CN1112181C - Application of tripterygium plant extract in preventing and curing diseases in nervous system - Google Patents
Application of tripterygium plant extract in preventing and curing diseases in nervous system Download PDFInfo
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Abstract
The present invention relates to application of a tripterygium wilfordii plant extract in preventing and curing nervous system diseases. The nervous system diseases comprise the Alzheimer's disease, the Parkinson disease, the Huntington's nervous retrograde disease, spinal cord injury and lateral spinal sclerosis. A monomer component in the tripterygium wilfordii plant extract has obvious immunosuppressive activity under the experiment conditions of monomer separation from bodies and monomer existence in bodies as well as has an obvious nutrition action on cultured DA energy neurons. The tripterygium wilfordii plant extract can promote the growth of midbrain nerve cells, and the protrusion and the extension of skin layer nerve cells of primary culture, and resist the injury action on nerve cells caused by environment toxins, endogenous toxins and excitability neurotoxins. The tripterygium wilfordii plant extract has an obvious protective action on cell survival.
Description
The present invention relates to the new purposes of Chinese medicine, the application of particularly a kind of tripterygium plant extract on preparation prevention and treatment nervous system disease medicine.
Described nervous system disease comprises Alzheimer, parkinson disease, Heng Tingdunshi neurodegenerative diseases.
Old neurodegenerative diseases is that what to occur with age growth is principal character with some neuron necrosis progrediens in the brain essence specific region, serve as a class nervous system disease that mainly shows with learning memory disorder or motion, behavior, mental maladjustment.Wherein common with Alzheimer (Alzheimer s disease is called for short AD) and parkinson disease (Parkinson s disease is called for short PD), the disability rate height, also maximum to senior health and fitness's influence.
Described PD is a kind of common nervous system extrapyramidal system degenerative disease that is mainly in person in middle and old age's phase, and patient has more now and trembles, symptoms such as muscular rigidity, bradykinesia.The main diseases Neo-Confucianism of PD is changed into the dopamine of substantia nigra of midbrain and nigrostriatum path, and (dopamine, DA) serotonergic neuron degeneration necrosis causes striatum DA level significantly to reduce.Therefore, precursor one levodopa (L-DOPA) of additional DA can be alleviated the symptom of PD.But L-DOPA itself can not delay the further necrosis of dopaminergic neuron, and other side effect is arranged, and therefore, people attempt to find a kind of degeneration necrosis that can delay the DA serotonergic neuron always, to the medicine of the nutritious protective effect of DA serotonergic neuron.
(neurotrophic factors is to keep and promote neurocyte normal existence, growth and differentiation NTFs) to neurotrophic factor, promotes its specific polypeptide of regenerated class or protein under the nerve injury situation.Comprise nerve growth factor (nerve growth factor, NCF), Brain Derived Neurotrophic Factor (brain-derived ncurotrophic factor, BDNF), glial cell line-derived neurotrophic factor (glial cell line-derived neurotrophic factor, GDNF), neurotrophic factor-3 (neurotrophic-3, NT-3) etc.Wherein GDNF nutrition comparatively specifically and protection DA serotonergic neuron, and can promote the regeneration of DA serotonergic neuron, become the focus of current prevention and treatment PD.
But NGF, GDNF, BDNF etc. are high molecular weight protein, can not see through blood brain barrier, and be unsuitable peripherally administered.The method that solves this difficult point has two: 1, by carriers such as engineering cell, adenoviruss the external source gdnf gene is imported in the brain, make its long-term expression, secretion gdnf protein, play nutrition, protection and repair; 2, seek the small-molecule substance that has neurotrophic effect or promote the endogenous neurotrophic factor expression.
Alzheimer claims alzheimer disease again, is a kind of person in middle and old age's of betiding constitutional cerebral retrogressive disease.Clinically mainly show as cognition and damage in learning and memory, even the defective of emotion or personality aspect.The main pathology of AD is changed into senile plaque (claiming the starch speckle again), neurofibrillary tangles and selectivity cholinergic neuron and synapse is lost.Pathological anatomy shows that the neuron at positions such as the neopallium of AD patient's brain, hippocampus, nbM and nucleus ceruleus is lost in a large number, and particularly the minimizing of the acetylcholine nerve of cortex and Hippocampus is particularly remarkable.
