Atropine can induce autophagy independent of the M3 muscarinic acetylcholine receptor

Background: No other effects of atropine other than as an antagonist of muscarinic acetylcholine receptor (mAChR) have been found. Methods: In this study, human kidneyepithelial cells were treated with different physiological regulators. Results: Subsequently, it was found that atropine could significantly induce autophagy as demonstrated by the appearance of autophagosome-like double- or single-membrane vesicles in the cytoplasm ofhost cells and the number of GFP-LC3 dots. In addition, increased conversion of the autophagy marker protein LC3-I and LC3-II and increased p62/SQSTM1 indicatedincomplete autophagy. In addition, atropine induced autophagosome levels in a dose-dependent manner within a certain concentration range in human kidney epithelial cells. In atropine-treated mouse skeletal muscle cells containing nicotinic acetylcholinereceptors and rat cardiac muscle cells containing mAchR, atropine induced autophagy in mouse skeletal muscle cells but not in rat cardiac muscle cells. Furthermore, atropine did not induce autophagy in tissue cells containing mAchR in vivo but did in tissue cells not containing mAchR. Conclusion: This study expands the application and understanding of atropine’s action mechanism in the field of medicine.

Distribution of Like-muscarinic Acetylcholine Receptor M2 in the Brain of Three Castes of Polyrhachis vicina

The cholinergic system plays an important role in the central nervous system of insects and is closely related to the complex behavior of insects. The immunohistochemical technique was performed to detect the expression of like-muscarinic acetylcholine receptor M2 in the brain of three castes of Polyrhachis vicina. A positive expression of like-muscarinic acetylcholine receptor M2 was observed in the mushroom body, central body and antennal lobes of the ant brain; but there is great diversity in their location and intensity among worker, queen and male ants. It is speculated that like-muscafinic acetylcholine receptor M2 plays a critical role in the central nervous system, in terms of projecting visual information and olfactory information into the protocerebrum and integrating many inputs.

基于α7nAChRs的针刺对慢性偏头痛大鼠抗炎作用的研究

目的探讨针刺对慢性偏头痛(Chronic Migraine,CM)大鼠α7烟碱型乙酰胆碱受体(α7 nicotinic acetylcholine receptors,α7nAChRs)影响炎症变化的作用。方法将成年雄性SD大鼠根据基础痛阈随机分为对照组(VEH组)、模型组(NTG组)、模型+针刺组(TA组)、模型+针刺+α7nAChRs拮抗剂MLA组(MLA组)、模型+α7nAChRs激动剂PNU-282987组(PNU组)。采用隔日颈背部皮下重复注射硝酸甘油(NTG)建立CM大鼠模型。TA组和MLA组于NTG注射前1 h针刺,20 min/天,连续9天。MLA组于针刺前0.5 h腹腔注射MLA,PNU组于NTG注射前0.5 h腹腔注射PNU-282987,连续9天。采用Von Frey和热辐射测痛仪检测各组大鼠足底机械痛阈值(Paw Withdrawal Mechanical Threshold,PWMT)和甩尾潜伏期(Tail-Flick Latency,TFL)的变化;ELISA法检测各组大鼠血清及TNC中炎性因子IL-1β、TNF-α和TGF-β含量;免疫荧光双标法检测TNC中GFAP和α7nAChRs的平均光密度和共表达情况。结果与VEH组相比,NTG组PWMT、TFL明显降低(P<0.05或P<0.01),血清和TNC中IL-1β(P<0.01)、TNF-α(P<0.01,P<0.05)含量明显增多,TNC中AS明显活化(P<0.01),而α7nAChRs平均光密度和GFAP与α7nAChRs共表达明显减少(P<0.05,P<0.01);与NTG组相比,TA组PWMT、TFL升高(P<0.05或P<0.01),血清和TNC中IL-1β(P<0.01)、TNF-α(P<0.01,P<0.05)含量增多,TNC中GFAP平均光密度明显降低(P<0.01),而α7nAChRs平均光密度和GFAP与α7nAChRs共表达明显增加(P<0.01);与MLA组比较,TA组和PNU组PWMT、TFL明显升高(P<0.05或P<0.01),TA组血清和TNC中IL-1β(P<0.05)、TNF-α(P<0.01)含量增多而TGF-β(P<0.05)含量减少,PNU组血清和TNC中IL-1β含量减少(P<0.01,P<0.05)而TGF-β(P<0.01)含量明显增多。结论针刺能够有效缓解CM炎症反应和痛觉超敏状态,其抗炎镇痛效应可能与上调α7nAChRs的表达有关。

