CN101084909A - New use of ginsenoside Rg1 - Google Patents
New use of ginsenoside Rg1 Download PDFInfo
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- CN101084909A CN101084909A CN 200710009171 CN200710009171A CN101084909A CN 101084909 A CN101084909 A CN 101084909A CN 200710009171 CN200710009171 CN 200710009171 CN 200710009171 A CN200710009171 A CN 200710009171A CN 101084909 A CN101084909 A CN 101084909A
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- ginsenoside
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Abstract
本发明涉及人参皂苷Rg1治疗/预防阿尔茨海默病的用途，提供了人参皂苷Rg1在治疗/预防Aβ1－42介导的神经毒性作用所引起的阿尔茨海默病的新用途。本发明提供了中药单体人参皂苷Rg1具有以下功能：可通过减轻寡聚态Aβ1－42介导的神经元应激损害，保护线粒体的功能，减少神经元凋亡；人参皂苷Rg1还可减轻Aβ1－42对PKA－CREB信号通路的抑制作用，有助于形成长时程增强、改善记忆功能。从而为人参皂苷Rg1应用于阿尔茨海默病的防治提供了直接有力的实验依据和理论基础，既有很强的科学性和创新性，又有很高的开发应用价值。The invention relates to the use of ginsenoside Rg1 in treating/preventing Alzheimer's disease, and provides a new use of ginsenoside Rg1 in treating/preventing Alzheimer's disease caused by neurotoxicity mediated by Aβ 1-42 . The invention provides the traditional Chinese medicine monomer ginsenoside Rg1 which has the following functions: it can protect the function of mitochondria and reduce neuron apoptosis by alleviating the neuron stress damage mediated by oligomeric Aβ 1-42 ; ginsenoside Rg1 can also relieve The inhibitory effect of Aβ 1-42 on the PKA-CREB signaling pathway contributes to the formation of long-term potentiation and improvement of memory function. Therefore, it provides a direct and powerful experimental basis and theoretical basis for the application of ginsenoside Rg1 in the prevention and treatment of Alzheimer's disease, which is not only highly scientific and innovative, but also has high development and application value.
Description
Technical field
The present invention relates to the purposes of ginsenoside Rg1's treatment/prevention Alzheimer, relate in particular to the ginsenoside Rg1 at treatment/prevention A β
1-42The new purposes of the caused Alzheimer of neurotoxic effect of mediation.
Background technology
Alzheimer (Alzheimer ' s disease, AD) be a kind of be the neurodegenerative disease of main clinical manifestation with carrying out property cognitive dysfunction, the neural inflammatory speckle (Neuriteplaques that the extracellular is gathered in a large number, NP), the beta amyloid peptide (β-amyloidpeptide of abnormal deposition in the cell, A β) and neurofibrillary tangles (neurofibrillary tangle NFT) forms and nerve synapse is lost or neuron loss is the main europathology feature of AD.Though the pathogenesis of AD is not illustrated as yet fully; but " A β cascade theory " accepted by Most scholars; it is the A β Developmental and Metabolic Disorder that a variety of causes causes; cause the abnormal accumulation of A β at cerebral tissue; and then triggered and AD Pathophysiology, biochemical relevant cascade reaction, finally cause neuronic infringement.A β has several existing waies of single aggressiveness, oligomer and condensed state (filamentous) in vivo, and the A β that discovers the oligomerization attitude has more neurotoxicity than the A β of filamentous, single aggressiveness, and is more extensive to the nervous physiology destruction that comprises learning and memory.
An important early stage pathophysiological change in the AD morbidity is stress infringement in the cell, mitochondrial function imbalance.Discover in the cerebral tissue of transgenic mouse of the cultivation neuron that is directly exposed to A β and high expressed APP, relevant nitrogen free radical Developmental and Metabolic Disorder and the neuron linear plastochondria functional defect of activation, NO that early stage stress response protein kinases JNK/p38-SAPK all occurred, unusual as electron transport, mitochondrial membrane potential changes, ROS increases etc.These study prompting: stress infringement in the cell that A β causes, mitochondrial function imbalance play an important role in the AD pathogenic process.Synapse plasticity and LTP and ability of learning and memory are closely related, what research report had synapse function that the A β of neurotoxic effect causes and LTP is another early stage key pathophysiological change of AD unusually, and wherein the cAMP/PKA/CREB signal path occupies an important position in the adjusting that participates in LTP.
Up to the present the treatment of AD still lacks effective medicine, though cholinesterase inhibitor and nmda receptor antagonist are current main medicines commonly used clinically, can improve patient's AD cognition and memory function, raising patient's quality of life to some extent, but can not stop the process of AD pathology development and delay the progress of conditions of patients.The ginsenoside Rg1 is the monomeric compound that contains 2 glycosyls that extracts from Radix Ginseng, and research in the past has been found that: 1, the ginsenoside Rg1 can obviously alleviate the learning memory disorder of kinds of experiments animal model, improves learning and memory in rats and mice; 2, can improve the regulatory function of choline system by the synthetic of increase acetylcholine and the activity that discharges, increases M cholinoceptor quantity, raising acetylcholine transferase; 3, the ginsenoside Rg1 can protect dopamine neuron by alleviating the inductive stress of MPTP/MPP+; 4, Rg1 also has the plasticity that strengthens long-term potential, raising synapse and improves the effects such as immunomodulating of old Mus.But the ginsenoside Rg1 is to oligomerization attitude A β
1-42Does neuron stress damage, the mitochondrial function that causes is unbalance and strengthen the early stage key pathophysiological change of relevant AD such as cAMP/PKA/CREB signal path with the long time journey have protective effect? also be not very clear at present.
Summary of the invention
The object of the present invention is to provide the new purposes of a kind of ginsenoside Rg1, be specially the ginsenoside Rg1 at preparation treatment/prevention A β
1-42Application in the medicine/health product of the caused Alzheimer of neurotoxic effect of mediation.
Described neurotoxic effect is meant A β
1-42The cortical neuron of mediation is transferred and is died.
Described neurotoxic effect is meant A β
1-42The early stage neuron of mediation stress damage.
Described neurotoxic effect is meant A β
1-42Inductive cortical neuron mitochondrial injury.
Described neurotoxic effect is meant A β
1-42Inhibitory action to protein kinase K-cAMP response element binding protein signal path.
The English name of protein kinase K-cAMP response element binding protein is: proteinkinase K-cAMP response element binding protein, be abbreviated as PKA-CREB.
Ginsenoside Rg1 of the present invention generally uses with the form of pharmaceutical composition, and this compositions contains the ginsenoside Rg1 and the pharmaceutically acceptable auxiliaries as active component for the treatment of effective dose.The pharmaceutical composition that contains the ginsenoside Rg1 can be made tablet, capsule, powder, granule, lozenge by oral, injection or mucosa delivery, or dosage form such as oral liquid is for clinical practice.The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.The nutriment that contains the ginsenoside Rg1 can be oral.