Modern study thinks that (acetycholine ACh) is the neurotransmitter that promotes learning and memory to acetylcholine, and the M-cholinergic synapse is the memory basis.And the degeneration of cholinergic neuron is considered to cause dull-witted important pathological factor.Therefore, the medicine that can stop cholinergic nerve to be degenerated is the first-selection of following AD treatment.Proved that NGF can effectively prevent to simulate the cholinergic neuron degeneration of animal basal forebrain and the death of AD pathological changes.But because of the NGF molecular weight is big, oral or injection is difficult to arrive brain.Someone attempts with giving NGF repeatedly in the tricorn intubate brain, and has received sure effect.In order to solve repeatedly the problem that administration brought in the brain, in recent years, the scientific research personnel is striving to find the alternative approach of NGF administration, and these approach comprise: 1, the genetically engineered NGF cellulation of intracerebral transplantation; 2, take and to promote the biosynthetic medicine of NGF in the brain.
Discovering in recent years, and the immunosuppressant cyclosporin A (cyclosporin A, CsA), FK506 and Rapamycin (RAPA) etc. also have neurotrophic effect.People's such as Snyder experiment in vitro studies show that, denier (ng level) immunosuppressant FK506 and RAPA can work in coordination with the short PC12 cell axon growth effects of neurotrophic factor (NGF), the valid density that makes NGF bring out best axon growth effects has reduced 20-50 doubly, and half optimum effect dosage is also reduced to 0.1ng/ml.On the Mus dorsal root ganglion cell of In vitro culture, FK506 uses separately just can produce tangible neurotrophic effect, thinks that its mechanism still is that the NGF that relies on the glial cells such as Schwann cell in the neuroganglion to produce plays a role.
Experiment shows that also CsA can slow down 6-hydroxy dopamine (6-hydroxydopamine, the regression of the DA serotonergic neuron that 6-OHDA) brings out in mouse brain in the body.Equally, FK506, CsA also can resist the exhaustion of DA in the inductive C57/BLACK Parkinson disease model of the MPTP mouse brain.CsA and FK506 can both obviously raise DA in the striatum and the content of DOPAC have remarkable meaning.Especially be outstanding with FK506 in the two.CsA can also protect ischemia one reperfusion injury of cerebral tissue.
Think that at present the mechanism of neurotrophic effect of immunosuppressant is, CsA, FK506, RAPA etc. act on intracellular receptor separately respectively, the CsA correspondence be the ring film (Cyclophilin, Cyp), FK506 and RAPA correspondence be FK506 conjugated protein (FK506 binding protein, FKBP).Immunosuppressant forms complex with FKBP, Cyp specifically, and (Calcineurin, CaN) combination have suppressed the activity of this enzyme catalysis albumen dephosphorization acid reaction with phospholipase 2B again.Discover that further CaN has influenced the phosphorylation process of two kinds of enzymes in brain: 1, nitric oxide synthetase (NOS), the phosphorylation form of NOS will suppress its catalytic activity, thereby the neurotoxic effect of excessive glutamic acid is blocked in the generation that immunosuppressant suppresses NO by the phosphorylation degree that improves NOS; 2, growth associated protein (GAP-43).GAP-43 has participated in neuronic growth course and its phosphorylation form can strengthen this kind growth promoting function.Axon can take place and prolong in the PC12 cell under the stimulation of NGF.Very the FK506 of low concentration (nmol level) just can improve the sensitivity of cell to NGF, brings out the axon growth effects of PC12 cell, the effect of NGF can be improved 100 times.Therefore, selectivity suppresses the medicine of some effect substrate of CaN, and the inhibitive factor that reacts as the GAP-43 dephosphorylation may have great potential therapeutic value in the treatment nerve retrograde affection.
Radix Tripterygii Wilfordii is Celastraceae (Celastraceae) tripterygium plant, (Tripterygum WilfordiiHook.f), tripterygium plant also comprises Tripterygium hypoglaucum [T.Hypoglacum (LevI) Hutch] and black climing (TRegelliSprague et Tak), all has medical value.Radix Tripterygii Wilfordii contains multiple active ingredients such as alkaloid, diterpene, triterpene, sesquiterpene.Wherein, Diterpenes is main active ingredient, and triterpene and alkaloid are also had an activity.Present known Radix Tripterygii Wilfordii monomer component comprises diterpene-kind compound and tripterine triterpenoid compound such as (tripterine) such as Radix Tripterygii Wilfordii lactone alcohol (triptolide), tripchlorolide (tripc hlorolide), NSC-163063 (tripdiolide), Triptolide triol, sees Fig. 1.