激活α7nAchR促进肥胖小鼠的脂肪稳态和米色脂肪生成及产热作用

目的观察肥胖小鼠脂肪组织形态学改变以及脂代谢、炎症等相关指标的异常表现,探索激活α7烟碱型乙酰胆碱受体(α7 nAchR)促进肥胖机体白色脂肪米色化产热的作用机制。方法取诱导成肥胖小鼠40只和10只低脂进食小鼠,分为空白组、高脂组、模型组、激动剂组、抑制剂组(10只/组)。苏木素-伊红(HE)染色观察小鼠附睾白色脂肪组织,评估细胞数量、大小及形态。ELISA检测白色脂肪组织肿瘤坏死因子(TNF-α)、白细胞介素-1(IL1β)、白细胞介素10(IL10)、转化生长因子-β(TGF-β)表达水平。qRT-PCR检测白色脂肪一氧化氮合酶(iNOS)、精氨酸酶1(Arg1)mRNA。PCR检测解偶联蛋白(UCP-1)、PR结构域蛋白16(PRDM-16)、线粒体生成的关键调节因子(PGC-1α)mRNA水平。Western blot检测白色脂肪核转录因子P65(NF-κB P65)、磷酸化蛋白酪氨基酸激酶2(p-JAK2)、磷酸化传导及转录激活因子3(p-STAT3)表达水平。结果与空白组比较,高脂组体质量明显增加(P<0.01),白色脂肪组织中出现较多脂肪空泡,脂滴明显增大,iNOS mRNA及TNF-α、IL-1β水平升高(P<0.01),而Arg-1 mRNA及IL-10、TGF-β水平降低(P<0.01);而与模型组相比,药物干预的3组体质量均有所减轻(P<0.05),白色脂肪中脂滴缩小。激动剂组白色脂肪中PRDM-16、PGC-1α、UCP-1 mRNA下降最为明显。而激动剂组TNF-α、IL-1β水平降低(P<0.05,P<0.01),IL-10、TGF-β水平升高(P<0.01),M1/M2巨噬细胞比值降低。结论激活α7 nAchR后可以改善应用β3受体激动剂产生的白色脂肪组织稳态受损,促进白色脂肪中M1型巨噬细胞向M2型巨噬细胞转化减轻白色脂肪炎症反应,促进白色脂肪组织米色化,提高米色化产热效能。

Anisodine hydrobromide alleviates oxidative stress caused by hypoxia/reoxygenation in human cerebral microvascular endothelial cells predominantly via inhibition of muscarinic acetylcholine receptor 4

Background:Anisodine hydrobromide(AT3),an anti-cholinergic agent,could be delivered to the brain across the blood-brain barrier and has been used clinically for the treatment of cerebral ischemia/reperfusion injury.Endothelial dysfunction can be caused by hypoxia/reoxygenation(H/R)via oxidative stress and metabolic alterations.The present study investigated whether AT3 regulates the production of nitric oxide(NO)and reactive oxygen species(ROS),and the HIF-1αpathway via regulation of muscarinic acetylcholine receptors(mAChRs)in brain microvascular endothelial cells after H/R exposure.Methods:Under H/R conditions,hCMEC/D3 cerebral microvascular endothelial cells were treated with AT3.Specific inhibitors of M2-and M4-mAChRs were used to explore the mechanism by which AT3 influences oxidative stress in endothelial cells.Then,mAChRs expression was detected by western blotting and NO production was detected by Greiss reaction.The intracellular ROS level was measured using DCFH-DA probes.The expression of hypoxia-inducible transcription factor 1α(HIF-1α)was also detected.Results:While H/R induced the expression of M2-and M4-mAChRs,AT3 suppressed the H/R-upregulated M2-and M4-mAChRs.H/R also induced the production of NO,ROS,and apoptosis.AT3 and M4-mAChR inhibitors inhibited the H/R-induced production of NO and ROS and apoptosis.HIF-1αwas induced by H/R,but was suppressed by AT3.Conclusion:Thus,the in vitro evidence shows that AT3 protects against H/R injury in cerebral microvascular endothelial cells via inhibition of HIF-1α,NO and ROS,predominantly through the downregulation of M4-mAChR.The findings offer novel understandings regarding AT3-mediated attenuation of endothelial cell apoptosis and cerebral ischemia/reperfusion injury.

