黄芪活性成分QST、QT对CHOnl细胞M1受体及其偶联G-蛋白结合活性的影响

目的:观察黄芪活性成分QST、QT对CHOml细胞M1受体及其偶G-蛋白结合活性的影响.方法:以放射配基结合分析法检测CHOml细胞M1受体的结合特性,用[35S]GTPγS结合活性检测M1受体偶联的C-蛋白的结合活性,并计算偶联效率.结果:QST对M1受体密度、[35S]GTPγS结合活性、偶联效率有上调作用(P＜0.01),而QT无作用(P＞0.05).结论:QST对M1受体的偶联功能有上调作用.     

黄芪活性成分QST、AT对CHOml细胞M1受体及其偶联G—蛋白结合活性的影响

目的观察黄芪活性成分QST、QT对CHOml细胞M1受体及其偶G-蛋白结合活性的影响.方法以放射配基结合分析法检测CHOml细胞M1受体的结合特性,用[35S]GTPγS结合活性检测M1受体偶联的C-蛋白的结合活性,并计算偶联效率.结果QST对M1受体密度、[35S]GTPγS结合活性、偶联效率有上调作用(P＜0.01),而QT无作用(P＞0.05).结论QST对M1受体的偶联功能有上调作用.     

黄芪活性成分QST、QT对CHOml细胞M1受体及其偶联G-蛋白结合活性的影响

目的 :观察黄芪活性成分QST、QT对CHOml细胞M1受体及其偶G 蛋白结合活性的影响。方法 :以放射配基结合分析法检测CHOml细胞M1受体的结合特性 ,用 [3 5S]GTPγS结合活性检测M1受体偶联的G 蛋白的结合活性 ,并计算偶联效率。结果 :QST对M1受体密度、[3 5S]GTPγS结合活性、偶联效率有上调作用 (P <0 0 1) ,而QT无作用 (P >0 0 5)。结论 :QST对M1受体的偶联功能有上调作用     

应用m2-Gi1α融合蛋白鉴别M2受体的特异性药物

 目的表达m2-Gilα融合蛋白,通过检测GDP与m2-Gilα融合蛋白的亲和力大小来鉴别m2受体的特异性激动剂、拮抗剂和反向激动剂。方法用两步PCR反应重组m2-Gilα融合蛋白cDNAs,并插入到pBacPAK9病毒载体中,利用Sf9细胞表达M2受体蛋白和m2-Gilα融合蛋白,通过[³H]QNB结合饱和实验及[³⁵S]GTPγS竞争性替代结合实验,检测受体表达水平及GDP 与m2-Gilα融合蛋白的亲和力。结果m2-Gilα融合蛋白表达水平是(18.14±0.17)pmol·mg(膜蛋白)^-1。M2-Gilα融合蛋白与[³H]QNB的结合量随着[³H]QNB浓度的升高而增加,10μmol·L^-1GTDγS使m2-Gilα融合蛋白与[³H]QNB的饱和结合曲线明显下移。Ach使m2-Gilα融合蛋白与[³⁵S]GTPγS竞争性替代结合曲线明显右移,Pilo稍次之,alcuronium,AF—DX116,Iso及atropine 则使曲线左移,GDP的IC₅₀值分别是168.4,152.3,6.84,6.12,5.59,4.52μmol·L^-1,无配体存在时为14.7μmol·L^-1。结论表达的m2-Gilα融合蛋白具备M2配体结合的特性及G蛋白耦联受体的信号转导功能,对于有基础活性的m2-Gilα融合蛋白,Ach, Pilo是完全激动剂;atropine,alcuronium,Iso和AF-DX116为拮抗剂。     

