Cholinergic dysfunction-induced insufficient activation of alpha7 nicotinic acetylcholine receptor drives the development of rheumatoid arthritis through promoting protein citrullination via the SP3/PAD4 pathway

Both cholinergic dysfunction and protein citrullination are the hallmarks of rheumatoid arthritis (RA), but the relationship between the two phenomena remains unclear. We explored whether and how cholinergic dysfunction accelerates protein citrullination and consequently drives the development of RA. Cholinergic function and protein citrullination levels in patients with RA and collagen-induced arthritis (CIA) mice were collected. In both neuron-macrophage coculture system and CIA mice, the effect of cholinergic dysfunction on protein citrullination and expression of peptidylarginine deiminases (PADs) was assessed by immunofluorescence. The key transcription factors for PAD4 expression were predicted and validated. Cholinergic dysfunction in the patients with RA and CIA mice negatively correlated with the degree of protein citrullination in synovial tissues. The cholinergic or alpha7 nicotinic acetylcholine receptor (α7nAChR) deactivation and activation resulted in the promotion and reduction of protein citrullination in vitro and in vivo, respectively. Especially, the activation deficiency of α7nAChR induced the earlier onset and aggravation of CIA. Furthermore, deactivation of α7nAChR increased the expression of PAD4 and specificity protein-3 (SP3) in vitro and in vivo. Our results suggest that cholinergic dysfunction-induced deficient α7nAChR activation, which induces the expression of SP3 and its downstream molecule PAD4, accelerating protein citrullination and the development of RA.

Effect and mechanism of PNU-282987 on cognitive function of temporal lobe epilepsy model rats / 中国药房

OBJECTIVE To investigate the effect and mechanism of α7 nicotinic acetylcholine receptor （α7nAChR） agonists PNU-282987 on cognitive function in temporal lobe epilepsy （TLE） model rats. METHODS Sixty rats were randomly divided into control group， model group， PNU-282987 group （3 mg/kg） and methyllycaconitine （MLA）+PNU-282987 group （6 mg/kg MLA+3 mg/kg PNU-282987）， with 15 rats in each group. Except for control group， the TLE model was established in the other groups. After the model was successfully established， each group was given relevant medicine or normal saline intraperitoneally， once a day， for two consecutive weeks. The epilepsy attack of rats was observed and scored， and the duration of seizures was recorded； the cognitive function of rats was detected； pathological morphology of neurons in CA1 region was observed； the levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6） and IL-1β in the hippocampus were detected； the positive expressions of ionized calcium-binding adapter molecule-1 （IBA-1）， α7nAChR， nuclear factor-κB （NF-κB） p65， p-NF-κB p65 in the hippocampus were detected. RESULTS Compared with model group， the score and duration of seizures， the number of IBA-1 positive cells， the levels of TNF- α， IL-6 and IL-1β， the expressions of NF- κB p65 and p-NF- κB p65 protein decreased significantly in the hippocampus （P＜0.05）； the escape latency time was shortened significantly （P＜0.05）， the time spent in the original platform quadrant and times of crossing the platform increased significantly （P＜0.05）； neuronal damage in the CA1 region of the hippocampus was significantly reduced； the expression of α7nAChR protein increased significantly in hippocampus （P＜0.05）. Compared with PNU-282987 group， the above indexes of rats in MLA+PNU-282987 group were reversed significantly （P＜0.05）. CONCLUSIONS PNU-282987 could improve cognitive dysfunction in TLE model rats， and its mechanism may be associated with inhibiting microglia-mediated inflammatory response through α7nAChR/NF- κB signaling pathway， thus reducing hippocampal neuronal damage.