Nicotinic acetylcholine signaling is required for motor learning but not for rehabilitation from spinal cord injury

Therapeutic intervention for spinal cord injury is limited,with many approaches relying on strengthening the remaining substrate and driving recovery through rehabilitative training.As compared with learning novel compensatory strategies,rehabilitation focuses on resto ring movements lost to injury.Whether rehabilitation of previously learned movements after spinal cord injury requires the molecular mechanisms of motor learning,or if it engages previously trained motor circuits without requiring novel learning remains an open question.In this study,mice we re randomly assigned to receive intrape ritoneal injection with the pan-nicotinic,non-competitive antagonist mecamylamine and the nicotinicα7 subunit selective antagonist methyllycaconitine citrate salt or vehicle(normal saline)prior to motor learning assays,then randomly reassigned after motor learning for rehabilitation study post-injury.Ce rvical spinal co rd dorsal column lesion was used as a model of in complete injury.Results of this study showed that nicotinic acetylcholine signaling was required for motor learning of the single pellet-reaching task but it was dispensable for the rehabilitation of the same task after injury.Our findings indicate that critical diffe rences exist between the molecular mechanisms supporting compensatory motor learning strategies and the restoration of behavior lost to spinal cord injury.

高原低氧暴露下海马中M1AChR表达对雄性大鼠学习和记忆的影响

目的 考察高原低氧暴露下海马中毒蕈碱型乙酰胆碱受体M1(muscarinic acetylcholine receptor M1,M1 AChR)表达对雄性大鼠学习和记忆的影响。方法 将大鼠随机分为常氧对照组(NC组)、常氧组(N组)、低氧组(H组)和低氧+TAK-071组(HT组)。采用定位巡航实验和空间探索实验分别考察大鼠的学习和空间记忆能力;采用苏木素-伊红染色(hematoxylin-eosin staining,H&E)技术观察大鼠额叶和海马的组织形态;采用免疫组织化学(immunohistochemical,IHC)染色和蛋白质印迹(Western Blot,WB)技术测定大鼠海马组织中M1 AchR的蛋白表达水平;采用荧光定量PCR(quantitative PCR,qPCR)技术测定大鼠海马组织中M1 AchR和叉头框蛋白P2(forkhead box protein P2,FOXP2)的基因表达水平。结果 经过Morris水迷宫(morris water maze,MWM)训练后,大鼠海马组织中M1 AChR的蛋白总体表达水平明显降低(P<0.05),但在海马CA1区、CA3区显著增加(P<0.05)。低氧环境会明显改变大鼠在定位巡航实验中的表现:逃避潜伏期时长增加;明显改变大鼠在空间探索实验中的表现:在目标象限的停留时间减少(P<0.05)。激活M1AChR后,大鼠海马组织中因低氧增加的M1 AChR明显降低(P<0.05)。结论 激活M1 AChR可以改善高原低氧对雄性大鼠的记忆损伤,减缓记忆衰退进程;对学习改善无影响。推测M1 AChR在雄性大鼠认知功能中的作用可能是保持已有记忆。

毕赤酵母表达重组红林蚁AChE高密度发酵工艺研究

【目的】优化毕赤酵母高密度生产重组红林蚁乙酰胆碱酯酶(AChE)的发酵条件,为AChE在农药残留检测中应用提供酶源。【方法】采用毕赤酵母表达系统,选用高表达AChE的重组子X33/pPICZαAAChE,对毕赤酵母高密度生产重组红林蚁AChE的发酵条件进行优化,确定最佳发酵pH值、温度、溶解氧、补料流加速率等因素。【结果】毕赤酵母表达重组红林蚁AChE高密度发酵工艺的最佳参数:pH5.0、溶解氧30%、温度29℃、转速500 r/min、罐压0.06 MPa,甲醇诱导72~96 h时发酵液上清AChE活性增长最快。从发酵结果看,发酵结束菌体湿重可达280 mg/mL,发酵罐内甲醇诱导96 h酶活性最高,达6 000 U/mL,是摇瓶诱导表达的8倍。【结论】毕赤酵母表达系统最佳发酵工艺能实现红林蚁AChE的高密度发酵生产,降低AChE提取成本。