The application provides the Chinese medicine monomer ginsenoside Rg1 to have following function: can alleviate oligomerization attitude A β
1-42The neuron of mediation stress damage, and protects mitochondrial function, reduces Neuron Apoptosis; The ginsenoside Rg1 also can alleviate A β
1-42To the inhibitory action of PKA-CREB signal path, help to form the long time journey and strengthen, improve memory function.Thereby the control that is applied to Alzheimer for the ginsenoside Rg1 provides direct strong experimental basis and theoretical basis, and existing very strong science and novelty have very high development and application values again.
Description of drawings
Fig. 1 .1: neuron TUNNEL dye (* 400).Cortical neuron is respectively through the oligomerization attitude A β of 0.5 μ M, 1.0 μ M, 5.0 μ M
1-42Effect 24h or 48h carry out TUNNEL dyeing then, and apoptotic cells nuclear is dyed the positive.A1:0.5uM A β
1-42Handle 24h, B1:0.5uMA β
1-42Handle 48h; A2:1.0uM A β
1-42Handle 24h, B1:1.0uM A β
1-42Handle 48h; A3:5.0uM A β
1-42Handle 24h, B3:5.0uM A β
1-42Handle 48h; A4: matched group, B4: matched group.
The neuronic quantity of Fig. 1 .2:TUNNEL stained positive.* P＜0.05 vs matched group.
Fig. 1 .3: neuron TUNNEL dye (* 400).Behind the Rg1 preincubate 24h of neuron through 2.5 μ M, 5.0 μ M, 10 μ M, adding final concentration again is the oligomerization attitude A β of 5 μ M
1-42Combined effect 48h carries out the positive staining of TUNNEL at last.A: matched group, B:5.0uM A β
1-42For 48h, C:2.5uM Rg1+A β
1-42, D:5.0uM Rg1+A β
1-42, E:10uM Rg1+A β
1-42, F: simple 10uM Rg1 processed group.
Fig. 1 .4: the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42Inductive Neuron Apoptosis, every hole are got five visual field counting TUNNEL positive neurons and are averaged.* P＜0.05vs matched group; #P＜0.05vs A β
1-42Processed group.
Fig. 1 .5: oligomerization attitude A β 1-42 induces the activity of cortical neuron caspase-3 to increase.Use the oligomerization attitude A β of 0.5 μ M, 1.0 μ M and 5 μ M respectively
1-42When acting on cortical neuron 24h or 48h, * P＜0.05vs matched group.
Fig. 1 .6: the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42Inductive caspase-3 activation.After using the Rg1 preincubate 24h of 2.5 μ M, 5.0 μ M and 10 μ M respectively, add 5.0 μ M oligomerization attitude A β again
1-42Act on cortical neuron 48h altogether, * P＜0.05vs matched group, #P＜0.05vs A β
1-42Processed group.
Fig. 2 .1: oligomerization attitude A β
1-42Induce cortical neuron ROS level to raise.The density analysis of ROS is figured, * p＜0.05vs matched group.
Fig. 2 .2: the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42The rising of inductive cortical neuron ROS.The level of ROS is figured, * p＜0.05vs A β 1-42 processed group.
Fig. 2 .3: oligomerization attitude A β
1-42Induce cortical neuron H
2O
2Generation.H
2O
2Density analysis represent * p＜0.05vs matched group with rectangular histogram.
Fig. 2 .4: the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42H in the inductive cortical neuron
2O
2Raise.H
2O
2Level represent * p＜0.05vs A β with rectangular histogram
1-42Processed group.
Fig. 2 .5: oligomerization attitude A β
1-42The rising of inductive cortical neuron NO level.Use the oligomerization attitude A β of 0.5 μ M, 1.0 μ M and three kinds of concentration of 5.0 μ M respectively
1-42Act on neuron 24h or 48h, * p＜0.05vs matched group.
Fig. 2 .6: the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42The rising of inductive cortical neuron NO level.The level of NO is represented with rectangular histogram, * p＜0.05vs A β
1-42Processed group.
Fig. 2 .7: oligomerization attitude A β
1-42The expression of interior p38 of neuron and p-p38 behind the processing different time.With the level of p38 and p-p38 in western-blot method (p38 and p-p38 antibody) the detection lysate, * p＜0.05vs matched group is represented in the dot density analysis of total p38 and p-p38 with rectangular histogram.
Fig. 2 .8: the ginsenoside Rg1 reduces the level of p-p38/p38, * p＜0.05vs A β
1-42Processed group.
Fig. 2 .9: oligomerization attitude A β
1-42The expression of interior JNK of neuron and p-JNK behind the processing different time.Detect JNK and p-JNK level in the lysate with the western-blot method, total JNK and p-JNK level are represented * p＜0.05vs matched group with rectangular histogram through the dot density analysis.
Fig. 2 .10: the ginsenoside Rg1 reduces the level of p-JNK/JNK.* p＜0.05vs A β
1-42Processed group.
Fig. 3 .1: oligomerization attitude A β
1-42Reduce mitochondrial membrane potential, the density analysis of mitochondrial membrane potential is represented with rectangular histogram, * p＜0.05vs matched group.
Fig. 3 .2: the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42The reduction of inductive neuron linear mitochondrial membrane potential.The level of mitochondrial membrane potential is represented with rectangular histogram, * p＜0.05vs A β
1-42Processed group.
Fig. 3 .3: oligomerization attitude A β
1-42Reduce the generation of cortical neuron ATP.The level of ATP is represented with rectangular histogram, * p＜0.05vs matched group.
Fig. 3 .4: the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42Reduce the effect that cortical neuron ATP produces.The level of ATP is represented with rectangular histogram, * p＜0.05vs A β 1-42 processed group.
Fig. 3 .5: oligomerization attitude A β
1-42Reduce the activity of mitochondrion composite I V.The activity of composite I V is represented with rectangular histogram, * p＜0.05vs matched group.
Fig. 3 .6: the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42To the active infringement of mitochondrion composite I V.The activity of composite I V is represented with rectangular histogram, * p＜0.05vs A β
1-42Processed group.
Fig. 3 .7: oligomerization attitude A β
1-42Induce the release of cortical neuron mitochondrial cytochrome c.The level of mitochondrial cytochrome c is represented with rectangular histogram, * p＜0.05vs matched group.
Fig. 3 .8: the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42Induce the release of neuron linear plastochondria cytochrome c.Behind ginsenoside Rg1's preincubate 24h of variable concentrations (2.5 μ M/5 μ M/10 μ M) with the A β of oligomerization attitude
1-42Hatch 48h altogether, measure the content of endochylema and line grain cylinder cell pigment c then, * p＜0.05vs A β 1-42 processed group.