Among the figure, (1) is Radix Tripterygii Wilfordii lactone alcohol, and (2) are tripchlorolide, and (3) are the Triptolide triol, and (4) are tripterygone, and (5) are NSC-163063, and (6) are 16-hydroxytriptolide, and (7) are tripterine.
It is its main pharmacologically active that Radix Tripterygii Wilfordii extract has multiple pharmacologically actives, particularly immunosuppressive action such as immunosuppressant, antiinflammatory, antitumor and antifertility.What immunosuppressive activity was the strongest in the monomer component that Radix Tripterygii Wilfordii is extracted is Radix Tripterygii Wilfordii lactone alcohol, and its immunosuppressant ED50 is 0.06mg/kg.Radix Tripterygii Wilfordii decoct, Radix Tripterygii Wilfordii " general glycoside " (TII) or Radix Tripterygii Wilfordii lactone alcohol and tripterine (concanavallin A, ConA) effect of inductive mouse boosting cell secretion IL-2 all has obvious inhibitory action to canavaline.
In addition, the tripchlorolide of one of main active of Radix Tripterygii Wilfordii has a kind of dual regulation of dose dependent to mice spleen NK cytoactive, shows that Radix Tripterygii Wilfordii is not only to have immunosuppressive action.
In the past, mainly the tripterygium total extract was used for the treatment of nephropathy such as rheumatoid arthritis and chronic glomerulonephritis, nephrotic syndrome, Henoch Schonlein purpura nephritis clinically.In addition, connective tissue diseases such as systemic lupus erythematosus (sle), polymyositis, dermatitis and some dermatosiss all there is certain curative effect.Yet as Neuroprotective Agents, any report is not seen in preparation prevention and treat the medicine of old neurodegenerative disease so far with tripterygium total extract or monomer whose composition.
The purpose of this invention is to provide the application of a kind of tripterygium plant extract in preparation prevention and treatment nervous system disease medicine.
Described tripterygium plant extract comprises one or more in Radix Tripterygii Wilfordii lactone alcohol, tripchlorolide, NSC-163063, Triptolide triol, 16-hydroxytriptolide, triptolidenol, triptophenolide and tripterine and the wilfortrine.
Described nervous system disease comprises Alzheimer, parkinson disease, Heng Tingdunshi neurodegenerative diseases and spinal cord injury, lateral spinal sclerosis disease.
Described tripterygium plant extract and neurotrophic factor are united use, also are used for the treatment of nervous system disease.Described neurotrophic factor comprises nerve growth factor, glial cell line-derived neurotrophic factor, Brain Derived Neurotrophic Factor and ciliary neurotrophic factor.
Has the immunosuppressant of neuroprotective and Nutrition in order from natural drug, to seek; multiple single agent that immunosuppressive activity is arranged and compound natural medicine to present clinical practice are estimated; found that the monomer component in the natural medicinal plant Radix Tripterygii Wilfordii extract all has significant immunosuppressive activity under the experiment condition in stripped and body.Show with in vitro study in the body that further as the main active in the Radix Tripterygii Wilfordii extract, tripchlorolide (hereinafter to be referred as 968) has tangible Nutrition to the DA serotonergic neuron of cultivating.Compare with the cyclosporin of organizing in contrast, 968 under low concentration (0.001ng/ml) and higher concentration (0.1ng/ml), and after the DMEM that contains 10% hyclone cultivated 4 days, the DA serotonergic neuron of survival was respectively than matched group high 87% and 29%.Be lower than 10 in addition
-13The growth of cranial nerve cell during 968 of mol/L just can promote, aixs cylinder length exceeds 113% than matched group.As if 968 neurotrophic effect is not limited to the DA serotonergic neuron, and 968 just can promote the projection of the former foster cortical neurogenic cell of being commissioned to train to prolong under extremely low concentration.968 this short aixs cylinder goes out the growth effect and rebuilds significant for the synapse between neurocyte in the diseases such as PD, AD and spinal cord injury.
Another important feature of tripchlorolide be can some neurotoxins of antagonism to the nerve cell damage effect.Environmental toxin, endogenous toxin and excitatory neuron toxin are one of important mechanisms of neurodegenerative diseases morbidities such as PD, AD.Find in the experiment: the tripchlorolide of (1), 1pM and Radix Tripterygii Wilfordii lactone alcohol (T
10) can both antagonism DA energy neurotoxin MPP
+To the toxic action of PC12, the survival of pair cell has significant protective effect; (2), 968 can resist the damaging action of heavy dose of irritability glutamic acid to former generation cortical neuron, help safeguarding the neurocyte shape integrity, reduce apoptosis by glutamate induction; (3), find that in the whole animal experiment 968 can effectively stop DA serotonergic neuron damage in the inductive mouse brain of DA energy neurotoxin MPTP.
