人参、石菖蒲及其药对防治阿尔茨海默症的药理作用研究进展

阿尔茨海默病(Alzheimer′s disease,AD)病因复杂,发病机制尚未完全阐明,现有药物仅能缓解其症状,亟待研发有效的治疗药物。作为补虚药及开窍药的代表药物,人参及石菖蒲均有提高记忆力、改善学习能力及减轻认知障碍的药理作用,是中医治疗痴呆症的常用药物。人参-石菖蒲药对联用可促进活性成分入脑发挥药效,并通过抗炎、抗氧化应激、调节神经元-突触可塑性等多途径延缓AD进程,具有多层次、多系统和多靶点的作用特点。该文尝试总结现有研究成果,为进一步探讨人参、石菖蒲的联用,实现协同增效的作用机制以及配伍的量效关系奠定基础,为研制防治AD的中药创新药物提供科学依据。

外泌体与阿尔茨海默病研究进展

外泌体是一类纳米级别的细胞外囊泡,其携带的遗传信息在细胞间信息交流中发挥重要作用。阿尔茨海默病(Alzheimer's disease,AD)作为一种神经退行性疾病,具有起病隐匿且逐渐进展的特点,是老年痴呆症的主要类型之一,目前尚无有效的治疗手段。研究显示,外泌体在AD的发生和进展中扮演着重要角色,尤其是外泌体携带的microRNAs(miRNA)在其中具有显著作用。miRNA不仅可以作为AD的生物标志物,还对改善AD患者的认知功能具有潜在益处。该文对外泌体的来源(包括间充质细胞源性外泌体和脑源性外泌体)及与AD治疗相关的药物进行全面综述,以期进一步探讨其在AD中的应用前景。

复肝汤HPLC特征图谱及波长切换法同时测定6种成分的含量

目的建立复肝汤HPLC特征图谱,并测定复肝汤中毛蕊花糖苷、芦丁、槲皮素、木犀草素、芹菜素、丹参酮Ⅱ_(A)6种主要成分的含量。方法采用高效液相色谱法,色谱柱为Agilent 5TC-C_(18)色谱柱(4.6 mm×250 mm,5μm),流动相为甲醇-水,梯度洗脱,流速为1.0 mL·min^(-1),检测波长分别为310 nm(毛蕊花糖苷)、370 nm(芦丁、槲皮素、木犀草素、芹菜素)、250 nm(丹参酮Ⅱ_(A)),柱温30℃,进样量20μL。结果确定复肝汤HPLC特征图谱13个共有峰,利用相似度软件对10批样品特征图谱进行分析,各批样品相似度均在0.95以上。毛蕊花糖苷、芦丁、槲皮素、木犀草素、芹菜素和丹参酮ⅡA的线性范围分别在4.394~87.880μg·mL^(-1)(r=0.9999)、1.501~30.020μg·mL^(-1)(r=0.9999)、0.475~9.505μg·mL^(-1)(r=0.9997)、0.547~10.945μg·mL^(-1)(r=0.9995)、1.028~20.550μg·mL^(-1)(r=0.9998)、0.854~17.070μg·mL^(-1)(r=1.000);平均加样回收率在92.1%~101.3%,RSD在0.78%~1.4%。结论该方法简便、准确、重复性高,并可同时测定复肝汤中6种成分的含量,为复肝汤质量控制提供了参考。