川芎嗪抗伤害性反应作用机制初探

目的　观察川芎嗪(TMP)对嘌呤2 X(P2 X)受体激动剂[三磷酸腺苷(ATP)和α,β-亚甲三磷酸腺苷(α,β- me ATP) ]、前列腺素E2 (PGE2 )及P物质(SP)所致大鼠足底急性伤害性反应的影响。方法　通过大鼠痛行为反应确定局部应用TMP对ATP等P2 X受体激动剂、PGE2 及SP所致大鼠足底急性伤害性反应和足底炎症水肿的影响。结果　TMP (10 mm ol/L )明显抑制ATP (1μm ol/L )或α,β- m e ATP (0 .6μm ol/L )引起的大鼠足底急性伤害性反应。TMP(10 m mol/L)可抑制PGE2 (5 μmol/L)或α,β- me ATP(0 .2 μm ol/L)加PGE2 (5 μmol/L )引起的伤害性反应。TMP (10 m mol/L )不影响α,β- me ATP (0 .2μmol/L )加SP (10μmol/L )引起的伤害性反应。TMP对PGE2 、SP或α,β- m e ATP分别加PGE2 或SP引起的大鼠足底炎症水肿无明显影响。结论　TMP主要通过抑制P2 X受体兴奋介导的伤害性信息传递产生抗伤害性反应作用。     

蕨麻正丁醇部位下调缺氧内皮细胞HIF-1α及ET-1表达

[目的]探讨蕨麻正丁醇部位对内皮细胞缺氧损伤的保护机制。[方法]采用人脐静脉内皮细胞(EA.hy926)建立缺氧损伤实验模型,实验设常氧对照组、缺氧模型组、高(3.00 g/L)、中(1.50 g/L)、低浓度(0.75 g/L)蕨麻正丁醇部位组,复方丹参(3.0 g/L)组。胎盘兰染色法测定各组细胞存活率;逆转录-聚合酶链反应(RT-PCR)技术检测各组缺氧诱导因子-1α(HIF-1α)m RNA和内皮素-1(ET-1)m RNA表达,免疫细胞化学染色及Western Blot方法检测HIF-1α蛋白表达。放免法测定培养基中ET-1的活性。[结果]与对照组比较,缺氧模型组细胞存活率显著降低,HIF-1α和ET-1m RNA及蛋白表达增加(P0.01);蕨麻正丁醇部位各剂量组与缺氧模型组比较,细胞存活率显著升高,高、中、低剂量蕨麻正丁醇部位组、复方丹参组细胞HIF-1α和ET-1 m RNA及蛋白水平显著降低(P0.01)。[结论]在缺氧状态下,蕨麻正丁醇部位可能通过HIF-1α途径调控靶基因的表达,从而发挥保护作用。     

芦丁拮抗血小板活化因子与受体结合的作用

目的　观察芦丁对血小板活化因子(PAF)与受体结合的拮抗作用。方法　以放射配基结合试验观察[3H]PAF与家兔血小板受体特异性结合的作用;体外以分光光度法测定PAF介导血小板黏附的强度;以Fura- 2荧光分光光度法测定PAF介导的兔多形核白细胞(PMNs)内钙离子浓度的升高。结果　芦丁可浓度依赖地抑制1、2、4 nm ol/L [3H]PAF与血小板受体的特异性结合,其IC50 分别为7.3、8.7、17.2 mm ol/L ;芦丁可明显抑制PAF介导的血小板黏附,IC50 为1.5 1mm ol/L;芦丁浓度为6 8.2～5 4 6 μm ol/L 时可浓度依赖地抑制PAF介导的PMNs内游离钙浓度的升高。结论　芦丁具有抗PAF作用,为一种新的PAF受体拮抗剂。     

茛菪烷衍生物的合成及其活性研究

目的 设计、合成并筛选具有胆碱能活性,且对M1受体有选择性的莨菪烷衍生物.方法 以3α-羟基-6β-乙酰氧基莨菪烷为原料,通过酰化反应制备3α-烃氧基乙酰氧基-6β-乙酰氧基莨菪烷类衍生物,通过核磁共振及质谱鉴定所合成的化合物.运用豚鼠离体回肠纵肌M受体动力学实验方法以及人源M1,M2受体亚型基因转染的CHO细胞系放射配基受体结合法,对合成的化合物进行活性筛选.结果 制备了6个新的3α-烃氧基乙酰氧基-6β-乙酰氧基莨菪烷a～f.化合物d,e对M受体有良好的亲和力及内在活性,并对M1受体具有亚型选择性.结论 化合物d,e为具有M1受体亚型选择性的胆碱能活性化合物.     