Fig. 4 .1: oligomerization attitude A β
1-42Reduce the phosphorylation level of the CREB of glutamic acid mediation.Oligomerization attitude A β
1-42Detect the protein level of interior CREB of neuron and p-CREB behind the processing different time, the ratio of p-CREB/CREB is represented with rectangular histogram, * p＜0.05vs glutamic acid matched group.
Fig. 4 .2: the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42Reduce the phosphorylation degree of CREB.Behind ginsenoside Rg1's preincubate 24h of variable concentrations (2.5 μ M/5 μ M/10 μ M) with the A β of oligomerization attitude
1-42Hatch 2h altogether, measure the protein level of interior CREB of neuron and p-CREB then, the ratio of p-CREB/CREB is represented with rectangular histogram, * p＜0.05vs glutamic acid matched group.
Fig. 4 .3: the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42Reduction to the PKA enzymatic activity.Behind ginsenoside Rg1's preincubate 24h of variable concentrations (2.5 μ M/5 μ M/10 μ M) with the A β of oligomerization attitude
1-42Hatch 2h altogether, the activity of PKA is represented with rectangular histogram, * p＜0.05vs matched group.
Fig. 4 .4: the ginsenoside Rg1 reduces the protein level of PKA II α subunit.Answer the mark method to detect with the immunity of PKA II Alpha antibodies, the ratio of PKA II α/β-actin is represented with rectangular histogram, * p＜0.05vs A β
1-42Processed group.
The specific embodiment
1.TUNNEL dyeing
(1) cell inoculation is on coverslip, through ginsenoside Rg1 and/or oligomerization attitude A β
1-42After the intervention, inhale and abandon culture medium, carefully wash with 1xPBS.
(2) 3.7% formaldehyde are at 18 ℃-24 ℃ fixing 10min.
(3) inhale and to abandon behind the formaldehyde, wash with 1xPBS with the fixing 20min of 100% ethanol.
(4) after soaking 10min under 18 ℃ of-24 ℃ of conditions, the careful suction abandoned ambient water to sample with 1xPBS.
(5) neuron punching agent (Neuropore) liquid of adding 50 μ L fully contacts in cell, acts on 15min-30min down for 18 ℃-24 ℃ at wet box.
(6) flushing of the water of no DNA enzyme is 2 times, each 2min.
(7) drip inactivator and [contain 3%H
2O
2Methanol] behind effect 5min under 18 ℃ of-24 ℃ of conditions, with 1xPBS in 18 ℃ of-24 ℃ of conditions flushing 1 time, about 2min.
(8) use TDT (TdT) labelling buffer at 18 ℃ of-24 ℃ of conditioning 5min.
(9) drip labeled reactant mixed liquor [TdT dNTP Mix; TdT Enzyme; Mn
2+], under 37 ℃ of conditions of wet box, act on 1h.
(10) with TdT stop buffer cessation reaction, at 18 ℃ of-24 ℃ of conditioning 5min.
(11) abandon cessation reaction liquid, 1xPBS flushing 2 times, each 2min.
(12) dry ambient water, drip the horseradish peroxidase (streptavidin-HRP) of Streptavidin sign, hatch 10min 18 ℃ of-24 ℃ of conditions.
(13) 1xPBS flushing is 2 times, behind each 2min, 3,3 '-diaminobenzidine (DAB) colour developing, behind the room temperature 2-10min, fully washing, after nuclear is redyed, fully washing, after ammonia returns indigo plant, water and dashes, the resinene mounting.
1.1 oligomerization attitude A β
1-42Induce cortical neuron apoptosis TUNNEL dyeing
The cortical neuron of former C57BL/6 mice of being commissioned to train foster is respectively through the oligomerization attitude A β of 0.5 μ M, 1.0 μ M, 5 μ M
1-42Effect 24h or 48h, each is organized neuron and carries out TUNNEL dyeing, carries out TUNNEL positive cell counting at last, the TUNNEL positive cell number between each group of statistics.Shown in Fig. 1 .1, the matched group neuron morphology is normal, and structural integrity is evenly distributed, and sees the neuron of a spot of TUNNEL stained positive, promptly shows as kytoplasm and disappears, and karyon is sepia.Variable concentrations (0.5 μ M, 1.0 μ M, 5 μ M) oligomerization attitude A β
1-42Effect different time (24h, 48h) back TUNNEL stained positive neuron has increase in various degree, with the oligomerization attitude A β of 5 μ M
1-42Its quantity of effect 48h increases the most obviously (p=0.0001), and the positive neuron count results is shown in table 1.1 and Fig. 1 .2.
Table 1.1 TUNNEL stained positive neuron
| Groups | Positive?neurons |
| control?24h control?48h 0.5uM?24h 1.0uM?24h 5.0uM?24h 0.5uM?48h | 16.53±2.47 16.74±2.18 18.80±1.48 24.60±6.56 35.80±4.82 * 26.40±8.14 |
| 1.0uM?48h 5.0uM?48h | 32.60±3.05 * 63.44±10.53 * |
1.2 the ginsenoside Rg alleviates oligomerization attitude A β
1-42Inductive cortical neuron apoptosis
Behind the Rg1 preincubate 24h through 2.5 μ M, 5.0 μ M, 10 μ M, adding final concentration again is the oligomerization attitude A β of 5 μ M
1-42Combined effect 48h carries out the positive staining of TUNNEL at last.The result is shown in Fig. 1 .3, and the matched group neuron morphology is normal, and structural integrity is evenly distributed, and sees a spot of TUNNEL stained positive neuron, promptly shows as kytoplasm and disappears, and karyon is sepia, with the oligomerization attitude A β of 5 μ M
1-42Effect 48h TUNNEL stained positive neuronal quantity showed increased (p=0.0001) is behind variable concentrations (2.5 μ M, 5.0 μ M, 10 μ M) ginsenoside Rg's preincubate 24h, again with the oligomerization attitude A β of 5 μ M
1-42Hatch 48h jointly, then TUNNEL stained positive neuron reduction in various degree, the positive neuron count results is shown in table 1.2 and Fig. 1 .4.
Table 1.2 TUNNEL stained positive neuron (* P＜0.05vs matched group; #P＜0.05vs A β
1-42Processed group)
| Groups | ?Positive?neurons |
| control 5.0uM?48h 2.5uM?Rg1 5.0uM?Rg1 10.0uM?Rg1 only?10uM?Rg1 | ?16.74±2.18 ?63.44±10.53 ?44.54±3.46 *#?34.98±3.98 *#?28.80±4.84 *#?14.20±3.05 # |
2. caspase 3 (caspase-3) determination of activity
(1) scrapes cell with cell scraper, and calculate cell number.