基于UPLC-Q-TOF-MS法分析百合珠芽化学成分及其薯蓣皂苷元抗肿瘤活性研究

为建立超高效液相色谱-四级杆飞行时间质谱(UPLC-Q-TOF-MS)快速鉴定百合珠芽中10种化学成分的分析方法,并对薯蓣皂苷元进行抗肿瘤研究。实验采用Thermo Hypersil Gold C_(18)柱(100 mm×2.1 mm,1.9μm);乙腈(A)-0.5%乙酸(B)为流动相,梯度洗脱;电喷雾离子源(ESI),正负离子同步监测。采用CCK8法进行薯蓣皂苷元抗肺肿瘤A549细胞、胃肿瘤HGC-27细胞的药效筛选。结果表明,通过二级高分辨质谱分析结合相关文献,共鉴定对香豆酸、薯蓣皂苷、槲皮素等10个化合物。药理研究显示,薯蓣皂苷元对肺癌A549细胞增殖只有微弱的抑制活性,而对胃癌HGC-27细胞增殖表现出较强的抑制作用。本试验证实UPLC-Q-TOF-MS/MS可从保留时间、紫外吸收光谱、精确相对分子质量、分子式和二级结构碎片等方面对百合珠芽的主要成分进行定性分析,为寻找百合珠芽中活性物质提供一种快速准确的分析方法,且揭示百合珠芽中鉴定得到的单体成分可能具有较强的抗肿瘤活性。

百合药材、种植土壤及灌溉水中农药及重金属残留分析

对湘产药食两用植物卷丹百合的鲜药、干燥药材、产地土壤及灌溉水的安全性进行评价。采用气相色谱法对总六六六、总滴滴涕、甲氰菊酯、毒死蜱、百菌清进行测定,并用AFS-AAS法测定As、Hg、Pb、Cd、Cr和Cu含量。结果显示,6个采样区的土壤、灌溉水中六六六、滴滴涕、甲氰菊酯、毒死蜱均未见残留,百菌清残留量为ND^0. 126 mg/kg;在百合鲜药、干燥药材中,六六六、滴滴涕、甲氰菊酯、毒死蜱、百菌清5种农药均未测出有残留,另除G-1、D-4样品中Cd残留量超标外,所有样品重金属均符合《植物和制剂对外贸易和贸易的绿色贸易标准》(WM/T2-2004)。根据污染指数评价,龙山具有优良的卷丹百合产地环境,适合生产符合现代质量要求的绿色百合。但研究发现百合对于土壤中镉的富集系数好,有必要加强百合主产区土壤改性进一步研究。

固相萃取-离子色谱法同时测定卷丹产地灌溉水中7种无机阴离子

建立了一种固相萃取-离子色谱法同时测定卷丹百合产地灌溉水中7种无机阴离子的简便、快速分析方法。将采集的灌溉水过SPE-C18小柱去除有机杂质,0.22μm微孔滤膜过滤后,取续滤液进样分析。采用IonPac AS19柱(250×4mm,5μm)为离子交换柱,IonPac AG19柱(50×4mm)为保护柱;以20.0mmol/L KOH溶液为流动相,流速为1.30mL/min,柱温为30℃,可在18min内完成对F^-、Cl^-、Br^-、NO_2^-、NO_3^-、SO_4^(2-)和PO_4^(3-)这7种阴离子的同时分离检测。7种阴离子在较宽范围内有良好的线性关系和精密度,7种阴离子线性范围分别为0.01～20、0.01～20、0.02～20、0.04～20、0.3～20、0.01～20、0.01～20mg/L,相关系数R大于0.9995。平均加标回收率为92.4%～101.9%,相对标准偏差为0.19%～4.47%(n=3)。该方法简便快速、灵敏准确,能同时测定农业灌溉水中上述7种无机阴离子。对卷丹百合产地环境监控具有重要意义。