Further describe the present invention below in conjunction with accompanying drawing and concrete experiment.
Fig. 1 shows is the chemical constitution of activated monomer main in the Radix Tripterygii Wilfordii extract.
What Fig. 2 showed is the influence of 968 pairs of former rat midbrain dopaminergic neuron survivals of being commissioned to train foster of variable concentrations.
The dopaminergic neuron of counting is meant TH immunohistochemistry positive cells.Every point data is the average counter of three groups of parallel laboratory tests.
What Fig. 3 showed is the influence of 968 pairs of former rat midbrain dopaminergic neuron percentage survival of being commissioned to train foster of variable concentrations.
What Fig. 5 showed is the influences of various dose 968 pretreatment to DA metabolic rate (DOPAC+HVA/DA) in the C57BL/6J mouse striaturn of MPTP damage.
The in vitro study of experiment one, tripchlorolide protection dopaminergic neuron
Get gestational age and be 14-17 days the pregnant Mus of SD, sacrificed by decapitation, get the mouse embryo midbrain the capable midbrain DA of 24 porocyte culture plates serotonergic neuron former be commissioned to train foster.Next day, changed blood serum medium or serum-free medium and added variable concentrations 968 (10,1,0.1,0.01,0.001ng/ml).TH SABC method is identified the existing state of DA serotonergic neuron.Experiment is found, add variable concentrations 968 after, in blood serum medium is arranged, cultivate the 4th day, 0.001,0.01, the cell number of 0.1ng/ml group is respectively 187%, 148%, 129% of matched group, the NEWMAN-KEULS one-factor analysis of variance has significance (table 1, Fig. 2, Fig. 3).Meansigma methods ± the standard error of classifying as in the table marks with * with the matched group significant difference, and * represents p＜0.05, and * * represents p＜0.01.Presentation of results 968 former generation dopaminergic neuron to In vitro culture in blood serum medium is arranged has clear and definite Nutrition.
The protective effect of 968 pairs of tire Mus of table 1 variable concentrations midbrain dopaminergic neuron (TH positive neuron).
Matched group 968 (ng/ml) n=4
Grouping
n＝4 0.001 0.01 0.1 1 10
TH+ god
223±8.9 478±9.6** 357±10.3
* 289±7.3
* 279±15.6 220±8.3
Through first number
In addition, do not find that in the serum-free medium in former generation 968 have the Nutrition of clear and definite DA serotonergic neuron, the neuron number of experimental group and matched group does not have the significance difference.This finds no Nutrition with CsA and FK506 in serum-free medium be consistent.Test studying of two tripchlorolide protection dopaminergic neuron at body.
MPTP is the neural toxin of species specificity damage DA energy, and peripheral injection can see through blood brain barrier, specificity damage black substance one striatum dopaminergic neuron.The C57BL/6J mice, ip MPTP 30mg/kg body weight connected and annotated three days every day, and the reduction of DA content in the striatum is reached more than 80%.
In the experiment of 968 pairs of C57/BLACK parkinson model mice midbrain dopaminergic neurons protective effect, we are divided into 8 groups: (1) N.S+N.S. group, as blank; (2) N.S+MPTP is as negative control; (3)-(7) group is 968 (10 of variable concentrations
-6μ g/kg body weight-10
-1μ g/kg body weight) experimental group; (8) CsA (10mg/kg body weight)+MPTP is as positive controls.
Injection one time 968 or normal saline under the previous day of MPTP damage; Every morning injection 968 or normal saline were injected MPTP or normal saline afternoon three days afterwards; Continue again to inject once a day 968 or normal saline to putting to death animal the previous day, inject 11d altogether.Experiment detects with the content of HPLC method to DA in the mouse striaturn and metabolite HVA (4-hydroxy-3-methoxy-.alpha.-toluic acid .), DOPAC (dihydroxyphenyl acetic acid), and the ratio of DA, (HVA+DOPAC)/DA has been carried out calculating and statistical analysis.