(2) get required cell number volume, abandon supernatant behind the centrifugal 5min of 400xg.
(3) according to 50 μ L/2 * 10
5Individual cell proportion adds lysate, cracking 10min on ice.
(4) under 4 ℃ of conditions with the centrifugal 5min of maximum centrifugal speed, get supernatant.
(5) dithiothreitol, DTT (DTT) that adds 1M according to 1: 100 ratio is made into 2x Reat ion Buffer/DTT liquid in 2x reaction buffer (2x Reat ion buffer).
(6) on 96 hole measurement plates, add the 2xReation Buffer/DTT liquid of 50 μ L, incubate pre-5min in advance in 37 ℃.
(7) cell pyrolysis liquid of adding 50 μ L, positive inhibitor group should be added 1 μ Lcaspase inhibitor in addition.
(8) hatch 1h, last machine testing fluorescence intensity in 37 ℃.Excitation wavelength: 380nm; Emission wavelength: 460nm.
2.1 oligomerization attitude A β
1-42Strengthen the activity of neuron caspase-3
In the apoptotic while of research, measure the activity of caspase-3 in the cortical neuron down with the same terms.The result is the oligomerization attitude A β of 0.5 μ M, 1.0 μ M and 5 μ M in concentration shown in Fig. 1 .5
1-42When acting on cortical neuron 24h or 48h, cortical neuron caspase-3 activity is along with A β
1-42Activity increases and increases.In addition, the A β of oligomerization attitude under the same concentrations condition
1-42Action time is long more, and the caspase-3 activity increases obvious more.This prompting oligomerization attitude A β
1-42Activate the caspase-3 activity and have certain concentration dependent.1.0 μ M oligomerization attitude A β wherein
1-42The oligomerization attitude A β of effect 48h, 5 μ M
1-42Effect 24h and 48h and matched group compare, caspase-3 active significantly raise (the p value is respectively 0.019,0.001,0.0001).
2.2 the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42Induce cortical neuron caspase-3 activation
After using the Rg1 preincubate 24h of 2.5 μ M, 5.0 μ M and 10 μ M respectively, add 5.0 μ M oligomerization attitude A β again
1-42Act on cortical neuron 48h altogether, measure caspase-3 activity in the cortical neuron then.Discover, separately A β
1-42Caspase-3 is active in the cortical neuron activity of effect obviously raises, yet the caspase-3 activity in the cortical neuron of each concentration group of process ginsenoside Rg1 preincubate but has reduction (compare with A β 1-42 effect group, the p value is respectively all less than 0.05) in various degree.This prompting ginsenoside Rg1 can alleviate oligomerization attitude A β
1-42Activation to caspase-3.Shown in Fig. 1 .6.
1. the level of fluorescence spectrometry active oxy group (ROS)
Different condition is handled respectively organizes cell, the trypsinization through 0.25%, and aseptic PBS carefully washs the back counting.Get the same cell number, directly add 1M 2,7-dichloro resorcinolphthalin. diester (DCFH-DA) solution, the adjustment final concentration is 10uM, leaves standstill 0.5h in the 5%CO2 incubator under 37 ℃ of conditions.After the careful washing of aseptic PBS 3 times, use spectrofluorophotometer, select 480nm excitation wavelength and 530nm emission wavelength, measure fluorescence intensity, the blank pipe is set simultaneously.
1.1 oligomerization attitude A β
1-42Inductive cortical neuron ROS level raises
The oligomerization attitude A β of 5 μ M
1-42After acting on former mouse cortex neuron of being commissioned to train foster, observe the level of ROS in different time points (3h, 6h, 12h, 24h, the 48h) cell, result of study is shown in Fig. 2 .1: oligomerization attitude A β
1-42Can induce the generation of ROS in the neuron, and along with oligomerization attitude A β
1-42The ROS level is more and more higher in the prolongation of action time, neuron, wherein oligomerization attitude A β
1-42When effect 24h and 48h, the ROS in the neuron raises the most remarkable.This prompting ROS may participate in oligomerization attitude A β
1-42To the neuron detrimental effect.
1.2 the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42The rising of inductive cortical neuron ROS
The front is discovered, oligomerization attitude A β
1-42Can induce the generation of ROS in the neuron, and along with the prolongation of action time, at 5 μ M oligomerization attitude A β
1-42Effect 24h and 48h, intracellular ROS obviously raises.On this basis, behind ginsenoside Rg1's preincubate 24h of employing variable concentrations (2.5/5/10 μ M), again with A β
1-42 Combined effect 24h or 48h measure intracellular ROS level.The result is shown in Fig. 2 .2, at A β
1-42Behind combined effect 24h or the 48h, the ginsenoside Rg1 of variable concentrations all can suppress oligomerization attitude A β
1-42Due to cell in the rising of ROS level.
2. fluorescence spectrometry H
2O
2Level
Be seeded in the cortical neuron of 96 orifice plates, after various conditions are handled, cellular exposure is in Amplex reagent that contains 50 μ M (Amplex Red reagent) and 0.1U/ml horseradish peroxidase (horseradish peroxydase, HRP) effect 60min, selective exitation ripple 540nm and transmitted wave 590nm measure fluorescence intensity with multi-functional fluorescence microplate reader.The blank group is set simultaneously.
2.1 oligomerization attitude A β
1-42Induce cortical neuron H
2O
2Generation
5 μ M oligomerization attitude A β
1-42Behind the effect cortical neuron, observe H in different time points (3h, 6h, 12h, 24h, the 48h) cell
2O
2Level, discover: along with oligomerization attitude A β
1-42H in the prolongation of action time, neuron
2O
2Level constantly raises, and shown in Fig. 2 .3, prompting constantly increases the weight of neuronic infringement, with oligomerization attitude A β
1-42Effect 24h and 48h H
2O
2The level rising is (p is respectively 0.007,0.002) significantly.
2.2 the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42H in the inductive cortical neuron
2O
2Raise
5 μ M oligomerization attitude A β are discovered in the front
1-42Hatch 24h or the 48h H in the neuron that can obviously raise
2O
2Level.So select oligomerization attitude A β
1-42Hatch 24h or 48h as the search time point, the ginsenoside Rg1 who observes variable concentrations is to H in the neuron
2O
2May influencing of level.Discover that ginsenoside Rg1's preincubate group is than A β
1-42 Hatch 24h or 48h group separately, the H in the neuron
2O
2Level all has reduction in various degree.Give the ginsenoside Rg1 separately and handle, H in the neuron
2O
2Level is similar to matched group, shown in Fig. 2 .4.
3.NO the mensuration of metabolite nitrite
(1) uses unazotized cell pyrolysis liquid, cell lysis.
(2) use high speed centrifuge centrifugal then, remove remaining fragment.
(3) reuse Bio-rad protein reagent box carries out protein quantification.