补阳还五汤类方对氧化应激PC12模型细胞CDK5及凋亡因子的影响

目的观察补阳还五汤类方对氧化应激模型细胞周期素依赖性蛋白激酶5(cyclin-dependent kinase 5,CDK5)及凋亡因子的影响。方法以0.3 mmol·L^-1的H 2O 2与PC12细胞共同作用2 h,成功复制脑缺血体外氧化应激细胞模型,将PC12细胞分成正常组、模型组、补阳还五汤组、补阳还五汤精简方组。酶联免疫法检测胞外培养液中L-LDH的含量和胞内GSH的含量,流式细胞术检测胞内活性氧(ROS)含量,细胞免疫荧光法检测胞内CDK5、Tau、Bax、Bcl-2蛋白的表达,qPCR法检测胞内凋亡因子caspase-3、Bax、Bcl-2 mRNA的表达。结果补阳还五汤类方能有效降低氧化应激模型细胞培养液中L-LDH的含量,增加胞内GSH、降低胞内ROS、抑制模型细胞内CDK5和Tau蛋白的过表达,下调凋亡因子caspase-3和Bax/Bcl-2。结论补阳还五汤类方可能通过CDK5信号转导途径有效抑制神经细胞凋亡,对脑缺血后神经细胞的损伤发挥保护作用。

玄参化学成分的研究

目的研究玄参Scropularia ningpoensis Hemsl.的化学成分。方法玄参80%甲醇提取物采用硅胶和Sephadex LH-20进行分离纯化,根据理化性质及波谱数据鉴定所得化合物的结构。结果从中分离得到13个化合物,分别鉴定为对羟基肉桂酸甲酯(1)、3, 4-二甲氧基肉桂酸(2)、对羟基苯甲醛(3)、3-羟基对甲氧基苯甲醛(4)、对羟基苯乙酮(5)、肉桂酸(6)、2-(3′,4′-二羟基苯基)-1,3-苯并二茂恶-5-甲醛(7)、3-羟基对甲氧基苯甲酸(8)、对羟基肉桂酸(9)、阿魏酸(10)、3-甲氧基对羟基肉桂醛(11)、对羟基苯乙醇(12)、5-羟甲基糠醛(13)。结论化合物2、4、7～8、11～12为首次从该植物中分离得到。

分散固相萃取HPLC测定玄参中五种指标性成分

目的建立分散固相萃取-高效液相色谱方法应用于玄参中5种指标性成分的快速提取、净化和测定的方法,与传统方法进行比较。方法用分散固相萃取-高效液相色谱方法测量玄参中桃叶瑚珊苷、哈巴俄苷、安格洛苷、哈巴苷、肉桂酸成分,采用C 18柱(250mm×4.6mm,5μm),流动相为0.01%磷酸水溶液(B)-乙腈(A),检测波长210nm及250nm,体积流量1mL·min-1,进样量20μL,柱温25℃。结果该方法五种成分测量回收率分别为RSD值为1.91%、1.23%、1.22%、1.84%、1.32%。结论分散固相萃取-高效液相色谱方法、传统方法都具有较好的精密度(RSD≤1.51%)、稳定性(RSD≤2.00%)和重现性(RSD≤3.11%)。与普通方法比较,分散固相萃取预处理方法减少了背景干扰,为复杂基质玄参活性成分分析研究提供了较好的方法。

黑质注射LPS构建亚急性帕金森病模型的评价

目的向黑质部位连续注入脂多糖(lipopolysaccha-ride,LPS)建立亚急性帕金森病(Parkinson’s disease,PD)小鼠模型。方法将60只雄性C57BL/6小鼠随机分为3组,模型组向双侧黑质注射LPS(2.5μg/侧),溶剂组以相同方法注射生理盐水,空白组不给予任何处理。连续注射5 d后,观察小鼠PD样行为学变化。免疫组化法观察黑质致密部多巴胺(DA)能神经元及活化小胶质细胞数目的变化,实时荧光定量PCR检测小鼠黑质内炎症因子的含量和West-ern blot观察不同形式α-突触核蛋白(α-synuclein,α-Syn)的变化。结果与溶剂组相比,模型组小鼠出现明显的PD样行为表现。与空白组相比,溶剂未对小鼠产生行为学障碍。模型组黑质致密部的DA能神经元显著减少,活化小胶质细胞显著增多,中脑内炎症因子mRNA的表达显著上调和三种形式的α-Syn的蛋白表达显著增加。结论经双侧黑质连续5 d注入LPS可诱导较为稳定的亚急性PD小鼠模型。