Isolation and characterization of five novel disulfide-poor conopeptides from Conus marmoreus venom

Background: Conopeptides from cone snail venom have aroused great interest related to the discovery of novel bioactive candidates, due to their excellent prospects for the treatment of various health problems such as pain, addiction, psychosis and epilepsy. In order to explore novel biopeptides, we investigated the structure and function of five novel conopeptides isolated from the venom of Conus marmoreus from South China Sea. Methods: C. marmoreus crude venom was prepared, fractionated and purified by HPLC system. The primary sequences of the five novel disulfide-poor conopeptides Mr-1 to Mr-5 were identified by comprehensive analysis of de novo MALDI-TOF tandem mass spectrometry and Edman degradation data. In order to investigate their function, these five conopeptides were synthesized by Fmoc-SPPS chemistry, and their biological effects at several heterologous rat nicotinic acetylcholine receptor (nAChR) subtypes (α1β1δε, α3β2, α3β4, α4β2) were determined by electrophysiological technique. Results: Five novel disulfide-poor conopeptides were identified and named as follows: Mr-1 (DWEYHAHPKPNSFWT), Mr-2 (YPTRAYPSNKFG), Mr-3 (NVIQAPAQSVAPP NTST), Mr-4 [KENVLNKLKSK(L/I)] and Mr-5 [NAVAAAN(L/I)PG(L/I)V]. None of them contains a disulfide bond. The sequences of conopeptides Mr-2 to Mr-5 do not belong to any category of the known disulfide-poor conopeptides. No significant activity against the above nAChR subtypes were observed for the five conopeptides at 100 µM. Conclusion: We purified and structurally characterized five novel disulfide-poor conopeptides from C. marmoreus crude venom and first investigated their nAChR inhibitory effects. This work expanded our knowledge on the structure and function of disulfide-poor conopeptides from this cone snail venom.(AU)

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.

Chinese Herbal Formula Ermiao Powder () Regulates Cholinergic Anti-inflammatory Pathway in Rats with Rheumatoid Arthritis / 中国结合医学杂志

OBJECTIVE@#To investigate the effect of Chinese herbal formula Ermiao Powder (, EMP) on the expression of cholinergic anti-inflammatory pathway in rats with rheumatoid arthritis (RA).@*METHODS@#Seventy-two rats were randomly divided into 6 groups according to body weight, including normal control group, collageninduced arthritis (CIA) group, three doses EMP groups, and methotrexate (MTX) group (n=12 per group). All of the rats except for those in the normal control group were given multipoint subcutaneous injection of bovine type II collagen to establish a CIA model. Three EMP groups received a high- (4.5 g/kg), medium- (3.0 g/kg), and low- (1.5 g/kg) doses of EMP by intragavage, respectively. MTX group was injected intraperitoneally MTX at 0.9 mg/kg once a week as the positive control. The administration was 3 consecutive weeks. Joint swelling, arthritis index, and body weight changes in different experimental groups of rats were tested. The joint damage was evaluated by masson staining. Quantitative real-time polymerase chain reaction, Western blot, and immunohistochemistry (IHC) were performed to evaluate the expression of CHRNA7, encoding α7 nicotinic acetylcholine receptor in the cholinergic anti-inflammatory pathway, in different tissues and their localization in the spleen and joints.@*RESULTS@#CHRNA7 expression levels were significantly higher in the joints and spleens of CIA group than those in normal control group (both P<0.05). Moreover, the CHRNA7 mRNA and protein levels in the spleen and joints of MTX and three doses of EMP groups were significantly lower than CIA group (all P<0.05). Compared with the MTX group, treatment with low-dose EMP resulted in significant reduction of CHRNA7 mRNA and protein expression levels (P<0.05 or P<0.01). IHC showed positive signals of CHRNA7 in the white pulp and red pulp of the spleens of rats; CHRNA7 was expressed on fibroblast-like synoviocytes, macrophages, and endothelial cells in the joints of rats, and the expression in the joints of low-dose EMP group was significantly lower than that in the CIA group (P<0.01).@*CONCLUSIONS@#Cholinergic anti-inflammatory pathway was involved in the generation of the inflammatory reaction in CIA rats, and EMP exerted therapeutic effect on RA through cholinergic anti-inflammatory pathway.