(4) albumen of getting same amount is measured the nitrite anions that contains in the albumen with the Griess method, is provided with standard curve simultaneously, calculates the concrete numerical value of nitrite anions: A by 50 microlitres/hole from curve, adds standard substance and sample in 96 orifice plates.B adds standard substance and sample by 50 microlitres/hole in 96 orifice plates.C adds room temperature Griess ReagentI by 50 microlitres/hole in each hole.D adds room temperature Griess Reagent II by 50 microlitres/hole in each hole.Go up machine testing after mixing 10min-15min under the E room temperature, wavelength is that 540nm measures absorbance.
3.1 oligomerization attitude A β
1-42Induce the rising of cortical neuron NO level
Present embodiment is with the oligomerization attitude A β of 0.5 μ M, 1.0 μ M and three kinds of concentration of 5.0 μ M
1-42, research A β
1-42The level of NO in the cell behind effect 24h and the 48h.Result of study is shown in Fig. 2 .5: three kinds of concentration all can increase intracellular NO level.A β
1-42The cell of effect 48h is compared with effect 24h, and the NO level raises more obvious in the cell; The A β of high concentration
1-42A β than low concentration
1-42Inductive NO level rising degree is wanted significantly.The A β of 5.0 μ M wherein
1-42Behind the effect 48h, the NO level has raise 4-5 doubly in the cell.
3.2 the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42The rising of inductive cortical neuron NO level
With ginsenoside Rg1's preincubate 24h of variable concentrations, adding final concentration then is 5.0 μ M oligomerization attitude A β earlier
1-42Combined effect 48h measures NO level in the cell at last, relatively the variation between each group.Result of study is shown in Fig. 2 .6: the level of NO all is lower than simple A β in ginsenoside Rg1's preincubate group cell of variable concentrations
1-42The effect group, it is similar to matched group to give the interior NO level of ginsenoside Rg1's cell separately.This prompting ginsenoside Rg1 can be in various degree minimizing oligomerization attitude A β
1-42The NO level raises in the inductive cell.
4.Western-blot detect the protein level of JNK, p38
Abandon culture fluid, use the PBS of pre-cooling to clean 2 times, every hole adds 100 μ l cell pyrolysis liquid (10mmol/L Tris (PH7.4), 100mmol/L NaCl, 1mmol/L EDTA, 1mmol/L EGTA, 1mmol/L NaF, 20mmol/L Na
4P
2O
7, 2mmol/L Na
3VO
4, 0.1%SDS, 0.5%Sodium deoxycholate, 1%Trion-X100,1mmol/LPMSF, 60 μ g/ml aprotinin, 10 μ g/ml leupeptin, 1 μ g/mlpepstatin), cracking 20min on the ice face, with cell scraper cell is scraped, mixing, in 14000 * g, 4 ℃ of centrifugal 10min, suct clear liquid to centrifuge tube, the Bradford method is carried out protein quantification.Get 20 μ g samples, add isopyknic 2 * sample-loading buffer (125mmol/L Tris (PH6.8), 10%g lycerol, 10%SDS, 0.006%bromophenol blue, 130mmol/L DTT) mixing, boil 90sec in 100 ℃ of boiling water, separate with the 10%SDS-polyacrylamide gel electrophoresis, with half-dried electrotransfer method albumen is transferred to pvdf membrane, under the room temperature with an anti-(dilution in 1: 1000 that behind the confining liquid sealing 1h film is moved into the confining liquid dilution, the polyclonal antibody JNK in rabbit source, p-JNK, the monoclonal antibody p-p38 in p38 and Mus source), 4 ℃ of overnight incubation, washing liquid washing 3 * 5min, 1 * 10min, IgG two anti-(dilution in 1: 1000) the reaction 2h of horseradish peroxidase coupling connection, washing liquid washing 3 * 5min, 1 * 10min, with the chemoluminescence method colour developing, the x-ray film exposure.β-actin is as internal reference.Experiment repeats 3 times.
4.1 oligomerization attitude A β
1-42Induce the increase of cortical neuron p38 phosphorylation level
Present embodiment is selected to study oligomerization attitude A β in the 1h
1-42To the former neuronic influence of foster mouse cortex of being commissioned to train, promptly in former mouse cortex cell of being commissioned to train foster, add the oligomerization attitude A β of 5 μ M
1-42Effect 5min, 15min, 30min and 60min measure the protein level of p-p38 and p38 in the cell then with western-blot.Shown in Fig. 2 .7, can see A β
1-42The ratio of p-p38/p38 obviously raises behind the effect 5min, this prompting A β
1-42Induce cortical neuron p38 that phosphorylation takes place, activated the activity of p38.But along with A β
1-42The prolongation of action time, the ratio of p-p38/p38 raise variation have also taken place, and descend gradually.
4.2 the ginsenoside Rg1 reduces oligomerization attitude A β
1-42Inductive cortical neuron p38 phosphorylation
The front is discovered as 5 μ M oligomerization attitude A β
1-42P-p38/p38 ratio reaches the highest during effect 5min.So we select 5min as oligomerization attitude A β
1-42Action time, research ginsenoside Rg1 may influence to the p38 phosphorylation.The result is shown in Fig. 2 .8: the p38 specific inhibitor SB203580 of 20 μ M can obviously suppress oligomerization attitude A β
1-42Inductive p38 phosphorylation, and the ginsenoside Rg1 of variable concentrations has the similar effect to p38 specific inhibitor SB203580, reduction p-p38/p38 ratio that can be in various degree.In addition, the ginsenoside Rg1 who gives 10 μ M separately handles the protein expression level that can reduce p-p38 significantly.This prompting ginsenoside Rg1 can suppress the phosphorylation of cortical neuron p38 and alleviate A β
1-42Induce p38 that phosphorylation takes place, thereby suppress the early stage stress of p38 mediation.
4.3 oligomerization attitude A β
1-42The increase of inductive cortical neuron c-Jun aminoterminal kinases (JNK) phosphorylation level
Present embodiment is with 5 μ M oligomerization attitude A β
1-42, the situation of change of (5min, 15min, 30min and 60min) JNK phosphorylation level in the research 1h.Shown in Fig. 2 .9, can see A β
1-42Can make the ratio of p-JNK/JNK raise, this prompting A β
1-42Induce the excessive phosphorylation of JNK of cortical neuron.But along with A β
1-42The prolongation of action time, variation has also taken place in the ratio of p-JNK/JNK.At A β
1-42When effect 15min and 30min, the ratio of p-JNK/JNK is the highest, along with the ratio of the prolongation p-JNK/JNK of time descends gradually.This prompting A β
1-42Induce cortical neuron JNK activity to increase to be free scope, and be that the activity that stress activates JNK has taken place in early days.