齐拉西酮与利培酮治疗精神分裂症对患者认知功能及血清GABA、INH、Ach水平的影响

目的分析精神分裂症患者应用齐拉西酮、利培酮治疗后其认知功能、γ-氨基丁酸(GABA)、抑制素(INH)、乙酰胆碱(Ach)等变化情况。方法采用随机数字表法将100例于2020年6月至2023年6月,从河北省唐山市第五医院精神科选取的精神分裂症患者分为试验组和对照组,各50例。试验组和对照组分别口服盐酸齐拉西酮片、利培酮片,疗程均为2个月。比较两组疗效(治疗2个月后)、精神症状评分、认知功能、神经递质水平(治疗前和治疗2个月后)及安全性(治疗期间)。结果试验组治疗2个月后总有效率为92.00%,高于对照组的76.00%,差异有统计学意义(χ^(2)=4.762,P<0.05)。两组治疗2个月后较治疗前比较,阳性、阴性、精神病理症状评分及总分和血清INH、Ach水平降低,试验组较对照组更低,差异有统计学意义(t=9.449、5.243、20.206、17.310、15.229、7.345,P<0.05);两组治疗2个月后较治疗前比较,即刻、延迟、视觉、延迟视觉记忆评分及总分和血清GABA水平升高,试验组较对照组更高,差异有统计学意义(t=10.657、4.413、3.655、6.451、25.120、11.356,P<0.05)。治疗期间,试验组不良反应发生率为5.00%,低于对照组的26.00%(χ^(2)=4.336,P<0.05)。结论相比利培酮,齐拉西酮可明显升高精神分裂症患者血清GABA水平,降低其血清INH、Ach水平,改善神经功能,同时可改善患者精神症状及认知功能,提高疗效,但二者安全性相当。

基于α7nAChR/NF-κB通路研究点按脾俞、胃俞穴对缓解慢性疲劳综合征大鼠外周炎症的效应机制

目的观察点按脾俞、胃俞穴对慢性疲劳综合征(chronic fatigue syndrome,CFS)模型大鼠疲劳状态、运动行为、外周炎症及α7烟碱型乙酰胆碱受体(α7 nicotinic acetylcholine receptor,α7nAChR)/核因子κB(nuclear factor-κB,NF-κB)信号表达的影响,探讨点按脾俞、胃俞穴对缓解CFS大鼠外周炎症的效应机制。方法先将32只SD大鼠随机分为空白组(8只)和造模组(24只)。造模组采用强迫负重游泳联合慢性应激刺激的方法建立CFS大鼠模型。造模成功后,将造模组大鼠随机分为模型组、点按组、α7nAChR激动剂组,每组8只。空白组、模型组予腹腔注射等体积生理盐水;点按组以自制按法刺激仪点按双侧脾俞、胃俞穴,20 min/次,并予腹腔注射等体积生理盐水;α7nAChR激动剂组予腹腔注射α7nAChR激动剂PNU-282987(每次2.4 mg/kg)。每组每日干预1次,连续14 d。记录各组大鼠一般情况半定量评分、力竭游泳时间和旷场实验运动距离;计算大鼠脾脏指数和胸腺指数;采用ELISA法检测大鼠血清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)的含量;Western blot检测大鼠脾脏组织α7nAChR、NF-κB p65、NF-κB p-p65、TNF-α的蛋白表达水平;采用qPCR法检测α7nAChR、NF-κB p65、TNF-α的mRNA表达水平。结果与空白组比较,模型组一般情况半定量评分,血清TNF-α、IL-1β、IL-6含量,脾脏组织中NF-κB p-p65、NF-κB p65、TNF-α蛋白表达水平及NF-κB p65、TNF-αmRNA表达量均升高(P<0.01);力竭游泳时间、旷场实验运动距离、脾脏指数、胸腺指数、脾脏组织中α7nAChR蛋白表达水平及mRNA表达量均降低(P<0.05,P<0.01)。与模型组比较,点按组与α7nAChR激动剂组一般情况半定量评分,血清TNF-α、IL-1β、IL-6含量,脾脏组织中NF-κB p-p65、NF-κB p65、TNF-α蛋白表达水平及NF-κB p65、TNF-αmRNA表达量均降低(P<0.05,P<0.01);力竭游泳时间、旷场实验运动距离、脾脏指数、胸腺指数、脾脏组织中α7nAChR蛋白表达水平及mRNA表达量均升高(P<0.05,P<0.01)。与点按组比较,α7nAChR激动剂组血清TNF-α、IL-1β、IL-6含量,脾脏组织中NF-κB p-p65、NF-κB p65、TNF-α蛋白表达水平及NF-κB p65、TNF-αmRNA表达量均降低(P<0.01),脾脏组织中α7nAChR蛋白表达水平及mRNA表达量均升高(P<0.05,P<0.01)。结论点按脾俞、胃俞穴可有效改善CFS大鼠的疲劳状态和运动行为,提高免疫水平并缓解外周炎症,这可能与激活α7nAChR的表达,从而抑制NF-κB下游炎性通路的活性相关。