Effects of sevoflurane on the cognitive function and the quantity of alpha 7 nicotinicacetycholine receptors in the hippocampus of elderly model rats / 医学研究生学报

ObjectiveSome studies reported that α7nAChR is closely related to the cognitive function. However, the elderly patients have become a high-risk group of postoperative cognitive dysfunction. The aim of this study was to explore the effect of sevoflurane inhalation on the cognitive function and the quantity of alpha 7nicotinic acetycholine receptors in the hippocampus of elderly model rats.MethodsAdult male Sprague-Dawley rats (N=72) were given subcutaneous injection of D-galactose on the neck for 6 weeks to establish elderly models. The model rats were divided into 4 groups randomly: control group (group Con, n=18) with 6h exposure carrier gas (2L/min Air+2L/min O2); Sevoflurane group (group Sev, n=18) with 6 h exposure to 3.2% sevoflurane through carrier gas. Sev+α7nAChR antagonist group (group Sev+M) injected with methyllycaconitine, after 24 h inhaled of 3.2% sevoflurane and carrier gas for 6 h. Sev+α7nAChR agonist group (group Sev+P, n=18) injected with PNU-282987, after 24 h inhaled of 3.2% sevoflurane and carrier gas for 6 h. Morris water maze experiments were conducted on 6 rats in each group 2 h, 1 week and 4 weeks after treatments, respectively. Every cycle after the behavioral test, the hippocampi were taken out. RT-qPCR method was used to detect α7nAChR mRNA expression. Western blotting was used to detect α7nAChR proteins expression.ResultsBehavioral test: compared with Con group at 2 h after awakening, indicators of working memory and spatial probe test in Sev group and Sev+M group decreased significantly (P0.05). RT-qPCR: compared with Con group at 2 h and 1 w after awakening, the expression of alpha 7nAChR mRNA in the other groups was down-regulated, while at 4 w it was up-regulated (P<0.05).Western blot: protein expression of alpha 7nAChR was down-regulated in the 2 h, 1 w Sev group and the Sev+M group after awakening, and up-regulated in the 4 w group after awakening (P<0.05).ConclusionInhalation of 3.2% sevoflurane for 6 h can cause 7nAChR metabolic disturbance in hippocampus of aging model rats and lead to a short-term (1 w) decline in learning and memory ability of the rats, but this effect is reversible. The PNU-282987 agonist can alleviate the temporary decrease of learning and memory caused by sevoflurane.

Effect of Shock Wave on NMJ and Muscle Morphology in Rabbits / 昆明医科大学学报

Objective To evaluate the AChE and nAChR in the NMJ and the morphology of the muscle in the bilateral triceps surae after the unilateral shock wave therapy. Method 60 male New Zealand rabbits weighing (2± 0.2) Kg were used in this study. Two thousand shock waves at an energy flux density of 1.5bar and the frequency of 10Hz were applied to their left calf muscles. Divided into six groups, both sides of the triceps muscle of calf were taken out on the day of the shock wave and the1,2,4,6 and 8 weeks after the treatment. HE staining was used to observe the morphological changes of muscle tissue and the average optical density was measured after AChE stain so as to calculate the receptor count after Acetylcholine receptor immunohistochemistry. Result No abnormal morphological abnormalities were observed in all rabbits. In the first five groups, the AChE was significantly higher in the side of the shockwave treatment compairing with the control side (<0.05),slow decrease after 1 week after the treatment. In the first five groups, the nAChR was significantly lower in the side of the shock wave treatment compairing with the control side ( <0.05), and gradually increased to normal after 8 weeks. Conslusion Suitable dose of shock wave will not have a greater impact on morphology of muscle tissue. After the shock wave treatment, the amount and degree of stimulate of muscle cells were decreased, and the production of action potentials was reduced. While the experimental side AchE and AchR in shock wave treatment day to 8 weeks after treatment showed a significant trend to normal, it shows that the effect of shock wave on NMJ is transient and reversible.