We find, 968 of 0.01ng/ml concentration group can make that the content of DA increases (Fig. 4) significantly in the mouse striaturn; Simultaneously, (HVA+DOPAC)/ratio of DA is in all groups minimum (Fig. 5).HVA and DOPAC are the final metabolite of DA in brain.(HVA+DOPAC)/and DA ratio raises, and illustrate that the metabolic rate of DA speeds, and the metabolite generation increases, when being more common in the DA serotonergic neuron and not exclusively damaging.968 can make the ratio of (HVA+DOPAC)/DA reduce, and illustrate that it can slow down the DA metabolic turnover speed in the MPTP damage Mus DA serotonergic neuron, thereby have protected neuronic survival effectively.The damage of the parkinson mice midbrain DA serotonergic neuron that MPTP is caused has the certain protection effect.
The influence of the table 2 968 pretreatment C57BL/6J mouse striaturn DOPAMINE CONTENT IN RABBIT that damage causes to MPTP
( x±SE)
968 (ng/kg) CsA grouping normal group matched group
0.01 1 10 100 0.001 (10mg/kg) striatum DOPAMINE CONTENT IN RABBIT 5.01 ± 0.32 0.98 ± 0.11 0.96 ± 0.03 0.97 ± 0.23 1.32 ± 0.21 2.44 ± 0.31
*1.37 ± 0.13 1.34 ± 0.11 (ng/mg)
968 of the 0.01-0.1 μ g/kg homergys that can keep DA as can be seen from Figure 4.
By the isolated cells experiment with in the body zoopery, we find that 968 have certain neurotrophy effect really.Different according to 968 effects in blood serum medium and serum-free medium are arranged infer that its mechanism may be to improve the sensitivity that neuron waits the nerve growth factor in the serum (NGF), thereby bring into play neurotrophic effect.Test the influence of cranial nerve cell synapse length in 3 968 pairs of former tire Mus of being commissioned to train foster.
The pregnant Mus of 16 days SD of gestational age is got the capable former generation neuron cultivation of tire Mus midbrain, and middle cerebral tissue is shredded, digests, dispels, and makes it to become individual cells, with 3 * 10
5The density of individual cells/well is planted in 24 well culture plates.The culture medium that adds 10% hyclone earlier with DMEM, 37 ℃, 5%CO
2Incubator was cultivated 24 hours, changed DMEM/F-12 (1: 1) culture medium then into, added N
2Additive continues to cultivate (mainly being to suppress glial cells hyperplasia), and other condition is constant.Add 10 simultaneously
-15-10
-6968 of mol/L handles each dosage group 4 hole.After 72 hours, observe down at inverted phase contrast microscope (200 *), and use the image processing system images acquired, 4 visuals field are gathered in every hole, with the aixs cylinder length of image processing system measurement neurocyte, aixs cylinder is defined as the longest projection of each cell, gets 8 the longest cells of aixs cylinder in each is looked.Each dosage group gained data merges, averages, and basis of calculation deviation, (table 3) is listed in the table below.Data are analyzed with ANOVA, and with Dunnet multiple comparisons check, and there was a significant difference marks * p＜0.05, * * p＜0.01 with * with matched group.The result shows 10
-15-10
-7The enation of cranial nerve cell during mol/L968 handles and can obviously promote, aixs cylinder length is than contrast group leader 213%-150%, difference significance.The axon growth that 968 pairs of neurocytes are described has facilitation, and its valid density wide ranges has increased the choice of clinical medicine dose in future and the safety of medication.Table 3 variable concentrations 968 is handled former being commissioned to train of tire Mus midbrain is reposed through the influence of cell process length (x ± SE)
Contrast 968 concentration of treatment (mol/L)
Tire Mus midbrain cultured method of former generation is with embodiment 3, and difference is to change into DMEM/F-12+1%N
2After the culture medium,, siphoned away culture fluid in the 8th day, and wash with PBS with 968 processing 7 days.Add the dyeing of 0.01% pyridine orange, observe down at fluorescence microscope (100 *), nucleus presents green fluorescence, and three visuals field are got in every hole, with manual count device counting.The data ANOVA analyzes, and with the check of Dunnet multiple comparisons, finds 10
-12-10
-11Mol/L 968 processed group survivaling cell numbers illustrate that apparently higher than matched group 968 help neuron survival.
Table 4 variable concentrations 968 is handled former being commissioned to train of tire Mus midbrain is reposed through the influence of cell survival (x ± SE)
968 concentration (mol/L)
Matched group
10
-1210
-1110
-1010
-910
-810
-710
-6Cell number 173 ± 16.9 427 ± 64.2
*355 ± 40.2
*201 ± 34.8 228 ± 29.4 258 ± 77.4 148 ± 13.5 17.8 ± 6.5 experiments 5 968 can antagonism dopaminergic nerve toxin MPP
+Toxicity to the PC12 cell.