点击化学制备AChE/MAO-B双抑制剂及活性评价

通过点击化学(Click)设计、合成了22个香豆素衍生物。多数化合物在微摩尔范围内表现出良好的AChE和MAO-B双抑制活性,具有良好的生物相容性。尤其是一种名为A2B5的化合物,对AChE和MAO-B的IC_(50)分别为(0.23±0.02)、(0.31±0.03)μmol/L,是最佳的AChE/MAO-B双抑制剂。实验结果表明香豆素基团是一类有效的AChE和MAO-B双抑制活性基团,羟基取代苯环的存在能够增强抑制活性。分子对接研究发现A2B5是双位点抑制剂,苯基部分结合到AChE的CAS位点,香豆素结合到PAS位点,三氮唑环占据两个活性位点的中间峡谷。A2B5的香豆素部分结合到MAO-B的入口空腔,苯基部分结合到底物空腔,并嵌入到Tyr435、Tyr398和FAD形成的“芳香笼”。总之,香豆素C7位置取代衍生物可以被开发为AChE/MAO-B双抑制剂,为进一步开发抗阿尔茨海默病的双靶点药物提供一个新的起点。

Clinical Measurement of Antibodies against Acetylcholine Receptor (AchR),SOD and LPO in Patients with Myasthenia Gravis (MG)before and after Thymectomy

The antibodies against acetylcholine receptor AchR and levels of SOD and LPO were measured in 11 patients with myasthenia gravis (MG),and the results were compared with normal controls and patients with diseases other than MG.The results showed that the antibodies against AchR were higher as compared with other groups before and after operation. The post-operative level of antibodies was obviously lower than the pre-operative value. An slight increase in SOD and significant decrease in mean value of LPO after surgery were noted. The possible mechanism was discussed.

香豆素衍生物AChE抑制剂的3D-QSAR研究

采用Topomer CoMFA方法研究了44种香豆素衍生物AChE抑制剂的三维定量构效关系D-QSAR模型。建立的Topmer CoMFA模型的交互验证相关系数q^(2)为0.606,拟合相关系r^(2)=0.996,标准估计误差SEE=0.046,Fisher检验值F=891.096,由此可见该模型是有效的。通过Topomer CoMFA模型三维等势图揭示了结构对活性的影响。并采用分子对接研究了配体和受体蛋白之间的结合。该模型为进一步探索黄酮类衍生物作为AChE抑制的构效关系,以及该类分子的结构设计奠定理论基础。

α7nAChR抑制MyD88依赖性Zonulin释放改善脑梗死大鼠肠道炎症和肠屏障损伤

目的:观察α7型烟碱乙酰胆碱受体(α7nAChR)激动剂PNU282987对脑梗死大鼠肠道炎症和肠屏障损伤的影响。方法:将36只雄性SD大鼠随机分为假手术(sham)组、中脑动脉栓塞(MCAO)组和PNU282987组。MCAO组和PNU282987组采用Longa改良线栓法制备MCAO模型,PNU282987组腹腔注射1 mg/kg PNU282987,连续7 d。苏木精—伊红(HE)染色观察肠道黏膜病理改变,酶联免疫吸附试验(ELISA)检测结肠组织肿瘤坏死因子(TNF-α)、白细胞介素(IL-6)水平及血清二胺氧化酶(DAO)、D-乳酸(DLA)、内毒素(ET)水平,免疫组化法检测结肠组织Toll样受体2(TLR2)蛋白表达,western blotting法检测结肠组织紧密连接关键蛋白咬合蛋白(Occludin)、闭合蛋白(Claudin-1)、连蛋白(Zonulin)、带状闭合蛋白(ZO-1)、TLR2、髓样分化因子88(MyD88)、核因子-κB(NF-κB)和磷酸化NF-κB(p-NF-κB)蛋白表达。结果:与sham组比较,MCAO组大鼠肠上皮脱落、肠绒毛排列紊乱及黏膜下层炎性浸润,血清DAO、DLA、ET水平及结肠组织TNF-α、IL-6含量显著增高,结肠组织Occludin、Claudin-1、ZO-1蛋白表达显著下调,Zonulin、TLR2、MyD88、p-NF-κB蛋白表达显著上调(均P<0.05)。与MCAO组比较,PNU282987组大鼠肠上皮损伤减轻,血清DAO、DLA、ET水平及结肠组织TNF-α、IL-6含量显著降低,结肠组织Occludin、Claudin-1、ZO-1蛋白表达显著上调,Zonulin、TLR2、MyD88、p-NF-κB蛋白表达显著下调(均P<0.05)。结论:PNU282987可以显著改善脑梗死大鼠肠道炎症及肠屏障损伤,其机制可能与抑制TLR2/MyD88/NF-κB信号通路有关。