Low-intensity pulsed ultrasound pretreatment inhibits HMGB1 expression and attenuates lung ischemia-reperfusion injury in rats the cholinergic anti-inflammatory pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effects of low-intensity pulsed ultrasound (LIPUS) pretreatment on pulmonary expression of high mobility group box-1 (HMGB1) in a rat model of lung ischemia-reperfusion (IR).</p><p><b>METHODS</b>Thirty-two male SpragueDawley rats weighing 250-300 g were randomly divided (=8) into sham-operated group, lung IR group, LIPUS pretreatment group and pretreatment with α7-nicotinic cholinergic receptor (α7nAChR) antagonist group. In the sham-operated group, the left pulmonary hilum was dissociated without occlusion; in the other 3 groups, the left pulmonary hilum was occluded for 45 min followed by reperfusion for 180 min; LIPUS pretreatment for 30 min and intraperitoneal injection of methyllycaconitine (2 mg/kg), an α7nAChR antagonist, were administered before the operation. The wet/dry weight ratio (W/D) and pulmonary permeability index (LPI) of the lung tissue were measured, and the lung histopathology was observed and scored. The contents of interleukin-1 (IL-1) and IL-6 in the lung tissues were measured using ELISA, and the pulmonary expression of HMGB1 protein was detected using immunofluorescence assay and Western blotting.</p><p><b>RESULTS</b>Compared with those in the sham-operated group, the W/D of the lung tissue, LPI, pathological scores, IL-1 and IL-6 contents in the lung tissue, and pulmonary HMGB1 expression all significantly increased in the other 3 groups ( < 0.05). LIPUS preconditioning significantly lowered the W/D values, LPI, pathological score, IL-1 and IL-6 contents and HMGB1 expression in the lung tissues following lung IR, and these effects were significantly inhibited by administration of methyllycaconitine.</p><p><b>CONCLUSIONS</b>LIPUS preconditioning can reduce lung IR injury possibly by activating α7nAChR-dependent cholinergic anti-inflammatory pathway to reduce lung tissue HMGB1 expression.</p>

Effects of nicotine on proliferation and chemosensitivity of A549 cells / 中国生化药物杂志

Objective To investigate the effect of nicotine on proliferation and chemosensitivity of A549 cells in vitro. Methods A549 cells was assessed by MTT assay to measure cell proliferation and was assessed by RT-PCR tomeasure chemosensitivity. Results 0.01~100μmol/L nicotine could promote the proliferation of A549 cells, the most marked proliferation at 1μmol/L, compared with the control group, the activity of A549 cells was increased by 1.85 times (P<0.01). When the concentration of nicotine above 1μmol/L, the proliferation of A549 cells had an decreasing tendency. When the concentration above 1000μmol/L, the proliferation of A549 cells can be inhibited. Nicotine can also reduce chemosensitivity of A549 cells to 5-FU, with the addition of nicotine, A549 cells survival rate increased significantly, the most marked at 1μmol/L, compared with the control group, the inhibitory rate of A549 cells was 9 % (P< 0.01). Nicotine significantly increased the expression level of α7 nAChR in A549 cells and decreased the expression of PTEN , in a concentration dependent manner. Compared with the control group, 1μmol/L of nicotine could increase the expression levels of α7 nAChR by 3.4 fold, and decrease the expression levels of PTEN by 60.36 % (P< 0.01). Conclusion Nicotinecan promote the growth of A549 cells and reduce chemosensitivity of A549 cells to 5-FU.