The PC12 cell adopts RPMI 1640+10% new-born calf serum to cultivate, then with 1.5 * 10
5Density plant equably in 96 orifice plates, treat that the good back of cell attachment adds 968 or T
10Pretreatment 2 hours adds dopaminergic nerve toxin MPP then in experimental group
+60 μ mol/L damage PC12 cell.We have proved MPP in other experiment
+Median lethal concentration (the LC of damage PC12 cell
50) be 60 μ mol/L.Siphon away culture medium after 72 hours, tetrazolium bromide (MTT) the 100 μ l that add 0.5mg/ml in every hole, hatched 4 hours for 37 ℃, this moment, original yellowish green MTT formed blue granule under the catalysis of the succinic acid enzyme that living cells had, add 100 μ l cosolvent (isopropyl alcohols: Triton X-100: water=5: 1: 4) in every hole, 37 ℃ of shaking tables spend the night blue particle are dissolved fully, with Bio-Rad ELISA plate reading machine, detect the absorbance in each hole under the 490nm wavelength.Absorbance is directly proportional with the cell number in each hole under the similarity condition.Experimental group (comprises matched group and 968, T
10Processed group) absorbance and MPP
+The ratio of not damage group is the percentage survival of cell.Table 5 is classified the meansigma methods ± standard deviation of the cell survival rate in every group of 8 holes as.* expression is analyzed through ANOVA, and succeeded by the check of Dunnet multiple comparisons, there was a significant difference with matched group, * P＜0.05, * * P＜0.01.The result shows: 10
-9-10
-7968 and the T of mol/L
10Processing has antagonism MPP
+To the toxic action of PC12, make the survival rate of cell improve about 30%.
Table 5 968 and T
10Under 10% serum condition to MPP
+The Cytotoxic antagonism of PC12 (x ± SE)
Medicine matched group drug level (log mol/L)
-12 -11 -10 -9 -8 -7
T
4(968) 74.3±2.74 74.1±2.49 89.3±3.20
** 92.4±4.57
* 88.3±1.50
**
59.9±4.59
T
10 80.1±3.00
*?73.8±7.11 72.9±7.24 83.6±1.34
** 80.3±4.17
* 80.9±1.33
*
Above-mentioned experiment is the result under RPMI1640+10% new-born calf serum condition, and in order to get rid of serum role therein, we have reduced the concentration of serum, reduces to 1% by 10%, and other condition is constant, has observed 968 and T
10Influence to the PC12 cell survival.Data are listed in table 6.Table 6 968 and T
10Under 1% serum condition to MPP
+The Cytotoxic antagonism of PC12 (x ± SE)
Concentration (log mol/L) medicine matched group
-13 -12 -11 -10 -9 -8 -7 -6 -5968 38.6 71.2± 80.2± 71.2± 61.7± 70.0± 78.6± 66.7± 59.9± 50.0±
2.57 11.7
** 10.9
** 10.4
** 9.44
* 10.0
** 10.1
** 9.82
** 8.74
* 7.26T
10 65.2± 80.4± 86.8± 122.3± 129.8± 113.2± 117.0± 110.5± 62.8±
5.88 16.3 14.2 12.1
** 11.2
**?10.0
* 11.2
* 14.0
* 4.44
The above results shows, under the condition of low concentration serum (1%), and 968 and T
10Still has antagonism MPP
+Toxic effect.Test the inhibitory action that 6 968 pairs of glutamic acid bring out tire Mus cerebral cortex neuronal apoptosis
Material and method
1, bed board: in the inoculation before 2-3 days, with 12.5 μ g/ml poly-l-lysine bed boards, 6 orifice plates, 500 μ L/ holes, dry naturally in 24 orifice plates, 400 μ L/ holes.
2, inoculated and cultured:
Aseptic condition separates pregnancy period 16-18d tire Mus cerebral cortex down and places the DMEM/F-12 culture medium of pre-cooling, careful pia mater encephali and the blood vessel of rejecting, cerebral tissue is moved in another small beaker that contains (containing volume fraction is l0% hyclone and 10% horse serum) among the ice DMEM/F-12, blow and beat 20-30 time gently with tip polished glass pipe, be dispersed into cell suspension until whole cerebral tissue, and remove by filter indigested piece of tissue through 200 order nylon mesh, filtrate is used the DMEM/F-12 culture medium, (10% hyclone, 10% horse serum, 100kU.L
-1Streptomycin, pH7.2-7.4), after the blue dyeing of platform dish, 500 cells of counting under inverted phase contrast microscope, cytochrome is dead cell, and adjusts density to 1 * 10
9Individual .L
-1The 0.5ml cell suspension inoculation in 6 well culture plates of handling that spend the night of 12.5 μ g/ml, is put 37 ℃, 5%CO
2Cultivate in the incubator, treat behind the 24h that cell attachment changes liquid and removes dead cell 1 time, later every 3-4d changes liquid once, and (final concentration is 10 μ mol.L to the 3rd day adding 5-fluorouracil
-1) cultivate 24h, to suppress non-neuronal propagation.Begin experiment after continuing to be cultured to 5d.