Chemokines play complex roles in cerebral ischemia

Ischemic stroke(IS) is a disease caused by deficiency of blood and oxygen in focal or complete brain,followed by inflammation cascade and other pathological reactions,which finally lead to irreversible damage to the cerebrum.For the inflammation is a key progress at the initiation of ischemia and poststroke,and chemokines work as vital cytokines in inflammation,we focus the roles of chemokines in IS.Studies have shown cerebral ischemia is associated with marked induction of both CXC and CC chemokines which resulting in extensive leukocyte infiltration in the ischemic brain,and neutrophil infiltration may increase cerebral edema inducing injury in the ischemic area.In addition,chemokines also shows other functions such as promote neuroblast migration,hematogenous cell recruitment and functional brain repair.Thus,a similar chemokine ligand/chemokine receptor pair can mediate both beneficial and detrimental effects depending on the window of observation and pathophysiological conditions.This manuscript reviews the studies about chemokine-mediated effects in cerebral ischemia/reperfusion and discusses the potential significance of these interactions in injury and repair of ischemic tissues.We also refer drug development based on the chemokines and clinical applications using chemokines as diagnostic or prognostic biomarkers in ischemic stroke.

Inhibition of chemokine-like factor 1 improves bloodbrain barrier dysfunction in rats following focal cerebral ischemia

OBJECTIVE To investigate the role of chemokine-like factor 1(CKLF1),a novel C-C chemokine,on brain-blood barrier(BBB)integrity in rat focal cerebral ischemia and reperfusion model.METHODS Antibodies against CKLF1 was applied to the rightcerebral ventricle immediately after transient middle cerebral artery occlusion.Brain water content,Evans blue leakage and the expression of aquaporin-4(AQP-4),matrix metalloproteinase-9(MMP-9),zonula occludens-1(ZO-1)and occludin were measured.RESULTS After treatment with antiCKLF1 antibody,brain water content and Evans blue leakage in ipsilateral hemisphere were decreased in a dose-dependent manner at 24 h after reperfusion,but not changed in contralateral hemisphere.Anti-CKLF1 antibody reduced the expression of AQP-4 and MMP-9,and upregulated the expression of ZO-1 and Occludin.These results suggest that CKLF1 is involved in BBB disruption after reperfusion.CONCLUSION Inhibition of CKLF1 protects against cerebral ischemia by maintaining BBB integrity,possibly via inhibiting the expression of AQP-4 and MMP-9,and increasing the expression of tight junction protein.

Ginsenoside Rg1 protects against ischemia reperfusion-induced neurotoxicity through miR-144/Nrf2/ARE pathway