α7 Nicotinic acetylcholine receptor agonistinhibits bone cement particles stimulated secretion of inflammatory cytokinesin peripheral blood monocytes of mice / 基础医学与临床

Objective To investigate the effect of α7 nicotinic acetylcholine receptor(α7nAChR) agonist agent PNU282987 on bone cement particles stimulated secretion of inflammatory cytokines in peripheral blood monocytes and its molecular mechanism.Methods Mouse peripheral blood monocytes were isolated and the inflammatory response were induced by PMMA particles.TNF-α, IL-1β and IL-6 concentration in culture supernatant were measured by ELISA.TNF-α, IL-1β and IL-6 mRNA expression were measured by RT-PCR.p-p65, p65, p-JAK2, JAK2, p-STAT3, STAT3, and β-actin expression were detected by Western blot.NF-κB DNA binding activity were measured by ELISA.ResultsAfter stimulation of PMMA particles, TNF-α, IL-1β and IL-6 concentration in culture supernatant was significantly increased(P<0.05), TNF-α, IL-1β and IL-6 mRNA expression was significantly increased (P<0.05), p-p65, p-JAK2 and p-STAT3 expression and NF-κB DNA binding activity was also increased significantly (P<0.05).However, after PNU282987 treatment, TNF-α, IL-1β and IL-6 concentration in culture supernatant decreased(P<0.05), TNF-α, IL-1β and IL-6 mRNA expression decreased in a concentration gradient way(P<0.05), p-p65, p-JAK2 and p-STAT3expression and NF-κB p65 DNA binding activity was also decreased(P<0.05).Conclusions α7nAChR agonist PNU282987 significantly inhibites PMMA bone cement particles induced secretion of inflammatory cytokines in peripheral blood monocytes of mice.

Effect of Sinomenine on Expression of COX2, α7nAChR and A2A in A549 Cells / 广州中医药大学学报

Objective To observe the effect of sinomenine (SIN) on the expression of cyclooxygenase (COX2),alpha-7 nicotinic acetylcholine receptor(α7nAChR) and adenosine receptor(A2A) in A549 cells,and to explore the relative mechanism for cell proliferation.Methods The effect of SIN and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on the proliferation of A549 cells was determined by methyl thiazolyl tetrazolium (MTT) assay.The effect of SIN and NNK on the migration of A549 cells was detected by cell wound scratch assay.The effect of SIN and NNK on COX2 expression in A549 cells was determined by Western blotting method.The effect of SIN and NNK on the expression of α7nAChR and A2A mRNA and protein was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting method.Results NNK increased the proliferation and migration of A549 cells,while SIN inhibited the proliferation and migration of A549 cells.COX2 expression level was increased in NNK group but was decreased in SIN group.The expression levels of α7nAChR and A2A were up-regulated in NNK group but were down-regulated in SIN group.Conclusion SIN plays a role in inhibiting the proliferation and migration of A549 cells by suppressing COX2 expression.SIN has an inhibitory effect on the expression of α7nAChR and A2A.

Effect of α7nAchR gene 713T>C mutation on cognitive function and Aβ expression in AD mice / 重庆医学

Objective To investigate the effect of α7nAchR gene 713T>C mutation on the cognitive function and Aβ expression in Alzheimer′s disease(AD)mice.Methods Twenty APPSwe transgenic APPa7KO mice(6 months old,α7nAchR gene knockout)were divided into the mutation type group and wild type group according to the random number table method,10 cases in each group.The mutation type and wild type of AVV-α7nAchR cDNA were respectively injected by lateral ventricle,once per month,for 6 times.The change of cognitive function in mice was examined by Morris water maze.The ELISA method was used to detect Aβ 40 and Aβ 42 expression levels.The Aβ plaque deposit situation was detected by the immunochemical method.Results Compared with the mice in the wild type group,the escape latency and the time of first time to find the platform of the mice in the mutation type group were significantly extended,while Hippocampal Aβ40 and Aβ42 expression levels were significantly increased,the difference was statistically significant(P＜0.05).Conclusion α7nAchR gene 713T>C mutation aggravates the cognitive function impairments in AD mice and hippocampal neuron Aβ expression level.