Experimental result:
1,968 in the culture medium of serum is arranged the former generation cortical neuron to In vitro culture tangible Nutrition is arranged
With 968 (10 of variable concentrations
-13-10
-7Mol/L) hatch the former generation cortical neuron (5d of In vitro culture at the DMEM/F-12 that serum (10% hyclone+10% horse serum) is arranged, Wistar neonatal rat 14-16d), fixing also pair cell number, projection length and cell space area carry out the microscopic image analysis behind the 24h.The result as seen, the cell space number does not have significant change in 24h, and cell space area and projection length are 10
-13-10
-8Each concentration all significantly increases.
Table 7 968 is having under the serum condition influence to tire Mus cerebral cortex neure growth situation (x ± SE)
968 concentration (log mol/L)
Matched group
-13 -12 -11 -10 -9 -8 -7
67.67 ± 3 66.40 ± 78.00 ± 74.00 ± 79.20 ± 85.3 ± 82.20 ± 42.67 ± cell number
84 13.38 3.56 10.30 6.30 6.72 12.92 12.28
40.27 ± 2 83.70 ± 120.83 ± 110.63 ± 103.92 ± 97.10 ± 68.36 ± 43.46 ± cell space area (μ m
2)
97 3.07
** 6.18
** 2.83
** 4.04
** 2.81
** 2.48
** 2.43
50.11 ± 8 73.98 ± 79.16 ± 83.78 ± 99.79 ± 97.11 ± 75.83 ± 68.19 ± synapse length (μ m)
91 13.10
** 10.44
** 25.16
** 18.02
** 9.20
** 6.31
** 15.38
**
Be chosen at the most significant three concentration 10 of neurotrophic effect in the culture medium that contains serum
-12, 10
-11, 968 of 10-10mol/L is hatched the DMEM/F-12 that does not contain serum and (is not also contained N
2Additive) the former generation cortical neuron (5b, Wistar neonatal rat 14-16d) in, fixing pair cell number, projection length and cell space area simultaneously carry out the microscopic image analysis behind the 24h.Find that cell number does not have significant change, and the cell space area reduces significantly, projection length also has certain shortening.Illustrate 968 in the culture medium of serum-free the former generation cortical neuron to In vitro culture do not have Nutrition.Infer that from mechanism 968 is the Nutrition performance functions by strengthening NGF the serum.
2,968 can obviously resist the inductive former generation cortical neurogenic cell damage of Glu
At first with 968 (10
-12-10
-10Mol/L) in the DMEM/F-12+10% hyclone, hatch 24h jointly with the former generation cortical cell (5d) of In vitro culture, to add 100ng/mlNGF as positive control.The Giu effect 30min that adds 250 μ mol/L then fixedly carries out the microscopic image analysis immediately.The excitatory neuron toxic action of Glu can make synapse cell shorten or disappearance, and the 968 cell space area of cortical neurogenic cell and the reducing of projection length that can significantly protect Glu to cause.
968 pairs of glutamic acid of table 8 cause the influence (x ± SE) of tire Corium Mus layer neural cell injury
Matched group
(100ng/ml) -12 -11 -10
Cell number 19.00 ± 4.73 42.20 ± 3.88
*28.67 ± 3.18 31.00 ± 4.01 32.33 ± 2.75
Cell space area (μ m
2) 50.98 ± 4.75 80.06 ± 1.64
*72.28 ± 2.75
*75.40 ± 2.56
*69.20 ± 2.36
*
Synapse length (μ m) 34.51 ± 2.35 59.94 ± 2.01
*49.02 ± 2.75
*49.19 ± 1.45
*39.49 ± 1.29
The lot of documents report, apoptosis is the principal mode of a large amount of neuronal deaths in the neurodegenerative diseases such as PD, AD, and heavy dose of Glu handles the apoptosis that can induce neurocyte, we add Glu 250 μ mol/L and hatch the 30min cell death inducing in former tire Mus cerebral cortex neurocyte of being commissioned to train foster, collecting cell, carry out propidium iodide (PI) fluorescence staining, detect, analyze the percentage rate of apoptotic cell with flow cytometer.Find that Glu can make 38.9% neurocyte generation apoptosis under given concentration and action time, and 10
-10The mol/L968 processed group has only 22.5% cell generation apoptosis, with the effect of the NGF of 100ng/ml quite (21.9%).