OBJECTIVE Ginsenoside Rg1(Rg1),a purified compound from Panax ginseng,has been well documented to be effective against ischemia/reperfusion(I/R) neurotoxicity.However,the underlying mechanism is stil obscure.METHODS The anti-I/R effect of Rg1 were investigated in vitro and in vivo,and the dynamics of nuclear accumulation and the transcriptional activity of NF-E2-related factor 2(Nrf2) determined by Western blotting and Dual Luciferase Reporter Assay,respectively.Nrf2 siRNA was employed to investigate Nrf2′s role in the protective effect of Rg1 against I/R.Furthermore,the role of miR-144,which could regulate post-translational Nrf2 levels,was investigated in the anti-I/R effect of Rg1 by injection of AAV-hypoxia-inducible factor miR-144-shRNA in the predicted ischemic penumbra.RESULTS It was found that the anti-I/R effect of Rg1 was related to its anti-oxidative capacity,which is mainly regulated by the Nrf2/antioxidant response element(ARE) pathway.Further study suggested that Rg1 contributes to the enhancement of the Nrf2/ARE pathway,as manifested by increasing the dynamic peak content of Nrf2,which prolonged the maintenance stage,and promoting the expression of ARE-target genes after oxygen glucose deprivation/reperfusion(OGD/R) in PC12 cells.Nrf2-siRNA application significantly reduced these changes.Furthermore,the enhancement of the Nrf2/ARE pathway by Rg1 was independent of disassociation from Keap1;rather it was a result of posttranslational regulations.It was found that Rg1 significantly reduced the expression of miR-144,which down-regulates Nrf2 production by targeting its 3′-untranslated region,after OGD/R.Knockdown of Nrf2 showed no effect on the expression of miR-144,indicating that miR-144 is an upstream regulator of Nrf2.Moreover,direct binding between Nrf2 and miR-144 in the PC12 cells was identified.Application of anti-miR-144 significantly reduced Rg1′s anti-OGD/R capacity.Final y,the role of miR-144 in Rg1′ s anti-I/R effect was tested by inhibiting miR-144 in the predicted ischemic penumbra when hypoxia-inducible-factor was activated.The results showed that loss of miR-144 abolished the anti-I/R effect of Rg1,which included reduced infarct volume,improved neurological scores,attenuated oxidative impairment,as well as activation of the Nrf2/ARE pathway.CONCLUSION Oxidative stress after I/R is alleviated by Rg1 through inhibition of miR-144 activity and subsequent promotion of the Nrf2/ARE pathway at the post-translational level.

Prion-like propagation of α-synuclein in gutbrain axis

Parkinson disease(PD) is a progressive degenerative disease of the nervous system,which is characterized by movement disorders,such as static tremor,rigidity,and bradykinesia in advanced patients.Gastrointestinal(GI) dysfunction,such as gastric dysmotility,constipation,and anorectic dysfunction,is common non-motor symptom in the early stage of PD.The progression of PD includes the degenerative loss of dopaminergic neurons and aggregation ofα-synuclein in the substantia nigra.Interestingly,both of them are also present in the enteric nervous system of PD patients.In this review,we describe the relationship between non-motor symptoms particularly GI dysfunction and the pathogenesis of PD,aiming to show the powerful evidences about the prion-like propagation of α-synuclein and support the hypothesis of gut-brain axis in PD.We then summarize the mechanism of the gut-brain axis and confirmα-synuclein as a potential target for drug design or new clinical treatment.

IMM-H004 therapy for permanent focal ischemic cerebral injury and cardiopulmonary complications via CKLF1/CCR4-mediated inflammation pathway

OBJECTIVE To investigate the effects of IMM-H004 on permanent focal cerebral ischemia injury and associated cardiopulmonary complications,further elucidating the molecular mechanisms.METHODS The effects of IMM-H004 were investigated in wild-type(WT) and CKLF1-/-rats.The effects of IMM-H004 on ischemic stroke injury and its cardiopulmonary complications were determined using 2,3,5-triphenyltetrazolium chloride(TTC) staining,behavior tests,magnetic resonance imaging(MRI)scans,enzyme-linked immunosorbent assay(ELISA),Nissl staining,and histo-pathological examination.Multiple molecular experiments including immunohistological staining,immunofluorescence staining,quantitative RT-PCR,Western blotting,and co-immunoprecipitation assays were used to elucidate the underlying mechanisms.RESULTS IMM-H004 treatment provided significant protection against ischemic stroke-induced brain injury and associated cardiopulmonary complications,through CKLF1-depedent-anti-inflammation pathway in rats.IMM-H004 downregulated the amount of CKLF1 and disturbed the combination between CKLF1 and C-C chemokine receptor type 4,suppressing the inflammatory response and protecting the damaged organs in ischemic setting.CONCLUSION This preclinical study established efficacy of IMM-H004 as a potential therapeutic medicine for ischemic stroke and associated cardiopulmonary complications.The protective effects of IMM-H004 may due to its specific mechanism through CKLF1.These results support further efforts to develop IMM-H004 for human clinical trials in acute cerebral ischemia,especially for patients who are not suitable for reperfusion therapy.