Effects of glucocorticoids on intracellular calcium in microglial cells / 中国药理学通报

Aim To explore the effects of hydrocortisone on intracellular calcium in microglial cells.Methods The intracellular calcium was measured by instantaneous scanning with confocal laser microscope(CLM) in BV-2 cells, and fluo3-AM was used to dye the intracellular calcium.Results Both hydrocortisone and nicotine could obviously increase intracellular calcium in BV-2 cells(P0.05);but the blocker of α7 nicotinic acetylcholine receptor(α7nAChR) methyllycaconitine could suppress the rising of intracellular calcium induced by hydrocortisone(P<0.05).Conclusion Hydrocortisone enhances intracellular calcium via α7nAChR in microglial cells, which not only demonstrates the non-genomic effect of glucocorticoid, but also suggests that glucocorticoid could serve as endogenous ligand of α7nAChR.

Effects of nicotine on proliferation and chemosensitivity of A549 cells / 中国生化药物杂志

Objective To investigate the effect of nicotine on proliferation and chemosensitivity of A549 cells in vitro. Methods A549 cells was assessed by MTT assay to measure cell proliferation and was assessed by RT-PCR tomeasure chemosensitivity. Results 0.01~100μmol/L nicotine could promote the proliferation of A549 cells, the most marked proliferation at 1μmol/L, compared with the control group, the activity of A549 cells was increased by 1.85 times (P<0.01). When the concentration of nicotine above 1μmol/L, the proliferation of A549 cells had an decreasing tendency. When the concentration above 1000μmol/L, the proliferation of A549 cells can be inhibited. Nicotine can also reduce chemosensitivity of A549 cells to 5-FU, with the addition of nicotine, A549 cells survival rate increased significantly, the most marked at 1μmol/L, compared with the control group, the inhibitory rate of A549 cells was 9 % (P< 0.01). Nicotine significantly increased the expression level of α7 nAChR in A549 cells and decreased the expression of PTEN , in a concentration dependent manner. Compared with the control group, 1μmol/L of nicotine could increase the expression levels of α7 nAChR by 3.4 fold, and decrease the expression levels of PTEN by 60.36 % (P< 0.01). Conclusion Nicotinecan promote the growth of A549 cells and reduce chemosensitivity of A549 cells to 5-FU.

Comparison of electroacupuncture at lower-sea points on HMGB 1 and nAchR α7 in rats with acute gastric mucosal lesion / 中国针灸

<p><b>OBJECTIVE</b>To observe the influence of electroacupuncture (EA) at lower-sea points of stomach, large intestine, small intestine and gallbladder on interleukin-1β (IL-1β), high mobility group protein 1 (HMGB 1) and alpha 7 nicotinic acetylcholine receptor (nAchR α7) in rats with acute gastric mucosal lesion (AGML), so as to explore whether there is relative specificity in treating gastric viscera disease by stimulating Zusanli (ST 36).</p><p><b>METHODS</b>Sixty healthy SD rats were randomly assigned into a blank group, a model group, a Zusanli group, a Shangjuxu group, a Xiajuxu group and a Yanglingquan group, ten rats in each one (half male and half female). The WRS method was applied to induce the AGML model except the rats in the blank group. The rats in the blank group were treated with routine diet; the rats in the model group were treated with immobilization at rat platform, 30 min per time; the rats in the Zusanli group, Shangjuxu group, Xiajuxu group and Yanglingquan group were treated with acupuncture and connected with EA device (dilatational wave 10 Hz/50 Hz, positive electrode on the left side and negative electrode on the right side, intensity was appropriate when rat hind leg slightly shook), 30 min per time. The treatment was given once a day. After consecutive 10-day treatment, the gastric tissue was collected and the damage of gastric mucosa was evaluated; ELISA method was applied to measure the content of serum IL-1β and tissue HMGB 1; the Western blot method was applied to measure the expression of nAchR α7 receptor.</p><p><b>RESULTS</b>(1) Compared with the model group, the ulcer index (UI) of gastric mucosa, serum IL-1β and tissue HMGB 1 were lower, and the expression of nAchR α7 was increased in the remaining groups (<0.05,<0.01). (2) Compared with the Zusanli group, the UI of gastric tissue, serum IL-1β and tissue HMGB 1 were higher in the Shangjuxu group, Xiajuxu group and Yanglingquan group (<0.05,<0.01), and the expression of nAchRα7 was reduced in the Yanglingquan group (<0.01).</p><p><b>CONCLUSIONS</b>(1) EA at digestive system-related lower-sea points, through IL-1β, HMGB 1 and nAchR α7, could regulate immune response, lighten inflammatory reaction and reduce mucosal injury, which could realize the intervention effect on AGML rats. (2) From the comparison, it is concluded the intervention effect of Zusanli group is superior to the other groups, partly indicating the relative specificity between Zusanli and stomach.</p>