The influence that table 9 variable concentrations 968 is handled the former tire Corium Mus layer neuronal apoptosis of being commissioned to train foster of glutamate induction
( x±SE)
The normal group matched group
(100ng/ml)-12-11-10 apoptotic cell percentage rate (%) 16.13 ± 1.44 38.91+1.92 21.94 ± 1.01
*25.2 ± 3.25
*29.91 ± 1.89
*22.45 ± 1.85
*
Claims (6)
1, the purposes of tripterygium plant extract is characterized in that the application that is used to prepare prevention and treats the medicine of nervous system disease.
2, purposes as claimed in claim 1 is characterized in that described tripterygium plant comprises Radix Tripterygii Wilfordii, Tripterygium hypoglaucum and black climing.
3, purposes as claimed in claim 1 is characterized in that described tripterygium plant extract comprises one or more in Radix Tripterygii Wilfordii lactone alcohol, tripchlorolide, NSC-163063, Triptolide triol, 16-hydroxytriptolide, triptolidenol, triptophenolide and tripterine and the wilfortrine.
4, purposes as claimed in claim 1 is characterized in that described nervous system disease comprises Alzheimer, parkinson disease, Heng Tingdunshi neurodegenerative diseases and spinal cord injury, lateral spinal sclerosis disease.
5, purposes as claimed in claim 1 is characterized in that tripterygium plant extract and neurotrophic factor unite use, is used to prepare the medicine for the treatment of nervous system disease.
6, purposes as claimed in claim 5 is characterized in that described neurotrophic factor comprises nerve growth factor, glial cell line-derived neurotrophic factor, Brain Derived Neurotrophic Factor and ciliary neurotrophic factor.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00107779 CN1112181C (en) | 2000-05-26 | 2000-05-26 | Application of tripterygium plant extract in preventing and curing diseases in nervous system |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 00107779 CN1112181C (en) | 2000-05-26 | 2000-05-26 | Application of tripterygium plant extract in preventing and curing diseases in nervous system |
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| Publication Number | Publication Date |
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| CN1276209A CN1276209A (en) | 2000-12-13 |
| CN1112181C true CN1112181C (en) | 2003-06-25 |
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| CN 00107779 Expired - Lifetime CN1112181C (en) | 2000-05-26 | 2000-05-26 | Application of tripterygium plant extract in preventing and curing diseases in nervous system |
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101288671B (en) * | 2008-04-17 | 2010-12-01 | 首都医科大学 | Use of Triperugium wilfordii monomeric compound for increasing gene expression efficiency mediated by adeno-associated virus vector and subsidiarily treating neurodegenerative disease |
| CN103082285A (en) * | 2012-12-26 | 2013-05-08 | 东莞市照燕生物科技有限公司 | Nutritional health-care product for preventing encephalatrophy |
| CN104258371B (en) * | 2014-09-25 | 2016-06-15 | 中山大学 | WWW tripeptides purposes in preparation treatment Alzheimer disease drug |
| CN104274817B (en) * | 2014-09-25 | 2016-06-15 | 中山大学 | WRW tripeptides purposes in preparation treatment Alzheimer disease drug |
| WO2017214709A1 (en) | 2016-06-15 | 2017-12-21 | NOISEUX, Nicolas | Reagents, compositions and methods for improving viability and function of cells, tissues and organs |
| CN110200975B (en) * | 2019-07-18 | 2022-06-28 | 北京大学 | Pharmaceutical composition and application thereof in preparing medicines for treating neurodegenerative diseases |
| CN112472792B (en) * | 2020-12-28 | 2022-12-13 | 河北师范大学 | Application of cyclosporine A and tripterine in preparation of medicine for treating lung cancer |
| CN115400133B (en) * | 2022-09-01 | 2024-02-06 | 扬州市职业大学(扬州开放大学) | Application of begonin in preparation of medicines for treating cardiac hypertrophy |
-
2000
- 2000-05-26 CN CN 00107779 patent/CN1112181C/en not_active Expired - Lifetime
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| CN1276209A (en) | 2000-12-13 |
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