Roles of calretinin interneurons in APP / PS1 mouse model

OBJECTIVE To explore the changes of calretinin interneurons in APP/PS1 mouse model and their correlation with the pathological features of Alzheimer disease.METHODS The morphological differences between the brain regions of control and transgenic(TG) mice were detected by Nissl staining.By immunofluorescence histochemistry and western blotting,the expression of related proteins was detected,including GAD65/67,calretinin and Aβ1-42.At the same time,in terms of behavioral experiments,the motor ability,the level of fear and cognitive level of control and transgenic mice were tested through open-field test,elevated plus maze and novel object recognition(NOR) experiment.RESULTS According to Nissl staining results,compared with the control group,the number of cells in glomerular layer(GL) and granule cell layer(GCL) of olfactory bulb in the TG group was significantly reduced.Meanwhile,in the hippocampus,compared with the control group,the ventral CA1 and CA3 is dysplastic in APP/PS1 group.Moreover,the morphology of subgranular zone(SGZ) layer cells in the dorsal DG significantly changed and the number of cells in hilar regions significantly decreased in the TG group.Other brain regions associated with cognition,including the piriform cortex,entorhinal cortex and the prefrontal cortex,showed no significant changes between the control and TG group.By Western blotting experiment,compared with the control mice,it was found that the expression of GAD65/67 and calretinin in the olfactory bulb and hippocampus was significantly decreased in the TG group.In detail,the level of the protein in the relevant brain regions was detected by immunofluorescence histochemistry(IHC).The expression of calretinin in GL of the olfactory bulb and the hippocampal DG region was significantly decreased,which was correlated with the distribution of Aβ1-42 by IHC.In the part of functional behavioral experiment,compared with the control mice,the total distance of movement in 6 M TG group mice was significantly increased in the open field test.In the elevated plus maze test,there were no significant differences between control and TG group.In addition,the mice of TG group lacked the ability to distinguish between old and new objects in the NOR experiment.CONCLUSION The number of calretinin interneurons in olfactory bulb and hippocampus decreased significantly in APP/PS1 mouse model,which is more correlated with the distribution of Aβ1-42 and functional disorders,including the enhancement of spontaneous locomotor activity and cognitive impairment.

CKLF1 aggravates focal cerebral ischemia injury at early stage partly by modulating microglia/macrophage toward M1 polarization

OBJECTIVE To investigate the CKLF1 mediated expression of microglia/macrophage phenotypes in vitro and in vivo,discussing the involved pathway.METHODS In vitro,primary microglia isolated from mice cortex were used to study the effects of CKLF1 by qPCR analysis and immunofluorescence staining.In vivo,WT C57 and CKLF1 deficient mice were used to explore the effects of CKLF1.TTC staining,MRI and Nissl staining were applied to examine the infarction or neuron loss.Zea longa test was used to evaluate the neurological deficit of mice.Western blotting was used to investigate the changes of specific protein and discuss the involved pathway.We also used qPCR analysis and immunofluorescence staining for polarization markers to determine the effects of CKLF1.RESULTS CKLF1 could drive primary microglia to M1 phenotype for 24 h stimulation in primary microglia.In mice transient ischemic stroke model,CKLF1 attenuated ischemic injury,and accompanied by promoting microglia/macrophage toward M1 polarization.Increased expression of pro-inflammatory cytokines and decreased expression of neurotropic factors and anti-inflammatory cytokines were observed in mice subjected to cerebral ischemia with C27.Moreover,NF-κB activation enhancement was detected in C27 modulated M1 polarization effects.CONCLUSION CKLF1 is an important mediator of driving M1 phenotype of microglia/macrophage at early stage of cerebral ischemic injury,contributing to aggravation of cerebral ischemia injury,which closely related to microglia/macrophage M1 polarization guided inflammatory response.Targeting CKLF1 has the potential to treat ischemic stroke.