莨菪烷衍生物的合成及其活性研究

目的 设计、合成并筛选具有胆碱能活性,且对M1受体有选择性的莨菪烷衍生物。方法以3α-羟基-6β-乙酰氧基莨菪烷为原料,通过酰化反应制备3α-烃氧基乙酰氧基-6β-乙酰氧基莨菪烷类衍生物,通过核磁共振及质谱鉴定所合成的化合物。运用豚鼠离体回肠纵肌M受体动力学实验方法以及人源M1,M2受体亚型基因转染的CHO细胞系放射配基受体结合法,对合成的化合物进行活性筛选。结果制备了6个新的3α-烃氧基乙酰氧基-6β-乙酰氧基莨菪烷a-f。化合物d,e对M受体有良好的亲和力及内在活性,并对M1受体具有亚型选择性。结论化合物d,e为具有M1受体亚型选择性的胆碱能活性化合物。     

葛根素减轻PM2.5对血管内皮细胞损伤的机制研究

探讨PM2.5对EA.hy926型人脐静脉内皮细胞损伤的影响及葛根素的保护作用及机制。采集大气PM2.5分别以0,20,200,400 mg·L~(-1)染毒EA.hy926细胞24 h,MTT法测细胞存活率,流式细胞术测细胞凋亡,Western blot法测p-ERK1/2,Bax,Bcl-2蛋白水平,ELISA法测肿瘤坏死因子-α(TNF-α)及白细胞介素-6(IL-6)含量,并测细胞丙二醛(MDA)含量、超氧化物歧化酶(SOD)及乳酸脱氢酶(LDH)活性;分别加入葛根素(10,50,100μmol·L~(-1))和ERK1/2通路特异性阻滞剂PD98059 20μmol·L~(-1)检测葛根素的干预作用及机制。检测发现与对照组比较,PM2.5染毒后呈剂量依赖性降低EA.hy926细胞存活率,上调p-ERK1/2蛋白水平及Bax/Bcl-2蛋白比率以促进细胞凋亡,诱导分泌TNF-α及IL-6含量增高,降低SOD活性,增加MDA含量及LDH活性(P0.05);葛根素呈剂量依赖性增加EA.hy926细胞的存活率,下调p-ERK1/2蛋白水平及Bax/Bcl-2蛋白比率以抑制细胞凋亡,降低TNF-α及IL-6含量,增加SOD活性,降低MDA含量及LDH活性(P0.05)。研究显示葛根素可能通过抑制ERK1/2通路减轻PM2.5对EA.hy926细胞的损伤。     

莱菔硫烷诱导HepG-2细胞G2/M期阻滞及其对Cdk1和CyclinB1蛋白表达的影响

目的：研究莱菔硫烷（SFN）在体外对人肝癌HepG-2细胞G2/M期的阻滞作用,并探讨其分子作用机制。方法：10、20、40μmol·L-1的SFN处理体外培养的HepG-2细胞株48h后,采用流式细胞仪检测SFN对HepG2细胞周期的影响;WesternBlot法检测SFN对HepG2细胞内Cdk1、p-Cdk1（Thr14）和CyclinB1蛋白表达的影响。结果：SFN作用于HepG-2细胞48h后,随着SFN浓度的增大,G2/M期细胞比例逐渐升高,当SFN浓度达到40μmol·L-1时,G2/M期细胞比例达到31.95%,且出现凋亡峰;随SFN浓度的增大,细胞内Cdk1和CyclinB1蛋白的表达量显著降低（P〈0.01或P〈0.05）,同时p-Cdk1（Thr14）的表达显著升高（P〈0.01或P〈0.05）。结论：SFN可诱导人肝癌HepG-2细胞发生G2/M期阻滞;SFN可通过下调HepG-2细胞内Cdk1和CyclinB1蛋白的表达、上调p-Cdk1（Thr14）的蛋白表达水平,进而抑制Cdk1-CyclinB1复合物的形成和活化使人肝癌HepG-2细胞阻滞在G2/M期。     