4.4 the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42Inductive cortical neuron JNK phosphorylation
Oligomerization attitude A β is discovered in the front
1-42Can make the ratio of p-JNK/JNK that change has taken place, when 15min and 30min, after the ratio of p-JNK/JNK reached top level, the ratio of p-JNK/JNK just descended gradually.So, select the time point of 15min as research, inquire into ginsenoside's may influence to the ratio of p-JNK/JNK.Result of study shown in Fig. 2 .10, the JNK phosphorylation specific inhibitor sp600125 oligomerization attitude capable of blocking A β of 10 μ M
1-42The ratio of inductive p-JNK/JNK raises, and the ginsenoside Rg1 of variable concentrations can alleviate oligomerization attitude A β in various degree
1-42The ratio of inductive p-JNK/JNK raises, and this prompting ginsenoside Rg1 can suppress the activity of JNK, thereby alleviates A β
1-42Stress damaging in early days of mediation.
1. the level of fluorescence spectrometry mitochondrial membrane potential
Different condition is handled respectively organizes cell, the trypsinization through 0.25%, the careful washing of aseptic HBSS (Hank s ' balanced saltsolution) back counting.Get the same cell number, directly add 1xJC-1 after 37 ℃ incubator is hatched 15min,, add isopyknic analysis buffer mixing gently, last machine testing fluorescence intensity (ex λ 488nm, em λ 525nm with HBSS washing three times; Ex λ 530nm, em λ 590nm).The inside and outside membrane potential difference of fluorescence intensity ratio reflection mitochondrion of gained: red fluorescence intensity (ex λ 530nm, em λ 590nm)/green fluorescence intensity (ex λ 488nm, em λ 525nm).
1.1 oligomerization attitude A β
1-42Destruction to the cortical neuron mitochondrial membrane potential
On the Normocellular mitochondrial membrane, there is potential difference in interior lateral surface, and it is required that it is that mitochondrion is kept the electron conduction process, if the potential difference on the mitochondrial membrane has weakened, this mitochondrial function will be restricted.Please find 5 μ M oligomerization attitude A β in this
1-42Can reduce the potential difference on the mitochondrial membrane, along with oligomerization attitude A β
1-42The prolongation of action time (3h, 6h, 12h, 24h, 48h), this mitochondrial membrane potential difference is more and more littler, shown in Fig. 3 .1.This prompting oligomerization attitude A β
1-42The neuron linear plastochondria had toxic action.
1.2 the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42Destruction to the neuron linear mitochondrial membrane potential
Oligomerization attitude A β is discovered in the front
1-42Effect 24h or 48h can obviously reduce mitochondrial membrane potential, therefore study in front on the basis, inquire into the ginsenoside Rg1 to oligomerization attitude A β
1-42May influencing of failure line mitochondrial membrane potential.Discover that the raising mitochondrial membrane potential that the ginsenoside Rg1 of variable concentrations can be in various degree alleviates oligomerization attitude A β
1-42Destruction to mitochondrial membrane potential.This prompting ginsenoside Rg1 may have the protective wire plastochondria, alleviates oligomerization attitude A β
1-42To the mitochondrial toxic action of neuron, the result is shown in Fig. 3 .2.
2. ATP level determination in the cell
Adopt the content of the interior ATP of ATP Bioluminescence As say Kit HS II detection cell of Roche company.Dissolving buffer in the ATP test kit (dilution buffer) transfers to 10 with each group cell quantity
4/ ml, the cell cracking agent (cell lysis reagent) that adds afterwards in the ATP test kit acts on 5min under 15 ℃ of-25 ℃ of conditions, add isopyknic fluorescence (element) enzyme reagent (luciferase reagent) at last, detect chemiluminescent intensity with chemiluminescence detector, ATP level in the cell more on the same group.
2.1 oligomerization attitude A β
1-42Reduce the level of cortical neuron ATP
ATP is that direct energy provides body in the cell, and it derives from mitochondrion.Whether and the extent of damage functional status of the direct response line plastochondria of the level of ATP also can point out mitochondrial infringement.Shown in Fig. 3 .3,5 μ M oligomerization attitude A β
1-42Can reduce the level of ATP in the cell, and the level reduction of ATP there is statistical significance (the p value is respectively 0.047,0.001,0.0004) when 12h, 24h and 48h, with A β
1-42The ATP level reduces the most obvious when effect 24h and 48h.This also points out oligomerization attitude A β
1-42But the energy-producing function of failure line plastochondria, and prolongation in time, its destruction is more and more serious.
2.2 the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42Reduce the effect that cortical neuron ATP produces
The A β of 5 μ M oligomerization attitudes is discovered in the front
1-42When acting on cortical neuron 24h and 48h, the ATP level in the neuron obviously descends.On this basis, the influence of ATP level in ginsenoside Rg1's pair cell of research variable concentrations.The result is shown in Fig. 2 .14: no matter at the A of oligomerization attitude β
1-42Act on cortical neuron 24h or 48h, the ginsenoside Rg1 of variable concentrations can alleviate the A β of oligomerization attitude
1-42Reduce the degree of ATP in the cell.This prompting ginsenoside Rg1 may have the protective wire plastochondria, alleviates oligomerization attitude A β
1-42To mitochondrial destruction.
3. intracellular plastochondria composite I V determination of activity
(1) oligomerization attitude A β
1-42Act on the cortical neuron different time, wash 2 times with the cold PBS of 1x (PH7.4).
(2) scrape (every group about 1 * 10 in cell with cell scraper
7Individual cell), centrifugal, abandon supernatant.
(3) with 50 μ L dissociating buffer (isolaton buffer:250mM sucrose; 20mMHEPES PH7.2 and 1mM EDTA) re-suspended cell gently.
(4) add 1x analysis buffer (1x assay buffer:10mM Tris-HCl PH7.0 and 120mM KCl) re-suspended cell gently.
(5) add 1x enzyme dissolving buffer (1x enzyme dilution buffer:10mM Tris-HCl PH7.0) 50 μ L re-suspended cell gently.The mixing liquid of sucking-off fraction carries out protein quantification.
(6) add 50 μ L cytochrome c substrate solutions (ferrocytochrome csubstrate solution) at last (0.22mM), measure its per 5 seconds light absorption value in 3min at wavelength 550nm.
3.1 oligomerization attitude A β
1-42To the active infringement of mitochondrion composite I V
Mitochondrion composite I V is a complex enzyme important on the mitochondrion electron transport chain, and its activity change is related to the generation that mitochondrion produces the ability of ATP and has influence on oxidation product.Present embodiment changes situation with the specific substrate cytochrome c of mitochondrion composite I V at the light absorption value of 550nm, indirectly the functional status of indication wire plastochondria composite I V.Discover that mitochondrion composite I V activity is subjected to oligomerization attitude A β
1-42Influence, the A β of oligomerization attitude
1-42Can reduce the activity of mitochondrion composite I V.At 5.0 μ M oligomerization attitude A β
1-42Effect 12h, 24h and 48h after, composite I V descends in various degree in the mitochondrion, especially oligomerization attitude A β
1-42Effect 24h and 48h after, in the mitochondrion composite I V descend the most obvious, shown in Fig. 3 .5.