Pharmacological effects of ginsenoside Rg1 in neuropsychopharmacology

Panax Ginseng has been used for thousands of years in traditional Chinese medicine(TCM)as a tonic to improver stamina and vitality.Ginsenoside Rg1(Rg1),a saponin extracted from Panax ginseng,is considered one of the most potent pharmacological candidates among TCM.In various diseases related to nervous system,Rg1 has shown excellent pharmacological activities.①Stroke:Rg1 has been well documented to be effective against ischemic/reperfusion(I/R)neuronal injury.A systematic review and meta-analysis revealed a marked efficacy of Rg1 in experi⁃mental acute ischemic stroke,as manifested by its ability to reduce infract volume and improve neurological score.The protective effects of Rg1 were abolished by injecting of AAV-HIF-miR-144-shRNA into the predicted ischemic penumbra.②Depression:In addition,Rg1 showed antidepressive effects in chronic unpredictable mild stress(CUMS)model of depression and in gonadectomized(GDX)model of neuroendocrine disturbance.Rg1 displayed antidepressant activity through the modulation of HPA and HPG axis,markedly alleviated depression-like behavior in rats.Long-term Rg1 treat⁃ment of CUS-exposed rats also significantly prevented the decrease in dye diffusion and improved the ultrastructure of astrocyte gap junctions in the PFC.Rg1 upregulated Cx43 expression in PFC reduced by CUS exposure,indicating beneficial effects on the functional activity of gap junction channels in the brain.③Parkinson disease(PD):Oral treatment with Rg1 significantly attenuated high MPTP-induced mortality,behavior defects,loss of dopamine neurons and abnormal unltrastructure changes in SNpc.It regulated MPTP-induced reactive astrocytes and microglia and decreased the release of cytokines such as TNF-alpha and IL-1βin SNpc.Rg1 also alleviated the unusual MPTP-induced increase in oligomeric,phosphorylated and disease-relatedα-synuclein in SNpc.④Alzheimer disease(AD):Okadaic acid(OKA)intracerebroventricular injection induced memory impairment,including changes in the ability of orientation navigate,spatial probe and relearning memory in behavioral test of Morris water maze(MWM).OKA treated rats showed memory impair⁃ment including increasing of phospho-tau,decreasing of phospho-GSK3βand the formation ofβ-amyloid in special brain regions,which were reversed by Rg1.The possible neuroprotective mechanism might be that Rg1 decreases OKAinduced memory impairment through GSK3β/tau signaling pathway and/or attenuating Aβformation.Meanwhile,Rg1 activated ERK/MAPK pathway by CaMKIIα,and the activation of CREB was not only dependent on ERK induced by Rg1.Additionally,Rg1 inhibited microglial activation by suppressing Iba1 expression.Rg1 inhibited the inflammation mediated by LPS through suppressing NF-κB and MAPK pathway,which provided the explanation for its therapeutic ef⁃fect on neurodegenerative diseases.