MTDLs Design on AChE （Acetylcholinesterase） and β-Secretase （BACE-1）： 3D-QSAR and Molecular Docking Studies

To find promising new multitargeted AD （Alzheimer＇s disease） inhibitors, the 3D-QSAR （three-dimensional quantitative structure-activity relationship） model for 32 AD inhibitors was established by using the CoMFA （comparative molecular field analysis） and CoMSIA （comparative molecular similarity index analysis） methods. Results showed that the CoMFA and CoMSIA models were constructed successfully with a good cross-validated coefficient （q2） and a non-cross-validated coefficient （R2）, and the binding modes obtained by molecular docking were in agreement with the 3D-QSAR results, which suggests that the present 3D-QSAR model has good predictive capability to guide the design and structural modification of novel multitargeted AD inhibitors. Meanwhile, we found that one side of inhibitory molecule should be small group so that it would be conductive to enter the gorge to interact with the catalytic active sites of AChE （acetylcholinesterase）, and the other side of inhibitory molecule should be large group so that it would be favorable for interaction with the peripheral anionic site of ACHE. Furthermore, based on the 3D-QSAR model and the binding modes of AChE and [3-secretase （BACE-1）, the designed molecules could both act on dual binding sites of AChE （catalytic and peripheral sites） and dual targets （ACHE and BACE-1）. We hope that our results could provide hints for the design of new multitargeted AD derivatives with more potency and selective activity.

Cloning and Sequence of Nicotinic Acetylcholine Receptor α Subunit from Chilo suppressalis

Nicotinic acetylcholine receptors (nAChRs) play a significant role in excitatory synaptic transmission in insects and are the target for chloronicotinyl and nereistoxin insecticides.In recent years,Chilo suppressalis,an economically important pest of rice,developed high resistance against monosultap,a nereistoxin insecticide acting on nAChR.In order to reveal the hypothesized target insensitive mechanism,studies on the molecular property of nAChR from Chilo suppressalis are required.In this study,the full length cDNA of nAChR α subunit from this pest was cloned by RT-PCR.Sequence analysis shows that it is a novel nAChR α subunit,which was named as Cs α 1(Genbank accession No.AF418987).It contains 1?997?bp nucleotides and involves an open reading frame (ORF) encoding a mature protein of 509 amino acids excluding a signal peptide of 24 amino acids.The deduced amino acid sequence was 52%-94% identical to the reported insect nAChR genes.