Nocardimicins A, B, C, D, E, and F, siderophores with muscarinic M3 receptor inhibiting activity from Nocardia sp. TP-A0674

In the screening for muscarinic M3 receptor binding inhibitors from microbial secondary metabolites, the extract of Nocardia sp. TP-A0674 was found to be highly active. Bioassay-guided fractionation of it led to the isolation of six new siderophores, nocardimicins A (1), B (2), C (3), D (4), E (5), and F (6), as active principles. Their chemical structures were determined by spectroscopic and degradation analysis. Of these congeners, nocardimicin B (2) inhibited the binding of tritium-labeled N-methylscopolamine to the muscarinic M3 receptor most potently with a Ki value of 0.13 microM. Compound 2 showed more selective activity to M3 and M4 receptors than other subtypes.     

橐吾中倍半萜对二氢吡啶类受体的作用研究

 目的：观察8个倍半萜类化合物对二氢吡啶类受体结合的作用和对离体血管张力的影响。方法：用［3H］尼群地平对猪心室肌微粒体膜受体结合方法和家兔离体血管条实验对化合物的体外作用进行研究。结果：化合物Ⅰ、Ⅱ、Ⅲ对尼群地平与受体结合有明显抑制作用，其IC₅₀分别为2.0×10^-5mol·L^-1，2.7×10^-5mol·L^-1和3.9×10^-5mol·L^-1，其余化合物作用较弱。这些化合物抑制受体结合的作用强度与结构中母核与取代基的改变有关，而取代基的变化对活性的影响更重要。化合物Ⅱ、Ⅲ对高K+引起的兔胸主动脉收缩有剂量依赖性的抑制作用。结论：这类化合物有钙拮抗作用，作用机制可能与影响二氢吡啶类受体有关；它们作用有量效关系和构效关系，提示有以此为先导进行深入结构改造和构效关系研究的价值。     

Decreased muscarinic M1 receptor gene expression in the cerebral cortex of streptozotocin-induced diabetic rats and Aegle marmelose leaf extract's therapeutic function

AIM: In the present study we have investigated the changes in the total muscarinic and muscarinic M1 receptor ([(3)H]QNB) binding and gene expression in the cerebral cortex of streptozotocin (STZ) induced diabetic, insulin and aqueous extract of Aegle marmelose leaf treated diabetic rats. MATERIALS AND METHODS: Diabetes was induced in rats by intrafemoral injection of streptozotocin. Aegle marmelose leaves was given orally to one group of rats at a dosage of 1g/kg body weight per day for fourteen days. Blood glucose and plasma insulin level were measured. Muscarinic and Muscarinic M1 receptor binding studies were done in the cerebral cortex of experimental rats. Muscarinic M1 receptor gene expression was studied using real-time PCR. RESULTS: Scatchard analysis for total muscarinic receptors in cerebral cortex showed that the B(max) was decreased significantly (p<0.001) in diabetic rats with a significant decrease (p<0.01) in the K(d) when compared to control group. Binding analysis of Muscarinic M1 receptors showed that B(max) was decreased significantly (p<0.001) in diabetic group when compared to control group. The K(d) also decreased significantly (p<0.01) when compared to control group. The binding parameters were reversed to near control by the treatment of diabetic rats with Aegle marmelose. Real-Time PCR analysis also showed a similar change in the mRNA levels of muscarinic M1 receptors. CONCLUSION: The results showed that there is decrease in total muscarinic and muscarinic M1 receptors during diabetes which is up regulated by insulin and Aegle marmelose leaf extract treatment. This has clinical significance in therapeutic management of diabetes.     