基于“肾藏精”理论探讨外泌体与阿尔茨海默病的防治

“肾藏精”理论是中医基础理论的重要组成部分,来源于《素问·六节藏象论》中的记载:“肾者主蛰,封藏之本,精之处也。”该理论认为,精是人体生命活动的根本物质,它与人体的生长发育有着密切关系。阿尔茨海默病(Alzheimer’s disease, AD)是常见的神经退行性疾病之一,其主要病理特征为β-淀粉样蛋白(amyloid β-protein, Aβ)沉积和Tau磷酸化,这些会激活神经毒性反应,最终导致神经元功能障碍和细胞死亡,严重损害患者的认知和记忆功能。目前针对AD在中医治疗已经取得了一定的成果,但AD发病机制复杂,能够彻底治疗的药物研发也存在一些困难。干细胞疗法是促进机体自我修复和再生的重要方法,骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)属于成体干细胞的一种,具有多向分化能力。该文通过整理与总结相关文献,以BMSCs为切入点探讨其与中医“肾藏精”理论的联系,并从现代分子生物学角度来探讨“肾藏精”理论的现代物质基础。研究表明,补肾中药治疗AD的机制与BMSCs密切相关。BMSCs产生的外泌体是影响AD的主要物质之一,外泌体主要含有核酸、蛋白质及脂质等,并参与细胞间通讯,调节细胞功能,可通过减少Aβ沉积、抑制Tau蛋白磷酸化和神经炎症、促进神经元再生等途径来影响AD。因此,基于“肾藏精”理论探讨外泌体与AD的防治,将为中医药治疗AD提供一种新的研究思路。

构建多巴胺传感器检测多巴胺受体激动剂和拮抗剂

帕金森病(Parkinson’s disease,PD)是一种黑质致密部多巴胺能神经元缺失或死亡的中枢神经系统退行性疾病。临床上,左旋多巴是治疗PD最有效也最常用的药物。然而,长期服用左旋多巴易出现运动并发症以及因外周多巴胺累积导致的其他不良反应,成为PD治疗亟待解决的问题。多巴胺受体激动剂与多巴胺功能相似,能直接刺激突触后多巴胺受体,尽可能延缓左旋多巴的使用,减少长期服用左旋多巴引起的并发症。因此,筛选有效的多巴胺受体激动剂成为研究和治疗PD的关键问题。为建立快速、稳定、可靠的多巴胺受体激动剂筛选方法,本研究将人2型多巴胺受体(dopamine receptor 2,DRD2)基因与环状绿色荧光报告基因(circular permuted EGFP,cpEGFP)融合构建重组基因,用慢病毒载体包装,该载体置换基因编码的G蛋白偶联受体(G protein-coupled receptor,GPCR)激活传感器的胞内3环(the third intracellular loop,ICL3)内部跨膜结构域,在不改变GPCR活性的基础上,通过多巴胺受体激动剂与GPCR相互作用介导的构象变化调节GPCR融合的cpEGFP荧光。病毒载体感染HEK293T细胞,通过嘌呤霉素筛选后,获得高表达DRD2的细胞株,并用多巴胺受体激动剂作为阳性药(包括多巴胺、甲磺酸溴隐亭、卡麦角林、普拉克索)摸索最佳筛选和检测条件,建立稳定的多巴胺受体激动剂筛选模型。结果显示,本研究建立的多巴胺受体激动剂筛选模型能够检测到多梯度细胞接种密度下的药物活性,而且在多巴胺、甲磺酸溴隐亭、卡麦角林和普拉克索浓度很低(最低约0.1μmol·L^(-1))的情况下也能检测到阳性信号。经多次实验显示,本研究构建的模型最佳筛选条件:细胞接种数为7×10^(4)个,多巴胺受体激动剂阳性药的有效浓度为1~100μmol·L^(-1)均有较好的检测效果。此外,本研究先加入10μmol·L^(-1)多巴胺受体拮抗剂(包括盐酸氯普噻吨、多潘立酮、舒必利),然后再加入10μmol·L^(-1)多巴胺受体激动剂后,则无法检测到荧光阳性信号,这表明多巴胺受体拮抗剂阻断了多巴胺受体激动剂的活性,因而无法引起多巴胺受体变构,说明该模型具有较好的特异性,同时也能用于多巴胺受体拮抗剂的筛选和检测。总之,该研究构建了稳定的多巴胺传感器检测体系,可有效筛选潜在的多巴胺受体激动剂,而且操作简便,检测系统稳定,可实现快速、大规模的筛选模式,大大提高了药物筛选的效率,为以DRD2为靶点的药物开发和PD治疗提供了关键的方法体系。