LPS group was significantly declined, and the protein expression of AChE was significantly increased. DEX could reverse the expressions of ChAT and AChT, however, α-BGT could reverse the protective effect of DEX [ChAT (gray value) in NS, LPS, DEX+LPS and α-BGT+DEX+LPS groups was 1.536±0.150, 0.381±0.138, 0.914±0.173, 0.628±0.088, respectively, and AChE (gray value) was 0.382±0.201, 1.843±0.325, 0.898±0.155, and 1.470±0.220, respectively, P < 0.05 or P<0.01]. Conclusions Delirium syndrome may occur in mice with endotoxemia. DEX could attenuate endotoxemia-associated delirium syndrome through transforming central neurotransmitter, and its mechanism maybe related with α7nAChR.
Реферат
Alpha7 nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated ion channel critical for cognition, learning and memory. Deficiency of neuronal α7 nAChR has been implicated in the cognitive deficits and neuropsychiatric disorders. Chemical activation of α7 nAChR improves neurological functions in animal models. In this study, we designed and synthesized a series of indolizine derivatives with various substitutions at different positions on the scaffold, and investigated their structure-activity relationships (SAR). All compounds were screened and evaluated for their agonist activity using the two-electrode voltage clamp recording system in Xenopus oocytes expressing human α7 nAChR. Compound 16c carrying 6-methylindolizine moiety activates α7 nAChR with EC50 at 1.60±0.19 μmol·L-1 and maximum effect (Emax) of 69.0%±2.8% compared with 3 mmol·L-1 ACh. Compound 17b with 8-cyclopropyl substitution shows an increased Emax of 81.1%±9.3% with EC50 at 2.74±0.74 μmol·L-1. The SAR of the series shows that introducing the small hydrophobic groups at 6- or 8- position can improve both potency and maximum effect.
Реферат
Objective To observe the protective effects of genistein sodium sulfonate(GSS)on mice chronic hepatic injury in-duced by carbon tetrachloride(CCl4 )and its influence on the protein expression of α7 nicotinic acetylcholine receptor(α7nAChR) and interleukin-1 beta (IL-1β)in liver tissue.Methods 60 SPF grade male Kunming mice were randomly divided into 5 groups,in-cluding the control group,model group,low and high doses GSS groups,and positive control group,12 cases in each group.Except for the control group,the other 4 groups were intra peritoneally injected by 10 % CCl4 with a volume of 0.1 mL/10 g for 6 weeks. The mice chronic liver injury was prepared.At the same time,the high and low doses DSS groups were given the different doses of GSS(0.30,0.10 mg/kg),the positive control group was given bifendate(DDB,2.5mg/kg),the control group and the model group were given the equal volume of normal saline for 6 consecutive weeks.The AST and ALT activity was detect and the ratio of ALT/AST was calculated;the Western blot method was used to detect the expression levels ofα7nAChR and IL-1βprotein in liver.Re-sults The serum levels of ALT and AST in the model group were increased obviously,and the expression level ofα7nAChR in the liver tissue was decreased,while the expression level of IL-1βwas increased;after the GSS treatment,the serum AST and ALT lev-els were significantly lower than those in the model group(P <0.05),while the expression level ofα7nAChR was increased (P <0.01)and the expression level of IL-1βwas decreased(P <0.05).Conclusion GSS might increase the expression ofα7nAChR in injured liver tissue,activates the cholinergic anti-inflammatory pathway,thus decreases the expression of inflammatory cytokines and antagonizes the mice chronic liver injury by inhibiting the inflammatory reaction.