3.2 the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42Activity infringement to mitochondrion composite I V
Oligomerization attitude A β is discovered in the front
1-42The activity that can suppress mitochondrion composite I V, oligomerization attitude A β
1-42Behind effect 24h and the 48h, composite I V is active in the mitochondrion obviously descends.So, on the research basis in front, inquire into the ginsenoside Rg1 to A β
1-42Suppress active may the influence of composite I V.The ginsenoside Rg1 of various concentration can alleviate A β in various degree
1-42To the active inhibitory action of composite I V, the prompting ginsenoside Rg1 can alleviate oligomerization attitude A β
1-42To the active inhibitory action of mitochondrial respiratory chain composite I V, shown in Fig. 3 .6.
4. the mensuration of the extraction of cell mitochondrial, cell cytosol and line grain cylinder cell pigment c
Extract the step of cell mitochondrial:
(1) variable concentrations oligomerization attitude A β
1-42Act on the cortical neuron different time, wash one time with cold 1x PBS (PH7.4).
(2) scrape (every group about 1 * 10 in cell with cell scraper
7Individual cell), 4 ℃ of centrifugal 5min of condition 600xg abandon supernatant, repeat twice.
(3) add 1ml endochylema buffer (1x cytosolic buffer), resuspended gently, place 15min on ice.
(4) use the homogenizer (KONTES of 1ml on ice, Fisher Scientific Company) after the manual homogenate, the centrifugal 20min of 800xg abandons precipitate (precipitate is nucleus, cell debris and complete cell) under 4 ℃ of conditions, and supernatant comprises mitochondrion and endochylema liquid.
(5) nucleus of the supernatant centrifugal 10min reject of 800xg remnants under 4 ℃ of conditions repeats 2 times.
(6) the centrifugal 20min of 10000xg under 4 ℃ of conditions, the gained supernatant is the endochylema composition, is precipitated as the mitochondrion composition.
(7) with the resuspended mitochondrion part of 1x endochylema buffer (1x cytosolic buffer), after the centrifugal 20min of 10000xg abandons supernatant under 4 ℃ of conditions, add complete mitochondrion buffer (complete mitochondrial buffer), on ice abundant mixing behind the cracking 15min.
(8) contain the supernatant liquid of endochylema composition, the centrifugal 30min of 16000xg abandons precipitation under 4 ℃ of conditions.
(9) measure protein concentration with Bio-Rad protein assay, be placed in-80 ℃ of preservations after packing.
Measure cell cytosol and line grain cylinder cell pigment c:
(1) the cytochrome c standard curve prepares and contain the sample dropping of cytochrome c: the cytochrome c with concentration known dilutes from 50ng/ml-0.781ng/ml with the sealing buffer successively according to gradient.The sample that contains cytochrome c is diluted to 20-100ng/ml concentration with the sealing buffer, and every hole adds 100 μ L volumes.
(2) combining of cytochrome c and antibody: after at room temperature the speed of 100rpm is hatched 2h, wash 3 times with 250 μ L washing liquids.
(3) cytochrome c detects the combination of antibody: require to cooperate required cytochrome c to detect antibody concentration to specifications, after the speed of 100rpm is hatched 1h under the room temperature, wash 3 times with 250 μ L washing liquids.
(4) streptavidin of HRP coupling connection detects antibodies with cytochrome c: require to specifications to cooperate desired concn, after the speed of 100rpm is hatched 1h under the room temperature, wash 4 times with 250 μ L washing liquids.
(5) colour developing, reading: after hatching 2-10min under the develping solution room temperature, add stop solution, at last at 450hm spectrophotometer reading.
4.1 oligomerization attitude A β
1-42Induce the release of cortical neuron mitochondrial cytochrome c
Cytochrome c mainly is present in the mitochondrion, is mitochondrion electron transport chain important substance, participates in the generation of mitochondrial oxidative phosphorylation and ATP.Cytochrome c is present in the mitochondrion under the normal condition, when mitochondrion suffers damage the mitochondrial pair of membrane structure permeability increase or film integrality destroyed, mitochondrial cytochrome c just is discharged in the endochylema, thereby activate the caspase cascade reaction, induce the dependent apoptosis of caspase to produce.Shown in Fig. 3 .7, the cytochrome c content of endochylema is seldom in the matched group, and most of cytochrome c is arranged in cell mitochondrial.Through 5.0 μ M oligomerization attitude A β
1-42After the effect, along with the prolongation of time, intracytoplasmic cytochrome c content increases gradually, and the content of Intramitochondrial cytochrome c constantly reduces.
4.2 the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42The release of inductive neuron linear plastochondria cytochrome c
Oligomerization attitude A β is discovered in the front
1-42Behind the effect 48h, intracytoplasmic cytochrome c content obviously increases, and the content of Intramitochondrial cytochrome c significantly reduces, and intracytoplasmic cytochrome c content surpasses Intramitochondrial cytochrome c content.On this basis, behind ginsenoside Rg1's preincubate 24h of research variable concentrations with the A β of oligomerization attitude
1-42Hatch 48h altogether, measure the content of endochylema and line grain cylinder cell pigment c then, the result is shown in Fig. 3 .8: the cytochrome c of ginsenoside Rg1's preincubate group of variable concentrations in can minimizing endochylema in various degree, improve Intramitochondrial cytochrome c content.This prompting ginsenoside has the certain protection effect to mitochondrion.
1.Western-blot detect the protein level of p-CREB/CREB and PKA II α subunit
Abandon culture fluid, use the PBs of pre-cooling to clean 2 times, every hole adds 100 μ l cell pyrolysis liquid (10mmol/L Tris (PH7.4), 100mmol/L NaCl, 1mmol/L EDTA, 1mmol/L EGTA, 1mmol/L NaF, 20mmol/L Na
4P
2O
7, 2mmol/L Na
3VO
4, 0.1%SDS, 0.5%Sodium deoxycholate, 1%Trion-X 100,1mmol/LPMSF, 60 μ g/ml aprotinin, 10 μ g/ml leupeptin, 1 μ g/mlpepstatin), cracking 20min on the ice face, with cell scraper cell is scraped, mixing, in 14000 * g, 4 ℃ of centrifugal 10min, suct clear liquid to the EP pipe, the Bradford method is carried out protein quantification.Get 20 μ g samples, add isopyknic 2 * sample-loading buffer (125mmol/L Tris (PH6.8), 10%g lycerol, 10%SDS, 0.006%bromophenol blue, 130mmol/L DTT) mixing, boil 90sec in 100 ℃ of boiling water, separate with the 10%SDS-polyacrylamide gel electrophoresis, with half-dried electrotransfer method albumen is transferred to pvdf membrane, under the room temperature with an anti-(polyclonal antibody CREB in rabbit source who behind the confining liquid sealing 1h film is moved into the confining liquid dilution, the polyclonal antibody PKA II α in the monoclonal antibody p-CREB in Mus source and rabbit source is dilution in 1: 1000 respectively), 4 ℃ of overnight incubation, washing liquid washing 3 * 5min, 1 * 10min, IgG two anti-(dilution in 1: 1000) the reaction 2h of horseradish peroxidase coupling connection, washing liquid washing 3 * 5min, 1 * 10min, with the chemoluminescence method colour developing, the x-ray film exposure.β-actin is as internal reference.Experiment repeats 3 times.