EFFECTS OF MOXIBUSTION ON CEREBRAL ACETYLCHOLINE CONTENT AND CHOLINE ACETYL TRANSFERASE ACTIVITY IN THE AGED RATS

Objective To observe the effect of moxibustion of Baihui（百会GV20） and Shenshu（肾俞 BL 23） on acetylcholine （AOh） content and choline acetyl transferase （CHAT） activity in the brain of aging rats so as to investigate its underlying mechanism in delaying brain aging in the rat. Methods Thirty Wistar rats including 10 young rats （young group） and 20 aged rats that were divided into aging group and moxibustion group evenly, were used in this study. For rats of moxibustion group, moxibustion was applied to Baihui （百会GV20） and Shenshu（肾俞 BL23） for 10 min, once a day, with 5 days being a course of treatment, 8 courses altogether. At the end of the experiments, the rats anesthetized with 6 % chloral hydrate were decapitated for taking brain tissue, then AOh content, CHAT and acetylcholinesterase （ACHE） activity were detected by using high performance liquid chromatography （HPLC）. Results Compared with young rat group, AOh contents of basal ganglia, hippocampus and cerebral cortex tissues in aging group and moxibustion group all decreased significantly （P〈 0.05, 0.01 ）, while compared with aging group, ACh contents in the 3 cerebral regions in moxibustion group all increased considerably （P〈0.01）. In comparison with young group, both CHAT activity and AChE activity of the brain tissue in aging group and moxibustion group lowered significantly （ P 〈 0.05, 0.01 ） ; comparison between aging group and moxibustion group showed that CHAT activity of the later group increased evidently （P〈 0.05） while AChE activity in moxibustion group decreased considerably （P〈0.01）, displaying that moxibustion could potentiate CHAT activity and further lower AChE activity of the brain in aged rats. Conclusion Moxibustion of Baihui（百会GV20） and Shenshu（肾俞 BL23） has effects in raising AOh contents and lowering CHAT and AChE activity, which may contribute to its efficacies in repairing the injured central cholinergic nerve system and delaying the aging process in the aged rats.

Stomatal Opening Induced by Acetylcholine Is Associated with Cytoskeletal Components

Acetylcholine (Ach) is a key component of animal cholinergic system. Recent experiments demonstrated that Ach, choline acetyltransferase, acetylcholinesterase and Ach receptors are present in all parts of plants and have many functions, including inducing stomatal movement. The authors' previous work has evidenced that microtubules and microfilaments are involved in regulating both stomatal closing and opening. The present investigation is to determine whether stomatal opening induced by Ach is associated with microtubules and microfilaments. The results showed that Ach could induce stomatal opening of Vicia faba L. with or without addition of KCl in the dark. Ach also stimulated protoplast swelling in a K +_free solution in the dark. However, the induction was partially suppressed when the strips and protoplasts were pretreated with either cytochalasin B, an inhibitor of F_actin polymerization, or oryzalin, an inhibitor of plant microtubule polymerization. Thus, our data suggest for the first time that stomatal opening induced by Ach is associated with the dynamics of microtubules and microfilaments.

Effect of acetylcholine on pain-related electric activities in hippocampal CA1 area of normal and morphinistic rats

Objective To examine the effect of acetylcholine（ACh）on the electric activities of pain-excitation neurons （PEN）and pain-inhibitation neurons（PIN）in the hippocampal CA1 area of normal rats or morphinistic rats,and to explore the role of ACh in regulation of pain perception in CA1 area under normal condition and morphine addiction.Methods The trains of electric impulses applied to sciatic nerve were set as noxious stimulation.The discharges of PEN and PIN in the CA l area were recorded extracellularly by glass microelectrode.We observed the influence of intracerebroventricular （i.c.v.）injection of ACh and atropine on the noxious stimulation-evoked activities of PEN and PIN in the CA1 area.Results Noxious stimulation enhanced the electric activity of PEN and depressed that of PIN in the CA1 area of both normal and addiction rats.In normal rats,ACh decrease the pain-evoked discharge frequency of PEN,while increased the frequency of PIN.These effects reached the peak value at 4 min after injection of ACh.In morphinistic rats,ACh also inhibited the PEN electric activity and potentialized the PIN electric activity,but the maximum effect appeared at 6 min after administration. The ACh-induced responses were significantly blocked by muscarinic receptor antagonist atropine.Conclusion Cholinergic neurons and muscarinic receptors in the hippocampal CA1 area are involved in the processing of nociceptive information and they may play an analgesia role in pain modulation.Morphine addiction attenuated the sensitivity of painrelated neurons to the noxious information.

Septic encephalopathy: When cytokines interact with acetylcholine in the brain

Sepsis-associated encephalopathy(SAE) is a brain dysfunction that occurs secondary to infection in the bo characterized by alteration of consciousness, ranging from delirium to coma, seizure or focal neurological signs. S involves a number of mechanisms, including neuroinflammation, in which the interaction between cytokines a acetylcholine results in neuronal loss and alterations in cholinergic signaling. Moreover, the interaction also occurs the periphery, accelerating a type of immunosuppressive state. Although its diagnosis is not specific in biochemis and imaging tests, it could potentiate severe outcomes, including increased mortality, cognitive decline, progress immunosuppression, cholinergic anti-inflammatory deficiency, and even metabolic and hydroelectrolyte imbalan Therefore, the bilateral communication between SAE and the multiple peripheral organs and especially the immu system should be emphasized in sepsis management.