Differential Effects of Quercetin and Quercetin Glycosides on Human α7 Nicotinic Acetylcholine Receptor-Mediated Ion Currents

Quercetin is a flavonoid usually found in fruits and vegetables. Aside from its antioxidative effects, quercetin, like other flavonoids, has a various neuropharmacological actions. Quercetin-3-O-rhamnoside (Rham1), quercetin-3-O-rutinoside (Rutin), and quercetin-3-(2(G)-rhamnosylrutinoside (Rham2) are mono-, di-, and tri-glycosylated forms of quercetin, respectively. In a previous study, we showed that quercetin can enhance α7 nicotinic acetylcholine receptor (α7 nAChR)-mediated ion currents. However, the role of the carbohydrates attached to quercetin in the regulation of α7 nAChR channel activity has not been determined. In the present study, we investigated the effects of quercetin glycosides on the acetylcholine induced peak inward current (I(ACh)) in Xenopus oocytes expressing the α7 nAChR. I(ACh) was measured with a two-electrode voltage clamp technique. In oocytes injected with α7 nAChR copy RNA, quercetin enhanced I(ACh), whereas quercetin glycosides inhibited I(ACh). Quercetin glycosides mediated an inhibition of I(ACh), which increased when they were pre-applied and the inhibitory effects were concentration dependent. The order of I(ACh) inhibition by quercetin glycosides was Rutin≥Rham1>Rham2. Quercetin glycosides-mediated I(ACh) enhancement was not affected by ACh concentration and appeared voltage-independent. Furthermore, quercetin-mediated I(ACh) inhibition can be attenuated when quercetin is co-applied with Rham1 and Rutin, indicating that quercetin glycosides could interfere with quercetin-mediated α7 nAChR regulation and that the number of carbohydrates in the quercetin glycoside plays a key role in the interruption of quercetin action. These results show that quercetin and quercetin glycosides regulate the α7 nAChR in a differential manner.

Anti-inflammatory mechanism of alpha 7 nicotinic acetylcholine receptors / 国际儿科学杂志

Alpha nicotinic acetylcholine receptors(α7nAChR) is a classic neurotransmitter receptor. Re-cent studies have found α7nAChR plays a key role in the cholinergic anti-inflammatory pathway. In this paper, biology characteristics ofα7nAChR molecular,the relationship with cholinergic anti-inflammatory pathway,anti-inflammatory effects of intracellular mechanisms,clinical application are reviewed.

New evidences of α7nAChR participating in 100 Hz EA regulating NK cells activity / 重庆医学

Objective On the basis of previous experiments ,we try further to seek the new evidences of α7nAChR participating in 100 Hz electroacupuncture (EA ) regulating natural killer (NK ) cells activity .Methods α7nAChR antibody and the specificα7nAChR antagonist α-bungarotoxin (α-Bgt) were used to study the effect of α7nAChR on NK cell activity in 100 Hz EA mice .Re-sults Compared with 100 Hz EA group ,100 Hz EA plus α7nAChR-Ab group showed a significant enhanced in NK cell activity (P<0 .05) .100 Hz EA treated with 1 .5 or 3 .0 μg α-Bgt groups had higher NK cell activity than 100 Hz EA group(P<0 .01) . Conclusion α7nAChR is involved in 100 Hz EA modulation for immune function ,which provide the theoretical and practical basis for further clarifying and expanding the clinical application of EA .