1.1 oligomerization attitude A β
1-42Reduce the phosphorylation degree of CREB
CREB has another name called the cAMP response element binding protein, is a kind of transcription regulaton factor, and CREB can make the CREB-cAMP conjugate strengthen the protein expression of downstream gene in Ser133 site phosphorylation.Result of study shown in Fig. 4 .1, oligomerization attitude A β
1-42Reduce the phosphorylation level of the CREB of glutamic acid mediation, at A β
1-42During effect 2h, the ratio of p-CREB/CREB reduces the most obvious.
1.2 the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42Reduce the phosphorylation degree of CREB
The front studies show that, oligomerization attitude A β
1-42Can reduce the phosphorylation of the CREB of glutamic acid mediation, at 5.0 μ M oligomerization attitude A β
1-42During effect 2h, the ratio of p-CREB/CREB reduces the most obvious.Therefore, select 2h as A β
1-42The time point of effect, research the ginsenoside Rg1 may influence it.The result is shown in Fig. 4 .2, and the increase p-CREB protein level that the ginsenoside Rg1 can be in various degree makes the ratio of p-CREB/CREB raise.
1.3 the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42The protein level of inductive PKA II α subunit
PKA II α subunit is the adjusting subunit of PKA, and it is the bonded group of cAMP and PKA, and the protein level of PKA II α subunit and the activity of PKA are inverse ratio.Present embodiment will be inquired into the oligomerization attitude A β of 5 μ M
1-42Behind the effect 2h, on the basis of the active variation of PKA, study and the active situation of inferring PKA indirectly from the angle of PKA subunit, and the possible protective effect mechanism of ginsenoside Rg1.The result is shown in Fig. 4 .4.
2. PKA determination of activity in the cell
(1) respectively organizes cell after handling, inhale and abandon supernatant, wash one time with 1 * ice-cold PBS.Add lysate buffer[20mM MOPS, 50mM β-glycerolphosphate, 50mMsodium fluoride, 1mM sodium vanadate, 5mM EGTA, 2mM EDTA, 1%NP40,1mM dithiothreitol, 1mM benzamidine, 1mM PMSF and 10 μ g/mL leupeptin and aprotinin];
(2) cracking on ice is 10 minutes, and cell scraper scrapes, and moves to the centrifuge tube of 1.5ml;
Centrifugal 15 minutes of (3) 13,000rpm;
(4) get supernatant, protein quantification.
(5) preparation of PKA zymolyte: a) calculate required hole count, the remaining drying sealing masking foil sack that is kept at 4 ℃; B) every hole added the kinase assay dilution buffer of 50 μ L, soaking at room temperature 10 minutes; C) the careful suction abandoned each hole liquid.
(6) add standard substance and sample: a) every hole adds the reactant liquor of 30 μ L: add pure active PKA or sample or blank well (having only kinase assay dilutionbuffer); B) except the blank group, every hole adds the ATP solution (with the dissolved 1mg/mL concentration of kinaseassay dilution buffer) of 10 μ L, avoids cross-contamination future, adds the tip head that renews behind the liquid in every hole; C) seal to the hole, hatched 90 minutes, preferably jiggled several times, so that abundant mixing every 20 minutes at 30 ℃; D) suction is abandoned every hole liquid with cessation reaction, and the back-off plate on clean paper, is patted Sptting plate.
(7) add specificity phosphoric acid substrate antibody: a) except blank well, every hole adds the phospho-secific substrate antibody of 40 μ L; B) sticking the film that seals of function with new having, seal plate, hatched under the room temperature 60 minutes, is every 20 minutes jogs at last, makes its abundant mixing.
(8) wash plate: a) inhale and abandon each hole liquid; B) every hole adds the washing liquid of 100 μ L, uses the volley of rifle fire (in order to reduce background, each washing liquid 1-2 minute at interval); C) repeat to wash 3 times, totally 4 times; D) the 4th, after washing liquid was abandoned in suction, the back-off plate on clean paper, was patted plate.
(9) add anti-rabbit Ig-G:HRP CONJUGATE:a) except blank well, every hole adds the anti-rabbit igg that showing of 40 μ L prepares: HRP Conjugate (1: 1000 antibody dilution buffer dilution); B) seal plate, at room temperature hatched 30 minutes,, make its abundant mixing every 10 minutes jogs; C) wash plate, step is with 4.
(10) adding TMB SUBSTRATE and ACID STOP SOLUTION:a) every hole adds the TMB SUBSTRATE of 60 μ L; B) hatch 30-60min under the room temperature, observe change color simultaneously; C) add the STOP SOLUTION of 20 μ L according to the order that adds TMB SUBSTRATE.
(11) measure light absorption value: extinction wavelength 450nm.
2.1 the ginsenoside Rg1 alleviates oligomerization attitude A β
1-42Reduction to the PKA enzymatic activity
Protein kinase A (PKA) is a kind of important protein kinases in the cell, and activatory PKA can make the serine or the threonine residues phosphorylation of some albumen (comprising CREB) in the cell, so change these proteic activity, further has influence on Expression of Related Genes.Discover the oligomerization attitude A β of 5 μ M in front
1-42Effect 2h can obviously reduce the phosphorylation level of CREB, and the topmost adjusting kinases of CREB is PKA, so present embodiment will be inquired into the oligomerization attitude A β of 5 μ M
1-42Behind the effect 2h, the active variation of PKA, and the possible protective effect of ginsenoside Rg1.As a result shown in Fig. 4 .3: the oligomerization attitude A β of 5 μ M
1-42Behind the effect 2h, the active of PKA significantly descends, and the ginsenoside Rg1 of each concentration can alleviate A β in various degree
1-42Inductive PKA is active to descend.The activity of the protection PKA that these promptings ginsenoside Rg1 possibility is direct or indirect alleviates A β
1-42Inhibitory action to PKA.
Claims (5)
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