NMR metabolomics for identification of adenosine A1 receptor binding compounds from Boesenbergia rotunda rhizomes extract

Ethnopharmacological relevance

Boesenbergia rotunda Linn. (Zingiberaceae) is traditionally used in many Asian countries as medicine for stomach pain and discomfort, viral and bacterial infection, inflammation, and as diuretic agent.

Aim of the study

The study aimed to identify adenosine A1 receptor binding compounds from Boesenbergia rotunda rhizome extract by using comprehensive extraction coupled to the NMR metabolomics method.

Materials and methods

Dried and powdered Boesenbergia rotunda rhizomes were extracted with the comprehensive extraction method to obtain several fractions with different polarity. Each fraction was divided into two: for NMR analysis and for adenosine A1 receptor binding test. Orthogonal projection to the least square analysis (OPLS) was used to study the correlation between metabolites profile and adenosine A1 receptor binding activity of the plant extracts. Based on Y-related coefficient and variable of important (VIP) value, signals in active area of OPLS loading plot were studied and the respective compounds were then elucidated

Results and discussions

Based on OPLS Y-related coefficient plot and variable of importance value plot, several characteristic signals were found to positively correlate to the binding activity. By using 1D and 2D NMR spectra of one of the most active fraction, pinocembrine and hydroxy-panduratin were identified as the possible active compounds. Two signals from ring C of pinocembrine flavanone skeleton with negative coefficient correlations possibly overlapped with those of non-active methoxylated flavanones which were also presence in the extract. NMR based metabolomics applied in this study was able to quickly identify bioactive compounds from plant extract without necessity to purify them. Further confirmation by isolating pinocembrine and hydroxy-panduratin and testing their adenosine A1 receptor binding activity to chemically validate the method are required.

Conclusion

Two flavonoid derivatives, pinocembrine and hydroxy-panduratin, have been elucidated as possible active compounds bind to adenosine A1 receptor. Flavonoid was reported to be one of natural antagonist ligand for adenosine A1 receptor while antagonistic activity to the receptor is known to associate with diuretic activity. Thus, the result of this research supports the traditional use of Boesenbergia rotunda rhizome extract as diuretic agent.     

小檗碱对胃癌SGC7901细胞增殖和凋亡的影响

目的:探讨小檗碱对胃癌细胞系SGC7901的抑制作用及相关机制。方法:采用MTT的方法观察不同浓度小檗碱对SGC7901细胞的24h和48h抑制率,并计算24h和48h的50%抑制浓度(IC50)值;采用流式细胞技术检测不同浓度小檗碱作用48h后胃癌细胞系SGC7901凋亡的发生率和细胞周期的变化。结果:小檗碱对胃癌细胞SGC7901生长的抑制作用呈现出时间和剂量依赖性。24h和48h IC50分别为74.16、36.84μmol/L。1μmol/L至120μmol/L浓度梯度的小檗碱干预48h后,SGC7901凋亡率发生率逐渐增加。其中5、10、25、50、75、120μmol/L与对照组比较有统计学差异(P0.05),120μmol/L时凋亡发生率达到35.43%;5、25、75μmol/L浓度的小檗碱作用后,SGC7901胃癌细胞G0/G1期细胞比例较对照组明显升高(P0.05),而S期细胞比例则随浓度增加呈现出下降趋势,其中25、75μmol/L浓度组与对照组比较,差异有统计学意义(P0.05),对G2/M期细胞,各组比例变化无明显差别。结论:小檗碱对胃癌细胞系SGC7901具有时间和浓度依赖性的抑制作用,抑制作用与阻滞细胞周期于G0/G1期,抑制DNA合成和诱导凋亡有关。     

Mechanism underpinning effects of Shichangpu(Rhizoma Acori Tatarinowii) on attention deficit hyperactivity disorder

OBJECTIVE: To investigate the mechanism underlying the effects of Shichangpu(Rhizoma Acori Tatarinowii) on attention deficit hyperactivity disorder(ADHD).METHODS: A network pharmacology approach integrating ingredients of Shichangpu(Rhizoma Acori Tatarinowii) and target with ADHD, network construction, molecular function interactions and pathway analysis was used.RESULTS: This approach successfully helped to identify 7 active ingredients of CN, interacting with21 key targets(ADRA1 A, ADRA1 B, ADRA2 A,ADRA2 B, ADRA2 C, ADRB1, ADRB2, CHRM1, CHRM2,CHRM3, PTGS1, SLC6 A2, SLC6 A3, SLC6 A4, DRD1,DRD5, HTR2 A, ADRA1 D, MAOB, GRIA2, HTR1 A). The molecular function interactions among candidate targets mainly consisted of four groups: G-protein coupled amine receptor activity, catecholamine binding, monoamine transmembrane transporter activity and neurotransmitter receptor activity. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that Shichangpu(Rhizoma Acori Tatarinowii)-regulated pathways were mainly classified into signal transduction and monoamine neurotransmitters.CONCLUSION: Our investigation revealed that Shichangpu(Rhizoma Acori Tatarinowii) could improve the symptoms of ADHD by regulating neurotransmitter, in multiple types of compounds-target-pathway, which may be implicated in the major pathological processes of ADHD.     

山柰酚拮抗血小板活化因子与其受体结合的作用

目的 :观察山柰酚对氚标记的血小板活化因子与血小板膜上受体结合作用的影响 ,试图证明该药为一新型血小板激活因子 (PAF)受体拮抗剂。方法 :以放射配基结合试验观察 [³H]PAF与家兔血小板受体的特异性结合 ;分光光度法测定PAF诱发的血小板黏附强度 ;Fura-2荧光分光光度法测定PAF介导的兔多形核白细胞 (PMNs)内钙离子浓度的升高。结果 :山柰酚可浓度依赖地抑制 1,2 ,4nmol·L^-1[³H]PAF与血小板受体的特异性结合 ,IC50 分别为 30 .8,74 .6 ,92 .0 μmol·L^-1;该药可明显抑制PAF诱发的兔血小板黏附及PMNs内游离钙升高 ,且均呈明显的量效关系 ,其抑制血小板黏附的IC50 为 65μmol·L^-1。结论 :山柰酚具抗PAF的作用 ,为一新的PAF受体拮抗剂。     

Melanin‐concentrating hormone‐1 receptor binding activity of pheophorbides isolated from morus alba leaves

The time‐resolved fluorescence technique based on melanin‐concentrating hormone (MCH) receptor subtype‐1 (MCH‐1 receptor) binding assay was adopted to carry out a bioassay‐guided fractionation of the methanol extract of Morus alba leaves. This fractionation and purification led to the isolation of two compounds identified as pheophorbide a methyl ester and 13²(S)‐hydroxypheophorbide a methyl ester. These active pheophorbides exhibited potent inhibitory activity in binding of europium‐labeled MCH to the human recombinant MCH‐1 receptor (IC₅₀ value; 4.03 and 0.33 ?M, respectively). Besides binding activity, the pheophorbides inhibited MCH‐mediated extracellular signal‐regulated kinase (ERK) phosphorylation in Chinese hamster ovary cells expressing human MCH‐1 receptor. These results suggest that pheophorbide a methyl ester and 13²(S)‐hydroxypheophorbide a methyl ester act as modulators of MCH‐1 receptor and MCH‐mediated ERK signaling. Copyright © 2009 John Wiley & Sons, Ltd.